WO2016005381A1 - Protéines de fusion pdgfrbêta-fc et leurs utilisations - Google Patents

Protéines de fusion pdgfrbêta-fc et leurs utilisations Download PDF

Info

Publication number
WO2016005381A1
WO2016005381A1 PCT/EP2015/065464 EP2015065464W WO2016005381A1 WO 2016005381 A1 WO2016005381 A1 WO 2016005381A1 EP 2015065464 W EP2015065464 W EP 2015065464W WO 2016005381 A1 WO2016005381 A1 WO 2016005381A1
Authority
WO
WIPO (PCT)
Prior art keywords
pdgfrbeta
fusion protein
linker
pdgf
less
Prior art date
Application number
PCT/EP2015/065464
Other languages
English (en)
Inventor
Lars Linden
Thomas Schlange
Andreas Wilmen
Mark Trautwein
Tibor SCHOMBER
Michael BÖTTGER
Jürgen KLAR
Simone Greven
Original Assignee
Bayer Pharma Aktiengesellschaft
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US14/328,487 external-priority patent/US20160009776A1/en
Application filed by Bayer Pharma Aktiengesellschaft filed Critical Bayer Pharma Aktiengesellschaft
Publication of WO2016005381A1 publication Critical patent/WO2016005381A1/fr

Links

Classifications

    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the Platelet-Derived Growth Factor (PDGF) family consists of four members (A-D) that share a cluster of 8 cysteine residues.
  • PDGFs form biologically active homodimers and one heterodimer (PDGF-AB) via disulfide bonds. They bind to two closely related receptor tyrosine kinases, PDGFRalpha and -beta, which homo- or heterodimerize upon ligand binding.
  • PDGFRalpha receptor tyrosine kinases
  • -beta receptor tyrosine kinases
  • the specificity of the different PDGF ligands for both receptors varies, with PDGF-BB and -DD binding PDGFRbeta, and PDGF-BB, PDGF-CC and -AA binding PDGFRalpha.
  • PDGF- BB, PDGF-AB and to a weaker extent PDGF-CC and -DD were also reported to bind and activate the heterodimeric PDGFRalpha/beta (Heldin and Westermark, Physiol Rev 1999; 79(4): 1283-1316; Reigstad et al., FEBS J 2005; 272: 5723-5741).
  • PDGFR activation p85 and Grb2 scaffolding proteins are recruited to the phosphorylated receptor and initiate the activation of the MAPK and PI3K/AKT signaling pathways.
  • PDGF signaling regulates proliferation, survival and migration of PDGFRalpha or -beta expressing cells. Both receptors are found on vascular smooth muscle cells and pericytes where PDGFRbeta stimulates migration, while PDGFRalpha appears to have an anti-migratory function (Yokote et al., J Biol Chem 1996; 271: 5101-5111). Anti-apoptotic and pro-proliferative functions have been attributed to both receptors (Yao and Cooper, Science 1995; 267: 2003-2006).
  • PDGFR beta Fc decoy receptors in vivo without a peptide linker inserted between receptor and Fc portion has been reported to reduce tumor microvascular density in a triple-negative breast cancer xenograft model (Shan et al., Cancer Sci 102 (10): 1904-1910, October 2011) but showed no impact on vessel density in ovarian xenograft models (Lu et al., Am J Obstet Gynecol 198: 477.el-477.el0, April 2008). None of the published reports on PDGF decoy receptors gives a hint to the effect on vascular integrity and permeability in the eye or in any other tissue or compartment.
  • Full IgG antibodies have an exceptionally long half-life, while small antibody derivatives or other biologies formats often suffer from rapid elimination from circulation. In order to improve administration and therapeutic efficacy, modifications to extend the plasma half-life have been developed and implemented in these biologies formats. Methods for half-life extension include PEGylation, HESylation, glycosylation and polysialation. Another approach to achieve longer half-life is fusion of target proteins to an immunoglobin Fc domain or to human serum albumin.
  • linker-containing PDGFRbeta-Fc fusion proteins according to the invention show a significant and unexpected increase in intravitreal terminal half-life compared to the linker-less variant. Additionally, PDGFRbeta-Fc fusion proteins show the ability to reduce neovascularization without increasing, and even decreasing, vascular leakage, either as a stand-alone therapy or a combination therapy together with a receptor tyrosine kinase signaling antagonist, which is in stark contrast to other known anti-PDGF therapeutics.
  • linker-containing PDGFRbeta-Fc fusion proteins according to the invention show superior properties both in reducing neovascularization and concerning vascular leakage, when compared to the linker- less variant or compared with other PDGF-directed therapeutics.
  • the disclosure relates to PDGFRbeta-Fc fusion proteins, or biologically active fragments thereof, that exhibit increased in vitro potency and extended vitreal half-life in vivo, which, in some embodiments, is useful for the treatment of neovascular or fibrotic disease especially of the eye, in particular of the retina, e.g., but not limited to, wet age- related macular degeneration and posterior vitreoretinopathy, either alone or in combination with other anti-angiogenic regimens such as, but not limited to, one or more of anti-VEGF, anti-VEGFR, anti-HGF, anti-HGFR, anti-FGF, anti-FGFR anti-IGF, or anti-IFGR agents.
  • anti-VEGF anti-VEGFR
  • anti-HGF anti-HGFR
  • anti-FGF anti-FGFR anti-IGF
  • anti-IFGR agents anti-IFGR agents
  • the disclosure provides a PDGFRbeta-Fc fusion protein comprising an extracellular domain of PDGFRbeta and a Fc domain, wherein the PDGFRbeta-Fc fusion protein binds one or more of PDGF ligands -BB, -DD, and -AB with a KD of less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 500 pM, less than 200 pM, less than 125pM, less than 100 pM, less than 50 pM, less than 30 pM or less than 10 pM .
  • the PDGFRbeta-Fc fusion protein binds one or more of PDGF ligands -BB, -DD, and -AB with a K D of between 1-100 pM, between 100-500 pM, between 500-1000 pM, between 1 nM and 100 nM, or between 100-500 nM . In some embodiments, the PDGFRbeta-Fc fusion protein does not substantially bind to platelet-derived growth factor ligands AA and CC.
  • the PDGFRbeta extracellular domain of the fusion protein binds one or more of the PDGF ligands -BB and - DD with a K D of 1 to 500 pM, 1 to 200 pM, 1 to 100 pM, 1 to 50 pM, 50 to 150 pM or 100 to 200 pM .
  • the fusion protein may further comprise a polypeptide linker domain connecting the PDGFRbeta extracellular domain and the Fc domain.
  • the linker has a length of at least 12, 15, 20, 25, 30 or 35 amino acids. I n other embodiments, the linker has the amino acid sequence (GGGGS)n, where n is > 3.
  • the PDGFRbeta extracellular domain of the fusion protein binds the PDGF ligand -BB with a KD of 1 to 500 pM, 1 to 200 pM, 1 to 100 pM, 1 to 50 pM, 50 to 150 pM or 100 to 200 pM.
  • the fusion protein may further comprise a polypeptide linker domain connecting the PDGFRbeta extracellular domain and the Fc domain.
  • the linker has a length of at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.
  • the linker has the amino acid sequence (GGGGS)n, where n is > 3.
  • the linker may also have the amino acid sequence (GGGGS)n where n is 3-10.
  • n may be 3-7.
  • n is 3, 4, 5, 6, 7, 8, 9 or 10.
  • n is 4.
  • the PDGFRbeta extracellular domain of the fusion protein binds the PDGF ligand -DD with a KD of 1 to 500 pM, 1 to 200 pM, 1 to 100 pM, 1 to 50 pM, 50 to 150 pM or 100 to 200 pM.
  • the PDGFRbeta portion is connected to the Fc portion by means of a linker.
  • the linkers are glycine and serine rich linkers. Other near neutral amino acids, such as, but not limited to, Thr, Asn, Pro and Ala, may also be used in the linker sequence.
  • the linker comprises various permutations of amino acid sequences containing Gly and Ser.
  • the linkers have a length of at least 12, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids.
  • the extracellular domain of PDGFRbeta comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ. ID NO: 2. In other embodiments of any of the foregoing or following, the extracellular domain of PDGFRbeta comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3.
  • the disclosure provides a PDGFRbeta-Fc fusion protein comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 15-25, or biologically active fragments thereof.
  • PDGFRbeta-Fc fusion protein comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO: 19, or biologically active fragments thereof.
  • the PDGFRbeta-Fc fusion protein or biologically active fragment thereof, has an ICso for phosphorylation of Protein Kinase B (AKT) that is 2-500, 2-200, 2 to 100, 2 to 50 fold, 2 to 25 fold, 5 to 15 fold, or 2 to 10 fold lower than that of a linker-less PDGFRbeta-Fc fusion protein in a PDGF-BB-mediated phosphorylation assay.
  • AKT Protein Kinase B
  • the disclosure also provides vectors and plasmids comprising a nucleic acid sequence encoding any of the PDGFRbeta-Fc fusions proteins, or biologically active fragments thereof, described herein.
  • the nucleic acids, vectors and/or plasmid may be provided in a kit including instructions for transfection and expression in a host cell or may be used in gene therapy.
  • a host cell comprising a nucleic acid encoding a PDGFRbeta- Fc fusion protein, or biologically active fragment thereof, as described herein. I n particular embodiments the host cell is a prokaryotic cell. I n other embodiments the host cell is a eukaryotic cell.
  • does not substantially increase vascular leakage means that the vascular leakage score determined by angiography increases by no more than 20%, 15%, 10%, 8%, 5%, or 2% as compared to vascular leakage observed in an untreated control subject.
  • the administration decreases vascular leakage as compared to to the vascular leakage observed in an untreated control subject. In some embodiments, the administration decreases vascular leakage by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to to the vascular leakage observed in an untreated control subject.
  • the disclosure provides methods for inhibiting ocular neovascularization, comprising administering to a subject any of the PDGFRbeta-Fc fusion proteins, or biologically active fragments thereof, described herein, wherein said administration does not substantially increase vascular leakage as compared to an untreated control subject.
  • the administration decreases vascular leakage as compared to an untreated control subject.
  • the administration decreases neovascularization as compared to an untreated control subject.
  • the ocular neovascular disorder is age-related macular degeneration, choroidal neovascularization, choroidal neovascular membrane, cystoid macula edema, epi-retinal membrane and macular hole, myopia-associated choroidal neovascularisation, vascular streaks, retinal detachment, diabetic retinopathy, diabetic macular edema, atrophic changes of the retinal pigment epithelium, hypertrophic changes of the retinal pigment epithelium, retinal vein occlusion, choroidal retinal vein occlusion, macular edema, macular edema due to retinal vein occlusion, proliferative vitreoretinopathy, familiar exudative vitreoretionpathy, retinitis pigmentosa, Stargardt's disease, retinopathy of prematurity, keratitis, corneal transplantation or keratoplasty, corneal angiogenesis due to hypoxia
  • Another aspect of the disclosure provides a method for treating an ocular neovascular disorder, comprising administering to a subject any of the PDGFRbeta-Fc fusion proteins, or biologically active fragments thereof, described herein; and a receptor tyrosine kinase signaling antagonist.
  • the receptor tyrosine kinase antagonist is a VEGF antagonist, IGF antagonist, FGF antagonist or a HGF antagonist.
  • the combined PDGFRbeta-Fc, or biologically active fragment thereof, and receptor tyrosine kinase treatment decreases neovascularization as compared to an untreated control subject or as compared to a subject receiving only a PDGFRbeta-Fc or biologically active fragment thereof treatment or as compared to only a receptor tyrosine kinase treatment.
  • the PDGFRbeta-Fc fusion protein (or biologically active fragment thereof) and/or the receptor tyrosine kinase signaling antagonist are administered orally, topically, intravitreally, intraocularly, intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally or peribulbarly.
  • the PDGFRbeta-Fc fusion protein (or biologically active fragment thereof) and/or the receptor tyrosine kinase signaling antagonist are administered to the eye topically, by intravitreal injection, or by intraocular insertion of a biodegradable or non-biodegradable drug delivery system.
