WO2024105116A1 - NANOCONJUGATES CONTAINING PDGFR-β LIGANDS AND USES THEREOF - Google Patents

NANOCONJUGATES CONTAINING PDGFR-β LIGANDS AND USES THEREOF Download PDF

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WO2024105116A1
WO2024105116A1 PCT/EP2023/081937 EP2023081937W WO2024105116A1 WO 2024105116 A1 WO2024105116 A1 WO 2024105116A1 EP 2023081937 W EP2023081937 W EP 2023081937W WO 2024105116 A1 WO2024105116 A1 WO 2024105116A1
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fusion protein
polypeptide
region
intervening
protein
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PCT/EP2023/081937
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French (fr)
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Esther Vázquez Gómez
Antonio Villaverde Corrales
Eric VOLTÀ DURÁN
Ramon Mangues Bafalluy
Lorena ALBA CASTELLÓN
Ugutz Unzueta Elorza
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Universitat Autònoma De Barcelona
Fundació Institut De Recerca De L´Hospital De La Santa Creu I Sant Pau
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Publication of WO2024105116A1 publication Critical patent/WO2024105116A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/49—Platelet-derived growth factor [PDGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle

Definitions

  • Metastatic cancer stem cells are particularly appealing targets for high precision nanomedicines as they are the origin of metastases, the main cause of patients’ death. So far, despite being theoretically promising, attempts of selectivity in cancer nanomedicines have not generically reached accumulation in target tissues over 1-2 % of the administered material. Many nanoscale materials, because of their physicochemical properties or their natural interactivity with biological receptors are in fact retained in liver or phagocyted, precluding the desired biodistribution of the carrier and the payload drug.
  • stroma refers to the tissue that surrounds epithelial cancer cells, including extracellular matrix, endothelial cells, immune cells and fibroblasts, which modulate and participate in cancer development.
  • fibroblasts synthesize and remodel the extracellular matrix being then responsible for the cancer tissue architecture and structural properties.
  • Fibroblasts are also responsible for the production of growth factors and cytokines that will be secreted within the tumor, supporting its development.
  • stromal PDGFR-P+ fibroblasts are a pivotal, highly interesting cell type for imaging, drug delivery or selective destruction in the context of cancer therapies.
  • the modest explorations have been so far limited to interfere signaling, but appropriate drugs or drug vehicles have not been yet developed and this type of approach has been essentially neglected.
  • the inventors herein have fully validated the fusion protein PDGFD-GFP-H6, among the starting candidates, as a potent prototype for molecular delivery, imaging and theranostics in cancer through the so far neglected targeting of CAF. Importantly, this is the first time that the bacterial production and the use of PDGFD as a targeting ligand are reported, what opens a spectrum of clinical avenues.
  • protein nanoparticles resulting from oligomeric self-assembling can be functionalized through intrinsic, biological active protein domains, by chemically coupling drugs or through combining both strategies.
  • this result supports proteins as editable, versatile and biodegradable building blocks suited to construct nanoscale oligomers for clinical purposes.
  • PDGFR-p ligand selected from the group of consisting of PDGFB (SEQ ID NO: 1) and PDGFD (SEQ ID NO: 2) or a functionally equivalent variant thereof,
  • a method comprising c) providing a fusion protein comprising i) a PDGFR-p ligand selected from the group of consisting of PDGFB (SEQ ID N0 : l), PDGFD (SEQ ID NO : 2) and a functionally equivalent variant thereof, ii) an intervening polypeptide region and iii) positively charged amino acid-rich region, wherein the PDGFR-p ligand and the positively charged amino acid-rich region are located at the ends of the protein and wherein the fusion protein is provided in an activated form, wherein said activated form of the fusion protein contains a reactive group in the intervening region and d) contacting said fusion protein with a therapeutic agent or an oligomeric form thereof, wherein said therapeutic agent contains a group which is capable of reacting with the reactive group in the fusion protein, wherein said contacting is carried out under conditions adequate for the formation of a bond between the reactive group in the fusion protein and the group in the therapeutic agent.
  • One more aspect of the invention relates to a vector comprising a polynucleotide according to the invention.
  • the invention relates to a host cell comprising a polynucleotide according to the invention or a vector according to the invention.
  • a further aspect of the invention relates to a method for preparing a nanoparticle comprising multiple copies of the fusion protein according to the invention comprising placing a preparation of said fusion protein in a suitable buffer.
  • Another aspect of the present invention relates to a nanoparticle comprising multiple copies of the fusion protein according to the invention or which has been obtained by a method according to the invention.
  • One more aspect of the present invention relates to the fusion protein according to the invention, the polynucleotide according to the invention, the vector according to the invention, the host cell according to the invention or the nanoparticle according to the invention for use in medicine.
  • a further aspect of the invention relates to the fusion protein according to the invention, the polynucleotide according to the invention, the vector according to the invention, the host cell according to the invention or the nanoparticle according to the invention wherein the intervening polypeptide is an antitumor peptide or wherein the intervening polypeptide is linked to an antitumor agent for use in the treatment of cancer.
  • FIG. 1 Representative FESEM images of isolated nanoparticles. The white bars represent 50 nm. Broader fields and TEM images are shown in the Figure 6.
  • Figure 2. Internalization of CAFs-directed nanoparticles. Two mesenchymal cell types, mouse embryonic fibroblast (MEFs) and mouse mesenchymal stem cells (MSCs), were exposed to 100 nM of different nanoparticles.
  • MEFs mouse embryonic fibroblast
  • MSCs mouse mesenchymal stem cells
  • A GFP, PDGFR-p and Tubulin immunoblotting of MEFs and MSCs whole cell extracts after 1 h exposure.
  • B Nanoparticle internalization analysed by flow cytometry in MEFs and MSCs upon 1 h or 24 h exposure. Values are expressed as mean fluorescence intensity fold change respect to untreated cells.
  • PDGFRp is required for internalization of nanoparticles. MEFs were incubated with 50 ng/mL PDGF-BB for 30 min following 100 nM PDGFB-GFP-H6 or PDGFD-GFP-H6 treatment for 1 h.
  • A Fixed cell were stained with DAPI and analysed by immunofluorescence.
  • B Whole cell extracts were used for detection of PDGFR p and GFP, by immunoblotting. Tubulin was used as loading control.
  • C Graphs represents densitometry quantification of 3 independent assays. * p ⁇ 0.05; ** p ⁇ 0.01 ; *** p ⁇ 0.001 Bar size: 50 nm.
  • FIG. 4 Biodistribution and in vivo uptake of PDGFB-GFP-H6 and PDGFD-GFP- H6.
  • A Immunohistochemistry detection of PDGFRp within tumors.
  • B GFP fluorescence detection by IVIS-Spectrum in tumors upon 2 h after PDGFB-GFP-H6 or PDGFD-GFP-H6 administration.
  • D Immunohistochemistry against GFP to detect CAFs-targeting nanoparticle in tumor, kidney and liver of treated mice. * p ⁇ 0.05. Bars Size: 50 pm.
  • FIG. 1 Integrity and assembling of protein building blocks.
  • A Western blot immunodetection of purified proteins after dialysis, using either anti-His or anti-GFP antibodies.
  • P1 refers to PDGFRP1-GFP-H6, Z to Z09591-GFP-H6, B to PDGFB-GFP- H6 and D to PDGFD-GFP-H6.
  • M indicates the marker line and the siding numbers the molecular mass of the markers in kDa.
  • B Particle size upon treatment of nanoparticles with 1 % SDS, for disassembling. Numbers indicate the mean peak size (in nm) for every building block.
  • FIG. Electron microscopy of assembled nanoparticles. Representative high- resolution images of electron microscopy (TEM and FESEM) of well-formed nanoparticles resulting from the assembly of the four GFP-H6 constructs with PDGFPR1 , Z09591 , PDGFB and PDGFD. Bars size: 50 nm.
  • FIG. 8 PDGFB-GFP-H6 and PDGFD-GFP-H6 dose response in MEFs. MEFs were exposed to different concentrations of either PDGFB-GFP-H6 or PDGFD-GFP-H6 nanoparticles for 1 h.
  • A Immunoblotting against GFP and PDGFR-p of whole cell extract from treated MEFs. Tubulin was used as loading control.
  • B Internalization of either PDGFB-GFP-H6 or PDGFD-GFP-H6 measured by GFP detection through flow cytometry. Values are expressed as mean fluorescence intensity fold change respect to untreated cells.
  • C Nanoparticle (GFP) and PDGFR-p (RED) detection by epifluorescence microscopy in MEFs exposed to nanoparticles at 3 different concentrations. *** p ⁇ 0.001. Scale bar: 50 mm.
  • CAFs-targeting nanoparticles are non-toxic for fibroblasts. Viability of MEFs and MSCs exposed to either PDGFB-GFP-H6 (A) or PDGFD-GFP-H6 (B) for 24 and 48 h. Protein nanoparticles were added to cell cultures at 100 nM, 500 nM and 1000 nM. Untreated cells (0) were considered as being 100 % viable.
  • PDGFD-FD-PE24-H6 nanotoxin alters the colorectal tumor microenvironment.
  • A nanotoxin is a self-assembly nanoparticle of 44.4nm as detected by DLS.
  • B Schematic representation of the experimental colorectal cancer mouse model administered with PDGFD-FD-PE24-H6 (PDGFD-NT-H6) nanotoxin.
  • C-E Quantification of immunohistochemistry in tumor sections against (C) blood vessel, CD31 ;
  • D Macrophages, F4/80;
  • E Lymphocytes CD3, CD4, and CD8.
  • nanotoxin administration caused an increase in the analyzed tumor microenvironment cell type. *p>0.05, **p>0.01 , ***p>0.001.
  • FIG. 12 Primary human CAFs isolated from head and neck tumors are sensitive to PDGFD-FD-PE24-H6 nanotoxin. Human CAFs were isolated from fresh tumor tissue surgically removed from head and neck squamous cell carcinoma patients.
  • A Isolated CAFs were exposed for 1 h to 50nM PDGFD-GFP-H6 nanoparticle. CAFs were analyzed using flow cytometry to quantify PDGFRp expression (PDGFRP+) and nanoparticle internalization (GFP+).
  • PDGFRP+ flow cytometry to quantify PDGFRp expression
  • GFP+ nanoparticle internalization
  • B CAFs sensitivity to PDGFD-FD-PE24-H6 was assessed by XTT upon cell exposure to 50nM PDGFD-FD-PE24-H6 for 48h. The viability of buffer- treated CAFs was considered to be 100 %. ***p>0.001.
  • FIG. 13 CAFs-targeted nanoparticles that integrate the C. diphteriae toxin domain are cytotoxic to PDGFRp-expressing fibroblasts.
  • A The GFP domain of the scaffold PDGFD-GFP-H6 monomer was replaced by a toxic domain from C. diphteriae, PDGFD-DITOX-H6. The monomers auto ensemble to form nanoparticles of 24.01 nm, assessed by DLS.
  • B Fibroblasts expressing PDGFRp (MSCs) were exposed to increase concentrations of PDGFD-DITOX-H6 for 48h. *p>0.05, **p>0.01.
  • FIG. 14 The monomethyl auristatin E (MMAE) nanoconjugate selectively eliminated PDGFRp-expressing fibroblasts.
  • MMAE monomethyl auristatin E
  • A PDGFD-GFP-H6 monomer was conjugated to MMAE, a potent microtubule inhibitor that triggers cell death. DLS confirms that once conjugated it self-assemble to form 43.7 nm nanoparticles.
  • B PDGFD-GFP- H6-MMAE nanoconjugate is able to induce cell death in MEFs and MSCs when exposed at 50nM for 72h. In both cases the use of the scaffold protein as a receptor competitor 1 h before nanoconjugate addition results in a strong inhibition of the fibroblasts death. **p>0.01 , ***p>0.001.
  • the inventors have developed a particular type of PDGFR-p-targeted nanoparticles based on biofabricated, self-assembling proteins, upon a hierarchical and iterative selective process starting from four initial candidates. These nanoparticles, produced by simple and fully scalable biofabrication processes, have been successfully validated as highly selective targeting agents, both in cell culture and in vivo, using multiple analytical procedures. The data fully supports the concept of selective drug carriers based on nanoscale protein materials to target the main architectonic agents in solid tumors for their further development and use in the clinical setting.
  • a first aspect of the present invention relates to a fusion protein comprising
  • PDGFR-p ligand selected from the group of consisting of PDGFB (SEQ ID N0: l) and PDGFD (SEQ ID NO: 2) or a functionally equivalent variant thereof,
  • fusion protein is well known in the art, referring to a single polypeptide chain artificially designed which comprises two or more sequences from different origins, natural and/or artificial.
  • the fusion protein, per definition, is never found in nature as such.
  • single polypeptide chain means that the polypeptide components of the fusion protein can be conjugated end-to-end but also may include one or more optional peptide or polypeptide ’’linkers” or “spacers” intercalated between them, linked by a covalent bond.
  • peptide or “polypeptide”, as used herein, generally refers to a linear chain of around 2 to 40 amino acid residues joined together with peptide bonds. It will be understood that the terms “peptide bond”, “peptide”, “polypeptide” and protein are known to the person skilled in the art. From here on, “peptide” and “polypeptide” will be used indistinctly.
  • amino acid residue refers to any naturally occurring amino acid, any amino acid derivative or any amino acid mimic known in the art.
  • residues of the protein or peptide are sequential, without any nonamino acid interrupting the sequence of amino acid residues.
  • the sequence may comprise one or more non-amino acid moieties.
  • sequence of residues of the protein or peptide may be interrupted by one or more non-amino acid moieties.
  • the fusion protein of the invention is devoid of disulfide bridges.
  • disulfide bridges refers to two cysteine residues which are adjacent in the three-dimensional structure of a protein and which can be oxidized to form a disulfide bond A.
  • the PDGFR-p ligand is selected from the group of consisting of PDGFB (SEQ ID NO: 1) and PDGFD (SEQ ID NO: 2) or a functionally equivalent variant thereof.
  • PDGFR-P refers to the protein “platelet-derived growth factor receptor beta” which is encoded by the PDGFRB gene in humans.
  • the PDGFRB gene encodes a typical receptor tyrosine kinase, which belongs to the type III tyrosine kinase receptor (RTK) family and is structurally characterized by five extracellular immunoglobulin-like domains, a single membrane-spanning helix domain, an intracellular juxtamembrane domain, a split tyrosine kinase domain and a carboxylic tail.
  • RTK tyrosine kinase receptor
  • PDGFR-p is essential for vascular development, and that PDGFB is responsible for activating PDGFR-p during embryogenesis.
  • PDGFR-p reduces the number of pericytes and vascular smooth muscle cells, and thereby compromises the integrity and/or functionality of the vasculature in multiple organs, including the brain, heart, kidney, skin and eye. Furthermore, PDGFR-p participates in cell differentiation, migration and proliferation processes in several neoplasias. PDGFR- P-expressing fibroblasts are positively involved in cancer progression and its infiltration in tumors compromises patient survival, especially in non-small cell lung, breast, pancreatic and colorectal cancer. Importantly, its expression levels in cancer-associated fibroblasts (CAFs) correlates with bad prognosis, relapse and drug resistance in several types of cancer.
  • CAFs cancer-associated fibroblasts
  • ligand denotes a bioactive molecule which binds to a cell-associated protein termed "receptor”. Both “receptor” and “ligand” are commonly known to those skilled in the art. Accordingly, a “PDGFR-p ligand” will be a molecule capable of binding to PDGFR-p.
  • functionally equivalent variants of the PDGFR-p ligands are those showing a degree of identity with respect to the PDGFB and/or the PDGFD peptides, according to their respective SEQ ID NOs, greater than 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%.
  • the degree of identity between two amino acid sequences can be determined by conventional methods, for example, by means of standard sequence alignment algorithms known in the state of the art, such as, for example BLAST (Altschul S.F. et al., J. Mol.
  • the PDGFR-p ligands of the invention may include post-translational modifications, such as glycosylation, acetylation, isoprenylation, myristoylation, proteolytic processing, etc.
  • the PDGFR-p ligand is PDGFB whose sequence consist of SEQ ID NO: 1.
  • the PDGFR-p ligand is PDGFD whose sequence consist of SEQ ID NO: 2.
  • suitable functional variants of the PDGFR-p ligands are those wherein one or more positions contain an amino acid which is a conservative substitution of the amino acid present in the PDGFB and/or the PDGFD peptides mentioned above.
  • "Conservative amino acid substitutions” result from replacing one amino acid with another having similar structural and/or chemical properties
  • the following six groups each contain amino acids that are conservative substitutions for 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).
  • a suitable assay for determining whether a given peptide can be seen as a functionally equivalent variant thereof is, for instance, the following assay: a putative PDGFB and/or the PDGFD peptide variant is fused in frame with a marker polypeptide (e.g. a fluorescent protein).
  • a marker polypeptide e.g. a fluorescent protein.
  • Such fusion proteins can be obtained by preparing a recombinant nucleic acid wherein the nucleic acids encoding the peptide and the fluorescent protein are fused in frame and expressed in an adequate host cell or organism. The fusion protein is then contacted with a culture of cells PDGFR-p (e.g.
  • the peptide is a functionally equivalent variant of the corresponding peptide, the marker protein will be internalized and presence of fluorescence in the cytoplasm of the cell will be visible. Furthermore, the performance of the functionally equivalent variant can be assayed by comparing the fluorescence microscopy image resulting from the fluorescent protein to that obtained with a known cytoplasmic stain (e.g. DAPI).
  • a known cytoplasmic stain e.g. DAPI
  • positively charged amino acid refers to a polypeptide sequence characterized in that it contains multiple positively charged amino acids.
  • the positively charged amino acid-rich region may be formed exclusively by positively charged amino acids or may contain other amino acids provided that the overall net charge of the region at pH 7 is positive.
  • the positively charged amino acid-rich region sequence may comprise 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 acids residues.
  • the positively charged amino acid-rich region may contain only one type of positively charged amino acid or may contain more than one type of positively charged amino acid.
  • the positively charged amino acid-rich region is a polyhistidine region.
  • the positively charged amino acid-rich region is a polyarginine region.
  • the positively charged amino acid-rich region comprises lysine and arginine residues.
  • the positively charged amino acid-rich region comprises lysine and histidine residues.
  • the positively charged amino acid-rich region comprises arginine and histidine residues.
  • the positively charged amino acid-rich region comprises lysine, arginine and histidine residues.
  • the positively charged amino acid-rich region comprises 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 acids residues, wherein the positively charged amino acid can be histidine, lysine, arginine or combinations thereof.
  • 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 less positively charged amino acids residues, wherein the positively charged amino acid can be histidine, lysine, arginine or combinations thereof.
  • the positively charged amino acid-rich region comprises between 2 and 50 amino acids, between 2 and 40 amino acids, between 2 and 30 amino acids, between 2 and 25 amino acids, between 2 and 20 amino acids, between 2 and 10 amino acids or between 2 and 8 amino acids.
  • the positively charged amino acid-rich region comprises between 3 and 50 amino acids, between 3 and 40 amino acids, between 3 and 30 amino acids, between 3 and 25 amino acids, between 3 and 20 amino acids, between 3 and 10 amino acids or between 3 and 8 amino acids. In some embodiments, the positively charged amino acid-rich region comprises between 4 and 50 amino acids, between 4 and 40 amino acids, between 4 and 30 amino acids, between 4 and 25 amino acids, between 4 and 20 amino acids, between 4 and 10 amino acids or between 4 and 8 amino acids. In some embodiments, the positively charged amino acid-rich region comprises between 5 and 50 amino acids, between 5 and 40 amino acids, between 5 and 30 amino acids, between 5 and 25 amino acids, between 5 and 20 amino acids, between 5 and 10 amino acids or between 5 and 8 amino acids.
  • the positively charged amino acid-rich region of the fusion protein of the invention is a polyhistidine region.
  • the polyhistidine region comprises between 2 and 10 contiguous histidine residues.
  • the positively charged acid-rich region is a polyhistidine region according to the sequence HHHHHH (S EQ ID NO: 3).
  • the positively charged amino acid-rich region of the fusion protein of the invention is a polyarginine region.
  • the polyarginine region comprises between 2 and 10 contiguous arginine residues.
  • the positively charged amino acid-rich region of the fusion protein of the invention is a polylysine region.
  • the polylysine region comprises between 2 and 10 contiguous polylysine residues.
  • the different elements of the fusion protein (PDGFR-p ligand, intervening polypeptide region, and positively charged amino acid-rich region) of the invention can be placed in any relative order provided that the PDGFR-p ligand and the positively charged amino acid-rich region are functional on any position of the fusion protein and also the intervening polypeptide region remains functional totally or in part.
  • the terms “N-terminal end”, “N-terminus”, and “amino-terminal end” of a polypeptide are indistinct.
  • the terms “C-terminal end”, “C-terminus”, and “carboxi-terminal end” are considered equivalent.
  • the terms are of common usage for the person skilled in the art regarding the free moieties of the amino acids at the ends of the polypeptide chains comprised by a protein.
  • the PDGFR-p ligand of the fusion protein is located at the N-terminal end of the protein, while the positively charged amino acidrich region of the fusion protein is located at the C-terminal end of the protein.
  • the positively charged amino acid-rich region of the fusion protein is located at the N-terminal end of the protein, while the PDGFR-p ligand of the fusion protein is located at the C-terminal end of the protein.
  • the intervening polypeptide region can be located at either the C-terminal end or the N-terminal end of the fusion protein, while the PDGFR-p ligand is in the middle position of the fusion protein and the positively charged amino acid-rich region is at the end of the fusion protein opposite the Intervening polypeptide region, or the positively charged amino acid-rich region is in the middle position of the fusion protein and the PDGFR-p ligand is located at the end of the fusion protein opposite the Intervening polypeptide region.
  • the relative order of the elements of the fusion protein according to the invention can be:
  • N-terminal end and C-terminal end do not mean that the components need to be directly conjugated end-to-end, but that they maintain that relative order of positions regardless of the presence of additional elements at the end of either component or intercalated between them, such as linkers/spacers.
  • the fusion protein of the invention comprises the aforementioned elements ((1) PDGFR-p ligand, (2) intervening polypeptide region, and (3) positively charged amino acid-rich region) and these can be conjugated end-to-end but also may include one or more optional peptide or polypeptide ’’linkers” or “spacers” intercalated between them, linked, preferably by peptidic bond.
  • the PDGFR-p ligand is connected to the intervening polypeptide via a first peptide linker and/or wherein the intervening polypeptide is connected to the positively charged amino acid-rich region via a second peptide linker.
  • the spacer or linker amino acid sequences can act as a hinge region between components (1) and (2) and (2) and (3), allowing them to move independently from one another while maintaining the three-dimensional form of the individual domains, such that the presence of peptide spacers or linkers does not alter the functionality of any of the components (1), (2) and (3).
  • a preferred intermediate amino acid sequence according to the invention would be a hinge region characterized by a structural ductility allowing this movement.
  • said intermediate amino acid sequence is a flexible linker. The effect of the linker region is to provide space between the components (1) and (2) and (2) and (3). It is thus assured that the secondary and tertiary structure of component (1), (2) or (3) is not affected by the presence of either of the others.
  • the spacer is of a polypeptide nature.
  • 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 approximately 100 amino acids.
  • the spacer or linker can be bound to components flanking the two components of the conjugates of the invention by means of covalent bonds, preferably by peptide bonds; and also preferably the spacer is essentially afunctional, and/or is not prone to proteolytic cleavage, and/or does not comprise any cysteine residue.
  • the three- dimensional structure of the spacer is preferably linear or substantially linear.
  • spacer or linker peptides include those that have been used to bind proteins without substantially deteriorating the function of the bound peptides or at least without substantially deteriorating the function of one of the bound peptides. More preferably the spacers or linkers used to bind peptides comprise coiled coil structures.
  • linker peptides comprise 2 or more amino acids selected from the group consisting of glycine, serine, alanine and threonine.
  • a preferred example of a flexible linker is a polyglycine linker.
  • the possible examples of linker/spacer sequences include GGSSGGS (SEQ ID NO: 4), SGGTSGSTSGTGST (SEQ ID NO : 5), AGSSTGSSTGPGSTT (SEQ ID NO: 6) or GGSGGAP (SEQ ID NO: 7). These sequences have been used for binding designed coiled coils to other protein domains [Muller, K.M., Arndt, K.M. and Alber, T., Meth.
  • linkers comprise the amino acid sequence GGGVEGGG (SEQ ID NO: 8), the sequence of 10 amino acid residues of the upper hinge region of murine lgG3 (PKPSTPPGSS, SEQ ID NO : 9), which has been used for the production of dimerized antibodies by means of a coiled coil (Pack, P. and Pluckthun, A., 1992, Biochemistry 31 :1579-1584), the peptide of sequence APAETKAEPMT (SEQ ID NO: 10), the peptide of sequence GAP, the peptide of sequence AAA and the peptide of sequence AAALE (SEQ ID NO: 11).
  • the components of the conjugates of the invention can be connected by peptides the sequence of which contains a cleavage target for a protease, thus allowing the separation of any of the components.
  • Protease cleavage sites suitable for their incorporation into the polypeptides of the invention include enterokinase (cleavage site DDDDK, SEQ ID NO: 12), factor Xa (cleavage site IEDGR, SEQ ID NO: 13), thrombin (cleavage site LVPRGS, SEQ ID NO: 14), TEV protease (cleavage site ENLYFQG, SEQ ID NO: 15), PreScission protease (cleavage site LEVLFQGP, SEQ ID NO: 16), inteins and the like.
  • the first peptide linker and/or the second peptide linker comprise a cleavable target site.
  • the PDGFR-p ligand is bound to the intervening polypeptide region through a first linker and the intervening polypeptide region is bound to the positively charged amino acid-rich region through a second linker also.
  • the linkers connecting the PDGFR-p ligand to the intervening polypeptide region and the intervening polypeptide region to the positively charged amino acid-rich region may comprise the same sequence or different ones with the aforementioned limitation that the presence and/or sequence of the linkers does not result in functional alterations of the PDGFR-p ligand, the intervening polypeptide region, and/or the positively charged amino acid-rich region (for instance, but not limited to, due to secondary or tertiary structure modifications of the fusion protein or formation of disulfide bonds).
  • the first linker and/or the second linker of the fusion protein of the invention comprise the sequence GGSSGGS (S EQ ID NO: 4).
  • intervening polypeptide region and “intervening region” are herein considered equivalent.
  • the intervening polypeptide region of the fusion proteins of the invention comprises a physiologically functional peptide, meaning that its interaction with the cellular components results in physiological changes.
  • the intervening polypeptide region does not need to be physiologically functional once it is incorporated into the fusion protein of the invention. Accordingly, linker regions connecting the different elements of the fusion protein according to the invention are not considered intervening regions.
  • 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.
  • the intervening polypeptide region is a physiologically functional peptide or a fragment or a mutant thereof with a reduced physiological function once it is incorporated into the fusion protein of the invention. In another embodiment, the intervening polypeptide region does not have any physiological function once incorporated into the fusion protein of the invention. In another preferred embodiment, the intervening polypeptide region is a fragment or a mutant of a physiologically functional polypeptide with an already reduced physiological function, as compared to the wild-type physiologically functional polypeptide before being incorporated into the fusion protein of the invention. More preferably, the intervening polypeptide region is a protein which does not have any physiological function already when not forming part of the fusion protein of the invention, due to the presence of inactivating mutations.
  • the intervening polypeptide of the fusion protein of the invention is a therapeutic agent or an imaging agent.
  • therapeutic as used herein in relation to the therapeutic agents, is used in a generic sense and includes treating agents, prophylactic agents, and replacement agents.
  • the nature of the intervening region is substantially polypeptidic, except where the therapeutic agent is concerned. It is intended that the therapeutic agent conjugated to the fusion protein is not limited in its chemical structure.
  • the intervening region of the fusion protein is selected from the group consisting of a fluorescent protein, albumin, nidogens, chorionic gonadotropin, and a cystatin.
  • Fluorescent protein relates to proteins whose atomic structure allows them to present fluorescence, which is a phenomenon well-known in the art.
  • Nonlimiting examples of commonly used fluorescent proteins suitable for the fusion protein of the invention are the green fluorescent protein (GFP, first discovered in Aequorea victoria), the red fluorescent protein (RFP), the yellow fluorescent protein (YFP), the blue fluorescent protein (BFP), the cyan fluorescent protein, 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],
  • fluorescent proteins suitable for the fusion protein of the invention are the enhanced green fluorescent protein (eGFP), enhanced cyan fluorescent protein CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilised 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), mRFPI, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, Phycobiliproteins and Phycobiliprotein conjugates including B- Phycoerythrin, R-Phycobiliprotein conjug
  • the intervening polypeptide is a fluorescent protein selected from the group consisting of the mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), and the like.
  • the fluorescent protein of the intervening region of the fusion protein of the invention is GFP (SEQ ID NO: 17).
  • albumin refers to the water-soluble, glycosylated globular proteins commonly found in the plasma of animals, especially mammals.
  • the protein of the intervening region of the fusion protein is albumin, more preferably human albumin (SEQ ID NO: 18).
  • Nedogen relates to any protein of the family of nidogens, formerly known as entactins, which are sulfated monomeric glycoproteins located in the basal lamina.
  • the protein of the intervening region of the fusion protein is selected from the group consisting of the human nidogen 1 (with identification number P14543-1 of the Uniprot Database (version dated July 7, 2009), SEQ ID NO: 19) and the human nidogen 2 (NID2, SEQ ID NO: 20).
  • the protein of the intervening region of the fusion protein of the invention is the nidogen-1 G2 domain.
  • G2 domain of nidogen-1 refers to the domain G2 of the protein nidogen 1 as defined above.
  • the nidogen-1 G2 domain is as shown in SEQ ID NO : 33 which corresponds to amino acid numbers 430 and 667 of the amino acid sequence of the nidogen-1 protein, with identification number P14543-1 of the Uniprot Database (version dated July 7, 2009) (SEQ ID NO: 19).
  • the domain G2 of nidogen 1 is as shown in SEQ ID NO: 34, which lacks the first two amino acids of SEQ ID NO: 33, and thus, corresponds to a region consisting on amino acid numbers 432 and 667 of the amino acid sequence of the nidogen-1 protein precursor with identification number P14543-1 of the Uniprot Database (version dated July 7, 2009) (SEQ ID NO: 19).
  • the G2 domain is flanked by short EGF-like domains.
  • the nidogen-1 G2 domain lacks EGF- like domains at the N-or at the C-terminus.
  • the protein of the intervening region of the fusion protein of the invention is a variant of the nidogen-1 G2 domain.
  • the variant of the nidogen-1 G2 domain contains the H459A and the R468N mutations.
  • the variant of the nidogen-1 G2 domain contains the H459A and the F639S mutations.
  • the variant of the nidogen-1 G2 domain contains the H459A and the R650A mutations.
  • the variant of the nidogen-1 G2 domain contains the R468N and the F639S mutations.
  • the variant of the nidogen-1 G2 domain contains the R468N and the R650A mutations. In one embodiment, the variant of the nidogen-1 G2 domain contains the H459A, the R468N, and the F639S mutations. In one embodiment, the variant of the nidogen-1 G2 domain contains the H459A, the R468N, and the R650A mutations. In one embodiment, the variant of the nidogen-1 G2 domain contains the R468N, the F639S and the R650A mutations. In one embodiment, the variant of the nidogen-1 G2 domain contains the H459A, the R468N, the F639S and the R650A mutations. In a preferred embodiment, the of the nidogen-1 G2 domain variant has a sequence as defined in SEQ ID NO: 34 or 35 (hereinafter referred to as NIDOmut2).
  • the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant as defined in any of the embodiments above and, in particular, the nidogen-1 G2 domain variant having the H459A, the R468N, the F639S and the R650A mutations which, in addition, comprises a mutation at a position selected from the group consisting of 543 (corresponding to histidine at position 114 in SEQ ID NO: 33) and position 545 (corresponding to histidine at position 116 in SEQ ID NO: 33).
  • position H543 is mutated to Lys.
  • position H545 is mutated to Asn.
  • the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain that comprises the H459A, the R468N, the F639S, the R650A and the H543K mutations.
  • the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain that comprises the H459A, the R468N, the F639S, the R650A and the H545N mutations.
  • the nidogen-1 G2 domain variant comprises a H543K mutation and a H545N mutation.
  • the nidogen-1 G2 domain variant comprises or consists of SEQ ID NO : 36 (hereinafter referred to NIDOmut3), which is characterized in that it contains the H459A, the R468N, the F639S, the R650A, the H543K and the H545N mutations.
  • the protein of the intervening region of the fusion protein of the invention is a variant of the nidogen-1 G2 domain as defined in any of the embodiments above and, in particular, the NIDOmut3 variant, which, in addition comprises a mutation selected from the group consisting of: a mutation at valine at position 449 (corresponding to position 20 in SEQ ID NO: 33).
  • the valine at position 449 is mutated to Thr.
  • the nidogen-1 G2 domain variant has a sequence as defined in SEQ ID NO: 37 (hereinafter referred to as NIDOmut3-V45T). a mutation at valine at position 525 (corresponding to position 96 in SEQ ID NO: 33).
  • the valine at position 449 is mutated to Gin.
  • the nidogen-1 G2 domain variant has a sequence as defined in SEQ ID NO : 38 (hereinafter referred to as NIDOmut3- V121Q). a mutation at the phenylalanine at position 561 (corresponding to position 142 in SEQ ID NO: 33).
  • the phenylalanine at position 561 is mutated to Glutamic acid.
  • the nidogen-1 G2 domain variant has a sequence as defined in SEQ ID NO: 39 (hereinafter referred to as NIDOmut3-F157E).
  • the valine at position 619 is mutated to threonine.
  • the nidogen-1 G2 domain variant has a sequence as defined in of SEQ ID NO: 40 (hereinafter referred to as NIDOmut3-V215T).
  • the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain as defined in any of the embodiments above, and in addition comprises the V449T, the V525Q, the F561 E and the V619T mutations.
  • the protein of the intervening region of the fusion protein of the invention is a variant nidogen G2 domain that comprises the H459A, the R468N, the F639S, the R650A, the H543K, the V449T, the V525Q, the F561 E and the V619T mutations.
  • the protein of the intervening region of the fusion protein of the invention is a variant nidogen G2 domain that comprises the H459A, the R468N, the F639S, the R650A, the V449T, the H545N, the V525Q, the F561 E and the V619T mutations.
  • the protein of the intervening region of the fusion protein of the invention is a variant nidogen G2 domain that comprises the H459A, the R468N, the F639S, the R650A, the H543K, the H545N, the V449T, the V525Q, the F561 E and the V619T mutations.
  • the variant nidogen G2 domain comprises or consists of the sequence as defined in SEQ ID NO: 42 (hereinafter referred to as NIDOmut4).
  • the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain as defined in any of the embodiments above and, in particular the NIDOmut4), which in addition comprises a mutation at the threonine at position 619 (corresponding to position 190 in SEQ ID NO: 33).
  • the threonine at position 619 is mutated to valine.
  • the protein of the intervening region of the fusion protein of the invention is a variant nidogen G2 domain as defined in any of the embodiments above in which the amino acid at position 619 (corresponding to position 190 in SEQ ID NO: 33) is the same residue that appear in the human nidogen G2 domain as defined in the UniProt database under accession number P14534), i.e. a Valine.
  • the protein of the intervening region of the fusion protein of the invention is a nidogen G2 domain variant having the H459A, the R468N, the F639S, the R650A, the H543K, the H545N, the V449T, the V525Q and the F561 E mutations.
  • the protein of the intervening region of the fusion protein of the invention is a nidogen G2 domain variant having the sequence of SEQ ID NO: 43 (hereinafter referred to as NIDOmut4_T215V).
  • the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain as defined in any of the embodiments above and, in particular, the NIDOMut4, which, in addition comprises a mutation at the cysteine at position 618 (corresponding to position 189 in SEQ ID NO : 33).
  • the cysteine at position 618 is mutated to serine.
  • the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the H459A, the R468N, the F639S, the R650A, the H543K, the H545N, the V449T, the V525Q, the V619T, the F561 E and the C618S mutations.
  • the protein of the intervening region of the fusion protein of the invention is a nidogen G2 domain variant having the sequence of SEQ ID NO: 44 (hereinafter referred to as NIDOmut5).
  • the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain as defined in any of the embodiments above and, in particular the NIDOMut3 variant, which in addition comprises a mutation selected from the group consisting of: a mutation at valine at position 580 (corresponding to position 151 in SEQ ID NO: 33).
  • the valine at position 580 is mutated to Thr.
  • the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the sequence of SEQ ID NO: 45 (hereinafter referred to as NIDOmut3-V176T).
  • a mutation at isoleucine at position 604 (corresponding to position 175 in SEQ ID NO: 33).
  • the isoleucine at position 604 is mutated to Thr.
  • the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the sequence of SEQ ID NO: 46 (hereinafter referred to as NIDOmut3-l200T).
  • NIDOmut3-l200T nidogen-1 G2 domain variant having the sequence of SEQ ID NO: 46 (hereinafter referred to as NIDOmut3-l200T).
  • a mutation at the valine at position 638 (corresponding to position 209 in SEQ ID NO: 33).
  • the valine at position 638 is mutated to tyrosine.
  • the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the sequence of SEQ ID NO: 47 (hereinafter referred to as NIDOmut3-V236Y). a mutation at the leucine at position 641 (corresponding to position 212 in SEQ ID NO: 33). Preferably, the leucine at position 641 is mutated to threonine.
  • the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the sequence of SEQ ID NO: 48 (hereinafter referred to as NIDOmut3-L237T).
  • the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the sequence of SEQ ID NO: 49 (hereinafter referred to as NIDOmut3-S65l).
  • NIDOmut3-S65l nidogen-1 G2 domain variant having the sequence of SEQ ID NO: 49
  • a mutation at arginine at position 518 (corresponding to position 89 in SEQ ID NO: 33).
  • the arginine at position 518 is mutated to lie.
  • the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the sequence of SEQ ID NO: 50 (hereinafter referred to as NIDOmut3-R1141). a mutation at the cysteine at position 618 (corresponding to position 189 in SEQ ID NO: 33). Preferably, the cysteine at position 618 is mutated to serine. Accordingly, in one embodiment, the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the sequence of SEQ ID NO: 51 (hereinafter referred to as NIDOmut3-C214S).
  • the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain as defined in any of the embodiments above and, in particular the NIDOmut3 variant, which, in addition, contains mutations at the 469 (preferably a S469I mutation) and at the 518 position (preferably a R518I mutation).
  • the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the H459A, the R468N, the F639S, the R650A, the H543K, the H545N, the S469I and the R518I mutations and corresponds to the sequence of SEQ ID NO: 52 (hereinafter referred to as NIDOmut3-S65l_R114l).
  • the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain as defined in any of the embodiments above and, in particular the NIDOmut5 variant, which, in addition, contains mutations at the 469 (preferably a S469I mutation) and at the 518 position (preferably a R518I mutation).
  • the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the H459A, the R468N, the F639S, the R650A, the H543K, the H545N, the V449T, the V525Q, the V619T, the F561 E, the S469I and the R518I mutations, as defined in SEQ ID NO: 53 (hereinafter referred to as NIDOmut5-S65l_R1141).
  • the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain as defined in any of the embodiments above and, in particular the NIDOmut5 variant, which, in addition, contains a mutation at serine at position 469 (corresponding to position 40 in SEQ ID NO: 33).
  • the serine at position 469 is mutated to lie.
  • the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the H459A, the R468N, the F639S, the R650A, the H543K, the H545N, the V449T, the V525Q, the V619T, the F561 E and the S469I mutations.
  • the protein of the intervening region of the fusion protein of the invention is a nidogen G2 domain variant having the sequence of SEQ ID NO: 54 (hereinafter referred to as NIDOmut5-S65l).
  • the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain as defined in any of the embodiments above and, in particular the NIDOmut5 variant, which, in addition, contains a mutation at arginine at position 518 (corresponding to position 89 in SEQ ID NO: 33).
  • the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the H459A, the R468N, the F639S, the R650A, the H543K, the H545N, the V449T, the V525Q, the V619T, the F561 E and the R518I mutations.
  • the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the sequence of SEQ ID NO: 55 (hereinafter referred to as NIDOmut5-R114l).
  • nidogen-1 G2 domain variants suitable for use in the present invention are summarized in the Table 1 below.
  • GC Choionic gonadotropin
  • Human chorionic gonadotropin presents two subunits, alpha (a) and beta (P). It is intended that any one of the two subunits separately and both together are suitable for the purposes of the invention.
  • the protein of the intervening region of the fusion protein is the chorionic gonadotropin.
  • the protein of the intervening region of the fusion protein is the human chorionic gonadotropin (hGC, SEQ ID NO: 21).
