Nanostmctured proteins and uses thereof

Abstract

The present invention relates to nanostructured proteins, more particularly to fusion proteins suitable for their selective delivery to specific cell and tissue types. It also relates to nanoparticles comprising such nanostructured proteins, as well as nucleic acids, vectors, cells comprising said proteins, and therapeutic uses thereof.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of nanostructured protein materials, more specifically to fusion proteins useful for therapy. BACKGROUND

[0002] Systemic administration of drugs in the form of nanocnjugates benefits from enhanced drug stability compared to free molecules. By virtue of the chemical incorporation of functional groups in the nanoscale carrier, valuable additional properties such as cell targeting can be incorporated into a given hybrid composite material, benefiting from the high surface / volume ratio of nanomaterials. The resulting drug-loaded conjugates of size between about 8 and 100 nm can escape filtration from the kidneys without aggregation in the lungs or other highly vascularized organs when administered systemically. This fact, combined with suitable physicochemical properties of the material, can lead to prolonged circulation times and prolonged drug exposure to the target organ, thus enhancing the therapeutic impact and benefits to the patient.

[0003] Among the diversity of materials investigated as drug carriers, including metals, ceramics, polymers and carbon nanotubes, proteins offer unique properties in terms of biocompatibility and biodegradability, making them particularly attractive in the context of an increasing number of nanotoxicology issues.

[0004] However, many protein species are themselves potent drugs useful for human therapy, as evidenced by more than 400 protein-based products approved by major drug agencies. Therefore, the engineering of protein drugs as self-organizing building blocks, which exhibit intrinsic therapeutic activity upon self-assembly into nanoparticles, constitutes a favorable concept. Thus, since the nanomaterial itself acts as a nanoscale drug (desirably between 8 and 100 nm), this approach does not require further activation and drug conjugation. In this way, chemically homogeneous protein nanoparticles exhibiting intrinsic therapeutic activity (as common protein species, e.g. hormones, growth factors, vaccines, etc. are currently used in human medicine) can be biologically produced in a single step (as nanoscale assembled entities). Since the material itself acts as a drug, the possibility of drug leakage during circulation, which is an undesirable possibility especially in the case of cytotoxic agents, can be completely eliminated, which is a significant advantage over the prior art.

[0005] The inventors have previously explored this field by applying the principle of nano-architectures based on the addition of a cationic N-terminal domain plus a C-terminal polyhistidine tag to a core protein. [Serna, N. et al. 2016. Nanomedicine, 12: 1241-51] It has been described in the art that these terminal tags and the charge balance generated throughout the fusion facilitate the self-assembly and oligomerization of the monomeric protein into robust ring-shaped nanoparticles that are stable in plasma [Cespedes, M. V. et al. 2014. ACS Nano., 8: 4166-4176] and, if functionalized with a cell-targeting peptide, have a high cell penetration. [Xu, Z. K. et al. 2015. Materials Letters, 154: 140-3] Nonetheless, the building blocks of these protein structures can also comprise functional peptides in modular organization fused to the stretch, such as cell-targeting agents, endosomolytic agents or nuclear localization signals.

[0006] Therefore, it would be highly beneficial to exploit this simple protein engineering, as there is still a need in the art for drug delivery systems with enhanced selectivity and biodisponibility. SUMMARY

[0007] In a first aspect, the present application relates to a fusion protein comprising

[0008] (i) a polycationic peptide,

[0009] (ii) an intervening polypeptide region, and

[0010] (iii) a positively charged amino acid-rich region,

[0011] wherein the intervening polypeptide region is not a fluorescent protein or a human p53 alone.

[0012] In a second aspect, the present application relates to a method of preparing a nanoparticle comprising a plurality of copies of the fusion protein according to the first aspect of the present application, the method comprising placing a preparation of said fusion protein in a low salt buffer.

[0013] In a further aspect, the present application relates to a polynucleotide encoding the fusion protein according to the first aspect of the present application, a vector comprising said polynucleotide, and a host cell comprising said polynucleotide or said vector.

[0014] In a further aspect, the present application relates to a nanoparticle comprising a plurality of copies of the fusion protein of the present application or a nanoparticle obtained by the method of the present application for preparing a nanoparticle.

[0015] In yet another further aspect, the present application relates to a fusion protein, a polynucleotide, a vector, a host cell, or a nanoparticle according to the present application for use in medicine. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Design and biochemical characterization of T22-BAK-GFP-H6 nanoparticles. A) Schematic representation of the CXCR4-binding T22-BAK-GFP-H6 building block indicating its modular composition. The amino acid sequences of the CXCR4 peptide ligand T22 and the therapeutic BH3 domain of the BAK protein are shown. The length of the modules is indicated here as an approximation. The linker sequence is GGSSRSSS. B) Mass spectrometry of the purified T22-BAK-GFP-H6 fusion indicates the experimental molecular weight (33,988.762 Da). Protein integrity is also shown by immunodetection of H6 in a Coomassie blue-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis gel (Co) and a Western blot (WB). C) Hydrodynamic size distribution of T22- enabled nanoparticles in the native state and under SDS-mediated un-assembly. Herein included are the un-assembled parental BAK-GFP-H6 and GFP-H6 proteins as well as the related T22-GFP-H6 particle (and SDS-mediated un-assembled monomers) for size comparison. All proteins were solubilized in their respective storage buffer. D) FESEM images of randomly selected fields of view show the ultrastructural morphology of T22-BAK-GFP-H6 nanoparticles. Bars represent 20 nm.

[0017] Figure 2 Cellular penetration of T22-BAK-GFP-H6 nanoparticles. A) Internalization of T22-BAK-GFP-H6 nanoparticles in cultured CXCR4+ HeLa and SW1417 cells after 24 hours of exposure. The intensity of the intracellular fluorescence was corrected by specific fluorescence, yielding arbitrary units (au) representing protein amounts. B) Time-dependent intracellular accumulation of nanoparticles (2 μΜ) by HeLa cells. The inset is the viability of CXCR4-SW1417 cells after 48 hours of exposure to 2 μΜ T22-BAK-GFP-H6 nanoparticles. C) Specificity of CXCR4-mediated T22-BAK-GFP-H6 nanoparticle internalization by using the CXCR4+ inhibitor AMD3100.

[0018] Figure 3Accumulation and organ biodistribution of T22-GFP-H6 and T22-BAK-GFP-H6 nanoparticles and unassembled BAK-GFP-H6 protein in CXCR4+ colorectal tumors. A) Representative ex vivo tumor fluorescence images (FLI) at 2h, 5h, 24h and 48h after intravenous administration of a 330 pg dose of each protein. B) Quantification of GFP fluorescence in tumors at 2h, 5h, 24h and 48h using the IVIS Spectrum system. GFP signal from protein-treated mice was divided by the autofluorescence signal of each organ from buffer-treated mice to calculate the FLI ratio. Bars with # p < 0.05 are statistically significant compared to the rest of the T22-BAK-GFP-H6-treated groups. Bars with * p < 0.05 are statistically significant between the indicated groups. C) Immunohistochemistry for the His-tag domain of the nanoparticles in tumors at 5h. D) Representative ex vivo images of the material accumulated in mouse brain, lung, heart, liver, kidney and bone marrow tissues after treatment. Note that there is no or residual fluorescence in these organs compared to the tumor. E) Representative H&E staining showing no changes in the structure in any of the organs. Abbreviations: H&E, hematoxylin and eosin staining; iv, intravenous; FLI, fluorescence imaging; NP, nanoparticle.

[0019] Figure 4 Decreased proliferation index, caspase-3 activation, proteolyzed PARP, apoptosis induction and necrosis rate in tumor-bearing mice 2h, 5h, 24h and 48h after administration of T22-BAK-GFP-H6 compared to buffer and T22-GFP-H6 and BAK-GFP-H6 control counterparts. The number of mitotic figures (mitotic activity index) in tumors was quantified by H&E staining (A) and by IHC both cleaved (active) caspase-3 (B) and proteolyzed PARP (C) positive tumor cells. Bars with #, & p < 0.05 are statistically significant compared to each T22-BAK-GFP-H6-treated group; bars with * p < 0.05 are statistically significant between the indicated groups. D) Apoptotic figures were counted by nuclear condensation after Hoechst staining. Bars with * p < 0.05 are statistically significant between the indicated groups. E) Total and necrotic areas (pm2) in tumor sections were measured using Cell D software at low power field magnification. Bars with #, & p < 0.05 are statistically significant compared to each T22-BAK-GFP-H6-treated group; bars with * p < 0.05 are statistically significant between the indicated groups. 2*p<0.05. Bars indicate that it was statistically significant between 2h and 5h treatment groups. All quantifications in panels A-D were obtained by counting 10 high power fields (x400) per sample. Data are expressed as mean ± SE. All statistical analyses were performed using Mann Whitney U-test. Abbreviations: H&E, hematoxylin and eosin staining.

[0020] Figure 5 Physical and biological characterization of T22-PUMA-GFP-H6 and T22-GWH1-GFP-H6 nanoparticles. Schematic representation of the building blocks based on PUMA (A) and GWH1 (B). The amino acid sequence of the therapeutic protein stretch is indicated, while the rest of the construct is as shown in Figure 1 DLS plot of the nanoparticles (green) and the building blocks after disassembly (red) with the peak in nm next to it. Representative FESEM images of the isolated nanoparticles are also shown. Bars represent 40 nm. C) Representative ex vivo tumor fluorescence images (FLI) and normal organs (brain, kidney, lung, heart and liver tissues) after intravenous administration of a 300 pg dose of each nanoparticle. D) Quantification of the fluorescence signal (radiance efficiency) in each organ. E and F) Quantification of the number of mitotic figures detected by nuclear condensation after Hoechst staining, the number of apoptotic figures and the quantification of necrotic areas in the tumor (H&E staining) 5h after administration of T22-PUMA-GFP-H6 or T22-GWH1-GFP-H6. Quantifications were performed as shown in Figure 4

[0021] Figure 6 Characterization of H6-GFP-R9 and H6-R9-GFP proteins by DLS. Hydrodynamic size distribution of H6-GFP-R9 and H6-R9-GFP nanoparticles was determined by DLS in three independent assays.

[0022] Figure 7 ​: Characterization of GWH1 -based protein nanoparticles. A) Schematic of recombinant proteins used in this study. Box lengths are representative only. T22-GFP-H6

[13] and T22-GWH1 -GFP-H6 (Serna et al. submitted) have been fully described elsewhere. B) Mass spectrometry analysis of recombinant GWH1 -based proteins following affinity chromatography. C) Visualization of purified proteins by TGX gel chemistry following PAGE. D) Size of GWH1 -GFP-H6 nanoparticles compared to parent GFP-H6. T22-GWH1 -GFP-H6 nanoparticles are 24.6 nm in size and will be fully described elsewhere (Serna et al. submitted). E) FESEM imaging of purified GWH1 -GFP-H6 nanoparticles at different magnifications. Bars represent 50 nm.

[0023] Figure 8 : Anti-bacterial activity of GWH1 -based protein nanoparticles. A) Cell viability of different bacterial species exposed to 1.25 mg / ml GWH1 -based protein nanoparticles for 24 hours (24 hours for M. luteus). T22-GFP-H6 included as a negative control. B) Dose-dependent anti-bacterial activity of GWH1 -GFP-H6 following 24 hours (48 hours for M. luteus) of incubation. C) Bacterial cell lysis following 24 hours (48 hours for M. luteus) of incubation with 1.25 mg / ml GWH1 -GFP-H6 nanoparticles monitored by light microscopy.

[0024] Figure 9 : Cytotoxic activity of GWH1 -based protein nanoparticles. A) Protein internalization monitored by intracellular GFP fluorescence 24 hours following exposure to nanoparticles. Data has been corrected by specific fluorescence values to allow comparison on a molar basis. B) HeLa cell viability following 24 hours of exposure to 10 μΜ protein nanoparticles. C) Light microscopy of cultured HeLa cells exposed to protein nanoparticles under the conditions of Figure B.

[0025] Figure 10: Design of T22-DITOX-H6 and T22-PE24-H6 nanoparticles. A. Natural structure of A-B toxins such as diphtheria toxin (Corynebacterium diphtheriae) or exotoxin A (Pseudomonas aeruginosa). The natural toxin is divided into two fragments (A and B). Fragment A includes the catalytic domain (C-domain), while fragment B includes the translocation and receptor binding domains (T- and R-domains). The selected domains used to construct the recombinant nanoparticles are colored in dark purple (T22-PE24-H6 construct does not include the T-domain). B. Modular organization of T22-DITOX-H6 and T22-PE24-H6, where T22 acts as a CXCR4 ligand and a building tag. Functional segments are crossed by linker regions (light blue) and furin cleavage sites (dark blue, bold). A natural furin cleavage site also occurs within DITOX (dark blue, underlined), which separates the amino-terminal catalytic domain from the carboxy-terminal translocation domain. A KDEL peptide has been incorporated near the H6 region of T22-PE24-H6. Box sizes are only referential. Two additional proteins were constructed for comparison purposes, namely T22-DITOX-H6 F- and T22-PE24-H6 F-, which lack the engineered furin cleavage site (bold, dark blue region) exactly. C. Expected route of the cytotoxins of T22-DITOX-H6 and T22-PE24-H6 nanoparticles after intracellular furin-mediated release of the protein domains that are useful for biodistribution and cell penetration steps but are not associated with cell killing within CXCR4+ target cells. Color images are available upon request.

[0026] Figure 11 : Nanostructure of toxin-based proteins T22-DITOX-H6 and T22-PE24-H6. Size and SDS-mediated disassembly of T22-DITOX-H6 and T22-PE24-H6 nanoparticles were determined by DLS. Values of the peak size (mode) are indicated in bold (in nm, ± SEM). Z-potential (Zp) values of the nanoparticles are also indicated. Molecular weight of the proteins after purification was shown by Western blot on PAGE-SDS. B. FESEM examination of purified T22-DITOX-H6 and T22-PE24-H6 material. Bars represent 50 nm. Color images are available upon request.

[0027] Figure 12: Internalization of toxin-based nanoparticles in CXCR4+ cells. A. Mass spectra of pure unlabelled and ATTO-labelled (*) T22-DITOX-H6 and T22-PE24-H6 proteins. B. Dose-dependent uptake of T22-DITOX-H6* and T22-PE24-H6* nanoparticles in CXCR4+ HeLa cells after 1 h exposure. C. Time course kinetics of intracellular internalization of T22-DITOX-H6* and T22-PE24-H6* nanoparticles (1 μΜ) in CXCR4+ HeLa cells. Note the short error bars in the graph. D. Inhibition of protein (100 nM) uptake by the CXCR4 antagonist AMD3100 (+) after 1 h exposure. Significant differences between relevant data pairs are indicated as § for p<0.01. All A, B and C data are presented as mean ± SEM (n=2). E. Confocal microscopy of HeLa cells exposed to T22-DITOX-H6* and T22-PE24-H6* nanoparticles (1 μΜ) for 5 h. Cell Mask membrane stain (red) was added with the nanoparticles to visualize endosomal membranes. Nanoparticles are visible in the green and blue nuclear regions. Yellow spots represent the merging of red and green signals. Inset, 3D Imaris reconstruction of confocal stack. Bars represent 5 μm. Color images can be provided on request.

[0028] Figure 13: Specific cytotoxicity of toxin-based nanoparticles in CXCR4+ cells. A. Intracellular T22-DITOX-H6 was detected by Western blot analysis of HeLa cell extracts after 24 hours of exposure of cell cultures to nanoparticles (1 μΜ protein). M indicates migration of molecular weight markers. B. Left: Cell death induced by T22-DITOX-H6 and T22-PE24-H6 nanoparticles (10 nM) in SW1417 CXCR4- cell line and different CXCR4+ cell lines, including the isogenic CXCR4+ version of SW1417, 48 hours after exposure (72 hours for SW1417 cell line). Significant differences between relevant pairs of data are indicated as 0.01 < p < 0.05 and § p < 0.01. Right: Inhibition of HeLa cell death (induced by 10 nM of protein nanoparticles) by the CXCR4 antagonist AMD3100 or by 2 μΜ protein T22-GFP-H6. Significant differences between relevant data are indicated as a change of letters from "a" to "b". All significant results are p < 0.01. All data are presented as mean ± SEM (n = 3). C. T22-DITOX-H6 F- and T22-PE24-H6 F- promoted HeLa cell death compared to relevant T22-DITOX-H6 and T22-PE24-H6, respectively. Cells were exposed to 10 nM of each protein for 48 hours. Data and statistics as in Figure B. D. Immunocytochemical staining showing lack of CXCR4 expression in isogenic SW1417 CXCR4- cells compared to high CXCR4 expression in SW1417 CXCR4+ cells. Bars represent 50 μm. E. Different CXCR4 protein expression in these cells was assessed by immunoblotting assay. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as protein loading control. Color images are available upon request.

[0029] Figure 14 : T22-DITOX-H6* and T22-PE24-H6* nanoparticles biodistribution kinetics in a CXCR4+ colorectal cancer mouse model. Emitted ex vivo fluorescence by subcutaneous tumors and relevant organs in buffer-administered (control) and T22-DITOX-H6*- and T22-PE24-H6*-treated mice at 5 hours, 24 hours, 48 hours and 72 hours after a single intravenous dose of 50 μg or 300 μg. Emission scale is shown as radiance efficiency units (see Materials and Methods for protein nanoparticles based on diphtheria toxin (DITOX) and Pseudomonas aeruginosa exotoxin (PE24)).

[0030] Figure 15Apoptosis induction in tumors by ATTO-labeled and unlabeled T22-DITOX-H6 (50 pg) and T22-PE24-H6 (300 pg) nanoparticles. A. Representative H&E staining of subcutaneous tumors showing apoptotic figures (black arrows). No significant cell apoptosis was detected in liver tissue at the study times. Few and small inflammatory foci were observed in this organ and are indicated by yellow arrows, inflammatory foci disappeared at 72 h, returning to histologically normal tissue parenchyma. Note that the kidney had no histological changes. Bars: 50 pm. B. For each nanoparticle, the number of apoptotic cell bodies in H&E tumor sections per ten high-power fields (400x magnification) is plotted. For the study times showing higher numbers of interest for the apoptotic damage, we also show representative Hoechst staining of subcutaneous tumors at different magnifications for animals treated with the unlabeled protein version. All data are presented as mean ± SEM (n = 3). Statistical significance: a p = 0.008; b p = 0.027; c p = 0.010; d,e,f p = 0.001.

[0031] Figure 16 Pharmacokinetics, antitumor effect and mouse weight after T22-DITOX-H6 and T22-PE24-H6 administration. A. Pharmacokinetics of T22-DITOX-H6* and T22-PE24-H6* after intravenous bolus administration of 50 pg or 300 pg, respectively. Fluorescence was recorded in plasma after blood centrifugation at times 0, 1, 2, 5, 24 and 48 h (n = 3 per time point). B. Antitumor effect of T22-DITOX-H6 and T22-PE24-H6 was measured by analyzing tumor volume and number of apoptotic bodies at the end of the experiment after repeated dosing administration of each nanoparticle (10 pg, three times per week, x 8 doses). C. Evolution of mouse weight after the described protein nanoparticle repeated dose regimen. ¥ for 0.01 < p < 0.05 and § for p < 0.01. All data are presented as mean ± SEM, n = 3.

[0032] Figure 17: Physico-chemical properties of T22-mRTA-H6. A. Modular scheme and amino acid sequence of T22-mRTA-H6. mRTA is a modified fragment A of ricin in which the Asn residue 132 has been replaced by Ala (underlined). The dimensions of the boxes are only referential. B. Fractionation between insoluble (I) and soluble (S) cellular fractions in total cellular extracts after 3 hours of protein production at 37°C. SDS-PAGE analysis of T22-mRTA-H6 after one step of affinity purification revealed by Coomassie blue (CB) staining and Western Blot (WB) using anti-histidine antibodies. U and AB represent un-stained and full blue marker, respectively (Bio-Rad, Refs 161-0363 and 161-0373) and 1, 2 and 3 represent the non-specific elution peak and two peaks with increasing level of purity, respectively. The protein in peak 3 was used for further experiments. C. Hydrodynamic size (and Z potential) of T22-mRTA-H6 nanoparticles spontaneously formed at purification, determined by DLS (red line). Pdi is the polydispersity index and all the graphs are expressed in nm. The size of the monomer, determined after 40 minutes of material disaggregation with 1% SDS, is also indicated (green line). D. FESEM imaging of T22-mRTA-H6 nanoparticles at different magnifications. The bars represent 20 nm. E. Far-UV CD of T22-mRTA-H6 measured at 25°C in carbonate-bicarbonate buffer at pH 8. F. ThT fluorescence emission spectra alone (black line) or in the presence of T22-mRTA-H6 (light grey line) and T22-mRTA-H6 previously heated at 100°C (dark grey line). λex= 450 nm. At the bottom of the graph, ThT fluorescence emission at 490 nm of T22-mRTA-H6 (black bar) and T22-mRTA-H6 previously heated at 100°C (grey bar). GF. Size of T22-mRTA-H6 nanoparticles dialysed against 51 mM sodium phosphate, 158.6 mM trehalose dehydrate, 0.01% polysorbate-20 buffer at different pH values, determined by DLS. The coloured images can be provided upon request.

[0033] Figure 18Cytotoxicity and CXCR4 specificity of T22-mRTA-H6 nanoparticles. A. Viability of cultured CXCR4+ HeLa cells after 72 hours of exposure to different concentrations of T22-mRTA-H6 nanoparticles, expressed as a dose-response curve. B. Inhibition of cell death mediated by the CXCR4 antagonist AMD3100 (always at a molar excess of 10:1) in HeLa cells exposed to different concentrations of T22-mRTA-H6 nanoparticles for 72 hours. C. CXCR4 membrane protein levels determined by flow cytometry in different cell lines (3T3, MV411, THP1 and HeLa), expressed as mean fluorescence intensity ratio ± SE. D. Degree of internalization of 100 nM T22-GFP-H6 in different cell lines after 172 hours of exposure. Results are expressed as mean fluorescence intensity ratio ± SE. E. Viability of cultured CXCR4- 3T3 cells after 48, 72 hours of exposure to different concentrations of T22-mRTA-H6 nanoparticles and the small-molecular-weight antitumor drug Ara-C. Commercial CXCR4- and CXCR4+ human AML cell lines (MV411 and THP1, respectively) are included as controls. Ara-C shows cytotoxicity above 100 nM (not shown). Standard errors are presented in all bars. Significance levels are indicated by superscripts (*p < 0.05, **p < 0.01).

[0034] Figure 19Cellular penetration and intracellular toxicity of T22-mRTA-H6 nanoparticles. A. Intracellular fluorescence in cultured HeLa cells exposed to 100 nM ATTO 488 labeled T22-mRTA-H6. Extracellular fluorescence was completely removed by a hash trypsin treatment as described (Richard, J. P. et al. The Journal of biochemistry 2003, 278(1): 585). B. Under the same conditions, externalized phosphatidylserine was detected in cells exposed to unlabeled T22-mRTA-H6 by Annexin V detection kit (APC, eBioscience). Dead cells were labeled with propidium iodide (PI). Quadrant Ql shows HeLa cells labeled with PI. Q2 shows cells labeled with Annexin V and PI. Q3 shows cells without PI and Annexin V. Q4 shows cells labeled with Annexin V. Thus, dead cells are shown in Ql and Q2, while live cells are in Q3 and Q4. Apoptotic cells are shown in Q4. At the bottom, Hoechst staining of HeLa cells under the above conditions. Images were obtained by fluorescence microscopy (x400). C. Loss of JC-1 red fluorescence in cells treated with T22-mRTA-H6 as described above, indicating changes in mitochondrial Δψ. D. Cellular ROS levels detected with a fluorescent microplate assay. HeLa cells were treated with buffer, T22-mRTA-H6 (100 nM for 15 or 24 hours) or 100 μΜ pyocyanin (1 hour) as a positive control. Values are expressed as relative fluorescence units ± SE. E. Inhibition of caspases with zVAD-fmk reverses the anti-tumor activity of T22-mRTA-H6 in HeLa cells. Cells were pre-treated with 100 μΜ zVAD-fmk for 1 hour and then exposed to 100 nM T22-mRTA-H6 for 48 hours. Cell viability is expressed as percentage of cell survival compared to controls. Values are mean ± SE. Vehicle indicates treatment with buffer. Significance levels are indicated (*p < 0.05, **p < 0.01).

[0035] Figure 20Anti-tumour activity of T22-mRTA-H6 in a disseminated AML mouse model. A. Follow-up of the bioluminescence emitted by mice treated with soluble T22-mRTA-H6 nanoparticles (T22mRTA), T22-mRTA-H6 IBs (IB-T22mRTA) or buffer (VEHICLE) during the 14 days experiment by IVIS spectral analysis. B. In vivo detection of the luminescence levels in tissues such as skeleton, hind limbs, liver and spleen infiltrated with leukaemia cells of mice treated with buffer (VEHICLE), T22-mRTA-H6 IBs (IB-T22mRTA) or soluble T22-mRTA-H6 (T22mRTA) in the IVIS spectrum. C. Detection of CD45 positive cells in spleen, liver and bone marrow of mice treated with buffer (VEHICLE), T22-mRTA-H6 IBs (IB-T22mRTA) or soluble T22-mRTA-H6 nanoparticles (T22mRTA) by IHQ. T22mRTA, mice treated with soluble T22-mRTA-H6; IB-T22mRTA, group of mice treated with T22-mRTA-H6 IBs; VEHICLE, group treated with vehicle. Bars represent 50 pm. Color images can be provided upon request.

[0036] Figure 21 Histopathology in a disseminated AML mouse model after treatment with T22-mRTA-H6. Normal (heart, lung, kidney) and leukaemia infiltrated organs (bone marrow, liver, spleen) were stained with hematoxylin and eosin. Images were taken with a microscope with a 20x objective and an Olympus DP72 digital camera. H&E, hematoxylin and eosin; T22mRTA, mice treated with soluble T22-mRTA-H6; IB-T22mRTA, group of mice treated with T22-mRTA-H6 IBs; VEHICLE, group of mice treated with buffer. Bars represent 50 pm. Color images can be provided upon request. DETAILED DESCRIPTION

[0037] The authors of the present invention have observed that fusion proteins comprising a polycationic peptide and a positively charged amino acid-rich region flanked by a biologically active intervening polypeptide can be assembled into nanoparticles in which the activity of the biologically active intervening polypeptide is preserved. By means of the affinity between the polycationic region and cell surface receptors, these nanoparticles can be delivered to specific cells, thus allowing the specific delivery of biologically active polypeptides to cells of interest.

[0038] Although fusion proteins with similar structure and intervening polypeptides that are fluorescent proteins have been described in the art, the results obtained by the inventors are unexpected, both as a function of the mechanisms involved in the biological activity of fluorescent proteins, which are intrinsically different, and as a function of the pro-apoptotic peptides, cytotoxic proteins and other therapeutic polypeptides that can exert curative activity in cancer or other pathologies.

[0039] In the case of GFP and other fluorescent proteins, they have biological activity (fluorescence emission) by virtue of an intrinsic activity (proper folding and conformational structure of the fluorophore) that does not require interaction with or the participation of any external agent. The protein itself is active without any cell or cellular structure.

