SOLUBLE FGFR3 DECOYS FOR TREATING SKELETAL GROWTH DISORDERS
20210009657 · 2021-01-14
Assignee
- Pfizer Inc. (New York, NY)
- Inserm (Institut National De La Sante Et De La Recherche Medicale) (Paris, FR)
- Université Côte d'Azur (Nice Cedex 2, FR)
Inventors
Cpc classification
C07K2319/33
CHEMISTRY; METALLURGY
A61B2503/06
HUMAN NECESSITIES
A61B5/4538
HUMAN NECESSITIES
A61P19/08
HUMAN NECESSITIES
C07K2319/30
CHEMISTRY; METALLURGY
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
C07K2319/70
CHEMISTRY; METALLURGY
A61K9/0019
HUMAN NECESSITIES
A61B5/4848
HUMAN NECESSITIES
C07K2319/32
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61B5/00
HUMAN NECESSITIES
A61K9/00
HUMAN NECESSITIES
Abstract
The invention features soluble FGF decoy polypeptides and fusion polypeptides comprising an FGF decoy polypeptide linked to a heterologous polypeptide, such as an aggrecan binding protein. Both soluble FGF decoy polypeptides and fusion polypeptides can be used to prevent or treat skeletal disorders, such as achondroplasia.
Claims
1-34. (canceled)
35. A soluble Fibroblast Growth Factor Receptor 3 (sFGFR3) polypeptide comprising an amino acid sequence at least 90% identical to amino acids 1 to 310 of SEQ ID NO: 1, but excluding amino acid residues 324 to 694 of SEQ ID NO: 1.
36. The sFGFR3 polypeptide of claim 35, wherein the sFGFR3 polypeptide comprises an amino acid sequence at least 90% identical to amino acids 1 to 323 of SEQ ID NO: 1.
37. The sFGFR3 polypeptide of claim 35, wherein the sFGFR3 polypeptide comprises an amino acid sequence of amino acids 1 to 323 of SEQ ID NO: 1.
38. The sFGFR3 polypeptide of claim 35, wherein the polypeptide binds to one or more of fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (FGF9), and fibroblast growth factor 18 (FGF18).
39. The sFGFR3 polypeptide of claim 35, wherein the sFGFR3 polypeptide further comprises a heterologous polypeptide.
40. The sFGFR3 polypeptide of claim 39, wherein the heterologous polypeptide comprises an Fc region.
41. The sFGFR3 polypeptide of claim 40, wherein the Fc region is a constant domain of an immunoglobulin selected from the group consisting of IgG-1, IgG-2, and IgG-3.
42. A nucleic acid molecule encoding the sFGFR3 polypeptide of claim 1.
43. A cell comprising the polypeptide of claim 35.
44. The cell of claim 43, wherein the cell is a HEK 293 cell or CHO cell.
45. A composition comprising the sFGFR3 polypeptide of claim 35.
46. The composition of claim 45, further comprising a pharmaceutically acceptable carrier.
47. The composition of claim 45, wherein the composition is formulated for subcutaneous or intravenous administration.
48. The composition of claim 47, wherein the composition is formulated to provide about 0.0002 mg/kg/day to about 20 mg/kg/day of the sFGFR3 polypeptide to a subject in need thereof.
49. The composition of claim 47, wherein the composition is formulated to provide about 0.001 mg/kg/day to about 7 mg/kg/day of the sFGFR3 polypeptide to the subject in need thereof.
50. The composition of claim 47, wherein the composition is formulated to provide about 0.2 mg/kg/day to about 3 mg/kg/day of the sFGFR3 polypeptide to the subject in need thereof.
51. A method of treating a sFGFR3-related skeletal growth retardation disorder in a subject in need thereof comprising administering to the subject the composition of claim 45.
52. The method of claim 51, wherein the subject is a human.
53. The method of claim 51, wherein the FGFR3-related skeletal disease is selected from the group consisting of achondroplasia, thanatophoric dysplasia type I (TOI), thanatophoric dysplasia type II (TOII), severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN), hypochondroplasia, and a craniosynostosis syndrome.
54. The method of claim 53, wherein the FGFR3-related skeletal disease is achondroplasia or hypochondroplasia.
Description
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0100] We have discovered that polypeptide variants of soluble FGFR3 fibroblast growth factor receptor 3 (sFGFR3) may be used to treat or prevent skeletal growth retardation disorders, such as achondroplasia, in a subject in need thereof (e.g., a human, particularly an infant or a child). The invention provides sFGFR3 polypeptide variants including a fragment of the extracellular domain
[0101] (ECD). In particular, sFGFR3 polypeptides of the invention include sFGFR3 deletion (Del) variants featuring a deletion of, e.g., amino acids 311 to 422 of SEQ ID NO: 6, to provide the following exemplary Del variants: sFGFR3_Del2 (amino acids 1 to 548 of SEQ ID NO: 1), sFGFR3_Del3 (amino acids 1 to 440 of SEQ ID NO: 1), and sFGFR3_Del4 (amino acids 1 to 323 of SEQ ID NO: 1). The invention also features fusion polypeptides including a sFGFR3 polypeptide fragment, such as a sFGFR3 polypeptide fragment including, e.g., amino acids 1 to 323 of SEQ ID NO: 1, fused to a heterologous polypeptide, such as an aggrecan-binding protein including human hyaluronan and proteoglycan link protein 1 (HPLN1) or fragments thereof (e.g., sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 4 with or without amino acid residues 1 to 8 of SEQ ID NO: 4 or SEQ ID NO: 4 with or without amino acid residues 1 to 8 of SEQ ID NO: 4 or SEQ ID NO: 33)). Methods for administering sFGFR3 polypeptides (e.g., Del variants as described herein) and fusion polypeptides (e.g., sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 4 with or without amino acid residues 1 to 8 of SEQ ID NO: 4)) to treat or prevent skeletal growth retardation disorders (e.g., achondroplasia) in a subject in need thereof (e.g., a human, particularly an infant or a child) are also described.