  • cystatin refers to a member of a family of protease inhibitors known as cystatins which are capable of inhibiting the activity of peptidase enzymes belonging to peptidase families C1 (papain family) and C13 (legumain family).
  • the cystatin is selected from the group consisting of cystatin A, cystatin B, cystatin C, cystatin D and cystatin M.
  • the cystatin is a cystatin A, also known as Stefin A.
  • Stefin A is of human origin having the sequence SEQ ID NO : 22.
  • the cystatin is a stefin A variant having one or more mutations selected from the group consisting of the G4W, the G4R, the V48D, the V48L, the G50S, the K71 N, the S72G, the L73P, the L82R, the T83S mutations.
  • the stefin A variant contains the following mutations with respect to the sequence shown in SEQ ID NO: 22:
  • G4R corresponding to the mutant defined as SUN mutant in Hoffman et al., supra).
  • suitable polypeptides and proteins that can be used as components of the intervening region include any polypeptide or protein without any physiological or biological activity on their own, as well as any biologically non-reactive peptide or protein.
  • the protein of the intervening region of the fusion protein is an inert protein.
  • inert protein refers to polypeptides or proteins or fragments or domains of proteins without known physiological or biological activity, or without the ability to specifically interact with other macromolecules for a biological function, and fragments or domains of proteins devoid of known therapeutic activity (e.g. antitumor activity).
  • the inert protein that is part of the fusion protein is non-reactive and functions as a physical structure for the binding of the therapeutic agents. It is intended that the inert proteins do not comprise any motifs that have intrinsic enzymatic, physiological, or biological activity on their own, nor do they present immune reactivity, meaning that they stimulate neither the adaptive, nor the innate immune responses.
  • any intrinsic activity of said protein is irrelevant for the purposes of the invention and does neither contribute, nor hinder the biological activity of the therapeutic agent.
  • the intervening polypeptide of the fusion proteins of the invention is a fragment of any of the proteins described in any of the embodiments of this section D.
  • the intervening polypeptide of the fusion protein of the invention is a mutant of any of the proteins described in any of the embodiments of this section D.
  • the intervening polypeptide of the fusion protein of the invention is a biologically active polypeptide.
  • biologically active polypeptide refers to a polypeptide that affects some parameter of biological systems or chemical reactions, for example by altering growth of an organism, by affecting association of molecules and/or cells and by slowing down or accelerating chemical reactions.
  • biologically active polypeptide also encompasses biologically active fragments thereof, as well as biologically active sequence analogues thereof.
  • the intervening polypeptide of the fusion protein of the invention is a biologically active polypeptide, preferably a therapeutic agent.
  • therapeutic agent is drawn to any compound, without chemical structure limitations, suitable for therapy and/or treatment of a condition, disorder or disease.
  • the nature of the therapeutic agent is not particularly limiting for the present invention provided it remains active in the fusion protein or can be activated once it is delivered to the inside of the cell. Accordingly, any therapeutic agent can be used in the fusion protein provided that it shows an activity or can reach an activity once it is delivered to the inside of the cell of at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50% or less of the activity of the unconjugated therapeutic agent.
  • the purpose of the invention is to facilitate the action of the therapeutic agent by increasing its selectivity and reducing its off-target effects, it is contemplated that the effects of the therapeutic agent conjugated to the fusion protein may be synergic and exceed the parametrized values already known for the specific therapeutic agent.
  • some embodiments of the therapeutic agent conjugated to the fusion protein of the invention also show at least 101 %, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 175%, at least 200%, at least 300%, at least 400%, at least 500%, at least 1000%, or more of the functionality of the therapeutic agent alone.
  • the intervening region of the fusion protein of the invention is a therapeutic agent selected from the group consisting of
  • cytotoxic polypeptide refers to an agent that is capable of inhibiting cell function.
  • the agent may inhibit proliferation or may be toxic to cells.
  • Any polypeptides that when internalized by a cell interfere with or detrimentally alter cellular metabolism or in any manner inhibit cell growth or proliferation are included within the ambit of this term, including, but not limited to, agents whose toxic effects are mediated when transported into the cell and also those whose toxic effects are mediated at the cell surface.
  • Useful cytotoxic polypeptides include proteinaceous toxins such as bacterial toxins.
  • proteinaceous cell toxins useful for incorporation into the conjugates according to the invention include, but are not limited to, type one and type two ribosome inactivating proteins (RIP).
  • Useful type one plant RIPs include, but are not limited to, dianthin 30, dianthin 32, lychnin, saporins 1-9, pokeweed activated protein (PAP), PAP II, PAP-R, PAP-S, PAP-C, mapalmin, dodecandrin, bryodin-L, bryodin, Colicin 1 and 2, luffin-A, luffin-B, luffin-S, 19K-protein synthesis inhibitory protein (PSI), 15K-PSI, 9K-PSI, alpha-kirilowin, beta-kirilowin, gelonin, momordin, momordin-ll, momordin-lc, MAP-30, alpha-momorcharin, beta-momorcharin, trichosanthin, TAP-29, tri
  • Useful type two RIPs include, but are not limited to, volkensin, ricin, nigrin-b, CIP- 29, abrin, modeccin, ebulitin-[alpha], ebulitin-[beta], ebultin-[gamma], vircumin, porrectin, as well as the 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).
  • bacterial toxins useful as cell toxins include, but are not limited to, shiga toxin and shiga-like toxins (i.e. , toxins that have the same activity or structure), as well as the catalytic subunits and biologically functional fragments thereof. These bacterial toxins are also type two RIPs (Sandvig and Van Deurs, 1996. Physiol. Rev. 76:949-66; Armstrong, 1995. J. Infect. Dis., 171 :1042-5; Kim et al., 1997. Microbiol. Immunol. 41 :805-8; and Skinner et al., 1998. Microb. Pathog. 24:117-22).
  • 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 enzymatic subunits also can be used as a cell toxin moiety (Pastan et al., Annu. Rev. Biochem. 61 :331-54; Brinkmann and Pastan, Biochim. et Biophys.
  • Targets include, but are not limited to the more than 34 described Colicin family of RNase toxins which include colicins A, B, D, E1-9, cloacin DF13 and the fungal RNase, [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).
  • anti-angiogenic polypeptide denotes a polypeptide capable of inhibiting angiogenesis.
  • Suitable antiangiogenic polypeptides include, without limitation, angiostatin, endostatin, anti-angiogenic anti-thrombin III, sFRP-4 as described in W02007115376, and an anti-VEGF antibody such as anibizumab, bevacizumab (avastin), Fab IMG 1121 and F200 Fab.
  • tumor suppressor is a gene or gene product that has a normal biological role of restraining unregulated growth of a cell.
  • the functional counterpart to a tumor suppressor is an oncogene — genes that promote normal cell growth may be known as “proto-oncogenes”
  • proto-oncogenes A mutation that activates such a gene or gene product further converts it to an "oncogene", which continues the cell growth activity, but in a dysregulated manner
  • tumor suppressor genes and gene products are well known in the literature and may include PTC, BRCA1 , BRCA2, p16, APC, RB, WTI, EXTI, p53, NFI, TSC2, NF2, VHL.ST7, ST14, PTEN, APC, CD95 or SPARC.
  • pro-apoptotic polypeptides refers to a protein which is capable of inducing cell death in a cell or cell population. The overexpression of these proteins involved in apoptosis displaces the careful balance between anti-apoptotic and pro-apoptotic factors towards an apoptotic outcome.
  • Suitable pro-apoptotic polypeptides include, without limitation, 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 homologs, 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-R1 and TRAIL-R2.
  • 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,
  • metastasis suppressor refers to a protein that acts to slow or prevent metastases (secondary tumors) from spreading in the body of an organism with cancer.
  • Suitable metastasis suppressor include, without limitation, proteins such as BRMS I, CRSP3, DRGI, KA11 , KISS-I, NM23, a TIMP-family protein and uteroglobin.
  • a “polypeptide encoded by a suicide gene” refers to a polypeptide the expression of which results in cell expressing it killing itself through apoptosis. This approach comprises the selective expression of the suicide gene only in particular cells, though the use of specific promoters, for instance, that would activate only in cells actually suffering the disease to be suppressed.
  • This approach comprises the use of pairs of enzyme and pro-drug, in which the enzyme is used to transform the target cells previously to the administration of the prodrug, which under the action of the enzyme becomes a product toxic for the cell that kickstarts the apoptotic process.
  • the enzymes of these systems of suicide gene therapy are usually not found in the same organism in which they are intended to be expressed, and so in mammals have been used enzymes obtained from bacteria, fungi or other organisms.
  • This strategy has several known examples [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, carboxypeptidase G2/Nitrogen mustard system, cytochrome P450/oxazaphosphorine system, purine nucleoside phosphorylase/6- methylpurine deoxyriboside (PNP/MEP), the horseradish peroxidase/indole-3-acetic acid system (HRP/IAA), and the carboxylesterase/irinotecan (CE/irinotecan) system, the truncated EGFR, inducible caspase ("iCasp”), the the E. coli gpt gene, the E. coli Deo gene and nitroreductase.
  • iCasp inducible caspase
  • chaperone polypeptide refers to a protein molecule that assists in folding or unfolding of protein molecules and/or assembly or disassembly of macromolecular structures.
  • Exemplary chaperones include, but are not limited to, ABCE1 ATP-binding cassette sub-family E member 1 ; AHSA1 Activator of 90 kDa heat shock protein ATPase homolog 1 ; ANP32B acidic leucine-rich nuclear phosphoprotein 32 family; BAG6 Large proline-rich protein BAG6; BCS1 L mitochondrial chaperone BCS1 ; CALR calreticulin; CANX calnexin; CCT2 T-complex protein 1 subunit beta CCT3 T-complex protein 1 subunit gamma CCT4 T-complex protein 1 subunit delta CCT5 T- complex protein 1 subunit epsilon CCT6A T-complex protein 1 subunit zbeta CCT7 T- complex protein 1 sub
  • an immunostimulatory polypeptide agent is a polypeptide encoded by a polynucleotide which is capable of activating or stimulating the immune response (including enhancing a pre-existing immune response) in a subject to whom it is administered, whether alone or in combination with another agent.
  • 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), and the like.
  • 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
  • the therapeutic agent may also be conjugated to the fusion protein of the invention. It is intended that the therapeutic agent, as aforementioned, is conjugated to the intervening region of the fusion protein without limitation of the position of the conjugation inside the intervening region with regards to the N-terminal and C-terminal ends. Accordingly, the therapeutic agent can be conjugated to the intervening polypeptide region in an equidistant position with respect to the N-terminal and C-terminal ends or it can be closer to either of them.
  • the therapeutic agent can be conjugated to the intervening polypeptide region at a distance of 100, 90, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 30, 25, 20, 15, 20, 10, or less amino acid residues from the N-terminal or C-terminal end, or at the same residue of the N-terminal or C-terminal end.
  • the therapeutic agent and the elements of the fusion protein are functional and the conjugation of the therapeutic agent does not interfere with the activity of either therapeutic agent or the fusion protein.
  • the therapeutic agent, the PDGFR-p ligand, and the positively charged amino acid-rich region conserve at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, preferably 95%, more preferably 99%, even more preferably 100% of their functionality with respect to the non-conjugated forms of the fusion protein and the therapeutic agent respectively.
  • the intervening polypeptide region of the fusion protein of the invention is conjugated to at least one therapeutic agent.
  • the intervening polypeptide region of the fusion protein of the invention is conjugated to a plurality of therapeutic agents, wherein said plurality of therapeutic agents are the same or different.
  • chemotherapeutic agents refers to anti-cancer agents.
  • an anti-cancer agent is an agent that at least partially inhibits the development or progression of a cancer, including inhibiting in whole or in part symptoms associated with the cancer even if only for the short term.
  • the therapy agent is a chemotherapy agent.
  • anti-cancer agents can be categorized as DNA damaging agents and these include topoisomerase inhibitors (e.g., etoposide, ramptothecin, topotecan, teniposide, mitoxantrone), DNA alkylating agents (e.g., cisplatin, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chorambucil, busulfan, thiotepa, carmustine, lomustine, carboplatin, dacarbazine, procarbazine), DNA strand break inducing agents (e.g., bleomycin, doxorubicin, daunorubicin, idarubicin, mitomycin C), anti-microtubule agents (e.g., vincristine, vinblastine), anti-metabolic agents (e.g., cytarabine, methotrexate, hydroxyurea, 5-fluorouracil,
  • anti-cancer agents include without limitation Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Axaitinib; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Bortezomib (VELCADE); Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calciprotiol; Calusterone; Caracemide; Carbetimer; Carboplatin (a platinum- containing regimen); Carmustine; Carubicin Hydroch
  • the anti-cancer agent is provided as an oligomer containing several units of the anti-cancer molecule.
  • the anti-cancer agent is a floxuridin poly- or oligonucleotide, which comprises several floxuridine molecules.
  • the floxuridine poly- or poligonucleotide contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more floxuridine molecules.
  • the floxuridine polynucleotide is a floxuridine pentanucleotide, i.e. a oligonucleotide containing 5 floxuridine molecules.
  • the anti-cancer agent is Monomethyl auristatin E.
  • the anti-cancer agent may be an enzyme inhibitor including without limitation tyrosine kinase inhibitor, a CDK inhibitor, a MAP kinase inhibitor, or an EGFR inhibitor.
  • the tyrosine kinase inhibitor may be without limitation Genistein (4', 5, 7- trihydroxyisoflavone), Tyrphostin 25 (3,4,5-trihydroxyphenyl), methylene]- propanedinitrile, Herbimycin A, Daidzein (4',7-dihydroxyisoflavone), AG-126, trans-1- (3'- carboxy-4'-hydroxyphenyl)-2-(2",5"-dihydroxy-phenyl)ethane, or HDBA (2- Hydroxy5- (2,5-Dihydroxybenzylamino)-2-hydroxybenzoic acid.
  • the CDK inhibitor may be without limitation p21 , p27, p57, pl5, pl6, pl8, or pl9.
  • the MAP kinase inhibitor may be without limitation KY12420 (C23H24O8), CNI-1493, PD98059, or 4-(4- Fluorophenyl)-2-(4- methylsulfinyl phenyl)-5-(4-pyridyl) IH-imidazole.
  • the EGFR inhibitor may be without limitation erlotinib (TARCEVA), gefitinib (IRESSA), WHI- P97 (quinazoline derivative), LFM-A12 (leflunomide metabolite analog), ABX-EGF, lapatinib, canertinib, ZD-6474 (ZACTIMA), AEE788, and AG1458.
  • the anti-cancer agent may be a VEGF inhibitor including without limitation bevacizumab (AVASTIN), ranibizumab (LUCENTIS), pegaptanib (MACUGEN), sorafenib, sunitinib (SUTENT), vatalanib, ZD-6474 (ZACTIMA), anecortave (RETAANE), squalamine lactate, and semaphorin.
  • AVASTIN bevacizumab
  • ranibizumab LCENTIS
  • MACUGEN pegaptanib
  • sorafenib sunitinib
  • SUTENT sunitinib
  • ZACTIMA ZACTIMA
  • anecortave squalamine lactate
  • semaphorin semaphorin
  • the anti-cancer agent may be an antibody or an antibody fragment including without limitation an antibody or an antibody fragment including but not limited to bevacizumab (AVASTIN), trastuzumab (HERCEPTIN), alemtuzumab (CAMPATH, indicated for B cell chronic lymphocytic leukemia,), gemtuzumab (MYLOTARG, hP67.6, anti-CD33, indicated for leukemia such as acute myeloid leukemia), rituximab (RITUXAN), tositumomab (BEXXAR, anti-CD20, indicated for B cell malignancy), MDX-210 (bispecific antibody that binds simultaneously to HER- 2/neu oncogene protein product and type I Fc receptors for immunoglobulin G (IgG) (Fc gamma Rl)), oregovomab (OVAREX, indicated for ovarian cancer), edrecolomab (PANOREX), daclizumab (ZENAPAX
  • a protein that acts as an angiogenesis inhibitor is targeted to a tumor.
  • agents include, in addition to the anti-angiogenic polypeptides mentioned above, Marimastat; AG3340; COL-3, BMS- 275291 , Thalidomide, Endostatin, SLI5416, SLI6668, EMD121974, 2-methoxyoestradiol, carboxiamidotriazole, CMIOI, pentosan polysulphate, angiopoietin 2 (Regeneron), herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM- 1470, platelet factor 4 or minocycline.
  • DNA cleaving agents suitable for inclusion as the cell toxin in the conjugates used in practicing the methods include, but are not limited to, anthraquinone-oligopyrrol-carboxamide, benzimidazole, leinamycin; dynemycin A; enediyne; as well as biologically active analogs or derivatives thereof (i.e., those having a substantially equivalent biological activity).
  • DNA cleaving agents suitable for inclusion as the cell toxin in the conjugates used in practicing the methods include, but are not limited to, anthraquinone-oligopyrrol-carboxamide, benzimidazole, leinamycin; dynemycin A; enediyne; as well as biologically active analogs or derivatives thereof (i.e., those having a substantially equivalent biological activity).
  • Known analogs and derivatives are disclosed, for examples in Islam et al., J. Med. Chem. 34 2954-61 , 1991 ; Skibo et al
  • the chemotherapy agent is an antimetabolite.
  • Antimetabolite refers to the compounds which inhibit the use of a metabolite that is part of normal metabolism. Antimetabolites are often similar in structure to the metabolite that they interfere with, such as the antifolates that interfere with the use of folic acid.
  • Non-limiting examples of antimetabolites include the following compounds: bleomycin, busulfan, capecitabine, carmustine, carboplatin, chlorodeoxyadenosine, cisplatin, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxorubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate mitomycin, mitoxantrone, oxaliplatin, paclitaxel, procarbazine, SN-38, thioguanine, thiotepa, teniposide, vinblastine, vincristine, and vinorelbine.
  • the antimetabolite is a pyrimidine analogue or an oligomeric form thereof.
  • pyrimidine refers to nitrogenous monocyclic heterocycles.
  • pyrimidine analog refers to a compound that has the same basic chemical structure as a naturally occurring pyrimidine.
  • the intervening region of the fusion protein of the invention corresponds to a protein that acts as an angiogenesis inhibitor which is targeted to a tumor.
  • agents include, Marimastat; AG3340; COL- 3, BMS-275291 , Thalidomide, Endostatin, SU5416, SU6668, EMD121974, 2- methoxyoestradiol, carboxiamidotriazole, CMIOI, pentosan polysulphate, angiopoietin 2 (Regeneron), herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM- 1470, platelet factor 4 or minocycline.
  • VEGF inhibitors including without limitation bevacizumab (AVASTIN), ranibizumab (LUCENTIS), pegaptanib (MACUGEN), sorafenib, sunitinib (SUTENT), vatalanib, ZD-6474 (ZACTIMA), anecortave (RETAANE), squalamine lactate, and semaphorin.
  • toxins refers to non-proteinaceous/non-polypeptidic cytotoxic compounds obtained from different organisms, as well as chemically modified derivatives of those same compounds and compounds obtained through chemical synthesis.
  • the compounds of this category with biological origin may be obtained from microorganisms (whether bacteria, archaea, protozoa or unicellular fungi) or pluricellular organisms (pluricellular fungi, plants, or animals, like mollusks).
  • toxins suitable for the invention are calicheamycin y1 , dolastatin 10, maytansinoid (DM1) and pyrrolobenzodiazepine dimer (PBD).
  • intervening polypeptide region of the fusion protein of the invention is conjugated to a therapeutic agent selected from the group consisting of: Vitamin D, Lorsatan, PAT-1251 , GKT137831 ; Nilotinib; Masitinib; Tofacitinib; Peficitinib; Verteporfin; Fasudil and Ripasudil.
  • a therapeutic agent selected from the group consisting of: Vitamin D, Lorsatan, PAT-1251 , GKT137831 ; Nilotinib; Masitinib; Tofacitinib; Peficitinib; Verteporfin; Fasudil and Ripasudil.
  • the intervening region of the fusion protein of the invention is conjugated to a therapeutic agent selected from the therapeutic agents indicated in the column “Therapeutic agent” of table 2 below.
  • Table 2 Suitable additional therapeutic agents to be used in the conjugates according to the present invention.
  • the disease or disorder to be treated by the therapeutic agent, or a group of therapeutic agents is provided in the same row as said therapeutic agent.
  • the present invention further contemplates the fusion protein of the invention as an imaging tool.
  • the ability to qualitatively or quantitatively display PDGFR-p in vivo or in vitro provides researchers and clinicians with important diagnostic and therapeutic tools. For example, the ability to image tumours, visually identify liver- related diseases such as liver fibrosis, liver cirrhosis, or abnormal liver function.
  • the ability to measure the expression of PDGF-Rp in patients with these conditions may also provide clinicians and researchers with assistance in diagnosing, predicting, and treating cancer and liver-related disease states.
  • the intervening polypeptide of the fusion protein of the invention is an imaging polypeptide.
  • the imaging polypeptide is a fluorescent protein.
  • the intervening polypeptide of the fusion protein of the invention is conjugated to at least one imaging agent.
  • the intervening polypeptide of the fusion protein of the invention is conjugated to a plurality of imaging agents, wherein said plurality of imaging agents are the same or different.
  • imaging agent refers to a molecule capable of providing a signal that can be detected by one or more detection techniques (eg, spectrometry, calorimetry, spectroscopy, or visual inspection). Suitable examples of detectable signals may include optical signals and electronic or radioactive signals. Examples of imaging agents include, for example: chromophores, fluorophores, Raman active tags, radioactive labels, enzymes, enzyme substrates, or combinations thereof. Suitable radioisotopes may include: H-3, C-11 , C-14, F-18, P-32, S-35, 1-123, 1-124, 1-125, 1-131 , Cr-51 , CI-36, Co-57, Fe-59, Se-75 and Eu-152.
  • Halogen isotopes such as chlorine, fluorine, bromine, and iodine
  • metals including technetium, yttrium, rhenium, and indium are also useful markers.
  • typical metal ions that can be used as signal generating agents include: Tc-99m, 1-123, In-111 , 1-131 , Ru-97, Cu-67, Ga-67, 1-125, Ga-68, As -72, Zr-89, Gd-153 and TI-201.
  • Radioisotopes used for in vivo imaging diagnosis by positron emission tomography (PET) include C-11 , F-18, Ga- 68, and 1-124.
  • paramagnetic label which may be a metal ion, exists in the form of metal complex or metal oxide particles.
  • Suitable paramagnetic isotopes can include Gd- 157, Mn-55, Dy-162, Cr-52 and Fe-56.
  • Paramagnetic label is synonymous of "paramagnetic metal ion", “paramagnetic ion” or “metal ion”, and refers to a metal ion that is magnetized parallel or antiparallel to a magnetic field to an extent proportional to the magnetic field. Usually these are metal ions with unpaired electrons.
  • paramagnetic metal ions include, but are not limited to: Gadolinium III (Gd3 + or Gd (III)), Iron III (Fe3 + or Fe (III)), Manganese II (Mn2 + or Mn (II)), Yttrium III (Yt3 + Or Yt (III)), dysprosium (Dy3 + or Dy (III)) and chromium (Cr (III) or Cr3 +).
  • radioactivity refers to the radiation, including alpha particles, beta particles, nucleons, electrons, positrons, neutrinos, and gamma rays, emitted by a radioactive substance.
  • Radionuclides suitable for use in the present invention include, but are not limited to, fluorine 18 ( 18 F), phosphorus 32 ( 32 P), scandium 47 ( 47 Sc), cobalt 55 ( 55 Co), copper 60 ( 60 Cu), copper 61 (61 Cu), copper 62 (62Cu), copper 64 (64Cu), gallium 66 (66Ga), copper 67 (67Cu), gallium 67 (67 Ga), gallium 68 (68Ga), rubidium 82 (82Rb), yttrium 86 (s6Y), yttrium 87 (87Y), strontium 89 (89Sr), yttrium 90 (90Y), rhodium 105 (05Rh), silver 111 (UtAg), indium 111 (" In), iodine 124 (1241), iodine 125 (1 asl), iodine 131 (1311), tin 117m (117 'Sn), technetium 99m (99mTc), promethium 149 (
  • fluorophore refers to a chemical compound that emits light (at a different wavelength) when it is excited by exposure to light of a specific wavelength. Fluorophores can be explained according to their emission characteristics or "color”. Green fluorophores (such as Cy3, FITC, and Oregon Green) can be characterized by their emission wavelengths typically between 515-540 nanometers. Red fluorophores (such as Texas Red, Cy5, and tetramethylrhodamine) can be characterized by their emission wavelengths typically between 590-690 nanometers.
  • fluorophores include, but are not limited to: 4-acetylamino-4 - isocyanothiostilbene-2,2'-disulfonic acid, acridine, acridine and acridine isothiocyanate derivatives, 5- (2'-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N- [3- (vinylsulfonyl) phenyl] naphthalimide-3,5-disulfonate (Fluorescent Yellow VS), N- (4- anilino-1-naphthyl) maleimide, 2-aminobenzamide (anthranilamide), bright yellow, coumarin, coumarin derivatives, 7- Amino-4-methyl coumarin (AMC, coumarin 120), 7- amino-trifluoromethyl coumarin (coumarin 151), cyanosine; 4 ', 6-diamididine 2-
  • rhodamine and derivatives Such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), Lisamine Rhodamine B Sulfonyl chloride, Rhodamine (Rhod), Rhodamine B, Rhodamine 123, Rhodamine X Sulfoyl chloride derivatives of thiocyanate, sulforhodamine B, sulforhodamine 101 and sulforhodamine 101 (Texas Red), N, N, N ', N'-tetramethyl-6 -Carboxy rhodamine (TAMRA), tetramethyl rhodamine, tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rose Acid and chelated lanthanide derivatives, quantum dots, cyanines, and squarylium (squaraine).
  • ROX 6-carboxy-X-rh
  • the fusion protein of the invention further comprises a reporter protein.
  • reporter protein refers to a protein resulting from the expression of a “reporter gene”.
  • Reporter proteins are well known and commonly used in the art as markers suitable for multiple purposes, such as location of the expression of the reporter genes in tissues, cells or subcellular locations, protein-protein interactions, transport across the plasmatic membranes or endomembranes, vesicular traffic, ligand-receptor interactions, etcetera.
  • reporter proteins in the context of the present invention include luciferase- 4-monooxygenase from Photinus pyralis, p-galactosidase, thymidine kinase, and the like.
  • the reporter proteins also include fluorescent proteins, which have already been discussed.
  • the reporter protein comprised by the fusion protein of the invention is directly adjacent to the positively charged amino acid-rich region or separated by a linker.
  • the relative position of the positively charged amino acid-rich region remains as per the the aforementioned considerations about the relative position of the elements of the fusion protein.
  • the fluorescent protein is always adjacent to it, either directly or separated by a linker.
  • the possible relative positions of the elements of the fusion protein of the invention would fit the following scheme (wherein RP refers to a reporter protein and the numbering stated above for the elements is retained: (1) PDGFR-p ligand, (2) intervening polypeptide region, (3) positively charged amino acid region):
  • the fusion protein of the invention further comprises a peptide that favours endosomal escape.
  • endosomal escape refers to the ability of the endosomal escape peptide to induce the release of the fusion proteins from the endosomal compartment after internalization by receptor-mediated endocytosis.
  • endosomal escape peptides are HA2 peptide, the CM 18 peptide, the S10 peptide, the KDEL peptide (SEQ ID NO: 26), and the polyhistidine peptide.
  • the HA2 peptide is a pH-sensitive amphiphilic peptide, and may include an amino acid sequence of SEQ ID NO : 23.
  • the CM18 peptide and the S10 peptide are amphipathic a-helical peptides, which may form transmembrane channels in cell membranes or may disrupt membranes by a carpet mechanism, and may include an amino acid sequence of SEQ ID NO : 24 or SEQ ID NO : 25, respectively.
  • the KDEL peptide is a target peptide sequence in mammals and plants located on the C-terminal end of the amino acid structure of a protein. The KDEL sequence prevents a protein from being secreted from the endoplasmic reticulum and facilitates its return if it is accidentally exported.
  • the peptide that favours endosomal escape is the KDEL peptide according to the sequence SEQ ID NO : 26.
  • the peptide that favours endosomal escape is a polyhistidine petpide of at least 2 histidines, at least 3 histidines, at least 4 histidines, at least 5 histidines, at least 6 histidines, at least 7 histidines, at least 8 histidines, at least 9 histidines, at least 10 histidines.
  • the number of therapeutic agents or imaging agents which are conjugated to the fusion protein of the invention will depend on the number of available residues in the intervening polypeptide which are available for chemical conjugation with the therapeutic agent or imaging agent. Since most conjugations occur via amino- or sulfhydryl groups present in the side chains of the amino acids forming part of the intervening polypeptide, the number of therapeutic/imaging agents conjugated to the fusion protein will depend on the number of lysine and arginine residues (for a conjugation via an amino groups in the side chains) or on the number of cysteine residues (for conjugation via sulfhydryl groups in the side chains) as well as on the yield of the conjugation reaction.
  • the fusion protein of the invention is conjugated to at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30 therapeutic agents and/or imaging agents.
  • the number of agents will also depend on the number of the monomers in the polymer.
  • the number of therapeutic agents in a given fusion protein will be the result of multiplying the number of oligomers attached to the fusion protein by the number of monomers.
  • preferred embodiments include fusion proteins comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 75, 85, 100, 125, 150 or more therapeutic agents per fusion protein, corresponding, respectively, to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25 or 30 Fdll pentamers conjugated per molecule.
  • the nanoparticles according to the invention result from the assembly of multiple copies of the fusion proteins of the invention.
  • the nanoparticle comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 17, 20, 25, more preferably at least 15 monomers of the fusion protein of the invention.
  • the total number of therapeutic agents attached to each nanoparticle will depend on (i) the number of therapeutic agents conjugated to each fusion protein, (ii) the oligomerization state of the therapeutic agents and (iii) the number of fusion proteins forming the nanoparticle.
  • the nanoparticle is conjugated to at least 30, 35, 40, 45, 50, 60, 65, 70, 57, 80, 85, 90, 59, 100, 125, 150, 175, 200, 225, 250, 275, 300 therapeutic agents.
  • the nanoparticle is conjugated to at least 30, 35, 40, 45, 50, 60, 65, 70, 57, 80, 85, 90, 59, 100, more preferably at least 60 molecules of Fdll pentamer.
  • the invention in a second aspect, relates to a method to prepare the fusion proteins of the invention comprising the steps of: a) providing a fusion protein comprising i) a PDGFR-p ligand selected from the group of consisting of PDGFB (SEQ ID NO: 1), PDGFD (SEQ ID NO: 2) and a functionally equivalent variant thereof, ii) an intervening polypeptide region and iii) a positively charged amino acid-rich region, wherein the PDGFR-p ligand and the positively charged amino acid-rich region are located at the ends of the protein and b) contacting said fusion protein with an activated form of a therapeutic agent or of an oligomeric form thereof or an activated form of a imaging agent or of an oligomeric form thereof wherein said activated form of a therapeutic agent or of an oligomeric form thereof or an activated form of a imaging agent or of an oligomeric form thereof contains a reactive group which is capable of reacting with at least one group in the intervening region
  • the invention relates to a method to prepare the fusion proteins of the invention comprising the steps of: a) providing a fusion protein comprising i) a PDGFR-p ligand selected from the group of consisting of PDGFB (SEQ ID NO: 1), PDGFD (SEQ ID N0: 2) and a functionally equivalent variant thereof, ii) an intervening polypeptide region and iii) a positively charged amino acid-rich region, wherein the PDGFR-p ligand and the positively charged amino acidrich region are located at the ends of the protein and wherein the fusion protein is provided in an activated form, wherein said activated form of the fusion protein contains a reactive group in the intervening region and b) contacting said fusion protein with a therapeutic agent or an oligomeric form thereof or with an imaging agent or an oligomeric form thereof, wherein said therapeutic agent or imaging agent contains a group which is capable of reacting with the reactive group in the fusion protein, wherein said contacting is carried out under conditions
  • reactive group refers to any moiety of a molecule which is capable of chemically reacting with another moiety from another molecule in such a fashion so as to bind the two molecules together, usually with the release of one or more additional molecules. Many such reactions are known in the art such as the formation of the peptide bond between a carboxyl and an amine group being one non-limiting example among them.
  • Activated refers to a modified version of the molecule which contains a chemical modification whereby said molecule is capable to chemically react in a manner not previously present in the molecule (for instance, the activation adds a moiety not present previously, allowing for a bond that was unfeasible before) or with an increased reactivity (meaning that the reaction of the molecule with another molecule requires a lower activation energy than in the inactivated state).
  • the present invention contemplates the possibility of activating the therapeutic agent or the imaging agent and then contacting the activated therapeutic agent or imaging agent with the fusion protein or of activating the fusion protein and then contacting the activated fusion protein with the therapeutic agent or imaging agent.
  • the activation of the fusion protein or of the therapeutic agent or imaging agent is usually carried out by reacting the molecule to be activated with a reagent that introduces the reactive group in a suitable moiety in the molecule to be activated.
  • a reagent that introduces the reactive group in a suitable moiety in the molecule to be activated examples include, but are not limited, to carboxyl, amine, imine, thiol, sulfone, hydroxyl, sulfate, and phosphate moieties, among many others which are commonly known to the person skilled in the art.
  • the activated form of the therapeutic agent or imaging agent is also herein referred to as the “activated therapeutic agent” or “activated imaging agent”.
  • the activated form of the fusion protein is also herein referred to as the “activated fusion protein”.
  • the reactive group or groups in the activated fusion protein is or are located in the intervening region, although it is not excluded that additional reactive groups can also be found in other regions of the fusion protein.
  • Activated as used herein in relation to the fusion protein also includes the incorporation of rare or non-natural amino acid (NNAA) into the fusion protein, preferably in the intervening region, wherein said NNAAs contain reactive groups which allow the conjugation of the fusion protein to a therapy agent or an imaging agent (Tsuchikama, K. and An, Z., 2018, Protein Cell, 9(1):33-46).
  • NNAA rare or non-natural amino acid
  • non-natural amino acid refers to an amino acid that is not one of the 20 common amino acids or pyrolysine or selenocysteine.
  • Other terms that may be used synonymously with the term “non-natural amino acid” is “non-naturally encoded amino acid,” “unnatural amino acid,” “non-naturally-occurring amino acid,” and variously hyphenated and non-hyphenated versions thereof.
  • non-natural amino acid includes, but is not limited to, amino acids which occur naturally by modification of a naturally encoded amino acid (including but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine) but are not themselves incorporated into a growing polypeptide chain by the translation complex.
  • the activated form of the fusion protein comprises at least one rare or non-natural amino acid selected from the group consisting of: selenocysteine, p-acetyl-phenylalanine, p-azidomethyl-L-phenylalanine and / ⁇ /6-((2- azidoethoxy)carbonyl)-L-lysine.
  • the activated form of the fusion protein comprises the at least one selenocysteine comprising the reactive selenium group and the therapeutic agent, preferably a chemotherapeutic agent, or the imaging agent comprises a maleimide or iodoacetamide group which is capable of reacting with the reactive group.
  • the formation of a bound between selenium and a maleimide or iodoacetamide group is normally carried out in acidic conditions as disclosed in Sochaj et al., (2015 Biotechnology Advances 33 775-784).
  • the activated form of the fusion protein comprises the at least one p-acetyl-phenylalanine comprising the reactive carbonyl group and the therapeutic agent or the imaging agent comprises a terminal alkoxyamine or hydrazide group which is capable of reacting with the reactive group.
  • the formation of an oxime or hydrazine bond wherein the activated fusion protein comprising an NNAA with an carbonyl group and the therapeutic or imaging agent comprising an alkoxyamine or hydrazide group can be performed as disclosed in Takimoto et al., (2009, Mol. BioSyst., 5, 931-934).
  • the activated form of the fusion protein comprises the at least one non-natural amino acid, wherein the non-natural amino acid is p-azidomethyl-L-phenylalanine or /V6-((2-azidoethoxy)carbonyl)-L-lysine, wherein the non — natural amino acid comprises azide as the reactive group and wherein the therapeutic agent or the imaging agent comprise an alkyne group which is capable of reacting with the reactive group.
  • the bond between an azide group and an alkyne group can be achieve by the use of click chemistry or biorthogonal chemistry.
  • click chemistry refers to powerful linking reactions that are able to generate covalent bonds quickly and reliably by joining small units comprising reactive groups together. Click chemistry reactions are typically modular, wide in scope, give high chemical yields, generate inoffensive byproducts, are stereospecific, and/or can be carried using readily available starting materials and reagents out under simple, physiological reaction conditions. In addition, click chemistry reactions preferably use no toxic solvents or use a solvent that is benign or easily removed (preferably water), and/or provides simple product isolation by non-chromatographic methods (crystallization or distillation).
  • Click chemistry reactions comprise, e.g., cycloaddition reactions, especially from the 1 ,3-dipolar family, hetero-Diels-Alder reactions; nucleophilic ring-opening reactions, e.g. of strained heterocyclic electrophiles, such as epoxides, aziridines, cyclic sulfates, cyclic sulfamidates, aziridinium ions and episulfonium ions; carbonyl chemistry of the non-aldol type (e.g. the formation of oxime ethers, hydrazones and aromatic heterocycles); and addition to carbon-carbon multiple bonds; e.g.
  • the invention relates to a method to prepare the fusion proteins of the invention comprising the steps of: a) providing a fusion protein comprising i) a PDGFR-p ligand selected from the group of consisting of PDGFB (SEQ ID N0: l), PDGFD (SEQ ID NO : 2) and a functionally equivalent variant thereof, ii) an intervening polypeptide region and iii) a positively charged amino acid-rich region, wherein the PDGFR-p ligand and the positively charged amino acid-rich region are located at the ends of the protein, and b) contacting said fusion protein with a therapeutic agent, or an oligomeric form thereof, or with an imaging agent, or an oligomeric form thereof, in the presence of an enzyme, wherein said therapeutic agent or imaging agent contains a reactive group which is recognized by the enzyme, said fusion protein contains a motif which is recognized by the enzyme, and wherein said contacting is carried out under conditions adequate for the formation of a bond between the reactive group in the
  • motif refers to any amino acid side chain, or group therein, or any specific sequence of amino acids which are recognized by the said enzyme and which function as the point of bond formation in the fusion protein, leading to an enzymatic conjugation.
  • enzymatic conjugation as used herein may be understood in the broadest sense as any means of a ligation that is catalyzed by an enzyme.
  • the enzymatic conjugation is a ligase- or a peptidase-based conjugation.
  • the fusion protein contains the LPXTG (X: any amino acid) motif (SEQ ID NO : 56), preferably in the intervening polypeptide, and the enzyme is a Sortase A transpeptidase, preferably the Staphylococcus aureus Sortase A.
  • “Sortase A” mediated transpeptidation is based on the Staphylococcus aureus Sortase A recognizing the LPXTG (X: any amino acid) motif (SEQ ID NO: 56) and cleaving the threonineglycine (T-G) bond, attaching an oligoglycine (oligo-G)-containing molecule.
  • Various cargo can be fused to the oligo-G for sortase A-mediated conjugation. For example, as disclosed in Beerli R.R. et al., (2015, PLoS One.
  • a fusion protein containing the LPXTG motif can be conjugated to an chemotherapy agent comprising which has been modified by addition of an pentaglycine peptide.
  • the fusion protein contains the LPXTG (X: any amino acid) motif (SEQ ID NO : 56), preferably in the intervening polypeptide, and the enzyme is a Sortase A transpeptidase, preferably the Staphylococcus aureus Sortase A, and the therapeutic agent, preferably a chemotherapy agent, or the imaging agent is modified with an oligo-glycine reactive group.
  • the enzyme is a microbial transglutaminase.
  • Microbial transglutaminase mediated transpeptidation is based on the transpeptidation catalyzed by microbial transglutaminases where a primary amine- containing linker is covalently attached to the primary amide side chain of a glutamine within the fusion protein of the invention.
  • microbial transglutaminases where a primary amine- containing linker is covalently attached to the primary amide side chain of a glutamine within the fusion protein of the invention.
  • Both non-specific and specific transglutaminases can be used, for example transglutaminases which recognize the small motif LLQG (SEQ ID NO : 57) are described in Strop et al., (Chem Biol 20:161- 167).
  • the fusion protein contains the LLQG (SEQ ID NO : 57) motif, preferably in the intervening polypeptide, the enzyme is a transglutaminases which recognizes the LLQG (SEQ ID NO : 57 motif and the therapeutic agent, preferably a chemotherapy agent, or the imaging agent is modified with or comprises an amine reactive group.
  • the enzyme is a transglutaminases which recognizes the LLQG (SEQ ID NO : 57 motif and the therapeutic agent, preferably a chemotherapy agent, or the imaging agent is modified with or comprises an amine reactive group.
