Fusion-proteins based on human ferritin and their use as multiple bioactive peptides delivery system

Abstract

The present invention relates to a fusion protein, nanoparticles composed of a plurality of monomers of said fusion protein, nucleic acids encoding said fusion protein, and therapeutic applications thereof. In particular, the fusion protein comprises a first domain based on human ferritin and a second domain conjugated to said first domain and located N-terminally with respect to said first domain, which second domain comprises at least one amino acid sequence of a programmed cell death ligand-1 (PD-L1) binding peptide and / or at least one amino acid sequence of a tumor-penetrating peptide.

Description

[0001] FUSION-PROTEINS BASED ON HUMAN FERRITIN AND THEIR USE AS MULTIPLE BIOACTIVE PEPTIDES DELIVERY SYSTEM

[0002] DESCRIPTION

[0003] Technical field of the invention

[0004] The present invention relates to a fusion protein, nanoparticles composed of a plurality of monomers of said fusion protein, nucleic acids encoding said fusion protein, and therapeutic applications thereof.

[0005] State of art

[0006] Despite significant advances in oncology drug discovery, survival expectations for patients with cancer are still disappointing. One of the main reasons for this unresolved challenge in cancer therapy are the poor drug penetration, chemotherapeutic resistance, short half-life, and side effects owing to cytotoxicity in healthy cells. The massive accumulation of therapeutic agents at diseased areas represents one of the most important challenges for improving the current therapies, especially for the anticancer therapy. The concentration of chemotherapeutics and / or immunotherapeutic s in deep regions of tumor beyond their initial penetration is significantly lower than the intended concentration, thus leading to unwanted relapse and drug resistance. The major reason for this is attributable to the structure and physiology of the tumor microenvironment (TME) that acts like a barrier for the complete penetration of therapeutic agents. For this reason, more efficient drug-delivery systems having better TME penetration ability have been developed or are in development by several groups.

[0007] For example, iRGD is a cyclic peptide composed of 9-amino acids (CRGDKGPDC, herein denoted as SEQ ID No. 18) including an Arg-Gly-Asp (RGD) motif, which has a high binding affinity to aVp3 and aVp5 integrins abundantly present in tumor vasculatures. iRGD- conjugated or co-administrated drugs showed enhanced distribution throughout the extra- vascular tumor parenchyma. Compared with RGD peptide (CRGDC, herein denoted as SEQ ID No. 19), the tumor targeting ability of iRGD is more intensified because iRGD can specifically bind to integrins and neuropilin- 1 (NRP-1) receptors that are overexpressed on various tumors. The cleaved form of iRGD (CRGDK, herein denoted as SEQ ID No. 20) binds to NRP-1, and subsequently triggers NRP-1 -dependent endocytosis, thus resulting in enhanced tumor penetration. iRGD shows attractive advantages in delivery systems, including low toxicity to normal cells, easy and low-cost synthesis, and targeted release.

[0008] Another recent important breakthrough in cancer therapy is the use of adjuvant molecules for restoring the body immunological response against cancer. In particular, cancer immunotherapy has made an extraordinary journey from bench to bedside. Tumor cells often overexpress immune checkpoint proteins to evade the host immune system by inhibiting T-cell attack. Exogenous antagonists can inhibit immune checkpoints and thus disrupt the immune- suppressing pathway, unleashing or enhancing pre-existing anti-cancer immune responses by T-cells able to destroy cancer cells. Preclinical and clinical data show that blockade of immune checkpoints by antibodies or peptides can indeed significantly enhance antitumor immunity. In particular, blockage of Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), or the interactions between programmed cell death protein 1 (PD-1) and its PD- L1 ligand, by specific antibodies is emerging as a promising immunotherapy for cancer treatment. For all their effectiveness, antibody drugs have some problems associated with production cost and immunogenicity. An alternative to the use of antibodies for cancer immunotherapy are peptides blocking the checkpoint interaction (e.g. PD-1 / PD-L1).

[0009] Screening a phage-displayed peptide library for peptides that selectively bind to PD-Ll-or PD-1 overexpressing cells identified peptides, CLQKTPKQC and CVRARTR (PD-LlPep- 1, herein denoted as SEQ ID No. 21, and PD-LlPep-2, herein denoted as SEQ ID No. 22, respectively) for PD-L1 and GNWDYNSQRAQLYNQ (herein denoted as SEQ ID No. 23) for PD-1, appeared to block PD-L1 or PD-1 and showed promising activity both in vitro and in vivo.

[0010] Recently, peptides that target next-generation immune checkpoints, such as T-cell immuno- globulin-3 (TIM-3), have attracted increasing attention. A TIM-3-binding peptide (GLIPLTTMHIGK, herein denoted as SEQ ID No. 24) that interferes with the binding of TIM-3 to Gal-9, the main ligand of TIM-3, thereby enhancing T-cell activity, was reported. As further therapeutic approach for treating cancer, and in particular cancer metastatization, is targeting the CD44 macromolecule. CD44 is a cell surface receptor involved in cell adhesion to the extracellular matrix. Although CD44 is expressed in normal cells, its alternative splicing isoforms, including CD44 variant 6 (CD44v6), are upregulated in tumor cells, contributing to tumor cell migration and metastasis by interacting with c-MET. By screening a phage-displayed random peptide library, two cyclic peptides, NLN (CNLNTIDTC, herein denoted as SEQ ID No. 25) and NEW (CNEWQKLSC, herein denoted as SEQ ID No. 26), which bind to CD44v6-expressing cells were selected. These peptides hindered c-MET activation, thereby inhibiting CD44v6-high tumor cell migration and invasion.

[0011] However, although peptide molecules have advantages, such as deep tissue penetration, lower immunogenicity, and lower cost for development, short peptides have some disadvantages, including weak affinity, low persistence in blood and susceptibility to degradation. In addition, very often a single bioactive peptide is not sufficient to elicit a strong long- persistence activity, especially in the case of tumor adjuvant-based therapies. In fact, to increase the rate of success of bioactive peptide -based therapies, the simultaneous delivery of different and multiple copies of bioactive peptides may be the key.

[0012] In this context, there is hence still a need to provide efficient carriers (nanovectors) for delivery of multiple bioactive peptides for therapeutic applications.

[0013] Summary of the Invention

[0014] The inventors surprisingly found that the simultaneous insertion of specific peptide amino acid sequences at the N-terminus region of a fusion protein based on the human H-type ferritin (HFt) confers it outstanding properties as adjuvant molecules for cancer chemotherapy and immunotherapy, as clearly showed from the experimental data reported in the examples and drawings of the present disclosure, thereby providing an efficient way to deliver different and multiple copies of bioactive peptides to a target site.

[0015] Human ferritin H-type ferritin (HFt)-based drugs have been recently attracting growing interest in the field of cancer therapy, due to their selectivity for cancer over normal cells, and binding to a large number of different human tumors. HFt is a highly symmetrical multimeric protein consisting of 24 subunits that assemble into a molecular structure with an essentially spherical shell, which encloses a cavity that is physiologically used for storing iron.