  • Fig. 2 summarizes the IC50 values (y-axis in both (A) and (B) is [M]) of PDGFRbeta-Fc constructs (x-axis) in cell-based assays on the Meso Scale ELISA platform.
  • A) shows data obtained with the pPDGFRbeta Meso Scale assay
  • B) shows data obtained with the pAKT Meso Scale assay.
  • PDGFRbetaln the pAKT assay reduction of the I C50 values by addition of a linker was even more pronounced, highlighted by a reduction 129 fold lower I C50 value for the 5xGGGGS linker variant.
  • Fig. 3 shows different treatment protocols for the rat laser-induced choroidal neovascularization model.
  • Fig. 5 shows the in vivo activity of PDGFRbeta-Fc constructs carrying no linker or a 3xGGGGS linker in the delayed short treatment protocol.
  • the 3xGGGGS linker variant of the PDGFRbeta- Fc fusion protein shows a reduction in vascular leakage that is more pronounced than in the anti-VEGF control group.
  • the PDGFRbeta-Fc variant without linker shows even a slight increase in vascular leakage. This demonstrates that the higher in vitro activity of PDGFRbeta-Fc fusion protein variants translates into in vivo activity that improves the therapeutic effect against CNV and the associated vision impairing vascular leakage.
  • Fig. 7 shows the PK profile in the rabbit vitreous humor after single intravitreal application of a PDGFRbeta-Fc fusion protein without linker (dashed line, triangles) and a 4xGGGGS linker variant (solid line, circles).
  • concentration (y-axis in ⁇ g/L]) of the 4xGGGGS linker variant declines less rapidly than that of the no linker variant (x-axis in [h]), resulting in an increased vitreal half-life of 7.21 d for the 4xGGGGS linker variant versus 4.75 d for the PDGFRbeta-Fc construct without linker.
  • Fig. 8 depicts the Sequence ID NOs 1-36. DETAILED DESCRIPTION OF THE DISCLOSURE
  • PDGF relates to the Platelet-Derived Growth Factor (PDGF) family of ligands of the two receptor tyrosine kinases PDGFRbeta and PDGFRalpha.
  • the PDGF family consists of four members (A, B, C, D) that share a cluster of 8 cysteine residues.
  • PDGFs form biologically active homodimers (PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD) and one heterodimer (PDGF-AB) via disulfide bonds.
  • PDGFRalpha and PDGFRbeta homo- or hetero- dimerize upon ligand binding.
  • PDGF-BB and -DD binding PDGFRbeta
  • PDGF-BB, PDGF-CC and -AA binding PDGFRalpha PDGF-BB, PDGF-AB and to a weaker extent PDGF-CC and -DD were also reported to bind and activate the heterodimeric PDGFRalpha/beta (Heldin and Westermark, Physiol Rev 1999; 79(4): 1283-1316; Reigstad et al., FEBS J 2005; 272: 5723-5741).
  • PDGFRbeta refers to the platelet-derived growth factor receptor beta, a 180-kDa transmembrane glycoprotein which binds PDGF BB with high affinity (Duan et al., J Biol Chem. 266: 413-418 1991).
  • This type I transmembrane glycoprotein comprises five NH2- terminal Ig domains, the extracellular domains D1-D5. It is anchored in the membrane by a single transmembrane domain and contains an intracellular receptor tyrosine kinase domain making it a member of the receptor tyrosine kinase family.
  • PDGFRbeta The extracellular domain of PDGFRbeta or fragments thereof are capable of binding one or more of the PDGF ligands - AA, -AB, -BB, -CC and -DD thereby blocking biological activities mediated by these ligands , for example its effects on vessel maturation. Unless explicitly stated otherwise, this term encompasses any of the PDGFRbeta variants or biologically active fragments disclosed herein. In certain embodiments, PDGFRbeta comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ. ID NO: 1, or biologically active fragments thereof.
  • PDGFRbeta-Fc PDGFRbeta-Fc fusion protein
  • PDGFRbeta-Fc protein fusion proteins comprising a PDGFRbeta portion and a Fc portion.
  • PDGFRbeta-Fc PDGFRbeta-Fc fusion protein
  • PDGFRbeta-Fc protein any of the PDGFRbeta-Fc protein variants or biologically active fragments disclosed herein.
  • the extracellular domain of a PDGFRbeta-Fc fusion protein comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any of SEQ ID NOs: 15-25, or biologically active fragments thereof.
  • extracellular ligand binding domain is defined as the portion of a receptor that, in its native conformation in the cell membrane, is oriented extracellularly where it can contact with its cognate ligand.
  • the extracellular ligand binding domain does not include the hydrophobic amino acids associated with the receptor's transmembrane region or any amino acids associated with the receptor's intracellular part.
  • the preceding 15-30, predominantly hydrophobic or apolar amino acids i.e., leucine, valine, isoleucine, and phenylalanine
  • the extracellular domain comprises the amino acids that precede the hydrophobic transmembrane stretch of amino acids, von Heijne has published detailed rules that are commonly referred to by skilled artisans when determining which amino acids of a given receptor belong to the extracellular, transmembrane, or intracellular parts (cf. von Heijne (1995) BioEssays 17:25).
  • the extracellular domain of PDGFRbeta comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ. ID NO: 2 or 3, or biologically active fragments thereof.
  • Fc The terms “Fc”, “Fc-part”, Fc-region, Fc-portion or “Fc-domain” denote the carboxy- terminal region of an immunoglobulin heavy chain of mammalian origin.
  • the immunoglobulin heavy chain constant region comprises three or four domains and a hinge region.
  • the domains are named sequentially as follows: CHl-hinge-CH2-CH3(-CH4).
  • the DNA sequences of the heavy chain domains have cross-homology among the immunoglobulin classes, e.g., the CH2 domain of IgG is homologous to the CH2 domain of IgA and IgD, and to the CH3 domain of IgM and IgE.
  • the terms, "Fc", “Fc-part” or “Fc domain” are understood to mean the carboxyl-terminal portion that contains at least a part of the hinge region, the CH2 domain and the CH3 domain.
  • the class of immunoglobulin from which the Fc-part is derived is IgG (Igy) ( ⁇ subclasses 1, 2, 3, or 4).
  • Other classes of immunoglobulin, IgA (Iga), IgD (Ig6), IgE ( ⁇ ge) and IgM ( ⁇ ) may be used.
  • IgA IgA
  • IgD IgD
  • IgE ⁇ ge
  • IgM IgM
  • a human IgGl heavy chain Fc-region extends from Asp 221, Cys226, or from Pro230, to the carboxyl- terminus of the heavy chain.
  • the Fc-region has the amino acid sequence of SEQ. ID NO: 14.
  • the C-terminal lysine (Lys447) of the Fc-region may or may not be present.
  • numbering of amino acid residues in the Fc-region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat, E.A., et al, Sequences of Proteins of I mmunological I nterest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, M D (1991), NIH Publication. Furthermore, it is contemplated that substitution or deletion of amino acids within the immunoglobulin heavy chain constant regions may be useful in the practice of the disclosure.
  • a "Fc part of an antibody” is a term well known to the skilled artisan and defined on the basis of papain cleavage of antibodies.
  • a “fusion protein” as used herein refers to an expression product resulting from the fusion of at least two genes.
  • An "Fc-fusion protein” or “Fc-fusion” is a chimeric polypeptide comprising the Fc- region, or constant region, of an antibody fused, or conjugated, to an unrelated protein or protein fragment either at the C- or the N-terminus or even both termini of the Fc part.
  • the Fc comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 14.
  • a “biologically active fragment thereof” is a fragment of protein or peptide that retains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99% of the biological activity of the reference protein or peptide.
  • a biologically active fragment of a PDGFRbeta or PDGFRbeta-Fc protein binds one or more of PDGF liga nds -BB, -DD, and -AB with a KD of less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 500 pM, less than 200 pM, less than 125pM, less than 100 pM, less than 50 pM, less than 30 pM or less than 10 pM .
  • a biologically active fragment of a PDGFRbeta or PDGFRbeta-Fc protein binds one or more of PDGF ligands -BB, -DD, and -AB with a KD of between 1-100 pM, between 100-500 pM, between 500-1000 pM, between 1 nM and 100 nM, or between 100-500 nM.
  • a biologically active fragment of a PDGFRbeta or PDGFRbeta-Fc protein binds one or more of the PDGF ligands -BB and -DD with a KD of less than 200 pM, or less than 125 pM, or less than 100 pM, or less than 50 pM, or less than 30 pM .
  • a biologically active fragment of a PDGFRbeta or PDGFRbeta-Fc protein binds one or more of the PDGF ligands -BB and -DD with a K D of 1 to 500 pM, 1 to 200 pM, 1 to 100 pM, 1 to 50 pM, 50 to 150 pM or 100 to 200 pM .
  • a “linker” refers to an amino acid sequence which is used to connect or fuse two or more different proteins or two or more different protein domains.
  • Linkers of this disclosure are used to link a PDGFRbeta part to a Fc-part to form an PDGFRbeta fusion protein according to the disclosure.
  • Binding affinity refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g. a receptor or a receptor fusion protein according to the disclosure and a ligand).
  • the dissociation constant "KD” is commonly used to describe the affinity between a molecule (such as receptor or a receptor fusion protein according to the disclosure) and its binding partner (such as a ligand) i.e. how tightly a ligand binds to a particular protein.
  • Ligand-protein affinities are influenced by non-covalent intermolecular interactions between the two molecules. Affinity can be measured by common methods known in the art, including those described herein.
  • the " KD” or “KD value” according to this disclosure is measured by using surface plasmon resonance assays using a Biacore T200 instrument (GE Healthcare Biacore, I nc.) with Series S Sensor Chips CM5.
  • binding assays were performed at 25 °C with assay buffer HBS- EP+ (including 1 mg/ml BSA, 500 nM NaCI, 0.05% NaN 3 in total). Fc fusions were captured with an anti-hlgG capture-Ab covalently coupled to the chip surface via amine coupling chemistry. Subsequently, PDGF antigens were used as analyte in various concentrations and after each association and dissociation phase the chip surface was regenerated (glycine HCI pH 2.0), followed by another capture and analyte injection cycle. Obtained sensorgrams were double-referenced, i.e. in-line reference cell correction followed by buffer sample subtraction.
  • KD values for PDGF -BB and -DD were calculated based on the ratio of dissociation (kd) and association (k a ) rate constants which were obtained by globally fitting sensorgrams with a first order 1:1 Langmuir binding model. Data for PDGF -AB were evaluated by a steady-state affinity plot.
  • Other suitable instruments are e.g. Biacore 2000, a Biacore 3000, Biacore 4000 or Biacore T100 (GE Healthcare Biacore, Inc.), ProteOn XPR36 instrument (Bio-Rad Laboratories, Inc.), IBIS MX96 (IBIS Technologies B.V.), or similar.
  • Percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence, respectively, is defined as the percentage of nucleic acid or amino acid residues, respectively, in a candidate sequence that are identical with the nucleic acid or amino acid residues, respectively, in the reference polynucleotide or polypeptide sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Conservative substitutions are not considered as part of the sequence identity. In some embodiments, the alignments are un-gapped alignments.
  • Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • the Fc fusion protein is "isolated".
  • An isolated biological component (such as a nucleic acid molecule or protein such as an antibody or Fc fusion) is one that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, e.g., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles.
  • Nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified by standard purification methods as described for example in J. Sambrook et al., 1989 (Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA) and R. K. Scopes et al. 1994 (Protein Purification, - Principles and Practice, Springer Science and Business Media LLC).
  • the term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • subject means an animal, including humans and non- human animals. In some embodiments, the subject is a human.
  • patient as used herein means a subject having any of the disorders disclosed herein.
  • Antagonist means a molecule that blocks, reduces or inhibits the expression of a protein of interest.