[0102] We have determined that smaller functional portions of sFGFR3 exhibit a similar mechanism of action relative to larger soluble FGFR3 fragments and full-length FGFR3 polypeptides while maintaining the capability of dimerization following fixation of free FGFs. Several FGFR3 polypeptide variants with shorter intra-cellular domains were designed. All variants bound human FGF2 with similar affinity relative to sFGFR3_Del1 (SEQ ID NO: 1). Therapeutic benefit was shown using even the shortest FGFR3 variant, sFGFR3_Del4 (amino acids 1 to 323 of SEQ ID NO: 1), which restored bone growth in transgenic Fgfr3.sup.ach/+ mice. Therapeutic efficacy in the treatment of achondroplasia was also observed when sFGFR3_Del4 was administered in a mouse model of this disease, thereby demonstrating the use of this FGFR3 fragment in treatment of growth disorders and for designing additional constructions, such as fusion proteins. The sFGFR3_Del4 construct was also used to validate the use of body weight and skull length monitoring as indexes of velocity of growth. Furthermore, the invention provides fusion proteins comprising the smaller functional portions of sFGFR3 and human hyaluronan and proteoglycan link protein 1 (HPLN1). These fusion proteins bind FGF, as well as aggrecan, and exhibit improved therapeutic benefit relative to previous sFGFR3 decoys.
Fibroblast Growth Factor Receptor 3
[0103] The present disclosure is not limited to a particular Fibroblast Growth Factor Receptor 3 (FGFR3) polypeptide or nucleic acid encoding a FGFR3. FGFR3 polypeptides encompass members of the fibroblast growth factor receptor (FGFR) family that mediate binding to fibroblast growth factors (FGFs) (e.g., FGF1, FGF2, FGF3, FGF4, FGFS, FGF6, FGF7, FGF8, FGF9, FGF10, or FGF18) and play a role in bone development and maintenance. In particular, a FGFR3 polypeptide can bind to FGF1, FGF2, FGF9 and/or FGF 18.
[0104] An FGFR3 polypeptide can include a polypeptide having the amino acid sequence of any one of the known FGFR3 polypeptides or a fragment thereof. FGFR3 polypeptides can include naturally occuring isoforms, such as FGFR3 produced by alternative splicing of the Ig3 domain of FGFR3, in which the C-terminal half of Ig3 is encoded by two separate exons, exon 8 (isoform 1; FGFR3 IIIb) and exon 9 (isoform 3; FGFR3 IIIc). In particular, a FGFR3 polypeptide can include the FGFR3 IIIc-type ECD with the C-terminal Ig3 half encoded by exon 9 (Accession No. NP_000133), which corresponds to FGFR3 transcript variant 1(Accession No. NM_000142.4). A FGFR3 polypeptide can also include the FGFR3 IIIb type ECD with the C-terminal half encoded by exon 8 (Accession No. NP_001156685), which corresponds to FGFR3 transcript variant 3 (Accession No. NM_001163213).
[0105] FGFR3 polypeptides may include not only the FGFR3 amino acid sequences described above, but any polypeptide having at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) amino acid sequence identity to these FGFR3 polypeptides (e.g., SEQ ID NO: 6) or an amino acid fragment of these FGFR3 amino acid sequences (e.g., at least 50, 100, 150, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, 600, 650, 700, 750, or more amino acid residues of an FGFR3 amino acid sequence (e.g., SEQ ID NO: 6)). In addition to the exemplary FGFR3 polypeptides discussed above, this disclosure also provides any FGFR3 polypeptide comprising the identical or similar FGF binding affinity for treating skeletal growth retardation disorders (e.g., achondroplasia) in a patient, e.g., a human.