[0016] Advantageously, in the quaternary structure, the N-terminus faces the external side of the spherical shell and can allow for the exposure of 24 copies of bioactive peptides. The outer diameter and the inner diameter are 12 and 8 nm, respectively. Such a shell-shaped molecular structure will be hereinafter designated as “nanoparticle” or “HFt nanoparticle”. Nanoparticles based on HFt show a number of advantages compared to other drug delivery systems, especially in connection with in vivo human applications. In fact, the HFt molecules are designed to cross the biological barriers (30 nm minor diameter) and are present both within cells and in blood under physiological conditions, although at low concentrations (approximately 20 pg / E). As anticipated above, because the ferritin structure is composed by 24 identical subunits, the weak affinity of small peptides can be overcome by displaying multiple copies on the surface of ferritin, prolonging their half-life as well as improving their biological effect.

[0017] Accordingly, a first object of the present invention is a fusion protein, comprising:

[0018] (a) a first domain comprising the amino acid sequence of the heavy chain of native human ferritin or a variant thereof having at least 90% identity with the amino acid sequence of the heavy chain of native human ferritin; and

[0019] (b) a second domain conjugated to said first domain and located N-terminally with respect to said first domain, which second domain comprises at least one amino acid sequence of a programmed cell death ligand- 1 (PD-L1) binding peptide and / or at least one amino acid sequence of a tumorpenetrating peptide.

[0020] In one preferred aspect of the invention, the fusion protein based on the heavy chain of human ferritin as herein described includes at the N terminus of the protein several bioactive peptides (BAP) amino acid sequences, most preferably from two to five peptide sequences, selected from the following: i) the amino acid sequence of a tumor-penetrating peptide with a linear structure able to bind either the integrin aVp3 / 5 and the neuropilin- 1 receptors and acting as tumor accumulation enhancer of co-administrated drugs; ii) a PD-L1 (programmed cell death ligand- 1) binding peptide with a linear structure; iii) a PD-1 (programmed cell death- 1) binding peptide with a linear structure; iv) a T-cell immunoglobulin- 3 (TIM-3) binding peptide with a linear structure; and v) a c-MET blocking peptide with a linear structure inhibiting development, progression and metastatization of tumors; in addition, a metalloproteinase cleavage sequence is present between the single BAP sequences, to allow them to be released in the target site.

[0021] Recently, to increase further the in vivo half-life and stability of native HFt, novel HFt-based constructs, named HFt-MP-PAS or HFt-MP-PASE, suitable for drug delivery were disclosed in W02016051340 Al and WO2019087155 Al. Both said patent applications, in particular all the sequences and all the embodiments described therein, are incorporated herein by reference in their entirety.

[0022] In these constructs the N-terminus of each HFt subunit is genetically fused to: i) a PAS or PASE polypeptide sequences, i.e., a sequence rich in proline (P), alanine (A) and serine (S) residues; and ii) a tumor- selective sequence (MP) responsive to proteolytic cleavage by tumor proteases (MMPs), inserted between each HFt subunit and the outer PAS polypeptide. The PAS / PASE shield was aimed at increasing the stability of modified protein variants. The presence of PAS / PASE is also capable of masking the protein surface and thus of extending its plasma half-life and target specificity.

[0023] Therefore, at the N-terminus of said fusion protein, a polypeptide that acts as a masking polymer increasing the protein stability and specificity may be advantageously included.

[0024] In particular, the inventors unexpectedly found that a new construct (named throughout the present specification as HFt-PASE@PDLl-NRPl) containing the amino acid sequences of two linear-structured peptides, i.e. LQKTPKQ (PD-L1 binding peptide, herein denoted as SEQ ID No. 9) and RGDKGPD (TME penetration enhancer, herein denoted as SEQ ID No. 10), respectively, can be used as tumor therapy adjuvant without loading the ferritin protein cavity with a chemotherapeutic drug. Moreover, the new construct HFt-PASE@PDLl- NRP1 showed significant tumor suppression when given in combination with different chemotherapeutic s drugs such as gemcitabine, encapsulated Genz-644282 (herein also named as HFt-Glu-MP-PASE-Genz or THE-0504), Antibody-drug conjugates (ADC) etc., enhancing the therapeutic activities of these chemotherapeutic drugs.

[0025] The present invention provides also compositions comprising the compounds of the invention as well as for specific uses in therapeutic applications. This and other objects are accomplished through the fusion protein as defined in appended claim 1. The other independent claims and the dependent claims relate to further aspects and specific embodiments of the invention, which form an integral part of the specification.

[0026] A further object of the present invention is an isolated nucleic acid encoding for a fusion protein or a nanoparticle according to any one of embodiments herein disclosed.

[0027] A further object of the present invention is a vector comprising said nucleic acids and a host cell comprising said nucleic acid or said vector.

[0028] Further features and advantages of the invention will appear from the following detailed description, which is provided for illustrative purposes only and not by way of limitation, with reference to the appended drawings, wherein:

[0029] Brief description of the drawings

[0030] Figure 1 is a schematic representation of the manufacture of HFt-based nanoparticles, wherein the N terminus of each of the 24 monomers is genetically bound to cleavable peptide sequences, bioactive peptides and to sequences essentially consisting of proline, alanine, serine and glutamate (PASE).

[0031] Figure 2 is a schematic representation of the HFt-PASE@PDLl-NRPl construct, wherein the N terminus of each of the 24 monomers is genetically bound to cleavable peptide sequences, two bioactive peptides and to a PASE sequence.

[0032] Figure 3 is a schematic representation of the HFt-PASE@PDLl-NRPl-cMET construct, wherein the N terminus of each of the 24 monomers is genetically bound to cleavable peptide sequences, three bioactive peptides and to a PASE sequence.

[0033] Figure 4 shows denaturing agarose gel electrophoresis for protein expression profiles. Panel A, HFt-PASE@PDLl-NRPl expression: lane 1, protein marker; lane 2, E.coli colony expression pattern without IPTG induction; lanes 3-5, expression pattern of different E.coli colonies after 2 hours of IPTG induction. Panel B, HFt-PASE@PDLl-NRPl-cMET expression: lane 1, protein marker; lane 2, E.coli colony expression pattern without IPTG induction; lanes 4-5, expression pattern of different E.coli colonies after 2 hours of IPTG induction.

[0034] Figure 5 shows the binding affinities of HFt-PASE@PDLl-NRPl compound for the human aVp3 receptor.

[0035] Figure 6 shows the binding affinities of HFt-PASE@PDLl-NRPl compound for the human PD-L1 receptor.

[0036] Figure 7 shows an internalization study on cultured cells (A375 Melanoma) of the antibody Trastuzumab. The presence of the construct HFt-PASE@PDLl-NRPl clearly determines an uptake enhancement of Trastuzumab up to four-fold.

[0037] Figure 8 shows the killing efficacy of the drug T-DXd (Trastuzumab deruxtecan) alone or in the presence of the construct HFt-PASE@PDLl-NRPl against the human cell line A375 Melanoma. Mean ± S.E.M. (n =3). The presence of the construct HFt-PASE@PDLl-NRPl clearly increases the killing ability of T-DXd.

[0038] Figure 9 shows the anti-tumor activity of the drug Gemcitabine alone or in the presence of the construct HFt-PASE@PDLl-NRPl in pancreatic KPC tumor-bearing mice. Tumorgrowth curves for each mouse group are indicated. Mice were sacrificed when the tumor had reached a volume in the range 1000-1500 mm3. The co-administration of Gemcitabine with the construct HFt-PASE@PDLl-NRPl clearly increases the antitumor activity of Gemcitabine.