  • An antagonist may also mean a molecule that blocks, reduces, or inhibits one or more natural activities of a protein of interest.
  • Antagonism of a receptor can also include reduction or making unavailable the ligands that would otherwise stimulate said receptor.
  • Antagonists include, but are not limited to small molecules, peptides, antibodies, receptor fusion proteins, aptamers, RNAi constructs and antisense constructs.
  • a "receptor tyrosine kinase signaling antagonist” or “RTK signaling antagonist” or “RTK antagonist” herein means a molecule that blocks, reduces or inhibits the expression or activity of any component in a receptor tyrosine kinase signaling cascade, including the transmembrane receptor with tyrosine kinase activity and downstream effector molecules or proteins.
  • RTK antagonists include, but are not limited to small molecules, peptides, antibodies, receptor fusion proteins, aptamers, RNAi constructs and antisense constructs.
  • the present disclosure relates to PDGFRbeta-Fc fusion proteins and methods for treating pathological neovascularization and fibrosis, e.g. cancer, ocular neovascular disorders, pulmonary fibrosis or nephropathies by providing PDGFRbeta-Fc fusion proteins.
  • the PDGFR-Fc fusion proteins of the present disclosure comprise an extracellular domain of PDGFRbeta and an Fc domain.
  • the disclosure provides a PDGFRbeta-Fc fusion protein comprising an extracellular domain of PDGFRbeta and a Fc domain, wherein the PDGFRbeta- Fc fusion protein binds one or more of PDGF ligands -BB, -DD, and -AB with a KD of less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 500 pM, less than 200 pM, less then 125pM, less than 100 pM, less than 50 pM, less than 30 pM or less than 10 pM .
  • the PDGFRbeta-Fc fusion protein binds one or more of PDGF ligands -BB, -DD, and -AB with a K D of between 1-100 pM, between 100-500 pM, between 500-1000 pM, between 1 nM and 100 nM, or between 100-500 nM .
  • the PDGFRbeta-Fc fusion binds one or more of the PDGF ligands -BB, -DD, and -AB with a KD of less than 125pM .
  • the PDGFRbeta-Fc fusion protein binds the PDGF ligand -BB with a KD of less than 30 pM . In further preferred embodiments, the PDGFRbeta-Fc fusion binds the PDGF ligands -BB and -DD with a K D of less than 30 pM, respectively.
  • the PDGFRbeta-Fc fusion protein does not substantially bind to platelet-derived growth factor ligands AA and CC.
  • the PDGFRbeta-Fc fusion protein may bind to PDGF ligands AA or CC with no meaningful binding affinity, such as it may bind to the ligands AA and CC with a KD of greater than 300 nM, greater than 400 nM, greater than 500 nM, or greater than 1 ⁇ .
  • the PDGFRbeta extracellular domain of the fusion protein binds one or more of the PDGF ligands -BB and -DD with a KD of less than 200 pM, or less than 125 pM, or less than 100 pM, or less than 50 pM, or less than 30 pM . In some embodiments, the PDGFRbeta extracellular domain of the fusion protein binds one or more of the PDGF ligands -BB and -DD with a K D of 1 to 500 pM, 1 to 200 pM, 1 to 100 pM, 1 to 50 pM, 50 to 150 pM or 100 to 200 pM.
  • PDGFRbeta-Fc fusion protein that comprise an N-terminal fragment of PDGFRbeta (SEQ. ID NO: 1), wherein the PDGFRbeta fragment comprises amino acids 1-500, 1-400, 1-300, 1-285 or 1-282 of SEQ ID NO: 1, and further wherein the fragment is able to bind one or more PDGF ligands -AA, - AB, -BB, -CC or -DD.
  • the N-terminal fragment is a biologically active fragment.
  • fusion proteins that comprise one or more extracellular domains of human PDGFRbeta (SEQ ID NO: 1), selected from Dl, D2, D3, D4 and D5.
  • it comprises the extracellular domains D1-D5 (EDC 1-5) of human PDGFRbeta as set forth in SEQ ID NO: 2.
  • the PDGFRbeta portion of the fusion protein comprises the extracellular domains D1-D3 (EDC 1- 3) of PDGFRbeta (SEQ ID NO: 3), or a biologically active fragment thereof.
  • the PDGFRbeta portion of the fusion protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2, or a biologically active fragment thereof. In yet other embodiments, the PDGFRbeta portion of the fusion protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3, or a biologically active fragment thereof.
  • the PDGFRbeta-Fc fusion proteins, or biologically active fragments thereof, according to the disclosure contain an Fc part derived from mammalian origin, e. g. but not limited to mouse, rat, monkey, pig or human.
  • the Fc-part is a human Fc and may be from human IgGl, lgG2, lgG3 or lgG4 subclass.
  • the Fc is derived from human IgGl subclass, e.g. as outlined in SEQ ID NO: 14.
  • the Fc- portion may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ. ID NO: 14, or fragments thereof.
  • the class of immunoglobulin from which the Fc-part is derived is IgG (Igy) ( ⁇ subclasses 1, 2, 3, or 4).
  • Other classes of immunoglobulin, IgA (Iga), IgD (Ig6), IgE (Igs) and IgM ( ⁇ ) may be used.
  • IgA IgA
  • IgD IgD
  • IgE IgE
  • IgM IgM
  • a human IgGl heavy chain Fc-region extends from Asp 221, Cys226, or from Pro230, to the carboxyl- terminus of the heavy chain.
  • the Fc-region has the amino acid sequence of SEQ ID NO: 14.
  • the C-terminal lysine (Lys447) of the Fc-region may or may not be present.
  • numbering of amino acid residues in the Fc-region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat, E.A., et a I, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication. Furthermore, it is contemplated that substitution or deletion of amino acids within the immunoglobulin heavy chain constant regions may be useful in the practice of the disclosure.
  • the Fc-portion comprises hybrid heavy chain constant regions, i.e., the Fc-portion comprises multiple heavy chain constant region domains selected from: a CHI domain, a CH2 domain, a CH3 domain, and a CH4 domain; wherein at least one of the constant region domains in the Fc is of a class or subclass of immunoglobulin distinct from the class or subclass of another domain in the Fc.
  • at least one of the constant region domains in the Fc is an IgG constant region domain
  • at least one of the constant region domains in the Fc is of a different immunoglobulin class, i.e., an IgA, IgD, IgE, or IgM constant region domain.
  • At least one of the constant region domains in the Fc is a IgGl constant region domain, and at least one of the constant region domains in the Fc is of a different IgG subclass, i.e., lgG2, lgG2, lgG3 or lgG4.
  • Suitable constant regions may be human or from another species (e.g., murine).
  • the PDGFRbeta-Fc fusion proteins of the disclosure are fusions of the extracellular domains D1-D3 of PDGFRbeta, derived from UNIPROT ID P09619 (SEQ ID NO: 2 and figure 1A) to a human IgGl Fcpart (SEQ ID NO: 14) connected via the C- terminal amino acid of the extracellular domain 3 to the N-terminal amino acid of the Fcpart.
  • connection can either be done directly (e.g., SEQ ID NO: 15; linker-less PDGFRbeta- Fc fusion protein) or via different amino acid linkers (e.g., SEQ ID NOs: 4-13) to generate the PDGFRbeta-Fc fusion proteins as outlined in SEQ ID NOs: 16-25.
  • the disclosure also contemplates PDGFRbeta-Fc fusion proteins comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 15-25, or biologically active fragments thereof.
  • the PDGFRbeta portion is connected to the Fc portion by means of a linker.
  • the linkers are glycine and serine rich linkers. Other near neutral amino acids, such as, but not limited to, Thr, Asn, Pro and Ala, may also be used in the linker sequence.
  • the linker comprises various permutations of amino acid sequences containing Glyand Ser.
  • the linkers have a length of at least 12, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids.
  • linkers according to the disclosure are outlined in SEQ ID NO: 7-13, i.e.
  • SEQ ID NO 12 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (6xGGGGS),
  • SEQ ID NO 13 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (7xGGGGS).
  • the PDGFRbeta-Fc fusion proteins of the present disclosure have an IC 5 o for phosphorylation of Protein Kinase B (AKT) that is 2-500, 2-200, 2 to 100, 2 to 50 fold, 2 to 25 fold, 5 to 15 fold, or 2 to 10 fold lower than that of a linker-less PDGFRbeta- Fc fusion protein in a PDGF-BB-mediated phosphorylation assay.
  • AKT Protein Kinase B
  • a fusion protein of the disclosure has an I C50 for phosphorylation of Protein Kinase B (AKT) that is at least 2, 3, 5, 7, 10, 12, 15, or 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 125, 130, 140, 150, 160, 170, 180, 190, or 200 fold lower than that of a linker-less PDGFRbeta-Fc fusion protein in a PDGF-BB-mediated phosphorylation assay.
  • AKT Protein Kinase B
  • the PDGFRbeta-Fc fusion proteins of the present disclosure have an I C50 for phosphorylation of Protein Kinase B (AKT) that is at least 10-fold lower than that of a linker-less PDGFRbeta-Fc fusion protein in a PDGF-BB-mediated phosphorylation assay.
  • AKT Protein Kinase B
  • the PDGFRbeta-Fc fusion proteins of the present disclosure also have an IC50 for PDGFRbeta auto-phosphorylation that 2 to 100, 2 to 50 fold, 2 to 25 fold, 5 to 15 fold, 2 to 5, or 3 fold lower than that of a linker-less PDGFRbeta-Fc fusion protein in a PDGF-BB-mediated phosphorylation assay.
  • a fusion protein of the disclosure has an I C50 for PDGFRbeta auto-phosphorylation that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 fold lower than that of a linker-less PDGFRbeta-Fc fusion protein in a PDGF-BB-mediated phosphorylation assay.
  • the PDGFRbeta-Fc fusion proteins of the present disclosure have an intravitreal half-life that is at least 10% to 60%, or 15% to 45%, or 20 to 50%, or 30% to 40% longer than that of a linker-less PDGFRbeta-Fc fusion protein.
  • a fusion protein of the disclosure has an intravitreal half-life that is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% longer than that of a linker-less PDGFRbeta-Fc fusion protein.
  • PDGFRbeta-Fc fusion proteins of the present disclosure are labeled with a detectable moiety.
  • the detectable moiety is a fluorophore, radioisotope, enzymatic label, chemiluminescent label, or biotin label.
  • the PDGFRbeta-Fc fusion protein is conjugated to an epitope tag.
  • the relative increase of in vitro potency measured as IC50 for phosphorylation of Protein Kinase B (AKT) in a PDGF-BB-mediated phosphorylation assay for any of the PDGFRbeta-Fc fusion proteins having a linker described herein as compared to a linker-less PDGFRbeta-Fc fusion protein is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18 or 20 times greater than the increase in binding affinity of the PDGFRbeta-Fc fusion protein for the PDGF ligand -BB compared to the linker-less PDGFRbeta-Fc fusion protein.
  • the relative increase of in vitro potency measured as IC50 for phosphorylation of Protein Kinase B (AKT) in a PDGF-BB-mediated phosphorylation assay for any of the PDGFRbeta-Fc fusion proteins having a linker described herein as compared to a linker-less PDGFRbeta-Fc fusion protein is at least 6 times greater than the increase in binding affinity of the PDGFRbeta-Fc fusion protein for the PDGF ligand -BB compared to the linker-less PDGFRbeta-Fc fusion protein.
  • PDGFRbeta-Fc fusion proteins of the disclosure are not limited to the specific peptide sequences provided herein. Rather, the disclosure also embodies variants of these polypeptides. With reference to the present disclosure and conventionally available technologies and references, the skilled worker will be able to prepare, test and utilize functional variants of the PDGFRbeta-Fc fusion proteins disclosed herein, while appreciating that such variants having the ability to bind to PDGF ligands fall within the scope of the present disclosure. Accordingly, the disclosure contemplates PDGFRbeta-Fc fusion proteins comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ. ID NOs: 15-25, or biologically active fragments thereof.
  • a variant can include, for example, a PDGFRbeta-Fc fusion protein that has at least one altered PDGFRbeta domain and/or Fc domain and/or linker sequence, vis-a-vis a peptide sequence disclosed herein.
  • variants may be obtained by using a gene encoding one PDGFRbeta or fusion protein thereof as starting point for optimization by diversifying one or more codons encoding amino acid residues in the PDGFRbeta-Fc fusion protein, and by screening a resulting collection of PDGFRbeta-Fc fusion protein variants for variants with improved properties. Diversification can be done by synthesizing a collection of DNA molecules using DNA mutagenesis techniques, which are well known in the art, for example "saturation mutatgenesis” techniques that rely on trinucleotide mutagenesis (TRIM) technology (Virnekas B. et al., Nucl. Acids Res. 1994, 22: 5600.).