Soluble FGFR3
[0106] sFGFR3 polypeptides of the invention include soluble (e.g., non-membrane-bound) forms of any of the FGFR3s described herein. The present disclosure is not limited to a particular sFGFR3 and may include any sFGFR3 polypeptide that binds one or more FGFs (e.g., FGF1 (SEQ ID NO: 7), FGF2 (SEQ ID NO: 8), FGF9 (SEQ ID NO: 9), and/or FGF 18 (SEQ ID NO: 10)), and accordingly, may be used as a decoy receptor for one or more FGFs to treat skeletal growth retardation disorders, e.g., achondroplasia. The invention further includes nucleic acids encoding the sFGFR3 polypeptides described herein that may be used to treat the conditions described herein, e.g., achondroplasia, in a subject in need thereof, such as SEQ ID NO: 5. The sFGFR3 polypeptide can be, for example, fragments of FGFR3 isoform 2 lacking exons 8 and 9 encoding the C-terminal half of the IgG3 domain and exon 10 including the transmembrane domain (i.e., sFGFR3_Del1; SEQ ID NO: 1 and Accession No. NP_075254), corresponding to FGFR3 transcript variant 2 (Accession No. NM_022965). Compositions including sFGFR3 are further described in PCT publication Nos: WO 2014/111744 and WO 2014/111467, which are each incorporated herein by reference in their entirety.
[0107] For example, sFGFR3 polypeptides can include fragments of the amino acid sequence of FGFR3 isoform 2 (e.g., at least amino acids 1 to 300, 1 to 310, 1 to 320, 1 to 330, 1 to 340, 1 to 350, 1 to 360, 1 to 370, 1 to 380, 1 to 390, 1 to 400, 1 to 410, 1 to 420, 1 to 430, 1 to 440, 1 to 440, 1 to 450, 1 to 460, 1 to 470, 1 to 480, 1 to 490, 1 to 500, 1 to 510, 1 to 520, 1 to 530, 1 to 540, 1 to 550, 1 to 560, 1 to 570, 1 to 580, 1 to 590, 1 to 600, 1 to 610, 1 to 620, 1 to 630, 1 to 640, 1 to 650, 1 to 660, 1 to 670, 1 to 680, or 1 to 690 of SEQ ID NO: 1). In particular, sFGFR3 polypeptides may include, but are not limited to, amino acids 1 to 323 of SEQ ID NO: 1 (sFGFR3_Del4), amino acids 1 to 440 of SEQ ID NO: 1 (sFGFR3_Del3), or amino acids 1 to 548 of SEQ ID NO: 1 (sFGFR3_Del2).
[0108] sFGFR3 polypeptides and fragments thereof can also include an N-terminal signal peptide sequence. The N-terminal signal peptide is present on the synthesized protein when it is synthesized, but is typically cleaved from the sFGFR3 polypeptide upon export of the polypeptide from the cell. The sFGFR3 polypeptides and sFGFR3 fusion polypeptides of the invention include both secreted (i.e., lacking the N-terminal signal) and non-secreted (i.e., having the N-terminal signal) forms thereof. One skilled in the art will appreciate that the position of the N-terminal signal peptide will vary in different sFGFR3 polypeptides and may include, for example, the first 5, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 30, or more amino acid residues on the N-terminus of the polypeptide. For example, an exemplary signal peptide can include, but is not limited to, amino acids 1 to 22 of SEQ ID NO: 1. Additionally, sFGFR3 polypeptides and fusion polypeptides, such as sFGFR3_Del2, sFGFR3_Del3, and sFGFR3_Del4 (corresponding to amino acids 1 to 548 of SEQ ID NO: 1, amino acids 1 to 440 of SEQ ID NO: 1, and amino acids 1 to 323 of SEQ ID NO: 1, respectively), or a FGFR3 fusion polypeptide (e.g., SEQ ID NO: 4 with or without amino acids 1 to 8 of SEQ ID NO: 4 or SEQ ID NO: 33), can include deletions of the N-terminal amino acids, e.g., at least the amino acids 1 to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 56 of SEQ ID NO: 1. One of skill in the art can predict the position of a signal sequence cleavage site, e.g., by an appropriate computer algorithm such as that described in Bendtsen et al. (J. Mol. Biol. 340(4):783-795, 2004) and available on the Web at www.cbs.dtu.dk/services/SignalP/.
sFGFR3 Fusion Polypeptides
[0109] sFGFR3 polypeptides of the invention can optionally be fused to a functional domain from a heterologous polypeptide (e.g., an aggrecan-binding protein) to provide a sFGFR3 fusion polypeptide, as described herein. In some sFGFR3 polypeptides, a flexible linker, may be included between the sFGFR3 polypeptide and the fusion polypeptide (e.g., an aggrecan-binding protein), such as a serine or glycine-rich sequence (e.g., a poly-glycine or a poly-glycine/serine linker). Further exemplary fusion proteins and linkers are described below.