[0039] Figure 10 shows the anti-tumor activity of the drug THE-0504 (encapsulated Genz-644282) alone or in the presence of the construct HFt-PASE@PDLl-NRPl or iRGD peptide in pancreatic KPC tumor-bearing mice. Tumor-growth curves for each mouse group are indicated. Mice were sacrificed when the tumor had reached a volume in the range 1000-1500 mm3. The co-administration of THE-0504 with the construct HFt-PASE@PDLl-NRPl clearly increases the antitumor activity of THE-0504. This effect is superior to that provided by the cyclic peptide iRGD when co-administrated with THE-0504.

[0040] Figure 11 shows the anti-tumor activity of the drug THE-0504 (Genz-644282) alone or in the presence of the construct HFt-PASE@PDLl-NRPl in pancreatic MiaPaca2 tumor-bearing mice. Tumor-growth curves for each mouse group are indicated. Mice were sacrificed when the tumor had reached a volume in the range 1000-1500 mm3. The co-administration of THE-0504 with the construct HFt-PASE@PDLl-NRPl clearly increases the antitumor activity of THE-0504.

[0041] Figure 12 shows the anti-tumor activity of the drug Immu-132 (sacituzumab govitecan) alone or in the presence of the construct HFt-PASE@PDLl-NRPl in pancreatic KPC tumorbearing mice. Tumor-growth curves for each mouse group are indicated. Mice were sacrificed when the tumor had reached a volume in the range 1000-1500 mm3. The co-administration of Immu-132 with the construct HFt-PASE@PDLl-NRPl clearly increases the antitumor activity of Immu-132.

[0042] Figure 13 shows the anti-tumor activity of the drug Immu-132 (sacituzumab govitecan) alone or in the presence of the construct HFt-PASE@PDLl-NRPl in pancreatic KPC tumorbearing mice. Tumor weights as well as residual tumors images for each mouse group are indicated. The co-administration of Immu-132 with the construct HFt-PASE@PDLl-NRPl clearly reduces the tumor weights up to twelve-fold in comparison to the Immu-132 alone.

[0043] Glossary As used herein, the term “conjugated” indicates that a domain or amino acid sequence according to any of the embodiments disclosed in the present specification or in the claims is linked to or chemically linked to another domain or amino acid sequence as disclosed herein, either directly or via a linker sequence.

[0044] As used herein, the expression “pharmaceutical active ingredient” or more simply “active ingredient” refers to any pharmaceutically active molecule (chemical compound, monoclonal antibody, peptide, etc.), for instance a molecule that can be used for cancer treatment.

[0045] As used herein, the term “subject” relates to animals, such as mammals, including human beings, cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, and the like.

[0046] As used herein, the term “treating” or “treatment” refers to evidence of success or improvement in the treatment of a certain disease, lesion, condition or symptom, or, in certain circumstances, the prevention of the onset of a symptom or condition.

[0047] In any point of the present specification or in the claims, the terms “comprising” or “com- prise(s)” can be replaced by the terms “consisting of’ or “consist(s) of’.

[0048] Detailed description of the invention

[0049] The fusion protein which is the subject matter of the present invention comprises at least two domains.

[0050] The first domain (a) comprises the amino acid sequence of the heavy chain of native human ferritin or a variant thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of the heavy chain of native human ferritin. Since the heavy chain of human ferritin has a length of 183 amino acids (SEQ ID NO: 1), a variant having at least 90% sequence identity contains up to 19 amino acid substitutions compared to the native sequence.

[0051] In particular, said variant having at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of the heavy chain of native human ferritin is a variant that has equivalent function or activity than that of said heavy chain of native human ferritin.

[0052] In one embodiment the heavy chain of human ferritin is a mutein wherein the cysteine residues from the protein surface are removed and / or at least one cysteine or negative (aspartate or glutamate) residue is inserted in the internal cavity of the protein, preferably two, three or four cysteine or aspartate or glutamate are inserted in the internal cavity of the protein. The native cysteine residues from the protein surface are replaced with a residue without a thiol reactive group, preferably the cysteine will be replaced with a serine. The protein surface of the human ferritin is herein defined as comprising any residues exposed to the solvent. The internal cavity of the human ferritin is herein defined as comprising any residues not exposed to the solvent.

[0053] In one preferred embodiment the heavy chain of human ferritin is the amino acid sequence set forth in SEQ ID No: 2 of the HFt variant (HFt-Cys) lacking the native cysteine residues, which represent an alternative variant. The amino acid sequence of HFt-Cys is characterized by three amino acid substitutions: Serine instead of Cysteine 90, Serine instead of Cysteine 102, Serine instead of Cysteine 130.

[0054] All the herein disclosed embodiments of the heavy chain of human ferritin may be used as first domain in the fusion protein according to the invention.

[0055] The amino acid sequence of the native HFt as well as the amino acid sequences of suitable variants of HFt that could be used as first domain in a fusion protein according to any of the embodiments disclosed herein are described in W02016051340 Al, at page 5 (lines 25 to 32) and page 6 (line 1), and in WO2019087155 Al, at page 8 and page 9 (lines 1 to 10), which are incorporated herein by reference in their entirety.

[0056] The second domain (b) of the fusion protein of the invention according to any of the embodiments disclosed in the present specification and in the claims, comprises at least one amino acid sequence of a programmed cell death ligand- 1 (PD-E1) binding peptide and / or at least one amino acid sequence of a tumor-penetrating peptide.

[0057] The second domain (b) of the fusion protein of the invention is conjugated to a first domain (a) according to any of the embodiments herein disclosed and located N-terminally with respect to said first domain.

[0058] In one embodiment, the second domain (b) of the fusion protein of the invention comprises at least one amino acid sequence of a PD-E1 binding peptide and at least one amino acid sequence of a tumor-penetrating peptide.

[0059] In one preferred embodiment, the second domain of the fusion protein comprises the amino acid sequence of a PD-E1 binding peptide EQKTPKQ herein denoted as SEQ ID No: 9.

[0060] According to the present invention, the second domain of the fusion protein particularly comprises the amino acid sequence of a tumor-penetrating peptide that binds to one or more receptors expressed by a cancer cell or tissue that mediates an active transport pathway, in particular a peptide capable of binding to aVp3 and aVp5 integrins and / or to neuropilin- 1, thereby acting as drug penetration and accumulation enhancer.

[0061] Preferably, the tumor penetrating peptide is a tumor vasculature-targeting peptide having a specific binding affinity to avP3 and avP5 integrins and neuropilin- 1.

[0062] In one embodiment, the second domain of the fusion protein of the invention comprises the amino acid sequence of a tumor-penetrating peptide selected from the following sequences:

[0063] - RGDKGPD herein denoted as SEQ ID No: 10;

[0064] - RGDRGPD herein denoted as SEQ ID No: 27;

[0065] - RGDKGPE herein denoted as SEQ ID No: 28 and

[0066] - RGDRGPE herein denoted as SEQ ID No: 29.

[0067] In one preferred embodiment, the second domain of the fusion protein of the invention comprises the amino acid sequence of the tumor-penetrating peptide RGDKGPD herein denoted as SEQ ID No: 10.

[0068] According to the invention, the second domain of a fusion protein according to any of the embodiments disclosed herein may further comprise the amino acid sequences of one or more additional bioactive peptides of interest.