[0040] However, pro-apoptotic peptides, cytotoxic proteins and other therapeutic polypeptides that can exert curative activity in cancer or other pathologies do require complex interactions with cellular structures and cellular proteins that allow reaching the appropriate cellular compartments (membrane crossings, etc.) at concentrations above a certain threshold (different between different therapeutic agents) that are able to trigger target cell death through complex signaling and metabolic cascades.

[0041] This means that it is not obvious or predictable that functional proteins other than fluorescent proteins can maintain biological activity and show therapeutic activity in vivo in the form of nanostructures, and can preserve this complex spectrum of activity based on specific protein-protein interactions. The activity of cytotoxic or pro-apoptotic proteins depends on living cells and correct performance in a complex intracellular cellular environment.

[0042] It is not predictable or expected that cytotoxic proteins organized into oligomeric nanostructures will maintain the entire set of interactions and biological activity intact to perform their therapeutic function.

[0043] On the other hand, it is not predictable that proteins other than GFP can be effectively produced in soluble form and be able to form stable, targeted and without any side-interactivity that would affect the desired biodistribution in vivo, nanoparticles inside the diseased tissue or cells.

[0044] Furthermore, the inventors have also generated nanostructured versions of toxins, where the protein toxin fragment is produced in bacteria with a polycationic peptide (such as the T22 peptide) and a positively charged amino acid-rich region (e.g. polyhistidine residues) on the side. These toxins are exotoxin of Pseudomonas aeruginosa, diphtheria toxin (both from bacteria) and the plant toxin ricin. All of these toxins irreversibly inhibit protein synthesis by acting as "Ribosome Inactivating Proteins" (RIPs), which are the most potent cytotoxic proteins in nature (especially ricin). These fusion proteins further comprise a protease cleavage site (e.g. furin cleavage site), so that during endosomal escape, the protein is cleaved in the endosome and released in its active toxin form with few additional amino acids. This design aims to release the most "natural" version of the active form in the cytoplasm of the target cell. The results obtained by the inventors with fusion proteins containing bacterial toxins are also completely unexpected, as they were not predictable a priori if:

[0045] • the selected segment of the toxin would work as a fusion protein,

[0046] • they would be produced in bacteria in soluble form and self-assemble,

[0047] • they would still work as regular oligomeric nanoparticles,

[0048] • the nanoparticles would be stable and selective in systemic administration,

[0049] • the protease active site would be active at this specific regulatory site,

[0050] • protease cleavage would allow the cytotoxic effect of the resulting toxin segment,

[0051] • the active toxin segment would reach its target inside the cell for proper interaction and ribosome inactivation.

[0052] Fusion proteins of the invention

[0053] In a first aspect, the present application relates to a fusion protein comprising

[0054] (i) a polycationic peptide,

[0055] (ii) an intervening polypeptide region, and

[0056] (iii) a positively charged amino acid-rich region,

[0057] wherein the intervening polypeptide region is not a fluorescent protein or human p53 alone.

[0058] The term "fusion protein" is well known in the art and refers to a single polypeptide chain that is artificially designed to include two or more sequences from different sources, natural and / or artificial. By definition, a fusion protein is never found in nature as such.

[0059] The term "single polypeptide chain" as used herein means that the polypeptide components of the fusion protein can be conjugated end-to-end, but can also include one or more optional peptide or polypeptide "linkers" or "spacers" inserted therebetween by covalent bonds.

[0060] As used herein, the terms "peptide" or "polypeptide" generally refer to a straight chain of about 2 to 40 amino acid residues linked together with peptide bonds. It will be appreciated that the terms "peptide bond," "peptide," "polypeptide," and protein are known to those skilled in the art. From here on, "peptide" and "polypeptide" will be used indistinguishably.

[0061] As used herein, "amino acid residue" refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimetic known in the art. In certain embodiments, the residues of a protein or peptide are contiguous, without any non-amino acid interrupting the sequence of amino acid residues. In other embodiments, the sequence can include one or more non-amino acid moieties. In specific embodiments, the sequence of residues of a protein or peptide can be interrupted by one or more non-amino acid moieties.

[0062] A. Polycationic Peptides

[0063] As used herein, the term "polycationic peptide" or "first positively charged amino acid-rich region" corresponds to a polypeptide sequence comprising multiple positively charged amino acids. The polycationic peptide can be formed of positively charged amino acids only, or can comprise other amino acids, provided that the overall net charge of the region is positive at pH 7.

[0064] It is well known in the art that amino acids and their corresponding amino acid residues have different properties depending on their side chains, and can be grouped according to those properties. Thus, at physiological pH, five amino acids display a charge: arginine, histidine, and lysine are positively charged, while aspartate and glutamate are negatively charged. Then, the skilled person will recognize that the polycationic peptides of the present invention correspond to polypeptides having a net charge of more than one positive charge under physiological pH conditions. Thus, the polycationic peptides of the present invention are not limited by the presence of one or more negatively charged amino acid residues, as long as there are always enough positively charged amino acid residues to produce two or more net positive charges.

[0065] Thus, in one embodiment of the present invention, the polycationic peptides of the present invention are selected from:

[0066] (i) an arginine-rich sequence,

[0067] (ii) a sequence capable of specific interaction with a receptor on the surface of a cell and promoting internalization of the fusion protein on said cell,

[0068] (iii) a GW-H1 peptide,

[0069] (iv) a CD44 ligand,

[0070] (v) a peptide capable of crossing the blood-brain barrier,

[0071] (vi) a cell-penetrating peptide, and

[0072] (vii) a nucleolin-binding peptide.

[0073] (i) an arginine-rich sequence

[0074] As previously mentioned, arginine amino acids and residues thereof exhibit a positive charge at physiological pH. It will be understood that an "arginine-rich sequence" refers to a polypeptide sequence comprising a plurality of arginine residues. Thus, a polypeptide sequence can include 33%, preferably 40%, preferably 45%, preferably 50%, preferably 55%, preferably 60%, preferably 65%, preferably 70%, preferably 75%, preferably 80%, preferably 85%, more preferably 90%, more preferably 95%, even more preferably 99%, yet even more preferably 100% of the amino acid residues of its complete sequence as arginine residues. It will be understood that whenever a sequence of an arginine-rich sequence includes less than 100% of the sequence as arginine residues, these residues need not all be adjacent or contiguous to each other.

[0075] The skilled person will recognize that a polypeptide having one or more arginine residues will be a polycationic peptide, provided that the total positive charge of the polypeptide at physiological pH is 2 or greater, not only from the positive charge of the arginine residues, but also from any other positively charged amino acids.

[0076] In an embodiment of the application, the polycationic peptide of the application is an arginine-rich sequence.

[0077] In a preferred embodiment of the application, the arginine-rich sequence of the polycationic peptide of the application is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

[0078] (ii) a sequence capable of specific interaction with a receptor on the surface of a cell and promoting internalization of the fusion protein on said cell

[0079] As used herein, the term "a sequence capable of specifically interacting with a receptor on the surface of a cell and promoting internalization of the fusion protein on said cell" refers to any polypeptide sequence that binds to a receptor on the surface of a cell, wherein the receptor undergoes endocytosis in response to the binding of said polypeptide sequence. This binding specificity allows the delivery of the polypeptide sequence and, as a part of it, the rest of the fusion protein to a cell, tissue or organ expressing said receptor. In this way, the fusion protein comprising said polypeptide sequence will be specific to said cell when administered to an animal or contacted with different types of cell populations in vitro.

[0080] The term "receptor" refers to a cell-associated protein that binds to a biologically active molecule called "ligand". Both "receptor" and "ligand" are generally known to those skilled in the art.

[0081] As used herein, "internalization" refers to the process by which a molecule or a construct comprising a molecule binds to a target element on the outer surface of a cell membrane and the resulting complex is internalized by the cell. Internalization can occur after the resulting complex is dissociated within the cytoplasm. The target element can then be localized to a specific cellular compartment together with the molecule or construct. Preferably, the polycationic peptide of the application will promote endosomal escape of the fusion protein in addition to promoting internalization.

[0082] In another preferred embodiment, the fusion protein of the application comprises a peptide that allows translocation of the protein to the cytosol and avoids its lysosomal degradation. In one embodiment, the peptide that allows translocation of the protein to the cytosol is a peptide comprising or consisting of the KDEL sequence (SEQ ID NO. 48). In a further preferred embodiment, the peptide that allows translocation of the protein to the cytosol is located at the C-terminal domain of the fusion protein.

[0083] A wide variety of uptake receptors and carriers and even a broader number of receptor-specific ligands are known in the art.

[0084] Non-limiting examples of receptors that can be targeted by the polycation of the application include angiotensin receptors, bombesin receptors, bradykinin receptors, calcitonin receptors, chemokine receptors, cholecystokinin receptors, corticotropin-releasing factor receptors, endothelin receptors, ephrin receptors, formyl peptide receptors, Frizzled receptors, galanin receptors, ghrelin receptors, neurokinin B receptors, melanocortin receptors, neuromedin FF / Neuromedin AF receptors, neuromedin S receptors, neuromedin W / Neuromedin B receptors, neuromedin Y receptors, neurotensin receptors, orexin receptors, peptide P518 receptors, somatostatin receptors, tachykinin receptors, Toll-like receptors, vasopressin and oxytocin receptors and VEGF receptors.

[0085] In preferred embodiments of the application, the polycationic peptide comprising a sequence capable of specifically interacting with a receptor on the surface of a cell and facilitating internalization of the fusion protein on said cell is a CXCR4 ligand.

[0086] As used herein, the term "CXCR4" refers to a G protein-coupled seven-transmembrane chemokine receptor. Like other chemokine receptors, CXCR4 plays an important role in immune and inflammatory responses by mediating the directed migration and activation of leukocytes. CXCR4 is expressed or overexpressed in a variety of cancer cell lines and tissues, including breast, prostate, lung, ovarian, colon, pancreatic, kidney, and brain, as well as non-Hodgkin's lymphoma and chronic lymphocytic leukemia. The only known ligand for CXCR4 is stromal cell-derived factor-1 (SDF-1 or CXCL12). The interaction between CXCR4 and SDF-1 plays an important role in multiple stages of tumorigenesis, including tumor growth, invasion, angiogenesis, and metastasis.

[0087] As used herein, the expression "specifically binds to CXCR4" refers to the ability of the conjugate of the application to bind to CXCR4 or a cell expressing it more frequently, more rapidly, more persistently, and / or with higher affinity than to alternative receptors or cells, without substantially binding to other molecules.

[0088] For example, binding affinity can be measured by the oil pad method [see Hesselgesset et al., 1998, J. Immunol., 160: 877-883] as described by Tamamura et al., which involves contacting the peptide with a CXCR4-transfected cell line (e.g., CHO cells) and a labeled CXCR4 ligand (e.g., I-SDF-1α), and measuring the percentage inhibition of binding of the labeled CXCR4 ligand by the targeting peptide. 125 I-SDF-1α) and measuring the percentage inhibition of binding of the labeled CXCR4 ligand by the targeting peptide.

[0089] For example, specific binding can be exhibited by a low affinity targeting agent having a Kd of at least about 10 -4 M. For example, if CXCR4 has more than one binding site for a ligand, a low affinity ligand can be used for targeting. Specific binding can also be exhibited by a high affinity ligand, for example, a ligand having a Kd of at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 - 10 M or a ligand having a Kd of at least about 10 -11 M or 10 -12 M or higher. Both low affinity and high affinity targeting ligands can be used in the conjugates of the application.

[0090] As used herein, the expression "promote endosomal escape" refers to the ability of a polycationic peptide or an endosomal escape peptide to induce release of the fusion protein from the endosomal compartment after internalization by receptor-mediated endocytosis.

[0091] In the case where the conjugate comprises a fluorescent protein, such as GFP, the ability of the conjugate of the application to be internalized by cells expressing CXCR4 can be conveniently determined by fluorescence methods. Such a fusion protein can be obtained by preparing a recombinant nucleic acid in which the nucleic acid encoding the T22 peptide and the fluorescent protein are fused in frame and expressed in a suitable host cell or organism. The fusion protein is then contacted with a culture of cells expressing CXCR4 or in vivo with a tissue expressing CXCR4 for a suitable amount of time, after which a fluorescence microscope can be used to determine whether the construct has penetrated the cell. The presence of fluorescence in the cytoplasm can be further investigated by comparing the fluorescence microscope image produced by the fluorescent protein to that obtained with a known cytoplasmic stain.

[0092] In an even more preferred embodiment of the application, the CXCR4 ligand is selected from the T22 peptide (SEQ ID NO: 5), the VI peptide (SEQ ID NO: 6), the CXCL12 peptide (SEQ ID NO: 7), the vCCL2 peptide (SEQ ID NO: 8) or a functionally equivalent variant thereof.

[0093] The T22 peptide corresponds to a peptide derived from the protein polyphemusin II (extracted from blood cell debris from Lymulus polyphemus). VCCL2 corresponds to the viral macrophage inflammatory protein-II, a homolog of the human chemokine CCL2 encoded by human herpesvirus 8. The VI peptide corresponds to residues 1-21 of the N-terminus of vCCL2. CXCL12, C-X-C motif chemokine 12, also known as stromal cell-derived factor 1 (SDF1), is a member of the chemokine family that acts as a proinflammatory mediator. All four peptides are known to have an interaction with the CXCR4 receptor as shown in Liang, X. 2008. Chem. Biol. Drug. Des. 72:91-110.

[0094] In one embodiment, the targeting peptide is selected from:

[0095] - the T140 peptide having the sequence RRX1CYRKX2PYRX3CR (SEQ ID NO: 9), wherein X1 is L-3-(2-naphthyl)alanine, X2 is D-Lys and X3 is L-citrulline.

[0096] — a TN14003 peptide having the sequence RRX1CYX2KX3PYRX4CR (SEQ ID NO: 10), wherein X1 is L-3-(2-naphthyl)alanine, X2 is L-citrulline, X3 is dLys and X4 is L- citrulline,

[0097] — a TC14012 peptide having the sequence RRX1CYEKX2PYRX3CR (SEQ ID NO: 11), wherein X1 is L-3-(2-naphthyl)alanine, X2 is D-citrulline and X3 is L- citrulline,

[0098] — a TE14011 peptide having the sequence RRX1CYX2KX3PYRX4CR (SEQ ID NO: 12), wherein X1 is L-3-(2-naphthyl)alanine, X2 is L-citrulline, X3 is D-Glu and X4 is L-citrulline, and

[0099] — a TZ14011 peptide having the sequence RRX1CYX2KX3PYRX4CR (SEQ ID NO: 13), wherein X1 is L-3-(2-naphthyl)alanine, X2 is L-citrulline, X3 is D-Lys and X4 is L-citrulline, or a variant thereof wherein the N-terminal arginine residue is acetylated (known as Ac-TZ14011).

[0100] The terms "functional variant" and "functionally equivalent variant" are interchangeable and are understood herein to mean all those peptides derived from the T22, V1, CXCL12 and / or vCCL2 peptides by modification, insertion and / or deletion of one or more amino acids, provided that the function of binding to CXCR4 and internalizing the fusion protein is substantially maintained.

[0101] In one embodiment, functionally equivalent variants of the cationic polypeptides are those showing a degree of identity of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% relative to the human T22, V1, CXCL12 and / or vCCL2 peptides according to their respective SEQ ID NO. The degree of identity between two amino acid sequences can be determined by conventional methods, for example, by standard sequence alignment algorithms known in the art, such as, for example, BLAST [Altschul S.F. et al., J. Mol. Biol.,. 1990 Oct 5; 215(3):403-10]. The cationic polypeptides of the application can include post-translational modifications, such as glycosylation, acetylation, isovalerylation, myristoylation, proteolytic processing, etc.

[0102] Alternatively, suitable functional variants of the cationic polypeptides are those in which one or more positions contain an amino acid that is a conservative substitution of an amino acid present in the T22, V1, CXCL12 and / or vCCL2 peptides mentioned above. A "conservative amino acid substitution" is one resulting from replacing one amino acid with another that has similar structural and / or chemical properties. For example, the following six groups each contain amino acids that are conservative substitutions of one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). Selection of such conservative amino acid substitutions is within the capability of those skilled in the art, and is described, for example, by Dordo et al. [J. Mol. Biol, 1999, 217; 721-739] and Taylor et al. [J. Theor. Biol., 1986, 119:205-218].

[0103] A suitable assay for determining whether a given peptide can be considered a functional equivalent variant thereof is, for example, the following assay: the putative T22, V1, CXCL12 or vCCL2 peptide variant is fused in frame with a marker polypeptide (e.g. a fluorescent protein). Such a fusion protein can be obtained by preparing a recombinant nucleic acid in which the nucleic acids encoding the peptide and the fluorescent protein are fused in frame and expressed in a suitable host cell or organism. The fusion protein is then contacted with a culture of cells CXCR4 (e.g. HeLa cells) for an appropriate time, after which time a fluorescence microscope can be used to determine whether the construct has penetrated the cell. If the peptide is a functionally equivalent variant of the corresponding peptide, the marker protein will be internalized and the presence of fluorescence will be visible in the cytoplasm of the cell. Furthermore, the performance of the functional equivalent variant can be analyzed by comparing the fluorescence microscope image produced by the fluorescent protein with the fluorescence microscope image obtained with a known cytoplasmic stain (e.g. DAPI).

[0104] (iii) GW-H1 peptide

[0105] The GW-H1 peptide was previously described by Chen and colleagues [Chen, Y-L.S. et al. 2012. Peptides, 36:257-265]. The GW-H1 peptide was first selected as an antimicrobial peptide, but it is also characterized by its ability to bind cell membranes, to internalize itself into the cytoplasm and to migrate to the nucleus of eukaryotic cells. Once inside the cell, GW-H1 is able to induce apoptosis. It has been proposed that GW-H1 exerts its cytolysic activity by folding into an amphipathic helix [Chen and colleagues, supra]. Thus, the peptide is thought to exert its cytolysic effect through two consecutive events, including binding to the cell membrane, followed by permeabilization.

[0106] In a preferred embodiment of the application, the polycationic peptide of the application is a GW-H1 peptide, having SEQ ID NO: 14.

[0107] (iv) CD44 ligand

[0108] CD44 is a cell surface transmembrane glycoprotein involved in cell-cell and cell-matrix interactions, cell adhesion and migration. CD44 has been implicated in inflammation and diseases such as cancer [Bajorath, J. 2000. Proteins. 39:103-111]. Many isoforms are known, which are expressed in a cell-specific manner and are also differentially glycosylated.

[0109] Thus, a "CD44 ligand" will be a molecule able to bind CD44. CD44 is the main surface receptor for hyaluronan, a component of the extracellular matrix, but it has other ligands such as chondroitin sulfate, fibronectin, osteopontin, tenascin, collagen and the heparin inhibitory domain of laminin. In addition, CD44 can also interact with metalloproteinases and selectins.

[0110] In an embodiment of the application, the polycationic peptide of the application is a CD44 ligand. In a preferred embodiment of the application, the CD44 ligand is selected from A5G27 (SEQ ID NO: 15) and FNI / II / V (SEQ ID NO: 16).

[0111] The peptide FNI / II / V corresponds to the HBFN-fragment V of fibronectin. The peptide A5G27 corresponds to a peptide of the alpha 5 chain of laminin [Pesarrodona, M et al. 2014. Int. J. of Pharmaceutics. 473:286-295].

[0112] (v) Peptide able to cross the blood-brain barrier

[0113] It is well known in the art that the main obstacle for the development of brain pathologies treatment methods is the blood brain barrier (BBB). The brain is protected from potentially toxic substances by the presence of two barrier systems: the blood brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). The BBB is considered the main route for the uptake of serum ligands as it has an approximately 5000-fold greater surface area than the BCSFB. The brain endothelial cells that constitute the BBB represent the main obstacle for the use of potential drugs against a variety of CNS disorders. Generally, only small lipophilic molecules can cross the BBB, i.e. from the blood circulation system to the brain. Many drugs with smaller size or higher hydrophobicity show promising results in animal studies for the treatment of CNS disorders.

[0114] Thus, a "peptide capable of crossing the blood brain barrier" will be a peptide capable of transporting itself and any molecule, preferably a protein, to which it binds, from the bloodstream to the CNS.

[0115] In 1983, it was reported that a peptide, β-casopain-5, can overcome the blood brain barrier [Ermisch, A. et al. 1983. J. of Neurochemistry. 41 : 1229-1233]. Since then, many other peptides with BBB penetration properties have been identified, characterized and classified, and in 2012 a comprehensive database was established, as reported by Van Dorpe et al. [Van Dorpe, S. et al. 2012. Brain Struct. Funct. 217: 687-718]. Most of the peptides listed in the aforementioned database are suitable for use in the fusion proteins of the present application.

[0116] In an embodiment of the present application, the polycationic peptide of the present application is a peptide capable of crossing the blood brain barrier. In a preferred embodiment of the present application, the peptide capable of crossing the blood brain barrier is selected from Seq-1-7 (SEQ ID NO: 17), Seq-1-8 (SEQ ID NO: 18) and Angiopep-2-7 (SEQ ID NO: 19).

[0117] (vi) Cell penetrating peptides (CPPs)

[0118] The term "cell penetrating peptide" (CPP) refers to a peptide, typically of about 5-60 amino acid residues in length, which can facilitate cellular uptake of a molecular cargo, particularly a moiety of a protein. A protein can present one or more CPPs. A CPP can also be characterized as being able to facilitate movement of or across one or more of a lipid bilayer, a cell membrane, an organelle membrane, a vesicle membrane, or a cell wall. CPPs herein will be polycationic. Examples of CPPs useful herein are disclosed in Schmidt et al. [2010. FEBS Lett. 584: 1806-1813], Holm et al. [2006. Nature Protocols 1 : 1001-1005], Yandek et al. [2007. Biophys. J. 92: 2434-2444], Morris et al. [2001. Nat. Biotechnol. 19: 1173-1176], and U.S. Patent Application Publication No. 2014 / 0068797, and further description of CPPs in general. CPPs do not rely on transporters or receptors, thereby facilitating direct transport of the protein of which they are a part through a lipid bilayer without the involvement of any other cellular components.

[0119] (vii) a nucleolin binding peptide

[0120] Nucleolin is a eukaryotic phosphoprotein involved in ribosomal synthesis and maturation. The protein is present in multiple cellular locations. How cell surface nucleolin is involved in signal transduction in cancer cells has been described [Reyes-Reyes, E. & Akiyama, S. K. 2008. Exp. Cell Res. 314: 2212-2223] as well as how the use of cell surface nucleolin antagonists inhibits tumor growth and angiogenesis [Destouches, D. et al. 2008. PLoS One. 3(6):e2518].

[0121] Accordingly, a "nucleolin binding peptide" is a peptide that is capable of binding to a nucleolin protein in a cell, preferably to a cell surface expressed fraction of nucleolin.

[0122] In an embodiment of the application, the polycationic peptide of the application is a nucleolin binding peptide.

[0123] International Patent Application Publication No. WO 2011 / 031477 A2 provides many examples of nucleolin binding peptides suitable for use in the fusion proteins of the application.

[0124] In a preferred embodiment of the application, the nucleolin binding peptide of the application is the peptide of sequence SEQ ID NO: 20.

[0125] B. Positively charged amino acid rich regions

[0126] As used herein, the term "positively charged amino acid" or "second positively charged amino acid-rich region" refers to a polypeptide sequence distinct from the polycationic region or the first positively charged amino acid-rich region, characterized in that it comprises a plurality of positively charged amino acids. In addition, the positively charged amino acid-rich region can be formed exclusively of positively charged amino acids, or can comprise other amino acids, so long as the overall net charge of the region is positive at pH 7. Thus, the positively charged amino acid-rich region sequence can include 33%, preferably 40%, preferably 45%, preferably 50%, preferably 55%, preferably 60%, preferably 65%, preferably 70%, preferably 75%, preferably 80%, preferably 85%, more preferably 90%, more preferably 95%, even more preferably 99%, yet even more preferably 100% of the amino acid residues of its complete sequence as positively charged amino acid residues.

[0127] The positively charged amino acid-rich region can comprise only one type of positively charged amino acid, or can comprise more than one type of positively charged amino acid. In one embodiment, the positively charged amino acid-rich region is a polyhistidine region. In one embodiment, the positively charged amino acid-rich region is a polyarginine region. In one embodiment, the positively charged amino acid-rich region is a polyhistidine region. In one embodiment, the positively charged amino acid-rich region includes lysine and arginine residues. In one embodiment, the positively charged amino acid-rich region includes lysine and histidine residues. In one embodiment, the positively charged amino acid-rich region includes arginine and histidine residues. In one embodiment, the positively charged amino acid-rich region includes lysine, arginine, and histidine residues.

[0128] In some embodiments, the positively charged amino acid-rich region includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 positively charged amino acid residues, wherein the positively charged amino acid can be histidine, lysine, arginine, or a combination thereof.

[0129] In some embodiments, the positively charged amino acid-rich region comprises fewer than 100, fewer than 90, fewer than 80, fewer than 70, fewer than 60, fewer than 50, fewer than 40, fewer than 30, fewer than 29, fewer than 28, fewer than 27, fewer than 26, fewer than 25, fewer than 24, fewer than 23, fewer than 22, fewer than 21, fewer than 20, fewer than 19, fewer than 18, fewer than 17, fewer than 16, fewer than 15, fewer than 14, fewer than 13, fewer than 12, fewer than 11, fewer than 10, or fewer positively charged amino acid residues, wherein the positively charged amino acid can be histidine, lysine, arginine, or a combination thereof.

[0130] In some embodiments, the positively charged amino acid-rich region comprises 2 to 50 amino acids, 2 to 40 amino acids, 2 to 30 amino acids, 2 to 25 amino acids, 2 to 20 amino acids, 2 to 10 amino acids, or 2 to 8 amino acids.

[0131] In some embodiments, the positively charged amino acid-rich region comprises 3 to 50 amino acids, 3 to 40 amino acids, 3 to 30 amino acids, 3 to 25 amino acids, 3 to 20 amino acids, 3 to 10 amino acids, or 3 to 8 amino acids. In some embodiments, the positively charged amino acid-rich region comprises 4 to 50 amino acids, 4 to 40 amino acids, 4 to 30 amino acids, 4 to 25 amino acids, 4 to 20 amino acids, 4 to 10 amino acids, or 4 to 8 amino acids. In some embodiments, the positively charged amino acid-rich region comprises 5 to 50 amino acids, 5 to 40 amino acids, 5 to 30 amino acids, 5 to 25 amino acids, 5 to 20 amino acids, 5 to 10 amino acids, or 5 to 8 amino acids.

[0132] In embodiments of the application, the positively charged amino acid-rich region of the fusion protein of the application is a poly-histidine region. In preferred embodiments of the application, the poly-histidine region comprises 2 to 10 consecutive histidine residues.

[0133] In embodiments of the application, the positively charged amino acid-rich region of the fusion protein of the application is a poly-arginine region. In preferred embodiments of the application, the poly-arginine region comprises 2 to 10 consecutive arginine residues.

[0134] In embodiments of the application, the positively charged amino acid-rich region of the fusion protein of the application is a poly-lysine region. In preferred embodiments of the application, the poly-lysine region comprises 2 to 10 consecutive poly-lysine residues.

[0135] C. Relative position of elements of the fusion protein and the linking element

[0136] The various elements of the fusion protein of the present application (the polycationic peptide, the intervening polypeptide region, and the positively charged amino acid-rich region) can be placed in any relative order, so long as the polycationic peptide and the positively charged amino acid-rich region function at either end of the fusion protein, and the intervening polypeptide region also maintains all or part of its function.