[0110] For example, the sFGFR3 polypeptides described above, such as fragments of sFGFR3_Dell (e.g., amino acids 1 to 323 of SEQ ID NO: 1 (sFGFR3_Del4), amino acids 1 to 440 of SEQ ID NO: 1 (sFGFR3_Del3), or amino acids 1 to 548 of SEQ ID NO: 1 (sFGFR3_Del2)), can be a fusion polypeptide including, e.g., an aggrecan-binding protein or any polypeptide that targets cartilage when administered to a subject (e.g., a human). In particular, any functional portion of a protein that binds to aggregan, e.g., any protein that interacts with the globular domain (G1, G2, or G3) of aggrecan, can be included in a sFGFR3 fusion polypeptide
[0111] Exemplary aggrecan-binding proteins that can be used to produce the SFGFR3 fusion polypeptides described herein include antibodies, fibulin-1, borrelial aggrecan-binding proteins (e.g., Borrelia glycosaminoglycan-binding protein (Bgp) and Borrelia burgdorferi high temperature requirement A (BbHtrA)), and cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSPS). For example, an aggrecan-binding protein may include human hyaluronan and proteoglycan link protein 1 (HPLN1) or fragments thereof. In particular, the HPLN1 fragment can include a cartilage link domain 1 (LK1; amino acids 158 to 252 of SEQ ID NO: 3), a cartilage link domain 2 (LK2; amino acids 259 to 349 of SEQ ID NO: 3), or both LK1 and LK2 domains (amino acids 158 to 349 of SEQ ID NO: 3). Additional sFGFR3 fusion polypeptides may include any polypeptide that has at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) amino acid sequence identity to the HPLN1 polypeptide (e.g., SEQ ID NO: 3) or an amino acid fragment thereof (e.g., at least 50, 100, 110, 120, 130, 140, 150, 150, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, or more amino acid residues of the entire length of the HPLN1 amino acid sequence (i.e., SEQ ID NO: 3)). For example, an aggrecan-binding protein or fragment thereof of a FGFR3 fusion polypeptide can include, e.g., both LK1 and LK2 domains (amino acids 158 to 349 of SEQ ID NO: 3), in combination with a sFGFR3 polypeptide or fragment thereof, such as amino acids 1 to 323 of SEQ ID NO: 1 (sFGFR3_Del4).
[0112] Additionally, the sFGFR3 polypeptides (e.g., sFGFR3_Del2, sFGFR3_Del3, and sFGFR3_Del4) can be a fusion polypeptide including, e.g., an Fc region of an immunoglobulin at the N-terminal or C-terminal domain. An immunoglobulin molecule has a structure that is well known in the art. It includes two light chains (23 kD each) and two heavy chains (50-70 kD each) joined by inter-chain disulfide bonds. Immunoglobulins are readily cleaved proteolytically (e.g., by papain cleavage) into Fab (containing the light chain and the VH and CH1 domains of the heavy chain) and Fc (containing the CH2 and CH3 domains of the heavy chain, along with adjoining sequences). Useful Fc fragments as described herein include the Fc fragment of any immunoglobulin molecule, including IgG, IgM, IgA, IgD, or IgE, and their various subclasses (e.g., IgG-1, IgG-2, IgG-3, IgG-4, IgA-1, IgA-2), from any mammal (e.g., human). For instance, the Fc fragment is human IgG-1. The Fc fragments of the invention may include, for example, the CH2 and CH3 domains of the heavy chain and any portion of the hinge region. The Fc region may optionally be glycosylated at any appropriate one or more amino acid residues known to those skilled in the art. An Fc fragment as described herein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, or more additions, deletions, or substitutions relative to any of the Fc fragments described herein.
[0113] The sFGFR3 fusion polypeptides described herein may include a peptide linker region between the SFGFR3 polypeptide or fragment thereof and the heterologous polypeptide or fragment thereof (e.g., an aggrecan-binding protein or fragment thereof or an Fc region). The linker region may be of any sequence and length that allows the sALP to remain biologically active, e.g., not sterically hindered. Exemplary linker lengths are between 1 and 200 amino acid residues, e.g., 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-90, 91-95, 96-100, 101-110, 111-120, 121-130, 131-140, 141-150, 151-160, 161-170, 171-180, 181-190, or 191-200 amino acid residues. For instance, linkers include or consist of flexible portions, e.g., regions without significant fixed secondary or tertiary structure. Preferred ranges are 5 to 25 and 10 to 20 amino acids in length. Such flexibility is generally increased if the amino acids are small and do not have bulky side chains that impede rotation or bending of the amino acid chain. Thus, preferably the peptide linker of the present invention has an increased content of small amino acids, in particular of glycines, alanines, serines, threonines, leucines and isoleucines.
[0114] Exemplary flexible linkers are glycine-rich linkers, e.g., containing at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% glycine residues. Linkers may also contain, e.g., serine-rich linkers, e.g., containing at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% serine residues. In some cases, the amino acid sequence of a linker consists only of glycine and serine residues. For example, the sFGFR3 fusion polypeptide (e.g., SEQ ID NO: 4 with or without amino acid residues 1 to 8 of SEQ ID NO: 4 or SEQ ID NO: 33) can include a glycine and serine linker, such as the amino acid sequence SGSGSGSGSGSGSGS (SEQ ID NO: 35). A linker may optionally be glycosylated at any appropriate one or more amino acid residues. Additionally, a linker as described herein may include any other sequence or moiety, attached covalently or non-covalently. The linker may also be absent, in which the FGFR3 polypeptide and heterologous polypeptide (e.g., an aggrecan-binding protein or fragment thereof or Fc region) are fused together directly, with no intervening residues.
[0115] Additional amino acid residues can be introduced into the FGFR3 fusion polypeptide according to the cloning strategy used to produce the FGFR3 fusion polypeptides. For instance, the additional amino acid residues do not provide a portion of the FGFR3 transmembrane domain in order to maintain the polypeptide in a soluble form. Furthermore, any such additional amino acid residues, when incorporated into the FGFR3 polypeptide or fusion polypeptide of the invention, do not provide a cleavage site for endoproteases of the host cell. The likelihood that a designed sequence would be cleaved by the endoproteases of the host cell can be predicted as described, e.g., by Ikezawa (Biol. Pharm. Bull. 25:409-417, 2002).