  • TAM trinucleotide mutagenesis
  • PDGFRbeta-Fc fusion proteins include molecules with modifications/variations including but not limited to modifications leading to altered pharmacokinetics (e.g. modification of the Fc part or attachment or removal of further molecules such as PEG or sialic acids), altered binding affinity, altered stability, ligand specifity, or altered potency.
  • polypeptide variants may be made that conserve the overall molecular structure of a PDGFRbeta-Fc fusion protein amino acid sequence described herein. Given the properties of the individual amino acids, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e., "conservative substitutions,” may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. In some embodiments, the polypeptide variants comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophane, and methionine;
  • polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine;
  • positively charged (basic) amino acids include arginine, lysine, and histidine; and
  • negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutions typically may be made within groups (a)-(d).
  • glycine and proline may be substituted for one another based on their ability to disrupt a-helices.
  • certain amino acids such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine and lysine are more commonly found in a-helices
  • valine, isoleucine, phenylalanine, tyrosine, tryptophan and threonine are more commonly found in ⁇ -pleated sheets.
  • Glycine, serine, aspartic acid, asparagine, and proline are commonly found in turns.
  • substitutions may be made among the following groups: (i) S and T; (ii) P and G; and (iii) A, V, L and I.
  • S and T amino acid sequence
  • P and G amino acid sequence
  • A, V, L and I amino acid sequence
  • substitutions may be made among the following groups: (i) S and T; (ii) P and G; and (iii) A, V, L and I.
  • the skilled scientist readily can construct DNAs encoding the conservative amino acid variants.
  • selected, random or complete subsets of amino acids can be selected at one or more positions in the protein that include non- conservative amino acid substitutions, and a subset of amino acid changes can be incorporated into the final protein that still allow adequate or even enhanced properties.
  • sequence identity between two polypeptide sequences, indicates the percentage of amino acids that are identical between the sequences.
  • sequence homology indicates the percentage of amino acids that either is identical or that represent conservative amino acid substitutions.
  • the present disclosure also relates to the nucleic acid molecules (also referred to as polynucleotides herein) that encode a PDGFRbeta-Fc fusion protein of the disclosure.
  • a nucleic acid molecule that encodes a PDGFRbeta-Fc fusion protein of the disclosure comprises a sequence selected from SEQ. ID NOs: 26-36, or fragments thereof that encode biologically active fragments of PDGFRbeta-Fc fusion protein.
  • the nucleic acids or polynucleotides are DNA. In other embodiments, the nucleic acids or polynucleotides are RNA.
  • nucleic acid molecules of the disclosure are not limited to the sequences disclosed herein, but also include variants thereof. Accordingly, in some embodiments, a nucleic acid molecule that encodes a PDGFRbeta-Fc fusion protein of the disclosure comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ. ID NOs: 26-36, or fragments thereof that encode biologically active fragments of PDGFRbeta-Fc fusion protein.
  • nucleic acids within the disclosure may be described by reference to their physical properties in hybridization.
  • DNA can be used to identify its complement and, since DNA is double stranded, its equivalent or homolog, using nucleic acid hybridization techniques. It also will be recognized that hybridization can occur with less than 100% complementarity.
  • hybridization techniques can be used to differentiate among DNA sequences based on their structural relatedness to a particular probe. For guidance regarding such conditions see, Sambrook et al., 1989 supra and Ausubel et al., 1995 (Ausubel, F. M ., Brent, R., guitarist, R. E., Moore, D. D., Sedman, J. G., Smith, J. A., & Struhl, K. eds. (1995). Current Protocols in Molecular Biology. New York: John Wiley and Sons).
  • Structural similarity between two polynucleotide sequences can be expressed as a function of "stringency" of the conditions under which the two sequences will hybridize with one another.
  • Tm of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatched base pairs.
  • ⁇ and ⁇ 2 are the ionic strengths of two solutions.
  • hybridization stringency is a function of many factors, including overall DNA concentration, ionic strength, temperature, probe size and the presence of agents which disrupt hydrogen bonding. Factors promoting hybridization include high DNA concentrations, high ionic strengths, low temperatures, longer probe size and the absence of agents that disrupt hydrogen bonding. Hybridization typically is performed in two phases: the "binding" phase and the “washing” phase.
  • nucleic acids within the scope of the disclosure may be described with reference to the product they encode.
  • These functionally equivalent polynucleotides are characterized by the fact that they encode the same peptide sequences, for example those found in SEQ. ID NOs: 15-25, due to the degeneracy of the genetic code.
  • the polynucleotides of the disclosure are functionally equivalent polynucleotides.
  • the polynucleotides are comprised of synthetic nucleic acids (e.g. synthetic DNAs). Methods of efficiently synthesizing oligonucleotides in the range of 20 to about 150 nucleotides are widely available. See Ausubel et al., section 2.11, Supplement 21 (1993). Overlapping oligonucleotides may be synthesized and assembled in a fashion first reported by Khorana et al., J. Mol. Biol. 72:209-217 (1971); see also Ausubel et al., supra, Section 8.2. In some embodiments, Synthetic nucleic acids are designed with convenient restriction sites engineered at the 5' and 3' ends of the gene to facilitate cloning into an appropriate vector.
  • synthetic nucleic acids are designed with convenient restriction sites engineered at the 5' and 3' ends of the gene to facilitate cloning into an appropriate vector.
  • a method of generating variants is to start with one of the nucleic acids disclosed herein and then to conduct site-directed mutagenesis. See Ausubel et al., supra, chapter 8, Supplement 37 (1997).
  • a target nucleic acid e.g., DNA
  • Single-stranded DNA is isolated and hybridized with an oligonucleotide containing the desired nucleotide alteration(s).
  • the complementary strand is synthesized and the double stranded phage is introduced into a host.
  • Some of the resulting progeny will contain the desired mutant, which can be confirmed using DNA sequencing.
  • various methods are available that increase the probability that the progeny phage will be the desired mutant. These and other methods of gene or DNA synthesis and modification are well known to those in the field and kits are commercially available for generating such mutants.
  • the present disclosure further provides recombinant nucleic acid constructs comprising one or more of the nucleotide sequences of the present disclosure.
  • the recombinant constructs of the present disclosure are used in connection with a vector, such as a plasmid, phagemid, phage or viral vector, into which a nucleic acid molecule encoding a PDGFRbeta-Fc fusion protein of the disclosure is inserted.
  • a PDGFRbeta-Fc fusion protein or derivative thereof provided herein can be prepared by recombinant expression of nucleic acid sequences encoding a PDGFRbeta-Fc fusion protein or portions thereof in a host cell.
  • a host cell can be transfected with one or more recombinant expression vectors carrying nucleic acid fragments encoding a PDGFRbeta-Fc fusion protein or portions thereof such that the PDGFRbeta-Fc fusion protein is expressed in the host cell.
  • Standard recombinant nucleic acid methodologies may be used to prepare and/or obtain nucleic acids encoding the PDGFRbeta-Fc fusion protein, incorporate these nucleic acids into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat. No. 4,816,397 by Boss et al.
  • nucleic acid expression methods can be used (see, for example, Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)).
  • a nucleic acid encoding the desired polypeptide can be inserted into an expression vector which is then transfected into a suitable host cell.
  • Suitable host cells are prokaryotic and eukaryotic cells. Examples for prokaryotic host cells are e.g. bacteria, examples for eukaryotic host cells are yeast, insect, plant or mammalian cells. It is understood that the design of the expression vector, including the selection of regulatory sequences is affected by factors such as the choice of the host cell, the level of expression of protein desired and whether expression is constitutive or inducible.
  • useful expression vectors for bacterial use are constructed by inserting a structural nucleic acid (e.g. DNA) sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
  • the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desirable, to provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.
  • bacterial vectors may be, for example, bacteriophage-, plasmid- or phagemid-based. These vectors can contain a selectable marker and bacterial origin of replication derived from commercially available plasmids typically containing elements of the well-known cloning vector pBR322 (ATCC 37017). Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is de-repressed/induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • appropriate means e.g., temperature shift or chemical induction
  • telomeres are typically harvested by centrifugation, discarded and the resulting supernatants are retained for further purification.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the protein being expressed. For example, when a large quantity of such a protein is to be produced, vectors which direct the expression to high levels of fusion protein products, that are readily purified, may be desirable.
  • PDGFRbeta-Fc fusion proteins of the present disclosure include products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic host, including, for example, E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus. Mammalian Expression & Purification
  • regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • the recombinant expression vectors can also include origins of replication and selectable markers (see e.g., U.S. 4,399,216, 4,634,665 and U.S. 5,179,017, by Axel et al.).
  • suitable selectable markers include genes that confer resistance to drugs such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate and the neo gene confers resistance to G418.
  • transfection of the expression vector into a host cell can be carried out using standard techniques such as electroporation, calcium-phosphate precipitation, and DEAE-dextran transfection.
  • an embodiment of the present disclosure is an expression vector comprising a nucleic acid sequence encoding for the PDGFRbeta-Fc fusion proteins of the present disclosure. See Example 1 for an exemplary description.
  • PDGFRbeta-Fc fusion proteins of the disclosure can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to ammonium sulfate or ethanol precipitation, acid extraction, Protein A chromatography, Protein G chromatography, anion or cation exchange chromatography, phospho-cellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, size exclusion chromatography, reversed phase chromatography and lectin chromatography.
  • the PDGFRbeta-Fc fusion proteins of the present disclosure include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a eukaryotic host, including, for example, yeast, plant, insect and mammalian cells.
  • a eukaryotic host including, for example, yeast, plant, insect and mammalian cells.
  • the PDGFRbeta-Fc fusion proteins of the present disclosure can be glycosylated or can be non-glycosylated.
  • Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20.