  • the contacting said fusion protein with a therapeutic agent, or an oligomeric form thereof, or with an imaging agent, or an oligomeric form thereof is done in at least two steps, wherein the fusion protein is first contacted with an enzyme which recognizes the motif present in the fusion protein, preferably in the intervening polypeptide, and modifies said motif, followed by the contacting of the fusion protein with the therapeutic agent or the imaging agent comprising a reactive group which presents reactivity towards the modified motif of the fusion protein.
  • the fusion protein contains the CXPXR (wherein X is any amino acid) motif, preferably in the intervening polypeptide, the enzyme is a formylglycine (FGIy)-generating enzyme which recognizes the CXPXR motif and modifies said motif, and the therapeutic agent, preferably a chemotherapy agent, or the imaging agent is modified with or comprises an aminooxy or hydrazide reactive group.
  • the enzyme is a formylglycine (FGIy)-generating enzyme which recognizes the CXPXR motif and modifies said motif
  • the therapeutic agent preferably a chemotherapy agent
  • the imaging agent is modified with or comprises an aminooxy or hydrazide reactive group.
  • FGIy formylglycine (FGIy)-generating enzyme
  • FGIy human enzymes present in the endoplasmic reticulum that catalyze the conversion of cysteine to formylglycine (fGly).
  • the fusion protein contains the VDSVEGEGEEEGEE motif (SEQ ID NO: 59), preferably in the intervening polypeptide, and the enzyme is tubulin tyrosine ligase (TTL).
  • TTL tubulin tyrosine ligase
  • tubulin tyrosine ligase or its acronym “TTL”, as used herein refers to an enzyme naturally involved in the intracellular regulation of microtubule stability recognizes a 14 amino acid recognition motif at the C- terminus of alpha-tubulin and posttranslationally attaches a terminal tyrosine residue. TTL can be used for the labelling known as Tub-tag.
  • Tub-tag refers to a novel approach for the site-specific modification of antibodies that combines the above mentioned use ofUAA incorporation with a highly efficient chemoenzymatic system.
  • the recognition motif (Tub-tag) allows the TTL- mediated attachment of non-natural tyrosine derivatives that carry uniquely reactive groups for chemoselective conjugation such as strain- promoted alkyne azide cycloadditions as disclosed in Schumacher et al. (2015, Angew. Chem. Int. Ed. 54, 13787 -13791).
  • the linking moiety is a bifunctional cross-linker and, more preferably, a heterobifunctional cross-linker, that reacts with the groups in the therapeutic agent/imaging agent and in the fusion protein, either sequentially (either reacting with the activated therapeutic/imaging agent first and then with the fusion protein, or first with the fusion protein and then with the activated therapeutic/imaging agent) or simultaneously, using among other linkages such as thioethers, amide bonds, carbonnitrogen double bonds, or linkages generated by cycloaddition as disclosed in Kalia J et al.
  • thiol-reactive functional groups include iodoacetamides, maleimides, and disulfides.
  • a protein can be treated with a small molecule or surface displaying an activated ester (e.g., an N-hydroxysuccinimidyl ester) to form amide bonds with the amino groups on lysine side chains and the N terminus.
  • the linking moiety is a heterobifunctional cross-linker which contains reactive groups capable of reacting with a thiol group and with an amino group.
  • the heterobifunctional cross-linker is 6-maleimidohexanoic acid N-hydroxysuccinimide ester.
  • the linking moiety reacts in a first step with the activated therapeutic agent or with the imaging agent and in a second step with the fusion protein. In another embodiment, the linking moiety reacts in a first step with the fusion protein and, in a second step, with the therapeutic agent or with the imaging agent.
  • the step of contacting the fusion protein of the invention with the activated form of the therapeutic/imaging agent is carried out in a medium which favors the reaction establishing the bond between them.
  • Media suitable for the reactions are commonly known to the person skilled in the art, including aqueous buffers and nonaqueous buffers.
  • solid supports can be used in conjunction with the media for any of the reaction steps conducing to the synthesis of the activated therapeutic/imaging agent and the conjugate of the fusion protein, the therapeutic/imaging agent, and also the linking moiety in the embodiments that include one.
  • the method for the preparation of the conjugates between the fusion protein and the therapeutic/imaging agent is not limited to the fusion protein, the activated therapeutic agent, the imaging agent and the linking moiety, but that some embodiments include also the use of one or more catalysts and co-factors in the reaction.
  • the activated form of the therapeutic agent or the activated form of the imaging agent contains a group which reacts with at least one of the side chains of a residue in a peptide region of the fusion protein, preferably in the intervening region of the fusion protein.
  • said residue is an external lysine or an external cysteine.
  • the group of the activated therapeutic agent, preferably the chemotherapeutic agent, or the activated imaging agent which reacts with the side chain of the intervening region of the fusion protein is a thiol group.
  • the group of the activated therapeutic agent, preferably the chemotherapeutic agent, or the activated imaging agent which reacts with the side chain of the intervening region of the fusion protein is an activated carboxylic acid group.
  • the activated therapeutic agent is an activated chemotherapeutic agent, more preferably a thiol-functionalized oligo-floxuridine.
  • the activated therapeutic agent is an activated chemotherapeutic agent, more preferably an activated carboxylic acid group-functionalized oligo-floxuridine.
  • the activated therapeutic agent is an activated chemotherapeutic agent, more preferably an activated perfluoroaryl-functionalized oligo- floxuridine.
  • the linking moiety is 4-maleimido hexanoic acid N-hydroxysuccinimide ester mediates the conjugation between the activated therapeutic agent or the imaging agent and the side chain of the residue of the peptide region of the fusion protein indicated in the previous embodiments of this section.
  • the linking moiety 4-maleimido hexanoic acid N- hydroxysuccinimide ester is bound in a first step to the therapeutic agent, preferably the activated Fdll, yet more preferably Fdll functionalized with a sulfhydryl, or the imaging agent and in a second step to the side chain in a residue of the fusion protein, more preferably to external lysines or external cysteines of the fusion protein, even more preferably to external lysines or external cysteines of the intervening region of the fusion protein.
  • the step of contacting the activated fusion protein of the invention with the therapeutic agent or the imaging agent is carried out in a medium which favors the reaction establishing the bond between them.
  • Media suitable for the reactions are commonly known to the person skilled in the art, including aqueous buffers and non-aqueous buffers.
  • solid supports can be used in conjunction with the media for any of the reaction steps leading to the synthesis of the conjugate of the fusion protein and the therapeutic agent or the imaging agent, and also the linking moiety in the embodiments that include one.
  • the method for the preparation of the conjugates between the fusion protein and the therapeutic agent or the imaging agent is not limited to the fusion protein, the activated therapeutic agent or the activated imaging agent, and the linking moiety, but that some embodiments include also the use of one or more catalysts and co-factors in the reaction.
  • the activated form of the fusion protein contains a group which reacts with at least one moiety in the therapeutic agent or in the imaging agent.
  • the group of the therapeutic agent, preferably the chemotherapeutic agent, or of the imaging agent which reacts with the activated fusion protein is a thiol group, an activated carboxylic acid group or a perfluoroaryl group.
  • the activated fusion protein agent is an amino functionalized fusion protein wherein one or more amino groups in the side chain of the amino acids forming part of the intervening polypeptide is modified with an activated group having thiol reactivity, activated carboxylic acid reactivity or perfluoroaryl reactivity.
  • the activated fusion protein agent is a fusion protein comprising at least one motif of the amino acid sequence FCPF (SEQ ID NO: 60), preferably in the intervening peptide, wherein said motif has perfluoroaryl reactivity.
  • the therapeutic agent is a chemotherapy agent and wherein the activated form thereof contains a group which reacts with at least one of the side chains in the intervening polypeptide region.
  • the group which reacts with at least one of the side chains in the intervening polypeptide region is a thiol group, an activated carboxylic acid group or an perfluoroaryl group.
  • the activated chemotherapeutic agent is thiol-functionalized oligo-floxuridine, an activated carboxylic acid functionalized oligo-fluxuridine or an activated perfluoroaryl functionalized oligo-fluxuridine.
  • the linking moiety is 4-maleimido hexanoic acid N-hydroxysuccinimide ester mediates the conjugation between an amino group in the fusion protein and a thiol group in the therapeutic agent or imaging agent.
  • the linking moiety 4-maleimido hexanoic acid N- hydroxysuccinimide ester is bound in a first step to the fusion protein, more preferably to external lysines of the fusion protein and in a second step to the therapeutic agent side chain in a residue of the fusion protein.
  • Nanoparticles of the invention and methods for preparing them with the fusion proteins of the invention are described in detail below.
  • the invention in another aspect, relates to a method to prepare nanoparticles comprising multiple copies of the fusion protein according to the first aspect of the invention comprising placing a preparation of said fusion protein in a suitable buffer.
  • nanoparticles are microscopic particles whose size is measured in nanometers.
  • the nanoparticles of the invention comprise the nanoparticles that result from the aggregation of multiple copies of the fusion protein of the invention as defined in the previous section.
  • the preparation of the fusion protein of the invention comprises the monomeric form of the fusion proteins of the invention, which are thermodynamically favored to form non-covalent electrostatic unions and spontaneously aggregate in the conditions of the low salt buffer.
  • the size of the nanoparticles can be in the 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 5 and 100 nm.
  • suitable buffer comprises any buffer solution resulting from the dissolution of one or more salts in water with the capability to moderate changes in pH, wherein the amount of dissolved salt or salts results in an osmolarity similar to that of the physiological fluids, such as the cytoplasm or the extracellular medium, for instance.
  • suitable buffer is understood to keep pH and osmolarity inside the range of physiological values and will be used inside the range of physiological temperatures.
  • the range of physiological temperatures can oscillate between 15 and 45° C, more preferably between 20 and 40°C , even more preferably between 25 and 39°C, yet even more preferably between 30 and 37°C
  • the osmolarity of the suitable buffer will be in the range between 100 and 400 milli-osmoles/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.
  • Buffers suitable for the invention are the Tris-dextrose buffer (20 mM Tris +5% dextrose, pH 7.4), the Tris-NaCI buffer (20 mM Tris, 500 NaCI, pH 7.4), the PBS-glycerol buffer (phosphate buffered saline, PBS, pH 7.4, which is well known in the art, +10% glycerol), Tris Buffered Saline (TBS)-dextrose (20 mM Tris-HCI buffer pH 7.5, well known in the art, 200NaCI, +5% dextrose), Tris Buffered Saline-Tween 20 (TBST) buffer (10 mM Tris-HCI pH 7.5, 200 mM NaCI, +0.01% Tween 20), sodium carbonate (166 NaHCO3 pH 8.0, 333 mM NaCI) or any physiological buffer known in the art with a pH not lower than 6.
  • Tris-dextrose buffer (20 mM Tris +5% dextrose, pH 7.4
  • the suitable buffer of the method of the invention is selected from the group consisting of a carbonate buffer, a Tris buffer and a phosphate buffer.
  • the suitable buffer of the method of the invention is a carbonate buffer that comprises sodium carbonate at a concentration between 100 and 300 nM.
  • the suitable buffer of the method of the invention is a Tris buffer that comprises Tris at a concentration of between 10 and 30 nM.
  • the suitable buffer of the invention is a phosphate buffer that comprises Na2HPC>4 and NaH2PO4 at a total concentration of between 5 mM and 20 mM.
  • the suitable buffer of the method of the invention further comprises dextrose and/or glycerol.
  • the suitable buffer of the method of the invention has a pH between 6.5 and 8.5.
  • the suitable buffer is a sodium carbonate at a concentration of between 100 and 300 mM further comprising salt at a concentration of 200 mM to 400 mM.
  • the suitable buffer of the method of the invention is 166 mM NaHCCh, 333 mM NaCI, pH 8.0.
  • the invention in another aspect of the invention, relates to nanoparticles comprising multiple copies of the fusion protein of the first aspect of the invention or prepared according to the method or the invention for preparing nanoparticles.
  • the nanoparticles of the invention comprise assembled complexes of multiple copies of the fusion proteins of the invention, which result from the electrostatic interaction between regions in their structures favoring their non-covalent binding and coupling in physiological conditions. Since the method of the invention for the preparation of nanoparticles comprises placing a preparation of the fusion protein of the invention in a low salt buffer, it is understood that the nanoparticles thus formed comprise also an assembled complex of multiple copies of the fusion protein.
  • the nanoparticles of the invention have a diameter between 5 and 100 nm.
  • the invention in another aspect of the invention, relates to a polynucleotide encoding the fusion protein of the first aspect invention, a vector comprising the aforementioned polynucleotide, and a host cell comprising the aforementioned polynucleotide or the aforementioned vector.
  • nucleic acid and “polynucleotide”, as used herein interchangeably, 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 via phosphodiester bonds, related naturally occurring structural variants and synthetic non-naturally occurring analogs thereof.
  • polynucleotide encodes the polypeptide or protein sequence of the fusion protein of the invention that corresponds to the first aspect of the invention.
  • the polynucleotide of the invention therefore comprises the sequence encoding all of the elements comprised in the fusion protein: the PDGFR-p ligand, the intervening peptide region, the positively charged amino acidrich region, and any other elements that may be part of the fusion protein such as the reporter protein, linkers, and so on and so forth.
  • nucleic acids or polynucleotides of the invention include coding regions and the adequate regulatory signals for promoting expression in cells to give rise to the biologically active fusion protein.
  • nucleic acids containing a coding region will be operably linked to appropriate regulatory sequences.
  • Such regulatory sequence will at least comprise a promoter sequence.
  • promoter refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA- dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.
  • a “constitutive” promoter is a promoter that is active under most physiological and developmental conditions.
  • An “inducible” promoter is a promoter that is regulated depending on physiological or developmental conditions.
  • a “tissue specific” promoter is only active in specific types of differentiated cells/tissues.
  • promoters suitable for the embodiment of the present invention include, without being necessarily limited to, constitutive promoters such as the derivatives of the genomes of eukaryotic viruses such as the polyoma virus, 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, retrovirus LTR regions, the promoter of the immunoglobulin gene, the promoter of the actin gene, the promoter of the EF-1 alpha gene as well as inducible promoters in which the expression of the protein depends on the addition of a molecule or an exogenous signal, such as the tetracycline system, the NFKB/UV light system, the Cre/Lo
  • the polynucleotides of the invention encoding the fusion protein of the invention can be part of a vector.
  • the invention relates to a vector comprising a polynucleotide of the invention.
  • a person skilled in the art will understand that there is no limitation as regards the type of vector which can be used because said vector can be a cloning vector suitable for propagation and for obtaining the polynucleotides or expression vectors in different heterologous organisms suitable for purifying the fusion proteins of the invention.
  • suitable vectors include expression vectors in prokaryotes such as pET (such as pET14b), pUC18, pUC19, Bluescript and their derivatives, mp18, mp19, pBR322, pMB9, ColEI, pCRI, RP4, phages and shuttle vectors such as pSA3 and pAT28, expression vectors in yeasts such as vectors of the type of 2 micron plasmids, integration plasmids, YEP vectors, centromeric plasmids and the like, expression vectors in insect cells such as the pAC series and pVL series vectors, expression vectors in plants such as vectors of expression in plants such as pl Bl, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series vectors and the like and expression vectors in superior eukaryotic cells based on viral vector
  • the vector of the invention can be used to transform, transfect, or infect cells which can be transformed, transfected or infected by said vector.
  • Said cells can be prokaryotic or eukaryotic.
  • the vector wherein said DNA sequence is introduced can be a plasmid or a vector which, when it is introduced in a host cell, is integrated in the genome of said cell and replicates together with the chromosome (or chromosomes) in which it has been integrated.
  • Said vector can be obtained by conventional methods known by the persons skilled in the art (Sambrook et al., 2001 , “Molecular cloning, to Laboratory Manual”, 2nd ed., Cold Spring Harbor Laboratory Press, N.Y. Vol 1-3 a).
  • the invention also relates to a cell comprising a polynucleotide or a vector of the invention, for which said cell has been able to be transformed, transfected or infected with a polynucleotide or vector provided by this invention.
  • the transformed, transfected or infected cells can be obtained by conventional methods known by persons skilled in the art (Sambrook et al., 2001 , mentioned above).
  • Host cells suitable for the expression of the conjugates of the invention include, without being limited to, mammal, plant, insect, fungal and bacterial cells.
  • Bacterial cells include, without being limited to, Gram-positive bacterial cells such as species of the Bacillus, Streptomyces, Listeria and Staphylococcus genera and Gram-negative bacterial cells such as cells of the Escherichia, Salmonella and Pseudomonas genera.
  • Fungal cells preferably include cells of yeasts such as Saccharomyces cereviseae, Pichia pastoris and Hansenula polymorpha.
  • Insect cells include, without being limited to, Drosophila and Sf9 cells.
  • Plant cells include, among others, cells of crop plants such as cereals, medicinal, ornamental or bulbous plants.
  • Suitable mammal cells in the present invention include epithelial cell lines (human, ovine, porcine, etc.), osteosarcoma cell lines (human, etc.), neuroblastoma cell lines (human, etc.), epithelial carcinomas (human, etc.), glial cells (murine, etc.), hepatic cell lines (from monkey, 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 the mESC line, human embryonic stem cells such as HS293, BGV01 , SHEF1 , SHEF2, HS181 , NIH3T3 cells, 293T, REH and MCF-7 and hMSC cells.
  • epithelial cell lines human, ovine, porcine
  • the polynucleotide, the vector, and the host cell of the invention are suitable for the expression of the biologically active form of the fusion protein of the invention.
  • the invention relates to a fusion protein, a polynucleotide, a vector, a host cell or a nanoparticle according to the invention for use in medicine.
  • the fusion protein, polynucleotide, vector, host cell, or nanoparticle of the invention can be administered to a patient in order to induce a therapeutic response.
  • the therapeutic response comprises the suppression, reduction or arrest of the causes of the pathological condition or the disease suffered by a patient; the elimination, reduction, arrest or amelioration of the symptoms of the condition or disease; or the extinction, arrest or slowing down of the progression of the condition or disease in the patient.
  • fusion protein, polynucleotide, vector, host cell or nanoparticle of the invention suitable for use in medicine may be presented accompanied by a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • Remington's Pharmaceutical Sciences. Ed. by Gennaro, Mack Publishing, Easton, Pa., 1995 discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.
  • compositions comprising the fusion protein, polynucleotide, vector, host cell, or nanoparticle of the invention and a pharmaceutically acceptable carrier are pharmaceutical compositions.
  • compositions of this invention can be administered to a patient by any means known in the art including oral and parenteral routes.
  • inventive compositions may be administered by injection (e.g., intravenous, subcutaneous or intramuscular, intraperitoneal injection), rectally, vaginally, topically (as by powders, creams, ointments, or drops), or by inhalation (as by sprays).
  • fusion protein Use of the fusion protein, the polynucleotide, the vector, the host cell, or the nanoparticle of the invention in the treatment of cancer.
  • Another embodiment of the invention relates to a fusion protein, a polynucleotide of the invention, the vector of the invention, the host cell of the invention comprising the vector or the polynucleotide and expressing the fusion protein, and the nanoparticle of the invention, or their corresponding pharmaceutical compositions, wherein the intervening polypeptide region is an antitumor peptide or wherein the intervening polypeptide is linked to an antitumor agent, for use in the treatment of cancer.
  • the terms “treat”, “treatment” and “treating” refer to the reduction or amelioration of the progression, severity and/or duration of cancer, or the amelioration of one or more symptoms (preferably, one or more discernible symptoms) of cancer.
  • the terms “treat”, “treatment” and “treating” also refer to the amelioration of at least one measurable physical parameter of cancer, such as growth of a tumor, not necessarily discernible by the patient.
  • “treat”, “treatment” and “treating” refer also to the inhibition of the progression of cancer, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both.
  • “Treat”, “treatment” and “treating” may refer, too, to the reduction or stabilization of tumor size or cancerous cell count.
  • cancer refers to a group of diseases involving abnormal, uncontrolled cell growth and proliferation (neoplasia) with the potential to invade or spread (metastasize) to other tissues, organs or, in general, distant parts of the organism; metastasis is one of the hallmarks of the malignancy of cancer and cancerous tumors.
  • the abnormal growth and/or proliferation of cancerous cells is the result of a combination of genetic and environmental factors that alter their normal physiology.
  • the growth and/or proliferation abnormalities of cancerous cells result in physiological disorders and, in many cases, death of the individual, due to the dysfunctionality or loss of functionality of the cell types, tissues and organs affected.
  • cancer includes, but is not restricted to, cancer of the breast, heart, small intestine, colon, spleen, kidney, bladder, head, neck, ovaries, prostate gland, brain, pancreas, skin, bone, bone marrow, blood, thymus, womb, testicles, hepatobiliary system and liver; in addition to tumors such as, but not limited to, adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma, hemangiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, hepatobiliary cancer, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma and
  • this term includes acrolentiginous melanoma, actinic keratosis adenocarcinoma, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamus carcinoma, astrocytic tumors, Bartholin gland carcinoma, basal cell carcinoma, bronchial gland carcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Ewing sarcoma, focal nodular hyperplasia, germ cell tumors, glioblastoma, glucagonoma, hemangioblastoma, hemagioendothelioma, hemagioma, hepatic adenoma
  • the fusion protein, the polynucleotide, the vector, the host cell, or the nanoparticle of the invention for use in the treatment of cancer wherein the cancer is a tumor, preferably a solid tumor.
  • the antitumor peptide of the fusion protein, the polynucleotide, the vector, the host cell, or the nanoparticle of the invention is selected from the group consisting of
  • the antitumor peptide of the fusion protein, the polynucleotide, the vector, the host cell, or the nanoparticle of the invention is selected from the group consisting of the BH3 domain of BAK, PUMA, GW- H1 , the Diphtheria toxin, the Pseudomonas exotoxin and Ricin.
  • the antitumor peptide of the fusion protein, the polynucleotide, the vector, the host cell, or the nanoparticle of the invention is a truncated form or a mutant of the peptide selected from the group indicated just before, preferably from the group consisting of the Diphtheria toxin, the Pseudomonas exotoxin and Ricin. Preferred sequences of said peptides are indicated above in the “Intervening polypeptide region" section.
  • the antitumor agent of the fusion protein, polynucleotide, vector, host cell or nanoparticle of the invention is selected from the group consisting of: (i) a chemotherapy agent,
  • the antitumor agent of the fusion protein, polynucleotide, vector, host cell or nanoparticle of the invention is selected is selected from a group consisting of: a floxuridine polynucleotide, pyrimidine analogue or an oligomeric form thereof, auristatin, and any of the agents indicated above in the “Intervening polypeptide region” section.
  • the cancer to be treated with the fusion protein, the polynucleotide, the vector, the host cell, or the nanoparticle of the invention is selected from the group consisting of non-small cell lung, breast, colon, liver, prostate, pancreatic or colorectal cancer.
  • the cancer to be treated with the fusion protein, the polynucleotide, the vector, the host cell, or the nanoparticle of the invention is characterized by comprising cancer cells that express or overexpress PDGFR-p.
  • overexpressed PDGFR-P or “overexpression of PDGFR-P” refers to an abnormal level of expression of PDGFR-p in epithelial and stromal cells within cancer tissues.
  • Overexpression of PDGFR-p can be determined by standard assays in the art, such as, immunohistochemistry assays, wherein histological sections of the tissue/cells of interest are incubated with primary antibodies against PDGFR-p, followed by secondary antibodies against the primary antibody. The presence of a label in the secondary antibody whose signal can be detected and quantify, allows to compare the signal amount from the tissue/cells of interest against a reference value of tissue/cells which are known to express PDGFR-p at physiological levels.
  • Tissues/cells which overexpress PDGFR-p have a higher signal than the control tissue/cells (Tsao A. S., et al, 2011 , Clin Lung Cancer; 12(6): 369-374).
  • Another method to determine if cells overexpress PDGFR-p is the use of the fusion protein of the invention wherein the intervening polypeptide is an imaging agent of the ones described previously. The incubation of the fusion protein with the cells of interest will allow the fusion protein of the invention to bind the PDGFR-p. After the removal of unbound fusion protein of the invention, the imaging agent can be detected and the quantity of imaging agent/fusion protein bound to the PDGFR-p expressing cells determined. If the value obtained is significantly higher than the value obtained for cells expressing PDGFR-p at physiological levels to which the procedure as exemplified was repeated, then the cells of interest do overexpress PDGFR-p.
  • fusion protein Use of the fusion protein, the polynucleotide, the vector, the host cell, or the nanoparticle of the invention in the treatment of diseases caused by alterations in PDGFR-/3 positive cells.
  • the invention relates to a fusion protein, a polynucleotide, a vector, a host cell or a nanoparticle according to the invention for use in the treatment of diseases caused by alterations in PDGFR-p positive cells.
  • diseases caused by alterations in PDGFR-p positive cells refers to diseases which can be characterized by the presence, participation or function of cells which express in their surface the PDGFR-p protein and therefore can be targeted by the fusion protein of the invention or the nanoparticles of the invention, in order to deliver therapeutic agents to said cells and treat the disease.
  • diseases are fibrosis, pancreatitis, neurological diseases, atherosclerosis and inflammation. It will be understood that the target within the PDGFR-p positive cells will depend on the specific disease to be treated and that a person skilled in the art would be able to select the therapeutic agent suitable for the disease to be treated.
  • the diseases caused by alterations in PDGFR-p positive cells are characterized by the presence of PDGFR-p positive cells.
  • the PDGFR-p positive cells are selected from a group consisting of: lung fibroblast, hepatic stellate cells, pancreatic stellate cells, mesenchymal cells, perivascular astrocytes, astrocytes, oligodendrocyte, vascular smooth muscle cell and fibroblast.
  • the diseases caused by alterations in PDGFR-p positive cells are selected from a group consisting of: pulmonary fibrosis, hepatic fibrosis, pancreatic fibrosis, pancreatitis; ischemic stroke, Parkinson, Alzheimer, neurofibrosmatosis, amyotrophic lateral sclerosis, systemic sclerosis, glaucoma and rheumatoid arthritis.
  • the disease caused by alterations in PDGFR-p positive cells is pulmonary fibrosis which is characterized by the presence of PDGFR-p positive lung fibroblasts.
  • pulmonary fibrosis refers to pulmonary fibrosis occurred due to various reasons, specifically, due to radiation exposure, drug therapy for anticancer treatment, smoking, or dusty work environments, etc., but is not limited thereto.
  • the pulmonary fibrosis above may be side effects of radiotherapy occurred by exposure of radiation to normal tissues during radiotherapy for cancer or may be side effects of drug therapy for anticancer treatment, but is not limited thereto.
  • the radiotherapy or drug therapy for cancer which is capable of inducing pulmonary fibrosis, includes treatment of a lung cancer, a breast cancer, or Hodgkin lymphoma, etc., but is not limited thereto.
  • therapeutic agents which can be conjugated to the fusion protein of the invention for the treatment of pulmonary fibrosis are, without limitation, nintedanib and imatinib.
  • the disease caused by alterations in PDGFR-p positive cells is hepatic fibrosis which is characterized by the presence of PDGFR-p positive hepatic stellate cells.
  • hepatic fibrosis refers to abnormal hyperplasia of connective tissue in the liver, excessive precipitation of diffuse extracellular matrix in the liver, pathological changes in the normal structure of the liver (lesion), which are caused by or accompanied by inflammation, infection (e.g. viral infection), immune response, ischemia, chemicals, radiation, oxidative stress and alcohol abuse, etc. Hepatic fibrosis further developing into cirrhosis is also covered by the term "hepatic fibrosis" in the present invention.
  • therapeutic agents which can be conjugated to the fusion protein of the invention for the treatment of hepatic fibrosis are, without limitation, imatinib, vitamin D, degreeatan, PAT-1251 , GKT137831 , calciprotiol and all the toxins previously mentioned in relation to the therapeutic agents which can be conjugated to the fusion protein of the invention as well as toxins in the form of polypeptides which can be the intervening polypeptide as described previously.
  • hepatic stellate cells refers to liver-specific mesenchymal cells that play vital roles in liver physiology and fibrogenesis. They are located in the space of Disse and maintain close interactions with sinusoidal endothelial cells and hepatic epithelial cells.
  • the disease caused by alterations in PDGFR-p positive cells is pancreatic fibrosis or pancreatitis which is characterized by the presence of PDGFR-p positive pancreatic stellate cells.
  • pancreatitis refers to an inflammation of the pancreas.
  • Pancreatitis can be either acute or chronic. Acute pancreatitis generally develops suddenly, and chronic pancreatitis is a long-term condition, which typically develops after multiple episodes of acute pancreatitis, leading to pancreatic fibrosis. Acute pancreatitis distinguishes between two different forms: Interstitial edematous acute pancreatitis with no local or systemic complications and acute necrotizing pancreatitis (ANP), associated with both local and systemic complications and a high risk of mortality.
  • Examples of therapeutic agents which can be conjugated to the fusion protein of the invention for the treatment of pancreatitis or pancreatic fibrosis are, without limitation, calciprotiol.
  • pancreatic stellate cells in the context of the present invention refers to myofibroblast-like cells that are located in exocrine regions of the pancreas. Pancreatic stellate cells are mediated by paracrine and autocrine stimuli and share similarities with the hepatic stellate cell.
  • the disease caused by alterations in PDGFR-p positive cells is renal fibrosis which is characterized by the presence of PDGFR-p positive mesenchymal cells.
  • the term "renal fibrosis" as used herein includes all diseases in which fibrosis occurs in the kidney due to various causes, and the fibrosis may include, but is not limited to, those caused by any one or more selected from the group consisting of: catheter installation, glomerulosclerosis, glomerulonephritis, nephritis, acute renal failure, chronic renal failure, end-stage renal disease, and metabolic disease.
  • therapeutic agents which can be conjugated to the fusion protein of the invention for the treatment of renal fibrosis are, without limitation, imatinib and all of the tyrosine kinase inhibitors previously described.
  • mesenchymal cells refers to a cell forming a mesenchymal tissue, such as osteoblat, chondrocyte, myoblast, adipocyte, stroma cell, tendon cell, and the like.
  • the disease caused by alterations in PDGFR-p positive cells is ischemic stroke which is characterized by the presence of PDGFR-p positive perivascular astrocytes.
  • ischemic stroke refers to cerebral ischemic stroke, caused by reduced blood flow to the brain or parts thereof which leads to a reduced delivery (undersupply) of oxygen to brain cells resulting in tissue damage due to brain cell death.
  • therapeutic agents which can be conjugated to the fusion protein of the invention for the treatment of ischemic stroke are, without limitation, imatinib and all of the tyrosine kinase inhibitors previously described.
  • perivascular astrocytes refers to cells in the brain which closely juxtapose blood vessels and are postulated to have important roles in the control of vascular physiology, including regulation of the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • the diseases caused by alterations in PDGFR-p positive cells is selected from a group consisting of: Parkinson, Alzheimer and neurofibrosmatosis, wherein said diseases are characterized by the presence of PDGFR-p positive astrocytes and oligodendrocyte.
  • Parkinson refers to a disease which is characterized by progressive degeneration of nigrostriatal dopamine neurons. In this sense, the term Parkinson's disease also comprises the term Parkinson's syndrome.
  • Alzheimerer refers to a disease in which losses of significant memory and other intellectual abilities occur enough to hinder patient's daily life, and means a neurodegenerative disease characterized by a histopathological overall atrophy of the brain, enlargement of the ventricle, multiple lesions of nerve fibers (nerve fiber twist), neuritic plaque and the like.
  • neurofibrosmatosis refers to a group of three conditions in which tumors grow in the nervous system. The three types are neurofibromatosis type I (NF1), neurofibromatosis type II (NF2), and schwannomatosis.
  • NF1 symptoms include light brown spots on the skin, freckles in the armpit and groin, small bumps within nerves, and scoliosis.
  • NF2 symptoms include light brown spots on the skin, freckles in the armpit and groin, small bumps within nerves, and scoliosis.
  • NF2 there may be hearing loss, cataracts at a young age, balance problems, flesh colored skin flaps, and muscle wasting.
  • schwannomatosis there may be pain either in one location or in wide areas of the body.
  • the tumors in NF are generally non-cancerous.
  • therapeutic agents which can be conjugated to the fusion protein of the invention for the treatment of Parkinson, Alzheimer and neurofibrosmatosis are, without limitation, imatinib, nilotinib, sunitinib, masitinib and all of the tyrosine kinase inhibitors previously described.
  • astrocytes also termed astroglia, refers to the cells which anchor neurons to their blood supply. Generally, astrocytes regulate the external chemical environment of neurons by removing excess ions, notably potassium, and recycling neurotransmitters released during synaptic transmission. Astrocytes may be the predominant "building blocks" of the blood-brain barrier. Astrocytes may regulate vasoconstriction and vasodilation by producing substances such as arachidonic acid, whose metabolites are vasoactive.
  • oligodendrocytes refers to myelinating cells of the central nervous system (CNS). They are the end product of a cell lineage which has to undergo a complex and precisely timed program of proliferation, migration, differentiation, and myelination to finally produce the insulating sheath of axons.
  • the diseases caused by alterations in PDGFR-p positive cells is selected from a group consisting of: amyotrophic lateral sclerosis, systemic sclerosis and atherosclerosis, wherein said diseases are characterized by the presence of PDGFR-p positive vascular smooth muscle cells.
  • amyotrophic lateral sclerosis refers to all types of the disease including calcified plaques, non-calcified plaques, fibrocalcified plaques and others.
  • amyotrophic lateral sclerosis refers to a progressive neurodegenerative disorder which affects the upper and lower motor neuron or the lower motor neuron alone or the upper motor neuron alone.
  • systemic sclerosis refers to a multisystem disorder characterized by inflammatory, vascular, and fibrotic changes of skin and various internal organ sytems (chiefly Gl tract, lungs, heart, and kidney). Primary event may be endothelial cell injury with eventual intimal proliferation, fibrosis, and vessel obliteration. Clinical manifestations include, but are not limited to, Raynaud's phenomenon, scleroderma (fibrosis of the skin), hypertension, and renal failure.
  • therapeutic agents which can be conjugated to the fusion protein of the invention for the treatment of amyotrophic lateral sclerosis, systemic sclerosis and atherosclerosis are, without limitation, imatinib, masitinib and all of the tyrosine kinase inhibitors previously described.
  • vascular smooth muscle cells refers to the smooth muscle that makes up most of the walls of blood vessels.
  • the diseases caused by alterations in PDGFR-p positive cells is selected from a group consisting of: inflammation, arthritis rheumatoid and glaucoma, wherein said diseases are characterized by the presence of PDGFR-p positive fibroblasts.
  • inflammation refers to any condition characterized by a localized protective response elicited by injury or destruction of tissues resulting from any of the causes mentioned hereinbefore, and which is manifest by heat, swelling, pain, redness, dilation of blood vessels and/or increased blood flow, invasion of the affected area by white blood cells, loss of function and/or any other symptoms known to be associated with the inflammatory condition.
  • the term will thus be understood to include inter alia acute, chronic, ulcerative, specific, allergic and necrotic inflammation, as well as all other forms of inflammation known to those skilled in the art.
  • arthritis rheumatoid refers to a long-term autoimmune disorder that primarily affects joints. It typically results in warm, swollen, and painful joints.
  • glaucoma refers to a group of ocular neurodegenerative disorders which represents the main cause of irreversible blindness worldwide whose incidence is constantly increasing.
  • Glaucoma is characterized by altered aqueous humor outflow with a consequent increase of the intraocular pressure, by the excavation of the optic nerve head (ONH) and by the slow progressive loss of retinal ganglion cells (RGCs).
  • therapeutic agents which can be conjugated to the fusion protein of the invention for the treatment of ischemic stroke are, without limitation, tofacitinib, peficitinib, verteporfin, fasudil and ripasudil.
  • fibroblasts refers to a cell constituting a component of fibrous connective tissue, and may be a cell of connective tissue of a mammal. Fibroblasts can produce extracellular matrix and collagen, and can serve to heal wounds, for example, skin scars, burns, pressure sores, or cut wounds.
  • the diseases caused by alterations in PDGFR-p positive cells are the diseases referenced to in the column entitled “Disease or disorder” in Table 2, (diseases or disorders) wherein the therapeutic agent for each disease is the therapeutic agent indicated in the column “Therapeutic agent” of Table 2 (therapeutic agent) and said therapeutic agent is conjugated to the intervening polypeptide of the fusion protein of the invention.
  • fusion protein Use of the fusion protein, the polynucleotide, the vector, the host cell, or the nanoparticle of the invention in the diagnosis of PDGFR-p positive cancer.
  • the fusion protein or the nanoparticle of the present invention may also be used to detect and/or measure PDGFR-p, or PDGFR-p-expressing cells in a sample, e.g., for diagnostic purposes.
  • the fusion protein or nanoparticle of the invention may be used to diagnose a condition or disease characterized by aberrant expression (e.g., over-expression, under-expression, lack of expression, etc.) of PDGFR-p.
  • Exemplary diagnostic assays for PDGFR-p may comprise, e.g., contacting a sample, obtained from a patient, with the fusion protein or the nanoparticle of the invention, wherein the intervening polypeptide of the fusion protein is an imaging polypeptide or conjugated to an imaging agent as previously described, or wherein the fusion protein further comprises a reporter molecule as previously described.
  • an unlabeled fusion protein or nanoparticle of the fusion protein can be used in diagnostic applications in combination with a secondary antibody specific for the fusion protein of the invention which is itself detectably labeled with an imaging agent as previously described.
  • Specific exemplary assays that can be used to detect or measure PDGFR-p in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).
  • a further aspect of the present invention relates to the use of the fusion protein or the nanoparticle of the invention for the detection and/or measurement of PDGFR-p in a sample.
  • Another aspect of the present invention relates to the fusion protein or the nanoparticle of the invention for the diagnosis of PDGFR-p positive cancer.
  • Samples that can be used in PDGFR-p diagnostic assays according to the present invention include any tissue or fluid sample obtainable from a patient which contains detectable quantities of PDGFR-p protein, or fragments thereof, under normal or pathological conditions.
  • levels of PDGFR-beta in a particular sample obtained from a healthy patient e.g., a patient not afflicted with a disease or condition associated with abnormal PDGFR-p levels or activity
  • This baseline level of PDGFR-p can then be compared against the levels of PDGFR-p measured in samples obtained from individuals suspected of having a PDGFR-p related disease or condition.
  • PDGFB SEQ ID NO: 1
  • PDGFD PDGFD
  • PDGFRP1 SEQ ID NO: 27
  • Z09591 SEQ ID NO : 28
  • the H6 tag allows both protein purification but it also promotes assembling through coordination with divalent cations.
  • the recombinant pET22b vectors were transformed into Escherichia coli by heat shock at 42 °C for 45 s.
  • PDGFRP1-GFP-H6 (SEQ ID NO : 29) and Z09591-GFP-H6 (SEQ ID NO : 30), lacking intramolecular or intermolecular disulfide bonds, were produced in BL21 (DE3), PDGFB-GFP-H6 (SEQ ID NO: 31) in Origami B (BL21 DE3, OmpT’, Lon’, TrxB”, Gor, Novagen) and PDGFD-GFP-H6 (SEQ ID NO: 32) in BL21 (DE3) previously engineered for the expression of sulfhydryl oxidase and DsbC (kindly provided by Prof.
  • PDGFB-GFP-H6 was produced overnight at 16 °C and 250 rpm in LB supplemented with 100 pg mL -1 ampicillin, 12.5 pg mL -1 tetracycline and 15 pg mL ’ 1 kanamycin, upon addition of 1 x 10' 4 M IPTG, at ODssonm 0.6-0.8 units. Cells were then harvested by centrifugation (15 min, 5000 g) and stored at -80 °C.
  • cells were resuspended in wash buffer (2 x 10' 2 M Tris-HCI, 5 x 10' 1 M NaCI, 1 x 10' 2 M imidazole, pH 8.0) in presence of protease inhibitors (complete EDTA-free, Roche Diagnostics) and disrupted in an EmulsiFlex-C5 system (Avestin) by 3 rounds at 8000 psi.
  • the soluble fraction was then collected by centrifugation (45 min at 15000 g) and proteins purified by an immobilized metal affinity chromatography (IMAC) using a HisTrap HP column (GE Healthcare) in an AKTA pure system (GE Healthcare).
  • IMAC immobilized metal affinity chromatography
  • Elution was achieved by a lineal increase of imidazole concentration (Elution Buffer, 2 x 10' 2 M Tris-HCI, 5 x 10’ 1 M NaCI, 5 x 10’ 1 M imidazole, pH 8.0).
  • Elution Buffer 2 x 10' 2 M Tris-HCI, 5 x 10’ 1 M NaCI, 5 x 10’ 1 M imidazole, pH 8.0.
  • bacteria were grown in LB supplemented with 100 pg mL -1 ampicillin and 34 pg mL -1 chloramphenicol at 37 °C until ODssonm reached 0.4-0.5 units. In that moment, the expression of sulfhydryl oxidase and DsbC was induced by adding 0.5 % (m/v) of L- arabinose and temperature was lowered to 30 °C.