[0116] The FGFR3 polypeptides and fusion polypeptides of the invention may be associated into dimers or tetramers. Additionally, the polypeptide or fusion polypeptide of the invention (e.g., a sFGFR3 polypeptide or fusion polypeptide) may be glycosylated or PEGylated.
Production of sFGFR3 Nucleic Acids and Polypeptides
[0117] Nucleic acids encoding sFGFR3 and sFGFR3 fusion polypeptides of the invention can be produced by any method known in the art. Typically, a nucleic acid encoding the desired fusion polypeptide is generated using molecular cloning methods, and is generally placed within a vector, such as a plasmid or virus. The vector is used to transform the nucleic acid into a host cell appropriate for the expression of the fusion polypeptide. Representative methods are disclosed, for example, in Maniatis et al. (Cold Springs Harbor Laboratory, 1989). Many cell types can be used as appropriate host cells, although mammalian cells are preferable because they are able to confer appropriate post-translational modifications. Host cells of the present invention may include, e.g., Human Embryonic Kidney 293 (HEK 293) cells, Chinese Hamster Ovary (CHO) cell, L cell, C127 cell, 3T3 cell, BHK cell, COS-7 cell or any other suitable host cell known in the art. For example, the host cell is a HEK 293 cells. Alternatively, the host cell can be a CHO cell.
Methods of Treatment
[0118] Provided herein are methods for treating a skeletal growth retardation disorder in a patient, such as a patient having achondroplasia (e.g., a human having achondroplasia). In particular, the patient may exhibit or may be likely to have one or more symptoms of a skeletal growth retardation disorder (e.g., achondroplasia). The method involves administering a sFGFR3 polypeptide, such as sFGFR3_Del2, sFGFR3_Del3, and sFGFR3_Del4 (corresponding to amino acids 1 to 548 of SEQ ID NO: 1, amino acids 1 to 440 of SEQ ID NO: 1, and amino acids 1 to 323 of SEQ ID NO: 1, respectively), or a FGFR3 fusion polypeptide (e.g., SEQ ID NO: 4 with or without amino acid residues 1 to 8 of SEQ ID NO: 4 or SEQ ID NO: 33) to the patient (e.g., a human). For example, the patient exhibits signs or symptoms of a skeletal growth retardation disorder, such as those described herein (e.g., achondroplasia), e.g., prior to administration of the sFGFR3 polypeptide or FGFR3 fusion polypeptide. Treatment with a sFGFR3 polypeptide of the invention, such as sFGFR3_Del2, sFGFR3_Del3, and sFGFR3_Del4 (e.g., amino acids 1 to 548 of SEQ ID NO: 1, amino acids 1 to 440 of SEQ ID NO: 1, and amino acids 1 to 323 of SEQ ID NO: 1, respectively), or a sFGFR3 fusion polypeptide of the invention (e.g., SEQ ID NO: 4 with or without amino acid residues 1 to 8 of SEQ ID NO: 4 or SEQ ID NO: 33) can also occur after a patient (e.g., a human) has been diagnosed with a skeletal growth retardation disorder, such as those described herein (e.g., achondroplasia), or after a patient exhibits signs or symptoms of a skeletal growth retardation disorder, such as those described herein (e.g., achondroplasia). In particular, the patient is treated with sFGFR3_Del4-LK1-LK2.
[0119] Treatment with a sFGFR3 polypeptide, such as sFGFR3_Del2, sFGFR3_Del3, and sFGFR3_Del4 (e.g., amino acids 1 to 548 of SEQ ID NO: 1, amino acids 1 to 440 of SEQ ID NO: 1, and amino acids 1 to 323 of SEQ ID NO: 1, respectively), or a sFGFR3 fusion polypeptide (e.g., SEQ ID NO: 4 with or without amino acid residues 1 to 8 of SEQ ID NO: 4 or SEQ ID NO: 33) can result in an improvement in a symptom of a skeletal growth retardation disorder, e.g., achondroplasia. The methods can be used to treat symptoms associated with a skeletal growth retardation disorder, e.g., achondroplasia, such that there is reversal or a reduction in the severity of symptoms of the skeletal growth retardation disorder, e.g., achondroplasia.
Skeletal Growth Retardation Disorder
[0120] Skeletal growth retardation disorders can be treated or prevented by administering a sFGFR3 polypeptide or a sFGFR3 fusion polypeptide as described herein. In particular, the method involves administering a sFGFR3 polypeptide, such as sFGFR3_Del2, sFGFR3_Del3, and sFGFR3_Del4 (e.g., amino acids 1 to 548 of SEQ ID NO: 1, amino acids 1 to 440 of SEQ ID NO: 1, and amino acids 1 to 323 of SEQ ID NO: 1, respectively), or a sFGFR3 fusion polypeptide (e.g., sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 4 with or without amino acid residues 1 to 8 of SEQ ID NO: 4 or SEQ ID NO: 33)) to a patient (e.g., a human). Skeletal growth retardation disorders that can be treated with the sFGFR3 polypeptides and sFGFR3 fusion polypeptides described herein are characterized by deformities and/or malformations of the bones and can include, but are not limited to, FGFR3-related skeletal diseases. In particular, the patient is treated with sFGFR3_Del4-LK1-LK2.
