Ferritin-borocaptate sodium nanocages for the treatment of tumors

Abstract

The present invention concerns h-ferritin complexes loaded with anti-tumoral drugs for the treatment of cancer through Boron Neutron Capture Therapy.

Description

[0001] " Ferri tin-Borocaptate sodium nanocages for the treatment of tumors"

[0002] DESCRIPTION

[0003] The technology of boron neutron capture therapy (BNCT ) is now re-emerging in the field of radiotherapy as a therapy against tumors .

[0004] Radiation therapy is still required in at least 50% of cancer patients .

[0005] It is known for the serious complications , especially in patients with recurrent cancer and symptoms caused by the radiotherapy treatment .

[0006] Boron neutron capture therapy (BNCT ) has been developed with the purpose of reducing the side ef fects of radiotherapy .

[0007] The unstable form ofnBoron is formed by irradiation of10Boron; the energy irradiated by decaying in the form of a particles (4He ) and7Li recoil particles accompanied by a small amount of gamma rays produces the death of the tumoral cells without severe side ef fects o f the surrounding tissues thanks to a very short radius of reaction activity .

[0008] The main advantages of this technology comprise the possibility of precise targeting and a low toxicity .

[0009] At present , it is considered for applications in the treatment of glioblastoma multi forme , head and neck cancer, malignant melanoma, and other cancers . There is still the need to develop innovative more efficient boron delivery agents for a more efficient Boron neutron capture therapy (BNCT) .

[0010] Brief description of the figures

[0011] Figure 1: Chromatogram of the DEAE experiment performed on DEAE Sepharose.

[0012] Figure 2: SDS-PAGE analysis.

[0013] Figure 3: Chromatogram of the SEC experiment performed on Superdex 200 10 / 300 column.

[0014] Figure 4: SPR based analysis of hFerritin towards transferrin receptor 1. (A) manual immobilization of

[0015] 1500 resonance unit of transferrin receptor and subsequent injection on 100 nM hFerritin; (B) Zoom-in on the SPR curve generated by hFerritin binding to the receptor .

[0016] Figure 5: SPR based analysis of hFerritin loaded with BSH towards transferrin receptor 1. (A) manual immobilization of 300 resonance unit of transferrin receptor and subsequent injection on 100 nM hFerritin- BSH complex; (B) Zoom-in on the SPR curve generated by hFerritin-BSH binding to the receptor.

[0017] Figure 6: Kinetic parameters calculated through SPR analysis. (A) kinetic analysis referring to the fitted curve of figure 6A; (B) steady state analysis referring to the plot of figure 6B .

[0018] Figure 7: SPR based analysis of hFerritin loaded with BSH towards transferrin receptor 1 in single cycle kinetic mode. (A) hFerritin loaded with BSH has been injected at five different concentrations, namely 62,5 nM, 125 nM, 250 nM, 500 nM, and 1 pM. (B) Steady state analysis of the hFerritin-BSH complex for the binding towards transferrin receptor 1.

[0019] Figure 8: SDS-PAGE analysis of BSH-ferritin samples that have been loaded onto SPR chip.

[0020] Summary of the invention

[0021] The inventors of the present application have surprisingly found that ferritin can provide nanocages suitable for the delivery of compounds for the treatment of tumors with the Boron Neutron Capture Technology (BNCT) .

[0022] Object of the invention

[0023] A first object of the invention is represented by nanocages of the heavy chain of ferritin for the medical use in the treatment of tumors with the Boron Neuron Capture Technology (BNCT) .

[0024] A second object of the invention is represented by a process for the preparation of the disclosed nanocages.

[0025] In an aspect, the preparation comprises the incubation of a solution of ferritin and a10Boron isotope at an acidic pH in the presence of iron (IT) or (ITT) salts, followed by the incubation at basic pH.

[0026] A third object is represented by the nanocages of the heavy chain of ferritin as such. A fourth obj ect is represented by a method for the treatment of tumor with the nanocages of the heavy chain of ferritin of the invention .

[0027] A further obj ect of the invention is represented by the use of nanocages of the heavy chain of ferritin comprising a gadolinium compound as a magnetic resonance imaging (MRI ) contrast media .