[0137] As used herein, the terms "N-terminal," "N-terminus," and "amino terminal" are used interchangeably with respect to polypeptides. Likewise, the terms "C-terminal," "C-terminus," and "carboxyl terminal" are considered equivalent. These terms are the customary usage of those skilled in the art with respect to the free portion of the amino acid at the end of a polypeptide chain included in a protein.

[0138] Thus, in one embodiment of the present application, the polycationic peptide of the fusion protein is at the N-terminus of the protein, and the positively charged amino acid-rich region of the fusion protein is at the C-terminus of the protein. In another embodiment of the present application, the positively charged amino acid-rich region of the fusion protein is at the N-terminus of the protein, and the polycationic peptide of the fusion protein is at the C-terminus of the protein. In another embodiment of the present application, the intervening polypeptide region can be at either the C-terminus or the N-terminus of the fusion protein, with the polycationic peptide in the middle of the fusion protein, and the positively charged amino acid-rich region at the end of the fusion protein opposite the intervening polypeptide region, or the positively charged amino acid-rich region in the middle of the fusion protein, and the polycationic peptide at the end of the fusion protein opposite the intervening polypeptide region.

[0139] Thus, the relative order of the elements of the fusion protein according to the present application can be:

[0140] ■N-polycationic peptide-intervening region polypeptide-positively charged amino acid-rich region-C;

[0141] ■N-positively charged amino acid-rich region-intervening region polypeptide-polycationic peptide-C;

[0142] ■N-polycationic peptide-positively charged amino acid-rich region-intervening region polypeptide-C;

[0143] ■N-positively charged amino acid-rich region-polycationic peptide-intervening region polypeptide-C;

[0144] ■N-intervening region polypeptide-polycationic peptide-positively charged amino acid-rich region-C; or

[0145] ■N-intervening region polypeptide-positively charged amino acid-rich region-polycationic peptide-C

[0146] The terms "N-terminal" and "C-terminal" do not imply that the components need to be conjugated directly end-to-end, but rather that they maintain the relative order of the positions, regardless of whether additional elements such as linkers / spacers are present at the ends of the components or inserted therebetween.

[0147] Thus, the fusion proteins of the present application comprise the above-mentioned elements ((1) a polycationic peptide, (2) an intervening polypeptide region, and (3) a positively charged amino acid-rich region), and these can be conjugated end-to-end, but can also comprise one or more optional peptide or polypeptide "linkers" or "spacers" inserted therebetween, preferably linked by peptide bonds.

[0148] According to the present application, the spacer or linker amino acid sequence can act as a hinge region between components (1) and (2), (2) and (3), and (1) and (3), which allows them to move independently of each other, while maintaining the three-dimensional form of the individual domains, such that the presence of the peptide spacer or linker does not alter the functionality of any of components (1), (2), and (3). In this sense, a preferred intermediate amino acid sequence according to the present application will be a hinge region, characterized by a structural flexibility that allows this movement. In a particular embodiment, the intermediate amino acid sequence is a flexible linker. The role of the linker region is to provide space between components (1) and (2), and (2) and (3). Thus, it is ensured that the secondary and tertiary structure of components (1), (2), or (3) is not affected by the presence of the other. The spacer has polypeptide properties. The linker peptide preferably comprises at least 2 amino acids, at least 3 amino acids, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, or about 100 amino acids.

[0149] The spacer or linker can be bound by covalent bonds, preferably by peptide bonds, flanking the components of the conjugate of the present application; and it is also preferred that the spacer is essentially functional, and / or does not tend to proteolytic cleavage, and / or does not comprise any cysteine residues. Similarly, the three-dimensional structure of the spacer is preferably linear or essentially linear.

[0150] Preferred examples of spacer or linker peptides include those that have been used to bind proteins without substantially reducing the function of the binding peptide or at least substantially reducing the function of one of the binding peptides. More preferably, the spacer or linker for binding peptides comprises a coiled coil structure.

[0151] Preferred examples of linker peptides include 2 or more amino acids selected from glycine, serine, alanine and threonine. A preferred example of a flexible linker is a polyglycine linker. Possible examples of linker / spacer sequences include SGGTSGSTSGTGST (SEQ ID NO: 21), AGSSTGSSTGPGSTT (SEQ ID NO: 22) or GGSGGAP (SEQ ID NO: 23). These sequences have been used to join designed coiled coils to other protein domains [Muller, K. M., Arndt, K. M. and Alber, T., Meth. Enzymology, 2000, 328: 261-281]. Further non-limiting examples of suitable linkers include the amino acid sequence GGGVEGGG (SEQ ID NO: 24), the sequence of 10 amino acid residues of the upper hinge region of murine IgG3 (PKPSTPPGSS, SEQ ID NO: 25), which has been used to generate dimeric antibodies by coiled coils [Pack, P. and Pluckthun, A., 1992, Biochemistry 31 : 1579-1584], a peptide of the sequence APAETKAEPMT (SEQ ID NO: 26), a peptide of the sequence GAP, a peptide of the sequence AAA and a peptide of the sequence AALE.

[0152] Alternatively, the components of the fusion proteins of the application can be linked by a peptide whose sequence contains a cleavage target site for a protease, thus allowing the separation of any component. Suitable protease cleavage sites for incorporation into the polypeptides of the application include enterokinase (cleavage site DDDDK, SEQ ID NO: 27), factor Xa (cleavage site IEDGR, SEQ ID NO: 28), thrombin (cleavage site LVPRGS, SEQ ID NO: 29), TEV protease (cleavage site ENLYFQG, SEQ ID NO: 30), PreScission protease (cleavage site LEVLFQGP, SEQ ID NO: 31), a furin protease (cleavage site GNRVRRSV, SEQ ID NO. 46 or RHRQPRGWEQL, SEQ ID NO. 47) inteins and the like. In a preferred embodiment, the target cleavage site is for the protease furin (cleavage site GNRVRRSV, SEQ ID NO. 46 or RHRQPRGWEQL, SEQ ID NO. 47).

[0153] In another preferred embodiment, the cleavage target site for a protease is located between any of the components of the fusion protein of the application. In a more preferred embodiment, the fusion protein comprises several cleavage target sites, each of which is comprised between different components of the fusion protein, either between the polycationic peptide and the intervening peptide, and / or between the intervening peptide and the positively charged amino acid-rich region, more particularly at the C-terminus of the polycationic peptide, at the N-terminus of the intervening peptide, at the C-terminus of the intervening peptide, and / or at the N-terminus of the positively charged amino acid-rich region. In an even more preferred embodiment, the cleavage target site is located between the polycationic peptide and the intervening peptide, and still more preferably at the N-terminus of the intervening polypeptide. In another preferred embodiment, it is located at the C-terminus of the polycationic peptide.

[0154] In another preferred embodiment, the cleavage site of the application is located at the C-terminus or N-terminus of the linking group as described herein, which is located between any of the components of the fusion protein of the application.

[0155] Thus, in embodiments of the application, the polycationic peptide is bound to the intervening polypeptide region by a linker. In another embodiment of the application, the intervening polypeptide region is bound to the positively charged amino acid-rich region by a linker. In yet another embodiment of the application, the polycationic peptide is bound to the intervening polypeptide region by a linker, and the intervening polypeptide region is also bound to the positively charged amino acid region by a linker.

[0156] As will be appreciated by the skilled person, the linker connecting the polycationic peptide to the intervening polypeptide region and the linker connecting the intervening polypeptide region to the positively charged amino acid-rich region can comprise the same sequence or different sequences, but with the proviso that the presence and / or sequence of the linker does not result in a functional alteration of the polycationic peptide, the intervening polypeptide region and / or the positively charged amino acid-rich region (for example, but not limited to, due to a modification of the secondary or tertiary structure of the fusion protein or the formation of disulfide bonds).

[0157] The above considerations regarding the relative position of the elements of the fusion protein from N-terminus to C-terminus also apply in the case where linkers are present between them, regardless of their number or what elements are placed between them. Thus, the possible combinations and relative order of the elements are as follows (where the above numbering of the elements is maintained: (1) polycationic peptide, (2) intervening polypeptide region, (3) positively charged amino acid-rich region):

[0158] ■N-(1)-(2)-(3)-C

[0159] ■N-(1)-linker-(2)-(3)-C

[0160] ■N-(1)-(2)-linker-(3)-C

[0161] ■ N-(1)-Linker-(2)-Linker-(3)-C

[0162] ■ N-(3)-(2)-(1)-C

[0163] ■ N-(3)-Linker-(2)-(1)-C

[0164] ■ N-(3)-(2)-Linker-(1)-C

[0165] ■ N-(3)-Linker-(2)-Linker-(3)-C

[0166] ■ N-(2)-(1)-(3)-C

[0167] ■ N-(2)-Linker-(1)-(3)-C

[0168] ■ N-(2)-(1)-Linker-(3)-C

[0169] ■ N-(2)-Linker-(1)-Linker-(3)-C

[0170] ■ N-(2)-(3)-(1)-C

[0171] ■ N-(2)-Linker-(3)-(1)-C

[0172] ■ N-(2)-(3)-Linker-(1)-C

[0173] ■ N-(2)-Linker-(3)-Linker-(1)-C

[0174] ■ N-(1)-(3)-(2)-C

[0175] ■ N-(1)-(3)-Linker-(2)-C

[0176] ■ N-(1)-Linker-(3)-(2)-C

[0177] ■ N-(1)-Linker-(3)-Linker-(2)-C

[0178] ■ N-(3)-(1)-(2)-C

[0179] ■ N-(3)-Linker-(1)-(2)-C

[0180] ■ N-(3)-(1)-Linker-(2)-C

[0181] ■ N-(3)-Linker-(1)-Linker-(2)-C

[0182] In a preferred embodiment of the application, the linker of the fusion protein of the application comprises the sequence GGSSRSS (SEQ ID NO: 32), the sequence of the GGGNS sequence (SEQ ID NO: 33).

[0183] The above considerations regarding the relative position of the elements of the fusion protein from N- to C-terminus also apply to the case where protease cleavage sites are present or comprised between them, regardless of their number or what elements are placed between them. Thus, the possible combinations and relative order of the elements are as follows (where the above numbering of the elements is maintained: (1) polycationic peptide, (2) intervening polypeptide region, (3) positively charged amino acid-rich region), and wherein the term "protease cleavage site" is understood as a polypeptide region consisting of or comprising a protease cleavage site:

[0184] ■N-(1)-(2)-(3)-C

[0185] ■N-(1)-protease cleavage site-(2)-(3)-C

[0186] ■N-(1)-(2)-protease cleavage site-(3)-C

[0187] ■N-(1)-protease cleavage site-(2)-protease cleavage site-(3)-C

[0188] ■N-(3)-(2)-(1)-C

[0189] ■N-(3)-protease cleavage site-(2)-(1)-C

[0190] ■N-(3)-(2)-protease cleavage site-(1)-C

[0191] ■N-(3)-protease cleavage site-(2)-protease cleavage site-(1)-C

[0192] ■N-(2)-(1)-(3)-C

[0193] ■N-(2)-protease cleavage site-(1)-(3)-C

[0194] ■N-(2)-(1)-protease cleavage site-(3)-C

[0195] ■N-(2)-protease cleavage site-(1)-protease cleavage site-(3)-C

[0196] ■N-(2)-(3)-(1)-C

[0197] ■N-(2)-protease cleavage site-(3)-(1)-C

[0198] ■N-(2)-(3)-protease cleavage site-(1)-C

[0199] ■N-(2)-protease cleavage site-(3)-protease cleavage site-(1)-C

[0200] ■N-(1)-(3)-(2)-C

[0201] ■N-(1)-(3)-protease cleavage site-(2)-C

[0202] ■N-(1)-protease cleavage site-(3)-(2)-C

[0203] ■N-(1)-protease cleavage site-(3)-protease cleavage site-(2)-C

[0204] ■N-(3)-(1)-(2)-C

[0205] ■N-(3)-protease cleavage site-(1)-(2)-C

[0206] ■N-(3)-(1)-protease cleavage site-(2)-C

[0207] ■N-(3)-protease cleavage site-(1)-protease cleavage site-(2)-C.

[0208] In an alternative embodiment, the fusion protein according to the application comprises linker regions connecting the elements of the fusion protein and protease cleavage sites between them, regardless of their number or what elements are placed between them. Thus, possible combinations and relative order of the elements are as follows (wherein the numbering of the above elements is maintained: (1) polycationic peptide, (2) intervening polypeptide region, (3) positively charged amino acid-rich region), and wherein the term "protease cleavage site" is to be understood as a polypeptide region consisting of or comprising a protease cleavage site:

[0209] ■N-(1)-(2)-(3)-C

[0210] ■N-(1)-linker-protease cleavage site-(2)-(3)-C

[0211] ■N-(1)-protease cleavage site-linker-(2)-(3)-C

[0212] ■N-(1)-linker-protease cleavage site-linker-(2)-(3)-C

[0213] ■N-(1)-(2)-protease cleavage site-linker-(3)-C

[0214] ■N-(1)-(2)-linker-protease cleavage site-(3)-C

[0215] ■N-(1)-linker-protease cleavage site-(2)-protease cleavage site-(3)-C

[0216] ■N-(1)-linker-protease cleavage site-(2)-protease cleavage site-(3)-C

[0217] ■N-(1)-linker-protease cleavage site-(2)-protease cleavage site-(3)-C

[0218] ■N-(1)-linker-protease cleavage site-(2)-protease cleavage site-(3)-C

[0219] ■N-(1)-linker-protease cleavage site-(2)-protease cleavage site-(3)-C

[0220] ■N-(1)-linker-protease cleavage site-(2)-protease cleavage site-(3)-C

[0221] ■N-(1)-linker-protease cleavage site-(2)-protease cleavage site-(3)-C

[0222] ■N-(1)-linker-protease cleavage site-(2)-protease cleavage site-(3)-C

[0223] ■N-(1)-linker-protease cleavage site-(2)-protease cleavage site-(3)-C

[0224] ■N-(1)-linker-protease cleavage site-(2)-protease cleavage site-(3)-C

[0225] ■N-(3)-linker-protease cleavage site-(2)-(1)-C

[0226] ■N-(3)-linker-protease cleavage site-(2)-(1)-C

[0227] ■N-(3)-linker-protease cleavage site-(2)-(1)-C

[0228] ■N-(3)-(2)-linker-protease cleavage site-(1)-C

[0229] ■N-(3)-(2)-linker-protease cleavage site-(1)-C

[0230] ■N-(3)-(2)-linker-protease cleavage site-(1)-C

[0231] ■N-(3)-Linker-Protease Cleavage Site-(2)-Protease Cleavage Site-(1)-C

[0232] ■N-(3)-Protease Cleavage Site-Linker-(2)-Protease Cleavage Site-(1)-C

[0233] ■N-(3)-Linker-Protease Cleavage Site-Linker-(2)-Protease Cleavage Site-(1)-C

[0234] ■N-(3)-Protease Cleavage Site-(2)-Linker-Protease Cleavage Site-(1)-C

[0235] ■N-(3)-Protease Cleavage Site-(2)-Protease Cleavage Site-Linker-(1)-C

[0236] ■N-(3)-Protease Cleavage Site-(2)-Linker-Protease Cleavage Site-Linker-(1)-C

[0237] ■N-(3)-Linker-Protease Cleavage Site-(2)-Linker-Protease Cleavage Site-(1)-C

[0238] ■N-(3)-Protease Cleavage Site-Linker-(2)-Linker-Protease Cleavage Site-(1)-C

[0239] ■N-(3)-Linker-Protease Cleavage Site-Linker-(2)-Linker-Protease Cleavage Site-(1)-C

[0240] ■N-(3)-Linker-Protease Cleavage Site-(2)-Protease Cleavage Site-Linker-(1)-C

[0241] ■N-(3)-Protease Cleavage Site-Linker-(2)-Protease Cleavage Site-Linker-(1)-C

[0242] ■N-(3)-Linker-Protease Cleavage Site-Linker-(2)-Protease Cleavage Site-Linker-(1)-C

[0243] ■N-(3)-Linker-Protease Cleavage Site-Linker-(2)-Linker-Protease Cleavage Site-Linker-(1)-C

[0244] ■N-(2)-Linker-Protease Cleavage Site-(1)-(3)-C

[0245] ■N-(2)-Protease Cleavage Site-Linker-(1)-(3)-C

[0246] ■N-(2)-Linker-Protease Cleavage Site-Linker-(1)-(3)-C

[0247] ■N-(2)-(1)-linker-protease cleavage site-(3)-C

[0248] ■N-(2)-(1)-protease cleavage site-linker-(3)-C

[0249] ■N-(2)-(1)-linker-protease cleavage site-linker-(3)-C

[0250] ■N-(2)-linker-protease cleavage site-(1)-protease cleavage site-(3)-C

[0251] ■N-(2)-protease cleavage site-linker-(1)-protease cleavage site-(3)-C

[0252] ■N-(2)-linker-protease cleavage site-linker-(1)-protease cleavage site-(3)-C

[0253] ■N-(2)-protease cleavage site-(1)-linker-protease cleavage site-(3)-C

[0254] ■N-(2)-protease cleavage site-(1)-protease cleavage site-linker-(3)-C

[0255] ■N-(2)-protease cleavage site-(1)-linker-protease cleavage site-linker-(3)-C

[0256] ■N-(2)-linker-protease cleavage site-(3)-(1)-C

[0257] ■N-(2)-protease cleavage site-linker-(3)-(1)-C

[0258] ■N-(2)-linker-protease cleavage site-linker-(3)-(1)-C

[0259] ■N-(2)-(3)-linker-protease cleavage site-(1)-C

[0260] ■N-(2)-(3)-protease cleavage site-linker-(1)-C

[0261] ■N-(2)-(3)-linker-protease cleavage site-linker-(1)-C

[0262] ■N-(2)-linker-protease cleavage site-(3)-protease cleavage site-(1)-C

[0263] ■N-(2)-protease cleavage site-linker-(3)-protease cleavage site-(1)-C

[0264] ■N-(2)-Linker-Protease Cleavage Site - Linker - (3)-Protease Cleavage Site - (1)-C

[0265] ■N-(2)-Protease Cleavage Site - (3)-Linker - Protease Cleavage Site - (1)-C

[0266] ■N-(2)-Protease Cleavage Site - (3)-Linker - Protease Cleavage Site - Linker - (1)-C

[0267] ■N-(2)-Protease Cleavage Site - (3)-Linker - Protease Cleavage Site - (1)-C

[0268] ■N-(1)- (3)-Linker - Protease Cleavage Site - (2)-C

[0269] ■N-(1)- (3)-Protease Cleavage Site - Linker - (2)-C

[0270] ■N-(1)- (3)-Linker - Protease Cleavage Site - Linker - (2)-C

[0271] ■N-(1)-Linker - Protease Cleavage Site - (3)- (2)-C

[0272] ■N-(1)-Protease Cleavage Site - Linker - (3)- (2)-C

[0273] ■N-(1)-Linker - Protease Cleavage Site - Linker - (3)- (2)-C

[0274] ■N-(1)-Linker - Protease Cleavage Site - (3)-Protease Cleavage Site - (2)-C

[0275] ■N-(1)-Protease Cleavage Site - Linker - (3)-Protease Cleavage Site - (2)-C

[0276] ■N-(1)-Linker - Protease Cleavage Site - Linker - (3)-Protease Cleavage Site - (2)-C

[0277] ■N-(1)-Protease Cleavage Site - (3)-Linker - Protease Cleavage Site - (2)-C

[0278] ■N-(1)-Protease Cleavage Site - (3)-Protease Cleavage Site - Linker - (2)-C

[0279] ■N-(1)-Protease Cleavage Site - (3)-Protease Cleavage Site - Linker - (2)-C

[0280] ■N-(3)-Linker - Protease Cleavage Site - (1)- (2)-C

[0281] ■N-(3)-protease cleavage site - linker - (1) - (2) - C

[0282] ■N-(3)-linker - protease cleavage site - linker - (1) - (2) - C

[0283] ■N-(3) - (1) - linker - protease cleavage site - (2) - C

[0284] ■N-(3) - (1) - protease cleavage site - linker - (2) - C

[0285] ■N-(3) - (1) - linker - protease cleavage site - linker - (2) - C

[0286] ■N-(3) - linker - protease cleavage site - (1) - protease cleavage site - (2) - C.

[0287] ■N-(3) - protease cleavage site - linker - (1) - protease cleavage site - (2) - C.

[0288] ■N-(3) - linker - protease cleavage site - linker - (1) - protease cleavage site - (2) - C.

[0289] ■N-(3) - linker - protease cleavage site - (1) - linker - protease cleavage site - (2) - C.

[0290] ■N-(3) - protease cleavage site - linker - (1) - linker - protease cleavage site - (2) - C.

[0291] ■N-(3) - linker - protease cleavage site - linker - (1) - linker - protease cleavage site - (2) - C.

[0292] ■N-(3) - linker - protease cleavage site - (1) - protease cleavage site - linker - (2) - C.

[0293] ■N-(3) - protease cleavage site - linker - (1) - protease cleavage site - linker - (2) - C.

[0294] ■N-(3) - linker - protease cleavage site - linker - (1) - protease cleavage site - linker - (2) - C.

[0295] ■N-(3) - linker - protease cleavage site - (1) - linker - protease cleavage site - linker - (2) - C.

[0296] ■N-(3) - protease cleavage site - linker - (1) - linker - protease cleavage site - linker - (2) - C.

[0297] ■N-(3)-Linker-Protease Cleavage Site - Linker-(1)-Linker-Protease Cleavage Site - Linker-(2)-C.

[0298] ■N-(3)-Protease Cleavage Site-(1)-Linker-Protease Cleavage Site-(2)-C.

[0299] ■N-(3)-Protease Cleavage Site-(1)-Protease Cleavage Site - Linker-(2)-C.

[0300] ■N-(3)-Protease Cleavage Site-(1)-Linker-Protease Cleavage Site - Linker(2)-C.

[0301] D. Intervening Polypeptide Region

[0302] The terms "intervening polypeptide region" and "intervening region" are considered equivalent herein.

[0303] The intervening polypeptide region of the fusion protein of the application comprises a physiologically functional peptide, which means that its interaction with a cellular component results in a physiological change. Thus, the linker region connecting the different elements of the fusion protein according to the application is not considered an intervening region. Thus, in preferred embodiments, the intervening region comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 or more amino acids.

[0304] In embodiments of the application, the intervening polypeptide region of the fusion protein of the application is a therapeutic agent.

[0305] The term "therapeutic" is used in the general sense and includes therapeutic, prophylactic and replacement agents.

[0306] The essence of the intervening region is a polypeptide, as it is part of the fusion protein of the application that has a polycationic peptide and a region rich in positively charged amino acids.

[0307] Suitable polypeptides that can be used as components of the intervening region include any polypeptide that is capable of promoting a decrease in the rate of cell proliferation.

[0308] Examples of therapeutic proteins suitable for use in the intervening region of the fusion protein of the application include, but are not limited to, cytotoxic polypeptides, anti-angiogenic polypeptides, polypeptides encoded by tumor suppressor genes, polypeptides encoded by polynucleotides capable of activating an immune response against a tumor.

[0309] Thus, in embodiments of the application, the therapeutic agent of the intervening region of the fusion protein of the application is selected from the group consisting of:

[0310] (i) a cytotoxic polypeptide,

[0311] (ii) an anti-angiogenic polypeptide,

[0312] (iii) a polypeptide encoded by a tumor suppressor gene,

[0313] (iv) a pro-apoptotic polypeptide,

[0314] (v) a polypeptide having anti-metastatic activity,

[0315] (vi) a polypeptide encoded by a polynucleotide capable of activating an immune response against a tumor,

[0316] (vii) a chemotherapeutic agent,

[0317] (viii) an anti-angiogenic molecule,

[0318] (ix) a polypeptide encoded by a suicide gene,

[0319] (x) a chaperone protein or protein aggregation inhibitor.

[0320] (i) a cytotoxic polypeptide

[0321] As used herein, the term cytotoxic polypeptide refers to an agent capable of inhibiting cellular function. The agent can inhibit proliferation or be toxic to the cell. Any polypeptide that, when internalized by a cell, interferes with or adversely alters cellular metabolism or inhibits cell growth or proliferation in any manner, including but not limited to agents that mediate their toxic effects when transported into the cell, as well as those that mediate their toxic effects at the cell surface, are included within the scope of this term. Useful cytotoxic polypeptides include protein toxins and bacterial toxins.

[0322] Examples of proteinaceous cytotoxins that can be used to incorporate into conjugates according to the present application include, but are not limited to, type one and type two ribosome inactivating proteins (RIPs). Useful type one plant RIPs include, but are not limited to, dianthin 30, dianthin 32, gossypol, saponin 1-9, pokeweed activated protein (PAP), PAP II, PAP-R, PAP-S, PAP-C, mapalmin, pokerrin, luffin-L, luffin, ebulin 1 and 2, cucumisin-A, cucumisin-B, cucumisin-S, 19K-protein synthesis inhibitory protein (PSI), 15K-PSI, 9K-PSI, alpha-kirilowin, beta-kirilowin, gelonin, momorcharin, momorcharin-II, momorcharin-Ic, MAP-30, alpha-momorcharin, beta-momorcharin, trichosanthin, TAP-29, calpains; barley RIP; flax RIP, malt-agglutinin, corn RIP, asparaginases 1 and 2 [Stirpe et al., 1992. Bio / Technology 10:405-12]. Useful type two RIPs include, but are not limited to, volkensin, ricin, securin-b, CIP-29, abrin, modeccin, ribosome inactivating protein-[alpha], ribosome inactivating protein-[beta], ribosome inactivating protein-[gamma], vircumin, porrectin, and biologically active enzymatic subunits thereof [Stirpe et al., 1992. Bio / Technology 10:405-12; Pastan et al., 1992. Annu. Rev. Biochem. 61 :331-54; Brinkmann and Pastan, 1994. Biochim. et Biophys. Acta 1198:27-45; and Sandvig and Van Deurs, 1996. Physiol. Rev. 76:949-66].

[0323] Examples of bacterial toxins that can be used as a cytotoxin include, but are not limited to, Shiga toxin and Shiga-like toxins (i.e., toxins with the same activity or structure), as well as catalytic subunits and biologically functional fragments thereof. Additional examples of useful bacterial toxins include, but are not limited to, Pseudomonas exotoxin and diphtheria toxin [Pastan et al., 1992. Annu. Rev. Biochem. 61 :331-54; and Brinkmann and Pastan, 1994. Biochim. et Biophys. Acta 1198:27-45]. Truncated forms and mutants of the toxin enzyme subunit can also be used as a cytotoxin moiety. Other targeting agents include, but are not limited to, the colicin family of more than 34 ribonuclease toxins described, including colicin A, B, D, E1-9, enterobactin DF13, and fungal ribonucleases, [alpha]-sarcin [Ogawa et al. 1999. Science 283:2097-100; Smarda et al., 1998. Folia Microbiol (Praha) 43:563-82; Wool et al., 1992. Trends Biochem. Sci., 17:266-69].

[0324] (ii) Anti-angiogenic polypeptides

[0325] The proliferation of tumor cells is largely dependent on extensive tumor vascularization that accompanies cancer progression. Thus, inhibition of neovascularization with anti-angiogenic agents and targeted destruction of existing blood vessels have been introduced as effective and relatively non-toxic methods of tumor therapy.