[0121] Administration of a sFGFR3 polypeptide or a sFGFR3 fusion polypeptide as described herein (such as, e.g., sFGFR3_Del4-LK1-LK2) can treat a skeletal growth retardation disorder including, but not limited to, achondroplasia, achondrogenesis, acrodysostosis, acromesomelic dysplasia, atelosteogenesis, camptomelic dysplasia, chondrodysplasia punctata, rhizomelic type of chondrodysplasia punctata, cleidocranial dysostosis, congenital short femur, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, Crouzonodermoskeletal syndrome, dactyly, brachydactyly, camptodactyly, polydactyly, syndactyly, diastrophic dysplasia, dwarfism, dyssegmental dysplasia, enchondromatosis, fibrochondrogenesis, fibrous dysplasia, hereditary multiple exostoses, hypophosphatasia, hypophosphatemic rickets, Jaffe-Lichtenstein syndrome, Kniest dysplasia, Kniest syndrome, Langer-type mesomelic dysplasia, Marfan syndrome, McCune-Albright syndrome, micromelia, metaphyseal dysplasia, Jansen-type metaphyseal dysplasia, metatrophic dysplasia, Morquio syndrome, Nievergelt-type mesomelic dysplasia, neurofibromatosis (such as type 1 (e.g., with bone manifestations or without bone manifestations), type 2, or schwannomatosis), osteoarthritis, osteochondrodysplasia, osteogenesis imperfecta, perinatal lethal type of osteogenesis imperfecta, osteopetrosis, osteopoikilosis, peripheral dysostosis, Reinhardt syndrome, Roberts syndrome, Robinow syndrome, short-rib polydactyly syndromes, short stature, spondyloepiphyseal dysplasia congenita, or spondyloepimetaphyseal dysplasia. For instance, administration of the sFGFR3 polypeptides or sFGFR3 fusion polypeptide described herein may resolve and/or prevent symptoms associated with any of the aforementioned disorders.
[0122] The sFGFR3 polypeptides and sFGFR3 fusion polypeptides of the present invention can be used to treat symptoms associated with a skeletal growth retardation disorder, such as a FGFR3-related skeletal disease (e.g., achondroplasia). Non-limiting examples of symptoms of skeletal growth retardation disorders that may be treated, e.g., with a sFGFR3 polypeptide or a sFGFR3 fusion polypeptide, include the following: short limbs and trunk, bowlegs, a waddling gait, skull malformations (e.g., a large head), cloverleaf skull, craniosynostosis (e.g., premature fusion of the bones in the skull), wormian bones (e.g., abnormal thread-like connections between the bones in the skull), anomalies of the hands and feet (e.g., polydactyly or extra fingers), hitchhiker thumbs and abnormal fingernails and toenails, and chest anomalies (e.g., pear-shaped chest or narrow thorax). Additional symptoms that can treated by administering sFGFR3 polypeptides and sFGFR3 fusion polypeptides can also include non-skeletal abnormalities in patients having skeletal growth retardation disorders, e.g., anomalies of the eyes, mouth, and ears, such as congenital cataracts, myopia, cleft palate, or deafness; brain malformations, such as hydrocephaly, porencephaly, hydranencephaly, or agenesis of the corpus callosum; heart defects, such as atrial septal defect, patent ductus arteriosus, or transposition of the great vessels; developmental delays; or mental retardation. Accordingly, adinistration of a sFGFR3 polypeptide or a sFGFR3 fusion polypeptide, as described herein, may result in an improvement in one or more symptoms of a skeletal growth retardation disorder.
[0123] Any skeletal growth retardation disorder that is a FGFR3-related skeletal disease (e.g., caused by or associated with overactivation of FGFR3 as result of a gain-of-function FGFR3 mutation) can be treated by administering a sFGFR3 polypeptide or a sFGFR3 fusion polypeptide as described herein to a patient (e.g., a human). For example, a sFGFR3 polypeptide or a sFGFR3 fusion polypeptide can be administered to treat FGFR3-related diseases, such as skeletal dysplasias and FGFR3-related craniosynostosis. FGFR3-related skeletal diseases can include, but are not limited to, achondroplasia, thanatophoric dysplasia type I (TDI), thanatophoric dysplasia type II (TDII), severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN), hypochondroplasia, and craniosynostosis (e.g., Muenke syndrome and Crouzon syndrome with acanthosis nigricans).
[0124] Patients (e.g., human patients) with mutations in the FGFR3 gene associated with different FGFR3-related skeletal disorders, such as achondroplasia, hypochondroplasia, SADDAN, TDI, and TDII, can also be treated with a sFGFR3 polypeptide, such as sFGFR3_Del2, sFGFR3_Del3, and sFGFR3_Del4, or a sFGFR3 fusion polypeptide, such as a fusion polypeptide including sFGFR3_Del4 and a fragment of an aggrecan-binding protein (e.g., sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 4 with or without amino acid residues 1 to 8 of SEQ ID NO: 4 or SEQ ID NO: 33)).