[0028] Detailed description of the invention

[0029] For the purposes of the present invention, nanocages of the heavy chain of ferritin shall be intended as nanocages of the 24-chain multimer of the human ferritin heavy chain .

[0030] As is it well-known, human heavy-chain (H) ferritin is in the form of a nanocage protein constituted by 24 identical subunits that can sel f-assemble providing a globular structure characteri zed by an internal cavity .

[0031] According to the first obj ect of the invention, there are disclosed nanocages of the heavy chain of ferritin for the medical use with the Boron Neuron Capture Technology (BNCT ) .

[0032] As per the present invention, the disclosed nanocages comprise a compound of the10Boron isotope .

[0033] In a preferred aspect , said compound can be selected from boronophenylalanine (BPA) or sodium mercaptoundecahydro-closo-dodecaborate (BSH) . Other compounds that can be used include derivatives of boronophenylalanine (BPA) or of sodium mercaptoundecahydro-closo-dodecaborate (BSH) .

[0034] The nanocages can be prepared from recombinant human ferritin heavy chain .

[0035] For instance , the heavy chain of the human ferritin can be obtained from Escheri chia coii using well-known recombinant technologies .

[0036] For the purposes of the present invention, the medical use is disclosed for the treatment of tumors .

[0037] Tumors comprise solid tumors .

[0038] In particular, solid tumors are selected from the group comprising glioblastoma multi forme , head and neck cancer, malignant melanoma .

[0039] According to a second obj ect , the present invention discloses a process for the preparation of the above nanocages .

[0040] In particular, said process comprises the preparation of a solution of the nanocages of the invention comprising a compound of the10Boron i sotope as above disclosed .

[0041] For said purposes , a suitable amount of the10Boron isotope compound is added to a suitable solution of the heavy chain of the human ferritin .

[0042] For the purposes of the present invention, a suitable amount of the10Boron isotope compound is in a ratio of about 1.000:1 with the protein (1:1.000 ratio10Boron isotope compound: protein) .

[0043] According to an embodiment of the invention, the10Boron isotope compound is in a ratio of about 50 eq. with respect to the protein (ferritin) .

[0044] For the purposes of the present invention, a suitable solution of the heavy chain of the human ferritin has a preferred concentration of about 5 mg / ml (calculated with the Bradford assay) .

[0045] According to an embodiment of the present invention, the solution may also comprise iron (II) or iron (III) salts .

[0046] In a preferred embodiment, the solution may comprise iron (II) salts.

[0047] For instance, an iron (II) salt is selected from the group comprising: ammonium sulphate ( (NH4 ) Fe ( SO4 ) 2 ) , nitrate or sulphate iron (II) .

[0048] Preferably, the iron salt is added to the solution in a hFerritin : iron salt molar ratio of about 10 to 100.

[0049] According to an embodiment of the invention, the iron salt is in a ratio of about 100 eq. with respect to the protein (ferritin) .

[0050] For the purposes of the present invention, once prepared, the solution of the heavy chain of the human ferritin admixed with the10Boron isotope compound, and optionally the iron (II) or (III) salt, is brought to a basic pH or to an acidic pH. For the purposes of the present invention, a basic pH is intended of about 7-9.

[0051] NaOH may be used.

[0052] For the purposes of the present invention, an acidic pH is intended of about 2-5.

[0053] HC1 may be used.

[0054] According to an embodiment of the invention, the solution of ferritin, iron (II) o (III) salt and the10Boron isotope compound, in a suitable buffer at basic pH, is brought to an acidic pH and left incubating for a period of up to 1 hour, and preferably of 30 minutes, at room temperature at an acidic pH such as pH 2; then, the solution is brought to a basic pH.

[0055] The solution is left for a suitable period of time, possibly under stirring and then the pH is adjusted to 7-8.

[0056] According to another embodiment of the invention, the solution of ferritin and iron (II) o (III) salt in a suitable buffer at basic pH is left incubating for a period of up to 1 hour, and preferably of 10 minutes, at room temperature; then the10Boron isotope compound is added and the solution is left incubating for a period of up to 1 hour, and preferably of 20 minutes, at room temperature .