  • proteins were dialyzed against an appropriate solution, namely sodium carbonate (166 mM NaCChH, pH 8.0) for PDGFRP1-GFP-H6 and sodium carbonate with salt (166 mM NaCO 3 H, 333 mM NaCI, pH 8.0) for Z09591-GFP-H6, PDGFB-GFP-H6 and PDGFD-GFP-H6.
  • sodium carbonate 166 mM NaCChH, pH 8.0
  • salt 166 mM NaCO 3 H, 333 mM NaCI, pH 8.0
  • ZnCh was added to pure solutions of PDGFRP1-GFP-H6 (3:1 molar ratio, Zn 2+ : Histidine residues in H6) and Z09591-GFP- H6 (1 :1 molar ratio, Zn 2+ : Histidine residues in H6) to promote protein assembling and nanoparticle formation.
  • Protein purity and integrity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and western blot immunodetection using an anti-His (Santa Cruz Biotechnology) and an anti-GFP (Santa Cruz Biotechnology) antibody. The same molar amount was used for each protein.
  • Protein stability in the in vitro culture media was measured by incubating the proteins at the maximum concentration used in in vitro experiments (1 x 10' 3 M), for the maximum time of exposure (24 h) at 37 °C, and separating the soluble and insoluble fractions by centrifugation (15 min, 15000 g). A protein electrophoresis gel, followed by an anti-His Western Blot, were finally performed.
  • Nanoscale morphometry size and shape of the different protein nanoparticles was visualized at nearly native state with two rapid high- resolution imaging techniques. Drops of 5 pL of samples diluted at 0.2 pg mL -1 in its buffer were deposited in silicon wafers (Ted Pella) and observed in a field emission scanning electron microscope (FESEM) Merlin (Zeiss) operating at 1 kV and equipped with an in-lens secondary electron detector.
  • FESEM field emission scanning electron microscope
  • Three-dimensional models and visualization In silico three-dimensional structure prediction of the four PDGFR-p targeted modular proteins was performed using AlphaFold Colab. ChimeraX software version 1.2 was used then for their 3D structure visualization. GraphPad Prism 8 was used for graphics and statistical tests.
  • MEFs and MSCs were plated in p100 dishes (3x10 5 cells/p100) and exposed to nanoparticles at different concentration (from 25 nM to 100 nM) for two time periods (1 h, 24 h). When indicated, cells were treated with 50 ng/mL PDGF-BB (Prepotech) 30 min prior exposure. Cells were washed with PBS, detached from the plate, and trypsinized (1 mg/ml trypsin, Life Technologies) for 15 min at 37°C to remove membrane binding of the nanoparticles. RIPA lysis buffer plus phosphatase and protease inhibitors (Roche) was used to obtain whole-cell extracts.
  • PDGF-BB Prepotech
  • Flow cytometry Internalization of nanoparticles was quantified by GFP fluorescence signal using FACS Calibur (BD Biosciences). MEFs and MSCs were exposed at 25, 50, 100, 250, 500 and 1000 nM for 1 h or 24 h. After treatment, cells were washed with PBS, detached from the plate and trypsinized for 15 min at 37 °C to eliminate non-internalized nanoparticle. Data were analyzed using the Flow Jo software and represented as mean fluorescence intensity (MFI) fold change with respect to buffer- treated cells. All experiments were performed in triplicate.
  • MFI mean fluorescence intensity
  • Cell viability assay Cell viability upon exposure to PDGFB-GFP-H6 or PDGFD- GFP-H6 was assessed with the Cell Proliferation Kit II (XTT) (Roche) according to the manufacturer’s instructions. Briefly, 1x10 3 cells were seeded in 96 well plates and treated with different concentrations of nanoparticles (0, 250, 500 and 1000 nM) for 24 or 48h. XTT reagent was added to the plate and further incubated at 37°C for 4h, then absorbance, which directly correlates to the number of viable cells, was measured using a multi-well spectrophotometer (FLUOstar Optima, BMG Labtech). All experiments were performed in triplicate.
  • XTT Cell Proliferation Kit II
  • mice and procedures for the administration of nanoparticles All mice and procedures were carried out in accordance with the EU regulations on animal research and approved by Catalonia’s Animal ethics committee (reference 9721). Eight-week-old female C57BL/6 were purchased from Charles River (France), housed in a specific pathogen-free (SPF) environment with sterile food and water ad libitum. The subcutaneous tumor model was generated by subcutaneous injection of 1 million MC38 cells mixed with 0.5 million MEFs in the flank of the animal. Mice bearing tumors around 100-200 mm 3 were administrated intravenously with 200 pg of either PDGFB-GFP-H6 or PDGFD-GFP-H6 nanoparticles.
  • Fluorescence intensity (FLI) data are expressed as average radiant efficiency and it has been calculated subtracting the FLI signal of buffer-treated mice to the FLI signal of nanoparticle treated animals.
  • PDGFB- and PDGFD-based constructs rendered particulate materials of 16 and 26 nm (with a secondary minor peak of around 100 nm in the case of PDGFB-GFP-H6, Figure 1 B, Table 4), as expected for modular constructs based on H6-tagged GFP proteins with cationic N-terminal domains.
  • the assembling was spontaneous and it did not required additional cation supply.
  • PDGFRP1-GFP-H6 and Z09591-GFP-H6 resulted in smaller nanoparticles of around 9 nm that were only formed when cationic Zn was added to the buffer.
  • Table 3 Main biophysical properties of the ligands (top) and ligand-derived constructs (bottom). Table 4. Mean hydrodynamic size of the protein constructs measured in their respective storage buffers.
  • Ligand-carrying protein oligomers benefit from multiple surface presentation of the ligand in a virus-like fashion and from the cooperativity in the receptor-binding and endosome formation, that results in the final cytoplasmic delivery of the engulfed material.
  • PDGFD-GFP-H6 resulted in stable nanoparticles with relatively low polydispersion, with a convenient size of around 26 nm and showing a high potency for receptor-dependent specific cell uptake (Figure 2, 3).
  • this nanoparticle was the candidate most efficiently internalized by target cells (Figure 2), that it also showed an extraordinarily internalized in mice ( Figure 4), with a very selective accumulation in tumoral tissues.
  • This construct was able to target, penetrate and selectively make PDGFR-p + CAF fluorescent ( Figure 4) because of the integrated GFP ( Figure 1), that remained bioactive despite the multiple biological barriers the construct was surpassing after their intravenous administration.
  • Nanotoxins were designed in house as a codon-optimized gene and subcloned into pET22b plasmids using Ndel and Hindi 11 restriction enzymes.
  • Geneart ThermoFisher
  • PDGFD-FD-PE24-H6 PDGFD was located at the N-terminus, followed by a furin cleavable site (GNRVRRSV - SEQ ID NO: 61) flanked by two flexible linkers (GGSSRSS - SEQ ID NO: 62), the catalytic domain of Pseudomonas aeruginosa exotoxin A (PE24), the H6 tag and the C-terminal subcellular location signal KDEL (SEQ ID NO: 26).
  • the recombinant pET22b vectors were transformed into Escherichia coli by heat shock at 42 °C for 45 s.
  • bacteria were grown in LB supplemented with 100 pg mL -1 ampicillin and 34 pg mL -1 chloramphenicol at 37 °C until QD550nm reached 0.4-0.5 units.
  • the expression of sulfhydryl oxidase and DsbC was induced by adding 0.5 % (m/v) of L-arabinose and temperature was lowered to 30 °C. 45 minutes later, temperature was lowered to 20 °C and 1 x 10-3M of IPTG was added to induce overnight expression.
  • Cells were then harvested by centrifugation (15 min, 5000 g) and an osmotic shock was performed to remove the metallophores from the periplasmic fraction.
  • cells were first resuspended in a hypertonic solution (20 % sucrose, 1 x 10-3M ethylenediaminetetraacetic acid (EDTA), 5 x 10-2 M 4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid (HEPES), pH 7.9) and centrifuged (30 min, 7000 g, 4 °C). Supernatant was discarded and cells were then resuspended in a hypotonic solution (5 x 10-3 M MgSO4), placed 10 min at 4 °C and subsequently centrifuged (15 min, 4500 g) to remove again the supernatant. Bacterial cells, now without periplasm, were finally resuspended in wash buffer in presence of protease inhibitors and stored at -80 °C
  • PDGFD-DITOX-H6 PDGFD was located at the N-terminus, followed by a furin cleavable site (GNRVRRSV— SEQ ID NO: 61) preceded by a flexible linker GGSSRSS (SEQ ID NO: 62), the catalytic and translocation domain of Corynebacterium Diphtheriae toxin (including its furin cleavable site GNRVRRSV [SEQ ID NO: 61] between them) and the C-terminal hexa-histidine tag.
  • the recombinant pET22b vector was transformed into Escherichia coli BL21 (DE3) Origami B.
  • PDGFD-DITOX-H6 was produced overnight at 20 °C and 250 rpm in LB supplemented with 100 pg/ml ampicillin, 12.5 pg/ml tetracycline and 15 pg/ml kanamycin, upon addition of 1 *10-4 M IPTG, at OD 550nm 0.6-0.8 units. Cells were then harvested by centrifugation (15 min, 5000 g ) and stored at -80 °C. For protein purification, cells were resuspended in wash buffer (2 *10-2 M Tris-HCI, 5 *10-1 M NaCI, 1 *10-2 M imidazole, pH 8.0) in presence of protease inhibitors (complete EDTA-free, Roche Diagnostics).
  • monomethyl Auristatin E (OH-Glu-Val-Cit-PAB-MMAE) was purchased at MedChemExpress (HY-148245), resuspended in N- Dimethylformamide (DMF) and activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, Sigma 03449) and N-Hydroxysulfosuccinimide sodium salt (NHS- sulfo, Sigma 56481) at 1 :2:2 molar ratio for 15 minutes (25% DMF, 75% MES) at room temperature.
  • DMF N- Dimethylformamide
  • EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • NHS- sulfo N-Hydroxysulfosuccinimide sodium salt
  • Protein functionalization took place at 1 :10 proteimdrug ratio in its own storage solution (166 mM NaCO3H, 333Mm NaCI, pH 8) O/N at 4°C. Finally, excess of non-conjugated MMAE molecules were completely removed by nanoconjugates IMAC re-purification using HisTrap HP 1 mL columns in an AKTA pure (Cytiva) chromatography system at 4°C and dialyzed again against its storage solution (166 mM NaCO3H, 333Mm NaCI, pH 8).
  • DLS Dynamic light scattering
  • mice and procedures were carried out in accordance with the Ell regulations on animal research and approved by Catalonia’s Animal ethics committee.
  • Eight-week- old female C57BL/6 or Swiss Nude were purchased from Charles River (France), housed in a specific pathogen-free (SPF) environment with sterile food and water ad libitum.
  • SPF pathogen-free
  • the subcutaneous tumor model was generated by subcutaneous injection of 1 million MC38 cells mixed with 0.5 million MEFs in the flank of the C57BL/6 animals. Mice with 20-30mm3 tumors were randomised and intravenously administered with 1mg/Kg of PDGFD-NT-H6 nanotoxin or NaHCO3, daily up to 7 doses. Tumors were harvested 72h after last dose and measure ex-vivo to calculate their volume. For head and neck tumor model, one million UM-SCC-74B cells were implanted into the tongue of Swiss Nude mice.
  • mice were randomised and intravenously administered with 1mg/Kg of PDGFD-NT-H6 nanotoxin, PDGFD-GFP-H6 or NaHCO3, daily up to 7 doses. Twenty-four hours after last dose, animals were euthanized and tumor were measured with a calliper. Tumour samples were paraffin-embedded histological analysis. Tumor volume was calculate as width2 x length x 0.5.
  • hCAFs Membrane expression of PDGFRp and Internalization (GFP) of nanoparticles in human CAFs were quantified using MACSQuantIO analyser (Milteny).
  • hCAFs were exposed to 50nM PDGFD-GFP-H6 for 1h. After treatment, cells were washed with PBS, detached from the plate and trypsinized for 15 min at 37 °C to eliminate non-internalized nanoparticle.
  • Anti-PDGFRp-PE antibody (Milteny) was added to the cell to detect membrane receptor expression. Fluorescence was detected and the percentage of positive cell was calculated. Untreated, unlabelled cells were considered as negative population. Data were analyzed using MACSQuantify software (Milteny) and represented as percentage of positive cells. All experiments were performed in triplicate. hCAFs isolation
  • Subcutaneous tumor-bearing immunocompetent mice were treated with PDGFD- FD-PE24-H6 nanotoxin, which formed nanoparticles of 44.4 nm (Figure 10A), daily for 7 days (Figure 10B). Histological tumor analysis revealed that nanotoxin treatment increase blood vessels density as well as macrophages and T-lymphocytes within the tumor, suggesting that depletion of PDGFR-expressing CAFs impact tumor microenvironment content (Figure 10C).
  • the PDGFD-FD-PE24-H6 nanotoxin do not only achieve an antitumor effect in a colorectal cancer model but also in a head and neck squamous cell carcinoma.
  • PDGFR- targeted nanotoxin treatment showed to be innocuous to UM-SCC-74B, a tongue squamous cell carcinoma human cell line ( Figure 11A-B).
  • UM-SCC-74B cells were orthotopically implanted in immunodeficient nude mice. Animals treated with CAFs-targeted nanotoxin developed smaller tumors than buffer-treated mice ( Figure 11C). In agreement with the smaller size, the proliferation marker, ki67, was downregulated in the treated group ( Figure 11 D), suggesting that CAFs targeting strategy impact cancer cell behavior.
  • PDGFD-FD-PE24-H6 nanotoxin another nanotoxin, PDGFD-DITOX-H6, that contains diphtheria toxin within their structure, was developed.
  • PDGFD-DITOX-H6 monomer self-assembled to form nanoparticle around 24nm ( Figure 13A).
  • PDGFD-DITOX-H6 nanotoxin showed to be cytotoxic to MSCs, a fibroblast cell line ( Figure 13B).
  • the scaffold protein PDGFD-GFP-H6 was used to chemically conjugate monomethyl auristatin E (MMAE), a highly toxic antitumor drug that blocks microtubule polymerization.
  • MMAE monomethyl auristatin E
  • PDGFD-GFP-H6-MMAE formed 43.7 nm nanoparticles ( Figure 14A) and exhibited cytotoxic activity when added to fibroblast cell line MEFs and MSCs ( Figure 14B).
  • PDGFD-GFP-H6 when cells were first exposed to the unconjugated nanoparticle, PDGFD-GFP-H6, the ability of the nanoconjugate to eliminate PDGFR-expressing fibroblast was impaired, indicating that PDGFD-GFP-H6-MMAE exerts it cytotoxic effect in a receptor dependent manner ( Figure 14B).

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Abstract

The present invention relates to the field of nanostructured protein materials, more specifically to therapeutic agents carrying fusion proteins which can be used for therapy.

Description

NANOCONJUGATES CONTAINING PDGFR-p LIGANDS AND USES THEREOF
FIELD OF THE INVENTION
The present invention relates to the field of nanostructured protein materials, more specifically to therapeutic agents carrying fusion proteins which can be used for therapy.
BACKGROUND OF THE INVENTION
Cancer represents a leading cause of death worldwide and an unmet clinical problem. It is so despite the substantial economic and human resources invested in antitumor drug discovery and the diversity of innovative therapeutic approaches and emerging materials intended for more effective drug delivery that are currently under evaluation. The generic lack of selectivity and high toxicity of most chemical drugs, the development of drug resistance and relapse and the multiple facets that support cancer development and progression make this goal especially complex. Increasing drug selectivity for target cells through novel smarter and selective drug vehicles and identifying complementary therapeutic targets should both support significant steps towards successful cancer therapies. In the context of these non-exclusive alternatives, high precision drug delivery is being explored mainly by the refinement of nanomedical tools and by developing biocompatible materials within the nanoscale as drug carriers. Their building blocks are to be functionalized with specific ligands of overexpressed tumoral markers for selective cell destruction. Metastatic cancer stem cells are particularly appealing targets for high precision nanomedicines as they are the origin of metastases, the main cause of patients’ death. So far, despite being theoretically promising, attempts of selectivity in cancer nanomedicines have not generically reached accumulation in target tissues over 1-2 % of the administered material. Many nanoscale materials, because of their physicochemical properties or their natural interactivity with biological receptors are in fact retained in liver or phagocyted, precluding the desired biodistribution of the carrier and the payload drug. Among other materials under exploration for high precision nanomedicines are lipids, carbon nanotubes, ceramics, metals and non-protein polymers, that might pose concerns regarding cell toxicity and permanence in the media. In addition, proteins, as main biological molecules, are excellent building blocks for the biofabrication of nanomaterials. Notably, production of clinically-oriented engineered proteins or their resulting protein materials is, in addition, supported by 40 years of experience in the large- scale biofabrication of protein drugs for human clinics. On the other hand, the identification of alternative or complementary targets for selective cell killing offers promising routes to designing innovative drugs. Looking at those other potential strategies, stroma refers to the tissue that surrounds epithelial cancer cells, including extracellular matrix, endothelial cells, immune cells and fibroblasts, which modulate and participate in cancer development. Among these elements, fibroblasts, synthesize and remodel the extracellular matrix being then responsible for the cancer tissue architecture and structural properties. Fibroblasts are also responsible for the production of growth factors and cytokines that will be secreted within the tumor, supporting its development. In particular, a specific subpopulation of this cell type, stromal PDGFR-P+ fibroblasts are a pivotal, highly interesting cell type for imaging, drug delivery or selective destruction in the context of cancer therapies. Despite the robust data supporting PDGFR-P+ fibroblasts as targets in cancer therapies, the modest explorations have been so far limited to interfere signaling, but appropriate drugs or drug vehicles have not been yet developed and this type of approach has been essentially neglected.
SUMMARY OF THE INVENTION
The inventors herein have fully validated the fusion protein PDGFD-GFP-H6, among the starting candidates, as a potent prototype for molecular delivery, imaging and theranostics in cancer through the so far neglected targeting of CAF. Importantly, this is the first time that the bacterial production and the use of PDGFD as a targeting ligand are reported, what opens a spectrum of clinical avenues. In this context, it is worthy to stress that protein nanoparticles resulting from oligomeric self-assembling can be functionalized through intrinsic, biological active protein domains, by chemically coupling drugs or through combining both strategies. In addition, this result supports proteins as editable, versatile and biodegradable building blocks suited to construct nanoscale oligomers for clinical purposes.
Therefore, in a first aspect, the invention relates to a fusion protein comprising
(i) a PDGFR-p ligand selected from the group of consisting of PDGFB (SEQ ID NO: 1) and PDGFD (SEQ ID NO: 2) or a functionally equivalent variant thereof,
(ii) an intervening polypeptide region and
(iii) a positively charged amino acid-rich region.
In a further aspect, the invention relates to a method for preparing a fusion protein according to the invention selected from: (i) A method comprising a) providing a fusion protein comprising i) a PDGFR-p ligand selected from the group of consisting of PDGFB (SEQ ID N0 : l), PDGFD (SEQ ID NO : 2) and a functionally equivalent variant thereof, ii) an intervening polypeptide region and iii) a positively charged amino acid-rich region, wherein the PDGFR-p ligand and the positively charged amino acid-rich region are located at the ends of the protein and b) contacting said fusion protein with an activated form of a therapeutic agent or of an oligomeric form thereof wherein said activated form of a therapeutic agent or of an oligomeric form thereof contains a reactive group which is capable of reacting with at least one group in the intervening region of the fusion protein and wherein the contacting is carried out under conditions adequate for the formation of a bond between the reactive group in the therapeutic agent and the group in the intervening polypeptide region or
(ii) a method comprising c) providing a fusion protein comprising i) a PDGFR-p ligand selected from the group of consisting of PDGFB (SEQ ID N0 : l), PDGFD (SEQ ID NO : 2) and a functionally equivalent variant thereof, ii) an intervening polypeptide region and iii) positively charged amino acid-rich region, wherein the PDGFR-p ligand and the positively charged amino acid-rich region are located at the ends of the protein and wherein the fusion protein is provided in an activated form, wherein said activated form of the fusion protein contains a reactive group in the intervening region and d) contacting said fusion protein with a therapeutic agent or an oligomeric form thereof, wherein said therapeutic agent contains a group which is capable of reacting with the reactive group in the fusion protein, wherein said contacting is carried out under conditions adequate for the formation of a bond between the reactive group in the fusion protein and the group in the therapeutic agent.
Another aspect of the invention relates to a polynucleotide encoding a fusion protein according to the invention.
One more aspect of the invention relates to a vector comprising a polynucleotide according to the invention.
Yet another aspect, the invention relates to a host cell comprising a polynucleotide according to the invention or a vector according to the invention.
A further aspect of the invention relates to a method for preparing a nanoparticle comprising multiple copies of the fusion protein according to the invention comprising placing a preparation of said fusion protein in a suitable buffer.
Another aspect of the present invention relates to a nanoparticle comprising multiple copies of the fusion protein according to the invention or which has been obtained by a method according to the invention.
One more aspect of the present invention relates to the fusion protein according to the invention, the polynucleotide according to the invention, the vector according to the invention, the host cell according to the invention or the nanoparticle according to the invention for use in medicine.
A further aspect of the invention relates to the fusion protein according to the invention, the polynucleotide according to the invention, the vector according to the invention, the host cell according to the invention or the nanoparticle according to the invention wherein the intervening polypeptide is an antitumor peptide or wherein the intervening polypeptide is linked to an antitumor agent for use in the treatment of cancer.
DESCRIPTION OF THE FIGURES
Figure 1. Design and production of PDGFR-p targeted-proteins. A. Protein gel electrophoresis, showing the purity of the obtained proteins and the proteolytic integrity after affinity purification and further dialysis. P1 refers to PDGFRP1-GFP-H6 (29.7 kDa), Z to Z09591-GFP-H6 (34.6 kDa), B to PDGFB-GFP-H6 (40.5 kDa) and D to PDGFD- GFP-H6 (42.2 kDa). M indicates the marker line and the siding numbers the molecular mass of the markers expressed in kDa. B. Particle size determined by DLS expressed in nm. Numbers indicate the mean peak size for every material. Additional data can be found in the Table 4. C. Representative FESEM images of isolated nanoparticles. The white bars represent 50 nm. Broader fields and TEM images are shown in the Figure 6. Figure 2. Internalization of CAFs-directed nanoparticles. Two mesenchymal cell types, mouse embryonic fibroblast (MEFs) and mouse mesenchymal stem cells (MSCs), were exposed to 100 nM of different nanoparticles. (A) GFP, PDGFR-p and Tubulin immunoblotting of MEFs and MSCs whole cell extracts after 1 h exposure. (B) Nanoparticle internalization analysed by flow cytometry in MEFs and MSCs upon 1 h or 24 h exposure. Values are expressed as mean fluorescence intensity fold change respect to untreated cells. (C) GFP detection by confocal microscopy upon 1 h exposure, Blue: DAPI. Selected fields are enlarged in the insets for a better visualization. ** p<0.01 ; *** p<0.001. Bars size: 50 nm.
Figure 3. PDGFRp is required for internalization of nanoparticles. MEFs were incubated with 50 ng/mL PDGF-BB for 30 min following 100 nM PDGFB-GFP-H6 or PDGFD-GFP-H6 treatment for 1 h. (A) Fixed cell were stained with DAPI and analysed by immunofluorescence. (B) Whole cell extracts were used for detection of PDGFR p and GFP, by immunoblotting. Tubulin was used as loading control. (C) Graphs represents densitometry quantification of 3 independent assays. * p<0.05; ** p<0.01 ; *** p<0.001 Bar size: 50 nm.
Figure 4. Biodistribution and in vivo uptake of PDGFB-GFP-H6 and PDGFD-GFP- H6. (A) Immunohistochemistry detection of PDGFRp within tumors. (B) GFP fluorescence detection by IVIS-Spectrum in tumors upon 2 h after PDGFB-GFP-H6 or PDGFD-GFP-H6 administration. (C) Quantification of fluorescence in buffer, PDGFB- GFP-H6 or PDGFD-GFP-H6 treated tumors n=4. (D) Immunohistochemistry against GFP to detect CAFs-targeting nanoparticle in tumor, kidney and liver of treated mice. * p<0.05. Bars Size: 50 pm.
Figure 5. Integrity and assembling of protein building blocks. A. Western blot immunodetection of purified proteins after dialysis, using either anti-His or anti-GFP antibodies. P1 refers to PDGFRP1-GFP-H6, Z to Z09591-GFP-H6, B to PDGFB-GFP- H6 and D to PDGFD-GFP-H6. M indicates the marker line and the siding numbers the molecular mass of the markers in kDa. B. Particle size upon treatment of nanoparticles with 1 % SDS, for disassembling. Numbers indicate the mean peak size (in nm) for every building block.
Figure 6. Electron microscopy of assembled nanoparticles. Representative high- resolution images of electron microscopy (TEM and FESEM) of well-formed nanoparticles resulting from the assembly of the four GFP-H6 constructs with PDGFPR1 , Z09591 , PDGFB and PDGFD. Bars size: 50 nm. Figure 7. Stability of protein nanoparticles. A. Intrinsic GFP-emission at 511 nm upon fluorophore excitation at 488 nm shown in relative percentage (100 % accounting for the intrinsic fluorescence of a non-targeted GFP-H6 fusion). B. Anti-His western blot showing protein integrity and stability upon incubation of the materials with MEM-Alpha during 24 h at 1000 nM. No precipitation or degradation was observed for any of the candidates. S and I indicate Soluble and Insoluble protein fractions, respectively.
Figure 8. PDGFB-GFP-H6 and PDGFD-GFP-H6 dose response in MEFs. MEFs were exposed to different concentrations of either PDGFB-GFP-H6 or PDGFD-GFP-H6 nanoparticles for 1 h. (A) Immunoblotting against GFP and PDGFR-p of whole cell extract from treated MEFs. Tubulin was used as loading control. (B) Internalization of either PDGFB-GFP-H6 or PDGFD-GFP-H6 measured by GFP detection through flow cytometry. Values are expressed as mean fluorescence intensity fold change respect to untreated cells. (C) Nanoparticle (GFP) and PDGFR-p (RED) detection by epifluorescence microscopy in MEFs exposed to nanoparticles at 3 different concentrations. *** p<0.001. Scale bar: 50 mm.
Figure 9. CAFs-targeting nanoparticles are non-toxic for fibroblasts. Viability of MEFs and MSCs exposed to either PDGFB-GFP-H6 (A) or PDGFD-GFP-H6 (B) for 24 and 48 h. Protein nanoparticles were added to cell cultures at 100 nM, 500 nM and 1000 nM. Untreated cells (0) were considered as being 100 % viable.
Figure 10. PDGFD-FD-PE24-H6 nanotoxin alters the colorectal tumor microenvironment. (A) nanotoxin is a self-assembly nanoparticle of 44.4nm as detected by DLS. (B) Schematic representation of the experimental colorectal cancer mouse model administered with PDGFD-FD-PE24-H6 (PDGFD-NT-H6) nanotoxin. (C-E) Quantification of immunohistochemistry in tumor sections against (C) blood vessel, CD31 ; (D) Macrophages, F4/80; (E) Lymphocytes CD3, CD4, and CD8. In all cases, nanotoxin administration caused an increase in the analyzed tumor microenvironment cell type. *p>0.05, **p>0.01 , ***p>0.001.
Figure 11. PDGFD-FD-PE24-H6 nanotoxin exerts antitumor activity in an orthotopic head and neck mouse tumor model. (A) Cell viability assay (XTT) was performed in PDGFRp-expressing fibroblasts (MSCs) and in a human head and neck squamous cell carcinoma cell line (UM-SCC-74B). Cell viability was quantified after cell exposure to 50nM PDGFD-NT-H6 for 48h. Only fibroblasts were sensitive to the nanotoxin. (B) Schematic representation of the UM-SCC-74B orthotopic mouse model used to evaluate the antitumor activity of the nanotoxin. (C) At the final point tumors were measured ex vivo and the final tumor volume was calculated. (D) Tumor sections were stained against Ki67 to quantify tumor cell proliferation. Tumors from treated mice showed a decrease in the proliferation compared to buffer treated counterparts. *p>0.05, ***p>0.001.
Figure 12. Primary human CAFs isolated from head and neck tumors are sensitive to PDGFD-FD-PE24-H6 nanotoxin. Human CAFs were isolated from fresh tumor tissue surgically removed from head and neck squamous cell carcinoma patients. (A) Isolated CAFs were exposed for 1 h to 50nM PDGFD-GFP-H6 nanoparticle. CAFs were analyzed using flow cytometry to quantify PDGFRp expression (PDGFRP+) and nanoparticle internalization (GFP+). (B) CAFs sensitivity to PDGFD-FD-PE24-H6 was assessed by XTT upon cell exposure to 50nM PDGFD-FD-PE24-H6 for 48h. The viability of buffer- treated CAFs was considered to be 100 %. ***p>0.001.
Figure 13. CAFs-targeted nanoparticles that integrate the C. diphteriae toxin domain are cytotoxic to PDGFRp-expressing fibroblasts. (A) The GFP domain of the scaffold PDGFD-GFP-H6 monomer was replaced by a toxic domain from C. diphteriae, PDGFD-DITOX-H6. The monomers auto ensemble to form nanoparticles of 24.01 nm, assessed by DLS. (B) Fibroblasts expressing PDGFRp (MSCs) were exposed to increase concentrations of PDGFD-DITOX-H6 for 48h. *p>0.05, **p>0.01.
Figure 14. The monomethyl auristatin E (MMAE) nanoconjugate selectively eliminated PDGFRp-expressing fibroblasts. (A) PDGFD-GFP-H6 monomer was conjugated to MMAE, a potent microtubule inhibitor that triggers cell death. DLS confirms that once conjugated it self-assemble to form 43.7 nm nanoparticles. (B) PDGFD-GFP- H6-MMAE nanoconjugate is able to induce cell death in MEFs and MSCs when exposed at 50nM for 72h. In both cases the use of the scaffold protein as a receptor competitor 1 h before nanoconjugate addition results in a strong inhibition of the fibroblasts death. **p>0.01 , ***p>0.001.
DETAILED DESCRIPTION OF THE INVENTION
The inventors have developed a particular type of PDGFR-p-targeted nanoparticles based on biofabricated, self-assembling proteins, upon a hierarchical and iterative selective process starting from four initial candidates. These nanoparticles, produced by simple and fully scalable biofabrication processes, have been successfully validated as highly selective targeting agents, both in cell culture and in vivo, using multiple analytical procedures. The data fully supports the concept of selective drug carriers based on nanoscale protein materials to target the main architectonic agents in solid tumors for their further development and use in the clinical setting.
Fusion proteins of the invention
Thus, a first aspect of the present invention relates to a fusion protein comprising
(i) a PDGFR-p ligand selected from the group of consisting of PDGFB (SEQ ID N0: l) and PDGFD (SEQ ID NO: 2) or a functionally equivalent variant thereof,
(ii) an intervening polypeptide region and
(iii) a positively charged amino acid-rich region.
The term “fusion protein” is well known in the art, referring to a single polypeptide chain artificially designed which comprises two or more sequences from different origins, natural and/or artificial. The fusion protein, per definition, is never found in nature as such.
The term “single polypeptide chain”, as used herein means that the polypeptide components of the fusion protein can be conjugated end-to-end but also may include one or more optional peptide or polypeptide ’’linkers” or “spacers” intercalated between them, linked by a covalent bond.
The term “peptide” or “polypeptide”, as used herein, generally refers to a linear chain of around 2 to 40 amino acid residues joined together with peptide bonds. It will be understood that the terms “peptide bond”, “peptide”, “polypeptide” and protein are known to the person skilled in the art. From here on, “peptide” and “polypeptide” will be used indistinctly.
As used herein, an "amino acid residue" refers to any naturally occurring amino acid, any amino acid derivative or any amino acid mimic known in the art. In certain embodiments, the residues of the protein or peptide are sequential, without any nonamino acid interrupting the sequence of amino acid residues. In other embodiments, the sequence may comprise one or more non-amino acid moieties. In particular embodiments, the sequence of residues of the protein or peptide may be interrupted by one or more non-amino acid moieties.
In order to avoid possible deleterious interactions functionally between different regions of the protein, the fusion protein of the invention is devoid of disulfide bridges. The term “disulfide bridges” as used herein refers to two cysteine residues which are adjacent in the three-dimensional structure of a protein and which can be oxidized to form a disulfide bond A. The PDGFR-p ligand
The PDGFR-p ligand is selected from the group of consisting of PDGFB (SEQ ID NO: 1) and PDGFD (SEQ ID NO: 2) or a functionally equivalent variant thereof.
The term “PDGFR-P”, as used herein, refers to the protein “platelet-derived growth factor receptor beta” which is encoded by the PDGFRB gene in humans. The PDGFRB gene encodes a typical receptor tyrosine kinase, which belongs to the type III tyrosine kinase receptor (RTK) family and is structurally characterized by five extracellular immunoglobulin-like domains, a single membrane-spanning helix domain, an intracellular juxtamembrane domain, a split tyrosine kinase domain and a carboxylic tail. PDGFR-p is essential for vascular development, and that PDGFB is responsible for activating PDGFR-p during embryogenesis. Eliminating either PDGFR-p, or PDGFB reduces the number of pericytes and vascular smooth muscle cells, and thereby compromises the integrity and/or functionality of the vasculature in multiple organs, including the brain, heart, kidney, skin and eye. Furthermore, PDGFR-p participates in cell differentiation, migration and proliferation processes in several neoplasias. PDGFR- P-expressing fibroblasts are positively involved in cancer progression and its infiltration in tumors compromises patient survival, especially in non-small cell lung, breast, pancreatic and colorectal cancer. Importantly, its expression levels in cancer-associated fibroblasts (CAFs) correlates with bad prognosis, relapse and drug resistance in several types of cancer.
The term “ligand” denotes a bioactive molecule which binds to a cell-associated protein termed "receptor". Both “receptor” and “ligand” are commonly known to those skilled in the art. Accordingly, a “PDGFR-p ligand” will be a molecule capable of binding to PDGFR-p.
The terms “functional variant” and “functionally equivalent variant” are interchangeable and are herein understood as all those peptides derived from the PDGFB and/or the PDGFD peptides by means of modification, insertion and/or deletion of one or more amino acids, provided that the function of binding to PDGFR-p and internalizing the fusion protein is substantially maintained.
In one embodiment, functionally equivalent variants of the PDGFR-p ligands are those showing a degree of identity with respect to the PDGFB and/or the PDGFD peptides, according to their respective SEQ ID NOs, greater than 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%. The degree of identity between two amino acid sequences can be determined by conventional methods, for example, by means of standard sequence alignment algorithms known in the state of the art, such as, for example BLAST (Altschul S.F. et al., J. Mol. Biol., 1990 Oct 5; 215(3): 403- 10). The PDGFR-p ligands of the invention may include post-translational modifications, such as glycosylation, acetylation, isoprenylation, myristoylation, proteolytic processing, etc.
In a particular embodiment the PDGFR-p ligand is PDGFB whose sequence consist of SEQ ID NO: 1. In another particular embodiment the PDGFR-p ligand is PDGFD whose sequence consist of SEQ ID NO: 2.
Alternatively, suitable functional variants of the PDGFR-p ligands are those wherein one or more positions contain an amino acid which is a conservative substitution of the amino acid present in the PDGFB and/or the PDGFD peptides mentioned above. "Conservative amino acid substitutions" result from replacing one amino acid with another having similar structural and/or chemical properties For example, the following six groups each contain amino acids that are conservative substitutions for 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 skill of one of ordinary skill in the art and is described, for example by Dordo et al. et al., (J. Mol. Biol, 1999, 217;721-739) and Taylor et al., (J. Theor. Biol., 1986, 119:205-218).
A suitable assay for determining whether a given peptide can be seen as a functionally equivalent variant thereof is, for instance, the following assay: a putative PDGFB and/or the PDGFD peptide variant is fused in frame with a marker polypeptide (e.g. a fluorescent protein). Such fusion proteins can be obtained by preparing a recombinant nucleic acid wherein the nucleic acids encoding the peptide and the fluorescent protein are fused in frame and expressed in an adequate host cell or organism. The fusion protein is then contacted with a culture of cells PDGFR-p (e.g. mouse embryonic fibroblast (MEFs) and mesenchymal stem cells (MSCs)) for an appropriate amount of time after which fluorescence microscopy may be used to determine whether the construct penetrated the cell. If the peptide is a functionally equivalent variant of the corresponding peptide, the marker protein will be internalized and presence of fluorescence in the cytoplasm of the cell will be visible. Furthermore, the performance of the functionally equivalent variant can be assayed by comparing the fluorescence microscopy image resulting from the fluorescent protein to that obtained with a known cytoplasmic stain (e.g. DAPI). B. Positively charged amino acid-rich region
The term “positively charged amino acid” as used herein, refers to a polypeptide sequence characterized in that it contains multiple positively charged amino acids. In addition, the positively charged amino acid-rich region may be formed exclusively by positively charged amino acids or may contain other amino acids provided that the overall net charge of the region at pH 7 is positive. Thus, the positively charged amino acid-rich region sequence may comprise 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 acids residues.
The positively charged amino acid-rich region may contain only one type of positively charged amino acid or may contain 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 comprises lysine and arginine residues. In one embodiment, the positively charged amino acid-rich region comprises lysine and histidine residues. In one embodiment, the positively charged amino acid-rich region comprises arginine and histidine residues. In one embodiment, the positively charged amino acid-rich region comprises lysine, arginine and histidine residues
In some embodiments, the positively charged amino acid-rich region comprises 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 acids residues, wherein the positively charged amino acid can be histidine, lysine, arginine or combinations thereof.
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 less positively charged amino acids residues, wherein the positively charged amino acid can be histidine, lysine, arginine or combinations thereof. In some embodiments, the positively charged amino acid-rich region comprises between 2 and 50 amino acids, between 2 and 40 amino acids, between 2 and 30 amino acids, between 2 and 25 amino acids, between 2 and 20 amino acids, between 2 and 10 amino acids or between 2 and 8 amino acids.
In some embodiments, the positively charged amino acid-rich region comprises between 3 and 50 amino acids, between 3 and 40 amino acids, between 3 and 30 amino acids, between 3 and 25 amino acids, between 3 and 20 amino acids, between 3 and 10 amino acids or between 3 and 8 amino acids. In some embodiments, the positively charged amino acid-rich region comprises between 4 and 50 amino acids, between 4 and 40 amino acids, between 4 and 30 amino acids, between 4 and 25 amino acids, between 4 and 20 amino acids, between 4 and 10 amino acids or between 4 and 8 amino acids. In some embodiments, the positively charged amino acid-rich region comprises between 5 and 50 amino acids, between 5 and 40 amino acids, between 5 and 30 amino acids, between 5 and 25 amino acids, between 5 and 20 amino acids, between 5 and 10 amino acids or between 5 and 8 amino acids.
In an embodiment of the invention, the positively charged amino acid-rich region of the fusion protein of the invention is a polyhistidine region. In a preferred embodiment of the invention, the polyhistidine region comprises between 2 and 10 contiguous histidine residues. In an embodiment of the invention, the positively charged acid-rich region is a polyhistidine region according to the sequence HHHHHH (S EQ ID NO: 3).
In an embodiment of the invention, the positively charged amino acid-rich region of the fusion protein of the invention is a polyarginine region. In a preferred embodiment of the invention, the polyarginine region comprises between 2 and 10 contiguous arginine residues.
In an embodiment of the invention, the positively charged amino acid-rich region of the fusion protein of the invention is a polylysine region. In a preferred embodiment of the invention, the polylysine region comprises between 2 and 10 contiguous polylysine residues.
C. Relative positions of the elements of the fusion proteins and linking elements
The different elements of the fusion protein (PDGFR-p ligand, intervening polypeptide region, and positively charged amino acid-rich region) of the invention can be placed in any relative order provided that the PDGFR-p ligand and the positively charged amino acid-rich region are functional on any position of the fusion protein and also the intervening polypeptide region remains functional totally or in part. As used herein, the terms “N-terminal end”, “N-terminus”, and “amino-terminal end” of a polypeptide are indistinct. Equally, the terms “C-terminal end”, “C-terminus”, and “carboxi-terminal end” are considered equivalent. The terms are of common usage for the person skilled in the art regarding the free moieties of the amino acids at the ends of the polypeptide chains comprised by a protein.