  • an embodiment of the present disclosure are also host cells comprising the vector or a nucleic acid molecule, whereby the host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, and may be a prokaryotic cell, such as a bacterial cell.
  • the host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, and may be a prokaryotic cell, such as a bacterial cell.
  • Another embodiment of the present disclosure is a method of using the host cell to produce a PDGFRbeta-Fc fusion protein, comprising culturing the host cell under suitable conditions and recovering said PDGFRbeta-Fc fusion protein.
  • the PDGFR-Fc fusion proteins of the present disclosure are coadministered with known medicaments, and in some embodiments the fusion protein might itself be modified.
  • PDGFRbeta-Fc fusion proteins of the present disclosure may be administered as the sole pharmaceutical agent or in combination with one or more additional therapeutic agents where the combination causes no unacceptable adverse effects.
  • this combination therapy includes administration of a single pharmaceutical dosage formulation which contains PDGFRbeta-Fc fusion protein of the disclosure and one or more additional therapeutic agents, as well as administration of a PDGFRbeta-Fc fusion protein of the disclosure and each additional therapeutic agent in its own separate pharmaceutical dosage formulation.
  • PDGFRbeta-Fc fusion proteins of the disclosure and a therapeutic agent may be administered to the patient together in a single dosage composition, or each agent may be administered in separate dosage formulations.
  • a PDGFRbeta-Fc fusion protein of the disclosure and one or more additional therapeutic agents may be administered at essentially the same time (e.g., concurrently) or at separately staggered times (e.g., sequentially).
  • compositions which comprise a PDGFR-Fc fusion protein, alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • the pharmaceutical compositions of the disclosure may also comprise, in addition to a PDGFRbeta-Fc fusion protein, a pharmaceutically active compound that is suitable to treat PDGFRbeta related diseases such as cancer, back-of-the-eye diseases such as age-related macular degeneration (AMD), choroidal neovascularization (CNV), choroidal neovascular membrane (CNVM), cystoid macula edema (CME), epi-retinal membrane (ERM) and macular hole, myopia-associated choroidal neovascularisation, vascular streaks, retinal detachment, diabetic retinopathy, diabetic macular edema (DME), atrophic changes of the retinal pigment epithelium (RPE), hypertrophic changes of the retinal pigment epithelium (RPE), retinal vein occlusion, choroidal retinal vein occlusion, macular edema, macular edema due to retinal vein occlusion
  • examples include but are not limited to angiogenesis in the front of the eye like corneal angiogenesis following e.g. keratitis, corneal transplantation or keratoplasty, corneal angiogenesis due to hypoxia (extensive contact lens wearing), pterygium conjunctivae, subretinal edema and intraretinal edema.
  • the described PDGFRbeta-Fc fusion proteins are useful for the treatment of nephropathies such as diabetic nephropathy and fibrotic disease such as pulmonary fibrosis. Any of these molecules can be administered to a patient alone, or in combination with other agents, drugs or hormones, in pharmaceutical compositions where it is mixed with excipient(s) or pharmaceutically acceptable carriers.
  • the present disclosure also relates to the administration of pharmaceutical compositions. Such administration may be accomplished orally or parenterally.
  • Methods of parenteral delivery include but are not limited to intravitreal, topical, intra-arterial (directly to the tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.
  • these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa.).
  • the application of the PDGFRbeta-Fc fusion protein to the eye is performed by intravitreal injection.
  • compositions for administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
  • Such carriers enable the pharmaceutical compositions to be formulated as e.g. tablets, pills, dragees, capsules, liquids, creams, gels including hydrogels, ointments, syrups, slurries, suspensions, sprays, aerosols, injectables, implants and the like.
  • Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl, cellulose, hydroxypropylmethylcellulose, or sodium carboxymethyl cellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross- linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • the pharmaceutical compositions are substantially pyrogen free. In some embodiments, the pharmaceutical compositions are pyrogen free.
  • Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e. dosage.
  • Push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
  • compositions for parenteral administration include aqueous or nonaqueous solutions, suspensions, or emulsions of active compounds.
  • the pharmaceutical compositions of the disclosure may be formulated in aqueous solutions.
  • the compositions are formulated in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline e.g. phosphate buffered saline.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline e.g. phosphate buffered saline.
  • Aqueous injection suspensions may contain substances that increase viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • An aqueous formulation for parenteral administration may contain 1-200 mg/ml of the PDGFRbeta-Fc fusion protein according to the disclosure.
  • a formulation for parenteral administration contains 1-200 mg/mL, 1-100 mg/ml, 5-50 mg/ml, 1-50 mg/ml or 1-25 mg/ml mg/ml of the PDGFRbeta-Fc fusion protein according to the disclosure.
  • a formulation for parenteral administration contains 8-30 mg/ml of the PDGFRbeta-Fc fusion protein according to the disclosure.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Kits
  • the disclosure further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the disclosure.
  • Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration.
  • kits may contain polynucleotide sequences encoding the PDGFRbeta-Fc fusion proteins of the disclosure.
  • the polynucleotide is RNA.
  • the polynucleotide is DNA.
  • the polynucleotide sequences encoding these fusion proteins are provided in a plasmid suitable for transfection into and expression by a host cell.
  • the plasmid may contain a promoter (often an inducible promoter) to regulate expression of the polynucleotide in the host cell.
  • the plasmid may also contain appropriate restriction sites to facilitate the insertion of other polynucleotide sequences into the plasmid to produce various proteins.
  • the plasmids may also contain numerous other elements to facilitate cloning and expression of the encoded proteins. Such elements are well known to those of skill in the art and include, for example, selectable markers, initiation codons, termination codons, and the like.
  • compositions of the present disclosure may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • the pharmaceutical composition may be provided as a salt and can be formed with acids, including by not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • This composition may be further enhanced by inclusion of sugars, such as but not limited to sucrose, trehalose, mannitol or glucose.
  • the composition may further contain other excipients like detergents, polyols or substances used in protein or antibody formulations by the skilled in the art.
  • the preparation may be a lyophilized powder that is combined with buffer prior to use.
  • compositions comprising a compound of the disclosure formulated in an acceptable carrier
  • they can be placed in an appropriate container and labeled for treatment of an indicated condition.
  • labeling would include amount, frequency and method of administration.
  • compositions suitable for use in the present disclosure include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose, i.e. treatment of a particular disease state characterized by PDGFRbeta expression.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g. using neoplastic cells, primary or immortalized fibroblasts or endothelial cells, or in animal models, usually mice, rats, rabbits, dogs, pigs or monkeys.
  • animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of PDGFRbeta fusion protein or biologically active fragment thereof, that ameliorate the symptoms or condition.
  • Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, ED50/LD50.
  • the pharmaceutical compositions exhibit large therapeutic indices.
  • the data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use.
  • the dosage of such compounds lies within a range of circulating concentrations what include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • PDGFRbeta-Fc fusion protein or biologically active fragment thereof to be administered to a subject is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors that may be taken into account include the severity of the disease state, e.g., tumor size and location; change in visual acuity, retinal thickness determined e.g. by Optical Coherence Tomography (OCT), functional retinal changes monitored e.g. in Electroretinograms (ER); age, weight and gender of the patient; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
  • OCT Optical Coherence Tomography
  • long acting pharmaceutical compositions might be administered every day, or every 2 days, or every 3 to 4 days, or every week, or once every two weeks, or once every month, or once every two months, or once every quarter of the year, or once every half of the year, or every year depending on half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 2 g, depending upon the route of administration and the factors mentioned above.
  • Guidance as to particular dosages and methods of delivery is provided in the literature. See U.S. Pat. No. 4,657,760; 5,206,344; or 5,225,212.
  • Those skilled in the art will employ different formulations for polynucleotides than for proteins or their inhibitors.
  • delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • the PDGFbeta-Fc fusion protein, or a biologically active fragment thereof, according to the disclosure is injected into the eye in volumes of about 5-150 ⁇ , of about 25-125 ⁇ , or about 10-50 ⁇ , wherein the concentration of the protein is about 1-200 mg/ml, about 5-50 mg/ml, or about 8-30 mg/ml.
  • the present disclosure also relates to a use of the pharmaceutical composition according to the disclosure to treat or prevent ophthalmological disorders.
  • the present disclosure also relates to a method for treating or preventing an ophthalmological disorder comprising administering to a subject a pharmaceutical composition containing a pharmaceutically effective amount of a PDGFRbeta-Fc fusion according to the present disclosure.
  • the administration of a PDGFRbeta-Fc fusion protein described herein decreases neovascularization.
  • the administration of a PDGFRbeta-Fc fusion protein decreases neovascularization by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 40%, or at least 50% compared to an untreated a control subject.
  • the administration does not substantially increase vascular leakage, where herein "does not substantially increase vascular leakage” means that the vascular leakage score determined by angiography increases by no more than 15%, 10%, 8%, 5%, 2% or 1% as compared to vascular leakage observed in an untreated control subject.
  • the administration decreases vascular leakage as compared to to the vascular leakage observed in an untreated control subject. In some embodiments, the administration decreases vascular leakage by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to to the vascular leakage observed in an untreated control subject.
  • the disclosure provides a method for inhibiting ocular neovascularization, comprising administering to a subject a PDGFRbeta-Fc fusion protein described herein, wherein said administration reduces vascular leakage.
  • the administration decreases vascular leakage by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to to the vascular leakage observed in an untreated control subject.
  • the administration decreases neovascularization.
  • the administration of a PDGFRbeta-Fc fusion protein decreases neovascularization by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 40%, or at least 50% compared to an untreated control subject.