[0057] In a preferred embodiment of the present invention, there are disclosed nanocages of the heavy chain of ferritin comprising sodium mercaptoundecahydro-closo- dodecaborate (BSH) for the medical use in the treatment of tumors with the Boron Neuron Capture Technology (BNCT ) .

[0058] According to a third obj ect , the present invention discloses nanocages of the heavy chain of ferritin comprising a compound of the10Boron isotope .

[0059] In a preferred embodiment , said10Boron isotope compound is selected from boronophenylalanine (BPA) or sodium mercaptoundecahydro-closo-dodecaborate (BSH) .

[0060] Other compounds that can be used include derivatives of boronophenylalanine (BPA) or of sodium mercaptoundecahydro-closo-dodecaborate (BSH) .

[0061] The nanocages may comprise recombinant human ferritin heavy chain .

[0062] For instance , the heavy chain of the human ferritin can be obtained from Escheri chia coii using well-known recombinant technologies .

[0063] In a preferred embodiment of the present invention, there are disclosed nanocages of the heavy chain of ferritin comprising sodium mercaptoundecahydro-closo- dodecaborate (BSH) .

[0064] According to a further obj ect of the invention, there are disclosed nanocages of the heavy chain of ferritin comprising a gadolinium compound .

[0065] According to a fourth obj ect , the present invention discloses a method for the treatment of tumor with the nanocages of the heavy chain of ferritin above disclosed . In particular, said treatment is for a solid tumor.

[0066] More in particular, said treatment concerns a tumor selected from glioblastoma multiforme, head and neck cancer, malignant melanoma.

[0067] For the purposes of the present invention, said method comprises the step of administering to a subject in need thereof of a pharmaceutically acceptable amount of the nanocages of 24-multimer of the Ferritin heavy chain comprising a10Boron isotope compound of the invention .

[0068] According to an even further embodiment, there is disclosed the use of nanocages of the heavy chain of ferritin comprising a gadolinium compound as a magnetic resonance imaging (MRI) contrast media.

[0069] In particular, gadolinium compounds can be selected from the group comprising: Gd-DOTA (Gadolinium (III) 1,4,7, 10-Tetraazacyclododecane-l, 4,7, 1 O-tetraacetate ; CAS number 72573-82-1) .

[0070] The invention will be further disclosed in the following experimental section.

[0071] Subcloning strategy

[0072] In order to express the target protein in a heterologous system (E. coll) , the appropriate construct DNAs were designed including codon optimization for the host strain. The resulting DNA sequence is described below. (Optimized Sequence Length: 549 bp, GC%:52,75. Ndel and

[0073] Notl restriction sites are underlined in the below SEQ.

[0074] ID no. 1) :

[0075] The nucleotide sequence has been subcloned in pETSOb vector under Ndel and Notl restriction sites.

[0076] The protein sequence resulting from the gene translation and the biophysical parameters of the final construct are described below:

[0077] Number of amino acids: 183

[0078] Molecular weight: 21225.64

[0079] Theoretical pl: 5.31

[0080] Expression and purification steps

[0081] In-house E. coll BL21 (DE3) cells were transformed by heat shock with the vectors pET30b-hFerritin . Cells were grown in 1 liter of 2XTY medium at 37 °C to reach an optical density (OD600) of 0.6 and subsequently induced by adding IPTG at the final concentration of 0.5 mM for 2.5 h at 37°C. Culture cells were harvested by centrifugation at 8000 rpm and 4°C for 10 minutes. Bacterial pellet was than washed with phosphate buffer and stored at -80°C.

[0082] Purification procedure

[0083] Induced E. coll BL21 (DE3) cells were resuspended in lysis buffer (MES 20 mM pH 6.0) and disrupted by ultrasonication. Upon the addition of a protease inhibitor cocktail, the insoluble material of the lysate was removed by centrifugation (14,000 xg for 45 min at 4°C) , and after heat treatment at 70 °C for 15 min, the supernatant was loaded onto DEAE Sepharose anion exchange resin, pre-equilibrated with MES 20 mM pH 6.0 The purified protein was eluted with a linear NaCl gradient, from 0 mM to 1 M, in the same buffer. The eluted fractions were analyzed through SDS-GE allowing us to select the samples containing the target.