[0326] As used herein, the term "anti-angiogenic polypeptide" means a polypeptide capable of inhibiting angiogenesis. Suitable anti-angiogenic polypeptides include, but are not limited to, angiostatin, endostatin, anti-angiogenic antithrombin III, sFRP-4 as described in WO2007115376, and anti-VEGF antibodies such as anibizumab, bevacizumab (Avastin), Fab IMC 1121, and F200 Fab.

[0327] (iii) Polypeptides encoded by tumor suppressor genes

[0328] As used herein, a "tumor suppressor" is a gene or gene product that has the normal biological role of suppressing the unregulated growth of cells. The functional counterpart of a tumor suppressor is an oncogene, a gene that promotes normal cell growth. Mutations that activate such a gene or gene product further transform it into an oncogene that continues cell growth activity, but in an unregulated manner. Examples of tumor suppressor genes and gene products are well known in the literature and can include PTC, BRCA1, BRCA2, pl6, APC, RB, WTl, EXTl, p53, NFl, TSC2, NF2, VHL, ST7, ST14, PTEN, APC, CD95, or SPARC.

[0329] (iv) pro-apoptotic polypeptides

[0330] As used herein, the term "pro-apoptotic polypeptide" refers to a protein that is capable of inducing cell death in a cell or population of cells. Overexpression of these proteins involved in apoptosis shifts the delicate balance between anti-apoptotic and pro-apoptotic factors to the outcome of apoptosis. Suitable pro-apoptotic polypeptides include, but are not limited to, pro-apoptotic members of the BCL-2 family of proteins such as BAX, BAK, BOK / MTD, BID, BAD, BIK / NBK, BLK, HRK, BIM / BOD, BNIP3, NIX, NOXA, PUMA, BMF, EGL-I, and viral homologues, caspases such as caspase-8, the adenovirus E4orf4 gene, p53 pathway genes, pro-apoptotic ligands such as TNF, FasL, TRAIL, and / or their receptors such as TNFR, Fas, TRAIL-Rl, and TRAIL-R2.

[0331] (v) polypeptides having anti-metastatic activity

[0332] As used herein, the term "metastasis suppressor" refers to a protein that acts to slow or prevent the spread of metastases (secondary tumors) within an organism having cancer. Suitable metastasis suppressors include, but are not limited to, proteins such as BRMS l, CRSP3, DRGl, KAI1, KISS-l, NM23, TIMP family proteins, and uteroglobin.

[0333] (vi) polypeptides encoded by polynucleotides capable of activating an immune response against a tumor

[0334] As used herein, an immunostimulatory polypeptide agent is a polypeptide encoded by a polynucleotide capable of activating or stimulating an immune response in a subject to whom it is administered, whether alone or in combination with another agent, including enhancing a pre-existing immune response. Suitable, non-limiting examples of immunostimulatory peptides include flagellin, muramyl dipeptide, cytokines including interleukins (e.g., IL-2, IL-7, IL-15 (or superagonist / mutant forms of these cytokines), IL-12, IFN-gamma, IFN-alpha, GM-CSF, FLT3-ligand, etc.), immunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD28, anti-CD3, or single chain / antibody fragments of these molecules), etc.

[0335] (vii) a chemotherapeutic agent

[0336] It is understood that the term "chemotherapeutic agent" refers to an anti-cancer agent.

[0337] As used herein, an anti-cancer agent is an agent that at least partially inhibits the development or progression of cancer, including inhibiting all or part of the symptoms associated with cancer, even if only for a short period of time.

[0338] Suitable anti-cancer agents include interferon alpha-2a; interferon alpha-2b; interferon alpha-nl; interferon alpha-n3; interferon beta-la; interferon gamma-l b.

[0339] The anti-cancer agent can be an enzyme inhibitor, including but not limited to a tyrosine kinase inhibitor, a CDK inhibitor, a MAP kinase inhibitor, or an EGFR inhibitor. The CDK inhibitor can be, but is not limited to, p21, p27, p57, pl5, pl6, pl8, or pl9.

[0340] The anti-cancer agent can be an antibody or antibody fragment, including but not limited to, bevacizumab (AVASTIN), trastuzumab (HERCEPTIN), alemtuzumab (CAMPATH, indicated for B-cell chronic lymphocytic leukemia), gemtuzumab ozogamicin (MYLOTARG, hP67.6, anti-CD33, indicated for leukemias such as acute myeloid leukemia), rituximab (RITUXAN), tositumomab (BEXXAR, anti-CD20, indicated for B-cell malignancies), MDX-210 (bispecific antibody that binds to both HER-2 / neu oncogene protein product and to the Fc receptor for immunoglobulin G (IgG) (Fc gamma Rl)), oregovomab (OVAREX, indicated for ovarian cancer), edrecolomab (Panorex), daclizumab (ZENAPAX), palivizumab (SYNAGIS, indicated for respiratory tract conditions such as RSV infection), ibritumomab tiuxetan (ZEVALIN, indicated for non-Hodgkin's lymphoma), cetuximab (ERBITUX), MDX-447, MDX-22, MDX-220 (anti-TAG-72), IOR-C5, IOR-T6 (anti-CD 1), IOR-EGF / R3, celogovab (ONCOSCINT OV 103), epratuzumab (LYMPHOCIDE), pertuzumab (THERAGYN), and Gliomab-H (indicated for brain cancer, melanoma).

[0341] (viii) Anti-angiogenic molecules

[0342] It is also contemplated that in certain embodiments, the intervening region of the fusion protein of the application corresponds to a protein that acts as an inhibitor of angiogenesis, which targets tumors. In addition to the anti-angiogenic polypeptides described above, these agents include marimastat; AG3340; COL-3, BMS-275291, thalidomide, endostatin, SU5416, SU6668, EMD121974, 2-methoxyestradiol, carboxyamidotriazole, CMlOl, pentosan polysulfate, angiopoietin 2 (Regeneron), herbimycin A, PNU145156E, 16K prolactin fragment, linomide, thalidomide, pentoxifylline, genistein, TNP470, endostatin, paclitaxel, accutin, angiostatin, cilofungin, vincristine, bleomycin, AGM-1470, platelet factor 4, or minocycline. Also included are VEGF inhibitors including, but not limited to, bevacizumab (AVASTIN), ranibizumab (LUCENTIS), pegaptanib (MACUGEN), sorafenib, sunitinib (SUTENT), vatalanib, ZD-6474 (ZACTIMA), anecortave (RETAANE), squalamine lactate, and netrins.

[0343] (ix) a polypeptide encoded by a suicide gene

[0344] In the context of the present application, "a polypeptide encoded by a suicide gene" refers to a polypeptide whose expression results in the cell in which it is expressed killing itself through apoptosis. This approach includes the selective expression of a suicide gene only in specific cells, although the use of specific promoters that are activated only in cells that actually have the disease, for example, is inhibited.

[0345] This approach involves the use of pairs of enzymes and prodrugs, where the enzyme is used to transform the target cell prior to the use of the prodrug, which under the action of the enzyme becomes a cytotoxic product that initiates the apoptotic process. Typically, the enzymes of these suicide gene therapy systems are not normally expected to be found in the same organism that expresses them, and thus enzymes obtained from bacteria, fungi or other organisms have been used in mammals. There are several known examples of this strategy [reviewed in Karjoo, Z. et al. 2016. Adv. Drug Deliv. Rev. 99 (Pt. A): 123-128], such as the thymidine kinase / ganciclovir system, the cytosine deaminase / 5-fluorocytosine system, the nitroreductase / CB1954 system, the carboxypeptidase G2 / nitrogen mustard gas system, the cytochrome P450 / oxazaphosphorine system, the purine nucleoside phosphorylase / 6-methylpurine deoxyriboside (PNP / MEP), the horseradish peroxidase / indole-3-acetic acid system (HRP / IAA) and the carboxylesterase / irinotecan (CE / irinotecan) systems, truncated EGFR, inducible caspases (“iCasp”), the E. coli gpt gene, the E. coli Deo gene and nitroreductase.

[0346] (x) chaperone and protein aggregation inhibitors

[0347] As used herein, "chaperone polypeptide" or "chaperone" refers to a protein molecule that aids in the folding or unfolding of protein molecules and / or the assembly or disassembly of macromolecular structures. Exemplary chaperones include, but are not limited to, ABCE1, ATP-binding cassette subfamily E member 1; AHSA1, activator of 90 kDa heat shock protein ATPase homolog 1; ANP32B, acidic leucine-rich nuclear phosphoprotein 32 family; BAG6, BAG cochaperone 6; BCS1L, mitochondrial chaperone BCS1; CALR, calreticulin; CANX calnexin; CCT2 T, complexin 1 subunit beta, CCT3 T, complexin 1 subunit gamma, CCT4 T, complexin 1 subunit delta, CCT5 T, complexin 1 subunit epsilon, CCT6A T complexin 1 subunit zeta, CCT7 T complexin 1 subunit beta, CD74 H-2 class II histocompatibility antigen gamma chain; CDC37 Hsp90 co-chaperone Cdc37; CLGN calegin; DNAJA1 DnaJ homolog subfamily A member 1; DNAJC1 DnaJ homolog subfamily C member 1; DNAJC11 DnaJ homolog subfamily C member 11; HSP90AA1 heat shock protein HSP 90-alpha HSP90AB1 heat shock protein HSP 90-beta HSP90B1 endoplasmin; HSPA1B heat shock 70kDa protein 1A / 1B; HSPA2 heat shock-related 70kDa protein 2; HSPA8 heat shock cognate 71kDa protein; HSPA9 stress-70 protein, mitochondrial; HSPD1 60kDa heat shock protein, mitochondrial; HYOU1 hypoxia up-regulated protein 1; NDUFAF2 Mimitin, mitochondrial; SCO1 protein SCO1 homolog, mitochondrial; SCO2 protein SCO2 homolog, mitochondrial; ST13 Hsc70-interacting protein; TBCD, tubulin-specific chaperone D; TCP1 T-complexin 1 subunit alpha, TIMMDC1, translocase of inner mitochondrial membrane domain; and TMEM126B, transmembrane protein 126B.

[0348] Thus, in embodiments of the application, the therapeutic agent of the intervening region of the fusion protein of the application is a cytotoxic polypeptide.

[0349] In preferred embodiments of the application, the cytotoxic polypeptide of the intervening region of the fusion protein is selected from the group consisting of the BH3 domain of BAK, PUMA GW-H1, diphtheria toxin, Pseudomonas exotoxin and ricin. In further preferred embodiments of the application, the cytotoxic polypeptide of the intervening region of the fusion protein is a truncated form or a mutant selected from the group consisting of the peptides just indicated above, preferably selected from the group consisting of diphtheria toxin, Pseudomonas toxin and ricin.

[0350] As used herein, "BAK" refers to the well-known pro-apoptotic factor belonging to the Bcl-2 protein family, which triggers programmed cell death through the caspase-dependent apoptosis pathway by inactivating anti-apoptotic proteins, permeabilizing the mitochondrial membrane, and thus releasing cytochrome C and other mitochondrial cell death factors. [See Llambi, F. et al. 2011. Mol. Cell, 44:517-31]. In one embodiment, BAK refers to the full-length BAK (SEQ ID NO: 34). In other embodiments, BAK refers to any truncated form thereof containing the functional BH3 domain (SEQ ID NO: 35). Experiments provided herein show that BH3 BAK still plays a role in assembling into cell-targeting nanoparticles.

[0351] As used herein, "PUMA" refers to the protein characterized by the full sequence corresponding to SEQ ID NO: 36, which is a (Bcl-2 Homology 3) BH3-only protein that triggers cell death by interacting with pro- and anti-apoptotic proteins of the Bcl-2 family.

[0352] As used herein, GW-H1 refers to the polypeptide having the sequence of SEQ ID NO: 14, which exerts its cytolysic activity by folding into an amphipathic helix. As shown in the examples of the present application, GW-H1 shows a more gentle action than other tested constructs, but in this form, the nanomaterial is considered to exert a cytolysic effect through two consecutive events, including binding to the cell membrane, followed by permeabilization.

[0353] As used herein, "diphtheria toxin" refers to the exotoxin of Corynebacterium diphtheriae, and "Pseudomonas exotoxin" refers to the exotoxin A of Pseudomonas aeruginosa, which belongs to the family of ADP-ribosylating toxins. Both toxins are proteins that act on the eukaryotic elongation factor 2 (eEF-2), essentially inhibiting the translation activity of the cell into which they are incorporated and inducing apoptosis. The structure of both toxins presents a receptor-binding domain (which binds to a surface receptor of the cell and induces endocytosis; in the case of diphtheria toxin, heparin-binding epidermal growth factor precursor; in the case of exotoxin A, CD91), a translocation domain, and a catalytic domain, also referred to herein as "active segment", which performs the action on eEF-2. The catalytic domain or active segment of diphtheria toxin corresponds to SEQ ID NO: 37, while the catalytic domain or active segment of exotoxin A of Pseudomonas aeruginosa corresponds to SEQ ID NO: 38 [a review is provided in Shapira, A. & Benhar, I., 2010, Toxins, 2:2519-2583].

[0354] In preferred embodiments, the diphtheria toxin of embodiments of the application is a truncated or mutated form of the exotoxin of Corynebacterium diphtheria. In further preferred embodiments, the diphtheria toxin of the application contains the translocation and catalytic domains of diphtheria toxin. Said diphtheria toxin is referred to herein as DITOX and has the sequence of SEQ ID NO. 43.

[0355] In another preferred embodiment, the Pseudomonas exotoxin of the application is a truncated or mutated form of exotoxin A of Pseudomonas aeruginosa. In further preferred embodiments, the Pseudomonas exotoxin of the application is a de-immunized catalytic domain based on exotoxin A of Pseudomonas aeruginosa, in which point mutations that disrupt B and T cell epitopes are incorporated. Said Pseudomonas exotoxin is referred to herein as PE24 and has the sequence of SEQ ID NO. 44.

[0356] As used herein, "ricin toxin" refers to a ribosome inactivating protein (RIP) originally extracted from the seeds of Ricinus communis of approximately 65 KDa, which consists of two chains connected by a disulfide bond: chain A with N-glycosidase enzyme activity and chain B with lectin properties that binds to carbohydrate ligands on the surface of target cells. In preferred embodiments, the ricin toxin of the application is a truncated or mutated form of ricin toxin extracted from the seeds of Ricinus communis. In further preferred embodiments, the ricin toxin of the application is a mutated form of the ricin A chain. In even more preferred embodiments, said mutated ricin A chain consists of a ricin A chain with the mutation N132A to suppress vascular leakage syndrome while maintaining cytotoxic activity upon administration. Said mutated ricin A chain is referred to herein as mRTA and has the sequence of SEQ ID NO. 45. In preferred embodiments, the ricin toxin of the application consists of mRTA.

[0357] In preferred embodiments, the intervening polypeptide is a bacterial toxin, the polycationic peptide is T22 and the positively charged amino acid-rich region is a polyhistidine, and more specifically a hexahistidine, wherein the T22 peptide and the bacterial toxin are connected by a linker having the sequence GGSSRSS and a furin cleavage site having the sequence GNRVRRSV. In preferred embodiments, the bacterial toxin is a modified diphtheria toxin comprising the T-domain of the A-fragment and the B-fragment, but lacking the R-domain of the B-fragment. In more preferred embodiments, the bacterial toxin is a modified diphtheria toxin corresponding to SEQ ID NO. 37, even more preferably the bacterial toxin is a modified diphtheria toxin DITOX corresponding to SEQ ID NO. 43. In another embodiment, the bacterial toxin is a Pseudomonas exotoxin. In more preferred embodiments, the bacterial toxin is a Pseudomonas exotoxin having SEQ ID NO. 38, even more preferably the bacterial toxin is a Pseudomonas exotoxin PE24 having SEQ ID NO. 44.

[0358] In preferred embodiments, the intervening polypeptide is a bacterial toxin, the polycationic peptide is T22 and the positively charged amino acid-rich region is a polyhistidine, and more specifically a hexahistidine, wherein the T22 peptide and the bacterial toxin are connected by a linker having the sequence GGSSRSS, a furin cleavage site having the sequence RHRQPRGWEQL and a second linker having the sequence GGS and further comprising a KDEL sequence at the C-terminus after the positively charged amino acid-rich region. In preferred embodiments, the bacterial toxin is a modified diphtheria toxin comprising the T-domain of the A-fragment and the B-fragment, but lacking the R-domain of the B-fragment. In more preferred embodiments, the bacterial toxin is a modified diphtheria toxin corresponding to SEQ ID NO. 37. In yet more preferred embodiments, the bacterial toxin is a modified diphtheria toxin DITOX corresponding to SEQ ID NO. 43. In another embodiment, the bacterial toxin is a Pseudomonas exotoxin. In more preferred embodiments, the bacterial toxin is a Pseudomonas exotoxin having SEQ ID NO. 38, even more preferably the bacterial toxin is a Pseudomonas exotoxin PE24 having SEQ ID NO. 44.

[0359] In a preferred embodiment, the intervening polypeptide is ricin, the poly-cationic peptide is T22, and the positively charged amino acid-rich region is a poly-histidine, and more specifically a hexa-histidine, and the C-terminus after the positively charged amino acid-rich region further comprises a KDEL sequence. In a preferred embodiment, the fusion protein further comprises a linker region at the C-terminus of the T22 peptide comprising the sequence GGSSRSS. In another embodiment, the fusion protein further comprises a furin cleavage site having the sequence RHRQPRGWEQL, which is connected to the C-terminus of the linker region and a second linker region having the sequence GGS. In a preferred embodiment, the intervening polypeptide is a modified ricin carrying the N132A mutation intended to suppress the vascular leakage syndrome. In another preferred embodiment, the intervening polypeptide is a ricin A chain. In another embodiment, the intervening polypeptide is a ricin A chain carrying the N132A mutation.

[0360] In the fusion proteins of the present application, the intervening polypeptide region is not a fluorescent protein or p53.

[0361] In preferred embodiments, the intervening polypeptide is not a fluorescent protein. It is understood that the fusion proteins of the application can still include one or more fluorescent proteins within their structure, provided that the fluorescent protein is not the intervening polypeptide. Thus, in one embodiment, if the fusion protein according to the application comprises a single intervening polypeptide, then the polypeptide is not a fluorescent protein. In another embodiment, if the fusion protein of the application comprises one or more additional polypeptides in addition to the intervening polypeptide, then the additional one or more polypeptides can be fluorescent proteins. The term "intervening polypeptide" does not include any linker region that forms part of the fusion protein and connects different elements of the fusion protein. The fluorescent protein is selected from the group consisting of green fluorescent protein (GFP) or a variant thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, TYFP, Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed- monomer, J-Red, dimer2, t-dimer2 (12), mRFP1, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindlinG protein, phycobiliproteins and phycobiliprotein conjugates, including B-phycoerythrin, R-phycoerythrin and allophycocyanin. In other embodiments, the intervening polypeptide is not a fluorescent protein selected from the group consisting of mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2: 905-909) and the like.

[0362] In preferred embodiments, the intervening polypeptide is not p53 or a p53 isoform encoded by the TP53 gene, such as p53a, p53p, p53y, A40p53a, A40p53p, A40p53y, A133p53a, A133p53p, A133p53y, A160p53a, A160p53p, A160p53y and the like.

[0363] E. Reporter Proteins

[0364] In another embodiment of the application, the fusion protein of the application further comprises a reporter protein. It is to be understood that, as used herein, a reporter protein is distinct from the intervening polypeptide.

[0365] The skilled person will recognize that the term "reporter protein" refers to a protein resulting from the expression of a "reporter gene". Reporter proteins are well known and are commonly used in the art as markers suitable for a variety of purposes, such as reporting the location of expression of a gene in a tissue, cellular or subcellular location, protein-protein interactions, transport across the plasma or inner membranes, vesicular transport, ligand-receptor interactions, etc.

[0366] Reporter proteins useful in the context of the application include luciferase-4-monooxygenase from Photinus pyralis, beta-galactosidase, thymidine kinase, etc. Preferred reporter proteins suitable for use in the fusion proteins of the application are also fluorescent proteins, such as green fluorescent protein (GFP, first discovered in Aequorea victoria), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP) or any other variant, examples of which can be found in Kremers et al. [Kremers, G-J- et al. 2011. J. Cell Sci. 124: 157-160].

[0367] Thus, in a preferred embodiment of the application, the reporter protein of the fusion protein of the application is a fluorescent protein.

[0368] The fluorescent protein comprised by the fusion protein of the application is directly adjacent to the positively charged amino acid-rich region or separated by a linker. However, the relative position of the positively charged amino acid-rich region remains in accordance with the aforementioned considerations regarding the relative position of the elements of the fusion protein. Thus, irrespective of the position of the fusion protein, the fluorescent protein is always adjacent thereto, either directly or through a linker.

[0369] Thus, in an embodiment of the application comprising a fluorescent protein, the possible relative positions of the elements of the fusion protein of the application will suit the following schemes (where FP refers to the fluorescent protein and the aforementioned numbering of the elements is maintained: (1) polycationic peptide, (2) intervening polypeptide region, (3) positively charged amino acid-rich region):

[0370] ■N-(1)-(2)-FP-(3)-C

[0371] ■N-(1)-linker-(2)-FP-(3)-C

[0372] ■N-(1)-(2)-linker-FP-(3)-C

[0373] ■N-(1)-Linker-(2)-FP-(3)-C

[0374] ■N-(3)-FP-(2)-(1)-C

[0375] ■N-(3)-FP-Linker-(2)-(1)-C

[0376] ■N-(3)-FP-(2)-Linker-(1)-C

[0377] ■N-(3)-FP-Linker-(2)-Linker-(3)-C

[0378] ■N-(1)-(2)-FP-Linker-(3)-C

[0379] ■N-(1)-Linker-(2)-FP-Linker-(3)-C

[0380] ■N-(1)-(2)-Linker-FP-Linker-(3)-C

[0381] ■N-(1)-Linker-(2)-Linker-FP-Linker-(3)-C

[0382] ■N-(3)-Linker-FP-(2)-(1)-C

[0383] ■N-(3)-Linker-FP-Linker-(2)-(1)-C

[0384] ■N-(3)-Linker-FP-(2)-Linker-(1)-C

[0385] ■N-(3)-Linker-FP-Linker-(2)-Linker-(3)-C

[0386] ■N-(2)-(1)-FP-(3)-C

[0387] ■N-(2)-Linker-(1)-FP-(3)-C

[0388] ■N-(2)-(1)-Linker-FP-(3)-C

[0389] ■N-(2)-Linker-(1)-Linker-FP-(3)-C

[0390] ■N-(2)-FP-(3)-(1)-C

[0391] ■N-(2)-(3)-FP-(1)-C

[0392] ■N-(2)-Linker-FP-(3)-(1)-C

[0393] ■N-(2)-Linker-(3)-FP-(1)-C

[0394] ■N-(2)-FP-(3)-Linker-(1)-C

[0395] ■N-(2)-(3)-FP-Linker-(1)-C

[0396] ■N-(2)-Linker-FP-(3)-Linker-(1)-C

[0397] ■N-(2)-Linker-(3)FP--Linker-(1)-C

[0398] ■N-(1)-FP-(3)-(2)-C

[0399] ■N-(1)-(3)-FP-(2)-C

[0400] ■N-(1)-FP-(3)-Linker-(2)-C

[0401] ■N-(1)-(3)-FP-Linker-(2)-C

[0402] ■N-(1)-Linker-FP-(3)-(2)-C

[0403] ■N-(1)-Linker-(3)-FP-(2)-C

[0404] ■N-(1)-Linker-FP-(3)-Linker-(2)-C

[0405] ■N-(1)-Linker-(3)-FP-Linker-(2)-C

[0406] ■N-FP-(3)-(1)-(2)-C

[0407] ■N-(3)-FP-(1)-(2)-C

[0408] ■N-FP-(3)-Linker-(1)-(2)-C

[0409] ■N-(3)-FP-Linker-(1)-(2)-C

[0410] ■N-FP-(3)-(1)-Linker-(2)-C

[0411] ■N-(3)-FP-(1)-Linker-(2)-C

[0412] ■N-FP-(3)-Linker-(1)-Linker-(2)-C

[0413] ■N-(3)-FP-Linker-(1)-Linker-(2)-C

[0414] Nanoparticles comprising multiple copies of fusion proteins of the invention and methods of making the same

[0415] In a second aspect, the present application relates to a method of preparing a nanoparticle comprising a plurality of copies of the fusion protein according to the first aspect of the present application, the method comprising placing a preparation of said fusion protein in a low salt buffer.

[0416] As the skilled person will recognize, a "nanoparticle" is a microscopic particle whose size is measured in nanometers. The nanoparticle of the present application comprises a nanoparticle resulting from the assembly of a plurality of copies of the fusion protein of the present application, as defined in the previous section. In the method of preparing a nanoparticle with the fusion protein of the present application, the preparation of the fusion protein of the present application comprises monomeric forms of the fusion protein of the present application, which under the conditions of a low salt buffer are thermodynamically favored to form non-covalent electrostatic bindings and spontaneously aggregate.

[0417] The skilled person will recognize that the size of the nanoparticle can range between 1 and 1000 nm, more preferably between 2.5 and 500 nm, even more preferably between 5 and 250 nm, and yet even more preferably between 10 and 100 nm.

[0418] It will be understood that the expression "low salt buffer" includes any buffered solution resulting from the dissolution in water of one or more salts, which has the ability to moderately change the pH, wherein the amount of dissolved one or more salts results in a molar osmotic concentration lower or equal to physiological fluids, such as cytoplasm or extracellular medium. Thus, a low salt buffer should be understood as maintaining the pH and molar osmotic concentration within the range of physiological values, and will be used within the range of physiological temperatures.

[0419] The skilled person will recognize that the range of physiological temperatures can fluctuate between 15 and 45 °C, more preferably between 20 and 40 °C, even more preferably between 25 and 39 °C, even more preferably between 30 and 37 °C. The skilled person will also recognize that the molar osmotic concentration of the low salt buffer will range between 100 and 400 milliosmole osmole / L (mOsm / L), preferably between 150 and 350 mOsm / L, more preferably between 200 and 300 mOsm / L, even more preferably between 225 and 275 mOsm / L.

[0420] Suitable low salt buffers for the present application are, for example, Tris- glucose buffer (20 mM Tris + 5% glucose, pH 7.4), Tris-NaCl buffer (20 mM Tris, 500 mM NaCl, pH 7.4), PBS-glycerol buffer (phosphate buffered saline, PBS, pH 7.4, well known in the art, + 10% glycerol), Tris buffered saline (TBS)- glucose (20 mM Tris-HCl buffer pH 7.5, well known in the art, 200 mM NaCl, + 5% glucose), Tris buffered saline-Tween 20 (TBST) buffer (10 mM Tris-HCl pH 7.5, 200 mM NaCl, + 0.01% Tween 20), or any physiological buffer known in the art with a pH not lower than 6.

[0421] In a preferred embodiment of the present application, the low salt buffer of the method of the present application is selected from the group consisting of carbonate buffer, Tris buffer and phosphate buffer.

[0422] In an especially preferred embodiment of the present application, the low salt buffer of the method of the present application is a carbonate buffer comprising sodium bicarbonate at a concentration comprised between 100 and 300 nM. In another especially preferred embodiment of the present application, the low salt buffer of the method of the present application is a Tris buffer comprising Tris at a concentration comprised between 10 and 30 nM. In another especially preferred embodiment of the method of the present application, the low salt buffer of the present application is a phosphate buffer comprising Na2HP04and NaH2P04at a total concentration comprised between 5 mM and 20 mM.