  • ophthalmological disorders include, but are not limited to retinal disorders, macular degeneration such as age-related macular degeneration (AMD), non-exudative and exudative age-related macular degeneration, choroidal neovascularization (CNV), choroidal neovascular membrane (CNVM), macular edema such as cystoid macular edema (CME) or diabetic macular edema (DME), epi-retinal membrane disorders (ERM), macular hole, myopia-associated choroidal neovascularisation, vascular streaks, retinal detachment, diabetic retinopathy, atrophic changes of the retinal pigment epithelium (RPE), hypertrophic changes of the retinal pigment epithelium (RPE), retinal arterial occlusive diseases, retinal vein occlusion such as central retinal vein occlusion (CVRO), branch retinal vein occlusion (BRVO), choroidal retinal vein occlusion
  • AMD age-
  • retinal dystrophies congenital stationary night blindness, cone dystrophies, fundus flavimaculatus, Bests disease, pattern dystrophy of retinal pigmented epithelium, X-linked retinoschisis, Sorsby funduns dystrophy, benign concentric maculopathy, Biett's crystalline dystrophy, pseudoxanthoma elasticum, congenital hypertrophy of RPE, posterior uveal melanoma, choroidal meangioma, choroidal osteoma
  • examples include but are not limited to angiogenesis in the front of the eye like corneal angiogenesis following e.g. keratitis, corneal transplantation or keratoplasty, corneal angiogenesis due to hypoxia (extensive contact lens wearing), pterygium conjunctivae, subretinal edema and intraretinal edema.
  • AMD age-related macular degeneration
  • AMD include but are not limited to dry or nonexudative AMD, or wet or exudative or neovascular AMD.
  • the method comprises administering a pharmaceutical composition containing a pharmaceutically effective amount of a PDGFRbeta-Fc fusion according to the present disclosure to treat age-related macular degeneration (AMD) such as dry AMD or wet AMD, choroidal neovascularization (CNV), choroidal neovascular membrane (CNVM), macular edema such as cystoid macular edema (CME) or diabetic macular edema (DME).
  • AMD age-related macular degeneration
  • CNV choroidal neovascularization
  • CNVM choroidal neovascular membrane
  • macular edema such as cystoid macular edema (CME) or diabetic macular edema (DME).
  • the method comprises treating AMD.
  • composition according to the disclosure can be administered as the sole pharmaceutical composition or in combination with one or more other pharmaceutical compositions or active agents where the combination causes no unacceptable adverse effects.
  • Combination means for the purposes of the disclosure not only a dosage form which contains all the active agents (so-called fixed combinations), and combination packs containing the active agents separate from one another, but also active agents which are administered simultaneously or sequentially, as long as they are employed for the prophylaxis or treatment of the same disease.
  • the combination according to the disclosure is well tolerated in an animal model and is potentially effective even in low dosages, a wide range of formulation approaches is possible.
  • one possibility is to formulate the individual active ingredients of the combination according to the disclosure either as a solution or mixture, as in a "single syringe" device, or separately. In the latter case, it is not absolutely necessary for the individual active ingredients to be taken at the same time; on the contrary, sequential intake may be advantageous to achieve optimal effects.
  • it is appropriate with such separate administration to combine the formulations of the individual active ingredients simultaneously together in a suitable primary packaging, or in a "device" that facilitates administration.
  • the active ingredients are then present in the primary packaging or device in one or separate containers which may be, for example, syringes, double- chamber syringes, tubes, bottles or blister packs.
  • separate packaging of the components in the joint primary packaging is also referred to as a kit.
  • the pharmaceutical compositions of the present disclosure can be combined with other ophthalmological agents.
  • ophthalmological agents include, but are not limited to carotenoids like lycopene, lutein, zeaxanthin, phytoene, phytofluene, carnosic acid and derivatives thereof like carnosol, 6,7-dehydrocarnosic acid, 7-ketocarnosic acid, a zink source like zinc oxide or a zinc salt like its chloride, acetate, gluconate, carbonate, sulphate, borate, nitrate or silicate salt, copper oxide, vitamin A, vitamin C, vitamin E and/or ⁇ - carotene.
  • carotenoids like lycopene, lutein, zeaxanthin, phytoene, phytofluene, carnosic acid and derivatives thereof like carnosol, 6,7-dehydrocarnosic acid, 7-ketocarnosic acid, a zink source like zinc oxide or a zinc salt like its chlor
  • compositions of the present disclosure can be combined with other signal transduction inhibitors targeting receptor kinases of e.g. VEGFR, IGFR, FGFR, HGFR and their respective ligands or other pathway inhibitors like VEGF- Trap (aflibercept), FGFR fusion proteins, regorafenib, pegaptanib, ranibizumab, pazopanib, bevasiranib, KH-902, mecamylamine, PF-04523655, E-10030, ACU-4429, volociximab, sirolismus, fenretinide, disulfiram, sonepcizumab and/or tandospirone.
  • VEGF- Trap e.g. VEGFR, IGFR, FGFR, HGFR and their respective ligands or other pathway inhibitors
  • FGFR fusion proteins e.g. VEGFR, IGFR, FGFR, HGFR and their respective ligand
  • agents include, by no way of limitation, antibodies such as Avastin (bevacizumab). These agents also include, by no way of limitation, small-molecule inhibitors such as STI-571 / Gleevec (Novartis), PTK-787 (Wood et al., Cancer Res. 2000, 60(8), 2178-2189), SU-11248 (Demetri et al., Proceedings of the American Society for Clinical Oncology 2004, 23, abstract 3001), ZD- 6474 (Hennequin et al., 92nd AACR Meeting, New Orleans, March 24-28, 2001, abstract 3152), AG-13736 (Herbst et al., Clin. Cancer Res.
  • small-molecule inhibitors such as STI-571 / Gleevec (Novartis), PTK-787 (Wood et al., Cancer Res. 2000, 60(8), 2178-2189), SU-11248 (Demetri et al., Proceedings of the American Society for Clinical Oncology 2004, 23,
  • the PDGFRbeta-Fc fusion proteins of the present disclosure are administered to a subject in combination with a receptor tyrosine kinase antagonist.
  • the receptor tyrosine kinase antagonist with which the PDGFRbeta-Fc fusion protein of the present invention can be combined is a VEGFR antagonist, IGFR antagonist, FGFR antagonist or a HGFR antagonist.
  • the PDGFRbeta-Fc fusion protein (or biologically active fragment thereof) and the receptor tyrosine kinase signaling antagonist are administered simultaneously or within 90 days of each other.
  • subsequent administration of PDGFRbeta-Fc fusion protein is given biweekly, monthly or bimonthly for 1, 2, 3, 4, 5, 6, 7, 8 9, 10, 11 or 12 months after the initial treatment, or continued for years up to the lifetime of the patient.
  • the PDGFRbeta-Fc fusion protein (or biologically active fragment thereof) and receptor tyrosine kinase signaling antagonists are administered in parallel, wherein the PDGFRbeta-Fc fusion protein or biologically active fragment thereof is administered at biweekly, monthly or bimonthly intervals for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or for years, and receptor tyrosine kinase antagonist is administered monthly, or biweekly, or weekly, or daily, or up to 5 times per day for 1 day, or 1 week, or 2 weeks, or 1 month, or 2 months, or 3 months, or 6 months, or 9 months, or 12 months, or for years.
  • the PDGFRbeta-Fc or biologically active fragment thereof and receptor tyrosine kinase signaling antagonists are administered in parallel, wherein the PDGFRbeta-Fc (or biologically active fragment thereof) is administered at biweekly, monthly or bimonthly intervals for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or years, and receptor tyrosine kinase agonist is administered once.
  • the receptor tyrosine kinase signaling antagonist is a sustained release formulation.
  • the pharmaceutical compositions of the present disclosure is combined with VEGF-Trap (aflibercept), regorafenib, FGFR fusion proteins, bevacizumab, pegaptanib, ranibizumab, pazopanib and/or bevasiranib.
  • VEGF-Trap aflibercept
  • regorafenib FGFR fusion proteins
  • bevacizumab pegaptanib
  • ranibizumab ranibizumab
  • pazopanib pazopanib
  • bevasiranib bevasiranib.
  • combinations include combination c-met fusion proteins or antibodies against c-met or HGF, antibodies or fusion proteins against IGFR-I and/or II, IGF-I and or -II antibodies, and/or FGFs or FGFRs, Integrins, Cadherins, Cathepsins, MMPs, ADAMs, Nicotinic receptors, vascular disrupting agents, proteins of the complement system, steroids, chemokine/cytokine antibodies or chemokine/cytokine receptor antagonists like e.g. but not limited to agents targeting MCP-1/CCL2, Eotaxin-l/CCL, Eotaxin-2/CCL, Eotaxin-3/CCL, SDF-lalpha/CXCL12, CCR2, CCR3, CXCR4.
  • MCP-1/CCL2 Eotaxin-l/CCL
  • Eotaxin-2/CCL Eotaxin-3/CCL
  • SDF-lalpha/CXCL12 CCR2, CCR3, CXCR
  • the receptor tyrosine kinase antagonist is a VEGF antagonist.
  • the VEGF antagonist is a small molecule, an antibody, a VEGF trap, an aptamer, a RNAi construct or an antisense construct.
  • the VEGF antagonist is regorafenib, a hydrate, solvate or pharmaceutical acceptable salt thereof or a polymorph thereof.
  • the regorafenib is administered in a sustained release formulation.
  • a sustained release formulation is a formulation that on a single administration will continue to provide or release an active agent over an extended period of time.
  • a sustained release composition is an implant which may be solid, semisolid or viscoelastic.
  • the active agent may be released from the implant by diffusion, erosion or degradation of the polymer matrix, dissolution or osmosis of the active agent or swelling of the polymer matrix.
  • the polymer matrix agents are pharmaceutically acceptable and biocompatible with the eye and can be biodegradable or non-biodegradable.
  • the polymer matrix agents are biodegradable.
  • the implant comprising a biodegradable polymer matrix agent may partially or completely disappear in the eye by degradation or erosion which can be e.g.
  • the impant may completely disappear in the eye by degradation or erosion.
  • the polymer matrix agent can be cross-linked or non-cross-linked. In some emboidments, the polymer matrix agent is biocompatible with the eye and does not cause any substantial interference with the functioning or physiology of the eye. Examples of polymer matrix agents include but are not limited to polymers of hydroxyaliphatic carboxylic acids, polysaccharides (e.g.
  • alginates, cellulose and derivatives thereof like carboxymethylcellulose and esters thereof polymers of lactic acid (either in the D- or L- form or as a racemic mixture) such as polylactides (PLA), polymers of glycolic acid such as polyglycolides (PGA), poly-lactide-co-glycolide (PLGA), polycaprolactone, polyesters, poly(ortho esters), poly(phosphazine), polyphosphate ester), natural polymers such as gelatin or collagen, or mixtures of the before mentioned agents.
  • PLA polylactides
  • PGA polymers of glycolic acid
  • PLGA polycaprolactone
  • polyesters poly(ortho esters), poly(phosphazine), polyphosphate ester
  • natural polymers such as gelatin or collagen, or mixtures of the before mentioned agents.
  • the sustained release composition provides a sustained, controlled and/or extended delivery of the active agent at a maintained level of 0.01 ⁇ g to 100 ⁇ g per day, 0.01 ⁇ g to 50 ⁇ g per day, or 0.1 ⁇ g to 25 ⁇ g per day, or 0.5 ⁇ g to 15 ⁇ g per day, or 1 ⁇ g to 10 ⁇ g per day. In some embodiments, it releases the active agent in a sustained manner for a period of time of 3 months, or 4 months, or 5 months, or 6 months, or 8 months, or 10 months, or 12 months, or 14 months or 16 months or 18 months after the implant is placed into the eye.
  • the injection or implantation may be done in parallel and at essentially the same time point with the present injection or sequentially.
  • the administration (e.g. injection) of the ophthalmological drug according to the present disclosure can be repeated in the first month, or 2 months, or 3 months, or 4 months, or 5 months, or 6 months after implantation of a sustained release composition containing a second ophthalmological drug on a monthly basis.
  • the PDGFRbeta-Fc fusion proteins of the present disclosure can be combined with an eye drop composition containing a second ophthalmological drug, e.g. but not limited to an inhibitor of the VEGF pathway, which can be topically delivered into the eye.