[0084] As showed in figure 1, hFerritin is present at high concentration in fractions from #8 to #19 obtaining 11 ml of the protein at 3.5 mg / ml (calculated by means of Bradford assay) . Total yield= 39 mg of protein for 1 L of bacterial culture.

[0085] Borocaptate sodium loading onto hFerritin nanocages Borocaptate sodium (BSH) (ratio 1:1000 protein / BSH) was added to hFerritin sample concentrated at 5 mg / mL using the buffer 20 mM Tris-HCl, 150 mM NaCl, pH 7.5 as mixing solution and shaken until completely solubilized.

[0086] The solution was then slowly brought to pH 2 by the addition of HC1 (0.1 M) and maintained at room temperature for 30 min with gentle shaking to allow complete disassembly of the hferritin cages into monomeric subunits. After 30 minutes, the pH was slowly brought back to pH 7.5 by the addition of NaOH (0.1 M) and the solution was kept under gentle agitation for 2 h to allow complete reassembly of the hferritin cages.

[0087] A size exclusion chromatography step was performed to remove free BSH in solution and ferritin in monomeric form (see figure 2) .

[0088] The fractions eluted from the SEC were pooled obtaining a final concentration of 4 mg / mL of hferritin loaded with BSH.

[0089] Biochemical characterization

[0090] In order to evaluate the oligomeric state of the recombinant protein as well as to remove the excess of BSH we performed a size exclusion chromatography (SEC) experiment on a pre-calibrated Superdex 200 10 / 300 column. As shown in figure 2 the protein eluted at 10,70 mL as elution volume corresponding to a calculated molecular weight of 440 kDa . Considering the molecular weight of the monomeric unit we can conclude that the recombinant hferritin exists in solution as a multimer of 24 chains, in agreement with literature data. SEC experiment has been performed in 20 mM Tris-HCl, 150mM

[0091] NaCl, pH 7.5 as mobile phase.

[0092] Interaction studies between Ferritin-BSH nanocages and Transferrin Receptor 1 by surface plasmon resonance The binding interactions has been analyzed using a Biacore X100 instruments (GE Healthcare) and the CM5 chip functionalized with an anti-His antibody, following standard procedures.

[0093] Transferrin receptor 1 receptor was purchased from ACROBiosystems (TFR-8243) (see supporting information)

[0094] 1) Transferrin Receptor 1 immobilization and manual injection of uncoupled hFerritin

[0095] Transferrin receptor 1 containing the 6x Histidine tag was immobilized on the surface of a single channel of the CM5 sensor chip, which had previously been functionalized with anti-His antibody. As showed in figure 3A the receptor binding resulted in signal of 1500 resonance unit (RU) . Uncoupled wild-type hFerritin was injected on the immobilized Transferrin receptor as positive control showing a canonical binding curve with a peculiar association and dissociation kinetic (figure 3B) .

[0096] 2) Transferrin Receptor 1 immobilization and manual injection of BSH-loaded hFerritin

[0097] Transferrin receptor 1 containing the 6x Histidine tag was immobilized on the surface of a single channel of the CM5 sensor chip, which had previously been functionalized with anti-His antibody. As showed in figure 4A the receptor binding resulted in signal of 300 resonance unit (RU) . BSH-loaded wild-type hferritin was injected on the immobilized Transferrin receptor as positive control showing a canonical binding curve with a peculiar association and dissociation kinetic (figure 4B) .

[0098] 3) Ferritin-BSH nanocages single cycle kinetic analysis In a single-cycle kinetics experiment, the BSH-loaded hFerritin nanocages solution was injected in five flushes at increasing concentrations from 1 pM to 62,5 nM over the Transferrin Receptor 1-functionalized sensor surface, using HBS-EP+ (Cytiva) as running buffer, with a contact time of 120 seconds. The reference flow cell was used as a control surface for refractive index change and nonspecific binding. A long dissociation phase (600 seconds) and a single regeneration step followed the last sample injection (NaOH 50 mM) , without regeneration between each sample injections.