[0069] Suitable bioactive peptides include, for example, peptides that are capable of targeting specific cancer cells by specifically binding to one or more receptors overexpressed on the surface of the cells, such as receptors involved in cell adhesion, as well as peptides acting as checkpoint inhibitors.

[0070] In one particular embodiment, the second domain (b) of the fusion protein of the invention further comprises at least one of the following amino acid sequences:

[0071] (bl) the amino acid sequence of a c-MET blocking peptide such as a CD44 variant 6 binding peptide, preferably the amino acid sequence NEWQKLS herein denoted as SEQ ID No. 11; (b2) the amino acid sequence of a programmed cell death- 1 (PD-1) binding peptide;

[0072] (b3) the amino acid sequence of a T-cell immunoglobulin-3 (TIM-3) binding peptide, such as that set forth in SEQ ID No. 24.

[0073] In one aspect, when the second domain (b) of a fusion protein according to any of the variants herein disclosed comprises more than one peptide amino acid sequence, said second domain further comprises one or more linker sequences linking said peptide amino acid sequences to each other. The fusion protein according to any of the embodiments disclosed herein may further comprise an additional linker sequence linking the second domain to the N terminal region of the first domain.

[0074] In one embodiment, said one or more linker sequences comprise at least one amino acid sequence of a matrix metalloproteinase (MMP) cleavage site according to any of the variants known in the art.

[0075] In one preferred embodiment, the second domain (b) of a fusion protein according to any of the variants herein disclosed further comprises at least one amino acid sequence of a matrix metalloproteinase (MMP) cleavage site conjugated to each of said peptide amino acid sequences.

[0076] In one particular aspect, the second domain (b) of a fusion protein according to any of the variants herein disclosed further comprises at least one amino acid sequence of a MMP cleavage site conjugated to said amino acid sequence of a PD-L1 binding peptide and at least one amino acid sequence of a MMP cleavage site conjugated to said amino acid sequence of a tumor-penetrating peptide, respectively.

[0077] A suitable amino acid sequence of said MMP cleavage site to be used in a fusion protein according to any of the variants herein disclosed is a sequence of MMP 2, MMP 3, MMP 7 or MMP 9. Non limiting examples of amino acid sequences of said MMP cleavage sites that could be used in a fusion protein according to any of the embodiments disclosed herein are described in W02016051340 Al, at page 6 (lines 3 to 17), and in WO2019087155 Al, at page 9 (lines 11 to 24), which are incorporated herein by reference in their entirety.

[0078] In one preferred embodiment, the fusion protein according to any of the variants herein disclosed further comprises a third domain (c) conjugated to the N-terminal of said second domain (b) and consisting of the amino acid sequence of a polypeptide of at least 20 amino acid residues, which essentially consists or consists of proline, serine, and alanine (referred to as “PAS” for the sake of brevity), or which essentially consists or consists of proline, serine, alanine and at least one negatively charged residue selected from glutamate or aspartate (referred to as “PASE” for the sake of brevity).

[0079] Most preferably, the third domain of the fusion protein of the invention, linked to the N terminus, essentially consists or consists of the amino acid sequence of a polypeptide which is rich in proline, serine, alanine and at least one negative amino acids such as glutamate or aspartate (“PASE”), having the aim of increasing the stability of the protein during the drug encapsulating process, especially with drugs that can promote protein aggregation, and of increasing the stability of the protein drug complex in comparison to the polypeptide lacking the negative amino acids (PAS).

[0080] The polypeptide PASE essentially consists of amino acid sequences rich in Pro, Ala and Ser, and at least one or more Glu and / or Asp which form a negatively charged unstructured polymer, the length of which is preferably lower than 80 amino acid residues, more preferably comprised between 20 and 80 amino acid residues, still more preferably comprised between 30 and 70 amino acid residues. In a preferred embodiment, the proline residues of the aforesaid polypeptide PASE amount to 40% of the total amino acid residues of the polypeptide PASE.

[0081] Non limiting examples of amino acid sequences of said PAS or PASE polypeptides that could be used in a fusion protein according to any of the embodiments disclosed herein are described in W02016051340 Al, at page 7 (lines 5 to 10), and in WO2019087155 Al, at page 10 (lines 10 to 32) and at page 11 (lines 1 to 18), which are incorporated herein by reference in their entirety.

[0082] With the term “the polypeptide essentially consists of amino acid sequences rich in Pro, Ala and Ser” in the present description is defined a polypeptide that form a stable random coil conformation that consists of Pro, Ala and Ser wherein from 1 to 5% of Pro, Ala and Ser residues are replaced with other amino acid, such for example glycine, that do not alter the stable random coil conformation of the polypeptide.

[0083] As previously mentioned, in one preferred embodiment the bioactive peptides sequences described in the present invention are added to the surface of HFt through a short peptide sequence that contains one or more metallo proteinase cleavage sites, so as to provide the fusion protein of the invention with a releasable activity of the peptides. In fact, bioactive peptides can be selectively released at the target tissues by extracellular matrix metalloproteinases (MMPs). MMP 2 and MMP 9 were shown to be key metalloproteinases that are overexpressed in the tumor microenvironment and are involved in angiogenesis, invasion, and tumor metastasis.

[0084] A preferred embodiment of the present invention particularly provides a fusion protein comprising the following domains:

[0085] (a) a first domain comprising the amino acid sequence of the heavy of native human ferritin, or a variant thereof having at least 90% or at least 95%, or at least 98% or at least 99% identity with the amino acid sequence of the heavy chain of native human ferritin;

[0086] (b) a second domain comprising:

[0087] (i) the amino acid sequence LQKTPKQ (SEQ ID No. 9) as PD-L1 binding peptide, which is conjugated to the N-terminal of said first domain through the amino acid sequence of a matrix metalloproteinase (MMP) cleavage site, and

[0088] (ii) the amino acid sequence RGDKGPD (SEQ ID No. 10) as tumor penetrating peptide, which is conjugated to said amino acid sequence LQKTPKQ through the amino acid sequence of a matrix metalloproteinase (MMP) cleavage site;

[0089] (c) a third N-terminal domain consisting of the amino acid sequence of a polypeptide of at least 20 amino acid residues and which consists of proline, serine, alanine and at least one negatively charged residue selected from glutamate or aspartate (PASE).

[0090] Another preferred embodiment of the present invention provides a fusion protein comprising the following domains:

[0091] (a) a first domain comprising the amino acid sequence of the heavy of native human ferritin, or a variant thereof having at least 90% or at least 95%, or at least 98% or at least 99% identity with the amino acid sequence of the heavy chain of native human ferritin;

[0092] (b) a second domain comprising:

[0093] (i) the amino acid sequence LQKTPKQ (SEQ ID No. 9) as PD-L1 binding peptide, which is conjugated to the N-terminal of said first domain through the amino acid sequence of a matrix metalloproteinase (MMP) cleavage site,

[0094] (ii) the amino acid sequence NEWQKLS (SEQ ID No. 11) as c-MET blocking peptide, which is conjugated to said amino acid sequence LQKTPKQ through the amino acid sequence of a matrix metalloproteinase (MMP) cleavage site, and

[0095] (iii) the amino acid sequence RGDKGPD (SEQ ID No. 10) as tumor penetrating peptide, which is conjugated to said amino acid sequence NEWQKLS through the amino acid sequence of a matrix metalloproteinase (MMP) cleavage site; and

[0096] (c) a third N-terminal domain consisting of the amino acid sequence of a polypeptide of at least 20 amino acid residues and which consists of proline, serine, alanine and at least one negatively charged residue selected from glutamate or aspartate (PASE). The conjugation of a domain comprising the amino acid sequences of different bioactive peptides, from two to five, to a single multimeric surface of ferritin within the scope of the present invention offers several advantages over the prior art. Considering that a single peptide is characterized by a single functionality, the possibility to deliver multiple different peptides at a pathological site can guarantee the success for a specific therapy. The delivery of multiple functionalities is achieved by the inventors using the fusion-proteins based on the human ferritin reported in the present invention.