[0423] In an even more preferred embodiment of the present application, the low salt buffer of the method of the present application further comprises glucose and / or glycerol.

[0424] In a still more preferred embodiment of the present application, the low salt buffer of the method of the present application has a pH comprised between 6.5 and 7.5.

[0425] In an even still more preferred embodiment of the present application, the low salt buffer of the method of the present application is selected from the group consisting of:

[0426] (i) 166 mM NaHC03, pH 7.4

[0427] (ii) 20 mM Tris, 500 mM NaCl, 5% glucose, pH 7.4

[0428] (iii) 140 mM NaCl, 7.5 mM Na2HP04, 2.5 mM NaH2P04, 10% glycerol, pH 7.4 (iv) 20 mM Tris, 500 mM NaCl, 0.01% Tween 20, pH 7.5.

[0429] In another aspect of the application, the present application relates to a nanoparticle comprising a plurality of copies of the fusion protein of the first aspect of the application or a nanoparticle prepared according to the method of the present application for preparing a nanoparticle.

[0430] Thus, the nanoparticle of the present application comprises an assembled complex of a plurality of copies of the fusion protein of the present application, which arises due to electrostatic interactions between the regions in their structure, which facilitates their non-covalent association and coupling under physiological conditions. Since the method of the present application for preparing a nanoparticle comprises placing a preparation of the fusion protein of the present application in a low salt buffer, it should be understood that the nanoparticle thus formed also comprises an assembled complex of a plurality of copies of the fusion protein.

[0431] In a preferred embodiment of the present application, the nanoparticle of the present application has a diameter between 10 and 100 nm.

[0432] Polynucleotides, vectors and host cells of the invention

[0433] In another aspect of the present application, the present application relates to a polynucleotide encoding the fusion protein of the first aspect of the present application, a vector comprising the aforementioned polynucleotide and a host cell comprising the aforementioned polynucleotide or the aforementioned vector.

[0434] The terms "nucleic acid" and "polynucleotide", as used interchangeably herein, refer to a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants and synthetic non-naturally occurring analogs thereof or combinations thereof) linked by phosphodiester bonds, related naturally occurring structural variants and synthetic non-naturally occurring analogs thereof.

[0435] The skilled person will recognize that the polynucleotide encodes the polypeptide or protein sequence of the fusion protein of the present application corresponding to the first aspect of the present application. Thus, the polynucleotide of the present application comprises a sequence encoding all elements included in the fusion protein: the polycationic polypeptide, the intervening peptide region, the positively charged amino acid-rich region and any other element that can be part of the fusion protein, such as a reporter protein, a linker, etc.

[0436] It should be understood that the nucleic acid or polynucleotide of the present application comprises a coding region and appropriate regulatory signals for facilitating expression in a cell to produce a biologically active fusion protein.

[0437] Generally, a nucleic acid containing a coding region will be operably linked to appropriate regulatory sequences. Such regulatory sequences will include at least a promoter sequence. As used herein, the term "promoter" refers to a nucleic acid segment that functions to control transcription of one or more genes, is located in the transcriptional orientation upstream from the transcriptional start site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, the transcription start site, and any other nucleotide sequences known to one of skill in the art to be present in a promoter that directly or indirectly affect transcription from the promoter. The term "promoter" does not encompass a "transcriptional terminator" sequence. A "constitutive" promoter is one that is active under most physiological and developmental conditions. An "inducible" promoter is one that is regulated depending on physiological or developmental conditions. A "tissue-specific" promoter is one that is active only in specific types of differentiated cells / tissues.

[0438] In principle, any promoter can be used in the gene constructs of the present application, provided that the promoter is compatible with the cell in which the polynucleotide is to be expressed. Thus, promoters suitable for use in embodiments of the present application include, but are not limited to, constitutive promoters such as genomic derivatives of eukaryotic viruses such as polyoma, adenovirus, SV40, CMV, avian sarcoma virus, hepatitis B virus, the promoter of the metallothionein gene, the promoter of the herpes simplex virus thymidine kinase gene, the retroviral LTR regions, the promoter of the immunoglobulin genes, the promoter of the actin gene, the promoter of the EF-1 alpha gene, and inducible promoters in which the expression of the protein depends on the addition of a molecule or exogenous signal such as the tetracycline system, the NFKB / UV light system, the Cre / Lox system and the promoters of heat shock genes, the regulatable promoters of RNA polymerase II described in WO / 2006 / 135436, and tissue-specific promoters.

[0439] The polynucleotide of the present application encoding the fusion protein of the present application can be a part of a vector. Thus, in another embodiment, the present application relates to a vector comprising the polynucleotide of the present application. One skilled in the art will understand that there is no limitation on the type of vector that can be used, as the vector can be a cloning vector suitable for propagation in different heterologous organisms and for obtaining the polynucleotide or expression vector suitable for purifying the fusion protein of the present application. Thus, suitable vectors according to the present application include expression vectors in prokaryotes such as pET (such as pET14b), pUC18, pUC19, Bluescript and its derivatives, mp18, mp19, pBR322, pMB9, CoIEl, pCRl, RP4, bacteriophages and shuttle vectors such as pSA3 and pAT28, expression vectors in yeast such as the following types of vectors: 2 micron plasmids, integrating plasmids, YEP vectors, centromeric plasmids, etc., expression vectors in insect cells such as pAC series and pVL series vectors, expression vectors in plants such as expression vectors in plants such as pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series vectors, etc., and expression vectors in higher eukaryotic cells based on viral vectors (adenoviruses, viruses related to adenoviruses as well as retroviruses and lentiviruses) and non-viral vectors such as pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1 / hyg pHCMV / Zeo, pCR3.1, pEFl / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAXl, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d and pTDTl.

[0440] The vectors of the present application can be used to transform, transfect or infect cells that can be transformed, transfected or infected by said vectors. The cells can be prokaryotic or eukaryotic. For example, the vector into which the DNA sequence is introduced can be a plasmid or vector that, when introduced into a host cell, is integrated into the genome of said cell and replicates together with the chromosome(s) into which it has been integrated. Said vectors can be obtained by conventional methods known to the person skilled in the art [Sambrook et al., 2001, "Molecular cloning, to Laboratory Manual", 2nd edition, Cold Spring Harbor Laboratory Press, N.Y. Vol 1-3a].

[0441] Accordingly, the present application also relates to a cell comprising a polynucleotide or a vector of the present application, which cell has been transformed, transfected or infected with a polynucleotide or a vector provided by the present application. The transformed, transfected or infected cell can be obtained by conventional methods known to the person skilled in the art [Sambrook et al., 2001, supra].

[0442] Suitable host cells for expressing the conjugates of the present application include, but are not limited to, mammalian, plant, insect, fungal and bacterial cells. Bacterial cells include, but are not limited to, gram-positive bacterial cells, such as species of Bacillus, Streptomyces, Listeria and Staphylococcus; and gram-negative bacterial cells, such as cells of the genera Escherichia, Salmonella and Pseudomonas. Fungal cells preferably include yeast cells, such as Saccharomyces cerevisiae, Pichia pastoris and Hansenula polymorpha. Insect cells include, but are not limited to, Drosophila and Sf9 cells. Plant cells include cells of crop plants such as cereals, medicinal, ornamental or tuber plants, etc. Suitable mammalian cells in the present application include epithelial cell lines (human, ovine, porcine, etc.), osteosarcoma cell lines (human, etc.), neuroblastoma cell lines (human, etc.), epithelial carcinoma (human, etc.), glial cells (murine, etc.), hepatocyte cell lines (from monkeys, etc.), CHO (Chinese hamster ovary) cells, COS cells, BHK cells, HeLa cells, 911, AT1080, A549, 293 or PER.C6, NTERA-2 human ECC cells, D3 cells of mESC lines, human embryonic stem cells, such as HS293, BGV01, SHEF1, SHEF2, HS181, NIH3T3 cells, 293T, REH and MCF-7 and hMSC cells.

[0443] In a preferred embodiment of the present application, the polynucleotides, vectors and host cells of the present application are suitable for expressing a biologically active form of the fusion proteins of the present application.

[0444] Use of fusion proteins, polynucleotides, vectors and nanoparticles of the invention in medicine

[0445] In another aspect, the present application relates to a fusion protein, polynucleotide, vector, host cell or nanoparticle according to the present application for use in medicine.

[0446] The person skilled in the art will understand that by use in medicine, the fusion proteins, polynucleotides, vectors, host cells or nanoparticles of the present application can be administered to a patient to induce a therapeutic response. A therapeutic response includes inhibiting, reducing or arresting the cause of a pathological condition or disease suffered by the patient; eliminating, reducing, arresting or ameliorating a symptom of the disorder or disease; or eradicating, arresting or slowing the disorder or disease in the patient.

[0447] Those skilled in the art will recognize that the fusion proteins, polynucleotides, vectors, host cells or nanoparticles of the application suitable for use in medicine can be provided with a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" means any type of nontoxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation agent which is generally safe, non-toxic and neither biologically nor otherwise undesirable. Various carrier formulations and known techniques for the formulation of pharmaceutical compositions are disclosed in Remington's Pharmaceutical Sciences. Ed. by Gennaro, Mack Publishing, Easton, Pa., 1995.

[0448] Accordingly, the compositions comprising the fusion proteins, polynucleotides, vectors, host cells or nanoparticles of the application and a pharmaceutically acceptable carrier are pharmaceutical compositions.

[0449] The pharmaceutical compositions of the application can be administered to a patient by any means known in the art, including oral and parenteral routes. According to such embodiments, the compositions of the application can be administered by injection (e.g., intravenous, subcutaneous or intramuscular, intraperitoneal injection), rectally, vaginally, topically (e.g., as by spray, cream, salve or drops) or by inhalation (e.g., as by spray).

[0450] A- Use of the fusion proteins, polynucleotides, vectors, host cells or nanoparticles of the application in the treatment of cancer

[0451] Another embodiment of the application relates to a fusion protein, a polynucleotide of the application, a vector of the application, a host cell of the application comprising the vector or polynucleotide and expressing the fusion protein, and a nanoparticle of the application, or a corresponding pharmaceutical composition thereof, wherein the polycationic peptide is a sequence capable of specifically interacting with a receptor on the surface of a cell, the sequence being capable of promoting the internalization of the fusion protein into the cell, wherein said cell expressing the receptor is a tumor cell present in a cancer, and wherein the intervening polypeptide region is an antitumor peptide for the treatment of cancer.

[0452] As used herein, the terms "treat", "treatment" and "treating" refer to the reduction or amelioration of the progression, severity and / or duration of a cancer, or the amelioration of one or more symptoms (preferably, one or more discernible symptoms) of a cancer. The term "treatment" also refers to the improvement of at least one measurable physical parameter of a patient, not necessarily discernible by the patient, such as growth of a tumor. In addition, "treatment" also refers to the inhibition of the progression of a cancer, either physically, e.g., by stabilization of a discernible symptom, physiologically, e.g., by stabilization of a physical parameter, or both. "Treatment" can also refer to a reduction or stabilization of tumor size or a reduction or stabilization of cancer cell count.

[0453] The term "cancer" refers to a group of diseases involving abnormal, uncontrolled cell growth and proliferation (neoplasia) that can invade or spread (metastasize) to other tissues, organs, or generally distant parts of the organism; metastasis is one of the hallmarks of malignant tumors of cancer and carcinomas. The abnormal growth and / or proliferation of cancer cells is the result of a combination of genetic and environmental factors that alter their normal physiology. The abnormal growth and / or proliferation of cancer cells results in physiological disease and, in many cases, death of the individual due to dysfunction or loss of function of the affected cell types, tissues, and organs.

[0454] The term "cancer" includes, but is not limited to, breast cancer, cardiac cancer, small intestinal cancer, colon cancer, splenic cancer, renal cancer, bladder cancer, head cancer, neck cancer, ovarian cancer, prostate cancer, brain cancer, pancreatic cancer, skin cancer, bone cancer, bone marrow cancer, blood cancer, thymus cancer, uterine cancer, testicular cancer, hepatobiliary system cancer, and liver cancer; in addition to tumors, such as, but not limited to, adenoma, angiosarcoma, astrocytoma, carcinoma, embryonal tumor, glioblastoma, glioma, hemangioendothelioma, hemangiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, cholangiocarcinoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and teratocarcinoma. In addition, the term includes acral lentiginous melanoma, actinic keratosis adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytoma, basaloid adenocarcinoma, bronchiolar adenocarcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrial adenocarcinoma, ependymal sarcoma, Ewing's sarcoma, focal nodular hyperplasia, germ cell tumor, glioblastoma, glucagonoma, hemangio-blastoma, hemagioendothelioma, hemangioma, hepatoadenoma, hepatic adenomastosis, hepatocellular carcinoma, hepatobiliary surgical cancer, islet cell tumor, intraepithelial neoplasm, squamous cell intraepithelial neoplasm, invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelioma, meduloblastoma, medulloepithelioma, mucoepidermoid carcinoma, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, papillary serous adenocarcinoma, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vasoactive intestinal peptide tumor, Wilms' tumor, brain cancer, head and neck cancer, rectal cancer, astrocytoma, glioblastoma, small cell carcinoma and non-small cell carcinoma, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer, and breast cancer.

[0455] Accordingly, in preferred embodiments of the application, the antitumor peptide of the fusion protein, polynucleotide, vector, host cell, or nanoparticle of the application is selected from the group consisting of:

[0456] (i) a cytotoxic polypeptide,

[0457] (ii) an anti-angiogenic polypeptide,

[0458] (iii) a polypeptide encoded by a tumor suppressor gene,

[0459] (iv) a pro-apoptotic polypeptide,

[0460] (v) a polypeptide having anti-metastatic activity,

[0461] (vi) a polypeptide encoded by a polynucleotide capable of activating an immune response against a tumor,

[0462] (vii) a chemotherapeutic agent,

[0463] (viii) an anti-angiogenic molecule, and

[0464] (ix) a polypeptide encoded by a suicide gene.

[0465] In a more preferred embodiment of the application, the anti-tumor peptide of the fusion protein, polynucleotide, vector, host cell or nanoparticle of the application is selected from the BH3 domain of BAK, PUMA, GW-H1, diphtheria toxin, Pseudomonas exotoxin and ricin. In a further preferred embodiment of the application, the anti-tumor peptide of the fusion protein, polynucleotide, vector, host cell or nanoparticle of the application is a truncated form or a mutant of a peptide selected from the peptides just indicated above, preferably selected from diphtheria toxin, Pseudomonas exotoxin and ricin. Preferred sequences of said peptides are indicated in the section "Interposed polypeptide region" above.

[0466] In an even more preferred embodiment of the application, the polycationic peptide of the fusion protein, polynucleotide, vector, host cell or nanoparticle of the application is a CXCR4 ligand and the targeted cancer to be treated with the fusion protein, polynucleotide, vector, host cell or nanoparticle of the application is characterized by comprising cells expressing the CXCR4 receptor.

[0467] In a still more preferred embodiment of the application, the CXCR4 ligand of the fusion protein, polynucleotide, vector, host cell or nanoparticle of the application is selected from the T22 peptide, the VI peptide, the CXCL12 peptide, the vCCL2 peptide or a functional equivalent variant thereof.

[0468] In another more preferred embodiment of the application, the cancer to be treated with the fusion protein, polynucleotide, vector, host cell or nanoparticle of the application is selected from pancreatic cancer and colorectal cancer.

[0469] The protein CD44 is another well-known key regulator of cancer cell development and metastasis (reviewed in Senbanjo, L.T. & Chellaiah, M.A. 2017. Front. Cell Dev. Biol. 5: 18).

[0470] Thus, in another preferred embodiment of the application, the cancer to be treated with the fusion protein, polynucleotide, vector, host cell or nanoparticle of the application is characterized by the expression of CD44.

[0471] Another more preferred embodiment of the application relates to the fusion protein, polynucleotide, vector, host cell or nanoparticle of the application for use in the treatment of cancer, wherein the cancer is characterized by the expression of CD44, wherein the intervening region polypeptide is an antitumor peptide selected from one of the groups already listed, wherein the polycationic peptide region is a CD44 ligand, and wherein the CD44 ligand is A5G27 or FNI / II / V.

[0472] Another even more preferred embodiment of the application relates to the fusion protein, polynucleotide, vector, host cell or nanoparticle of the application for use in the treatment of cancer, wherein the cancer is characterized by the expression of CD44, wherein the intervening region polypeptide is an antitumor peptide, wherein the polycationic peptide region is a CD44 ligand selected between A5G27 and FNI / II / V, and wherein the cancer is colon cancer, liver cancer, prostate cancer or breast cancer.

[0473] Another preferred embodiment of the application relates to the fusion protein, polynucleotide, vector, host cell or nanoparticle of the application, wherein the polycationic peptide is a peptide capable of crossing the blood brain barrier, and wherein the intervening region polypeptide is an antitumor peptide for the treatment of cancer of the central nervous system.

[0474] Another more preferred embodiment of the application relates to the fusion protein, polynucleotide, vector, host cell or nanoparticle of the application, wherein the polycationic peptide is a peptide capable of crossing the blood brain barrier, and wherein the antitumor peptide is selected from one of the groups already listed, for the treatment of cancer of the central nervous system.

[0475] An even more preferred embodiment of the application relates to the fusion protein, polynucleotide, vector, host cell or nanoparticle of the application, wherein the polycationic peptide is a peptide capable of crossing the blood brain barrier selected from Seq-1-7, Seq-1-8 and Angiopep-2-7, and wherein the antitumor peptide is selected from one of the groups already listed, for the treatment of cancer of the central nervous system.

[0476] Yet an even more preferred embodiment of the application relates to the fusion protein, polynucleotide, vector, host cell or nanoparticle of the application, wherein the polycationic peptide is a peptide selected from Seq-1-7, Seq-1-8 and Angiopep-2-7, and wherein the antitumor peptide is selected from one of the groups already listed, for the treatment of cancer of the central nervous system, wherein the cancer of the central nervous system is a glioma.

[0477] B. Use of the fusion protein, polynucleotide, vector, host cell, or nanoparticle of the present invention in the treatment of a bacterial infection

[0478] Another embodiment of the present invention relates to the fusion protein, polynucleotide, vector, host cell, or nanoparticle of the present invention for use in the treatment of a disease caused by a bacterial infection.

[0479] As used herein, the term "treatment" refers to the reduction or amelioration of the progression, severity, and / or duration of a bacterial infection, or the amelioration of one or more symptoms, preferably one or more discernible symptoms, of a bacterial infection. The term "treatment" also refers to the amelioration of at least one measurable physical parameter of a bacterial infection that is not necessarily discernible by the patient, such as the presence of bacterial toxins. In addition, "treatment" can also refer to the inhibition of the progression of a bacterial infection, either physically, e.g., by stabilization of a discernible symptom, physiologically, e.g., by stabilization of a physical parameter, or both. "Treatment" can also refer to a reduction or stabilization of bacterial cell counts.

[0480] As used herein, the term "bacteria" refers to prokaryotes of the domain Bacteria. Non-limiting examples of bacterial genera that can be used in the methods of the present invention include: Actinomyces, Bacillus, Bacteroides, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Coxiella, Ehrlichia, Enterococcus, Eschericia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Nocardia, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptobacillus, Streptococcus, Treponema, Ureaplasma, Vibrio, and Yersinia. Individual prokaryotes of the domain Bacteria are designated bacteria.

[0481] The present invention contemplates the use of the fusion protein, polynucleotide, vector, host cell or nanoparticle for the treatment of infection by bacteria such as Neisseria gonorrhoeae and Neisseria meningitidis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus mutans; Haemophilus ducreyi; Moraxella, including M. catarrhalis, also known as Branhamella catarrhalis Bordetella spp., including B. pertussis, B. parapertussis and B. bronchiseptica, Mycobacterium, including M. tuberculosis, M. bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Legionella, including L. pneumophila, Escherichia, including E. coli enterotoxigenic, E. coli enterohemorrhagic and E. coli enteropathogenic, Vibrio, including V. cholerae, Shigella, including S. sonnei, Streptococcus dysgalactiae, Francisella tularensis; Yersinia, including Y. enterocolitica, Y. pestis, Y. pseudotuberculosis; Campylobacter, including C. jejuni, Salmonella, including S. typhi, S. enteritidis and Bongori Streptomyces; Listeria, including L. monocytogenes; Helicobacter, including H. pylori, Pseudomonas, including P. aeruginosa; Staphylococcus, including S. aureus, S. epidermidis; Enterococcus, including E. faecalis, E. faecium; Clostridium, including C. tetani, C. botulinum, C. difficile, Bacillus, including B. anthracis; Corynebacterium, including C. diphtheriae, Borrelia, including B. burgdorferi, B. garinii, B. afzelii, B. andersonfi, B. hermsii; Ehrlichia, including E. equi and E. chaffeensis; Rickettsia, including R. rickettsii; Chlamydia, including C. trachomatis, C. pneumoniae, C. psittaci; Leptospira, including L. interrogans; Treponema, including T. pallidum, T. denticola, T. hyodysenteriae, Mycobacterium tuberculosis, Streptococcus pneumoniae, Haemophilus, including H. influenzae type B and non-typeable H. influenzae, and the like, but not limited thereto.

[0482] C- Use of the fusion protein, polynucleotide, vector, host cell or nanoparticle of the present invention in the treatment of viral infections

[0483] Another embodiment of the application relates to the fusion protein, polynucleotide, vector, host cell or nanoparticle of the application, wherein the polycationic peptide is capable of specific interaction with a receptor on the cell surface of a cell infected with a virus causing the infection; and wherein the intervening polypeptide region is an antiviral agent for treating a disease caused by a viral infection.

[0484] As used herein, the term "treatment" refers to the reduction or amelioration of the progression, severity and / or duration of a viral infection, or the amelioration of one or more symptoms, preferably one or more discernible symptoms, of a viral infection. The term "treatment" also refers to the improvement of at least one measurable physical parameter of a bacterial infection that the patient does not necessarily perceive as an improvement, such as viral titer. In addition, "treatment" can also refer to the inhibition of the progression of a viral infection, either physically, e.g., by stabilization of a discernible symptom, physiologically, e.g., by stabilization of a physical parameter, or both. "Treatment" can also refer to a decrease or stabilization of viral titer.

[0485] As used herein, the term "virus" refers to a small pathogenic agent that can only replicate within living cells of an organism. Non-limiting examples of virus families that can be used in the methods of the application include Adenoviridae, African swine fever-like virus, Arenaviridae, Arteriviridae, Astroviridae, Baculoviridae, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Deltavirus, Filoviridae, Flaviviridae, Hepadnaviridae, Hepeviridae, Herpesviridae, Orthomyxoviridae, Paramyxoviridae, Picomaviridae, Poxyviridae, Reoviridae, Retroviridae, and Rhabdoviridae.

[0486] Examples of viral infections that the fusion protein, polynucleotide, vector, host cell or nanoparticle of the application are suitable to treat include human immunodeficiency virus (HIV-1), human herpes viruses such as HSV1 or HSV2, cytomegalovirus, in particular human, Epstein-Barr virus, varicella zoster virus, hepatitis viruses such as hepatitis B virus, hepatitis C virus, paramyxoviruses such as respiratory syncytial virus, parainfluenza virus, rubella virus, measles virus, mumps virus, human papillomavirus, flaviviruses (e.g., yellow fever virus, dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus), influenza virus, rotavirus, and the like.

[0487] In an even more preferred embodiment of the application, the antiviral agent of the fusion protein, polynucleotide, vector, host cell or nanoparticle of the application is selected from the group consisting of:

[0488] (i) a cytotoxic polypeptide,

[0489] (ii) a pro-apoptotic polypeptide,

[0490] (iii) a polypeptide encoded by a suicide gene; and

[0491] (iv) an anti-retroviral polypeptide

[0492] The cytotoxic polypeptide (i), the pro-apoptotic polypeptide (ii) and the polypeptide encoded by a suicide gene have been discussed in correspondence with the part of the fusion protein.

[0493] Anti-retroviral agents are one homotype of the class of antimicrobials that are antiviral. Anti-retroviral agents are specifically used to treat viral infections caused by retroviruses. Retroviruses include the family of viruses of the Retroviridae family, which includes genera such as alpharetrovirus, betaretrovirus and lentivirus. They are characterized by single-stranded, positive-sense RNA genomes. Retroviruses produce a double-stranded DNA copy of their genome by their own reverse transcriptase, which integrates into the genome of their host cell. The skilled person will recognize that an "anti-retroviral agent" includes any molecule or compound that is able to interfere with the normal replication cycle of a retrovirus at any stage. Thus, an anti-retroviral polypeptide (iv) as used herein refers to a polypeptide with anti-retroviral properties.

[0494] Anti-retroviral polypeptides suitable for use in the present application are for example "entry inhibitors", also known as "fusion inhibitors", which are peptides that interfere with the binding, fusion and entry of a retrovirus into a host cell. Examples of this group are efuvirtide, a biomimetic peptide that competes with the HIV-1 fusion machinery, and peptide T, a peptide that blocks the chemokine receptors CCR2 and CCR5.

[0495] Also included as entry inhibitors are antibodies specific for receptors used by retroviruses for cell fusion. Non-limiting examples of such receptors suitable to be blocked by antibodies are CD4, CCR2, CCR5 and CXCR4.

[0496] As used herein, the term "antibody" refers to a glycoprotein that exhibits specific binding activity for a particular protein known as an "antigen". The term "antibody" includes intact monoclonal antibodies or polyclonal antibodies or fragments thereof, and includes human antibodies, humanized antibodies, chimeric antibodies, and antibodies of non-human origin. "Monoclonal antibodies" are homogenous, highly specific antibodies to a single site or "determinant" of an antigen. "Polyclonal antibodies" include a heterogeneous population of antibodies to different antigenic determinants.

[0497] As used herein, antibodies suitable for use in the present application include not only full-length antibodies (e.g., IgG), but also antigen-binding fragments thereof, such as Fab, Fab', F(ab')2, Fv fragments, human antibodies, humanized antibodies, chimeric antibodies, antibodies of non-human origin, recombinant antibodies, and polypeptides derived from immunoglobulins produced by genetic engineering techniques, such as single chain Fv (scFv), diabodies, heavy chains or fragments thereof, light chains or fragments thereof, VH or dimers thereof, VL or dimers thereof, Fv fragments stabilized by disulfide bonds (dsFv), molecules with single chain variable region domains (Abs), minibodies, scFv-Fc, and any other modified configuration of an immunoglobulin that includes an antigen recognition site with the desired specificity. Antibodies of the present application can also be bispecific antibodies. Antibody fragments can refer to antigen-binding fragments. Antibodies include antibodies of any class, i.e., IgA, IgD, IgE, IgG (or sub-class thereof), and IgM, and the antibody need not be of any particular class.

[0498] Thus, a still more preferred embodiment of the present application relates to the fusion protein, polynucleotide, vector, host cell or nanoparticle of the present application, wherein the polycationic peptide is a CXCR4 ligand, and wherein the cell is an HIV infected cell for the treatment of HIV infection.

[0499] A still even more preferred embodiment of the present application relates to the fusion protein, polynucleotide, vector, host cell or nanoparticle of the present application, wherein the CXCR4 ligand is selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 or a functionally equivalent variant thereof, for the treatment of viral infection.