  • a second ophthalmological drug e.g. but not limited to an inhibitor of the VEGF pathway
  • These eye drops can be administered one or more times per day, up to 5 times per day, or up to 3 times per day.
  • the injection of the present ophthalmological drug can be done each 1, or 2, or 3, or 4, or 5, or up to 6 months in parallel with the treatment with the eye drops containing a second ophthalmological drug administered up to 5 times a day.
  • the eye drop composition contains regorafenib, a hydrate, solvate or pharmaceutical acceptable salt thereof or a polymorph thereof as particularly described in WO 2013/000917.
  • Example 1 Generation of different recombinant PDGFRbeta Fc constructs.
  • SEQ ID NO 12 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (6x GGGGS),
  • SEQ ID NO:s 4-6. i.e SEQ ID NO 4: EPKSC, SEQ ID NO 5: EPKSS or SEQ ID NO 6: GGGGG.
  • the gene of the respective Fc fusion protein was cloned into a suitable expression vector based on a CMV promoter system for protein expression in mammalian cells.
  • HEK293 6E cells were transiently transfected and the cell culture scale was up to 1.5 L in a shake flask or 10 L in a Cultibag fermenter (Sartorius). Expression was done at 37°C for 5- 6 days.
  • medium F17 LifeTechnologies
  • Tryptone TNI Organotechnie
  • 1 % reconcilFCS ultra low IgG" (Invitrogen) and 0.5 mM valproic acid was used.
  • the PDGFRbeta-Fc fusion proteins were purified from mammalian cell supernatants. Cells were removed by a suitable technique like centrifugation or filtration. The clarified cell supernatant was applied to a protein A column equilibrated in DPBS pH 7.4 (Sigma/Aldrich). The column was washed with ten column volumes of DPBS pH 7.4 + 500 mM sodium chloride. PDGFRbeta-Fc fusion proteins were eluted in 50 mM sodium acetate pH 3.5 + 500 mM sodium chloride. PDGFRbeta-Fc fusion proteins were further purified employing a size exclusion chromatography step on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4.
  • Binding affinities (KD values) of PDGFRbeta-Fc fusion proteins were measured by using surface plasmon resonance assays. Experiments were performed using a Biacore T200 instrument (GE Healthcare Biacore, Inc.) with Series S Sensor Chips CM5 (GE Healthcare Biacore, Inc.). Binding assays were carried out at 25 °C with assay buffer HBS-EP+ supplemented with BSA (Sigma) and NaN3 (10 mM HEPES pH 7.4, 500 nM NaCI, 1 mg/ml BSA, 0.05% SP20, 0.05% NaN3). Fc fusions were captured with an anti-hlgG capture antibody covalently immobilized to the chip surface via amine coupling chemistry.
  • Reagents for amine coupling (l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N- hydroxysuccinimide (NHS), ethanolamine-HCI pH 8.5) were used from the Amine Coupling Kit (GE Healthcare, product code BR-1000-50).
  • anti-hlgG capture antibody and immobilization buffer (10 mM sodium acetate pH 5.0) was used from the Human Antibody Capture Kit (GE Healthcare, BR-1008-39).
  • the sensor chip surface was activated with a freshly prepared solution of 0.2 M EDC and 0.05 M NHS passed over the chip surface for 420 s at a flow rate of 10 ⁇ /min, followed by an injection of anti-hlgG capture antibody (dissolved to 25 ⁇ g/ml in immobilization buffer) for 180 s at a flow rate of 5 ⁇ /min. Excess of activated groups were blocked with a 1 molar solution of ethanolamine injected at a flow rate of 10 ⁇ /min for 420 s.
  • hPDGF- AA -AB-BB -CC -DD obtained from R&D Systems were used as analyte to determine /CDS.
  • PDGF-AA Recombiant Human PDGF-AA, CF, Cat. No. 221-AA-010
  • 1.6 and 200 nM of PDGF-AB Recombiant Human PDGF-AB, CF, Cat.No. 222-AB-010
  • 0.01 and 1.5 nM of PDGF BB Recom binant Human PDGF-BB, CF, R&D Systems, Cat.No. 220-BB-010
  • 3.9 and 500 nM of PDGF-CC Recombinant Human PDGF-CC, CF, R&D Systems, Cat.No.
  • PDGF DD Recombinant Human PDGF-DD; R&D Systems; Cat.No. 1159-SB- 025/CF
  • assay buffer assay buffer
  • Table 1 KD values of different PDGFRbeta Fc constructs and the respective ligands BB and DD, [nM].
  • Example 3 Cell-based assays to determine in vitro potency of PDGFRbeta-Fc fusion proteins
  • PDGFRbeta-Fc fusion proteins have been characterized in vitro using Normal Human Dermal Fibroblasts (NHDF) (NHDF juvenile foreskin, Promocell, Catalog Number: C-12300, cultivated according to provider's manual,
  • NHDF Normal Human Dermal Fibroblasts
  • NHDFs were starved for 3h with serum-free culture medium, stimulated for 30 min with 6.3 ng/ml (for pAKT assays) and 50 ng/ml (for pPDGFRbeta assays) PDGF-BB (R&D Systems, Recombinant Human PDGF-BB, CF; 220-BB-010), which was pre-incubated with varying concentrations between 4.0xl0 ⁇ 8 to 8.6x10 12 M of the different PDGFRbeta-Fc fusion proteins for 30 min at room temperature, followed by cell lysis using the manufacturer provided lysis buffer for further analysis. The sample measurement and the data analysis were performed on the Meso Scale platform according to manufacturer's instructions.
  • Table 2 shows that in the pPDGFRbeta assay IC50 values declined from 3.18xl0 ⁇ 9 M for the variant containing no linker to 2.51x10 10 M for the 4xGGGGS linker, corresponding to a factor of 12.7 ( Figure 2A).
  • Figure 2A the reduction of the I C50 values by the addition of a linker was even more pronounced by a reduction 129-fold lower IC50 value for the 5xGGGGS linker variant.
  • the I C50 values for the variants carrying a linker of > 3xGGGGS were not statistically different from each other. This increase in in vitro potency was unexpectedly high when compared to the increases seen in binding affinity in the BIAcore assay.
  • the rat laser Choroidal-Neovascularization (CNV) model was used for the in vivo pharmacological characterization of PDGFRbeta-Fc fusion proteins. Ingrowth of vessels from the choroidea into the retina was induced by applying a laser burn to the choroid layer.
  • Three different treatment schedules were used to assess the efficacy of therapeutic agents on neovascularization and vascular leakage ( Figure 3).
  • Figure 3A the therapeutic agent was administered intravitreally by a single injection of 2 ⁇ of a PBS solution of the test compound one day after the laser burn. Animals were sacrificed on day 23 after the laser procedure.
  • a fluorescence angiography was performed in order to assess the vascular leakage at the lesion site by scoring the amount of fluorescence emanating from the newly formed vessels.
  • the vascular leakage score was determined using a fluorescence fundus camera (Kowe). After anaesthesia and pupillary dilation, 10% sodium fluorescein dye was injected subcutaneously, and images were recorded 2 and 10 min after dye injection. The vascular leakage of the fluorescein on the angiograms was evaluated by three different examiners who were blinded for group allocation (test compound versus vehicle), and scored with 0 (no leakage) to 3 (strongly stained).
  • the retinae were then collected on day 23 of the experiment for immunohistochemical analysis of the vessels to allow quantification of the neovascular area at the laser-induced lesion.
  • the neovascular area is determined as follows: On day 23, animals were sacrificed, and eyes were harvested and fixed in 4% paraformaldehyde solution for 1 hour at room temperature. After washing, the retina was carefully peeled, washed, blocked and stained with a FITC-isolectin B4 antibody in order to visualize the vasculature. Then, the sclera-choroids were flat-mounted and examined under a fluorescence microscope (Keyence Biozero) at 488 nm excitation wavelength. The area (in ⁇ 2 ) of choroidal neovascularization was measured using ImageTool software.
  • the PDGFRbeta-Fc fusion protein with a 4xGGGGS linker was tested in the delayed treatment protocol with long treatment duration as stand-alone and in combination with a VEGFRl-Fc fusion protein (R&D Systems, Recombinant Human VEGF Rl/Flt-1 Fc Chimera, CF, 321-FL-050/CF).
  • the 4xGGGGS linker PDGFRbeta-Fc fusion protein confirmed the findings from the short delayed treatment protocol with the linker-less PDGFRbeta-Fc fusion protein and the 3xGGGGS linker PDGFRbeta fusion protein on the reduction of neovascularization.
  • An additive effect on neovascularisation in combination with anti-VEGF was not observed, which, without being bound by theory, could be due to the ceiling effect mentioned above ( Figure 6A).
  • Vascular leakage is not significantly reduced by either the VEGFRl-Fc fusion protein or the 4x GGGGS linker PDGFRbeta-Fc fusion protein alone in this experiment.
  • the combination of the VEGFRl-Fc fusion protein and the 4x GGGGS linker PDGFRbeta-Fc, fusion protein resulted in a significant reduction of vascular leakage, demonstrating the benefit of such a combination ( Figure 6B).
  • Example 5 Pharmacokinetics and drug metabolism
  • Pharmacokinetic data within this application is used to describe characteristics of a class of receptor-Fc-fusion proteins targeting PDGF ligands.
  • the percentage exposure (area under the curve, AUC, a measure for the amount of substance present in the analyzed compartment) of the compounds was analyzed within the three eye compartments, as well as plasma samples.
  • the 4x GGGGS linker PDGFRbeta-Fc fusion protein, as representative for all tested PDGFRbeta-Fc fusion proteins, as well as Lucentis ® show similar results (see Table 3).
  • the partial distribution, calculated based on AUC values of the single compartments, reveals the majority ( ⁇ 90%) of administered compound within the vitreous, whereas only a minor part of the compound is located in the aqueous humor ( ⁇ 8 %) and the retinal compartment ( ⁇ 2%). Neither Lucentis ® nor any of the PDGFRbeta-Fc proteins were detected in rabbit plasma.
  • vitreous humor as compartment of interest was used as sole matrix for consideration in all following discussions.
  • 6.2 Comparison of pharmacokinetics of different PDGFRbeta-Fc fusion protein versus Lucentis ®
  • MRT Mean Residence Time
  • the linker-less PDGFRbeta- Fc fusion protein shows a longer vitreal terminal half-life of about 4.8 days and an MRT of 7.3 days after administration of 625 ⁇ g into the vitreous. 3 hours after administration, 68% of the linker-less PDGFRbeta-Fc fusion protein was found within the total eye. At least 62 % of it was recovered from the vitreous.
  • PK parameters were calculated from Figure 7 (solid line - 4xGGGGS linker PDGFRbeta- Fc fusion protein; dashed line -linker-less PDGFRbeta-Fc fusion protein variant).
  • This property is highly desirable to enable a reduction in intravitreal dosing frequency, which reduces the risk of adverse events associated with intravitreal application such as infection, the physical and psychological burden and the cost of more frequent intravitreal injections for the patient.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des protéines de fusion PDGFRbêta-Fc ou des fragments biologiquement actifs correspondants, comprenant un domaine extracellulaire de PDGFRbêta ou un fragment biologiquement actif correspondant, un lieur et un domaine Fc, ladite protéine de fusion se liant à un ou plusieurs des ligands du facteur de croissance dérivé des plaquettes avec une affinité élevée. Les protéines de fusion PDGFRbêta-Fc, ou des fragments biologiquement actifs correspondants, peuvent par conséquent être utilisés pour traiter une néovascularisation et une fibrose pathologiques, par exemple un cancer, des troubles néovasculaires oculaires ou des néphropathies. L'invention concerne également des procédés pour le traitement de troubles néovasculaires oculaires à l'aide de ces protéines de fusion sans augmenter la fuite vasculaire. De telles protéines de fusion PDGFRbêta-Fc, ou des fragments biologiquement actifs correspondants, présentent une demi-vie terminale accrue dans l'oeil. L'invention concerne également des séquences d'acide nucléique codant pour les protéines de fusion PDGFRbêta-Fc ci-dessus ou des fragments biologiquement actifs correspondants, des vecteurs les contenant, des compositions pharmaceutiques et des kits accompagnés d'instructions d'utilisation.
PCT/EP2015/065464 2014-07-10 2015-07-07 Protéines de fusion pdgfrbêta-fc et leurs utilisations WO2016005381A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP14176542 2014-07-10
EP14176542.0 2014-07-10
US14/328,487 US20160009776A1 (en) 2014-07-10 2014-07-10 Pdgfrbeta-fc fusion proteins and uses thereof
US14/328,487 2014-07-10