[0097] Stable fusion proteins containing several peptides sequences have been surprisingly and unexpectedly achieved by the authors of the present invention, who constructed nanoparticles based on the heavy chain of human ferritin (HFt), by using both the gene fusion technology and the production technology of recombinant proteins. In particular, as will be described in detail in the section related to the examples, genetic constructs were made, which, in one single nucleic acid sequence (for instance DNA), encode for the six or seven sequences set forth in Figure 2 or Figure 3: i) HFt; ii) short peptide sequences (MP) cleavable by MMP 2 / 9; iii) a PD-L1 binding peptide; iv) short peptide sequences (MP) cleavable by MMP 2 / 9; v) a c-Met blocking peptide; vi) short peptide sequences (MP) cleavable by MMP 2 / 9; vii) an integrin aVp3 / 5 as well as neuropilinl binding peptide; viii) unstructured polypeptide sequences rich in Pro, Ser, Ala and Glu (PASE) preferably with a length comprised between 20 and 80 residues. Sequences from ii) to viii) are bound to the N terminus of HFt for a reversible masking thereof.

[0098] As already stated above, the HFt fusion proteins obtained by the present inventors spontaneously form HFt nanoparticles capable of carrying therapeutics (chemical compounds, monoclonal antibodies, peptides, etc.) (Figure 1).

[0099] In one embodiment, different bioactive peptides are present on the surface of the HFt nanoparticle, preferably from two to five. The stability of the fusion protein carrying such different bioactive peptides can be increased thanks to the presence of the PAS or PASE polypeptides. The homogeneity of the material obtained is a highly desirable property in the pharmaceutical field, as it indicates the absence of negative effects, such as precipitation, clustering, and loss of the final product carrying the therapeutic molecule.

[0100] A therapeutic molecule is for example a pharmaceutical active ingredient. In therapeutic applications, the HFt nanoparticles of the present invention, which act as targeted carrier or adjuvant systems, can be administered to a subject or patient through any suitable administration route, for instance orally, parenterally, intravenously, intraperitoneally, intramuscularly, as a suppository, intralesionally, intranasally or subcutaneously, in- trathecally, intralymphatically, through inhalation of microdroplets, or by implant of a slow release device, for instance an osmotic pump.

[0101] In therapeutic applications, the HFt nanoparticles of the invention are used for the administration of a therapeutically effective dose of a pharmaceutical active ingredient. “Therapeutically effective dose” is intended to mean a dose that produces the therapeutic effect for which it is administered. The exact dose will depend on a number of factors, including the aim of the treatment, the subject, the disease to be treated, etc., and can easily be determined by a person of ordinary skill in the art by using per se known methodologies (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1 3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0102] It forms part of the invention also a pharmaceutical composition comprising a fusion protein or a nanoparticle according to any of the embodiments herein disclosed, in combination with at least one pharmaceutically acceptable excipient, carrier, or diluent.

[0103] The HFt nanoparticles or pharmaceutical compositions thereof of the invention may be used for treating any disease that requires the administration of a pharmaceutical ingredient, for instance by sequestering the active ingredient within the cavity of the nanoparticle or by covalently binding it to the nanoparticle surface.

[0104] The HFt nanoparticle or pharmaceutical compositions thereof of the present invention can be administered to a subject for the treatment of any disease, preferably a hyperproliferative disease, including cancer, for example: carcinomas, gliomas, mesotheliomas, melanomas, sarcomas, lymphomas, leukaemias, adenocarcinomas, breast cancer, ovary cancer, cervical cancer, glioblastoma, leukaemia, lymphoma, prostate cancer, Burkitt’s lymphoma, head and neck cancer, colon cancer, colorectal cancer, non small cell lung cancer, small cell lung cancer, oesophagus cancer, stomach cancer, pancreatic cancer, hepatobiliary cancer, bladder cancer, small intestine cancer, rectal cancer, kidney cancer, gall bladder cancer, penile cancer, urethra cancer, testicular cancer, cervix cancer, vaginal cancer, uterine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, endocrine pancreatic cancer, carcinoid tumor, bone cancer, skin cancer, retinoblastomas, multiple mielomas, Hodgkin lymphoma, non Hodgkin lymphoma (see CANCER: PRINCIPLES AND PRACTICE (De Vita, V. T. et al. 2008 Edition) for other types of cancer).

[0105] The following embodiments are objects of the present invention:

[0106] Embodiment 1. A fusion protein comprising:

[0107] (a) a first domain comprising the amino acid sequence of the heavy chain of native human ferritin or a variant thereof having at least 90% identity with the amino acid sequence of the heavy chain of native human ferritin; and

[0108] (b) a second domain conjugated to said first domain and located N-terminally with respect to said first domain, which second domain comprises at least one amino acid sequence of a programmed cell death ligand- 1 (PD-L1) binding peptide and / or at least one amino acid sequence of a tumorpenetrating peptide.

[0109] Embodiment 2. The fusion protein according to Embodiment 1, wherein said second domain (b) comprises at least one amino acid sequence of a PD-L1 binding peptide and at least one amino acid sequence of a tumor-penetrating peptide.

[0110] Embodiment 3. The fusion protein according to Embodiments 1 or 2, wherein said at least one amino acid sequence of a PD-L1 binding peptide comprises or consists of the amino acid sequence set forth in SEQ ID No. 9.

[0111] Embodiment 4. The fusion protein according to any one of Embodiments 1 to 3, wherein said at least one amino acid sequence of a tumor-penetrating peptide comprises or consists of an amino acid sequence selected from SEQ ID No: 10, SEQ ID No: 27, SEQ ID No: 28 and SEQ ID No: 29, preferably comprises or consists of the amino acid sequence set forth in SEQ ID No. 10.

[0112] Embodiment 5. The fusion protein according to any one of Embodiments 1 to 4, wherein said second domain (b) further comprises at least one amino acid sequence of a matrix metalloprotein- ase (MMP) cleavage site conjugated to each of said at least one amino acid sequence of a programmed cell death ligand-1 (PD-L1) binding peptide and / or of said at least one amino acid sequence of a tumor-penetrating peptide; in particular wherein said amino acid sequence of the matrix metalloproteinase (MMP) cleavage site is selected from the group consisting of SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5 SEQ ID No: 6, SEQ ID No: 7 or SEQ ID No: 8.

[0113] Embodiment 6. The fusion protein according to any one of Embodiments 1 to 5, further comprising:

[0114] (c) a third domain conjugated to the N-terminal of said second domain and consisting of the amino acid sequence of a polypeptide of at least 20 amino acid residues, which essentially consists or consists of proline, serine, and alanine (PAS) or which essentially consists or consists of proline, serine, alanine and at least one negatively charged residue selected from glutamate or aspartate (PASE).