[0500] D- Use of the fusion protein, polynucleotide, vector, host cell or nanoparticle of the present application in the treatment of a neurodegenerative disease

[0501] Protein aggregation is a biological phenomenon that results from the accumulation of misfolded proteins, either intracellularly or extracellularly. The resulting protein aggregates can initiate disease and, in fact, they have been found to be involved in a wide range of diseases called amyloidosis. Amyloidosis includes several well-studied neurodegenerative diseases such as ALS, Alzheimer's disease, Parkinson's disease and prion diseases.

[0502] Aggregation occurs as a result of errors in the physiological folding of a protein into its native three-dimensional conformation, which is thermodynamically the most favorable (also known as the "native state"). The hydrophobic parts of the protein tend to shield themselves from the cell's hydrophilic environment by becoming buried inside the protein, thus driving the folding process. As a result, the outside of the protein is typically hydrophilic, while the inside is typically hydrophobic. The protein structure is then stabilized by non-covalent electrostatic interactions and disulfide bonds, which create the secondary and tertiary structure of the protein, which are well known to those skilled in the art.

[0503] Errors that cause a protein to misfold or unfold can be caused by changes in the amino acid sequence of the protein. If these errors are not corrected, for example by "chaperone" proteins (as will be known to those skilled in the art, chaperone or "protein chaperone" proteins are scaffolds that assist other proteins to fold correctly into their correct conformation and tertiary or three-dimensional structure), the misfolded or unfolded proteins will aggregate due to their natural interaction with each other through their hydrophobic regions, as a way to limit their exposure to the cell's hydrophilic environment [Roberts, C.J., 2007. Biotechnology & Bioengineering, 98(5):927-938].

[0504] Accordingly, another embodiment of the present application relates to the fusion protein, polynucleotide, vector or nanoparticle of the present application, wherein the polycationic peptide is a peptide capable of crossing the blood-brain barrier, and wherein the intervening polypeptide region is a chaperone protein or a protein aggregation inhibitor for the treatment of a neurodegenerative disease.

[0505] Suitable chaperone or protein aggregation inhibitors are as defined above. Diseases that can be treated using the fusion protein, nanoparticle, vector or host cell according to the present application include Alzheimer's disease, Pick's disease, alpha 1 -antitrypsin deficiency, Parkinson's disease and other synucleinopathies, Creutzfeldt-Jakob disease, retinal ganglion cell degeneration in glaucoma, cerebral beta-amyloid angiopathy, prion disease, proteinopathy, frontotemporal lobar degeneration, type II diabetes, amyotrophic lateral sclerosis, Huntington's disease and other trinucleotide repeat disorders, familial Danish dementia, familial English dementia, hereditary cerebral hemorrhage with amyloidosis, Alexander disease, Seipinopathies, familial amyloid neuropathy, senile systemic amyloidosis, lysozyme amyloidosis, fibrinogen amyloidosis, dialysis amyloidosis, inclusion body myositis / myopathy, cataract, retinitis pigmentosa with mutations in rhodopsin, medullary carcinoma of the thyroid, cardiac atrial amyloidosis, pituitary prolactinomas, Hereditary lattice corneal dystrophy, cutaneous lichen amyloidosis, Mallory bodies, corneal lactotransserrin amyloidosis, Pulmonary alveolar proteinosis of the lung, odontogenic tumor amyloid, seminal vesicle amyloid, Apolipoprotein C2 amyloidosis, Apolipoprotein C3 amyloidosis, Lect2 amyloidosis, insulin amyloidosis, Galectin-7 amyloidosis (primary local cutaneous amyloidosis), corneal claudin amyloidosis, enfuvirtide amyloidosis, cystic fibrosis, sickle cell disease, hereditary cerebral hemorrhage with amyloidosis, AL amyloidosis, AH amyloidosis, AA amyloidosis, medial amyloidosis of the large arteries, ApoAI amyloidosis, ApoAII amyloidosis, ApoAIV amyloidosis and familial amyloidosis of the Finnish type.

[0506] Thus, a preferred embodiment of the present application relates to the fusion protein, polynucleotide, vector, host cell or nanoparticle of the present application, wherein the intervening polypeptide region is a chaperone or protein aggregation inhibitor for the treatment of a neurodegenerative disease, wherein the poly-cationic peptide capable of crossing the blood-brain barrier is selected from Seq-1-7, Seq-1-8 and Angiopep-2-7.

[0507] The present application is described below by the following examples, which are merely exemplary and do not limit the scope of the present application.

[0508] Example

[0509] Materials and methods for fusion proteins T22-BAK-GFP-H6, T22-GFP-H6, T22-GWH1-GFP-H6 and T22-PUMA GFP-H6Figure 1

[0510] Protein design, production and purification

[0511] Engineered fusion proteins were named according to their modular organization Materials and methods for protein nanoparticles based on diphtheria toxin (DITOX) and Pseudomonas aeruginosa exotoxin (PE24) , 5 ; T22-BAK-GFP-H6, T22-GFP-H6, T22-GWH1-GFP-H6 and T22-PUMA GFP-H6). Synthetic genes were designed in-house and obtained from GeneArt inserted into the pET-22b vector for prokaryotic expression. The encoded proteins were produced in E. coli Origami B (BL21, OmpT-, Lon-, TrxB-, Gor-, Novagen) cells harboring the plasmids, which were grown in 2 L shake flasks with 500 ml LB medium and 100 pg / ml ampicillin, 15 pg / ml kanamycin and 12.5 pg / ml tetracycline at 37 °C. After addition of 0.1 mM isopropyl- -d-thiogalactopyronaside (IPTG), recombinant gene expression was induced at an OD550 of about 0.5-0.7 and the bacterial cells were then kept growing at 37 °C for 3 h for T22-BAK-GFP-H6 fusion protein production and at 20 °C overnight for T22-GFP-H6, T22-GWH1-GFP-H6 and T22-PUMA-GFP-H6 production.

[0512] Bacterial cells were then harvested by centrifugation at 5000 g for 15 min at 4 °C in the presence of protease inhibitors without EDTA (Complete EDTA-free; Roche, Basel, Switzerland) and resuspended in wash buffer (20 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole, pH 8.0). Cells were disrupted in a French Press (Thermo FA-078A) at 1200 psi and the lysate was centrifuged for 45 min (15,000 g at 4 °C).

[0513] All proteins were purified by His-tag affinity chromatography using a HiTrap Chelating HP 1 ml column (GE Healthcare, Piscataway, NJ, USA) by AKTA purifier FPLC (GE Healthcare). After filtration of the soluble fraction, the sample was loaded onto the column and washed with 10 column volumes of wash buffer. Elution was achieved by a linear gradient of 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 8.0 and the purified fractions were collected and analyzed by SDS-PAGE and Western blot with an anti-His monoclonal antibody (Santa Cruz Biotechnology, Heidelberg, Germany) to visualize the protein of interest.

[0514] Proteins were dialyzed against salt-containing sodium bicarbonate buffer (166 mM NaHC03pH 7.4 + 333 mM NaCl) overnight at 4°C. These buffers were the final solvents for further experiments. Protein integrity and purity were checked by mass spectrometry (MALDI-TOF) and quantified by the Bradford test.

[0515] Fluorescence measurements, dynamic light scattering (DLS) and field emission scanning electron microscopy (FESEM)

[0516] Fluorescence of fusion proteins was measured with a Varian Cary Eclipse Fluorometer (Agilent Technologies, Palo Alto, CA, USA) at 510 nm using an excitation wavelength of 450 nm. Volume size distribution of nanoparticles and monomeric GFP protein fusions was determined by DLS at 633 nm (Zetasizer Nano ZS, Malvern Instruments Limited, Malvern, UK).

[0517] For fluorescence measurements, protein samples were diluted to 0.5 mg / ml in the respective storage buffer to a final volume of 100 μΐ. For DLS analysis, proteins (stored at -80°C) were thawed and 50 μΐ per sample were used. Qualitative analysis by field emission scanning electron microscopy (FESEM) was performed on a Zeiss Merlin (Zeiss, Oberkochen, Germany) field emission scanning electron microscope operated at 1 kV and equipped with a high-resolution in-lens secondary electron detector. Droplets of diluted purified proteins were deposited on silicon wafer surfaces (Ted Pella, Reading, CA, USA), air-dried and observed immediately.

[0518] Cell culture and flow cytometry

[0519] The CXCR4+ HeLa cell line (ATCC-CCL-2) was cultured in Eagle's Minimum Essential medium (Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum and incubated at 37°C and humidified atmosphere with 5% C02. In the meantime, the SW1417 cell line was maintained in Dulbecco's Modified Eagle Medium (DMEM: GlutaMAX TM , Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum and incubated at 37°C and humidified atmosphere with 10% C02. The HeLa and SW1417 cell lines were cultured on 24-well plates at 33 x 10 4 and 12 x 10 4 cells / well, respectively, for 24 hours until reaching 70% confluence.

[0520] Twenty-four hours before flow cytometry analysis, nanoparticles and monomeric proteins were added to cell cultures at different concentrations (ranging from 0.1 to 2 mM) in the presence of Optipro medium Cell samples were analyzed on a FACSCanto system (Becton Dickinson, Franklin Lakes, NJ, USA) using a 15W air-cooled argon ion laser at 488 nm excitation. GFP fluorescence emission was measured with detector D (530 / 30 nm band-pass filter) after 15 minutes of treatment with 1 mg / ml trypsin

[0521] AMD3100 / CXCR4+ inhibitor (octahydrochloride hydrate, Sigma-Aldrich, Steinheim, Germany) was used to measure the specific internalization of nanoparticles. For this experiment, T22-BAK-GFP-H6 was labeled with ATTO488 (41698, Sigma-Aldrich) at room temperature for 1 hour in the dark to obtain more fluorescent protein. T22-BAK-GFP-H6-ATTO488 was added at 25 nM during the 1 -hour incubation in the presence of a 1 : 10 ratio of AMD3100.

[0522] Confocal microscopy

[0523] ​​HeLa cells were grown on Mat-Tek dishes (MatTek Corporation, Ashland, MA, USA). The medium was removed and cells were washed with DPBS and OptiPro medium supplemented with L-glutamine and proteins were added 24 hours before staining at 2 mM. Nuclei were labeled with 0.2 pg / ml Hoechst 33342 (Molecular Probes, Eugene, OR, USA) and plasma membrane was labeled with 2.5 pg / ml CellMask™ Deep Red (Molecular Probes) in the dark for 10 minutes. Live cells were recorded using a Plan Apo 63x / 1.4 (oil HCxPL APO lambdablue) objective by a TCS-SP5 confocal laser scanning microscope (Leica Microsystems, Heidelberg, Germany).

[0524] To determine the location of the particles inside the cells, a stack of 10-20 sections was collected with a Z interval of 0.5 pm, pinhole set at 1 Airy unit. Images were processed and 3-D reconstruction was generated using Imaris version 7.2.1.0 software (Bitplane, Zurich, Switzerland).

[0525] Biodistribution

[0526] Five-week-old female Swiss nu / nu mice (Charles River, L’Arbresle, France) weighing between 18 and 20 grams and maintained under SPF conditions were used for in vivo studies. All in vivo procedures were approved by the Animal Ethics Committee of the Hospital de Sant Pau and were performed according to the directives of the European Council.

[0527] To generate a subcutaneous (SC) mouse model, we obtained 10 mg of SP5 CCR tumor tissue from donor animals and implanted it subcutaneously in the lower flank of Swiss nu / nu mice. When tumors approximately reached 500 mm3 3 Mice were randomly assigned and administered T22-BAK-GFP-H6, BAK-GFP-H6 and T22-GFP-H6 nanoparticles at a dose of 330 pg / mouse.

[0528] Short (2 and 5 hours) and long (24 and 48 hours) time points were tested to explore the biological effects of the administered nanoparticles. To this end, mice were euthanized and tumors and brain, pancreas, lung and heart, kidney, liver and bone marrow were collected and processed for histology and immunohistochemistry. Ex vivo GFP fluorescence was investigated in a Spectrum device (PerkinElmer Inc, Waltham, MA, USA) respectively. The fluorescent signal (FLI) was first digitized, displayed as a pseudo-color overlay and expressed as radiance efficiency. The FLI signal from protein-treated mice was divided by the FLI spontaneous fluorescence signal from control mice to calculate the FLI ratio.

[0529] Finally, all organs were collected and fixed in 4% formaldehyde phosphate buffer solution for 24 hours. Then these samples were embedded in paraffin for histological and immunohistochemical analysis, and determination of mitosis and apoptosis index and evaluation of necrosis.

[0530] Histopathology and immunohistochemistry analysis

[0531] Four-micrometer-thick sections were stained with hematoxylin and eosin (H&E) and complete histopathological analysis was performed by two independent observers. The presence and location of His-tag in the protein material and the presence and location of proteolyzed PARP and active cleaved caspase 3 proteins in the tissue sections were evaluated by immunohistochemistry using DAKO immunostaining system equipment and standard protocols. After incubation with the secondary antibody in the tumor tissue for 2, 5, 24 and 48 hours, the primary antibody against His-tag (1 : 1000; MBL International, Woburn, MA, USA), anti-PARP p85 fragment pAb (1 : 300; Promega, Madison, WI, USA) or anti-active caspase 3 antibody (1 : 300, BD PharMigen, San Diego, CA, USA) was incubated for 25 minutes. The number of stained cells was quantified by two independent blind counters, which recorded the number of positive cells per 10 high-power fields (magnification 400x). Representative pictures were taken using Cell^B software (Olympus Soft Imaging v 3.3, Nagano, Japan).

[0532] Evaluation of mitosis, apoptosis, necrosis rate

[0533] Proliferative capacity was assessed by counting the number of mitotic figures per ten high-power fields (magnification x 400) in H&E-stained tumor sections. Apoptosis induction was assessed by the presence of cell death bodies in H&E and also by Hoechst staining in tumor sections. Hoechst 33258 (Sigma-Aldrich, Steinheim, Germany) staining was performed on permeabilized sections in Triton X-100 (0.5%). Slides were then stained with Hoechst 33258 (1:5000 in PBS) for 1 hour, washed with water, fixed, and analyzed under a fluorescence microscope (λex = 334 nm / λem = 465 nm).

[0534] The number of apoptotic bodies was quantified using two independent blind recordings—the number of coagulated and / or fragmented nuclei per 10 high-power fields (400x magnification). Necrotic areas in the tumor were quantified using Cell∧B software at 15x magnification, and representative images were taken using the same Cell∧B software at 400x magnification.

[0535] Figure 10 Figure 10

[0536] Protein design, production and purification

[0537] We designed our own synthetic genes encoding the self-assembly module proteins T22-DITOX-H6 and T22-PE24-H6, respectively. Figure 10 A), provided by Geneart (ThermoFisher). DITOX contains the translocation domain and catalytic domain of diphtheria toxin from Corynebacterium diphtheriae. PE24 is based on the deimmunization catalytic domain of exotoxin A from Pseudomonas aeruginosa, incorporating point mutations that disrupt B and T cell epitopes. Furthermore, a KDEL sequence has been added to the C-terminus of T22-PE24-H6, enabling more efficient binding to the KDEL receptor in the Golgi apparatus during subsequent intracellular transport. A furin cleavage site has been inserted between the CXCR4 ligand T22 and the functional toxin. Figure 10A), which releases the amino-terminal peptide once internalized into the target cell. This structure has been designed so that both natural forms of the two toxins act on the free amino-terminal end and the recombinant forms, which have proven to be active in the absence of additional peptide segments, also show this end. Both gene fusions were inserted into the plasmid pET22b and the recombinant version of this vector was transformed by heat shock in Escherichia coli Origami B (BL21, OmpT-, Lon-, TrxB-, Gor-, Novagen, Darmstadt, Germany). The transformed cells were grown overnight at 37°C in LB medium supplemented with 100 μg / ml ampicillin, 12.5 μg / ml tetracycline and 15 μg / ml kanamycin. When the OD550of the cell culture was about 0.5-0.7, 0.1 and 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) were added in T22-DITOX-H6 and T22-PE24-H6, respectively, and the encoded proteins were produced overnight at 20°C. The bacterial cells were centrifuged for 15 min (at 4°C, 5000 g) and kept at -80°C until use. The pellet was thawed and resuspended in wash buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 10 mM imidazole) in the presence of protease inhibitors (Complete EDTA-free, Roche Diagnostics, Indianapolis, IN, USA). Cell disruption was performed by French press (Thermo FA-078A) at 1200 psi. The lysate was then centrifuged for 45 min (at 4°C, 15,000 g) and the soluble fraction was filtered using a 0.2 μm pore size. The proteins were then purified by His-tag using immobilized metal affinity chromatography (IMAC) using a HiTrap Chelating HP 1 ml column (GE Healthcare, Piscataway, NJ, USA) and an AKTA purifier FPLC (GE Healthcare). Elution was achieved using a linear gradient of elution buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl and 500 mM imidazole). The eluted fractions were collected, dialyzed against carbonate buffer (166 mM NaCO3H pH 8) and centrifuged for 15 min (at 4°C, 15,000 g) to remove insoluble aggregates. The integrity and purity of the proteins were analyzed by mass spectrometry (MALDI TOF), SDS-PAGE and Western blot using an anti-His monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein concentration was determined by the Bradford test. A nomenclature for the fusion proteins has been established according to the modular organization of the fusion proteins.

[0538] Furin cleavage design and detection

[0539] To facilitate the release of the ligand-free toxins of the T22-DITOX-H6 and T22-PE24-H6 fusion proteins inside the cell, two different furin cleavage sites were included in T22-DITOX-H6 and T22-PE24-H6, which naturally function in the respective toxin precursors to activate translocation Materials and methods for protein nanoparticles based on recombinant ricin (mRTA) A). The cleavage efficiency in the platform was assessed in T22-DITOX-H6, as the expected fragments should exhibit fully distinguishable molecular weights suitable for quantitative analysis. To this end, HeLa cell extracts exposed to 1 mM of protein for 24 hours were subjected to Western blot analysis. After protein incubation, cells were collected, centrifuged, suspended in DPBS, and disrupted by sonication. Western blot bands were quantified using Image Lab software version 5.2.1. Two additional modular proteins were also constructed, namely T22-DITOX-H6 F- and T22-PE24-H6 F-, which did not include these engineered furin cleavage sites. Their amino acid sequences were fully matching those of the equivalent constructs T22-DITOX-H6 and T22-PE24-H6, except for the bolded dark blue peptides Figure 17 A) corresponding to the protease target sites. These non-cleavable constructs were used for comparative analysis of the protein cytotoxins.

[0540] Fluorescent labeling and dynamic light scattering

[0541] For fluorescent labeling and dynamic light scattering of the T22-DITOX-H6 and T22-PE24-H6 fusion proteins, the fusion proteins were labeled with ATTO488 (Sigma Aldrich, Buchs, Switzerland) to track their internalization in in vitro and in vivo experiments. Conjugation was performed in a 1 :2 molar ratio at room temperature in the dark. The reaction mixture was gently stirred once every 15 minutes for 1 hour, centrifuged for 15 minutes (at 4°C, 15,000 g), and dialyzed overnight in the original buffer (166 mM NaCO3H pH 8) to eliminate free ATTO.

[0542] The fluorescence of 0.1 mg / mL nanoparticles was measured at 523 nm using a 488 nm excitation wavelength on a Varian Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Mulgrave, Australia). For comparative analysis, fluorescence intensity was corrected for protein content to provide specific emission values. The stability of the dye conjugation was assessed by incubating T22-DITOX-H6* at 37 °C for 48 h with gentle agitation at a final concentration of 0.5 μg / μl in human serum (S2257-5 mL, Sigma, St Louis, MO, USA). The sample was then dialyzed in 300 mL of carbonate buffer (166 mM NaCO3H, pH 8) for 2 h to remove any free ATTO that may have been released from the nanoparticles. Simultaneously (in parallel), a positive control containing the same amount of free ATTO was dialyzed. The fluorescence of the buffer obtained after dialyzeation was measured using a fluorometer. The volume size distribution of all nanoparticles was determined by dynamic light scattering (DLS) at 633 nm (ZetasizerNano ZS, Malvern Instruments Limited, Malvern, Worcestershire, UK).

[0543] Ultrastructure characterization

[0544] The size and shape of near-native T22-DITOX-H6 and T22-PE24-H6 nanoparticles were evaluated using a field emission scanning electron microscope (FESEM) operated at 1 kV by Zeiss Merlin (Zeiss, Oberkochen, Germany). Droplets of 3 μl of each protein sample were directly deposited onto a silicon wafer (Ted Pella Inc., Reading, CA, USA) for 1 minute, excess was removed with No. 1 Whatman filter paper (GE Healthcare, Piscataway, NJ, USA), air-dried, and observed without a secondary electron detector coated within a high-resolution lens. Representative images of different fields of view were acquired at magnifications ranging from 120,000× to 200,000× for each sample.

[0545] Cell culture and flow cytometry

[0546] The performance of recombinant proteins (HeLaATCC-CCL-2, SW1417ATCC-CCL-238, and Panc-1ATCC-CCL-1469) in vitro was investigated using CXCR4+ cervical cancer, colorectal cancer, and pancreatic cancer cell lines. HeLa cells were maintained in Eagle's minimum essential medium (ESM). In Rockville, MD, USA, SW1417 and Panc-1 were maintained in Dulbecco modified Eagle medium, while SW1417 and Panc-1 were maintained in Rockville, MD, USA. They are all supplemented with 10% fetal bovine serum. They were incubated at 37°C in a humid atmosphere with 5% CO2 (10% for SW1417 cells).

[0547] To monitor protein internalization, HeLa cells were sputtered at 3 x 10⁻⁶ m². 4 Cells / well were cultured in 24-well plates for 24 hours until 70% confluence was achieved. In the presence of L-glutamine supplemented with OptiPRO... TM In the case of SFM, the protein was incubated for 1 hour at different concentrations (100, 500, and 1000 nM). Specific internalization via the CXCR4 receptor was demonstrated by adding the specific antagonist AMD3100, which is expected to inhibit interaction with T22. This chemical inhibitor was added at a 1:10 ratio 1 hour before protein incubation. Furthermore, internalization kinetics were assessed at a concentration of 1 μM after different incubation periods (0, 20, 30, 60, 120, and 240 minutes). Following protein exposure, trypsin-EDTA at 1 mg / ml was administered at 37°C. Cells were isolated for 15 minutes, a stringent experimental protocol designed to remove externally attached proteins (Richard JP et al., J. Biol. Chem. 2003, 278: 585-590). Samples were analyzed using a 15 mW air-cooled argon-ion laser excited at 488 nm via a FACS-Canto system (Becton Dickinson, Franklin Lakes, NJ, USA). Experiments were performed in duplicate.

[0548] Confocal laser scanning microscope

[0549] For confocal microscopy, HeLa cells were grown on Mat-Tek plates (MatTek Corporation, Ashland, MA, USA). After exposure to the material, cell nuclei were labeled with 5 μg / ml Hoechst 33342 (ThermoFischer, Waltham, MA, USA) at room temperature, and plasma membranes were labeled with 2.5 μg / ml CellMask. TMDeep Red (ThermoFischer) labeling for 10 min. Cells were then washed in PBS buffer (Sigma-Aldrich, Steinheim, Germany). Confocal images of HeLa cells were collected on an inverted TCS SP5 Leica spectral confocal microscope (Leica Microsystems, Wetzlar, Germany) using a 63x (1.4 NA) oil immersion objective. Excitation was achieved via a 405 nm blue diode laser (nucleic acids), a 488 nm line argon ion laser (nanoparticles) and a 633 nm line HeNe laser (cell membrane). Optimized emission detection bandwidth configurations were set to avoid inter-channel crosstalk and a multi-track sequential acquisition setup was used. The confocal pinhole was set to 1 Airy unit and the z-stack acquisition interval was chosen to meet the Nyquists sampling criterion. Three-dimensional images were processed using the Surpass module in Imaris X64 v.7.2.1 software (Bitplane, Zurich, Switzerland).

[0550] Cell viability assay

[0551] Use A luminescent cell viability assay (Promega, Madison, WI, USA) was used to determine the cytotoxicity of T22-DITOX-H6, T22-PE24-H6, T22-DITOX-H6 F- and T22-PE24-H6 F- nanoparticles on HeLa, SW1417 CXCR4+ or SW1417 CXCR4- cell lines. Cells were cultured in opaque-walled 96-well plates at 3500 or 6000 cells / well for 24 hours at 37°C until reaching 70% confluence. Depending on the cell line used, all protein incubations were performed in the corresponding culture medium. One hour before protein incubation, AMD3100 (a chemical antagonist of CXCR4) was added at a 1 : 10 ratio to analyze the inhibition of cell death. T22-GFP-H6, a non-functional T22-carrying protein, was also used as a competitor of T22-empowered toxins at a final concentration of 2 μΜ. After protein incubation, single reagents provided by the manufacturer were added to the cultured cells, which prompted cell lysis and the production of a luminescent signal proportional to the amount of ATP in the sample. The ATP produced was directly related to the number of viable cells remaining in the well. Then, the plates were measured in a conventional luminescence meter Victor3 (Perkin Elmer, Waltham, MA, USA). Under the same experimental conditions, another non-fluorescent kit (EZ4U) was used to determine the viability of Panc-1 cells overexpressing luciferase. Cell viability assays were performed in triplicate.

[0552] Biodistribution, pharmacokinetics, and apoptosis induction in a CXCR4+ colorectal cancer mouse model following single-dose administration of nanoparticles

[0553] All in vivo experiments were approved by the Animal Ethics Committee of São Paulo Hospital. We used 5-week-old female Swiss Nu / Nu mice, weighing 18–20 g (Charles River, L'Abresle, France), kept under specific pathogen-free conditions. To generate a subcutaneous (SC) mouse model, we subcutaneously implanted 10 mg of patient-derived M5 colorectal cancer (CCR) tumor tissue from donor animals into the subcutaneous tissue of the mice. On day 15, when the tumor reached approximately 500 mm... 3 Mice received either a single intravenous bolus of 50 μg NaCO3H in pH 8 buffer (T22-DITOXH6*) (n=3) or a single intravenous bolus of 300 μg NaCO3H in pH 8 buffer (T22-PE24-H6*) (n=3). Control animals received the same buffer (n=3) or 0.25 μg free ATTO 488 (n=2). Mice were euthanized at 5, 24, and 48 hours, and subcutaneous tumors and organs (brain, lung, liver, kidney, and heart) were collected. [The text abruptly ends here, likely due to an incomplete sentence or missing information.] The fluorescence emitted in ex vivo tissue sections (3 mm thick) was measured using a spectroscopy platform (Perkin Elmer, Santa Clara, CA, USA) to determine the biodistribution of ATTO-labeled nanoparticles in tumor and non-tumor organs. First, the fluorescence signal (FLI)—correlated with the amount of protein accumulated in each tissue—was digitized, displayed as a pseudo-color overlay, and expressed as radiometric efficiency [(p / s / cm² / sr) / μW / cm²]. The FLI value was calculated by subtracting the FLI signal of experimental mice from the FLI autofluorescence of control mice. Samples were first fixed with 4% formaldehyde in PBS for 24 hours and then embedded in paraffin for histopathological evaluation and apoptosis index analysis. Pharmacokinetic analysis was performed after a single intravenous bolus administration of T22-PE24-H6* in 12 Swiss nude mice, or after a single bolus administration of 50 μg T22-DITOX-H6* in 12 animals.