[0099] The resulting SPR sensorgrams revealed good binding between Ferritin-BSH nanocages and Transferrin Receptor 1 with a dissociation constant (KD) of 4.8 nM, calculated as ratio between dissociation kinetic constant (Kd) and the association kinetic constant (Ka) (figure 5A and 6A) . The steady state analysis revealed a KD of 80.9 nM

[0100] (figure 5B and 6B) . The purity of the samples fluxed in SPR chip have been validated through SDS-PAGE analysis (figure 7) .

[0101] Borocaptate sodium loading onto hFerritin nanocages - additional protocols

[0102] Loading of BSH

[0103] Protocol 1: with opening of the cage

[0104] A solution of 5 mg / mL ferritin in TRIS HC1 buffer at pH 7.5 was sequentially added with a solution of FeSCh (100 eq. with respect to ferritin) and a solution of BSH (50 eq. with respect to ferritin) . The solution was brought to pH 2 by adding 0.1 M HC1 and the mixture was incubated for 30 minutes at room temperature.

[0105] The solution was brought back to pH 7.5 by adding a solution of 0.1 M NaOH and left for 2 hours at room temperature .

[0106] Ferritin was purified by passage on a sephadex G-25 column isolating the protein with a yield of 12%, an encapsulation efficiency of 52 ± 4% and a loading of 12 ± 2.9%.

[0107] Protocol 2: without opening the cage

[0108] A solution of 5 mg / mL ferritin in TRIS HC1 buffer at pH 7.5 was sequentially added with a solution of FeSO4 (100 eq. with respect to ferritin) , left for 10 minutes at 37°C, then a solution of BSH (50 eq. with respect to ferritin) and the mixture was incubated for 20 minutes at 37°C. Ferritin was purified by passage on a sephadex column isolating the protein with a yield of 48%, an encapsulation efficiency of 25 ± 3% and a loading of 4.6 ± 2.3%.

[0109] Loading of BPA

[0110] Protocol 1: with cage opening

[0111] A solution of FeSO4 (100 eq. with respect to ferritin) and a solution of BSH (50 eq. with respect to ferritin) were added sequentially to a 5 mg / mL ferritin solution in TRIS HC1 buffer at pH 7.5. The solution was adjusted to pH 2 by adding 0.1 M HC1 and the mixture was incubated for 30 minutes at room temperature.

[0112] The solution was adjusted to pH 7.4 by adding a 0.1 M NaOH solution.

[0113] The ferritin was purified by passage on a sephadex column, isolating the protein with a yield of 15%, an encapsulation efficiency of 42 ± 4% and a loading of 6 ± 2.7%.

[0114] Protocol 2: without opening the cage

[0115] A solution of 5 mg / mL ferritin in TRIS HC1 buffer at pH 7.5 was sequentially added with a solution of FeSCh (100 eq. with respect to ferritin) , left for 10 minutes at 37°C, then a solution of BSH (50 eq. with respect to ferritin) and the mixture was incubated for 30 minutes at 37°C.

[0116] The ferritin was purified by passage on a sephadex column isolating the protein with a yield of 79%, an encapsulation efficiency of 27 ± 3% and a loading of 3.3

[0117] ± 2.1%.

[0118] From the above reported description, the advantages of the present invention will be evident to the person skilled in the art.

[0119] First of all, HFn can be easily produced as a recombinant protein in E. coll; therefore, the present invention makes use of widespread used technology.

[0120] Also, hFn is characterized by a low immunogenicity since it is produced starting from human cDNA of H- f erritin .

[0121] A cave sphere structure as little as 12 nm can form from self- assembled 24 HFn units.

[0122] In addition, the complex has a suitable thermal and chemical stability and a high stability has also been seen in biological fluids thanks to its protein nature; therefore, the complex of the invention represents a very promising candidate for the cancer therapy.

[0123] HFN can deliver anticancer drugs specifically and selectively to tumoral cells through TfRl-mediated targeting and the subsequent receptor-mediated endocytosis, even when no further ligand functionalization occurs.

[0124] -k ~k ~k

Claims

CLAIMS1 . Nanocages of 24 -multimer of the Ferritin heavy chain for the medical use in the treatment of tumor with the Boron Neutron Capture Technology .2 . The nanocages according to the preceding claim comprising a10Boron isotope compound .