[0115] Embodiment 7. The fusion protein according to Embodiment 6, wherein said third domain amino acid sequence is selected from SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16 and SEQ ID No: 17.

[0116] Embodiment 8. The fusion protein according to any one of Embodiments 1 to 7, wherein said second domain (b) further comprises at least one of the following amino acid sequences:

[0117] (bl) the amino acid sequence of a c-MET blocking peptide, preferably the amino acid sequence set forth in SEQ ID No. 11 ;

[0118] (b2) the amino acid sequence of a programmed cell death- 1 (PD-1) binding peptide;

[0119] (b3) the amino acid sequence of a T-cell immunoglobulin-3 (TIM-3) binding peptide.

[0120] Embodiment 9.The fusion protein according to any one of Embodiments 1 to 7, wherein the first domain comprises the amino acid sequence of the heavy chain of native human ferritin of SEQ ID NO: 1 or the amino acid sequence of the heavy chain variant of human ferritin of SEQ ID NO: 2 or a variant of the heavy human ferritin with at least one cysteine or aspartate or glutamate in the internal cavity of the protein, preferably with two, three or four cysteines or aspartate or glutamate in the internal cavity. Embodiment lO.The fusion protein according to any one of embodiments 1 to 9, wherein said fusion protein comprises:

[0121] (a) a first domain comprising the amino acid sequence of the heavy of native human ferritin, or a variant thereof having at least 90% identity with the amino acid sequence of the heavy chain of native human ferritin;

[0122] (b) a second domain comprising:

[0123] (i) the amino acid sequence set forth in SEQ ID No. 9 as PD-L1 binding peptide, which is conjugated to the N-terminal of said first domain through the amino acid sequence of a matrix metalloproteinase (MMP) cleavage site, and

[0124] (ii) the amino acid sequence set forth in SEQ ID No. 10 as tumor penetrating peptide, which is conjugated to said amino acid sequence set forth in SEQ ID No. 9 through the amino acid sequence of a matrix metalloproteinase (MMP) cleavage site;

[0125] (c) a third N-terminal domain consisting of the amino acid sequence of a polypeptide of at least 20 amino acid residues and which consists of proline, serine, alanine and at least one negatively charged residue selected from glutamate or aspartate (PASE).

[0126] Embodiment 11. The fusion protein according to any one of embodiments 1 to 9, wherein said fusion protein comprises:

[0127] (a) a first domain comprising the amino acid sequence of the heavy of native human ferritin, or a variant thereof having at least 90% identity with the amino acid sequence of the heavy chain of native human ferritin;

[0128] (b) a second domain comprising:

[0129] (i) the amino acid sequence set forth in SEQ ID No. 9 as PD-L1 binding peptide, which is conjugated to the N-terminal of said first domain through the amino acid sequence of a matrix metalloproteinase (MMP) cleavage site,

[0130] (ii) the amino acid sequence set forth in SEQ ID No. 11 as c-MET blocking peptide, which is conjugated to said amino acid sequence set forth in SEQ ID No. 9 through the amino acid sequence of a matrix metalloproteinase (MMP) cleavage site, and

[0131] (iii) the amino acid sequence set forth in SEQ ID No. 10 as tumor penetrating peptide, which is conjugated to said amino acid sequence set forth in SEQ ID No. 11 through the amino acid sequence of a matrix metalloproteinase (MMP) cleavage site; and

[0132] (c) a third N-terminal domain consisting of the amino acid sequence of a polypeptide of at least 20 amino acid residues and which consists of proline, serine, alanine and at least one negatively charged residue selected from glutamate or aspartate (PASE).

[0133] Embodiment 12. The fusion protein according to any one of Embodiments 1 to 11, which protein is linked to an active ingredient and / or imaging agent.

[0134] Embodiment 13. A nanoparticle comprising a plurality of monomers of a fusion protein according to any one of Embodiments 1 to 12, preferably 24 monomers.

[0135] Embodiment 14. A nanoparticle according to Embodiment 13 wherein an active ingredient selected from doxorubicin, mitoxantrone, pixantrone, Genz 644282, paclitaxel, auristatins, camptothecins, gemcitabine and platinum based is linked to or encapsulated in said nanoparticle.

[0136] Embodiment 15. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 12, a nanoparticle according to Embodiments 13 or 14, in combination with at least one pharmaceutically acceptable excipient, carrier, or diluent.

[0137] Embodiment 16. The pharmaceutical composition according to Embodiment 15, for use as a medicament, in particular for use in a therapeutic treatment and / or diagnosis of a tumor.

[0138] The following examples are provided for illustrative purposes and not as a limitation of the scope of the invention as defined in the appended claims.

[0139] EXAMPLES

[0140] Example 1

[0141] Construction of expression vectors for HFt-PASE@PDLl-NRPl fusion proteins

[0142] As an attempt to deliver multiple copies of different bioactive peptides, it was decided to introduce specific peptides in the outer shield forming sequence fused to each HFt subunit. The HFt-PASE@PDLl-NRPl gene was achieved by combining six different sequences into one single sequence: HFt (SEQ ID NO: 1), MP (SEQ ID NO: 5), PDL1 (SEQ ID NO: 9), MP (SEQ ID NO: 5), NRP1 (SEQ ID NO: 10) and PASE (SEQ ID NO: 13). The schematic representation of the HFt-PASE@PDLl-NRPl construct is reported in Figure 2.

[0143] The pET 27b expression vector containing the HFt-PASE@PDLl-NRPl gene was synthesized by using GENEART AG (Germany). Gene synthesis was carried out taking into consideration the codon optimization for high levels of expression in Escherichia coli.

[0144] The fusion protein HFt-PASE@PDLl-NRPl was obtained via recombinant protein technology and purified from the cellular soluble fraction at high yield (about 150 mg per liter of E. coli cell culture). The relative protein expression is shown in Figure 4, panel A. Specific protein band after induction with IPTG is indicated by an arrow in the SDS-gel electrophoresis assay.

[0145] Example 2

[0146] Construction of expression vectors for HFt-PASE@PDLl-NRPl-cMET fusion proteins

[0147] As an attempt to deliver multiple copies of different bioactive peptides, it was decided to introduce specific peptides in the outer shield forming sequence fused to each HFt subunit. The HFt-PASE@PDLl-NRPl gene was achieved by combining eight different sequences into one single sequence: HFt (SEQ ID NO: 1), MP (SEQ ID NO: 5), PDL1 (SEQ ID NO: 9), MP (SEQ ID NO: 5), c-MET (SEQ ID NO: 11), MP (SEQ ID NO: 5), NRP1 (SEQ ID NO: 10) and PASE (SEQ ID NO: 13). The schematic representation of the HFt- PASE@PDLl-NRPl-cMET construct is reported in Figure 3. The pET 27b expression vector containing the HFt-PASE@PDLl-NRPl-cMET gene was synthesized by using GENEART AG (Germany). Gene synthesis was carried out taking into consideration the codon optimization for high levels of expression in Escherichia coli.

[0148] The fusion protein HFt-PASE@PDLl-NRPl-cMET was obtained via recombinant protein technology and purified from the cellular soluble fraction at high yield (about 150 mg per liter of E. coli cell culture). The relative protein expression is shown in Figure 4, panel B. Specific protein band after induction with IPTG is indicated by an arrow in the SDS-gel electrophoresis assay.