Thus, in an embodiment of the invention, the PDGFR-p ligand of the fusion protein is located at the N-terminal end of the protein, while the positively charged amino acidrich region of the fusion protein is located at the C-terminal end of the protein. In another embodiment of the invention, the positively charged amino acid-rich region of the fusion protein is located at the N-terminal end of the protein, while the PDGFR-p ligand of the fusion protein is located at the C-terminal end of the protein. In another embodiment of the invention, the intervening polypeptide region can be located at either the C-terminal end or the N-terminal end of the fusion protein, while the PDGFR-p ligand is in the middle position of the fusion protein and the positively charged amino acid-rich region is at the end of the fusion protein opposite the Intervening polypeptide region, or the positively charged amino acid-rich region is in the middle position of the fusion protein and the PDGFR-p ligand is located at the end of the fusion protein opposite the Intervening polypeptide region.
Accordingly, the relative order of the elements of the fusion protein according to the invention, can be:
■ N- PDGFR-p ligand-intervening region polypeptide- positively charged amino acid-rich region-C;
■ N- positively charged amino acid-rich region-intervening region polypeptide- PDGFR-p ligand -C;
■ N-PDGFR-p ligand - positively charged amino acid-rich region-intervening region polypeptide-C;
■ N- positively charged amino acid-rich region- PDGFR-p ligand -Intervening region polypeptide-C;
■ N-lntervening region polypeptide- PDGFR-p ligand - positively charged amino acid-rich region-C; or
■ N-lntervening region polypeptide- positively charged amino acid-rich region- PDGFR-p ligand -C
The terms “N-terminal end” and “C-terminal end” do not mean that the components need to be directly conjugated end-to-end, but that they maintain that relative order of positions regardless of the presence of additional elements at the end of either component or intercalated between them, such as linkers/spacers.
Therefore, the fusion protein of the invention comprises the aforementioned elements ((1) PDGFR-p ligand, (2) intervening polypeptide region, and (3) positively charged amino acid-rich region) and these can be conjugated end-to-end but also may include one or more optional peptide or polypeptide ’’linkers” or “spacers” intercalated between them, linked, preferably by peptidic bond.
Thus, in an embodiment of the invention, the PDGFR-p ligand is connected to the intervening polypeptide via a first peptide linker and/or wherein the intervening polypeptide is connected to the positively charged amino acid-rich region via a second peptide linker.
According to the invention, the spacer or linker amino acid sequences can act as a hinge region between components (1) and (2) and (2) and (3), allowing them to move independently from one another while maintaining the three-dimensional form of the individual domains, such that the presence of peptide spacers or linkers does not alter the functionality of any of the components (1), (2) and (3). In this sense, a preferred intermediate amino acid sequence according to the invention would be a hinge region characterized by a structural ductility allowing this movement. In a particular embodiment, said intermediate amino acid sequence is a flexible linker. The effect of the linker region is to provide space between the components (1) and (2) and (2) and (3). It is thus assured that the secondary and tertiary structure of component (1), (2) or (3) is not affected by the presence of either of the others. The spacer is of a polypeptide nature. 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 approximately 100 amino acids.
The spacer or linker can be bound to components flanking the two components of the conjugates of the invention by means of covalent bonds, preferably by peptide bonds; and also preferably the spacer is essentially afunctional, and/or is not prone to proteolytic cleavage, and/or does not comprise any cysteine residue. Similarly, the three- dimensional structure of the spacer is preferably linear or substantially linear.
The preferred examples of spacer or linker peptides include those that have been used to bind proteins without substantially deteriorating the function of the bound peptides or at least without substantially deteriorating the function of one of the bound peptides. More preferably the spacers or linkers used to bind peptides comprise coiled coil structures.
Preferred examples of linker peptides comprise 2 or more amino acids selected from the group consisting of glycine, serine, alanine and threonine. A preferred example of a flexible linker is a polyglycine linker. The possible examples of linker/spacer sequences include GGSSGGS (SEQ ID NO: 4), SGGTSGSTSGTGST (SEQ ID NO : 5), AGSSTGSSTGPGSTT (SEQ ID NO: 6) or GGSGGAP (SEQ ID NO: 7). These sequences have been used for binding 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 comprise the amino acid sequence GGGVEGGG (SEQ ID NO: 8), the sequence of 10 amino acid residues of the upper hinge region of murine lgG3 (PKPSTPPGSS, SEQ ID NO : 9), which has been used for the production of dimerized antibodies by means of a coiled coil (Pack, P. and Pluckthun, A., 1992, Biochemistry 31 :1579-1584), the peptide of sequence APAETKAEPMT (SEQ ID NO: 10), the peptide of sequence GAP, the peptide of sequence AAA and the peptide of sequence AAALE (SEQ ID NO: 11).
Alternatively, the components of the conjugates of the invention can be connected by peptides the sequence of which contains a cleavage target for a protease, thus allowing the separation of any of the components. Protease cleavage sites suitable for their incorporation into the polypeptides of the invention include enterokinase (cleavage site DDDDK, SEQ ID NO: 12), factor Xa (cleavage site IEDGR, SEQ ID NO: 13), thrombin (cleavage site LVPRGS, SEQ ID NO: 14), TEV protease (cleavage site ENLYFQG, SEQ ID NO: 15), PreScission protease (cleavage site LEVLFQGP, SEQ ID NO: 16), inteins and the like. As such, in an embodiment of the invention the first peptide linker and/or the second peptide linker comprise a cleavable target site.
In another embodiment of the invention, the PDGFR-p ligand is bound to the intervening polypeptide region through a first linker and the intervening polypeptide region is bound to the positively charged amino acid-rich region through a second linker also.
As the person skilled in the art will acknowledge, the linkers connecting the PDGFR-p ligand to the intervening polypeptide region and the intervening polypeptide region to the positively charged amino acid-rich region may comprise the same sequence or different ones with the aforementioned limitation that the presence and/or sequence of the linkers does not result in functional alterations of the PDGFR-p ligand, the intervening polypeptide region, and/or the positively charged amino acid-rich region (for instance, but not limited to, due to secondary or tertiary structure modifications of the fusion protein or formation of disulfide bonds).
The aforementioned considerations regarding the relative positions from the N- terminal end to the C-terminal end of the elements of the fusion protein apply also in the presence of linkers between them, independently of the number of them or what elements they are placed between. Therefore, the possible combinations and relative orders of elements will be the following (wherein the numbering stated above for the elements is retained: (1) PDGFR-p ligand, (2) intervening polypeptide region, (3) positively charged amino acid-rich region):
■ N-(1)-(2)-(3)-C
■ N-(1)-linker-(2)-(3)-C
■ N-(1)-(2)-linker-(3)-C
■ N-(1)-linker-(2)-linker-(3)-C
■ N-(3)-(2)-(1)-C
■ N-(3)-linker-(2)-(1)-C
■ N-(3)-(2)-linker-(1)-C
■ N-(3)-linker-(2)-linker-(1)-C
■ N-(2)-(1)-(3)-C
■ N-(2)-linker-(1)-(3)-C
■ N-(2)-(1)-linker-(3)-C
■ N-(2)-linker-(1)-linker-(3)-C
■ N-(2)-(3)-(1)-C
■ N-(2)-linker-(3)-(1)-C
■ N-(2)-(3)-linker-(1)-C
■ N-(2)-linker-(3)-linker-(1)-C
■ N-(1)-(3)-(2)-C
■ N-(1)-(3)-linker-(2)-C
■ N-(1)-linker-(3)-(2)-C
■ N-(1)-linker-(3)-linker-(2)-C
■ N-(3)-(1)-(2)-C
■ N-(3)-linker-(1)-(2)-C
■ N-(3)-(1)-linker-(2)-C
■ N-(3)-linker-(1)-linker-(2)-C
In a preferred embodiment of the invention, the first linker and/or the second linker of the fusion protein of the invention comprise the sequence GGSSGGS (S EQ ID NO: 4). D. Intervening polypeptide region
The terms “intervening polypeptide region” and “intervening region” are herein considered equivalent.
The intervening polypeptide region of the fusion proteins of the invention comprises a physiologically functional peptide, meaning that its interaction with the cellular components results in physiological changes. However, the intervening polypeptide region does not need to be physiologically functional once it is incorporated into the fusion protein of the invention. Accordingly, linker regions connecting the different elements of the fusion protein according to the invention are not considered intervening regions. 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.
In a preferred embodiment the intervening polypeptide region is a physiologically functional peptide or a fragment or a mutant thereof with a reduced physiological function once it is incorporated into the fusion protein of the invention. In another embodiment, the intervening polypeptide region does not have any physiological function once incorporated into the fusion protein of the invention. In another preferred embodiment, the intervening polypeptide region is a fragment or a mutant of a physiologically functional polypeptide with an already reduced physiological function, as compared to the wild-type physiologically functional polypeptide before being incorporated into the fusion protein of the invention. More preferably, the intervening polypeptide region is a protein which does not have any physiological function already when not forming part of the fusion protein of the invention, due to the presence of inactivating mutations.
In a preferred embodiment the intervening polypeptide of the fusion protein of the invention is a therapeutic agent or an imaging agent.
The term "therapeutic", as used herein in relation to the therapeutic agents, is used in a generic sense and includes treating agents, prophylactic agents, and replacement agents.
As it is part of the fusion protein of the invention with the PDGFR-p ligand and the positively charged amino acid-rich region, the nature of the intervening region is substantially polypeptidic, except where the therapeutic agent is concerned. It is intended that the therapeutic agent conjugated to the fusion protein is not limited in its chemical structure. In an embodiment of the invention, the intervening region of the fusion protein is selected from the group consisting of a fluorescent protein, albumin, nidogens, chorionic gonadotropin, and a cystatin.
“Fluorescent protein”, as used herein, relates to proteins whose atomic structure allows them to present fluorescence, which is a phenomenon well-known in the art. Nonlimiting examples of commonly used fluorescent proteins suitable for the fusion protein of the invention, are the green fluorescent protein (GFP, first discovered in Aequorea victoria), the red fluorescent protein (RFP), the yellow fluorescent protein (YFP), the blue fluorescent protein (BFP), the cyan fluorescent protein, 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],
Additional non-limiting examples of fluorescent proteins suitable for the fusion protein of the invention are the enhanced green fluorescent protein (eGFP), enhanced cyan fluorescent protein CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilised 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), mRFPI, 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 a fluorescent protein selected from the group consisting of the mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), and the like.
In a preferred embodiment, the fluorescent protein of the intervening region of the fusion protein of the invention is GFP (SEQ ID NO: 17).
“Albumin”, as used herein, refers to the water-soluble, glycosylated globular proteins commonly found in the plasma of animals, especially mammals. In a preferred embodiment, the protein of the intervening region of the fusion protein is albumin, more preferably human albumin (SEQ ID NO: 18).
“Nidogen”, as used herein, relates to any protein of the family of nidogens, formerly known as entactins, which are sulfated monomeric glycoproteins located in the basal lamina. In a more preferred embodiment of the invention, the protein of the intervening region of the fusion protein is selected from the group consisting of the human nidogen 1 (with identification number P14543-1 of the Uniprot Database (version dated July 7, 2009), SEQ ID NO: 19) and the human nidogen 2 (NID2, SEQ ID NO: 20).
In another preferred embodiment of the invention, the protein of the intervening region of the fusion protein of the invention is the nidogen-1 G2 domain. The term “G2 domain of nidogen-1”, as used herein, refers to the domain G2 of the protein nidogen 1 as defined above. The nidogen-1 G2 domain is as shown in SEQ ID NO : 33 which corresponds to amino acid numbers 430 and 667 of the amino acid sequence of the nidogen-1 protein, with identification number P14543-1 of the Uniprot Database (version dated July 7, 2009) (SEQ ID NO: 19). In another embodiment, the domain G2 of nidogen 1 is as shown in SEQ ID NO: 34, which lacks the first two amino acids of SEQ ID NO: 33, and thus, corresponds to a region consisting on amino acid numbers 432 and 667 of the amino acid sequence of the nidogen-1 protein precursor with identification number P14543-1 of the Uniprot Database (version dated July 7, 2009) (SEQ ID NO: 19). In the native nidogen-1 sequence, the G2 domain is flanked by short EGF-like domains. However, for the purposes of the present invention, the nidogen-1 G2 domain lacks EGF- like domains at the N-or at the C-terminus.
In another preferred embodiment of the invention, the protein of the intervening region of the fusion protein of the invention is a variant of the nidogen-1 G2 domain. In a preferred embodiment the variant of the nidogen-1 G2 domain contains the H459A and the R468N mutations. In one embodiment, the variant of the nidogen-1 G2 domain contains the H459A and the F639S mutations. In one embodiment, the variant of the nidogen-1 G2 domain contains the H459A and the R650A mutations. In one embodiment, the variant of the nidogen-1 G2 domain contains the R468N and the F639S mutations. In one embodiment, the variant of the nidogen-1 G2 domain contains the R468N and the R650A mutations. In one embodiment, the variant of the nidogen-1 G2 domain contains the H459A, the R468N, and the F639S mutations. In one embodiment, the variant of the nidogen-1 G2 domain contains the H459A, the R468N, and the R650A mutations. In one embodiment, the variant of the nidogen-1 G2 domain contains the R468N, the F639S and the R650A mutations. In one embodiment, the variant of the nidogen-1 G2 domain contains the H459A, the R468N, the F639S and the R650A mutations. In a preferred embodiment, the of the nidogen-1 G2 domain variant has a sequence as defined in SEQ ID NO: 34 or 35 (hereinafter referred to as NIDOmut2).
In another embodiment, the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant as defined in any of the embodiments above and, in particular, the nidogen-1 G2 domain variant having the H459A, the R468N, the F639S and the R650A mutations which, in addition, comprises a mutation at a position selected from the group consisting of 543 (corresponding to histidine at position 114 in SEQ ID NO: 33) and position 545 (corresponding to histidine at position 116 in SEQ ID NO: 33). In another embodiment, position H543 is mutated to Lys. In another embodiment, position H545 is mutated to Asn. In some embodiments, the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain that comprises the H459A, the R468N, the F639S, the R650A and the H543K mutations. In some embodiments, the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain that comprises the H459A, the R468N, the F639S, the R650A and the H545N mutations. In another embodiment, the nidogen-1 G2 domain variant comprises a H543K mutation and a H545N mutation. In one embodiment, the nidogen-1 G2 domain variant comprises or consists of SEQ ID NO : 36 (hereinafter referred to NIDOmut3), which is characterized in that it contains the H459A, the R468N, the F639S, the R650A, the H543K and the H545N mutations.
In another embodiment, the protein of the intervening region of the fusion protein of the invention is a variant of the nidogen-1 G2 domain as defined in any of the embodiments above and, in particular, the NIDOmut3 variant, which, in addition comprises a mutation selected from the group consisting of: a mutation at valine at position 449 (corresponding to position 20 in SEQ ID NO: 33). Preferably, the valine at position 449 is mutated to Thr. In a preferred embodiment, the nidogen-1 G2 domain variant has a sequence as defined in SEQ ID NO: 37 (hereinafter referred to as NIDOmut3-V45T). a mutation at valine at position 525 (corresponding to position 96 in SEQ ID NO: 33). Preferably, the valine at position 449 is mutated to Gin. In a preferred embodiment, the nidogen-1 G2 domain variant has a sequence as defined in SEQ ID NO : 38 (hereinafter referred to as NIDOmut3- V121Q). a mutation at the phenylalanine at position 561 (corresponding to position 142 in SEQ ID NO: 33). Preferably, the phenylalanine at position 561 is mutated to Glutamic acid. In a preferred embodiment, the nidogen-1 G2 domain variant has a sequence as defined in SEQ ID NO: 39 (hereinafter referred to as NIDOmut3-F157E). a mutation at the valine at position 619 (corresponding to position 190 in SEQ ID NO : 33). Preferably, the valine at position 619 is mutated to threonine. In a preferred embodiment, the nidogen-1 G2 domain variant has a sequence as defined in of SEQ ID NO: 40 (hereinafter referred to as NIDOmut3-V215T).
In another embodiment, the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain as defined in any of the embodiments above, and in addition comprises the V449T, the V525Q, the F561 E and the V619T mutations. In some embodiments, the protein of the intervening region of the fusion protein of the invention is a variant nidogen G2 domain that comprises the H459A, the R468N, the F639S, the R650A, the H543K, the V449T, the V525Q, the F561 E and the V619T mutations. In some embodiments, the protein of the intervening region of the fusion protein of the invention is a variant nidogen G2 domain that comprises the H459A, the R468N, the F639S, the R650A, the V449T, the H545N, the V525Q, the F561 E and the V619T mutations. In some embodiments, the protein of the intervening region of the fusion protein of the invention is a variant nidogen G2 domain that comprises the H459A, the R468N, the F639S, the R650A, the H543K, the H545N, the V449T, the V525Q, the F561 E and the V619T mutations. In another embodiment, the variant nidogen G2 domain comprises or consists of the sequence as defined in SEQ ID NO: 42 (hereinafter referred to as NIDOmut4).
In another embodiment, the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain as defined in any of the embodiments above and, in particular the NIDOmut4), which in addition comprises a mutation at the threonine at position 619 (corresponding to position 190 in SEQ ID NO: 33). Preferably, the threonine at position 619 is mutated to valine. In another embodiment, the protein of the intervening region of the fusion protein of the invention is a variant nidogen G2 domain as defined in any of the embodiments above in which the amino acid at position 619 (corresponding to position 190 in SEQ ID NO: 33) is the same residue that appear in the human nidogen G2 domain as defined in the UniProt database under accession number P14534), i.e. a Valine. Accordingly, in one embodiment, the protein of the intervening region of the fusion protein of the invention is a nidogen G2 domain variant having the H459A, the R468N, the F639S, the R650A, the H543K, the H545N, the V449T, the V525Q and the F561 E mutations. In one embodiment, the protein of the intervening region of the fusion protein of the invention is a nidogen G2 domain variant having the sequence of SEQ ID NO: 43 (hereinafter referred to as NIDOmut4_T215V).
In another embodiment, the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain as defined in any of the embodiments above and, in particular, the NIDOMut4, which, in addition comprises a mutation at the cysteine at position 618 (corresponding to position 189 in SEQ ID NO : 33). Preferably, the cysteine at position 618 is mutated to serine. Accordingly, in one embodiment, the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the H459A, the R468N, the F639S, the R650A, the H543K, the H545N, the V449T, the V525Q, the V619T, the F561 E and the C618S mutations. In one embodiment, the protein of the intervening region of the fusion protein of the invention is a nidogen G2 domain variant having the sequence of SEQ ID NO: 44 (hereinafter referred to as NIDOmut5).
In another embodiment, the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain as defined in any of the embodiments above and, in particular the NIDOMut3 variant, which in addition comprises a mutation selected from the group consisting of: a mutation at valine at position 580 (corresponding to position 151 in SEQ ID NO: 33). Preferably, the valine at position 580 is mutated to Thr. In one embodiment, the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the sequence of SEQ ID NO: 45 (hereinafter referred to as NIDOmut3-V176T). a mutation at isoleucine at position 604 (corresponding to position 175 in SEQ ID NO: 33). Preferably, the isoleucine at position 604 is mutated to Thr. In one embodiment, the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the sequence of SEQ ID NO: 46 (hereinafter referred to as NIDOmut3-l200T). a mutation at the valine at position 638 (corresponding to position 209 in SEQ ID NO: 33). Preferably, the valine at position 638 is mutated to tyrosine. In one embodiment, the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the sequence of SEQ ID NO: 47 (hereinafter referred to as NIDOmut3-V236Y). a mutation at the leucine at position 641 (corresponding to position 212 in SEQ ID NO: 33). Preferably, the leucine at position 641 is mutated to threonine. In one embodiment, the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the sequence of SEQ ID NO: 48 (hereinafter referred to as NIDOmut3-L237T). a mutation at serine at position 469 (corresponding to position 40 in SEQ ID NO : 33). Preferably, the serine at position 469 is mutated to lie. In one embodiment, the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the sequence of SEQ ID NO: 49 (hereinafter referred to as NIDOmut3-S65l). a mutation at arginine at position 518 (corresponding to position 89 in SEQ ID NO: 33). Preferably, the arginine at position 518 is mutated to lie. In one embodiment, the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the sequence of SEQ ID NO: 50 (hereinafter referred to as NIDOmut3-R1141). a mutation at the cysteine at position 618 (corresponding to position 189 in SEQ ID NO: 33). Preferably, the cysteine at position 618 is mutated to serine. Accordingly, in one embodiment, the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the sequence of SEQ ID NO: 51 (hereinafter referred to as NIDOmut3-C214S).
In some embodiments, the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain as defined in any of the embodiments above and, in particular the NIDOmut3 variant, which, in addition, contains mutations at the 469 (preferably a S469I mutation) and at the 518 position (preferably a R518I mutation). Accordingly, in one embodiment, the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the H459A, the R468N, the F639S, the R650A, the H543K, the H545N, the S469I and the R518I mutations and corresponds to the sequence of SEQ ID NO: 52 (hereinafter referred to as NIDOmut3-S65l_R114l).
In some embodiments, the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain as defined in any of the embodiments above and, in particular the NIDOmut5 variant, which, in addition, contains mutations at the 469 (preferably a S469I mutation) and at the 518 position (preferably a R518I mutation). Accordingly, in one embodiment, the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the H459A, the R468N, the F639S, the R650A, the H543K, the H545N, the V449T, the V525Q, the V619T, the F561 E, the S469I and the R518I mutations, as defined in SEQ ID NO: 53 (hereinafter referred to as NIDOmut5-S65l_R1141).
In some embodiments, the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain as defined in any of the embodiments above and, in particular the NIDOmut5 variant, which, in addition, contains a mutation at serine at position 469 (corresponding to position 40 in SEQ ID NO: 33). Preferably, the serine at position 469 is mutated to lie. Accordingly, in one embodiment, the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the H459A, the R468N, the F639S, the R650A, the H543K, the H545N, the V449T, the V525Q, the V619T, the F561 E and the S469I mutations. In one embodiment, the protein of the intervening region of the fusion protein of the invention is a nidogen G2 domain variant having the sequence of SEQ ID NO: 54 (hereinafter referred to as NIDOmut5-S65l).
In some embodiments, the protein of the intervening region of the fusion protein of the invention is a variant nidogen-1 G2 domain as defined in any of the embodiments above and, in particular the NIDOmut5 variant, which, in addition, contains a mutation at arginine at position 518 (corresponding to position 89 in SEQ ID NO: 33). Preferably, the arginine at position 518 is mutated to lie Accordingly, in one embodiment, the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the H459A, the R468N, the F639S, the R650A, the H543K, the H545N, the V449T, the V525Q, the V619T, the F561 E and the R518I mutations. In one embodiment, the protein of the intervening region of the fusion protein of the invention is a nidogen-1 G2 domain variant having the sequence of SEQ ID NO: 55 (hereinafter referred to as NIDOmut5-R114l).
The nidogen-1 G2 domain variants suitable for use in the present invention are summarized in the Table 1 below.
Table 1: Nigogen-1 G2 domain variants.
Figure imgf000025_0001
Figure imgf000026_0001
“Chorionic gonadotropin” (GC), as used herein, refers to the glycoprotein and hormone produced in the placenta of mammals after the zygote implantation. Human chorionic gonadotropin presents two subunits, alpha (a) and beta (P). It is intended that any one of the two subunits separately and both together are suitable for the purposes of the invention.
Thus, in another embodiment of the invention, the protein of the intervening region of the fusion protein is the chorionic gonadotropin.
In another preferred embodiment of the invention, the protein of the intervening region of the fusion protein is the human chorionic gonadotropin (hGC, SEQ ID NO: 21). As used herein, the term cystatin refers to a member of a family of protease inhibitors known as cystatins which are capable of inhibiting the activity of peptidase enzymes belonging to peptidase families C1 (papain family) and C13 (legumain family). In a preferred embodiment, the cystatin is selected from the group consisting of cystatin A, cystatin B, cystatin C, cystatin D and cystatin M. In yet another preferred embodiment, the cystatin is a cystatin A, also known as Stefin A. In a preferred embodiment, Stefin A is of human origin having the sequence SEQ ID NO : 22. In yet another embodiment, the cystatin is a stefin A variant having one or more mutations selected from the group consisting of the G4W, the G4R, the V48D, the V48L, the G50S, the K71 N, the S72G, the L73P, the L82R, the T83S mutations. In other embodiments, the stefin A variant contains the following mutations with respect to the sequence shown in SEQ ID NO: 22:
G4W, V48D, K71 N, S72G and L73P, corresponding to the mutant defined as STM mutant in Woodward et al. (J. Mol. Biol. (2005) 352, 1118-1133) and in Hoffman et al., Protein Engineering, Design & Selection vol. 23 no. 5 pp. 403- 413, 2010).
- G4R, V48L, G50S, K71 N, S72G, L73P, L82R and T83S, corresponding to the mutant defined as SQM mutant in Hoffman et al., supra.).
G4R corresponding to the mutant defined as SUN mutant in Hoffman et al., supra).
- V48L and G50S corresponding to the mutant defined as SUM mutant in Hoffman et al., supra.).
K71 N, S72G, L73P, L82R and T83S corresponding to the mutant defined as SUC mutant in Hoffman et al., supra.).
- V48L, G50S, K71 N, S72G, L73P, L82R and T83S, corresponding to the mutant defined as SDM mutant in Hoffman et al., supra.).
Other suitable polypeptides and proteins that can be used as components of the intervening region include any polypeptide or protein without any physiological or biological activity on their own, as well as any biologically non-reactive peptide or protein.
In another embodiment of the invention the protein of the intervening region of the fusion protein is an inert protein.
As used herein, “inert protein” refers to polypeptides or proteins or fragments or domains of proteins without known physiological or biological activity, or without the ability to specifically interact with other macromolecules for a biological function, and fragments or domains of proteins devoid of known therapeutic activity (e.g. antitumor activity). The inert protein that is part of the fusion protein is non-reactive and functions as a physical structure for the binding of the therapeutic agents. It is intended that the inert proteins do not comprise any motifs that have intrinsic enzymatic, physiological, or biological activity on their own, nor do they present immune reactivity, meaning that they stimulate neither the adaptive, nor the innate immune responses.
In general, wherein the protein of the intervening region of the fusion protein is concerned, it is intended that any intrinsic activity of said protein is irrelevant for the purposes of the invention and does neither contribute, nor hinder the biological activity of the therapeutic agent.
In a particular embodiment, the intervening polypeptide of the fusion proteins of the invention is a fragment of any of the proteins described in any of the embodiments of this section D.
In another preferred embodiment, the intervening polypeptide of the fusion protein of the invention is a mutant of any of the proteins described in any of the embodiments of this section D.
In another preferred embodiment, the intervening polypeptide of the fusion protein of the invention is a biologically active polypeptide.
The term “biologically active polypeptide” as used herein refers to a polypeptide that affects some parameter of biological systems or chemical reactions, for example by altering growth of an organism, by affecting association of molecules and/or cells and by slowing down or accelerating chemical reactions. The term biologically active polypeptide also encompasses biologically active fragments thereof, as well as biologically active sequence analogues thereof. In another preferred embodiment, the intervening polypeptide of the fusion protein of the invention is a biologically active polypeptide, preferably a therapeutic agent.
The term “therapeutic agent”, as used herein, is drawn to any compound, without chemical structure limitations, suitable for therapy and/or treatment of a condition, disorder or disease.
The nature of the therapeutic agent is not particularly limiting for the present invention provided it remains active in the fusion protein or can be activated once it is delivered to the inside of the cell. Accordingly, any therapeutic agent can be used in the fusion protein provided that it shows an activity or can reach an activity once it is delivered to the inside of the cell of at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50% or less of the activity of the unconjugated therapeutic agent. Alternatively, since the purpose of the invention is to facilitate the action of the therapeutic agent by increasing its selectivity and reducing its off-target effects, it is contemplated that the effects of the therapeutic agent conjugated to the fusion protein may be synergic and exceed the parametrized values already known for the specific therapeutic agent. Accordingly, it is intended that some embodiments of the therapeutic agent conjugated to the fusion protein of the invention also show at least 101 %, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 175%, at least 200%, at least 300%, at least 400%, at least 500%, at least 1000%, or more of the functionality of the therapeutic agent alone.
In an embodiment of the invention, the intervening region of the fusion protein of the invention is a therapeutic agent selected from the group consisting of
(i) a cytotoxic polypeptide,
(ii) an antiangiogenic polypeptide,
(iii) a polypeptide encoded by a tumor suppressor gene,
(iv) a pro-apoptotic polypeptide,
(v) a polypeptide having anti-metastatic activity,
(vi) a polypeptide encoded by a suicide gene,
(vii) a chaperone polypeptide, and
(viii) a polypeptide which is capable of activating the immune response towards a tumor.
(i) Cytotoxic polypeptides
As used herein, the term “cytotoxic polypeptide” refers to an agent that is capable of inhibiting cell function. The agent may inhibit proliferation or may be toxic to cells. Any polypeptides that when internalized by a cell interfere with or detrimentally alter cellular metabolism or in any manner inhibit cell growth or proliferation are included within the ambit of this term, including, but not limited to, agents whose toxic effects are mediated when transported into the cell and also those whose toxic effects are mediated at the cell surface. Useful cytotoxic polypeptides include proteinaceous toxins such as bacterial toxins.
Examples of proteinaceous cell toxins useful for incorporation into the conjugates according to the invention include, but are not limited to, type one and type two ribosome inactivating proteins (RIP). Useful type one plant RIPs include, but are not limited to, dianthin 30, dianthin 32, lychnin, saporins 1-9, pokeweed activated protein (PAP), PAP II, PAP-R, PAP-S, PAP-C, mapalmin, dodecandrin, bryodin-L, bryodin, Colicin 1 and 2, luffin-A, luffin-B, luffin-S, 19K-protein synthesis inhibitory protein (PSI), 15K-PSI, 9K-PSI, alpha-kirilowin, beta-kirilowin, gelonin, momordin, momordin-ll, momordin-lc, MAP-30, alpha-momorcharin, beta-momorcharin, trichosanthin, TAP-29, trichokirin; barley RIP; flax RIP, tritin, corn RIP, Asparin 1 and 2 (Stirpe et al., 1992. Bio/Technology 10:405- 12). Useful type two RIPs include, but are not limited to, volkensin, ricin, nigrin-b, CIP- 29, abrin, modeccin, ebulitin-[alpha], ebulitin-[beta], ebultin-[gamma], vircumin, porrectin, as well as the 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).
Examples of bacterial toxins useful as cell toxins include, but are not limited to, shiga toxin and shiga-like toxins (i.e. , toxins that have the same activity or structure), as well as the catalytic subunits and biologically functional fragments thereof. These bacterial toxins are also type two RIPs (Sandvig and Van Deurs, 1996. Physiol. Rev. 76:949-66; Armstrong, 1995. J. Infect. Dis., 171 :1042-5; Kim et al., 1997. Microbiol. Immunol. 41 :805-8; and Skinner et al., 1998. Microb. Pathog. 24:117-22). 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 enzymatic subunits also can be used as a cell toxin moiety (Pastan et al., Annu. Rev. Biochem. 61 :331-54; Brinkmann and Pastan, Biochim. et Biophys. Acta 1198:27-45, 1994; Mesri et al., J. Biol. Chem. 268:4852-62, 1993; Skinner et al., Microb. Pathog. 24:117-22, 1998; and U.S. Pat. No. 5,082,927). Other targeted agents include, but are not limited to the more than 34 described Colicin family of RNase toxins which include colicins A, B, D, E1-9, cloacin DF13 and the fungal RNase, [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).
(77) Antiangiogenic polypeptides
Proliferation of tumor cells relies heavily on extensive tumor vascularization, which accompanies cancer progression. Thus, inhibition of new blood vessel formation with anti-angiogenic agents and targeted destruction of existing blood vessels have been introduced as effective and relatively non-toxic approaches to tumor treatment.
The term "anti-angiogenic polypeptide", as used herein, denotes a polypeptide capable of inhibiting angiogenesis. Suitable antiangiogenic polypeptides include, without limitation, angiostatin, endostatin, anti-angiogenic anti-thrombin III, sFRP-4 as described in W02007115376, and an anti-VEGF antibody such as anibizumab, bevacizumab (avastin), Fab IMG 1121 and F200 Fab.
(Hi) Polypeptides encoded by a tumor suppressor gene
As used herein, a "tumor suppressor" is a gene or gene product that has a normal biological role of restraining unregulated growth of a cell. The functional counterpart to a tumor suppressor is an oncogene — genes that promote normal cell growth may be known as "proto-oncogenes” A mutation that activates such a gene or gene product further converts it to an "oncogene", which continues the cell growth activity, but in a dysregulated manner Examples of tumor suppressor genes and gene products are well known in the literature and may include PTC, BRCA1 , BRCA2, p16, APC, RB, WTI, EXTI, p53, NFI, TSC2, NF2, VHL.ST7, ST14, PTEN, APC, CD95 or SPARC.
(iv) Pro-apoptotic polypeptides
The term “pro-apoptotic polypeptides”, as used herein, refers to a protein which is capable of inducing cell death in a cell or cell population. The overexpression of these proteins involved in apoptosis displaces the careful balance between anti-apoptotic and pro-apoptotic factors towards an apoptotic outcome. Suitable pro-apoptotic polypeptides include, without limitation, 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 homologs, 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-R1 and TRAIL-R2.
(v) Polypeptides with anti-metastatic activity
The term “metastasis suppressor” as used herein, refers to a protein that acts to slow or prevent metastases (secondary tumors) from spreading in the body of an organism with cancer. Suitable metastasis suppressor include, without limitation, proteins such as BRMS I, CRSP3, DRGI, KA11 , KISS-I, NM23, a TIMP-family protein and uteroglobin.
(vi) Polypeptide encoded by a suicide gene
In the context of the invention, a “polypeptide encoded by a suicide gene” refers to a polypeptide the expression of which results in cell expressing it killing itself through apoptosis. This approach comprises the selective expression of the suicide gene only in particular cells, though the use of specific promoters, for instance, that would activate only in cells actually suffering the disease to be suppressed.
This approach comprises the use of pairs of enzyme and pro-drug, in which the enzyme is used to transform the target cells previously to the administration of the prodrug, which under the action of the enzyme becomes a product toxic for the cell that kickstarts the apoptotic process. Usually, the enzymes of these systems of suicide gene therapy are usually not found in the same organism in which they are intended to be expressed, and so in mammals have been used enzymes obtained from bacteria, fungi or other organisms. This strategy has several known examples [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, carboxypeptidase G2/Nitrogen mustard system, cytochrome P450/oxazaphosphorine system, purine nucleoside phosphorylase/6- methylpurine deoxyriboside (PNP/MEP), the horseradish peroxidase/indole-3-acetic acid system (HRP/IAA), and the carboxylesterase/irinotecan (CE/irinotecan) system, the truncated EGFR, inducible caspase ("iCasp"), the the E. coli gpt gene, the E. coli Deo gene and nitroreductase.
(vii) Chaperone polypeptide
As used herein, “chaperone polypeptide” or “chaperon” refers to a protein molecule that assists in folding or unfolding of protein molecules and/or assembly or disassembly of macromolecular structures. Exemplary chaperones include, but are not limited to, ABCE1 ATP-binding cassette sub-family E member 1 ; AHSA1 Activator of 90 kDa heat shock protein ATPase homolog 1 ; ANP32B acidic leucine-rich nuclear phosphoprotein 32 family; BAG6 Large proline-rich protein BAG6; BCS1 L mitochondrial chaperone BCS1 ; CALR calreticulin; CANX calnexin; CCT2 T-complex protein 1 subunit beta CCT3 T-complex protein 1 subunit gamma CCT4 T-complex protein 1 subunit delta CCT5 T- complex protein 1 subunit epsilon CCT6A T-complex protein 1 subunit zbeta CCT7 T- complex protein 1 subunit beta CD74 H-2 class II histocompatibility antigen gamma chai; CDC37 Hsp90 co-chaperone Cdc37; CLGN calmegin; 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; HSPA1 B Heat shock 70 kDa protein 1A/1 B; HSPA2 Heat shock-related 70 kDa protein 2; HSPA8 Heat shock cognate 71 kDa protein; HSPA9 Stress-70 protein, mitochondrial; HSPD1 60 kDa heat shock protein, mitochondrial; HYOLI1 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-complex protein 1 subunit alpha TIMMDC1 Translocase of inner mitochondrial membrane domain; and TM EM 126B Transmembrane protein 126B. (viii) Polypeptides capable of activating the immune response towards a tumor
As used herein, an immunostimulatory polypeptide agent is a polypeptide encoded by a polynucleotide which is capable of activating or stimulating the immune response (including enhancing a pre-existing immune response) in a subject to whom it is administered, whether alone or in combination with another agent. 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), and the like.
E. The Therapeutic Agent
The therapeutic agent may also be conjugated to the fusion protein of the invention. It is intended that the therapeutic agent, as aforementioned, is conjugated to the intervening region of the fusion protein without limitation of the position of the conjugation inside the intervening region with regards to the N-terminal and C-terminal ends. Accordingly, the therapeutic agent can be conjugated to the intervening polypeptide region in an equidistant position with respect to the N-terminal and C-terminal ends or it can be closer to either of them. Hence, the therapeutic agent can be conjugated to the intervening polypeptide region at a distance of 100, 90, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 30, 25, 20, 15, 20, 10, or less amino acid residues from the N-terminal or C-terminal end, or at the same residue of the N-terminal or C-terminal end.
The only intended limitation in the conjugation position of the therapeutic agent is that the therapeutic agent and the elements of the fusion protein are functional and the conjugation of the therapeutic agent does not interfere with the activity of either therapeutic agent or the fusion protein. So, the therapeutic agent, the PDGFR-p ligand, and the positively charged amino acid-rich region conserve at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, preferably 95%, more preferably 99%, even more preferably 100% of their functionality with respect to the non-conjugated forms of the fusion protein and the therapeutic agent respectively.
Hence, in a preferred embodiment the intervening polypeptide region of the fusion protein of the invention is conjugated to at least one therapeutic agent. In another preferred embodiment the intervening polypeptide region of the fusion protein of the invention is conjugated to a plurality of therapeutic agents, wherein said plurality of therapeutic agents are the same or different.
In yet another preferred embodiment the intervening polypeptide region of the fusion protein of the invention is conjugated to a therapeutic agent selected from the group consisting of
(i) a chemotherapy agent,
(ii) an antiangiogenic molecule, and
(iii) a toxin.
(i) Chemotherapy agent
It will be understood that the term “chemotherapeutic agents” refers to anti-cancer agents.
As used herein, an anti-cancer agent is an agent that at least partially inhibits the development or progression of a cancer, including inhibiting in whole or in part symptoms associated with the cancer even if only for the short term. In a preferred embodiment the therapy agent is a chemotherapy agent.
Several anti-cancer agents can be categorized as DNA damaging agents and these include topoisomerase inhibitors (e.g., etoposide, ramptothecin, topotecan, teniposide, mitoxantrone), DNA alkylating agents (e.g., cisplatin, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chorambucil, busulfan, thiotepa, carmustine, lomustine, carboplatin, dacarbazine, procarbazine), DNA strand break inducing agents (e.g., bleomycin, doxorubicin, daunorubicin, idarubicin, mitomycin C), anti-microtubule agents (e.g., vincristine, vinblastine), anti-metabolic agents (e.g., cytarabine, methotrexate, hydroxyurea, 5-fluorouracil, floxuridine, 6-thioguanine, 6-mercaptopurine, fludarabine, pentostatin, chlorodeoxyadenosine), anthracyclines, vinca alkaloids, or epipodophyllotoxins.
Additional examples of anti-cancer agents include without limitation Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Axaitinib; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Bortezomib (VELCADE); Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calciprotiol; Calusterone; Caracemide; Carbetimer; Carboplatin (a platinum- containing regimen); Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin (a platinum-containing regimen); Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Dasatinib; Daunorubicin; Decitabine; Dexormaplatin; Dezaguanine; Diaziquone; Docetaxel (TAXOTERE); Doxorubicin; Droloxifene; Dromostanolone; Duazomycin; Edatrexate; Eflornithine; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin; Erbulozole; Erlotinib (TARCEVA), Esorubicin; Estramustine; Etanidazole; Etoposide; Etoprine; Fadrozole; Fazarabine; Fenretinide; Floxuridine; Fludarabine; 5-Fluorouracil; Flurocitabine; Fosquidone; Fostriecin; Gefitinib (IRESSA), Gemcitabine; Hydroxyurea; Idarubicin; Ifosfamide; llmofosine; Imatinib mesylate (GLEEVAC); Interferon alpha-2a; Interferon alpha-2b; Interferon alpha-nl; Interferon alpha-n3; Interferon beta-l a; Interferon gamma-l b; Iproplatin; Irinotecan; Lanreotide; Lenalidomide (REVLLMID, REVIMID); Letrozole; Leuprolide; Lenvatinib; Liarozole; Lometrexol; Lomustine; Losoxantrone; Masoprocol; Maytansine; Mechlorethamine; Megestrol; Melengestrol; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone; Mycophenolic Acid; Nintedanib; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pemetrexed (ALIMTA), Pegaspargase; Peliomycin; Pentamustine; Pentomone; Peplomycin; Perfosfamide; Pipobroman; Piposulfan; Piritrexim Isethionate; Piroxantrone; Plicamycin; Plomestane; Ponatinib; Porfimer; Porfiromycin; Prednimustine; Procarbazine; Puromycin; PT630; Pyrazofurin; Regorafenib; Riboprine; Rogletimide; Safingol; Semustine; Simtrazene; Sitogluside; Sparfosate; Sparsomycin; Spirogermanium; Spiromustine; Spiroplatin; SOM230; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Tamsulosin; Taxol; Taxotere; Tecogalan; Tegafur; Teloxantrone; Temoporfin; Temozolomide (TEMODAR); Teniposide; Teroxirone; Testolactone; Thalidomide (THALOMID) and derivatives thereof; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan; Toremifene; All trans Retinoic acid (ATRA); Trestolone; Triciribine; Trimetrexate; Triptorelin; Tubulozole; Uracil; Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine; Vincristine; Vindesine; Vinepidine; Vinglycinate; Vinleurosine; Vinorelbine; Vinrosidine; Vinzolidine; Vorozole; Zeniplatin; Zinostatin; Zorubicin.