[0125] For example, the sFGFR3 polypeptide or sFGFR3 fusion polypeptide can be administered to treat achondroplasia resulting from the G380R, G375C, G346E or S279C mutations of the FGFR3 gene. Administration of the sFGFR3 polypeptides and sFGFR3 fusion polypeptides may be used to treat the following exemplary FGFR3-related skeletal disorders: hypochondroplasia resulting from the G375C, G346E or S279C mutations of the FGFR3 gene; TDI resulting from the R248C, S248C, G370C, S371C, Y373C, X807R, X807C, X807G, X8075, X807W and K650M mutations of the FGFR3 gene; TDII resulting from the K650E mutation of the FGFR3 gene; and SADDAN resulting from the K650M mutation of the FGFR3 gene. Thus, a patient treated with the sFGFR3 polypeptides or sFGFR3 fusion polypeptides disclosed herein may have, e.g., a mutation in the FGFR3 gene.
[0126] Any of the aforementioned mutations in the FGFR3 gene (e.g., the G380R mutation of the FGFR3 gene) can be detected in a sample from the patient (e.g., a human with achondroplasia, hypochondroplasia, SADDAN, TDI, and TDII) prior to or after treatment (e.g., treatment with a sFGFR3 polypeptide, such as sFGFR3_Del2, sFGFR3_Del3, and sFGFR3_Del4, or a sFGFR3 fusion polypeptide (e.g., sFGFR3_Del4-LK1-LK2). Additionally, the parents of the patient and/or fetal samples (e.g., fetal nucleic acid obtained from maternal blood, placental, or fetal samples) may be tested by methods known in the art for the mutation.
Achondroplasia
[0127] Achondroplasia is the most common cause of dwarfism in humans and can be treated or prevented by administering a sFGFR3 polypeptide or a sFGFR3 fusion polypeptide as described herein. In particular, achondroplasia may be treated by administering a FGFR3 polypeptide, such as sFGFR3_Del2, sFGFR3_Del3, and sFGFR3_Del4 (e.g., amino acids 1 to 548 of SEQ ID NO: 1, amino acids 1 to 440 of SEQ ID NO: 1, and amino acids 1 to 323 of SEQ ID NO: 1, respectively) or a sFGFR3 fusion polypeptide (e.g., sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 4 with or without amino acid residues 1 to 8 of SEQ ID NO: 4 or SEQ ID NO: 33)) to a patient (e.g., a human). Administration of a sFGFR3 polypeptide or a sFGFR3 fusion polypeptide, as described herein, may result in an improvement in symptoms including, but not limited to, growth retardation, skull deformities, orthodontic defects, cervical cord compression (with risk of death, e.g., from central apnea or seizures), spinal stenosis (e.g., leg and lower back pain), hydrocephalus (e.g., requiring cerebral shunt surgery), hearing loss due to chronic otitis, cardiovascular disease, neurological disease, respiratory problems, fatigue, pain, numbness in the lower back and/or spin, and obesity.
[0128] Patients treated using the sFGFR3 polypeptides or the sFGFR3 fusion polypeptides described herein may include, e.g., infants, children, and adults with achondroplasia. Infants are often diagnosed with achondroplasia at birth, and thus, treatment with a sFGFR3 polypeptide or sFGFR3 fusion protein, as described herein, may begin as early as possible in the patient's life, e.g., shortly after birth, or prior to birth (in utero).
[0129] Symptoms of achondroplasia in patients (e.g., humans) may also be monitored prior to or after a patient is treated with a sFGFR3 polypeptide, such as sFGFR3_Del2, sFGFR3_Del3, and sFGFR3_Del4 (e.g., amino acids 1 to 548 of SEQ ID NO: 1, amino acids 1 to 440 of SEQ ID NO: 1, and amino acids 1 to 323 of SEQ ID NO: 1, respectively), or a sFGFR3 fusion polypeptide (e.g., sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 4 with or without amino acid residues 1 to 8 of SEQ ID NO: 4 or SEQ ID NO: 33)). For instance, symptoms of achondroplasia may be monitored prior to treatment to assess the severity of achondroplasia and condition of the patient prior to performing the methods. The methods of the invention may include diagnosis of achondroplasia in a patient and monitoring the patient for changes in the symptoms of achondroplasia, such as changes in body weight, skull length and/or skull width of the patient based on changes monitored over a period of time, e.g., 1, 2, 3, 4 or more times per month or per year or approximately every 1, 2, 3, 4, 5, 6, 7, 8, 12 or 16 weeks over the course of treatment with the sFGFR3 polypeptide or the sFGFR3 fusion polypeptide of the present invention. Body weight and/or skull size of the patient or changes thereof can also be determined at treatment specific events, e.g. before and/or after administration of the sFGFR3 polypeptide or sFGFR3 fusion polypeptide as described herein. For example, body weight and/or skull size are measured in response to administration of the sFGFR3 polypeptide or sFGFR3 fusion polypeptide of the present invention.