[0149] Example 3

[0150] Testing of the HFt-PASE@PDLl-NRPl fusion protein binding ability

[0151] To investigate the binding affinity between the construct HFt-PASE@PDLl-NRPl (analyte) and its specific receptors (ligands) aVp3 and PD-L1, Surface Plasmon Resonance (SPR) experiments were performed. The experiment was made immobilizing the his-tagged-aVp3 or PD-L1 on the NTA sensor chip, followed by the injection of HFt-PASE@PDLl-NRPl at different concentrations. As can be seen in Figures 5 and 6, a clear concentration-dependent binding between the integrin (Figure 5) or PD-E1 (Figure 6) and HFt-PASE@PDEl-NRPl occurred. Indeed, as the concentration of analyte increased, a significant increase in response units is observed, indicating the formation of the ligand-analyte complex. Furthermore, during the dissociation phase, a gradual decrease in RU was observed. Both measured dissociation constants (KD) indicate a very strong and robust affinity for the analyte to aVp3 and PD-E1 ligands.

[0152] Example 4

[0153] Testing of the HFt-PASE@PDLl-NRPl fusion protein activity on cultured cells: uptake

[0154] For intemalization / uptake experiments, the antibody trastuzumab was conjugated with a pH- sensitive dye by the pHrodo Deep Red Antibody Eabeling kit (#P35355, Invitrogen, Thermofisher scientific), according to the manufacturer's instructions. A375 cells (human melanoma) were seeded at a density of 3xl03cells / well in 96-well plates one day before addition of Trastuzumab-pHrodo and HFt-PASE@PDEl-NRPl, alone or combined, in the indicated amounts showed in Figure 7. Following Antibody -pHrodo addition, cells were cultured under standard conditions inside an Incucyte S3 live-cell analysis System (Sartorius). Phase contrast and fluorescence images were captured at 20X magnification every 60 min for 48 hours, and mounted in the time-lapse mode by on-board software. Red integrated fluorescence (from pHrodo-labeled antibodies internalized in acidic intracellular compartments) was also quantitatively captured by the integrated, automated Incucyte software, and quantitated after background subtraction. Data were plotted and analysed by onboard software. Internalization was expressed as per cent of baseline (time 0) fluorescence over incubation time.

[0155] Testing of the HFt-PASE@PDLl-NRPl fusion protein activity on cultured cells: antiproliferative effects

[0156] To evaluate the potential enhancement in the antiproliferative activity by HFt- PASE@PDL1-NRP1, A375 cells (human melanoma) were seeded at a density of 2.5xl03cells / well in 96-well plates (Costar #3595). On the following day, Trastuzumab Deruxtecan (T-DXD) and HFt-PASE@PDLl-NRPl, alone or combined, were added to cultured cells (Figure 8). Cells were then grown under standard conditions (37 °C, 5% CO2) in in Roswell Park Memorial Institute (RPMI) 1640 culture medium, supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 1% glutamine. Cell viability was detected by the CellTiter-Glo 2.0 kit (Promega, Madison, WI, USA). Each experimental condition was performed in triplicate. After 96 hours from treatment, 100 pl of CellTiter-Glo reagent were added to each well. Plates were incubated for 10 minutes at room temperature in the dark, and luminescence was determined on Synergy / LX multimode reader (Biotek), using the Gen5 3.10 Imager software. Results were plotted as mean + / - SD and analysed by the GraphPad Prism 9.

[0157] Example 5

[0158] Anti proliferative effects of Gemcitabine in combination with HFt-PASE@PDLl-

[0159] NRP1 in vivo

[0160] Five-week-old female Balb / cmice (Charles River Laboratories, Lecco, Italy) were injected subcutaneously (i.e., right flank) with 4xl06pancreatic KPC cells resuspended in 200 pl of DMEM medium plus 1% BSA. When tumors had reached a volume of about 100-200 mm3, mice were randomized in groups of six animals and injected with 200 pL of physiological saline (i.v.), Gemcitabine (100 mg / Kg; i.p.) or Gemcitabine (100 mg / Kg; i.p.) plus HFt- PASE@PDL1-NRP1 (130 mg / Kg; i.v.). HFt-PASE@PDLl-NRPl was injected 24 hours before the treatments with Gemcitabine. Mice were injected twice a week for two weeks; tumor volume was measured twice a week with a digital caliper and mouse weight was monitored. When the tumor of mice had reached a volume >1000 mm3, animals were sacrificed. In Figure 9 is reported the tumor growth curves after about three weeks from the start of treatments. In this figure is evident the ability of HFt-PASE@PDLl-NRPl to enhance the therapeutic activity of co-administered Gemcitabine drug. In fact, tumor growth is significantly reduced in the presence of HFt-PASE@PDLl-NRPl.

[0161] Example 6

[0162] Anti proliferative effects of THE-0504 (Genz-644282) in combination with HFt- PASE@PDL1-NRP1 or iRGD peptide in vivo

[0163] Five-week-old female Balb / c mice (Charles River Laboratories, Lecco, Italy) were injected subcutaneously (i.e., right flank) with 4xl06pancreatic KPC cells resuspended in 200 pl of DMEM medium plus 1% BSA. When tumors had reached a volume of about 250 mm3, mice were randomized in groups of six animals and injected i.v. with 200 pL of physiological saline (i.v.), THE-0504 (1.0 mg / kg), THE-0504 (1.0 mg / kg) plus iRGD peptide (3.0 mg / Kg) or THE-0504 (1.0 mg / kg) plus HFt-PASE@PDLl-NRPl (130 mg / Kg). Mice were injected twice a week for three weeks; tumor volume was measured twice a week with a digital caliper and mouse weight was monitored. When the tumor of mice had reached a volume >1000 mm3, animals were sacrificed. In Figure 10 is reported the tumor growth curves after about three weeks from the start of treatments. In this figure is evident the ability of HFt- PASE@PDL1-NRP1 to enhance the therapeutic activity of co-administered THE-0504 drug. The effect is superior to the original cyclic iRGD peptide (CRGDKGPDC). In fact, tumor growth reduction is significantly more pronounced in the presence of HFt-PASE@PDLl- NRP1 in comparison to the iRGD peptide.

[0164] Anti proliferative effects of THE-0504 (Genz-644282) in combination with HFt- PASE@PDL1-NRP1 in vivo

[0165] Four- week-old female Athimic nude mice (Envigo , Italy) were injected subcutaneously (i.e., right flank) with 5xl06pancreatic MiaPaca2 cells resuspended in 200 pl of DMEM medium plus 1% BSA. When tumors had reached a volume of about 80 mm3, mice were randomized in groups of six animals and injected i.v. with 200 pL of physiological saline (i.v.), THE- 0504 (1.0 mg / kg), or THE-0504 (1.0 mg / kg) plus HFt-PASE@PDLl-NRPl (130 mg / Kg). Mice were injected twice a week for three weeks; tumor volume was measured twice a week with a digital caliper and mouse weight was monitored. When the tumor of mice had reached a volume > 1000 mm3, animals were sacrificed. In Figure 11 is reported the tumor growth curves after about three weeks from the start of treatments. In this figure is evident the ability of HFt-PASE@PDEl-NRPl to enhance the therapeutic activity of co-administered THE- 0504 drug. In fact, tumor growth is significantly reduced in the presence of HFt- PASE@PDL1-NRP1.