In one embodiment, the anti-cancer agent is provided as an oligomer containing several units of the anti-cancer molecule. In one embodiment, the anti-cancer agent is a floxuridin poly- or oligonucleotide, which comprises several floxuridine molecules. The floxuridine poly- or poligonucleotide contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more floxuridine molecules. In a preferred embodiment the floxuridine polynucleotide is a floxuridine pentanucleotide, i.e. a oligonucleotide containing 5 floxuridine molecules. In one embodiment, the anti-cancer agent is Monomethyl auristatin E.
The anti-cancer agent may be an enzyme inhibitor including without limitation tyrosine kinase inhibitor, a CDK inhibitor, a MAP kinase inhibitor, or an EGFR inhibitor. The tyrosine kinase inhibitor may be without limitation Genistein (4', 5, 7- trihydroxyisoflavone), Tyrphostin 25 (3,4,5-trihydroxyphenyl), methylene]- propanedinitrile, Herbimycin A, Daidzein (4',7-dihydroxyisoflavone), AG-126, trans-1- (3'- carboxy-4'-hydroxyphenyl)-2-(2",5"-dihydroxy-phenyl)ethane, or HDBA (2- Hydroxy5- (2,5-Dihydroxybenzylamino)-2-hydroxybenzoic acid. The CDK inhibitor may be without limitation p21 , p27, p57, pl5, pl6, pl8, or pl9. The MAP kinase inhibitor may be without limitation KY12420 (C23H24O8), CNI-1493, PD98059, or 4-(4- Fluorophenyl)-2-(4- methylsulfinyl phenyl)-5-(4-pyridyl) IH-imidazole. The EGFR inhibitor may be without limitation erlotinib (TARCEVA), gefitinib (IRESSA), WHI- P97 (quinazoline derivative), LFM-A12 (leflunomide metabolite analog), ABX-EGF, lapatinib, canertinib, ZD-6474 (ZACTIMA), AEE788, and AG1458.
The anti-cancer agent may be a VEGF inhibitor including without limitation bevacizumab (AVASTIN), ranibizumab (LUCENTIS), pegaptanib (MACUGEN), sorafenib, sunitinib (SUTENT), vatalanib, ZD-6474 (ZACTIMA), anecortave (RETAANE), squalamine lactate, and semaphorin. The anti-cancer agent may be an antibody or an antibody fragment including without limitation an antibody or an antibody fragment including but not limited to bevacizumab (AVASTIN), trastuzumab (HERCEPTIN), alemtuzumab (CAMPATH, indicated for B cell chronic lymphocytic leukemia,), gemtuzumab (MYLOTARG, hP67.6, anti-CD33, indicated for leukemia such as acute myeloid leukemia), rituximab (RITUXAN), tositumomab (BEXXAR, anti-CD20, indicated for B cell malignancy), MDX-210 (bispecific antibody that binds simultaneously to HER- 2/neu oncogene protein product and type I Fc receptors for immunoglobulin G (IgG) (Fc gamma Rl)), oregovomab (OVAREX, indicated for ovarian cancer), edrecolomab (PANOREX), daclizumab (ZENAPAX), palivizumab (SYNAGIS, indicated for respiratory conditions such as RSV infection), ibritumomab tiuxetan (ZEVALIN, indicated for NonHodgkin's lymphoma), cetuximab (ERBITUX), MDX-447, MDX-22, MDX-220 (anti-TAG- 72), I0R-C5, 10R-T6 (anti-CD 1), IOR EGF/R3, celogovab (ONCOSCINT OV 103), epratuzumab (LYMPHOCIDE), pemtumomab (THERAGYN), and Gliomab-H (indicated for brain cancer, melanoma).
It is contemplated that in certain embodiments of the invention a protein that acts as an angiogenesis inhibitor is targeted to a tumor. These agents include, in addition to the anti-angiogenic polypeptides mentioned above, Marimastat; AG3340; COL-3, BMS- 275291 , Thalidomide, Endostatin, SLI5416, SLI6668, EMD121974, 2-methoxyoestradiol, carboxiamidotriazole, CMIOI, pentosan polysulphate, angiopoietin 2 (Regeneron), herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM- 1470, platelet factor 4 or minocycline.
Other suitable active agents are DNA cleaving agents. Examples of DNA cleaving agents suitable for inclusion as the cell toxin in the conjugates used in practicing the methods include, but are not limited to, anthraquinone-oligopyrrol-carboxamide, benzimidazole, leinamycin; dynemycin A; enediyne; as well as biologically active analogs or derivatives thereof (i.e., those having a substantially equivalent biological activity). Known analogs and derivatives are disclosed, for examples in Islam et al., J. Med. Chem. 34 2954-61 , 1991 ; Skibo et al., J. Med. Chem. 37:78-92, 1994; Behroozi et al., Biochemistry 35:1568-74, 1996; Helissey et al., Anticancer Drug Res. 11 :527-51 , 1996; Unno et al., Chem. Pharm. Bull. 45:125-33, 1997; llnno et al., Bioorg. Med. Chem., 5:903-19, 1997; llnno et al., Bioorg. Med. Chem., 5: 883-901 , 1997; and Xu et al., Biochemistry 37:1890-7, 1998). Other examples include, but are not limited to, endiyne quinone imines (U.S. Pat. No. 5,622,958); 2,2r-bis (2-aminoethyl)-4-4'-bithiazole (Lee et al., Biochem. Mol. Biol. Int. 40:151-7, 1996); epilliticine-salen. copper conjugates (Routier et al., Bioconjug. Chem., 8: 789-92, 1997).
Some of the aforementioned chemotherapy agents can be grouped together under a common category as antimetabolites. In a preferred embodiment the chemotherapy agent is an antimetabolite. “Antimetabolite” as used herein, refers to the compounds which inhibit the use of a metabolite that is part of normal metabolism. Antimetabolites are often similar in structure to the metabolite that they interfere with, such as the antifolates that interfere with the use of folic acid. Non-limiting examples of antimetabolites include the following compounds: bleomycin, busulfan, capecitabine, carmustine, carboplatin, chlorodeoxyadenosine, cisplatin, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxorubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate mitomycin, mitoxantrone, oxaliplatin, paclitaxel, procarbazine, SN-38, thioguanine, thiotepa, teniposide, vinblastine, vincristine, and vinorelbine.
In a preferred embodiment the antimetabolite is a pyrimidine analogue or an oligomeric form thereof. The term "pyrimidine" as used herein refers to nitrogenous monocyclic heterocycles. The term “pyrimidine analog" refers to a compound that has the same basic chemical structure as a naturally occurring pyrimidine.
(II) Antiangiogenic molecules
It is also contemplated that in certain embodiments the intervening region of the fusion protein of the invention corresponds to a protein that acts as an angiogenesis inhibitor which is targeted to a tumor. These agents include, Marimastat; AG3340; COL- 3, BMS-275291 , Thalidomide, Endostatin, SU5416, SU6668, EMD121974, 2- methoxyoestradiol, carboxiamidotriazole, CMIOI, pentosan polysulphate, angiopoietin 2 (Regeneron), herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM- 1470, platelet factor 4 or minocycline. Also included are VEGF inhibitors including without limitation bevacizumab (AVASTIN), ranibizumab (LUCENTIS), pegaptanib (MACUGEN), sorafenib, sunitinib (SUTENT), vatalanib, ZD-6474 (ZACTIMA), anecortave (RETAANE), squalamine lactate, and semaphorin.
(Hi) Toxins
As used herein, the term “toxins” refers to non-proteinaceous/non-polypeptidic cytotoxic compounds obtained from different organisms, as well as chemically modified derivatives of those same compounds and compounds obtained through chemical synthesis. The compounds of this category with biological origin may be obtained from microorganisms (whether bacteria, archaea, protozoa or unicellular fungi) or pluricellular organisms (pluricellular fungi, plants, or animals, like mollusks). It is intended that the chemical composition and structure of these toxins is not limited in any way beyond their non-polypeptidic nature, therefore one or more amino acids may be part of their structure, whether as part of their basic composition or as result of chemical derivation, as long as all the amino acids participating in the structure are not bound together by peptide bonds.
Examples of toxins suitable for the invention are calicheamycin y1 , dolastatin 10, maytansinoid (DM1) and pyrrolobenzodiazepine dimer (PBD).
In another embodiment of the intervening polypeptide region of the fusion protein of the invention is conjugated to a therapeutic agent selected from the group consisting of: Vitamin D, Lorsatan, PAT-1251 , GKT137831 ; Nilotinib; Masitinib; Tofacitinib; Peficitinib; Verteporfin; Fasudil and Ripasudil. Additional therapeutic agents
In a particular embodiment the intervening region of the fusion protein of the invention is conjugated to a therapeutic agent selected from the therapeutic agents indicated in the column “Therapeutic agent” of table 2 below.
Table 2: Suitable additional therapeutic agents to be used in the conjugates according to the present invention. The disease or disorder to be treated by the therapeutic agent, or a group of therapeutic agents is provided in the same row as said therapeutic agent.
Figure imgf000039_0001
F. The Imaging Agent
The present invention further contemplates the fusion protein of the invention as an imaging tool. The ability to qualitatively or quantitatively display PDGFR-p in vivo or in vitro provides researchers and clinicians with important diagnostic and therapeutic tools. For example, the ability to image tumours, visually identify liver- related diseases such as liver fibrosis, liver cirrhosis, or abnormal liver function. The ability to measure the expression of PDGF-Rp in patients with these conditions may also provide clinicians and researchers with assistance in diagnosing, predicting, and treating cancer and liver-related disease states.
In a preferred embodiment, the intervening polypeptide of the fusion protein of the invention is an imaging polypeptide. In a preferred embodiment the imaging polypeptide is a fluorescent protein.
In another preferred embodiment, the intervening polypeptide of the fusion protein of the invention is conjugated to at least one imaging agent. In yet another polypeptide the intervening polypeptide of the fusion protein of the invention is conjugated to a plurality of imaging agents, wherein said plurality of imaging agents are the same or different.
The term "imaging agent" as used herein refers to a molecule capable of providing a signal that can be detected by one or more detection techniques (eg, spectrometry, calorimetry, spectroscopy, or visual inspection). Suitable examples of detectable signals may include optical signals and electronic or radioactive signals. Examples of imaging agents include, for example: chromophores, fluorophores, Raman active tags, radioactive labels, enzymes, enzyme substrates, or combinations thereof. Suitable radioisotopes may include: H-3, C-11 , C-14, F-18, P-32, S-35, 1-123, 1-124, 1-125, 1-131 , Cr-51 , CI-36, Co-57, Fe-59, Se-75 and Eu-152. Halogen isotopes (such as chlorine, fluorine, bromine, and iodine) and metals including technetium, yttrium, rhenium, and indium are also useful markers. Examples of typical metal ions that can be used as signal generating agents include: Tc-99m, 1-123, In-111 , 1-131 , Ru-97, Cu-67, Ga-67, 1-125, Ga-68, As -72, Zr-89, Gd-153 and TI-201. Radioisotopes used for in vivo imaging diagnosis by positron emission tomography (PET) include C-11 , F-18, Ga- 68, and 1-124. The paramagnetic label, which may be a metal ion, exists in the form of metal complex or metal oxide particles. Suitable paramagnetic isotopes can include Gd- 157, Mn-55, Dy-162, Cr-52 and Fe-56.
In a preferred embodiment the imaging agent from the fusion protein of the invention is selected from a group consisting of:
(i) a paramagnetic label,
(ii) a radionuclide, and
(iii) a fluorophore.
(I) Paramagnetic label As used herein, the term “paramagnetic label” is synonymous of "paramagnetic metal ion", "paramagnetic ion" or "metal ion", and refers to a metal ion that is magnetized parallel or antiparallel to a magnetic field to an extent proportional to the magnetic field. Usually these are metal ions with unpaired electrons. Examples of suitable paramagnetic metal ions include, but are not limited to: Gadolinium III (Gd3 + or Gd (III)), Iron III (Fe3 + or Fe (III)), Manganese II (Mn2 + or Mn (II)), Yttrium III (Yt3 + Or Yt (III)), dysprosium (Dy3 + or Dy (III)) and chromium (Cr (III) or Cr3 +). In certain embodiments, the paramagnetic ion is the lanthanide atom Gd (III) because of its high magnetic moment (u2 = 63BM2) and symmetric electron ground state (S8).
(77) Radionuclide
The term “radionuclide” as used herein refers to a nuclide that exhibits radioactivity. A "nuclide" refers to a type of atom specified by its atomic number, atomic mass, and energy state, such as carbon 14 (14C). "Radioactivity" refers to the radiation, including alpha particles, beta particles, nucleons, electrons, positrons, neutrinos, and gamma rays, emitted by a radioactive substance. Radionuclides suitable for use in the present invention include, but are not limited to, fluorine 18 (18F), phosphorus 32 (32P), scandium 47 (47Sc), cobalt 55 (55Co), copper 60 (60Cu), copper 61 (61 Cu), copper 62 (62Cu), copper 64 (64Cu), gallium 66 (66Ga), copper 67 (67Cu), gallium 67 (67 Ga), gallium 68 (68Ga), rubidium 82 (82Rb), yttrium 86 (s6Y), yttrium 87 (87Y), strontium 89 (89Sr), yttrium 90 (90Y), rhodium 105 (05Rh), silver 111 (UtAg), indium 111 (" In), iodine 124 (1241), iodine 125 (1 asl), iodine 131 (1311), tin 117m (117 'Sn), technetium 99m (99mTc), promethium 149 (149Pm), samarium 153 (153Sm), holmium 166 (166Ho), lutetium 177 (177Lu), rhenium 186 (86Re), rhenium 188 (88Re), thallium 201 (ao1T1), astatine 211 (21 'At), and bismuth 212 (12Bi).
(Hi) Fluorophore
The term "fluorophore" as used herein refers to a chemical compound that emits light (at a different wavelength) when it is excited by exposure to light of a specific wavelength. Fluorophores can be explained according to their emission characteristics or "color". Green fluorophores (such as Cy3, FITC, and Oregon Green) can be characterized by their emission wavelengths typically between 515-540 nanometers. Red fluorophores (such as Texas Red, Cy5, and tetramethylrhodamine) can be characterized by their emission wavelengths typically between 590-690 nanometers. Examples of fluorophores include, but are not limited to: 4-acetylamino-4 - isocyanothiostilbene-2,2'-disulfonic acid, acridine, acridine and acridine isothiocyanate derivatives, 5- (2'-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N- [3- (vinylsulfonyl) phenyl] naphthalimide-3,5-disulfonate (Fluorescent Yellow VS), N- (4- anilino-1-naphthyl) maleimide, 2-aminobenzamide (anthranilamide), bright yellow, coumarin, coumarin derivatives, 7- Amino-4-methyl coumarin (AMC, coumarin 120), 7- amino-trifluoromethyl coumarin (coumarin 151), cyanosine; 4 ', 6-diamididine 2- phenylindole (DAPI), 5 ', 5 ’’-dibromopyrogallol-sulfopeptide (bromopyrogallol red), 7- diethylamino-3- (4'-iso (Cyanothiophenyl) 4-methylcoumarin, 4,4'-diisocyanothiodihydro- stilbene-2,2'-disulfonic acid, 4,4'-diisocyanothiostilbene- 2,2'-disulfonic acid, 5- [dimethylamino] naphthalene-1 -sulfonyl chloride (DNS, dansyl chloride), eosin, eosin derivatives (such as eosin isothiocyanate), red Moss red and erythrosine derivatives (such as erythrosin B and erythrosine isothiocyanate); ethidium; Lumin and derivatives, such as 5-carboxyfluorescein (FAM), 5- (4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF), 2'7'-dimethoxy-4 '5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), QFITC (XRITC); fluorescein derivatives (reaction with amine to produce fluorescence); IR144; IR1446; malachite green isothiocyanate; 4- methylumbelliferone; o-cresolphthalein; nitrotyrosine; basic by-product red; phenol red, B-phycoerythrin; o-phthalaldehyde derivatives ( Fluorescence after reaction with amine); pyrene and derivatives, such as pyrene, pyrene butyrate and succinyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron.RTM. Brilliant Red 3B-A), rhodamine and derivatives , Such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), Lisamine Rhodamine B Sulfonyl chloride, Rhodamine (Rhod), Rhodamine B, Rhodamine 123, Rhodamine X Sulfoyl chloride derivatives of thiocyanate, sulforhodamine B, sulforhodamine 101 and sulforhodamine 101 (Texas Red), N, N, N ', N'-tetramethyl-6 -Carboxy rhodamine (TAMRA), tetramethyl rhodamine, tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rose Acid and chelated lanthanide derivatives, quantum dots, cyanines, and squarylium (squaraine).
G. Reporter proteins
In another embodiment of the invention, the fusion protein of the invention further comprises a reporter protein.
The person skilled in the art will acknowledge the term “reporter protein” as referring to a protein resulting from the expression of a “reporter gene”. Reporter proteins are well known and commonly used in the art as markers suitable for multiple purposes, such as location of the expression of the reporter genes in tissues, cells or subcellular locations, protein-protein interactions, transport across the plasmatic membranes or endomembranes, vesicular traffic, ligand-receptor interactions, etcetera.
Useful reporter proteins in the context of the present invention include luciferase- 4-monooxygenase from Photinus pyralis, p-galactosidase, thymidine kinase, and the like. The reporter proteins also include fluorescent proteins, which have already been discussed.
The reporter protein comprised by the fusion protein of the invention is directly adjacent to the positively charged amino acid-rich region or separated by a linker. The relative position of the positively charged amino acid-rich region, however, remains as per the the aforementioned considerations about the relative position of the elements of the fusion protein. Hence, independently of the position of the fusion protein, the fluorescent protein is always adjacent to it, either directly or separated by a linker.
Accordingly, in the embodiments of the invention comprising a fluorescent protein, the possible relative positions of the elements of the fusion protein of the invention would fit the following scheme (wherein RP refers to a reporter protein and the numbering stated above for the elements is retained: (1) PDGFR-p ligand, (2) intervening polypeptide region, (3) positively charged amino acid region):
■ N-(1)-(2)-RP-(3)-C
■ N-(1)-linker-(2)-RP-(3)-C
■ N-(1)-(2)-linker-RP-(3)-C
■ N-(1)-linker-(2)-linker-RP-(3)-C
■ N-(3)-RP-(2)-(1)-C
■ N-(3)-RP-linker-(2)-(1)-C
■ N-(3)-RP-(2)-linker-(1)-C
■ N-(3)-RP-linker-(2)-linker-(1)-C
■ N-(1)-(2)-RP-linker-(3)-C
■ N-(1)-linker-(2)-RP-linker-(3)-C
■ N-(1)-(2)-linker-RP-linker-(3)-C
■ N-(1)-linker-(2)-linker-RP-linker-(3)-C
■ N-(3)-linker-RP-(2)-(1)-C
■ N-(3)-linker-RP-linker-(2)-(1)-C
■ N-(3)-linker-RP-(2)-linker-(1)-C
■ N-(3)-linker-RP-linker-(2)-linker-(1)-C
■ N-(2)-(1)-RP-(3)-C
■ N-(2)-linker-(1)-RP-(3)-C ■ N-(2)-(1)-linker-RP-(3)-C
■ N-(2)-linker-(1)-linker-RP-(3)-C
■ N-(2)-RP-(3)-(1)-C
■ N-(2)-(3)-RP-(1)-C
■ N-(2)-linker-RP-(3)-(1)-C
■ N-(2)-linker-(3)-RP-(1)-C
■ N-(2)-RP-(3)-linker-(1)-C
■ N-(2)-(3)-RP-linker-(1)-C
■ N-(2)-linker-RP-(3)-linker-(1)-C
■ N-(2)-linker-(3)RP-linker-(1)-C
■ N-(1)-RP-(3)-(2)-C
■ N-(1)-(3)-RP-(2)-C
■ N-(1)-RP-(3)-linker-(2)-C
■ N-(1)-(3)-RP-linker-(2)-C
■ N-(1)-linker-RP-(3)-(2)-C
■ N-(1)-linker-(3)-RP-(2)-C
■ N-(1)-linker-RP-(3)-linker-(2)-C
■ N-(1)-linker-(3)-RP-linker-(2)-C
■ N-RP-(3)-(1)-(2)-C
■ N-(3)-RP-(1)-(2)-C
■ N-RP-(3)-linker-(1)-(2)-C
■ N-(3)-RP-linker-(1)-(2)-C
■ N-RP-(3)-(1)-linker-(2)-C
■ N-(3)-RP-(1)-linker-(2)-C
■ N-RP-(3)-linker-(1)-linker-(2)-C
■ N-(3)-RP-linker-(1)-linker-(2)-C
H. Peptide that favours endosoma I escape
In another embodiment of the invention, the fusion protein of the invention further comprises a peptide that favours endosomal escape.
The expression “favours endosomal escape”, as used herein, refers to the ability of the endosomal escape peptide to induce the release of the fusion proteins from the endosomal compartment after internalization by receptor-mediated endocytosis. Examples of endosomal escape peptides are HA2 peptide, the CM 18 peptide, the S10 peptide, the KDEL peptide (SEQ ID NO: 26), and the polyhistidine peptide. The HA2 peptide is a pH-sensitive amphiphilic peptide, and may include an amino acid sequence of SEQ ID NO : 23. The CM18 peptide and the S10 peptide are amphipathic a-helical peptides, which may form transmembrane channels in cell membranes or may disrupt membranes by a carpet mechanism, and may include an amino acid sequence of SEQ ID NO : 24 or SEQ ID NO : 25, respectively. The KDEL peptide is a target peptide sequence in mammals and plants located on the C-terminal end of the amino acid structure of a protein. The KDEL sequence prevents a protein from being secreted from the endoplasmic reticulum and facilitates its return if it is accidentally exported. In a preferred embodiment of the invention, the peptide that favours endosomal escape is the KDEL peptide according to the sequence SEQ ID NO : 26. In another preferred embodiment of the invention the peptide that favours endosomal escape is a polyhistidine petpide of at least 2 histidines, at least 3 histidines, at least 4 histidines, at least 5 histidines, at least 6 histidines, at least 7 histidines, at least 8 histidines, at least 9 histidines, at least 10 histidines.
Stoichiometry of the fusion protein and nanoconjugates of the invention
The number of therapeutic agents or imaging agents which are conjugated to the fusion protein of the invention, while not being particularly limitative, will depend on the number of available residues in the intervening polypeptide which are available for chemical conjugation with the therapeutic agent or imaging agent. Since most conjugations occur via amino- or sulfhydryl groups present in the side chains of the amino acids forming part of the intervening polypeptide, the number of therapeutic/imaging agents conjugated to the fusion protein will depend on the number of lysine and arginine residues (for a conjugation via an amino groups in the side chains) or on the number of cysteine residues (for conjugation via sulfhydryl groups in the side chains) as well as on the yield of the conjugation reaction. Thus, in a particular embodiment of the invention, the fusion protein of the invention is conjugated to at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30 therapeutic agents and/or imaging agents.
It will be understood that, in the particular case wherein the therapeutic agent or imaging agent is provided as a polymer, the number of agents will also depend on the number of the monomers in the polymer. In the particular case of a FdU oligomer, the number of therapeutic agents in a given fusion protein will be the result of multiplying the number of oligomers attached to the fusion protein by the number of monomers. In the preferred case of a FdU pentamer, preferred embodiments include fusion proteins comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 75, 85, 100, 125, 150 or more therapeutic agents per fusion protein, corresponding, respectively, to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25 or 30 Fdll pentamers conjugated per molecule.
In addition, the nanoparticles according to the invention result from the assembly of multiple copies of the fusion proteins of the invention. In preferred embodiments, the nanoparticle comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 17, 20, 25, more preferably at least 15 monomers of the fusion protein of the invention.
Accordingly, the total number of therapeutic agents attached to each nanoparticle will depend on (i) the number of therapeutic agents conjugated to each fusion protein, (ii) the oligomerization state of the therapeutic agents and (iii) the number of fusion proteins forming the nanoparticle. In preferred embodiments, the nanoparticle is conjugated to at least 30, 35, 40, 45, 50, 60, 65, 70, 57, 80, 85, 90, 59, 100, 125, 150, 175, 200, 225, 250, 275, 300 therapeutic agents. In a further preferred embodiment, the nanoparticle is conjugated to at least 30, 35, 40, 45, 50, 60, 65, 70, 57, 80, 85, 90, 59, 100, more preferably at least 60 molecules of Fdll pentamer.
Method for preparing the fusion proteins of the invention
In a second aspect, the invention relates to a method to prepare the fusion proteins of the invention comprising the steps of: a) providing a fusion protein comprising i) a PDGFR-p ligand selected from the group of consisting of PDGFB (SEQ ID NO: 1), PDGFD (SEQ ID NO: 2) and a functionally equivalent variant thereof, ii) an intervening polypeptide region and iii) a positively charged amino acid-rich region, wherein the PDGFR-p ligand and the positively charged amino acid-rich region are located at the ends of the protein and b) contacting said fusion protein with an activated form of a therapeutic agent or of an oligomeric form thereof or an activated form of a imaging agent or of an oligomeric form thereof wherein said activated form of a therapeutic agent or of an oligomeric form thereof or an activated form of a imaging agent or of an oligomeric form thereof contains a reactive group which is capable of reacting with at least one group in the intervening region of the fusion protein and wherein the contacting is carried out under conditions adequate for the formation of a bond between the reactive group in the therapeutic agent or imaging agent and the group in the intervening polypeptide region.
In another embodiment, the invention relates to a method to prepare the fusion proteins of the invention comprising the steps of: a) providing a fusion protein comprising i) a PDGFR-p ligand selected from the group of consisting of PDGFB (SEQ ID NO: 1), PDGFD (SEQ ID N0: 2) and a functionally equivalent variant thereof, ii) an intervening polypeptide region and iii) a positively charged amino acid-rich region, wherein the PDGFR-p ligand and the positively charged amino acidrich region are located at the ends of the protein and wherein the fusion protein is provided in an activated form, wherein said activated form of the fusion protein contains a reactive group in the intervening region and b) contacting said fusion protein with a therapeutic agent or an oligomeric form thereof or with an imaging agent or an oligomeric form thereof, wherein said therapeutic agent or imaging agent contains a group which is capable of reacting with the reactive group in the fusion protein, wherein said contacting is carried out under conditions adequate for the formation of a bond between the reactive group in the fusion protein and the group in the therapeutic agent or the imaging agent.
The person skilled in the art will recognize that “reactive group”, as used herein, refers to any moiety of a molecule which is capable of chemically reacting with another moiety from another molecule in such a fashion so as to bind the two molecules together, usually with the release of one or more additional molecules. Many such reactions are known in the art such as the formation of the peptide bond between a carboxyl and an amine group being one non-limiting example among them.
“Activated”, as used herein when referring to a molecule, refers to a modified version of the molecule which contains a chemical modification whereby said molecule is capable to chemically react in a manner not previously present in the molecule (for instance, the activation adds a moiety not present previously, allowing for a bond that was unfeasible before) or with an increased reactivity (meaning that the reaction of the molecule with another molecule requires a lower activation energy than in the inactivated state). The present invention contemplates the possibility of activating the therapeutic agent or the imaging agent and then contacting the activated therapeutic agent or imaging agent with the fusion protein or of activating the fusion protein and then contacting the activated fusion protein with the therapeutic agent or imaging agent. In both cases, the activation of the fusion protein or of the therapeutic agent or imaging agent is usually carried out by reacting the molecule to be activated with a reagent that introduces the reactive group in a suitable moiety in the molecule to be activated. Examples of reactive groups that allow the therapeutic agent, imaging agent or fusion proteins to be activated include, but are not limited, to carboxyl, amine, imine, thiol, sulfone, hydroxyl, sulfate, and phosphate moieties, among many others which are commonly known to the person skilled in the art. The activated form of the therapeutic agent or imaging agent is also herein referred to as the “activated therapeutic agent” or “activated imaging agent”. The activated form of the fusion protein is also herein referred to as the “activated fusion protein”. The reactive group or groups in the activated fusion protein is or are located in the intervening region, although it is not excluded that additional reactive groups can also be found in other regions of the fusion protein.
“Activated” as used herein in relation to the fusion protein also includes the incorporation of rare or non-natural amino acid (NNAA) into the fusion protein, preferably in the intervening region, wherein said NNAAs contain reactive groups which allow the conjugation of the fusion protein to a therapy agent or an imaging agent (Tsuchikama, K. and An, Z., 2018, Protein Cell, 9(1):33-46). The term “rare amino acid” as used herein refers to amino acids which while occurring natural in nature are rarely incorporated into proteins. Examples are pyrolysine and selenocysteine, the latter of which only 25 selenoproteins are known in mammals. The term "non-natural amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrolysine or selenocysteine. Other terms that may be used synonymously with the term "non-natural amino acid" is "non-naturally encoded amino acid," "unnatural amino acid," "non-naturally-occurring amino acid," and variously hyphenated and non-hyphenated versions thereof. The term "non-natural amino acid" includes, but is not limited to, amino acids which occur naturally by modification of a naturally encoded amino acid (including but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine) but are not themselves incorporated into a growing polypeptide chain by the translation complex.
In a preferred embodiment of the invention the activated form of the fusion protein comprises at least one rare or non-natural amino acid selected from the group consisting of: selenocysteine, p-acetyl-phenylalanine, p-azidomethyl-L-phenylalanine and /\/6-((2- azidoethoxy)carbonyl)-L-lysine.
In a preferred embodiment of the invention the activated form of the fusion protein comprises the at least one selenocysteine comprising the reactive selenium group and the therapeutic agent, preferably a chemotherapeutic agent, or the imaging agent comprises a maleimide or iodoacetamide group which is capable of reacting with the reactive group. The formation of a bound between selenium and a maleimide or iodoacetamide group is normally carried out in acidic conditions as disclosed in Sochaj et al., (2015 Biotechnology Advances 33 775-784).
In a more preferred embodiment of the invention the activated form of the fusion protein comprises the at least one p-acetyl-phenylalanine comprising the reactive carbonyl group and the therapeutic agent or the imaging agent comprises a terminal alkoxyamine or hydrazide group which is capable of reacting with the reactive group. The formation of an oxime or hydrazine bond wherein the activated fusion protein comprising an NNAA with an carbonyl group and the therapeutic or imaging agent comprising an alkoxyamine or hydrazide group can be performed as disclosed in Takimoto et al., (2009, Mol. BioSyst., 5, 931-934).
In another more preferred embodiment of the invention the activated form of the fusion protein comprises the at least one non-natural amino acid, wherein the non-natural amino acid is p-azidomethyl-L-phenylalanine or /V6-((2-azidoethoxy)carbonyl)-L-lysine, wherein the non — natural amino acid comprises azide as the reactive group and wherein the therapeutic agent or the imaging agent comprise an alkyne group which is capable of reacting with the reactive group. The bond between an azide group and an alkyne group can be achieve by the use of click chemistry or biorthogonal chemistry. The term “click chemistry” refers to powerful linking reactions that are able to generate covalent bonds quickly and reliably by joining small units comprising reactive groups together. Click chemistry reactions are typically modular, wide in scope, give high chemical yields, generate inoffensive byproducts, are stereospecific, and/or can be carried using readily available starting materials and reagents out under simple, physiological reaction conditions. In addition, click chemistry reactions preferably use no toxic solvents or use a solvent that is benign or easily removed (preferably water), and/or provides simple product isolation by non-chromatographic methods (crystallization or distillation).
Click chemistry reactions comprise, e.g., cycloaddition reactions, especially from the 1 ,3-dipolar family, hetero-Diels-Alder reactions; nucleophilic ring-opening reactions, e.g. of strained heterocyclic electrophiles, such as epoxides, aziridines, cyclic sulfates, cyclic sulfamidates, aziridinium ions and episulfonium ions; carbonyl chemistry of the non-aldol type (e.g. the formation of oxime ethers, hydrazones and aromatic heterocycles); and addition to carbon-carbon multiple bonds; e.g. oxidation reactions, such as epoxidation , di hydroxylation, aziridination, and nitrosyl and sulfenyl halide additions but also certain Michael addition reactions. General principles of click chemistry reactions are disclosed in Thirumurugan et al. (2013, Chem. Rev. 113, 4905-4979). It is within the knowledge of the person skilled in the art to select a click chemistry reaction that is suitable for linking the fusion protein to a therapeutic agent or an imaging agent.
In another embodiment, the invention relates to a method to prepare the fusion proteins of the invention comprising the steps of: a) providing a fusion protein comprising i) a PDGFR-p ligand selected from the group of consisting of PDGFB (SEQ ID N0: l), PDGFD (SEQ ID NO : 2) and a functionally equivalent variant thereof, ii) an intervening polypeptide region and iii) a positively charged amino acid-rich region, wherein the PDGFR-p ligand and the positively charged amino acid-rich region are located at the ends of the protein, and b) contacting said fusion protein with a therapeutic agent, or an oligomeric form thereof, or with an imaging agent, or an oligomeric form thereof, in the presence of an enzyme, wherein said therapeutic agent or imaging agent contains a reactive group which is recognized by the enzyme, said fusion protein contains a motif which is recognized by the enzyme, and wherein said contacting is carried out under conditions adequate for the formation of a bond between the reactive group in the therapeutic agent or the imaging agent and the motif in the fusion protein.
The term “motif” as used herein refers to any amino acid side chain, or group therein, or any specific sequence of amino acids which are recognized by the said enzyme and which function as the point of bond formation in the fusion protein, leading to an enzymatic conjugation. The term “enzymatic conjugation” as used herein may be understood in the broadest sense as any means of a ligation that is catalyzed by an enzyme. Preferably, the enzymatic conjugation is a ligase- or a peptidase-based conjugation. Examples of enzymatic conjugation are, without limitation, Sortase A (EC no.: 3.4.22.70) mediated transpeptidation and microbial transglutaminase (EC no.: 2.3.2.13) mediated transpeptidation. In a preferred embodiment of the invention, the fusion protein contains the LPXTG (X: any amino acid) motif (SEQ ID NO : 56), preferably in the intervening polypeptide, and the enzyme is a Sortase A transpeptidase, preferably the Staphylococcus aureus Sortase A.
“Sortase A” mediated transpeptidation is based on the Staphylococcus aureus Sortase A recognizing the LPXTG (X: any amino acid) motif (SEQ ID NO: 56) and cleaving the threonineglycine (T-G) bond, attaching an oligoglycine (oligo-G)-containing molecule. Various cargo can be fused to the oligo-G for sortase A-mediated conjugation. For example, as disclosed in Beerli R.R. et al., (2015, PLoS One. 10(7): e0131177), a fusion protein containing the LPXTG motif can be conjugated to an chemotherapy agent comprising which has been modified by addition of an pentaglycine peptide. Hence, in a preferred embodiment of the invention, the fusion protein contains the LPXTG (X: any amino acid) motif (SEQ ID NO : 56), preferably in the intervening polypeptide, and the enzyme is a Sortase A transpeptidase, preferably the Staphylococcus aureus Sortase A, and the therapeutic agent, preferably a chemotherapy agent, or the imaging agent is modified with an oligo-glycine reactive group.
In a preferred embodiment of the invention, the enzyme is a microbial transglutaminase.
Microbial transglutaminase mediated transpeptidation is based on the transpeptidation catalyzed by microbial transglutaminases where a primary amine- containing linker is covalently attached to the primary amide side chain of a glutamine within the fusion protein of the invention. Both non-specific and specific transglutaminases can be used, for example transglutaminases which recognize the small motif LLQG (SEQ ID NO : 57) are described in Strop et al., (Chem Biol 20:161- 167). In a preferred embodiment of the invention, the fusion protein contains the LLQG (SEQ ID NO : 57) motif, preferably in the intervening polypeptide, the enzyme is a transglutaminases which recognizes the LLQG (SEQ ID NO : 57 motif and the therapeutic agent, preferably a chemotherapy agent, or the imaging agent is modified with or comprises an amine reactive group. In a preferred embodiment of the invention the contacting said fusion protein with a therapeutic agent, or an oligomeric form thereof, or with an imaging agent, or an oligomeric form thereof, is done in at least two steps, wherein the fusion protein is first contacted with an enzyme which recognizes the motif present in the fusion protein, preferably in the intervening polypeptide, and modifies said motif, followed by the contacting of the fusion protein with the therapeutic agent or the imaging agent comprising a reactive group which presents reactivity towards the modified motif of the fusion protein. In a more preferred embodiment of the invention, the fusion protein contains the CXPXR (wherein X is any amino acid) motif, preferably in the intervening polypeptide, the enzyme is a formylglycine (FGIy)-generating enzyme which recognizes the CXPXR motif and modifies said motif, and the therapeutic agent, preferably a chemotherapy agent, or the imaging agent is modified with or comprises an aminooxy or hydrazide reactive group.
In the present context, the term “formylglycine (FGIy)-generating enzyme” refers to human enzymes present in the endoplasmic reticulum that catalyze the conversion of cysteine to formylglycine (fGly).
In a preferred embodiment of the invention, the fusion protein contains the VDSVEGEGEEEGEE motif (SEQ ID NO: 59), preferably in the intervening polypeptide, and the enzyme is tubulin tyrosine ligase (TTL). The term “tubulin tyrosine ligase”, or its acronym “TTL”, as used herein refers to an enzyme naturally involved in the intracellular regulation of microtubule stability recognizes a 14 amino acid recognition motif at the C- terminus of alpha-tubulin and posttranslationally attaches a terminal tyrosine residue. TTL can be used for the labelling known as Tub-tag. The term “Tub-tag” as used herein refers to a novel approach for the site-specific modification of antibodies that combines the above mentioned use ofUAA incorporation with a highly efficient chemoenzymatic system. When present in a fusion protein, the recognition motif (Tub-tag) allows the TTL- mediated attachment of non-natural tyrosine derivatives that carry uniquely reactive groups for chemoselective conjugation such as strain- promoted alkyne azide cycloadditions as disclosed in Schumacher et al. (2015, Angew. Chem. Int. Ed. 54, 13787 -13791).
In those embodiments of the invention wherein a linking moiety mediates the bond between the fusion protein and the therapeutic agent or between the fusion protein and the imaging agent, the linking moiety is a bifunctional cross-linker and, more preferably, a heterobifunctional cross-linker, that reacts with the groups in the therapeutic agent/imaging agent and in the fusion protein, either sequentially (either reacting with the activated therapeutic/imaging agent first and then with the fusion protein, or first with the fusion protein and then with the activated therapeutic/imaging agent) or simultaneously, using among other linkages such as thioethers, amide bonds, carbonnitrogen double bonds, or linkages generated by cycloaddition as disclosed in Kalia J et al. (Advances in bioconjugation. Curr Org Chem 2010 January, 14(2): 138- 147). As a way of example typical thiol-reactive functional groups include iodoacetamides, maleimides, and disulfides. In addition, a protein can be treated with a small molecule or surface displaying an activated ester (e.g., an N-hydroxysuccinimidyl ester) to form amide bonds with the amino groups on lysine side chains and the N terminus. In another embodiment, the linking moiety is a heterobifunctional cross-linker which contains reactive groups capable of reacting with a thiol group and with an amino group. In one embodiment, the heterobifunctional cross-linker is 6-maleimidohexanoic acid N-hydroxysuccinimide ester.
In a preferred embodiment, the linking moiety reacts in a first step with the activated therapeutic agent or with the imaging agent and in a second step with the fusion protein. In another embodiment, the linking moiety reacts in a first step with the fusion protein and, in a second step, with the therapeutic agent or with the imaging agent.