Publications (1)

Publication Number Publication Date
WO2016005381A1 true WO2016005381A1 (fr) 2016-01-14

Family

ID=53514190

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2015/065464 WO2016005381A1 (fr) 2014-07-10 2015-07-07 Protéines de fusion pdgfrbêta-fc et leurs utilisations

Country Status (1)

Country Link
WO (1) WO2016005381A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113493519A (zh) * 2020-03-19 2021-10-12 浙江道尔生物科技有限公司 一种半衰期显著延长的治疗眼部血管新生疾病的融合蛋白
RU2821574C1 (ru) * 2023-08-18 2024-06-25 Общество с ограниченной ответственностью "Пальмира Биофарма" (ООО Пальмира Биофарма) Нуклеотидная последовательность, кодирующая слитый белок, состоящий из растворимого внеклеточного фрагмента человеческого PDGFRa и константной части тяжелой цепи человеческого IgG4

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060234347A1 (en) * 2005-04-13 2006-10-19 Harding Thomas C Targeting multiple angiogenic pathways for cancer therapy using soluble tyrosine kinase receptors
WO2009105669A2 (fr) * 2008-02-20 2009-08-27 Genzyme Corporation Inhibition de l’angiogenèse
CN102311502A (zh) * 2010-07-10 2012-01-11 成都康弘生物科技有限公司 一种抑制血管新生或生长的融合蛋白及其医疗应用
WO2012097019A1 (fr) * 2011-01-13 2012-07-19 Regeneron Pharmaceuticals, Inc. Utilisation d'antagoniste de vegf pour traiter des troubles oculaires angiogéniques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060234347A1 (en) * 2005-04-13 2006-10-19 Harding Thomas C Targeting multiple angiogenic pathways for cancer therapy using soluble tyrosine kinase receptors
WO2009105669A2 (fr) * 2008-02-20 2009-08-27 Genzyme Corporation Inhibition de l’angiogenèse
CN102311502A (zh) * 2010-07-10 2012-01-11 成都康弘生物科技有限公司 一种抑制血管新生或生长的融合蛋白及其医疗应用
WO2012097019A1 (fr) * 2011-01-13 2012-07-19 Regeneron Pharmaceuticals, Inc. Utilisation d'antagoniste de vegf pour traiter des troubles oculaires angiogéniques

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113493519A (zh) * 2020-03-19 2021-10-12 浙江道尔生物科技有限公司 一种半衰期显著延长的治疗眼部血管新生疾病的融合蛋白
CN113493519B (zh) * 2020-03-19 2022-12-27 浙江道尔生物科技有限公司 一种半衰期显著延长的治疗眼部血管新生疾病的融合蛋白
RU2821574C1 (ru) * 2023-08-18 2024-06-25 Общество с ограниченной ответственностью "Пальмира Биофарма" (ООО Пальмира Биофарма) Нуклеотидная последовательность, кодирующая слитый белок, состоящий из растворимого внеклеточного фрагмента человеческого PDGFRa и константной части тяжелой цепи человеческого IgG4

Similar Documents

Publication Publication Date Title
JP6250714B2 (ja) Ｆｇｆ２１突然変異体及びその使用
JP6457431B2 (ja) 抗アクチビンａ抗体およびその使用
JP5823954B2 (ja) Ｆｇｆ２１変異体及びその使用
US20140323396A1 (en) Fgf21 mutants and uses thereof
JP2014502260A (ja) Ａｃｔｒｉｉａ結合剤およびその使用
AU2017299581A1 (en) Humanized monoclonal antibodies that target ve-ptp (hptp-β)
JP2020524661A (ja) 胃抑制ペプチド受容体（ｇｉｐｒ）に対するアンタゴニスト結合タンパク質／ｇｌｐ−１受容体アゴニスト融合タンパク質を使用した代謝障害の治療又は寛解方法
US20160009776A1 (en) Pdgfrbeta-fc fusion proteins and uses thereof
WO2023016449A1 (fr) Polypeptide de fusion bispécifique et son application
WO2016005381A1 (fr) Protéines de fusion pdgfrbêta-fc et leurs utilisations
WO2023219653A1 (fr) Composition pharmaceutique de protéine de fusion vegfr
WO2024114641A1 (fr) Molécules de liaison bispécifiques c5/vegf
WO2023092324A1 (fr) Molécule de liaison de ligand multispécifique et son utilisation
AU2021227958A1 (en) Method of enhancing aqueous humor outflow and reducing intraocular pressure
AU2014202582A1 (en) FGF21 mutants and uses thereof
CN114423788A (zh) 抗nrp1a抗体及其用于治疗眼或眼部疾病的用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15734393

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15734393

Country of ref document: EP

Kind code of ref document: A1