3. The nanocages according to the preceding claim 1 or 2 , wherein said10Boron isotope compound is selected from the group comprising : boronophenylalanine (BPA) or sodium mercaptoundecahydro-closo-dodecaborate (BSH) , or derivatives thereof .4 . The nanocages according to any one of the preceding claims , wherein said compound is represented by sodium mercaptoundecahydro-closo-dodecaborate (BSH) .5 . The nanocages according to any one of the preceding claims , wherein said Ferritin heavy chain is a recombinant form of heavy-chain ferritin expressed in Escheri chia coli .

6. The nanocages for the medical use in the treatment of tumors with the Boron Neutron Capture Technology according to any one of the preceding claims , wherein said tumor is a solid tumor .7 . The nanocages for the medical use in the treatment of tumors with the Boron Neutron Capture Technology according to any one of the preceding claims , wherein said tumor is selected from glioblastoma multi forme , head and neck cancer, malignant melanoma .8 . A process for the preparation of the nanocages of 24 - multimer of the Ferritin heavy chain comprising the step of admixing a solution comprising said nanocages with a10Boron isotope compound .

9. The process according to the preceding claim wherein said10Boron isotope compound is selected from the group comprising : boronophenylalanine (BPA) or sodium mercaptoundecahydro-closo-dodecaborate (BSH) , or derivatives thereof .10 . The process according to the preceding claim 8 or 9 wherein said10Boron isotope compound is represented by sodium mercaptoundecahydro-closo-dodecaborate (BSH) .11 . The process according to any one of the preceding claims 8 to 10 , wherein said solution further comprises an iron ( I T ) salt .12 . The process according to any one of the preceding claims 8 to 11 , wherein said solution comprising the nanocages and the10Boron isotope compound is brought to a basic or acidic pH .

13. The process according to any one of the preceding claims 8 to 11 , wherein said solution comprising the nanocages and the10Boron isotope compound is brought to and incubated at an acidic pH and then brought to a basic pH .14 . The process according to the preceding claim 12 or 13 comprising a further step of adj usting the pH to 7- 8 .15 . The process according to any one of the preceding claims 8 to 14 , comprising the further step of removing thefree sodium mercaptoundecahydro-closo-dodecaborate BSH and / or the monomeric hFerritin .

16. The process according to the preceding claim, wherein said step of removing is performed by si ze exclusion chromatography .17 . Nanocages of 24-multimer of the Ferritin heavy chain comprising a10Boron isotope compound .18 . Nanocages of 24-multimer of the Ferritin heavy chain comprising a10Boron isotope compound, wherein said nanocages are prepared according to the process of any one of the claims 8 to 16 .

19. Nanocages of 24 -multimer of the Ferritin heavy chain according to the preceding claim, wherein said10Boron isotope compound is selected from the group comprising : oronophenylalanine (BPA) or sodium mercaptoundecahydro- closo-dodecaborate (BSH) , or derivatives thereof .20 . Nanocages of 24 -multimer of the Ferritin heavy chain according to the preceding claim, wherein said10Boron isotope compound is represented by sodium mercaptoundecahydro-closo- odecaborate (BSH) .21 . Use of nanocages of 24 -multimer of the Ferritin heavy chain comprising a gadolinium compound as a magnetic resonance imaging (MRI ) contrast media .22 . Use of nanocages of 24 -multimer of the Ferritin heavy chain as a magnetic resonance imaging (MRI ) contrast media according to the preceding claim, wherein said gadolinium compound is Gd-DOTA.

23. Use of nanocages of 24 -multimer of the Ferritin heavy chain comprising a gadolinium compound as a magnetic resonance imaging (MRI ) contrast media according to claim 21 or 22 , wherein said nanocages are prepared according to the process of any one of claims 8 to 16 .24 . A method for the treatment of tumor comprising the step of administering to a subj ect in need thereof of a pharmaceutically acceptable amount of the nanocages of 24- multimer of the Ferritin heavy chain comprising a10Boron isotope compound according to claim 17 or 18 .25 . The method for the treatment of tumor according to the preceding claim, wherein said tumor is a solid tumor .

26. The method for the treatment of tumor according to the preceding claim 24 or 25 , wherein said tumor is selected from glioblastoma multi forme , head and neck cancer, malignant melanoma .

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