It is intended that the step of contacting the fusion protein of the invention with the activated form of the therapeutic/imaging agent is carried out in a medium which favors the reaction establishing the bond between them. Media suitable for the reactions are commonly known to the person skilled in the art, including aqueous buffers and nonaqueous buffers. It is also intended that solid supports can be used in conjunction with the media for any of the reaction steps conducing to the synthesis of the activated therapeutic/imaging agent and the conjugate of the fusion protein, the therapeutic/imaging agent, and also the linking moiety in the embodiments that include one. Furthermore, it is intended that the method for the preparation of the conjugates between the fusion protein and the therapeutic/imaging agent is not limited to the fusion protein, the activated therapeutic agent, the imaging agent and the linking moiety, but that some embodiments include also the use of one or more catalysts and co-factors in the reaction.
Thus, in one embodiment of the invention, the activated form of the therapeutic agent or the activated form of the imaging agent contains a group which reacts with at least one of the side chains of a residue in a peptide region of the fusion protein, preferably in the intervening region of the fusion protein.
In another preferred embodiment said residue is an external lysine or an external cysteine. In a further preferred embodiment of the invention, the group of the activated therapeutic agent, preferably the chemotherapeutic agent, or the activated imaging agent which reacts with the side chain of the intervening region of the fusion protein is a thiol group. In yet another preferred embodiment of the invention, the group of the activated therapeutic agent, preferably the chemotherapeutic agent, or the activated imaging agent which reacts with the side chain of the intervening region of the fusion protein is an activated carboxylic acid group. In an even more preferred embodiment of the invention, the activated therapeutic agent is an activated chemotherapeutic agent, more preferably a thiol-functionalized oligo-floxuridine. In another more preferred embodiment of the invention, the activated therapeutic agent is an activated chemotherapeutic agent, more preferably an activated carboxylic acid group-functionalized oligo-floxuridine. In an even more preferred embodiment of the invention, the activated therapeutic agent is an activated chemotherapeutic agent, more preferably an activated perfluoroaryl-functionalized oligo- floxuridine.
In a further preferred embodiment, the linking moiety is 4-maleimido hexanoic acid N-hydroxysuccinimide ester mediates the conjugation between the activated therapeutic agent or the imaging agent and the side chain of the residue of the peptide region of the fusion protein indicated in the previous embodiments of this section. In a yet more preferred embodiment, the linking moiety 4-maleimido hexanoic acid N- hydroxysuccinimide ester is bound in a first step to the therapeutic agent, preferably the activated Fdll, yet more preferably Fdll functionalized with a sulfhydryl, or the imaging agent and in a second step to the side chain in a residue of the fusion protein, more preferably to external lysines or external cysteines of the fusion protein, even more preferably to external lysines or external cysteines of the intervening region of the fusion protein.
It is also intended that the step of contacting the activated fusion protein of the invention with the therapeutic agent or the imaging agent is carried out in a medium which favors the reaction establishing the bond between them. Media suitable for the reactions are commonly known to the person skilled in the art, including aqueous buffers and non-aqueous buffers. It is also intended that solid supports can be used in conjunction with the media for any of the reaction steps leading to the synthesis of the conjugate of the fusion protein and the therapeutic agent or the imaging agent, and also the linking moiety in the embodiments that include one. Furthermore, it is intended that the method for the preparation of the conjugates between the fusion protein and the therapeutic agent or the imaging agent is not limited to the fusion protein, the activated therapeutic agent or the activated imaging agent, and the linking moiety, but that some embodiments include also the use of one or more catalysts and co-factors in the reaction.
Thus, in one embodiment of the invention, the activated form of the fusion protein contains a group which reacts with at least one moiety in the therapeutic agent or in the imaging agent. In a further preferred embodiment of the invention, the group of the therapeutic agent, preferably the chemotherapeutic agent, or of the imaging agent which reacts with the activated fusion protein is a thiol group, an activated carboxylic acid group or a perfluoroaryl group.
In an even more preferred embodiment of the invention, the activated fusion protein agent is an amino functionalized fusion protein wherein one or more amino groups in the side chain of the amino acids forming part of the intervening polypeptide is modified with an activated group having thiol reactivity, activated carboxylic acid reactivity or perfluoroaryl reactivity. In a more preferred embodiment of the invention, the activated fusion protein agent is a fusion protein comprising at least one motif of the amino acid sequence FCPF (SEQ ID NO: 60), preferably in the intervening peptide, wherein said motif has perfluoroaryl reactivity.
In a more preferred embodiment of the invention, the therapeutic agent is a chemotherapy agent and wherein the activated form thereof contains a group which reacts with at least one of the side chains in the intervening polypeptide region. In another preferred embodiment of the invention, the group which reacts with at least one of the side chains in the intervening polypeptide region is a thiol group, an activated carboxylic acid group or an perfluoroaryl group. In a further preferred embodiment of the invention, the activated chemotherapeutic agent is thiol-functionalized oligo-floxuridine, an activated carboxylic acid functionalized oligo-fluxuridine or an activated perfluoroaryl functionalized oligo-fluxuridine.
In a further preferred embodiment, the linking moiety is 4-maleimido hexanoic acid N-hydroxysuccinimide ester mediates the conjugation between an amino group in the fusion protein and a thiol group in the therapeutic agent or imaging agent. In a yet more preferred embodiment, the linking moiety 4-maleimido hexanoic acid N- hydroxysuccinimide ester is bound in a first step to the fusion protein, more preferably to external lysines of the fusion protein and in a second step to the therapeutic agent side chain in a residue of the fusion protein.
Nanoparticles of the invention and methods for preparing them with the fusion proteins of the invention
In another aspect, the invention relates to a method to prepare nanoparticles comprising multiple copies of the fusion protein according to the first aspect of the invention comprising placing a preparation of said fusion protein in a suitable buffer.
As the person skilled in the art will recognize, “nanoparticles” are microscopic particles whose size is measured in nanometers. The nanoparticles of the invention comprise the nanoparticles that result from the aggregation of multiple copies of the fusion protein of the invention as defined in the previous section. In the method for preparing nanoparticles with the fusion proteins of the invention, the preparation of the fusion protein of the invention comprises the monomeric form of the fusion proteins of the invention, which are thermodynamically favored to form non-covalent electrostatic unions and spontaneously aggregate in the conditions of the low salt buffer.
The person skilled in the art will acknowledge that the size of the nanoparticles can be in the 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 5 and 100 nm.
It will be understood that the expression “suitable buffer” comprises any buffer solution resulting from the dissolution of one or more salts in water with the capability to moderate changes in pH, wherein the amount of dissolved salt or salts results in an osmolarity similar to that of the physiological fluids, such as the cytoplasm or the extracellular medium, for instance. Thus, the suitable buffer is understood to keep pH and osmolarity inside the range of physiological values and will be used inside the range of physiological temperatures.
The person skilled in the art will recognize that the range of physiological temperatures can oscillate between 15 and 45° C, more preferably between 20 and 40°C , even more preferably between 25 and 39°C, yet even more preferably between 30 and 37°C The person skilled in the art will also acknowledge that the osmolarity of the suitable buffer will be in the range between 100 and 400 milli-osmoles/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.
Buffers suitable for the invention, for instance, are the Tris-dextrose buffer (20 mM Tris +5% dextrose, pH 7.4), the Tris-NaCI buffer (20 mM Tris, 500 NaCI, pH 7.4), the PBS-glycerol buffer (phosphate buffered saline, PBS, pH 7.4, which is well known in the art, +10% glycerol), Tris Buffered Saline (TBS)-dextrose (20 mM Tris-HCI buffer pH 7.5, well known in the art, 200NaCI, +5% dextrose), Tris Buffered Saline-Tween 20 (TBST) buffer (10 mM Tris-HCI pH 7.5, 200 mM NaCI, +0.01% Tween 20), sodium carbonate (166 NaHCO3 pH 8.0, 333 mM NaCI) or any physiological buffer known in the art with a pH not lower than 6.
In a preferred embodiment of the invention, the suitable buffer of the method of the invention is selected from the group consisting of a carbonate buffer, a Tris buffer and a phosphate buffer. In a particularly preferred embodiment of the invention, the suitable buffer of the method of the invention is a carbonate buffer that comprises sodium carbonate at a concentration between 100 and 300 nM. In another particularly preferred embodiment of the invention, the suitable buffer of the method of the invention is a Tris buffer that comprises Tris at a concentration of between 10 and 30 nM. In another particularly preferred embodiment of the method of the invention, the suitable buffer of the invention is a phosphate buffer that comprises Na2HPC>4 and NaH2PO4 at a total concentration of between 5 mM and 20 mM.
In an even more preferred embodiment of the invention, the suitable buffer of the method of the invention further comprises dextrose and/or glycerol.
In a yet more preferred embodiment of the invention, the suitable buffer of the method of the invention has a pH between 6.5 and 8.5.
In one more preferred embodiment of the invention, the suitable buffer is a sodium carbonate at a concentration of between 100 and 300 mM further comprising salt at a concentration of 200 mM to 400 mM.
In an even yet more preferred embodiment of the invention, the suitable buffer of the method of the invention is 166 mM NaHCCh, 333 mM NaCI, pH 8.0.
In another aspect of the invention, the invention relates to nanoparticles comprising multiple copies of the fusion protein of the first aspect of the invention or prepared according to the method or the invention for preparing nanoparticles.
Thus, the nanoparticles of the invention comprise assembled complexes of multiple copies of the fusion proteins of the invention, which result from the electrostatic interaction between regions in their structures favoring their non-covalent binding and coupling in physiological conditions. Since the method of the invention for the preparation of nanoparticles comprises placing a preparation of the fusion protein of the invention in a low salt buffer, it is understood that the nanoparticles thus formed comprise also an assembled complex of multiple copies of the fusion protein.
In a preferred embodiment of the invention, the nanoparticles of the invention have a diameter between 5 and 100 nm.
Polynucleotide, vector, and host cells of the invention
In another aspect of the invention, the invention relates to a polynucleotide encoding the fusion protein of the first aspect invention, a vector comprising the aforementioned polynucleotide, and a host cell comprising the aforementioned polynucleotide or the aforementioned vector. The terms “nucleic acid” and “polynucleotide”, as used herein interchangeably, 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 via phosphodiester bonds, related naturally occurring structural variants and synthetic non-naturally occurring analogs thereof.
The person skilled in the art will acknowledge that the polynucleotide encodes the polypeptide or protein sequence of the fusion protein of the invention that corresponds to the first aspect of the invention. The polynucleotide of the invention therefore comprises the sequence encoding all of the elements comprised in the fusion protein: the PDGFR-p ligand, the intervening peptide region, the positively charged amino acidrich region, and any other elements that may be part of the fusion protein such as the reporter protein, linkers, and so on and so forth.
It is understood that the nucleic acids or polynucleotides of the invention include coding regions and the adequate regulatory signals for promoting expression in cells to give rise to the biologically active fusion protein.
Generally, nucleic acids containing a coding region will be operably linked to appropriate regulatory sequences. Such regulatory sequence will at least comprise a promoter sequence. As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA- dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most physiological and developmental conditions. An "inducible" promoter is a promoter that is regulated depending on physiological or developmental conditions. A "tissue specific" promoter is only active in specific types of differentiated cells/tissues.
In principle, any promoter can be used for the gene constructs of the present invention provided that said promoter is compatible with the cells in which the polynucleotide is to be expressed. Thus, promoters suitable for the embodiment of the present invention include, without being necessarily limited to, constitutive promoters such as the derivatives of the genomes of eukaryotic viruses such as the polyoma virus, 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, retrovirus LTR regions, the promoter of the immunoglobulin gene, the promoter of the actin gene, the promoter of the EF-1 alpha gene as well as inducible promoters in which the expression of the protein depends on the addition of a molecule or an exogenous signal, such as the tetracycline system, the NFKB/UV light system, the Cre/Lox system and the promoter of heat shock genes, the regulatable promoters of RNA polymerase II described in WO/2006/135436 as well as tissue-specific promoters.
The polynucleotides of the invention encoding the fusion protein of the invention can be part of a vector. Thus, in another embodiment, the invention relates to a vector comprising a polynucleotide of the invention. A person skilled in the art will understand that there is no limitation as regards the type of vector which can be used because said vector can be a cloning vector suitable for propagation and for obtaining the polynucleotides or expression vectors in different heterologous organisms suitable for purifying the fusion proteins of the invention. Thus, suitable vectors according to the present invention include expression vectors in prokaryotes such as pET (such as pET14b), pUC18, pUC19, Bluescript and their derivatives, mp18, mp19, pBR322, pMB9, ColEI, pCRI, RP4, phages and shuttle vectors such as pSA3 and pAT28, expression vectors in yeasts such as vectors of the type of 2 micron plasmids, integration plasmids, YEP vectors, centromeric plasmids and the like, expression vectors in insect cells such as the pAC series and pVL series vectors, expression vectors in plants such as vectors of expression in plants such as pl Bl, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series vectors and the like and expression vectors in superior eukaryotic cells based on viral vectors (adenoviruses, viruses associated to adenoviruses as well as retroviruses and lentiviruses) as well as non-viral vectors such as pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1/hyg pHCMV/Zeo, pCR3.1 , pEFI/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAXI, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1 , pML2d and pTDTI.
The vector of the invention can be used to transform, transfect, or infect cells which can be transformed, transfected or infected by said vector. Said cells can be prokaryotic or eukaryotic. By way of example, the vector wherein said DNA sequence is introduced can be a plasmid or a vector which, when it is introduced in a host cell, is integrated in the genome of said cell and replicates together with the chromosome (or chromosomes) in which it has been integrated. Said vector can be obtained by conventional methods known by the persons skilled in the art (Sambrook et al., 2001 , “Molecular cloning, to Laboratory Manual”, 2nd ed., Cold Spring Harbor Laboratory Press, N.Y. Vol 1-3 a).
Therefore, the invention also relates to a cell comprising a polynucleotide or a vector of the invention, for which said cell has been able to be transformed, transfected or infected with a polynucleotide or vector provided by this invention. The transformed, transfected or infected cells can be obtained by conventional methods known by persons skilled in the art (Sambrook et al., 2001 , mentioned above).
Host cells suitable for the expression of the conjugates of the invention include, without being limited to, mammal, plant, insect, fungal and bacterial cells. Bacterial cells include, without being limited to, Gram-positive bacterial cells such as species of the Bacillus, Streptomyces, Listeria and Staphylococcus genera and Gram-negative bacterial cells such as cells of the Escherichia, Salmonella and Pseudomonas genera. Fungal cells preferably include cells of yeasts such as Saccharomyces cereviseae, Pichia pastoris and Hansenula polymorpha. Insect cells include, without being limited to, Drosophila and Sf9 cells. Plant cells include, among others, cells of crop plants such as cereals, medicinal, ornamental or bulbous plants. Suitable mammal cells in the present invention include epithelial cell lines (human, ovine, porcine, etc.), osteosarcoma cell lines (human, etc.), neuroblastoma cell lines (human, etc.), epithelial carcinomas (human, etc.), glial cells (murine, etc.), hepatic cell lines (from monkey, 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 the mESC line, human embryonic stem cells such as HS293, BGV01 , SHEF1 , SHEF2, HS181 , NIH3T3 cells, 293T, REH and MCF-7 and hMSC cells.
In a preferred embodiment of the invention, the polynucleotide, the vector, and the host cell of the invention are suitable for the expression of the biologically active form of the fusion protein of the invention.
Uses in medicine of the fusion protein, the polynucleotide, the vector and the nanoparticle of the invention
In another aspect, the invention relates to a fusion protein, a polynucleotide, a vector, a host cell or a nanoparticle according to the invention for use in medicine.
It will be understood by the person skilled in the art that by use in medicine, the fusion protein, polynucleotide, vector, host cell, or nanoparticle of the invention can be administered to a patient in order to induce a therapeutic response. The therapeutic response comprises the suppression, reduction or arrest of the causes of the pathological condition or the disease suffered by a patient; the elimination, reduction, arrest or amelioration of the symptoms of the condition or disease; or the extinction, arrest or slowing down of the progression of the condition or disease in the patient.
The person skilled in the art will acknowledge that the fusion protein, polynucleotide, vector, host cell or nanoparticle of the invention suitable for use in medicine may be presented accompanied by a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Remington's Pharmaceutical Sciences. Ed. by Gennaro, Mack Publishing, Easton, Pa., 1995 discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.
Accordingly, the compositions comprising the fusion protein, polynucleotide, vector, host cell, or nanoparticle of the invention and a pharmaceutically acceptable carrier are pharmaceutical compositions.
The pharmaceutical compositions of this invention can be administered to a patient by any means known in the art including oral and parenteral routes. According to such embodiments, inventive compositions may be administered by injection (e.g., intravenous, subcutaneous or intramuscular, intraperitoneal injection), rectally, vaginally, topically (as by powders, creams, ointments, or drops), or by inhalation (as by sprays).
Use of the fusion protein, the polynucleotide, the vector, the host cell, or the nanoparticle of the invention in the treatment of cancer.
Another embodiment of the invention relates to a fusion protein, a polynucleotide of the invention, the vector of the invention, the host cell of the invention comprising the vector or the polynucleotide and expressing the fusion protein, and the nanoparticle of the invention, or their corresponding pharmaceutical compositions, wherein the intervening polypeptide region is an antitumor peptide or wherein the intervening polypeptide is linked to an antitumor agent, for use in the treatment of cancer.
As used herein, the terms "treat", "treatment" and "treating" refer to the reduction or amelioration of the progression, severity and/or duration of cancer, or the amelioration of one or more symptoms (preferably, one or more discernible symptoms) of cancer. The terms "treat", "treatment" and "treating" also refer to the amelioration of at least one measurable physical parameter of cancer, such as growth of a tumor, not necessarily discernible by the patient. Furthermore, "treat", "treatment" and "treating" refer also to the inhibition of the progression of cancer, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. "Treat", "treatment" and "treating" may refer, too, to the reduction or stabilization of tumor size or cancerous cell count.
The term “cancer” refers to a group of diseases involving abnormal, uncontrolled cell growth and proliferation (neoplasia) with the potential to invade or spread (metastasize) to other tissues, organs or, in general, distant parts of the organism; metastasis is one of the hallmarks of the malignancy of cancer and cancerous tumors. The abnormal growth and/or proliferation of cancerous cells is the result of a combination of genetic and environmental factors that alter their normal physiology. The growth and/or proliferation abnormalities of cancerous cells result in physiological disorders and, in many cases, death of the individual, due to the dysfunctionality or loss of functionality of the cell types, tissues and organs affected.
The term “cancer” includes, but is not restricted to, cancer of the breast, heart, small intestine, colon, spleen, kidney, bladder, head, neck, ovaries, prostate gland, brain, pancreas, skin, bone, bone marrow, blood, thymus, womb, testicles, hepatobiliary system and liver; in addition to tumors such as, but not limited to, adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma, hemangiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, hepatobiliary cancer, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma and teratoma. Furthermore, this term includes acrolentiginous melanoma, actinic keratosis adenocarcinoma, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamus carcinoma, astrocytic tumors, Bartholin gland carcinoma, basal cell carcinoma, bronchial gland carcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Ewing sarcoma, focal nodular hyperplasia, germ cell tumors, glioblastoma, glucagonoma, hemangioblastoma, hemagioendothelioma, hemagioma, hepatic adenoma, hepatic adenomastosis, hepatocellular carcinoma, hepatobilliary cancer, insulinoma, intraepithelial neoplasia, squamous cell intraepithelial neoplasia, invasive squamous-cell carcinoma, large cell carcinoma, leiomyosarcoma, melanoma, malignant melonoma, malignant mesothelial tumor, meduloblastoma, medulloepithelioma, mucoepidermoid carcinoma, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, papillary serous adenocarcinoma, pituitary tumors, plasmacytoma, pseudosarcoma, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, microcytic carcinoma, soft tissue carcinoma, somatostatin secreting tumor, squamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vipoma, Wilm tumor, intracerebral cancer, head and neck cancer, rectal cancer, astrocytoma, glioblastoma, microcytic cancer and non-microcytic cancer, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer and breast cancer.
Hence, in a preferred embodiment of the invention, the fusion protein, the polynucleotide, the vector, the host cell, or the nanoparticle of the invention for use in the treatment of cancer, wherein the cancer is a tumor, preferably a solid tumor.
Thus, in a preferred embodiment of the invention, the antitumor peptide of the fusion protein, the polynucleotide, the vector, the host cell, or the nanoparticle of the invention is selected from the group consisting of
(i) a cytotoxic polypeptide,
(ii) an antiangiogenic polypeptide,
(iii) a polypeptide encoded by a tumor suppressor gene,
(iv) a pro-apoptotic polypeptide,
(v) a polypeptide having anti-metastatic activity,
(vi) a polypeptide which is capable of activating the immune response towards a tumor,
(vii) a polypeptide encoded by a suicide gene.
In a more preferred embodiment of the invention, the antitumor peptide of the fusion protein, the polynucleotide, the vector, the host cell, or the nanoparticle of the invention is selected from the group consisting of the BH3 domain of BAK, PUMA, GW- H1 , the Diphtheria toxin, the Pseudomonas exotoxin and Ricin. In a further preferred embodiment of the invention, the antitumor peptide of the fusion protein, the polynucleotide, the vector, the host cell, or the nanoparticle of the invention is a truncated form or a mutant of the peptide selected from the group indicated just before, preferably from the group consisting of the Diphtheria toxin, the Pseudomonas exotoxin and Ricin. Preferred sequences of said peptides are indicated above in the “Intervening polypeptide region" section.
In another preferred embodiment of the invention, the antitumor agent of the fusion protein, polynucleotide, vector, host cell or nanoparticle of the invention is selected from the group consisting of: (i) a chemotherapy agent,
(ii) an antiangiogenic molecule
(iii) a toxin.
In another preferred embodiment of the invention, the antitumor agent of the fusion protein, polynucleotide, vector, host cell or nanoparticle of the invention is selected is selected from a group consisting of: a floxuridine polynucleotide, pyrimidine analogue or an oligomeric form thereof, auristatin, and any of the agents indicated above in the “Intervening polypeptide region" section.
In another more preferred embodiment of the invention, the cancer to be treated with the fusion protein, the polynucleotide, the vector, the host cell, or the nanoparticle of the invention is selected from the group consisting of non-small cell lung, breast, colon, liver, prostate, pancreatic or colorectal cancer.
Thus, in another preferred embodiment of the invention, the cancer to be treated with the fusion protein, the polynucleotide, the vector, the host cell, or the nanoparticle of the invention is characterized by comprising cancer cells that express or overexpress PDGFR-p.
As used herein, the term “overexpressed PDGFR-P” or “overexpression of PDGFR-P” refers to an abnormal level of expression of PDGFR-p in epithelial and stromal cells within cancer tissues. Overexpression of PDGFR-p can be determined by standard assays in the art, such as, immunohistochemistry assays, wherein histological sections of the tissue/cells of interest are incubated with primary antibodies against PDGFR-p, followed by secondary antibodies against the primary antibody. The presence of a label in the secondary antibody whose signal can be detected and quantify, allows to compare the signal amount from the tissue/cells of interest against a reference value of tissue/cells which are known to express PDGFR-p at physiological levels. Tissues/cells which overexpress PDGFR-p have a higher signal than the control tissue/cells (Tsao A. S., et al, 2011 , Clin Lung Cancer; 12(6): 369-374). Another method to determine if cells overexpress PDGFR-p is the use of the fusion protein of the invention wherein the intervening polypeptide is an imaging agent of the ones described previously. The incubation of the fusion protein with the cells of interest will allow the fusion protein of the invention to bind the PDGFR-p. After the removal of unbound fusion protein of the invention, the imaging agent can be detected and the quantity of imaging agent/fusion protein bound to the PDGFR-p expressing cells determined. If the value obtained is significantly higher than the value obtained for cells expressing PDGFR-p at physiological levels to which the procedure as exemplified was repeated, then the cells of interest do overexpress PDGFR-p.
Use of the fusion protein, the polynucleotide, the vector, the host cell, or the nanoparticle of the invention in the treatment of diseases caused by alterations in PDGFR-/3 positive cells.
In another aspect, the invention relates to a fusion protein, a polynucleotide, a vector, a host cell or a nanoparticle according to the invention for use in the treatment of diseases caused by alterations in PDGFR-p positive cells.
The expression “diseases caused by alterations in PDGFR-p positive cells” as used herein refers to diseases which can be characterized by the presence, participation or function of cells which express in their surface the PDGFR-p protein and therefore can be targeted by the fusion protein of the invention or the nanoparticles of the invention, in order to deliver therapeutic agents to said cells and treat the disease. Examples of such diseases are fibrosis, pancreatitis, neurological diseases, atherosclerosis and inflammation. It will be understood that the target within the PDGFR-p positive cells will depend on the specific disease to be treated and that a person skilled in the art would be able to select the therapeutic agent suitable for the disease to be treated.
In a particular embodiment of the invention, the diseases caused by alterations in PDGFR-p positive cells are characterized by the presence of PDGFR-p positive cells. In another preferred embodiment of the invention the PDGFR-p positive cells are selected from a group consisting of: lung fibroblast, hepatic stellate cells, pancreatic stellate cells, mesenchymal cells, perivascular astrocytes, astrocytes, oligodendrocyte, vascular smooth muscle cell and fibroblast.
In a preferred embodiment of the invention, the diseases caused by alterations in PDGFR-p positive cells are selected from a group consisting of: pulmonary fibrosis, hepatic fibrosis, pancreatic fibrosis, pancreatitis; ischemic stroke, Parkinson, Alzheimer, neurofibrosmatosis, amyotrophic lateral sclerosis, systemic sclerosis, glaucoma and rheumatoid arthritis.
It will be of the understanding of the person skilled in the art that each treatment will be associated with a specific therapeutic agent which is targeted to the positive PDGFR-p cells that are altered in the disease. Therefore, in a preferred embodiment of the invention the disease caused by alterations in PDGFR-p positive cells is pulmonary fibrosis which is characterized by the presence of PDGFR-p positive lung fibroblasts. The term "pulmonary fibrosis" as used herein, refers to pulmonary fibrosis occurred due to various reasons, specifically, due to radiation exposure, drug therapy for anticancer treatment, smoking, or dusty work environments, etc., but is not limited thereto. Further, the pulmonary fibrosis above may be side effects of radiotherapy occurred by exposure of radiation to normal tissues during radiotherapy for cancer or may be side effects of drug therapy for anticancer treatment, but is not limited thereto. The radiotherapy or drug therapy for cancer, which is capable of inducing pulmonary fibrosis, includes treatment of a lung cancer, a breast cancer, or Hodgkin lymphoma, etc., but is not limited thereto. Examples of therapeutic agents which can be conjugated to the fusion protein of the invention for the treatment of pulmonary fibrosis are, without limitation, nintedanib and imatinib.
In a preferred embodiment of the invention the disease caused by alterations in PDGFR-p positive cells is hepatic fibrosis which is characterized by the presence of PDGFR-p positive hepatic stellate cells. In the present context, the term “hepatic fibrosis” refers to abnormal hyperplasia of connective tissue in the liver, excessive precipitation of diffuse extracellular matrix in the liver, pathological changes in the normal structure of the liver (lesion), which are caused by or accompanied by inflammation, infection (e.g. viral infection), immune response, ischemia, chemicals, radiation, oxidative stress and alcohol abuse, etc. Hepatic fibrosis further developing into cirrhosis is also covered by the term "hepatic fibrosis" in the present invention. Examples of therapeutic agents which can be conjugated to the fusion protein of the invention for the treatment of hepatic fibrosis are, without limitation, imatinib, vitamin D, lorsatan, PAT-1251 , GKT137831 , calciprotiol and all the toxins previously mentioned in relation to the therapeutic agents which can be conjugated to the fusion protein of the invention as well as toxins in the form of polypeptides which can be the intervening polypeptide as described previously.
The term “hepatic stellate cells” as used herein refers to liver-specific mesenchymal cells that play vital roles in liver physiology and fibrogenesis. They are located in the space of Disse and maintain close interactions with sinusoidal endothelial cells and hepatic epithelial cells.
In a preferred embodiment of the invention the disease caused by alterations in PDGFR-p positive cells is pancreatic fibrosis or pancreatitis which is characterized by the presence of PDGFR-p positive pancreatic stellate cells.
The term "pancreatitis" as used herein refers to an inflammation of the pancreas. Pancreatitis can be either acute or chronic. Acute pancreatitis generally develops suddenly, and chronic pancreatitis is a long-term condition, which typically develops after multiple episodes of acute pancreatitis, leading to pancreatic fibrosis. Acute pancreatitis distinguishes between two different forms: Interstitial edematous acute pancreatitis with no local or systemic complications and acute necrotizing pancreatitis (ANP), associated with both local and systemic complications and a high risk of mortality. Examples of therapeutic agents which can be conjugated to the fusion protein of the invention for the treatment of pancreatitis or pancreatic fibrosis are, without limitation, calciprotiol.
The term “pancreatic stellate cells” in the context of the present invention refers to myofibroblast-like cells that are located in exocrine regions of the pancreas. Pancreatic stellate cells are mediated by paracrine and autocrine stimuli and share similarities with the hepatic stellate cell.
In a preferred embodiment of the invention the disease caused by alterations in PDGFR-p positive cells is renal fibrosis which is characterized by the presence of PDGFR-p positive mesenchymal cells. The term "renal fibrosis" as used herein includes all diseases in which fibrosis occurs in the kidney due to various causes, and the fibrosis may include, but is not limited to, those caused by any one or more selected from the group consisting of: catheter installation, glomerulosclerosis, glomerulonephritis, nephritis, acute renal failure, chronic renal failure, end-stage renal disease, and metabolic disease. Examples of therapeutic agents which can be conjugated to the fusion protein of the invention for the treatment of renal fibrosis are, without limitation, imatinib and all of the tyrosine kinase inhibitors previously described.
The term “mesenchymal cells” as used herein refers to a cell forming a mesenchymal tissue, such as osteoblat, chondrocyte, myoblast, adipocyte, stroma cell, tendon cell, and the like.
In a preferred embodiment of the invention the disease caused by alterations in PDGFR-p positive cells is ischemic stroke which is characterized by the presence of PDGFR-p positive perivascular astrocytes. The term “ischemic stroke” as used herein refers to cerebral ischemic stroke, caused by reduced blood flow to the brain or parts thereof which leads to a reduced delivery (undersupply) of oxygen to brain cells resulting in tissue damage due to brain cell death. Examples of therapeutic agents which can be conjugated to the fusion protein of the invention for the treatment of ischemic stroke are, without limitation, imatinib and all of the tyrosine kinase inhibitors previously described.
The term “perivascular astrocytes” as used herein refers to cells in the brain which closely juxtapose blood vessels and are postulated to have important roles in the control of vascular physiology, including regulation of the blood-brain barrier (BBB). In a preferred embodiment of the invention the diseases caused by alterations in PDGFR-p positive cells is selected from a group consisting of: Parkinson, Alzheimer and neurofibrosmatosis, wherein said diseases are characterized by the presence of PDGFR-p positive astrocytes and oligodendrocyte. The term “Parkinson” as used herein refers to a disease which is characterized by progressive degeneration of nigrostriatal dopamine neurons. In this sense, the term Parkinson's disease also comprises the term Parkinson's syndrome. The term “Alzheimer” refers to a disease in which losses of significant memory and other intellectual abilities occur enough to hinder patient's daily life, and means a neurodegenerative disease characterized by a histopathological overall atrophy of the brain, enlargement of the ventricle, multiple lesions of nerve fibers (nerve fiber twist), neuritic plaque and the like. The term “neurofibrosmatosis” as used herein refers to a group of three conditions in which tumors grow in the nervous system. The three types are neurofibromatosis type I (NF1), neurofibromatosis type II (NF2), and schwannomatosis. In NF1 symptoms include light brown spots on the skin, freckles in the armpit and groin, small bumps within nerves, and scoliosis. In NF2, there may be hearing loss, cataracts at a young age, balance problems, flesh colored skin flaps, and muscle wasting. In schwannomatosis there may be pain either in one location or in wide areas of the body. The tumors in NF are generally non-cancerous. Examples of therapeutic agents which can be conjugated to the fusion protein of the invention for the treatment of Parkinson, Alzheimer and neurofibrosmatosis are, without limitation, imatinib, nilotinib, sunitinib, masitinib and all of the tyrosine kinase inhibitors previously described.
The term “astrocytes”, also termed astroglia, refers to the cells which anchor neurons to their blood supply. Generally, astrocytes regulate the external chemical environment of neurons by removing excess ions, notably potassium, and recycling neurotransmitters released during synaptic transmission. Astrocytes may be the predominant "building blocks" of the blood-brain barrier. Astrocytes may regulate vasoconstriction and vasodilation by producing substances such as arachidonic acid, whose metabolites are vasoactive.
The term “oligodendrocytes” as used herein refers to myelinating cells of the central nervous system (CNS). They are the end product of a cell lineage which has to undergo a complex and precisely timed program of proliferation, migration, differentiation, and myelination to finally produce the insulating sheath of axons.
In a preferred embodiment of the invention the diseases caused by alterations in PDGFR-p positive cells is selected from a group consisting of: amyotrophic lateral sclerosis, systemic sclerosis and atherosclerosis, wherein said diseases are characterized by the presence of PDGFR-p positive vascular smooth muscle cells. The term “atherosclerosis” as used herein refers to all types of the disease including calcified plaques, non-calcified plaques, fibrocalcified plaques and others. The term “amyotrophic lateral sclerosis” as used herein refers to a progressive neurodegenerative disorder which affects the upper and lower motor neuron or the lower motor neuron alone or the upper motor neuron alone. The term “systemic sclerosis” refers to a multisystem disorder characterized by inflammatory, vascular, and fibrotic changes of skin and various internal organ sytems (chiefly Gl tract, lungs, heart, and kidney). Primary event may be endothelial cell injury with eventual intimal proliferation, fibrosis, and vessel obliteration. Clinical manifestations include, but are not limited to, Raynaud's phenomenon, scleroderma (fibrosis of the skin), hypertension, and renal failure. Examples of therapeutic agents which can be conjugated to the fusion protein of the invention for the treatment of amyotrophic lateral sclerosis, systemic sclerosis and atherosclerosis are, without limitation, imatinib, masitinib and all of the tyrosine kinase inhibitors previously described.
The term “vascular smooth muscle cells” as used herein refers to the smooth muscle that makes up most of the walls of blood vessels.
In a preferred embodiment of the invention the diseases caused by alterations in PDGFR-p positive cells is selected from a group consisting of: inflammation, arthritis rheumatoid and glaucoma, wherein said diseases are characterized by the presence of PDGFR-p positive fibroblasts. The term “inflammation” as used herein refers to any condition characterized by a localized protective response elicited by injury or destruction of tissues resulting from any of the causes mentioned hereinbefore, and which is manifest by heat, swelling, pain, redness, dilation of blood vessels and/or increased blood flow, invasion of the affected area by white blood cells, loss of function and/or any other symptoms known to be associated with the inflammatory condition. The term will thus be understood to include inter alia acute, chronic, ulcerative, specific, allergic and necrotic inflammation, as well as all other forms of inflammation known to those skilled in the art. The term “arthritis rheumatoid” as used herein refers to a long-term autoimmune disorder that primarily affects joints. It typically results in warm, swollen, and painful joints. The term “glaucoma” as used herein refers to a group of ocular neurodegenerative disorders which represents the main cause of irreversible blindness worldwide whose incidence is constantly increasing. Glaucoma is characterized by altered aqueous humor outflow with a consequent increase of the intraocular pressure, by the excavation of the optic nerve head (ONH) and by the slow progressive loss of retinal ganglion cells (RGCs). Examples of therapeutic agents which can be conjugated to the fusion protein of the invention for the treatment of ischemic stroke are, without limitation, tofacitinib, peficitinib, verteporfin, fasudil and ripasudil.
The term “fibroblasts” as used herein refers to used herein refers to a cell constituting a component of fibrous connective tissue, and may be a cell of connective tissue of a mammal. Fibroblasts can produce extracellular matrix and collagen, and can serve to heal wounds, for example, skin scars, burns, pressure sores, or cut wounds.
In another preferred embodiment of the invention the diseases caused by alterations in PDGFR-p positive cells are the diseases referenced to in the column entitled “Disease or disorder” in Table 2, (diseases or disorders) wherein the therapeutic agent for each disease is the therapeutic agent indicated in the column “Therapeutic agent” of Table 2 (therapeutic agent) and said therapeutic agent is conjugated to the intervening polypeptide of the fusion protein of the invention.
Use of the fusion protein, the polynucleotide, the vector, the host cell, or the nanoparticle of the invention in the diagnosis of PDGFR-p positive cancer.
The fusion protein or the nanoparticle of the present invention may also be used to detect and/or measure PDGFR-p, or PDGFR-p-expressing cells in a sample, e.g., for diagnostic purposes. For example, the fusion protein or nanoparticle of the invention may be used to diagnose a condition or disease characterized by aberrant expression (e.g., over-expression, under-expression, lack of expression, etc.) of PDGFR-p. Exemplary diagnostic assays for PDGFR-p may comprise, e.g., contacting a sample, obtained from a patient, with the fusion protein or the nanoparticle of the invention, wherein the intervening polypeptide of the fusion protein is an imaging polypeptide or conjugated to an imaging agent as previously described, or wherein the fusion protein further comprises a reporter molecule as previously described. Alternatively, an unlabeled fusion protein or nanoparticle of the fusion protein can be used in diagnostic applications in combination with a secondary antibody specific for the fusion protein of the invention which is itself detectably labeled with an imaging agent as previously described. Specific exemplary assays that can be used to detect or measure PDGFR-p in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).
Therefore, in a further aspect of the present invention relates to the use of the fusion protein or the nanoparticle of the invention for the detection and/or measurement of PDGFR-p in a sample. Another aspect of the present invention relates to the fusion protein or the nanoparticle of the invention for the diagnosis of PDGFR-p positive cancer.
Samples that can be used in PDGFR-p diagnostic assays according to the present invention include any tissue or fluid sample obtainable from a patient which contains detectable quantities of PDGFR-p protein, or fragments thereof, under normal or pathological conditions. Generally, levels of PDGFR-beta in a particular sample obtained from a healthy patient (e.g., a patient not afflicted with a disease or condition associated with abnormal PDGFR-p levels or activity) will be measured to initially establish a baseline, or standard, level of PDGFR-p. This baseline level of PDGFR-p can then be compared against the levels of PDGFR-p measured in samples obtained from individuals suspected of having a PDGFR-p related disease or condition.
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The invention is described below by way of the following examples which are to be taken as merely illustrative and not limiting the scope of the invention.
EXAMPLES
Example 1
Materials and Methods
Protein description: Four peptide ligands of PDGFR-p were selected from literature and described hereafter. PDGFB (SEQ ID NO: 1) and PDGFD (SEQ ID NO: 2) are both human ligands of PDFGR-p. PDGFRP1 (SEQ ID NO : 27) is a small cationic peptide isolated by phage display (G2 version), while Z09591 (SEQ ID NO : 28) is an anionic affibody also identified by phage display and commonly used for radiolabeling and imaging of PDGFR-p expression in different cancers.