[0554] Three mice were sacrificed at each time point 0, 1, 2, 5, 24 and 48 hours after drug administration and about 1 ml of blood was collected in EDTA anticoagulant tubes. The precise volume of the obtained plasma was measured and the fluorescence emission at each time point was measured and the concentration of nanoparticles was called emitted fluorescence and calculated concentration of the administered dose. Apoptosis induction analysis was performed on 4 pm sections of tumors and normal organs (liver, lung, spleen, heart, kidney and brain) stained with hematoxylin and eosin (H&E), which were analyzed by two independent observers for histopathological analysis. Apoptosis induction was assessed by the presence of cell death bodies in both tumor sections stained with H&E and Hoechst. Sections permeabilized with X-100 (0.5%) were then stained with Hoechst 33258 (Sigma-Aldrich) diluted 1 :5000 in PBS for 1 hour, washed with water, mounted and analyzed under a fluorescence microscope (l ex = 334 nm / l em = 465 nm). The number of apoptotic cell bodies was quantified by recording the number of condensed and / or fragmented nuclei recorded per 10 high power fields (magnification 400x) in blind samples evaluated by two independent researchers using Cell & B.

[0555] Anti-tumor effect in CXCR4+ CRC model after repeated dose administration of nanoparticles

[0556] To generate a CXCR4+ colorectal xenograft mouse model, we used M5 colorectal tumor tissue derived from a patient. 10 mg fragments obtained from the donor animal were implanted in the lower flank of Swiss nu / nu mice to generate subcutaneous (SC) tumors (n = 9) as described above. Once tumors reached approximately 120 mm3, mice were randomized into control, T22-PE24-H6 and T22-DITOX-H6 groups and received intravenous administration of T22-PE24-H6 or T22-DITOX-H6, both in a repeated dosing regimen: 10 pg, 3 times per week, 8 doses each. The control group received buffer using the same dosing regimen. Mouse body weight was recorded 3 times per week during the experiment. Seventeen days after starting nanoparticle administration, mice were euthanized and subcutaneous tumors were measured to measure their final tumor volume and count the number of apoptotic figures in H&E stained tumor sections as described above in 5 high power fields (magnification 400x). 3 , mice were randomized into control, T22-PE24-H6 and T22-DITOX-H6 groups and received intravenous administration of T22-PE24-H6 or T22-DITOX-H6, both in a repeated dosing regimen: 10 pg, 3 times per week, 8 doses each. The control group received buffer using the same dosing regimen. Mouse body weight was recorded 3 times per week during the experiment. Seventeen days after starting nanoparticle administration, mice were euthanized and subcutaneous tumors were measured to measure their final tumor volume and count the number of apoptotic figures in H&E stained tumor sections as described above in 5 high power fields (magnification 400x).

[0557] Statistical analysis

[0558] Specificity of nanoparticle-facilitated cell death and pairwise data comparisons were examined by one-way ANOVA and Tukey's test, respectively. Pairwise dispersion of internalization and cell death were assessed using Student's t-test, while pairwise comparisons of the number of apoptotic bodies were made using the Mann-Whitney U test. Differences between groups were considered significant at p < 0.05, and differences between related data are indicated in the figures by letters, or for 0.01 < p < 0.05 as ©, and for p < 0.01 as §. All statistical analyses were performed using the SPSS version 11.0 package (IBM, NY, USA), and numerical values are expressed as mean ± standard error of the mean (SEM).

[0559] Example 1 : Design and characterization of T22-BAK-GFP-H6 fusion protein and nanoparticles

[0560] Genetic design and protein production

[0561] Recombinant protein T22-mRTA-H6 Figure 1 A) Designed to include the highly specific CXCR4 ligand T22 at the amino terminus, followed by a mutated form of ricin A chain, and a hexahistidine tail at the carboxy terminus. The mutation N132A was introduced to inhibit vascular leakage syndrome in potential future in vivo administration, while maintaining the cytotoxic activity. In addition, a furin cleavage site was also incorporated to allow release of the auxiliary N-terminal region and intracellular activity of ricin in the form of the quasi-native sequence in the endosome. A KDEL motif was also incorporated to facilitate endosomal escape. The plasmid construct pET22b-T22-mRTA-H6 encoding the protein under the control of the bacteriophage T7 promoter was generated by GeneArt and transformed into E. coli Origami B cells.

[0562] Production and purification of soluble protein

[0563] Recombinant bacteria were grown in lysogeny broth (LB) medium containing 100 pg / ml ampicillin, 15 pg / ml kanamycin and 12.5 pg / ml tetracycline at 37 °C and 250 rpm. When the OD of the culture reached a value between 0.5 and 0.7, recombinant gene expression was induced by the addition of 0.1 mM isopropyl-beta-thiogalactopyranoside (IPTG). The culture was then incubated overnight at 20 °C and 250 rpm. Cells were harvested and centrifuged (5,000 g, 15 min, 4 °C). The cell pellet was resuspended in wash buffer (51 mM sodium phosphate buffer, pH=8, 158.6 mM trehalose dihydrate, 0.01 % polysorbate-20, 15 mM imidazole, 300 mM NaCl) in the presence of complete EDTA-free protease inhibitor cocktail (Roche). Bacterial cells were sonicated twice, once at 10% amplitude and once at 15% amplitude, for 10 minutes each round, centrifuged (15,000 g, 45 min, 4 °C), and the soluble fraction was purified by affinity chromatography using a HiTrap Chelating HP column in an AKTA purifier FPLC (GE Healthcare). After the sample was filtered (0.22 pm) and injected into the column, the fraction to be collected was eluted at about 30% of the elution buffer (51 mM sodium phosphate, pH=8, 158.6 mM trehalose dihydrate, 0.01 % polysorbate-20, 500 mM imidazole, 300 mM NaCl). Buffer exchange was performed in Centricon Centrifugal Tubes Ultracel 10,000 NMWL. T22-mRTA-H6 was found to be highly stable in 51 mM sodium phosphate pH=6.2, 60 mg / ml a-trehalose dehydrate, 0.01 % polysorbate-20. Protein purity was analyzed by SDS electrophoresis on TGX unstained gels (Bio-Rad) followed by western blot analysis using an anti-His monoclonal antibody (Santa Cruz Biotechnology). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on TGX unstained gels (Bio-Rad) to analyze the protein. Samples were diluted in a molar ratio of 3: 1 in denaturing buffer (0.53 M Tris base, 5.52 M glycerol, 0.27 M SDS, 2.84 M beta-mercaptoethanol, 7.99 M urea), boiled at 96 °C for 10 minutes, and loaded into the gel lanes. For western blotting, an anti-His monoclonal antibody (Santa Cruz Biotechnology) was used, followed by a goat anti-mouse IgG (H+L)-HRP secondary antibody (Ref: 170-6516) conjugate (Bio-Rad, Ref: 170-6516).Images were observed using a ChemiDoc Touch imaging system. Protein production has been partially performed by the Protein Production Platform of ICTS "NANBIOSIS", more specifically by CIBER-BBN / IBB (http: / / www.nanbiosis.es / unit / u1-protein-production-platform-ppp / ).

[0564] Quantitative protein analysis

[0565] Protein purity was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a Chemi Doc Touch Imaging System (Bio-Rad). Briefly, both soluble and insoluble samples were mixed in a 3:1 ratio in denaturing buffer (0.53 M Tris base, 5.52 M glycerol, 0.27 M sodium dodecyl sulfate (SDS), 2.84 M beta-mercaptoethanol, 7.99 M urea), boiled for 5 or 45 minutes, respectively, and loaded onto the gel. For Western blotting, an anti-His monoclonal antibody (Santa Cruz Biotechnology) was used, followed by a goat anti-mouse IgG (H+L)-HRP secondary antibody conjugate (Bio-Rad). Gels were scanned at high resolution and bands were quantified with Quantity One Software (Bio-Rad) using known soluble recombinant T22-mRTA-H6 protein standards.

[0566] Quantitative and qualitative analysis of soluble proteins

[0567] Protein molecular weight was verified by mass spectrometry (MALDI-TOF) and concentration was determined by the Bradford assay (Dye Reagent Concentrate Bio-Rad kit). The volume size distribution of the protein nanoparticles was determined by dynamic light scattering (DLS). For this, a 50 μΙ aliquot (stored at -80 °C) was thawed and immediately the volume size distribution of the nanoparticles was determined at 633 nm (Zetasizer Nano ZS, Malvern Instruments Limited). Far-UV circular dichroism (CD) was measured at 25 °C with a Jasco J-715 spectrophotometer to assess the secondary structure of T22-mRTA-H6, which was dissolved at 0.35 mg / ml in 166 mM sodium bicarbonate buffer at pH 8. The CD spectra were obtained in a 1 mm path length cuvette in the wavelength range of 190-260 nm at a scan rate of 50 nm / min, a response of 1 s and a bandwidth of 1 nm. Six scans were accumulated. The size of the secondary structure was analyzed using the JASCO Spectrum manager analysis software. To investigate potential intermolecular beta-sheet structures in the protein nanoparticles, the regular method for Thioflavin T (ThT) staining was adapted.

[0568] Briefly, a protein aliquot (10 μΙ) was added to 90 μΙ, 50 μΜ (Sigma Aldrich) phosphate buffered saline (PBS) at pH 7.4 and stirred for 1 min. The final protein concentration was 0.17 mg / ml. ThT was excited at 450 nm and the fluorescence emission spectrum was recorded in the range of 460 to 565 nm using a Varian Cary Eclipse spectrophotofluorometer. Cross-beta sheet structures were monitored by enhancing the fluorescence emission of the free dye.

[0569] Cell culture and determination of cell viability and apoptosis

[0570] HeLa cells (ATCC-CCL-2) were cultured in MEM-alpha medium supplemented with 10% fetal bovine serum (Gibco Thermo Fisher Scientific (TFS)) at 37 °C in a 5% CO2 humidified atmosphere. They were seeded in opaque 96-well plates (3 x 10 4cells were exposed to soluble T22-mRTA-H6 for 24, 48 and 72 hours. Cells were also exposed to the insoluble protein form for 24, 48, 72, 96, 120 and 144 hours. Cell viability was determined by performing CellTiterGlo luminescent cell viability assay (Promega) in a Multilabel Plater Reader Victor3 (Perkin Elmer). For CXCR4 specificity assays, CXCR4 antagonist AMD3100 was added at a molar ratio of 10:1 one hour prior to the incorporation of the protein. The final volume of 10 μΐ of antagonist and protein was incubated, which was mixed with 90 μΐ of culture medium. All soluble protein experiments were performed in triplicate and insoluble protein in sextuplicate. On the other hand, AML cell lines THP1 (ACC-16) and MV411 (ACC-102) and 3T3 mouse fibroblasts (ACC-173) were purchased from DSMZ (Leibniz Institute DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). THP1 were cultured in RPMI-1640 medium supplemented with 10% FBS, 10 mmol / l L glutamine, 100 U / ml penicillin, 10 mg / ml streptomycin and 0.45 μg / ml amphotericin B. (Gibco, TFS). 3T3 cells were cultured with DMEM medium with the same supplements. Cells were maintained at 37°C in a humidified atmosphere of 5% CO2. Cell viability assays were performed on these cell lines using the XTT cell viability kit II (Roche Diagnostics) and absorbance was read at 490 nm with a spectrophotometer (BMG Labtech). Cells seeded on 96-well plates were pre-treated for 1 hour (100 μΜ zVAD-fmk) and then exposed to 100 nM T22-mRTA-H6 for 48 hours to assess the effect of the caspase inhibitor zVAD-fmk. The antitumoral drug Ara-C (cytosine β-D-arabinofuranoside hydrochloride) was purchased from Sigma Aldrich. To allow follow-up of AML in mice, the THP1 AML cell line was transfected with a plasmid encoding a luciferase gene that can confer bioluminescence (BLI) to cells for non-invasive imaging.Briefly, THP1 cells were harvested in 24-well plates, treated with 0.5 pg DNA plasmid and mixed with Lipofectamine LTX and PLUS reagents (A12621, Invitrogen, TFS) in Opti-MEM reduced serum medium (Gibco, TFS) following the manufacturer's instructions. After 48 hours, cells were incubated with luciferin in an IVIS Spectrum in vivo imaging system (PerkinElmer, Waltham, MA, USA) to test BLI levels. Finally, transfected cells were selected with 1.5 mg / mL geneticin (G418 sulfate, Gibco, TFS) and BLI was periodically analyzed to check plasmid preservation in cells, which were called THP1-Luci cells. Internalization of T22-GFP-H6 in 3T3, MV411, THP1 and HeLa was determined by fluorescence-activated cell sorting (FACS Calibur, BD). Cells were exposed to T22-GFP-H6 at a concentration of 100 nM for 1 hour. Then, cells were washed with PBS and trypsinized (1 mg / ml trypsin, Life Technologies) to remove non-specific binding of nanoparticles to the cell membrane. Finally, the level of intracellular GFP fluorescence was quantified by flow cytometry. The mean fluorescence intensity ratio is given as the mean fluorescence intensity of the treated sample divided by the mean fluorescence intensity of the vehicle.

[0571] To assess apoptosis, nuclear staining was performed with Hoescht 3342 dye (Sigma- Aldrich) in HeLa cells exposed to 100 nM T22-mRTA-H6 or buffer for different times. At the end of the incubation, the culture medium was collected and centrifuged to obtain the suspended cells. They were washed with PBS and centrifuged again. Adherent cells were trypsinized and pulled together with the previously obtained cells. These cells were fixed (3.7% paraformaldehyde in PBS, pH 7.4) for 10 min at -20 °C, washed with PBS and resuspended in 10 pl of PBS. Finally, cells were mounted on glass slides with ProLong Gold Antifade Mountant with DAPI and the appearance of the nuclei was observed under a fluorescence microscope. Moreover, cells exposed to the outside phosphatidylserine protein were detected by Annexin V detection kit (APC, eBioscience) according to the supplier's instructions, while dead cells were spotted with propidium iodide (PI). Intracellular internalization was monitored using ATTO-labeled proteins as described elsewhere. TM Gold Antifade Mountant and the appearance of the nuclei was observed under a fluorescence microscope. Moreover, cells exposed to the outside phosphatidylserine protein were detected by Annexin V detection kit (APC, eBioscience) according to the supplier's instructions, while dead cells were spotted with propidium iodide (PI). Intracellular internalization was monitored using ATTO-labeled proteins as described elsewhere.

[0572] Determination of ROS levels and mitochondrial damage

[0573] On the other hand, the level of cellular ROS was measured with the Cell ROS Assay Kit (Abeam). Briefly, HeLa cells were exposed to 100 nM T22-mRTA-H6 (15 or 24 hours) or buffer. Then, cells were washed and incubated with the ROS detection solution for 1 hour in the dark at 37°C, adding 100 mM pyocyanin to the positive control (1 hour). Then, the fluorescence level was read with a microplate reader (BMG Labtech) at Ex = 488 nm and Em = 520 nm. Values were expressed as relative fluorescence units after subtracting the background fluorescence of the blank. Finally, to measure the mitochondrial membrane potential (Ap), we used the Mitochondrial Potential Assay Kit (BD MitoScreen, BD Biosciences) according to the manufacturer’s instructions. Labeled cells were analyzed by flow cytometry and data were expressed as percentage of cells containing depolarized mitochondria (loss of JC-1 red fluorescence).

[0574] Flow cytometry

[0575] CXCR4 membrane expression was determined by fluorescence-activated cell sorting (FACSCalibur, BD). Cells were washed with PBS 0.5% BSA and incubated with PE-Cy5 mouse anti-CXCR4 monoclonal antibody (BD Biosciences) as control or PE-Cy5 mouse IdG2a isotype (BD Biosciences). Results of fluorescence emission were analyzed with the software Cell Quest Pro and expressed as the ratio between the mean fluorescence intensity of each sample and the isotype value.

[0576] Electron microscopy

[0577] Ultrastructure of soluble (in the form of nanoparticles) and insoluble (in the form of IBs) T22-mRTA-H6 was observed by field emission scanning electron microscopy (FESEM). Insoluble proteins were resuspended in PBS and sonicated at 10% amplitude, 0.5 sec on / off for 1 minute. Drops of 10 pL of soluble proteins in storage buffer or insoluble proteins in PBS were deposited on silicon chips (Ted Pella) during 1 minute, excess liquid was removed and air-dried. Samples without coating were observed with an in-lens detector running at 1 kV in a FESEM Zeiss Merlin (Zeiss). Representative images were obtained at a wide range of magnifications (100,000x to 450,000x).

[0578] Antitumor effect in a disseminated acute myeloid leukemia (AML) mouse model

[0579] Five-week-old female NSG (NOD-scid IL2Rgammanull) mice were obtained from Charles River Laboratories (Wilmington, MA, USA) and housed in miniature isolators with free access to sterile food and water. After one week of isolation, the NSG mice were intravenously (IV) injected with luciferase-transfected THP1 cells (THP1-Luci; 1×10⁻⁶). 6 Mice were randomly assigned to three different experimental groups (n=3, n=1, n=200, n=1, n=1, n=1, n=1, n=10 ... Tissue was excised and organ BLI levels were analyzed ex vivo. Subsequently, the organs were preserved in 3.7% formaldehyde and embedded in paraffin for further immunohistochemical analysis. BLI analysis and detection were performed using radiation photons in LivingImage 4.4 software for both in vivo and ex vivo studies. All procedures were performed in accordance with guidelines approved by the Animal Ethics Committee of São Paulo Hospital.

[0580] Histopathology and Immunohistochemical Staining

[0581] Sections of paraffin-embedded samples of infiltrated (liver, spleen, hind limbs, and skeleton) and normal (lung, heart, and kidney) organs were stained with hematoxylin and eosin (H&E) and the presence of toxicity was analyzed. In addition, immunohistochemical analysis of anti-human CD45 antibody (DAKO) was performed in paraffin-embedded tissue samples to detect AML cells in infiltrated tissue. Staining was performed in Dako Autostainer Link 48 according to the manufacturer's instructions. All samples were evaluated by two independent observers using an Olympus BX51 microscope (Olympus). Images were acquired using an Olympus DP72 digital camera and processed using CellDimaging 3.3 software (Olympus).

[0582] Statistical analysis

[0583] Quantitative data are expressed as mean ± standard error (SE). Normality and homogeneity of variance for all variables were tested using Shapiro-Wilk and Levene's test, respectively, prior to performing statistical analysis. Tukey's test was used to compare cytotoxic effects and competition assays of soluble proteins. Meanwhile, protein cytotoxic assays were evaluated by Mann-Whitney U test. Significance was accepted at p < 0.05.

[0584] Figure 1

[0585] The inventors designed a fusion protein comprising the cationic peptide T22, a potent CXCR4-ligand for the BAK BH3 domain, for the construction of BAK-like building blocks. GFP was incorporated into the fusion platform to facilitate monitoring of the localization of the material and to explore the potential use of the material in diagnostics as well as therapy (or for therapeutic diagnostics). A schematic representation of the fusion protein can be seen in Figure 1 A.

[0586] The chimeric protein was biologically produced in E. coli and purified by conventional procedures (as indicated in the Materials and Methods section) as a unique and stable molecular species with the expected quality ( Figure 1 B). As expected, the protein spontaneously assembled into discrete monodisperse material with a diameter of about 13.5 nm, which disassembled into >7 nm building blocks when treated with SDS ( Example 2: Functional analysis of T22-BAK-GFP-H6 fusion protein nanoparticles C). The T22-BAK-GFP-H6 monomer was slightly larger than the BAK-GFP-H6 protein (<7 nm) due to the absence of the cationic T22, which did not assemble. No disassembly was observed when incubated in Optipro complex medium (not shown) for 5 h, indicating the stability of the nanoparticle in complex physiological medium.

[0587] Furthermore, the T22-BAK-GFP-H6 nanoparticle was fluorescent, exhibiting a specific green fluorescence emission of 306.7 ± 7.8 units / μg, suitable for quantitative imaging. High resolution scanning electron microscopy revealed these materials to be planar objects with regular morphometric measurements ( Figure 2 D).

[0588] Figure 2

[0589] In terms of functional analysis, the inventors first determined the ability of the protein nanoparticle to bind and penetrate CXCR4+ cells in a receptor-dependent manner. Indeed, the assembled T22-BAK-GFP-H6 protein efficiently penetrated CXCR4+ HeLa and SW1417 cells ( Figure 2A). The kinetics of accumulation are compatible with receptor-mediated endocytosis Figure 2 B), while the uptake is CXCR4-dependent, AMD3100, an inhibitor of the T22-CXCR4 interaction, [Unzueta, U. et al. 2012. Int. J. Nanomedicine. 7:4533-44.] significantly reduced the intracellular fluorescence in both cell lines after exposure.

[0590] Control constructs without T22 were unable to enter the cells Figure 2 C). The efficient penetration of T22-BAK-GFP-H6 was confirmed by the ubiquitous occurrence of fluorescence in most of the exposed cells Figure 2 D, and the intracellular accumulation of material in the perinuclear region Example 3: In vivo accumulation and distribution of T22-BAK-GFP-H6 fusion protein nanoparticles E). T22-BAK-GFP-H6 was essentially non-toxic, as CXCR4-cell viability remained unchanged after prolonged exposure Figure 3 B, inset).

[0591] Figure 3

[0592] Considering the high CXCR4-linked cell penetration of T22-BAK-GFP-H6 nanoparticles, the inventors tested the new material in a mouse model of CXCR4+ colorectal cancer, in terms of biodistribution and ability of the material to induce selective apoptosis in tumor tissues. Systemic administration of T22-BAK-GFP-H6 nanoparticles through the tail vein allowed the transient accumulation of the material in the tumor, reaching a peak at 5 hours, as determined by ex vivo fluorescence images and IHC Figure 3 A-3C).

[0593] Other relevant organs, such as the kidney, showed only residual levels of fluorescence emission Table 1. D), not only confirming the desired localization of the material, but also the lack of significant kidney filtration, accumulation in the lung or detectable toxicity over time Table 1 D, 3E). In particular, the lack of protein in the kidney indicates the high stability of the oligomer, as monomeric or fragmented proteins, even those targeting specific tumor markers, accumulate in the kidney [Cespedes, M. V. et al. 2014. ACS Nano, 8:4166-76].

[0594] At 24 hours, but not at 48 hours, the tumor still showed detectable fluorescence (Table 1), indicating the prolonged persistence of the nanoparticles in the target organ.

[0595] Example 4: Effect of T22-BAK-GFP-H6 fusion protein nanoparticles on apoptosis and cell cycle

[0596]

[0597] Figure 4 Quantification of GFP fluorescence signal expressed as the proportion of fluorescence in brain, lung and heart, liver, kidney and bone marrow tissues. This was calculated by dividing the fluorescence of each organ in the protein-treated mice by the spontaneous fluorescence measured in the buffer-treated mice of the respective organ in the time course experiment. Data are expressed as mean ± SE.

[0598] Figure 4

[0599] T22-BAK-GFP-H6 induced significantly reduced mitotic figures compared to the parental T22-GFP-H6 or the non-targeted BAK-GFP-H6 protein Figure 4 A). This correlated with caspase-3 activation, proteolysis of PARP, the occurrence of apoptotic bodies and increased necrotic areas in tumor tissues soon after material administration in mice (2 hours) Figure 4 B-4F). Tumor cell apoptosis peaked at 5 hours and remained for at least 48 hours Figure 3 A).

[0600] In contrast, the non-targeted BAK-GFP-H6 protein produced only negligible levels of caspase-3 activation or cell apoptosis in the tumor, as it did not differ from the background of buffer-treated tumors Example 5: Physical and biological characterization of nanoparticles of T22-PUMA-GFP-H6 and T22-GW-H1-GFP-H6 fusion proteins F). No histological changes were observed in any of the non-target organs explored Figure 5 E). These observations not only confirmed the molecular availability of the BAK BH3 domain when delivered as a regular nanoparticle, however, as hypothesized, the T22-BAK-GFP-H6 nanoparticle exhibited intrinsic biological activity.

[0601] Figure 5 Figure 5

[0602] At this stage, the inventors further investigated the options this platform based on therapeutic protein nanoparticles only had. Thus, the inventors tested the formation of functional nanoscale materials based on modulators of p53 upregulated modulator of apoptosis PUMA [Zhang, Y et al. 2009. Mol Biol Cell., 20:3077-87] and the antimicrobial peptide GWH1 [Chen, Y-L.S. et al. 2012. Peptides, 36:257-65], both also induce apoptosis when internalized in cancer cells. According to the same modular approach as for T22-BAK-GFP-H6, T22-PUMA-GFP-H6 Figure 5 A) and T22-GWH1-GFP-H6 Example 6: Characterization of H6-GFP-R9 and H6-R9-GFP fusion proteinsB) 20 nm and 24 nm nanoparticles were formed, respectively, as the GFP fluorescence was retained in the three BAK-based constructs (not shown). Both nanoparticles accumulated in the tumor when administered in vivo 40-70 nm C-5D), with little occurrence of T22-GWH1-GFP-H6 in the kidney. Both types of nanoparticles significantly reduced the mitotic rate and even with some variability, the carpet material tended to induce cell death and promote selective necrosis in the tumor tissue, an effect that was evident in the case of the PUMA-based material Figure 6 E-5F).

[0603] Example 7: Characterization of GWH1 -based protein nanoparticles

[0604] The inventors designed fusion proteins including a hexahistidine region, GFP and a polyarginine sequence in the following order (H6-GFP-R9 fusion protein) and a hexahistidine region, a polyarginine sequence and GFP (H6-R9-GFP fusion protein).

[0605] Chimeric proteins were biologically produced in E. coli and purified by conventional procedures (as described in the Materials and Methods section) in the form of unique and stable molecular species of the expected quality. In the case of the H6-GFP-R9 fusion protein, the protein spontaneously assembled into discrete monodisperse materials of about Figure 1 60-90 nm (d) in diameter in the case of the H6-R9-GFP fusion protein. Figure 7 ).

[0606] Figure 7

[0607] GWH1-GFP-H6 (A) was successfully produced in recombinant E. coli without apparent signs of toxicity. Its purification in a single step by His-affinity chromatography resulted in a protein species of the expected molecular mass of 30.2 kDa (B, C). Since the GWH1 peptide is highly cationic and the combination of a terminal cationic peptide plus a polyhistidine promotes protein self-assembly, we tested the potential of this protein to form oligomers. Indeed, the spontaneous formation of nanoparticles was detected by DLS in the pure preparations of the protein (D), indicating the good performance of GWH1 as a nanoscale tissue tag. Those nanoparticles peaking at 47 nm were fully resolved by 0.1% SDS, yielding 5 nm building blocks that matched the size of the parent unassembled GFP-H6 (D). The formation of regular GWH1-GFP-H6 nanoparticles was fully assessed by FESEM (E). Figure 7 Figure 1 Example 8: Anti-bacterial activity of GWH1 -based protein nanoparticles Figure 8 Figure 8

[0608] ​​​​​Figure 8

[0609] To test whether GWH1-GFP-H6 retains its antibacterial activity when assembled into protein nanoparticles, we exposed cultures of several bacterial species to the material. As observed (…). Figure 8 A), GWH1-GFP-H6 showed potent antibiotic activity in three of the four species, which was clearly dose-dependent. Figure 8 B). The protein's activity against Pseudomonas aeruginosa remained significant, but weaker than in the remaining targets.