[0166] Example 7

[0167] Anti proliferative effects of Immu-132 (sacituzumab govitecan) in combination with HFt-PASE@PDLl-NRPl in vivo

[0168] Five-week-old female Balb / cmice (Charles River Laboratories, Lecco, Italy) were injected subcutaneously (i.e., right flank) with 4xl06pancreatic KPC cells resuspended in 200 pl of DMEM medium plus 1% BSA. When tumors had reached a volume of about 100 mm3, mice were randomized in groups of six animals and i.v. injected with 200 pL of physiological saline (i.v.), Immu-132 (45 mg / Kg) or Immu-132 (45 mg / Kg) plus HFt-PASE@PDLl- NRP1 (130 mg / Kg). HFt-PASE@PDLl-NRPl was injected 10 minutes before the treatments with Immu-132. Mice were injected every 3-4 days for three times; tumor volume was measured twice a week with a digital caliper and mouse weight was monitored. When the tumor of mice had reached a volume >1000 mm3, animals were sacrificed. In Figure 12 is reported the tumor growth curves after about two weeks from the start of treatments. In Figure 13 are reported the tumor weights and tumor residuals at the end of the experiment. Both figures 12 and 13 clearly demonstrated that the addition of HFt-PASE@PDLl-NRPl significantly increase the therapeutic activity of co-administered Immu-132 drug. Declaration according to Art. 170bis

[0169] In compliance with Art. 170bis of the Italian code of industrial property, the applicant of the present patent application declares that for the biological material, containing microorgan- isms or genetically modified organisms, object or used in the aforementioned patent application, the obligations deriving from national or Community regulations, and in particular, from the provisions referred to in paragraph 6 of the Legislative Decree of 12 April 2001 n.206 and 8 July 2003 n. 224, 10 concerning these modifications, have been respected.

Claims

CLAIMS1. A fusion protein comprising:(a) a first domain comprising the amino acid sequence of the heavy chain of native human ferritin or a variant thereof having at least 90% identity with the amino acid sequence of the heavy chain of native human ferritin; and(b) a second domain conjugated to said first domain and located N-terminally with respect to said first domain, which second domain comprises at least one amino acid sequence of a programmed cell death ligand- 1 (PD-L1) binding peptide and / or at least one amino acid sequence of a tumorpenetrating peptide.

2. The fusion protein according to claim 1, wherein said second domain (b) comprises at least one amino acid sequence of a PD-L1 binding peptide and at least one amino acid sequence of a tumorpenetrating peptide.

3. The fusion protein according to claims 1 or 2, wherein said at least one amino acid sequence of a PD-L1 binding peptide comprises or consists of the amino acid sequence set forth in SEQ ID No. 9.

4. The fusion protein according to any one of claims 1 to 3, wherein said at least one amino acid sequence of a tumor-penetrating peptide comprises or consists of an amino acid sequence selected from SEQ ID No: 10, SEQ ID No: 27, SEQ ID No: 28 and SEQ ID No: 29, preferably comprises or consists of the amino acid sequence set forth in SEQ ID No. 10.

5. The fusion protein according to any one of claims 1 to 4, wherein said second domain (b) further comprises at least one amino acid sequence of a matrix metalloproteinase (MMP) cleavage site conjugated to each of said at least one amino acid sequence of a programmed cell death ligand- 1 (PD-L1) binding peptide and / or of said at least one amino acid sequence of a tumor-penetrating peptide; in particular wherein said amino acid sequence of the matrix metalloproteinase (MMP) cleavage site is selected from the group consisting of SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5 SEQ ID No: 6, SEQ ID No: 7 or SEQ ID No: 8.

6. The fusion protein according to any one of claims 1 to 5, further comprising:(c) a third domain conjugated to the N-terminal of said second domain and consisting of the amino acid sequence of a polypeptide of at least 20 amino acid residues, which essentially consists or consists of proline, serine, and alanine (PAS) or which essentially consists or consists of proline, serine, alanine and at least one negatively charged residue selected from glutamate or aspartate (PASE).

7. The fusion protein according to claim 6, wherein said third domain amino acid sequence is selected from SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16 and SEQ ID No: 17.

8. The fusion protein according to any one of claims 1 to 7, wherein said second domain (b) further comprises at least one of the following amino acid sequences:(bl) the amino acid sequence of a c-MET blocking peptide, preferably the amino acid sequence set forth in SEQ ID No. 11 ;(b2) the amino acid sequence of a programmed cell death- 1 (PD-1) binding peptide; (b3) the amino acid sequence of a T-cell immunoglobulin-3 (TIM-3) binding peptide.

9. The fusion protein according to any one of claims 1 to 8, wherein the first domain comprises the amino acid sequence of the heavy chain of native human ferritin of SEQ ID NO: 1 or the amino acid sequence of the heavy chain variant of human ferritin of SEQ ID NO: 2 or a variant of the heavy human ferritin with at least one cysteine or aspartate or glutamate in the internal cavity of the protein, preferably with two, three or four cysteines or aspartate or glutamate in the internal cavity.

10. The fusion protein according to any one of claims 1 to 9, wherein said fusion protein comprises:(a) a first domain comprising the amino acid sequence of the heavy of native human ferritin, or a variant thereof having at least 90% identity with the amino acid sequence of the heavy chain of native human ferritin;(b) a second domain comprising:(i) the amino acid sequence set forth in SEQ ID No. 9 as PD-L1 binding peptide, whichis conjugated to the N-terminal of said first domain through the amino acid sequence of a matrix metalloproteinase (MMP) cleavage site, and(ii) the amino acid sequence set forth in SEQ ID No. 10 as tumor penetrating peptide, which is conjugated to said amino acid sequence set forth in SEQ ID No. 9 through the amino acid sequence of a matrix metalloproteinase (MMP) cleavage site;(c) a third N-terminal domain consisting of the amino acid sequence of a polypeptide of at least 20 amino acid residues and which consists of proline, serine, alanine and at least one negatively charged residue selected from glutamate or aspartate (PASE).

11. The fusion protein according to any one of claims 1 to 9, wherein said fusion protein comprises:(a) a first domain comprising the amino acid sequence of the heavy of native human ferritin, or a variant thereof having at least 90% identity with the amino acid sequence of the heavy chain of native human ferritin;(b) a second domain comprising:(i) the amino acid sequence set forth in SEQ ID No. 9 as PD-L1 binding peptide, which is conjugated to the N-terminal of said first domain through the amino acid sequence of a matrix metalloproteinase (MMP) cleavage site,(ii) the amino acid sequence set forth in SEQ ID No. 11 as c-MET blocking peptide, which is conjugated to said amino acid sequence set forth in SEQ ID No. 9 through the amino acid sequence of a matrix metalloproteinase (MMP) cleavage site, and(iii) the amino acid sequence set forth in SEQ ID No. 10 as tumor penetrating peptide, which is conjugated to said amino acid sequence set forth in SEQ ID No. 11 through the amino acid sequence of a matrix metalloproteinase (MMP) cleavage site; and(c) a third N-terminal domain consisting of the amino acid sequence of a polypeptide of at least 20 amino acid residues and which consists of proline, serine, alanine and at least one negatively charged residue selected from glutamate or aspartate (PASE).

12. A nanoparticle comprising a plurality of monomers of a fusion protein according to any one of claims 1 to 11, preferably 24 monomers, optionally further comprising an active ingredient linked to or encapsulated in said nanoparticle.