Protein design, production and purification: The sequences of four green- fluorescent PDGFR-p targeted proteins (Figure 1A) were designed in house as codon- optimized genes and subcloned into pET22b plasmids using Ndel and Hindi 11 restriction enzymes. Geneart (ThermoFisher) provided the recombinant plasmids. PDGFR-p targeted domains were envisaged as placed at the N-terminus of each protein, followed by a flexible linker (GGSSGGS - SEQ ID NO: 4), the green fluorescent protein (GFP) (SEQ ID NO: 17) and the hexa-histidine tag H6 (SEQ ID NO: 3). The H6 tag allows both protein purification but it also promotes assembling through coordination with divalent cations. The recombinant pET22b vectors were transformed into Escherichia coli by heat shock at 42 °C for 45 s. PDGFRP1-GFP-H6 (SEQ ID NO : 29) and Z09591-GFP-H6 (SEQ ID NO : 30), lacking intramolecular or intermolecular disulfide bonds, were produced in BL21 (DE3), PDGFB-GFP-H6 (SEQ ID NO: 31) in Origami B (BL21 DE3, OmpT’, Lon’, TrxB“, Gor, Novagen) and PDGFD-GFP-H6 (SEQ ID NO: 32) in BL21 (DE3) previously engineered for the expression of sulfhydryl oxidase and DsbC (kindly provided by Prof. A. de Marco). Protein production was carried out at the optimal conditions for each candidate. In this sense, PDGFRP1-GFP-H6 and Z09591-GFP-H6 were produced overnight at 20 °C and 250 rpm in Luria Broth (LB) supplemented with 100 pg mL -1 ampicillin, upon addition of 1 x 10'4 M of isopropyl-p-d-thiogalactopyronaside (IPTG), at ODssonm 0.6-0.8 units. PDGFB-GFP-H6 was produced overnight at 16 °C and 250 rpm in LB supplemented with 100 pg mL -1 ampicillin, 12.5 pg mL -1 tetracycline and 15 pg mL ’ 1 kanamycin, upon addition of 1 x 10'4 M IPTG, at ODssonm 0.6-0.8 units. Cells were then harvested by centrifugation (15 min, 5000 g) and stored at -80 °C. For protein purification, cells were resuspended in wash buffer (2 x 10'2 M Tris-HCI, 5 x 10'1 M NaCI, 1 x 10'2 M imidazole, pH 8.0) in presence of protease inhibitors (complete EDTA-free, Roche Diagnostics) and disrupted in an EmulsiFlex-C5 system (Avestin) by 3 rounds at 8000 psi. The soluble fraction was then collected by centrifugation (45 min at 15000 g) and proteins purified by an immobilized metal affinity chromatography (IMAC) using a HisTrap HP column (GE Healthcare) in an AKTA pure system (GE Healthcare). Elution was achieved by a lineal increase of imidazole concentration (Elution Buffer, 2 x 10'2 M Tris-HCI, 5 x 10’1 M NaCI, 5 x 10’1 M imidazole, pH 8.0). For PDGFD-GFP-H6 gene expression, bacteria were grown in LB supplemented with 100 pg mL -1 ampicillin and 34 pg mL -1 chloramphenicol at 37 °C until ODssonm reached 0.4-0.5 units. In that moment, the expression of sulfhydryl oxidase and DsbC was induced by adding 0.5 % (m/v) of L- arabinose and temperature was lowered to 30 °C. 45 minutes later, temperature was lowered to 16 °C and 1 x 10'3 M of IPTG was added to induce PDGFD-GFP-H6 overnight expression. Cells were then harvested by centrifugation (15 min 5000 g) and an osmotic shock was performed to remove the metallophores from the periplasmic fraction. For that, cells were first resuspended in a hypertonic solution (20% sucrose, 1 x 10'3 M ethylenediaminetetraacetic acid (EDTA), 5 x 10'2 M 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES), pH 7.9) and centrifuged (30 min, 7000 g, 4 °C). Supernatant was discarded and cells were then resuspended in a hypotonic solution (5 x 10’3 M MgSO4), placed 10 min at 4 °C and subsequently centrifuged (15 min, 4500 g) to remove again the supernatant. Bacterial cells, now without periplasm, were finally resuspended in wash buffer in presence of protease inhibitors and stored at -80 °C. Disruption and purification were carried out as stated for the other proteins. For comparison purposes, GFP-H6 was produced and purified as previously described (E.
Volta-Duran et al., Sicence China Materials 2019).
Physicochemical characterization of proteins and protein materials: After purification, proteins were dialyzed against an appropriate solution, namely sodium carbonate (166 mM NaCChH, pH 8.0) for PDGFRP1-GFP-H6 and sodium carbonate with salt (166 mM NaCO3H, 333 mM NaCI, pH 8.0) for Z09591-GFP-H6, PDGFB-GFP-H6 and PDGFD-GFP-H6. A particular amount of ZnCh was added to pure solutions of PDGFRP1-GFP-H6 (3:1 molar ratio, Zn2+: Histidine residues in H6) and Z09591-GFP- H6 (1 :1 molar ratio, Zn2+: Histidine residues in H6) to promote protein assembling and nanoparticle formation. Protein purity and integrity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and western blot immunodetection using an anti-His (Santa Cruz Biotechnology) and an anti-GFP (Santa Cruz Biotechnology) antibody. The same molar amount was used for each protein. Protein concentration was determined by the Bradford assay (Bio-Rad). Dynamic light scattering (DLS) was used to determine the volume size distribution of each type of nanoparticle. Measurements were conducted in a Zetasizer Advanced Pro Blue (Malvern Instruments Limited) at 25 °C and 633 nm (n=3) using a quartz cuvette. Also, the GFP fluorescence was evaluated in a Varian Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies). Protein concentration was set to 5 pM, the excitation wavelength to 488 nm and the emission recorded at 511 nm. The percentage of intrinsic fluorescence was calculated in comparison to the non-targeted control GFP-H6. Protein stability in the in vitro culture media (Dulbecco’s Modified Eagle Medium, DMEM, Gibco; supplemented with fetal bovine serum, FBS, Gibco) was measured by incubating the proteins at the maximum concentration used in in vitro experiments (1 x 10'3 M), for the maximum time of exposure (24 h) at 37 °C, and separating the soluble and insoluble fractions by centrifugation (15 min, 15000 g). A protein electrophoresis gel, followed by an anti-His Western Blot, were finally performed.
Ultrastructural characterization: Nanoscale morphometry (size and shape) of the different protein nanoparticles was visualized at nearly native state with two rapid high- resolution imaging techniques. Drops of 5 pL of samples diluted at 0.2 pg mL-1 in its buffer were deposited in silicon wafers (Ted Pella) and observed in a field emission scanning electron microscope (FESEM) Merlin (Zeiss) operating at 1 kV and equipped with an in-lens secondary electron detector. Drops of samples diluted at 0.2 pg mL-1 in its buffer were negatively stained with 2 % uranyl acetate (Merck) in 400 mesh carbon- coated copper grids (Electron Microscopy Sciences) and observed in a transmission electron microscope (TEM) JEM 1400 (Jeol) operating at 80 kV and equipped with an Orius SC200 CCD camera (Gatan). Representative images of general fields and nanoparticles details were obtained at three magnifications.
Three-dimensional models and visualization: In silico three-dimensional structure prediction of the four PDGFR-p targeted modular proteins was performed using AlphaFold Colab. ChimeraX software version 1.2 was used then for their 3D structure visualization. GraphPad Prism 8 was used for graphics and statistical tests.
Cell lines and cell culture: Mouse Embryonic Fibroblast (MEFs) and Mesenchymal Stem Cells (MSCs) were kindly provided by Prof. Antonio Garcia de Herreros. MEFs, MSCs, mouse colorectal cancer cell line MC-38 (Kerafast) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) 4.5g/L glucose, supplemented with 10 % Fetal Bovine Serum (FBS), 100 U/rnL penicillin, 100 mg/mL streptomycin and 2 mM glutamine (Life Technologies) and incubated at 37°C and 5 % CO2 in a humidified atmosphere.
Immunodetection: MEFs and MSCs were plated in p100 dishes (3x105 cells/p100) and exposed to nanoparticles at different concentration (from 25 nM to 100 nM) for two time periods (1 h, 24 h). When indicated, cells were treated with 50 ng/mL PDGF-BB (Prepotech) 30 min prior exposure. Cells were washed with PBS, detached from the plate, and trypsinized (1 mg/ml trypsin, Life Technologies) for 15 min at 37°C to remove membrane binding of the nanoparticles. RIPA lysis buffer plus phosphatase and protease inhibitors (Roche) was used to obtain whole-cell extracts. Protein extracts were loaded into 12 % acrylamide SDS-PAGE gel and transferred to a nitrocellulose membrane to detect GFP (Santa Cruz Technologies, sc-8334), PDGFR-p (Cell Signaling, 3169) and tubulin (Cell Signaling, 2148) by western blotting. Bands were visualized using ChemiDoc XRS+ imaging system (Biorad). Bands densitometry quantitation was performed with Imaged software. All experiments were performed in triplicate.
Flow cytometry: Internalization of nanoparticles was quantified by GFP fluorescence signal using FACS Calibur (BD Biosciences). MEFs and MSCs were exposed at 25, 50, 100, 250, 500 and 1000 nM for 1 h or 24 h. After treatment, cells were washed with PBS, detached from the plate and trypsinized for 15 min at 37 °C to eliminate non-internalized nanoparticle. Data were analyzed using the Flow Jo software and represented as mean fluorescence intensity (MFI) fold change with respect to buffer- treated cells. All experiments were performed in triplicate.
Confocal microscopy: For microscopy imaging, 1x104 cells were seeded on a coverslip in 24-well plate and exposed to 25, 50 or 100 nM nanoparticles for 1 h. When indicated, 50 ng/mL PDGF-BB (Prepotech) was added 30 min before exposure to assess competition. After incubation with nanoparticles, cells were washed, fixed with 4 % formaldehyde (ThermoFisher Scientific) and blocked with 3 % BSA. Then, nuclei were stained with DAPI and coverslips were mounted with Prolong™ Gold Antifade Mountant (ThermoFisher Scientific). Samples were visualized with SP5 Leica Microscopy.
Cell viability assay: Cell viability upon exposure to PDGFB-GFP-H6 or PDGFD- GFP-H6 was assessed with the Cell Proliferation Kit II (XTT) (Roche) according to the manufacturer’s instructions. Briefly, 1x103 cells were seeded in 96 well plates and treated with different concentrations of nanoparticles (0, 250, 500 and 1000 nM) for 24 or 48h. XTT reagent was added to the plate and further incubated at 37°C for 4h, then absorbance, which directly correlates to the number of viable cells, was measured using a multi-well spectrophotometer (FLUOstar Optima, BMG Labtech). All experiments were performed in triplicate.
Mice and procedures for the administration of nanoparticles: All mice and procedures were carried out in accordance with the EU regulations on animal research and approved by Catalonia’s Animal ethics committee (reference 9721). Eight-week-old female C57BL/6 were purchased from Charles River (France), housed in a specific pathogen-free (SPF) environment with sterile food and water ad libitum. The subcutaneous tumor model was generated by subcutaneous injection of 1 million MC38 cells mixed with 0.5 million MEFs in the flank of the animal. Mice bearing tumors around 100-200 mm3 were administrated intravenously with 200 pg of either PDGFB-GFP-H6 or PDGFD-GFP-H6 nanoparticles. Controls animals were administrated with the stock buffer for nanoparticles. Animals were euthanized 2 h post-administration and an ex vivo measurement of fluorescence intensity (FLI) of tumors was performed using IVIS® Spectrum 200 (PerkinElmer). Tumors and organs were collected, fixed in 4 % formaldehyde solution and paraffin-embedded. Fluorescence intensity (FLI) data are expressed as average radiant efficiency and it has been calculated subtracting the FLI signal of buffer-treated mice to the FLI signal of nanoparticle treated animals.
Histopathology: Paraffin-embedded 4 pm tissue sections were used for immunohistochemical analysis in DAKO Autostainer Link48 following the manufacturer’s instructions. Antigen Retrieval was done in PT Link with pH high solution. The antibodies used were PDGFR-p (1 :100, Cell Signaling 3169) and GFP (1 :200, Santa Cruz sc-8334). Representative images were captured using an Olympus DP73 digital camera and processed with the Olympus CellD Imaging 3.3 software.
Statistical analysis: Statistical analyses were performed using the GraphPad Prism 5 software (GraphPad Software, San Diego, California USA). Results were analyzed by Student t-test. Differences were considered statistically significant when p-values<0.05.
Results and discussion
Four modular GFP-based proteins were designed for recombinant production in which each one carried a specific PDFGR-p ligand, either PDGFRP1 , Z09591 , PDGFB or PDGFD. H6 tags were incorporated into the constructs to promote self-assembling as regular protein-only nanoparticles, because of the interactivity of the imidazole ring of the His residues with divalent cations. In silico modeling of the constructs did not anticipate any structural constraint for the solvent display of the ligand nor any steric impediment for the proper folding of the domains (not shown), as they were separated by a peptide linker (grey box, in Figure 1A). The recombinant production of these constructs in bacteria rendered good yields of full-length protein species (between 5 and 120 mg/L), that upon purification resulted in discrete bands of the expected molecular weights (Figure 1A). The integrity and high level of purity of all these constructs were further assessed by PAGE-SDS and independent Western blot analyses directed against alternative epitopes (Figure 1 B and Figure 5A). The main biophysical profile of the ligands and the resulting fusions, as well as production and optimal storage conditions are depicted in the Table 3. Upon dialysis, PDGFB- and PDGFD-based constructs rendered particulate materials of 16 and 26 nm (with a secondary minor peak of around 100 nm in the case of PDGFB-GFP-H6, Figure 1 B, Table 4), as expected for modular constructs based on H6-tagged GFP proteins with cationic N-terminal domains. As in the case of related protein constructs, the assembling was spontaneous and it did not required additional cation supply. PDGFRP1-GFP-H6 and Z09591-GFP-H6 resulted in smaller nanoparticles of around 9 nm that were only formed when cationic Zn was added to the buffer. All these materials were disassembled in presence of detergent (Figure 5B), confirming that they were oligomers formed by the recruitment of monomeric building blocks. By electron microcopy the assembled materials were visualized as discrete entities with sizes compatible to those obtained by DLS (Figure 1C). A detailed morphometric exploration of the materials in wide and narrow fields revealed a regular, virus-like geometry and a notable structural robustness (Figure 6).
Table 3. Main biophysical properties of the ligands (top) and ligand-derived constructs (bottom).
Figure imgf000077_0001
Figure imgf000077_0002
Table 4. Mean hydrodynamic size of the protein constructs measured in their respective storage buffers.
Figure imgf000078_0001
Envisaging functional analyses of all these materials once exposed to cells and whole bodies, their fluorescence emission was compared to that of the parental GFP-H6 and assessed as a suitable monitoring tool. Noteworthy, all the constructs, in the assembled form, were fluorescent and appropriate for further tracking in cell culture and in vivo, in the worst case reaching 70 % of the GFP-H6 emission (Figure 7A). Also, proteins remained proteolytically stable and soluble in complex culture media to be used for further analysis (Figure 7B), indicating again that they might be moved into next studies. In these analysyes, a first internalization screening of the nanoparticles into two PDGFR-p+ mesenchymal cell types, namely mouse embryonic fibroblast (MEFs) and mesenchymal stem cells (MSCs), showed a differential uptake (Figure 2). The PDGFB- and PDGFD-based constructs (especially PDGFD-GFP-H6) showed an excellent cell penetrability at low exposure times (1 h), determined by either GFP immunodetection (Figure 2A), flow cytometry (Figure 2B) and confocal microscopy (Figure 20). The smaller PDGFRP1-GFP-H6 and Z09591-GFP-H6 particles, in contrast, rendered internalization values indistinguishable from the background shown by the parental GFP- H6 (Figure 2B). The amount of detected protein was slightly higher at 1 h when compared to 24 h (Figure 2B) probably because of a combination of fast and efficient cell entry and a moderate lysosomal proteolysis of the engulfed material. In this regard, the dotted distribution of the PDGFD-GFP-H6 fluorescence (Figure 20) was indicative of an endosomal internalization route and a receptor- mediated uptake. Interestingly in none of the taken analyses exposed cells showed signs of death or cytotoxicity (Figure 2). For the two internalizing nanoparticles, namely PDGFB-GFP-H6 and PDGFD-GFP-H6, a dose-dependent entry was also demonstrated (Figure 8), and no plateau was observed at least up to 1 pM (Figure 8A, B). This was indicative of the high internalization capacity of both tested nanoparticles and the cell tolerance to the uptake. Already at 100 nM, the GFP fluorescence of both nanoparticles was widely distributed through the cytoplasm of the target cells (Figure 80). Again, at high doses of exposed nanoparticles, cells did not exhibit any symptom of toxicity at different times of exposure to high doses of PDGFB- GFP-H6 (Figure 9A) or PDGFD-GFP-H6 (Figure 9B). This was consistent with the intrinsic biocompatibility of proteins as building blocks of nanoscale materials and encouraged us to further analyze the performance of these protein materials focusing on clinically oriented applications.
In the context of the excellent uptake of both tested nanoparticles (Figure 2) it was required a clear confirmation of the specificity in such process and of the requirement of the PDGFR-p as a functional receptor. In this regard, the soluble version of the ligand, added before exposure to nanoparticles, blocked their uptake as determined visually by confocal microscopy (Figure 3A) or analytically, by immunoblotting (Figure 3B, C). These competition experiments confirmed that PDGFB-GFP-H6 and PDGFD-GFP-H6 nanoparticles bind and penetrate target cells upon selective attachment to PDGFR-p.
To evaluate the in vivo performance of PDGFB-GFP-H6 and PDGFD-GFP-H6 nanoparticles, C57BL/6 mice were subcutaneously implanted with the syngeneic colorectal cell line MC38 and MEFs to generate a convenient cancer model, in which PDGFR-p was expressed in tumor stromal compartment, where activated fibroblasts are located (Figure 4A). The high homology existing between human and mouse PDGFR-p (86 %) and PDGFR-p ligands (89 % for PDGFB and 91 % for PDGFD) allowed the testing of the nanoparticles containing human ligands in mice models. Two hours after PDGFB- GFP-H6 or PDGFD-GFP-H6 intravenous administration and in concordance with in vitro data both PDGFD-GFP-H6 nanoparticles accumulated in tumor (Figure 4B,C). Concomitantly with its better performance in cell culture (Figure 2) PDGFD-GFP-H6 showed a higher retention in target tissues than PDGFB-GFP-H6 (Figure 4B,C). The immunohistochemistry of the material in tumor and off-target main organs confirmed the high tumor accumulation and excellent CAF tumor-selectivity of PDGFD-GFP-H6, that was absent in liver and kidney (Figure 4D). In contrast, significant amounts of PDGFB- GFP-H6 nanoparticles, whose internalization into PDGFR-p+ fibroblasts was moderate (Figure 2, Figure 4A,B), were unexpectedly retained in kidney (Figure 4D). This observation suggested that PDGFB-GFP-H6, even showing an excellent targeting in cell culture (Figure 3) might be unsuitable as a PDGFR-p+ CAF-targeting agent since its biodistribution was not satisfactory. No signs of systemic toxicity was observed in any case (not shown). In summary, by addressing the need of developing non-toxic, non-xenobiotic and highly selective materials for delivery into tumor-associated PDGFR-p+ CAF, we have constructed and characterized a set of 4 modular proteins, targeting such receptor via alternative peptidic ligands, that organize as stable and regular oligomeric nanoparticles (Figure 1). Ligand-carrying protein oligomers benefit from multiple surface presentation of the ligand in a virus-like fashion and from the cooperativity in the receptor-binding and endosome formation, that results in the final cytoplasmic delivery of the engulfed material. Being protein-based development of cell-targeted nanoparticles a matter of assay and error, PDGFRP1-GFP-H6 and Z09591-GFP-H6 particles failed in an efficient and selective cell binding (Figure 2). Probably their small size around 9 nm (Figure 1 , 6) contributed to this issue, as larger nanoparticles ranging from ~ 15 to 80 nm have been repeatedly observed as optimal for receptor-dependent cell uptake. Noteworthy, none of the tested materials exhibited detectable toxicity upon exposure to target cells (Figure 9). Both PDGFB-GFP-H6 and PDGFD-GFP-H6 nanoparticles, ranging from 16 to 26 nm, selectively targeted and entered PDGFR-p+ CAF (Figure 3), keeping the fluorescence of the integrated GFP (Figure 2, 3, 4). However, PDGFB-GFP-H6, upon in vivo administration in a convenient mouse cancer model, did not show a good biodistribution, being majorly found in kidney (Figure 4). Despite its good receptor targeting and selectivity in cell culture (Figure 3), the protein was unable to perform in vivo regarding the desired biodistribution. This can be attributed to its slight structural instability determined by DLS, as this protein separated in two main populations of distinct sizes (Table 4). In contrast, PDGFD-GFP-H6 resulted in stable nanoparticles with relatively low polydispersion, with a convenient size of around 26 nm and showing a high potency for receptor-dependent specific cell uptake (Figure 2, 3). In fact, this nanoparticle was the candidate most efficiently internalized by target cells (Figure 2), that it also showed an exquisite biodistribution in mice (Figure 4), with a very selective accumulation in tumoral tissues. This construct was able to target, penetrate and selectively make PDGFR-p+ CAF fluorescent (Figure 4) because of the integrated GFP (Figure 1), that remained bioactive despite the multiple biological barriers the construct was surpassing after their intravenous administration.
Example 2
Materials and Methods
Protein production and purification Nanotoxins were designed in house as a codon-optimized gene and subcloned into pET22b plasmids using Ndel and Hindi 11 restriction enzymes. Geneart (ThermoFisher) provided the recombinant plasmids. For PDGFD-FD-PE24-H6, PDGFD was located at the N-terminus, followed by a furin cleavable site (GNRVRRSV - SEQ ID NO: 61) flanked by two flexible linkers (GGSSRSS - SEQ ID NO: 62), the catalytic domain of Pseudomonas aeruginosa exotoxin A (PE24), the H6 tag and the C-terminal subcellular location signal KDEL (SEQ ID NO: 26). The recombinant pET22b vectors were transformed into Escherichia coli by heat shock at 42 °C for 45 s. bacteria were grown in LB supplemented with 100 pg mL -1 ampicillin and 34 pg mL -1 chloramphenicol at 37 °C until QD550nm reached 0.4-0.5 units. In that moment, the expression of sulfhydryl oxidase and DsbC was induced by adding 0.5 % (m/v) of L-arabinose and temperature was lowered to 30 °C. 45 minutes later, temperature was lowered to 20 °C and 1 x 10-3M of IPTG was added to induce overnight expression. Cells were then harvested by centrifugation (15 min, 5000 g) and an osmotic shock was performed to remove the metallophores from the periplasmic fraction. For that, cells were first resuspended in a hypertonic solution (20 % sucrose, 1 x 10-3M ethylenediaminetetraacetic acid (EDTA), 5 x 10-2 M 4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid (HEPES), pH 7.9) and centrifuged (30 min, 7000 g, 4 °C). Supernatant was discarded and cells were then resuspended in a hypotonic solution (5 x 10-3 M MgSO4), placed 10 min at 4 °C and subsequently centrifuged (15 min, 4500 g) to remove again the supernatant. Bacterial cells, now without periplasm, were finally resuspended in wash buffer in presence of protease inhibitors and stored at -80 °C
For PDGFD-DITOX-H6, PDGFD was located at the N-terminus, followed by a furin cleavable site (GNRVRRSV— SEQ ID NO: 61) preceded by a flexible linker GGSSRSS (SEQ ID NO: 62), the catalytic and translocation domain of Corynebacterium Diphtheriae toxin (including its furin cleavable site GNRVRRSV [SEQ ID NO: 61] between them) and the C-terminal hexa-histidine tag. The recombinant pET22b vector was transformed into Escherichia coli BL21 (DE3) Origami B. PDGFD-DITOX-H6 was produced overnight at 20 °C and 250 rpm in LB supplemented with 100 pg/ml ampicillin, 12.5 pg/ml tetracycline and 15 pg/ml kanamycin, upon addition of 1 *10-4 M IPTG, at OD 550nm 0.6-0.8 units. Cells were then harvested by centrifugation (15 min, 5000 g ) and stored at -80 °C. For protein purification, cells were resuspended in wash buffer (2 *10-2 M Tris-HCI, 5 *10-1 M NaCI, 1 *10-2 M imidazole, pH 8.0) in presence of protease inhibitors (complete EDTA-free, Roche Diagnostics). For both proteins, PDGFD-FD-PE24-H6 and PDGFD-DITOX-H6, bacterial cells were disrupted in an EmulsiFlex-C5 system (Avestin) by 3 rounds at 8000 psi. The soluble fraction was then collected by centrifugation (45 min at 15000 g) and proteins purified by an immobilized metal affinity chromatography (IMAC) using a HisTrap HP column (GE Healthcare) in an AKTA pure system (GE Healthcare). Elution was achieved by a lineal increase of imidazole concentration (Elution Buffer, 2 *10-2 M Tris-HCI, 5 x10-1 M NaCI, 5 xio-1 M imidazole, pH 8.0). After purification, proteins were dialyzed against carbonate (166 mM NaCO3H, pH 8.0) w/o salt (333mM NaCI) solution, filtered at 0.22pm and stored at -80°C.
For the nanoconjugate, monomethyl Auristatin E (OH-Glu-Val-Cit-PAB-MMAE) was purchased at MedChemExpress (HY-148245), resuspended in N- Dimethylformamide (DMF) and activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, Sigma 03449) and N-Hydroxysulfosuccinimide sodium salt (NHS- sulfo, Sigma 56481) at 1 :2:2 molar ratio for 15 minutes (25% DMF, 75% MES) at room temperature. Protein functionalization took place at 1 :10 proteimdrug ratio in its own storage solution (166 mM NaCO3H, 333Mm NaCI, pH 8) O/N at 4°C. Finally, excess of non-conjugated MMAE molecules were completely removed by nanoconjugates IMAC re-purification using HisTrap HP 1 mL columns in an AKTA pure (Cytiva) chromatography system at 4°C and dialyzed again against its storage solution (166 mM NaCO3H, 333Mm NaCI, pH 8).
Dynamic light scattering (DLS) was used to determine the volume size distribution of each type of nanoparticle. Measurements were conducted in a Zetasizer Advanced Pro Blue (Malvern Instruments Limited) at 25 °C and 633 nm (n=3) using a quartz cuvette.
Mice and in vivo procedure
All mice and procedures were carried out in accordance with the Ell regulations on animal research and approved by Catalonia’s Animal ethics committee. Eight-week- old female C57BL/6 or Swiss Nude were purchased from Charles River (France), housed in a specific pathogen-free (SPF) environment with sterile food and water ad libitum.
The subcutaneous tumor model was generated by subcutaneous injection of 1 million MC38 cells mixed with 0.5 million MEFs in the flank of the C57BL/6 animals. Mice with 20-30mm3 tumors were randomised and intravenously administered with 1mg/Kg of PDGFD-NT-H6 nanotoxin or NaHCO3, daily up to 7 doses. Tumors were harvested 72h after last dose and measure ex-vivo to calculate their volume. For head and neck tumor model, one million UM-SCC-74B cells were implanted into the tongue of Swiss Nude mice. One day after, mice were randomised and intravenously administered with 1mg/Kg of PDGFD-NT-H6 nanotoxin, PDGFD-GFP-H6 or NaHCO3, daily up to 7 doses. Twenty-four hours after last dose, animals were euthanized and tumor were measured with a calliper. Tumour samples were paraffin-embedded histological analysis. Tumor volume was calculate as width2 x length x 0.5.
Histopathology
Four-pm paraffin-embedded tissue sections were used for immunohistochemical staining in the DAKO Autostainer Link48 following the manufacturer’s instructions. Antigen Retrieval was done in PT Link with pH high solution. Slides were scanned using Pannoramic Scan II (3D-Histech), and the quantification was performed using DensitoQuant or the NuclearQuant software (3D-Histec).
Cell viability assay
Cell viability was assessed with the Cell Proliferation Kit II (XTT) (Roche) according to the manufacturer’s instructions. Briefly, 1x103 cells were seeded in 96 well plates and treated with different nanoparticle for 48h or 72h, then XTT reagent was added to the plate and further incubated at 37°C for 4h, then absorbance, which directly correlates to the number of viable cells, was measured using a multi-well spectrophotometer (FLUOstar Optima, BMG Labtech). All experiments were performed in triplicate.
Flow Cytometry
Membrane expression of PDGFRp and Internalization (GFP) of nanoparticles in human CAFs were quantified using MACSQuantIO analyser (Milteny). hCAFs were exposed to 50nM PDGFD-GFP-H6 for 1h. After treatment, cells were washed with PBS, detached from the plate and trypsinized for 15 min at 37 °C to eliminate non-internalized nanoparticle. Anti-PDGFRp-PE antibody (Milteny) was added to the cell to detect membrane receptor expression. Fluorescence was detected and the percentage of positive cell was calculated. Untreated, unlabelled cells were considered as negative population. Data were analyzed using MACSQuantify software (Milteny) and represented as percentage of positive cells. All experiments were performed in triplicate. hCAFs isolation
Fresh tumor samples were desegregated using the human Tumor Dissociation Kit and the gentleMACS™ Octo Dissociator with Heaters (130-095-929, Milteny) to obtain a single cell suspention. Cells were labelled with human anti-CD326 microbeads (130- 061-101 Milteny) to mark epithelial cells. A negative selection was done by magnetic separation using an autoMACS® Pro Separator. Unlabelled cells were seeded in 6-well plate in Fibroblast basal media (CC-3132, Lonza). After 3 days, cells were trypsinized for 1 min and place in a new plate, allowing the enrichment of fibroblast. CAFs from passages 2-4 were used for the experiments.
Results
Subcutaneous tumor-bearing immunocompetent mice were treated with PDGFD- FD-PE24-H6 nanotoxin, which formed nanoparticles of 44.4 nm (Figure 10A), daily for 7 days (Figure 10B). Histological tumor analysis revealed that nanotoxin treatment increase blood vessels density as well as macrophages and T-lymphocytes within the tumor, suggesting that depletion of PDGFR-expressing CAFs impact tumor microenvironment content (Figure 10C).
The PDGFD-FD-PE24-H6 nanotoxin do not only achieve an antitumor effect in a colorectal cancer model but also in a head and neck squamous cell carcinoma. PDGFR- targeted nanotoxin treatment showed to be innocuous to UM-SCC-74B, a tongue squamous cell carcinoma human cell line (Figure 11A-B). To test the antitumor activity on a head and neck tumor model, UM-SCC-74B cells were orthotopically implanted in immunodeficient nude mice. Animals treated with CAFs-targeted nanotoxin developed smaller tumors than buffer-treated mice (Figure 11C). In agreement with the smaller size, the proliferation marker, ki67, was downregulated in the treated group (Figure 11 D), suggesting that CAFs targeting strategy impact cancer cell behavior.
In order to confirm that the cytotoxic effect observed in mouse derived fibroblast was also occurring in a human model we isolated CAFs from tumor tissues from patient with head and neck squamous carcinoma. These isolated primary CAFs demonstrated to express PDGFR in their membrane. The scaffold nanoparticle, PDGFD-GFP-H6, successfully internalized in the PDGFR-expressing CAFs (Figure 12A). Importantly, human primary CAFs were sensitive to PDGFD-FD-PE24-H6 nanotoxin in vitro (Figure 12B).
Concomitant with the development of PDGFD-FD-PE24-H6 nanotoxin another nanotoxin, PDGFD-DITOX-H6, that contains diphtheria toxin within their structure, was developed. PDGFD-DITOX-H6 monomer self-assembled to form nanoparticle around 24nm (Figure 13A). PDGFD-DITOX-H6 nanotoxin showed to be cytotoxic to MSCs, a fibroblast cell line (Figure 13B).
Finally, the scaffold protein PDGFD-GFP-H6 was used to chemically conjugate monomethyl auristatin E (MMAE), a highly toxic antitumor drug that blocks microtubule polymerization. PDGFD-GFP-H6-MMAE formed 43.7 nm nanoparticles (Figure 14A) and exhibited cytotoxic activity when added to fibroblast cell line MEFs and MSCs (Figure 14B). In addition, when cells were first exposed to the unconjugated nanoparticle, PDGFD-GFP-H6, the ability of the nanoconjugate to eliminate PDGFR-expressing fibroblast was impaired, indicating that PDGFD-GFP-H6-MMAE exerts it cytotoxic effect in a receptor dependent manner (Figure 14B).

Claims

CLAIMS A fusion protein comprising
(i) a PDGFR-p ligand selected from the group of consisting of PDGFB (SEQ ID N0: l) and PDGFD (SEQ ID NO: 2) or a functionally equivalent variant thereof,
(ii) an intervening polypeptide region and
(iii) a positively charged amino acid-rich region. The fusion protein according to claim 1 wherein the positively charged amino acid-rich region is a polyhistidine region. The fusion protein according to claim 2 wherein the polyhistidine region comprises between 2 and 10 contiguous histidine residues. The fusion protein according to claim 3 wherein the polyhistidine region is the HHHHHH sequence (SEQ ID NO: 3). The fusion protein according to claims 1 to 4 wherein the intervening polypeptide is a therapeutic agent or an imaging agent. The fusion protein according to claim 5 wherein the intervening polypeptide is a therapeutic agent selected from the group consisting of
(i) a cytotoxic polypeptide,
(ii) an antiangiogenic polypeptide,
(iii) a polypeptide encoded by a tumor suppressor gene,
(iv) a pro-apoptotic polypeptide,
(v) a polypeptide having anti-metastatic activity,
(vi) a polypeptide encoded by a suicide gene,
(vii)a chaperone polypeptide,
(viii) a polypeptide which is capable of activating the immune response towards a tumor, and
(ix) a toxin.
7. The fusion protein according to claim 5 wherein the intervening polypeptide is fluorescent protein.
8. The fusion protein according to claim 7 wherein the fluorescent protein is GFP.
9. The fusion protein according to any of claims 1 to 8 wherein the PDGFR-p ligand is connected to the intervening polypeptide via a first peptide linker and/or wherein the intervening polypeptide is connected to the positively charged amino acid-rich region via a second peptide linker.
10. The fusion protein according to claim 9 wherein the first peptide linker and/or the second peptide linker comprises the GGSSGGS sequence (SEQ ID NO: 4).
11. The fusion protein according to claim 9 or 10 wherein the first peptide linker and/or the second peptide linker comprise a cleavable target site.
12. The fusion protein according to any of claims 1 to 11 wherein the fusion protein is devoid of disulfide bridges.
13. The fusion protein according to any of claims 1 to 12 further comprising a reporter protein.
14. The fusion protein according to any of claims 1 to 19 further comprising a peptide that favours endosomal escape.
15. The fusion protein according to any of claims 1 to 13 wherein the intervening polypeptide region is conjugated to at least one therapeutic agent.
16. The fusion protein according to claim 14 wherein the intervening polypeptide is conjugated to a plurality of therapeutic agents, wherein said plurality of therapeutic agents are the same or different.
17. The fusion protein according to claim 14 or 15 wherein the therapeutic agent is selected from the group consisting of
(i) a chemotherapy agent, (ii) an antiangiogenic molecule and
(iii) a toxin. The fusion protein according to claim 17 wherein the therapeutic agent is a chemotherapy agent. The fusion protein according to claim 18 wherein the chemotherapy agent is an antimetabolite. The fusion protein according to claim 19 wherein the antimetabolite is a pyrimidine analogue or an oligomeric form thereof. A method for preparing a fusion protein according to any of claims 15 to 20 selected from:
(i) A method comprising a) providing a fusion protein comprising i) a PDGFR-p ligand selected from the group of consisting of PDGFB (SEQ ID N0 : l), PDGFD (SEQ ID NO : 2) and a functionally equivalent variant thereof, ii) an intervening polypeptide region and iii) a positively charged amino acid-rich region, wherein the PDGFR-p ligand and the positively charged amino acid-rich region are located at the ends of the protein and b) contacting said fusion protein with an activated form of a therapeutic agent or of an oligomeric form thereof wherein said activated form of a therapeutic agent or of an oligomeric form thereof contains a reactive group which is capable of reacting with at least one group in the intervening region of the fusion protein and wherein the contacting is carried out under conditions adequate for the formation of a bond between the reactive group in the therapeutic agent and the group in the intervening polypeptide region or
(ii) A method comprising a) providing a fusion protein comprising i) a PDGFR-p ligand selected from the group of consisting of PDGFB (SEQ ID N0 : l), PDGFD (SEQ ID NO : 2) and a functionally equivalent variant thereof, ii) an intervening polypeptide region and iii) positively charged amino acid-rich region, wherein the PDGFR-p ligand and the positively charged amino acidrich region are located at the ends of the protein and wherein the fusion protein is provided in an activated form, wherein said activated form of the fusion protein contains a reactive group in the intervening region and b) contacting said fusion protein with a therapeutic agent or an oligomeric form thereof, wherein said therapeutic agent contains a group which is capable of reacting with the reactive group in the fusion protein, wherein said contacting is carried out under conditions adequate for the formation of a bond between the reactive group in the fusion protein and the group in the therapeutic agent or
(iii) A method comprising a) providing a fusion protein comprising i) a PDGFR-p ligand selected from the group of consisting of PDGFB (SEQ ID N0 : l), PDGFD (SEQ ID NO : 2) and a functionally equivalent variant thereof, ii) an intervening polypeptide region and iii) a positively charged amino acid-rich region, wherein the PDGFR-p ligand and the positively charged amino acid-rich region are located at the ends of the protein, and b) contacting said fusion protein with a therapeutic agent, or an oligomeric form thereof, or with an imaging agent, or an oligomeric form thereof, in the presence of an enzyme, wherein said therapeutic agent or imaging agent contains a reactive group which is recognized by the enzyme, said fusion protein contains a motif which is recognized by the enzyme, and wherein said contacting is carried out under conditions adequate for the formation of a bond between the reactive group in the therapeutic agent or the imaging agent and the motif in the fusion protein.
22. The method according to claim 21 wherein the therapeutic agent is a chemotherapy agent and wherein the activated form thereof contains a group which reacts with at least one of the side chains in the intervening polypeptide region.
23. The method according to claim 22 wherein the group which reacts with at least one of the side chains in the intervening polypeptide region is a thiol group.
24. The method according to claim 22 or 23 wherein the activated chemotherapeutic agent is thiol-functionalized oligo-floxuridine.
25. A polynucleotide encoding a fusion protein according to any of claims 1 to 20.
26. A vector comprising a polynucleotide as defined in claim 25.
27. A host cell comprising a polynucleotide according to claim 25 or a vector according to claim 26.
28. A method for preparing a nanoparticle comprising multiple copies of the fusion protein according to any of claims 1 to 20 comprising placing a preparation of said fusion protein in a suitable buffer.
29. The method according to claim 27 wherein the buffer is selected from the group consisting of a carbonate buffer, a Tris buffer and a phosphate buffer.
30. The method according to claim 29 wherein the carbonate buffer is sodium carbonate at a concentration of between 100 and 300 mM.
31. The method according to claim 30 wherein the sodium carbonate buffer further comprises salt at a concentration of 200 mM to 400 mM. The method according to any of claims 28 to 31 wherein the pH of the buffer is between 6.5 and 8.5. The method according to any of claims 28 to 32 wherein the buffer is 166 mM NaHCO3, 333 mM NaCI, pH 8.0. A nanoparticle comprising multiple copies of the fusion protein according to any of claims 1 to 20 or which has been obtained by a method according to any of claims 28 to 33. The nanoparticle according to claim 34 having a diameter of between 5 nm and 100 nm. The fusion protein according to any of claims 1 to 20, the polynucleotide according to claim 25, the vector according to claim 26, the host cell according to claim 27 or the nanoparticle according to claims 34 or 35 for use in medicine. The fusion protein according to any of claims 1 to 20, the polynucleotide according to claim 25, the vector according to claim 26, the host cell according to claim 27 or the nanoparticle according to claim 34 or 35 wherein the intervening polypeptide is an antitumor peptide or wherein the intervening polypeptide is linked to an antitumor agent for use in the treatment of cancer. The fusion protein, polynucleotide, vector, host cell or nanoparticle for use according to claim 36 wherein the antitumor peptide is selected from the group consisting of
(i) a cytotoxic polypeptide,
(ii) an antiangiogenic polypeptide,
(iii) a polypeptide encoded by a tumor suppressor gene,
(iv) a pro-apoptotic polypeptide,
(v) a polypeptide having anti-metastatic activity,
(vi) a polypeptide which is capable of activating the immune response towards a tumor,
(vii)a polypeptide encoded by a suicide gene.
39. The fusion protein, polynucleotide, vector, host cell or nanoparticle for use according to claim 36 wherein the antitumor agent which is linked to the intervening polypeptide is selected from the group consisting of:
(i) a chemotherapy agent,
(ii) an antiangiogenic molecule.
(iii) a toxin.
40. The fusion protein, polynucleotide, vector, host cell or nanoparticle for use according to claim 37 to 39 wherein the tumor is a solid tumor.
41. The fusion protein, polynucleotide, vector, host cell or nanoparticle for use according to any of claims 37 to 40 wherein the cancer is characterized by comprising cancer cells that express PDGFR-p.
42. The fusion protein, polynucleotide, vector, host cell or nanoparticle for use according to any of claims 37 to 41 wherein the cancer is non-small cell lung, breast, colon, liver, prostate, pancreatic or colorectal cancer.
43. The fusion protein according to any of claims 1 to 20, the polynucleotide according to claim 25, the vector according to claim 26, the host cell according to claim 27 or the nanoparticle according to claim 34 or 35 wherein the intervening polypeptide is a cytotoxic polypeptide or wherein the intervening polypeptide is linked to an antifibrotic agent agent for use in the treatment of fibrosis.
44. The fusion protein according to any of claims 1 to 20, the polynucleotide according to claim 25, the vector according to claim 26, the host cell according to claim 27 or the nanoparticle according to claim 34 or 35 for use according to claim 43 wherein the fibrosis is pulmonary fibrosis, hepatic fibrosis, pancreatic fibrosis.
45. The fusion protein according to any of claims 1 to 20, the polynucleotide according to claim 25, the vector according to claim 26, the host cell according to claim 27 or the nanoparticle according to claim 34 or 35 wherein the intervening polypeptide is linked to an agent as defined in the column “Therapeutic agent” in Table 2 for use in the treatment of a disease as defined in the column “Disease or disorder” in Table 2.
PCT/EP2023/081937 2022-11-15 2023-11-15 NANOCONJUGATES CONTAINING PDGFR-β LIGANDS AND USES THEREOF WO2024105116A1 (en)

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