[0610] In all cases, bacterial death was clearly associated with cell lysis. Example 9: Cytotoxic activity of GWH1 -like protein nanoparticles C) strongly suggests that antibacterial activity is achieved through the conventional membrane activity of GWH1. Because the necessity of the free AMP N-terminus for this activity (and what would happen in the case of other AMPs) has not been previously described, and considering further potential designs for more complex GWH1-like recombinant constructs, we also tested T22-GWH1-GFP-H6 nanoparticles in the same assay. T22 is a cationic ligand for the cytokine receptor CXCR4, which may be clinically associated with HIV infection (as this protein is a co-receptor of the virus) and with several human cancers overexpressing this receptor, such as pancreatic cancer, metastatic melanoma, or osteosarcoma [19-23]. As observed, the T22-GWH1-GFP-H6 nanoparticles remained active against the target bacterial cells, but with reduced efficiency. Figure 9 A). T22 did not provide antimicrobial properties for these materials because the associated oligomeric construct T22-GFP-H6 did not show any biological effects. Figure 9 A).

[0611] Figure 9

[0612] We were interested in understanding whether GWH1-GFP-H6 nanoparticles would also exhibit cytotoxic potential. This is important because any residual anticellular activity of GWH1 nanoparticles would hinder their potential as antimicrobial agents. Because GWH1-GFP-H6 exhibited fluorescence representing approximately 50% of the specificity (not shown) of His-tagged GFP, we were able to monitor potential internalization in cultured human cells. As observed, GWH1-GFP-H6 did not internalize in HeLa cells, but two constructs carrying the CXCR4 ligand T22 were able to penetrate these cultured cells. Example 10: Protein nanoparticles based on diphtheria toxin (DITOX) and Pseudomonas aeruginosa exotoxin (PE24) A). GWH1-GFP-H6 nanoparticles were also inefficient in promoting HeLa cell death, as was the case with the control T22-GFP-H6. Figure 10B). However, strong cytotoxicity in cells exposed to T22-GWH1-GFP-H6 nanoparticles was evident Figure 10 B), indicating that the combination of intracellular targeting agent (T22) and AMPs is effective for cell killing.

[0613] Figure 11 Figure 11

[0614] Active fragments of diphtheria toxin (DITOX) and exotoxin of Pseudomonas aeruginosa (PE24) were produced in E. coli Figure 12 A, B), as modular fusion proteins T22-DITOX-H6 and T22-PE24-H6, aimed at inducing targeted cell death by the activity of catalytic fragments of protein drugs Figure 12 C). The cationic peptide T22, placed at the amino terminus of the complete construct and cooperating with the carboxy-terminal histidine, promotes both oligomerization into regular nanoparticles and binding to the cell surface chemokine receptor CXCR4, overexpressed in many aggressive human cancers. In this way, it has been demonstrated to be effective in facilitating endosomal penetration of the payload GFP and IRFP into CXCR4+ cancer stem cells. Then, T22-DITOX-H6 self-assembled into 38 and 90 nm nanoparticles (Pdi = 0.25 ± 0.01 nm) and T22-PE24-H6 self-assembled into ~ 60 nm nanoparticles (Pdi = 0.22 ± 0.01, Figure 12 A), always in the size range considered optimal for efficient cellular uptake. In the case of T22-PE24-H6, a secondary population of protein material was observed, always in the minority. Nanoparticles were effectively disassembled by 0.1% SDS, resulting in monodisperse building blocks peaking at ~ 6 nm (Pdi = 0.60 ± 0.01 and 0.30 ± 0.07, respectively), in agreement with the expected size of monomeric proteins. However, both protein nanoparticles were very stable in several physiological buffers incubated at the corner of the culture medium and also very stable when exposed to high salt content buffers (up to 1 M NaCl, not shown), which prompted us to expect high stability in vivo. In addition, nanoparticles were found to be stable after storage at -80°C for one year and when repeated cycles of freezing and thawing (not shown). The assembled proteins appeared as toroidal material Figure 12 B), which confirmed the size range observed by DLS. The same regular structure has been previously described for the related T22-GFP-H6 construct, in which the GFP-based subunits (molecular size similar to T22-DITOX-H6 and T22-PE24-H6) organized as toroidal entities, whose organization has been simulated by computer and confirmed by sophisticated analytical methods such as SAXS or high-resolution electron microscopy imaging techniques.

[0615] After chemical labeling with the fluorescent dye ATTO 488 (marked with *, Figure 12 A), purified T22-DITOX-H6 and T22-PE24-H6 nanoparticles were tested for their internalization into cultured CXCR4+ cells. Both labeled nanoparticles Figure 12 A) penetrated target HeLa cells Figure 11 B) in a dose-dependent manner and accumulated intracellularly with a characteristic of receptor-mediated uptake kinetics (in the case of T22-PE24-H6* with a faster slope, Figure 13 C). Their penetration was confirmed to be CXCR4-specific Figure 13 D) by inhibition by the CXCR4 antagonist AMD3100. Internalized nanoparticles were observed to be engulfed into endosomes, particularly in the cytoplasmic region close to the cell membrane, but they tended to be visualized as membrane-free entities Figure 13 E) when approaching the perinuclear region, indicating important endosomal escape. No extracellular fluorescence attached to cells was observed in any case.

[0616] Once internalization was assessed, we tested whether the furin cleavage sites introduced in the construct to release the toxin segment from the building block were active in the oligomer. The expected intracellular hydrolysis should enhance the cytotoxic properties of the toxin domain, which can then benefit from the lower load of surplus protein sequence. To this end, we explored the susceptibility of multiple cleavage sites in the construct T22-DITOX-H6, which upon intracellular digestion can provide completely distinguishable protein fragments. Unlike the extracellular protein, which appears as one single protein species Figure 10 A and 12A), immunodetection of the His tag of the protein engulfed by the cells showed that the protein was digested by different alternative sites, matching the molecular weight of the expected products of each furin cleavage site. In particular, the release of the T22 peptide Figure 13 A) by the de novo integrated cleavage sites was demonstrated in the protein internalized by the cells in vivo after analysis of cell extracts 24 hours after exposure to the nanoparticles, by the shift from the 48.65 kDa full-length protein to the 44.21 kDa fragment. The remaining fragments correspond to progressive digestion intermediates that still retain the carboxy-terminal label by which the protein can be immunodetected. The natural cleavage of the internal furin site that releases the catalytic domain from the translocation domain was also demonstrated by the occurrence of the main 20.60 kDa segment. Thus, the catalytic fragment is expected to occur in reasonable amounts inside the target cells and in other biologically active forms.

[0617] In exploring the cytotoxic effect, both T22-DITOX-H6 and T22-PE24-H6 were able to efficiently kill cultured HeLa cells with low IC50 values (0.78 nM and 0.99 nM, respectively, not shown). Cytotoxic effect was clearly detectable in several CXCR4-expressing cell lines, including SW1417 CXCR4+, but not in the isogenic SW1417 CXCR4- line Figure 13 B, left). Cytotoxicity was largely abolished by AMD3100 and by T22-GFP-H6, which displays the biological intrinsic protein of T22 ( Figure 14 B, right), thus confirming again the specificity of nanoparticle entry, the intracellular nature of nanoparticle-mediated toxicity and the expected CXCR4 receptor mediation in cell killing. Moreover, a reduction of T22-DITOX-H6 (90%) has been observed when chloroquine was added, which inhibits endosomal acidification (not shown). This fact confirms that the mechanism of action is pH-dependent as described above ( Figure 14 ). In this context, the relevance of the removal of the auxiliary protein segment (mediated by furin protease) on the cytotoxicity of the nanoparticle was also evaluated. To this end, versions of T22-DITOX-H6 and T22-PE24-H6 were constructed that did not contain the engineered cleavage site (labeled F-), and their biological activity was tested. Comparative analysis of HeLa cell death mediated by these proteins revealed a sharp decrease in the cytotoxicity of T22-DITOX-H6 F- and T22-PE24-H6 F- nanoparticles compared to the original material ( Figure 15 C). On the other hand, differential expression of CXCR4 in isogenic SW1417 cells was adequately evaluated by immunocytochemistry and Western blot ( Figure 15 D, E). Interestingly, the ability of T22-DITOX-H6 and T22-PE24-H6 to promote cell death was not lost after one year of storage at -80°C, nor after 4 cycles of freezing and thawing (not shown). Since a high CXCR4+ specific cytotoxicity was observed in cell culture, we next tested the performance of the toxin-based materials in vivo using a CXCR4-associated disease model. To this end, we explored the biodistribution, antitumor activity and potential toxic side effects of both T22-DITOX-H6* and T22-PE24-H6* nanoparticles in a subcutaneous colorectal cancer model overexpressing CXCR4. As expected, protein material was accumulated in the tumor after a single intravenous injection administration within the time frame of the study ( Figure 15). Other organs, such as the brain, lungs or heart, were completely free of fluorescence. However, significant levels of emission associated with both nanoparticles were found in the liver and kidneys. To discard the release of significant amounts of ATTO from the nanoparticles during blood circulation and the generation of artifacts in the biodistribution analysis, we evaluated the stability of the dye in T22-DITOX-H6* nanoparticles incubated in commercial serum. At 48 hours, the nanoparticles released only a small amount of fluorescence (5%). Moreover, the administration of free ATTO did not produce a detectable accumulation in the tumor, and the absence of dye signal in the main organs indicates a rapid urinary excretion (as expected for small molecules of 981 Da). These data fully support the biodistribution of labeled nanoparticles shown in Figure 6. Figure 13 .

[0618] The observation of the presence of nanoparticles in the liver deserves a more in-depth analysis, since the occurrence and damage of the liver is a serious problem in conventional and innovative cancer therapies, even in nanocomplexes or antibody-based drugs showing tissue-specific targeting. Then, since it is crucial to distinguish the fluorescence only occurring in these organs and the damage induced by the toxin, we comparatively studied the cellular damage in the tumor, liver and kidneys. In this regard, we observed high levels of apoptosis induced by both nanoparticles in the tumor tissue, which were particularly intense in animals treated with T22-PE24-H6* at 48 hours after administration Figure 15 . In contrast, apoptosis was not detected in the liver or kidneys Figure 16 , and most of the liver tissue was normal in its tissue structure, except for a few and scattered inflammatory foci Figure 16 , which can be attributed to the non-specific extracellular retention of the drug in off-target tissues. This modification was resolved after 72 hours of recovery of normal tissue. The intracellular activation of the toxin, possibly facilitated by the release of the auxiliary peptide mediated by furin Figure 16 , does not occur in liver tissue, which does not overexpress CXCR4. To discard ATTO, which can have a positive effect on the cytotoxicity of the tumor material after a single dose administration, we examined the local apoptosis in animals treated with the non-labeled protein forms T22-DITOX-H6 and T22-PE24-H6. The efficiency was highest at the times tested (24 hours and 48 hours, respectively). As observed, local apoptosis was still present Figure 16), a value even higher than the one induced by the labeled protein form. This result indicates that the observed anti-tumor effect is intrinsically linked to the protein material. Then, the data support the view that despite the presence of the protein drug in the liver and kidney, this did not translate into a relevant uptake of either of the two nanoparticles in the parenchyma of these tissues. Our observations indicate that both labeled protein drugs transiently circulate through fenestrated sinusoids and glomeruli, despite their nanometer size (compared to other normal tissues) as reported for other nanoparticles. Moreover, since their CXCR4 expression is negligible compared to e.g. spleen or bone marrow, which despite showing low nanoparticle accumulation express CXCR4, their lack of toxicity in the kidney or liver indicates that they cannot internalize into the parenchymal cells of these organs. This finding is similar to the findings reported for CXCR4-targeted imaging agents. Then, both T22-DITOX-H6 and T22-PE24-H6 seem to have a sufficiently high therapeutic index to validate (i) their potential use for the treatment of CXCR4+ tumors, but more importantly, (ii) the broad applicability of the lateral concept supports self-assembly self-driven protein drugs based on chemically uniform building blocks. To further assess the therapeutic potential of the engineered toxins and support the concept of the design of toxin-based nanoparticles, we also assessed the pharmacokinetics in blood mouse samples after a single dose of 50 pg T22-DITOX-H6* or 300 pg T22-PE24-H6*. This was done by recording their fluorescence emission at 0h, 1 h, 2h, 5h, 24h and 48h post administration. We observed a biphasic decrease in plasma concentration from Cmax, with a fast nanoparticle biodistribution of both tested proteins limited to the plasma compartment (Vd = 3.9 ml for T22-PE-H6* and Vd = 3.2 ml for T22-DITOX-H6*). This fast biodistribution was followed by a second and slow elimination phase with a half-life of t1 / 2 = 30h for both nanoparticles Figure 16 A). This kinetic behavior is similar to the one reported previously for pharmacologically inactive protein nanoparticles and also similar to the one described for antibody drug conjugates or large nanometer-sized size therapeutic proteins, which exhibit a similar behavior to the unconjugated antibody. In a further step, we assessed the anti-tumor effect of each nanoparticle in a CXCR4+ subcutaneous CRC mouse model after repeated dose administration. After a dose regimen of 10 pg T22-DITOX-H6, 3 times per week, 8 doses each, we observed a 5.8-fold reduction in tumor volume at the end of the experiment compared to buffer-treated mice (p = 0.05). This was associated with a 3.0-fold increase in apoptosis in the tumor tissue (p < 0.001) Example 11 : Protein nanoparticles based on recombinant ricin (mRTA)B) No significant difference in body weight between toxin treated and control groups Figure 17 C) Similarly, and after a dose regimen of 10 pg T22-PE24-H6, 3 times per week, 8 doses each, we observed a 2.3-fold reduction in tumor volume at the end of the experiment (p=0.034) compared to buffer treated mice, which was associated with a 3.8-fold increase in the number of apoptotic-like cells in the tumor tissue (p=0.001) Figure 17 B) Again, no significant difference in body weight between experimental and control groups was observed Figure 17 C).

[0619] Figure 17

[0620] Recombinant T22-mRTA-H6 Figure 17 A) was successfully produced in E. coli Origami B, purified by one-step affinity chromatography based on histidine, and detected as a single protein species with an expected molecular weight of 35.91 kDa, which was well confirmed by mass spectrometry (not shown) Figure 17 B) Directly observable by both DLS and FESEM, the pure protein is a ~11 nm entity appearing in the storage buffer without further treatment Figure 17 C, D), which indicates the spontaneous formation of self-assembled nanoparticles. This is an expected result, since the combination of a cationic peptide at the amino terminus and a polyhistidine at the carboxyl terminus has been proven optimal for promoting protein oligomerization into regular nanostructures, independently of the core protein segment (ricin, in the case of T22-mRTA-H6, Figure 17 A) treatment of the material with SDS gives 5.5 nm monomers Figure 18 C), which represent possible building blocks of the nanoparticle. In the related self-assembling protein T22-GFP-H6, both the size of the building blocks and the assembly form are identical to those of T22-mRTA-H6, and analysis methods using small-angle X-ray scattering and other sophisticated methods as well as computer simulations have revealed that the nanoparticle is formed from approximately 10 monomers. It is estimated that this picture is also appropriate for T22-mRTA-H6. Analysis of T22-mRTA-H6 nanoparticles by circular dichroism (CD) revealed a structural composition in which a-helices dominate (29.2%, Figure 18 E) However, the thioflavin T (Th T) assay also revealed the occurrence of intermolecular b-sheet interactions Figure 18 F), which can contribute to the stability of the protein nanoparticle and is also compatible with the significant degree of b-sheet structure found in the CD Figure 18E). Since the nanostructured ricin is intended for delivery into tumor tissue, we wanted to know if the nanoparticles could remain stable under the anomalous pH values ​​observed in the tumor environment, reportedly ranging from approximately 6.3 (intracellular) to 7.4 (extracellular). As observed, T22-mRTA-H6 maintained complete assembly under these conditions. Figure 18 F), from a stability perspective, this supports the usability of the build.

[0621] To test the function of this assembled form of recombinant ricin, cultured CXCR4+ HeLa cells were exposed to different concentrations of ricin-based nanoparticles. These materials exhibited potent, dose-dependent cytotoxicity, essentially eliminating cell viability at 100 nM. Figure 18 A). At 72 hours after exposure, the IC50 was measured to be 13 ± 0.5 nM. To confirm whether, as expected, T22-mRTA-H6-mediated cell death depends on its cell binding and internalization of the protein via the cell surface receptor CXCR4 and its ligand T22, we tested whether cell viability could be restored at a molar ratio of 10:1 when the potent CXCR4 antagonist AMD3100 could be used as a competitor to the toxin. As observed ( Figure 19 B) AMD3100 significantly enhanced cell viability in T22-mRTA-H6-treated cells, demonstrating nanoparticle-specific, receptor-mediated penetration into target cells. To further confirm this precise cell entry mechanism, we exposed non-tumor (CXCR4-)3T3 cells, as well as representative CXCR4- and CXCR4+ tumor cell lines, to T22-mRTA-H6, along with cytosine arabinoside (Ara-C), a conventional chemotherapeutic agent used to treat several cancer types, particularly acute myeloid leukemia (AML). These cell lines with different levels of CXCR4 expression ( Figure 19 C) Supports different levels of protein internalization mediated by specific interactions between T22 and CXCR4. Figure 19 D). This was determined by the uptake of T22-GFPH6, a self-assembled fluorescent protein closely associated with T22-mRTA-H6, which contains the same CXCR4 ligand hosted at the N-terminus of the polypeptide. It is important to note that, as expected, CXCR4 expression and T22-mediated protein internalization exhibit parallel behavior (comparison). Figure 19C and D). Then, when finally compared to them in a test, the ricin-based protein nanoparticle only promoted specific cell death in Crax C4+ cancer cells, but not in normal cells, while at this dose (100 nM) Ara-C did not show any toxic effect on either of these cell lines Figure 19 E). This observation demonstrates that not only protein drugs can be effectively targeted, but also with superior cytotoxicity compared to equimolar doses of model chemical drugs.

[0622] At this stage, we wanted to confirm that the T22-mRTA-H6-promoted cytotoxicity was related to the uptake of the nanoparticle inside the CXCR4+ cells and was triggered from the inside. This was achieved by exposing HeLa cells to ATTO-labeled nanoparticles and monitoring internalization. As observed Figure 20 A), the nanoparticles were internalized by the cells for at least 24 hours. As expected for the active form of ricin, apoptosis was detected by annexin affinity test and by Hoechst staining Figure 20 B), the number of apoptotic cells seemed to reach a peak about 15-24 hours after exposure. Moreover, 15 and 24 hours after treatment with T22-mRTA-H6, mitochondrial damage was confirmed by a significant increase in the number of cells with reduced JC-1 red fluorescence Figure 21 C), indicating that depolarization of mitochondrial ΔΨ was associated with apoptosis induction. Interestingly, the occurrence of cell damage was not associated with an increase in reactive oxygen species (ROS, Figure 20 D), while the formation of apoptotic bodies in HeLa cells exposed to ricin was apparently caspase-dependent Figure 20 E). The combination of these data indicates that T22-mRTA-H6-mediated cell death occurs through the classical caspase-dependent apoptosis pathway.

[0623] The anti-tumor effect of both T22-mRTA-H6 soluble nanoparticles and T22-mRTA-H6 IB was evaluated in a disseminated AML animal model. NSG mice were injected with THP1-Luci cells to generate leukemia spread in mice. Two days after the intravenous tail cell injection, a single dose of 1 mg of T22-mRTA-H6 IB was injected subcutaneously (SC) in the mice of two mice (IB-T22mRTA group). In different groups of mice, one mouse was intravenously administered with 10 μg of soluble T22-mRTA-H6 daily (T22mRTA group) or three mice were intravenously administered with buffer alone (VEHICLE group) for a total of 10 doses. No effect on mice weight was observed during the treatment (data not shown). The progression and dissemination of leukemia were evaluated by BLI using IVIS Spectrum monitoring. From day 6 until the end of the experiment, mice treated with soluble T22-mRTA-H6 (T22mRTA) showed lower luminescence emission than the VEHICLE group (Fig. 10A). Thus, as measured by BLI, treatment with soluble T22-mRTA-H6 inhibited the spread of AML cells in mice after each dose of 10 μg of T22-mRTA-H6, 4th, 6th, 8th, 10thdose, compared to the VEHICLE group (corresponding to day 6, 8, 10 or 13 after cell injection, respectively). In contrast, no difference in BLI was found between mice treated with T22-mRTA-H6 IB (IB-T22mRTA) and control VEHICLE mice (Fig. 10A). Figure 21 A). Thus, as measured by BLI, treatment with soluble T22-mRTA-H6 inhibited the spread of AML cells in mice after each dose of 10 μg of T22-mRTA-H6, 4th, 6th, 8th, 10thdose, compared to the VEHICLE group (corresponding to day 6, 8, 10 or 13 after cell injection, respectively). In contrast, no difference in BLI was found between mice treated with T22-mRTA-H6 IB (IB-T22mRTA) and control VEHICLE mice (Fig. 10A). ​ A). Thus, as measured by BLI, treatment with soluble T22-mRTA-H6 inhibited the spread of AML cells in mice after each dose of 10 μg of T22-mRTA-H6, 4th, 6th, 8th, 10thdose, compared to the VEHICLE group (corresponding to day 6, 8, 10 or 13 after cell injection, respectively). In contrast, no difference in BLI was found between mice treated with T22-mRTA-H6 IB (IB-T22mRTA) and control VEHICLE mice (Fig. 10A).

[0624] Next, when mice showed advanced disease signs, the anti-tumor activity of nanoparticles was analyzed in infected ex vivo organs 14 days after cell injection. Analysis of IVIS Spectrum showed that treatment with soluble T22-mRTA-H6 nanoparticles (T22mRTA) reduced BLI in bone marrow (skeleton and hind limbs), liver and spleen, in contrast to the findings in mice treated with buffer alone (VEHICLE) (Fig. 10B). However, treatment with T22-mRTA-H6 IB (IB-T22mRTA) did not show changes in BLI in the same tissues compared to control mice (VEHICLE) (Fig. 10B). ​ B). Thus, as measured by BLI, treatment with soluble T22-mRTA-H6 inhibited the spread of AML cells in mice after each dose of 10 μg of T22-mRTA-H6, 4th, 6th, 8th, 10thdose, compared to the VEHICLE group (corresponding to day 6, 8, 10 or 13 after cell injection, respectively). In contrast, no difference in BLI was found between mice treated with T22-mRTA-H6 IB (IB-T22mRTA) and control VEHICLE mice (Fig. 10A). ​ B). Thus, as measured by BLI, treatment with soluble T22-mRTA-H6 inhibited the spread of AML cells in mice after each dose of 10 μg of T22-mRTA-H6, 4th, 6th, 8th, 10thdose, compared to the VEHICLE group (corresponding to day 6, 8, 10 or 13 after cell injection, respectively). In contrast, no difference in BLI was found between mice treated with T22-mRTA-H6 IB (IB-T22mRTA) and control VEHICLE mice (Fig. 10A).

[0625] Furthermore, the dissemination of leukemic cells in the organs of infected animals was also evaluated by IHC of CD45, a human leukocyte marker that detects AML THP1 cells. The results associated with the BLI analysis indicated that, unlike the soluble T22-mRTA-H6 registered after the detection of T22-mRTA-H6 IB, the reduction in the number of CD45-positive cells in the bone marrow, liver and spleen of mice treated with soluble T22-mRTA-H6 reduced the dissemination in infiltrated tissues ​ C). Finally, H&E staining was performed on infiltrated organs and other organs not infected with leukemic cells. No signs of toxicity were observed in any of the infected or non-infected tissues that were not treated with soluble T22-mRTA-H6 or T22-mRTA-H6 IB ​ Since it occurs in vitro, IB would cause a slight biological effect, if any.

Claims

1. A fusion protein, wherein the components of the fusion protein are: (i) The polycationic peptide shown in SEQ ID NO:5 (ii) an intercalation polypeptide region, wherein the intercalation polypeptide region is a therapeutic agent of a cytotoxic polypeptide having the amino acid sequence shown in SEQ ID NO: 43 or SEQ ID NO: 44, and (iii) A positively charged, amino acid-rich region, which is a polyhistidine region of 6 histidine residues. The polycationic peptide is located at the N-terminus of the fusion protein, and the positively charged, amino acid-rich region is located at the C-terminus of the fusion protein; and The components of the fusion protein are linked by peptide linkers.

2. The fusion protein of claim 1, wherein the polycationic peptide is connected to the intercalation polypeptide region via a first peptide linker, and / or wherein the intercalation polypeptide region is connected to the positively charged amino acid-rich region via a second peptide linker.

3. The fusion protein according to claim 2, wherein the first peptide linker comprises the peptide shown in SEQ ID NO: 32 or SEQ ID NO:

33.

4. A fusion protein, wherein the components of the fusion protein are: (i) The polycationic peptide shown in SEQ ID NO:5 (ii) an intercalated polypeptide region, wherein the intercalated polypeptide region is a therapeutic agent of a cytotoxic polypeptide having the amino acid sequence shown in SEQ ID NO: 43 or SEQ ID NO:

44. (iii) A positively charged, amino acid-rich region, which is a polyhistidine region of 6 histidine residues, and (iv) Reporter proteins, The polycationic peptide is located at the N-terminus of the fusion protein, and the positively charged, amino acid-rich region is located at the C-terminus of the fusion protein; and The components of the fusion protein are linked by peptide linkers.

5. The fusion protein of claim 1, wherein the peptide linker includes a target cleavage site for a protease.

6. The fusion protein of claim 5, wherein the peptide linker targeting the protease cleavage site is located between the polycationic peptide and the intercalation polypeptide region.

7. The fusion protein according to claim 5, wherein the target cleavage site for the protease is selected from furin, enterokinase, factor Xa, thrombin, TEV protease and PreScission protease.

8. The fusion protein according to claim 7, wherein the target cleavage site for the furin protease is as described in SEQ ID NO. 46 or SEQ ID NO. 47, the target cleavage site for the enterokinase protease is as described in SEQ ID NO. 27, the target cleavage site for protease factor Xa is as described in SEQ ID NO. 28, the target cleavage site for the thrombin protease is as described in SEQ ID NO. 29, the target cleavage site for the TEV protease is as described in SEQ ID NO. 30, and the target cleavage site for the PreScission protease is as described in SEQ ID NO.

31.

9. A fusion protein, wherein the components of the fusion protein are: (i) The polycationic peptide shown in SEQ ID NO:5 (ii) an intercalated polypeptide region, wherein the intercalated polypeptide region is a therapeutic agent of a cytotoxic polypeptide having the amino acid sequence shown in SEQ ID NO: 43 or SEQ ID NO:

44. (iii) A positively charged, amino acid-rich region, which is a polyhistidine region of 6 histidine residues, and (iv) Peptides that facilitate the escape of endogenous substances The polycationic peptide is located at the N-terminus of the fusion protein, and the positively charged, amino acid-rich region is located at the C-terminus of the fusion protein; and The components of the fusion protein are linked by peptide linkers.

10. The fusion protein of claim 9, wherein the peptide that facilitates endosome escape is shown in SEQ ID NO.

48.

11. The fusion protein of claim 9, wherein the peptide that facilitates endosome escape is located in the C-terminal domain of the fusion protein.

12. A nanoparticle comprising multiple copies of the fusion protein according to any one of claims 1 to 11.

13. The nanoparticles according to claim 12, having a diameter between 10 and 100 nm.

14. Use of the fusion protein according to any one of claims 1 to 11 or the nanoparticles according to claim 12 or 13 in the preparation of a pharmaceutical.

15. Use of the fusion protein according to any one of claims 1 to 11 or the nanoparticle according to claim 12 or 13 in the preparation of a medicament for treating cancer, wherein the cancer cells express CXCR4.

16. The use according to claim 15, wherein the cancer is pancreatic cancer or colorectal cancer.

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