[0130] Body weight can be measured simply be weighing the subject on a scale, preferably in a standardized manner, e.g. with the same (in particular for humans) or no clothes or at a certain time of the day, preferably in a fasting state (for example in the morning before breakfast is taken, or after at least 1, 2, 3, 4, 5 or more hours of fasting).
[0131] Skull size is preferably represented by length, height, width and/or circumference. Measurements can be taken by any known or self-devised standardized method. For a human subject, the measurement of skull circumference is preferred. It is usually taken with a flexible and non-stretchable material such as a tape, which is wrapped around the widest possible circumference of the head (though not around the ears or the facial area below and including the eyebrows), e.g. from the most prominent part of the forehead around to the widest part of the back of the head. Another preferred measurement for a human subject can determine the height of the skull, for example from the underside of the chin to the uppermost point of the head. For a rodent subject, the measurement of the length of the skull (e.g. tip of the nasal bone to back of the occipital bone) is preferred. Alternatively, also the width of the skull (e.g. widest points of the parietal bone) or the height of the skull (e.g. lowest point of the angular process of lower jaw to frontal bone) are preferred. Preferably, any measurement is taken more than once, e.g. at least 3 times, and the largest number us taken as the length, height, width and/or circumference.
Pharmaceutical Compositions and Formulations
[0132] A composition of the present invention (e.g., including a sFGFR3 polypeptide, such as sFGFR3_Del2, sFGFR3_Del3, and sFGFR3_Del4 (e.g., amino acids 1 to 548 of SEQ ID NO: 1, amino acids 1 to 440 of SEQ ID NO: 1, and amino acids 1 to 323 of SEQ ID NO: 1, respectively), or a sFGFR3 fusion polypeptide (e.g., sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 4 with or without amino acid residues 1 to 8 of SEQ ID NO: 4 or SEQ ID NO: 33)) can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. The route of administration can depend on a variety of factors, such as the environment and therapeutic goals. In particular, the sFGFR3 polypeptides and sFGFR3 fusion polypeptides described herein (e.g., sFGFR3_Del4-LK1-LK2) can be administered by any route known in the art, e.g., subcutaneous (e.g., by subcutaneous injection), intravenously, orally, nasally, intramuscularly, sublingually, intrathecally, or intradermally. By way of example, pharmaceutical compositions of the invention can be in the form of a liquid, solution, suspension, pill, capsule, tablet, gelcap, powder, gel, ointment, cream, nebulae, mist, atomized vapor, aerosol, or phytosome.
Dosage
[0133] Any amount of a pharmaceutical composition (e.g., including a sFGFR3 polypeptide, such as sFGFR3_Del2, sFGFR3_Del3, and sFGFR3_Del4 (e.g., amino acids 1 to 548 of SEQ ID NO: 1, amino acids 1 to 440 of SEQ ID NO: 1, and amino acids 1 to 323 of SEQ ID NO: 1, respectively), or a sFGFR3 fusion polypeptide (e.g., sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 4 with or without amino acid residues 1 to 8 of SEQ ID NO: 4 or SEQ ID NO: 33)) can be administered to a patient, such as a patient with a skeletal growth retardation disorder (e.g., a patient with achondroplasia). The dosages will depend on many factors including the mode of administration and the age of the patient. Typically, the amount of the composition (e.g., including a sFGFR3 polypeptide or a sFGFR3 fusion polypeptide) contained within a single dose will be an amount that is effective to treat a condition (e.g., achondroplasia) as described herein without inducing significant toxicity.
[0134] For example, the sFGFR3 polypeptides, such as sFGFR3_Del2, sFGFR3_Del3, and sFGFR3_Del4 (e.g., amino acids 1 to 548 of SEQ ID NO: 1, amino acids 1 to 440 of SEQ ID NO: 1, and amino acids 1 to 323 of SEQ ID NO: 1, respectively), or sFGFR3 fusion polypeptides (e.g., sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 4 with or without amino acids 1 to 8 of SEQ ID NO: 4 or SEQ ID NO: 33)) described herein can be administered to patients (e.g., patients with achondroplasia) in individual doses ranging, e.g., from 0.0002 mg/kg to about 20 mg/kg (e.g., from 0.002 mg/kg to 20 mg/kg, from 0.01 mg/kg to 2 mg/kg, from .2 mg/kg to 20 mg/kg, from 0.01 mg/kg to 10 mg/kg, from 10 mg/kg to 100 mg/kg, from 0.1 mg/kg to 50 mg/kg, 0.5 mg/kg to 20 mg/kg, 1.0 mg/kg to 10 mg/kg, 1.5 mg/kg to 5 mg/kg, or 0.2 mg/kg to 3 mg/kg). In particular, the sFGFR3 polypeptide or sFGFR3 fusion polypeptides as described herein can be administered in individual doses of, e.g., 0.001 mg/kg to 7 mg/kg. These doses can be administered one or more times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more times) per day, week, month, or year.
[0135] Exemplary doses of the sFGFR3 polypeptides of fragments thereof, such as sFGFR3_Del2, sFGFR3_Del3, and sFGFR3_Del4 (e.g., amino acids 1 to 548 of SEQ ID NO: 1, amino acids 1 to 440 of SEQ ID NO: 1, and amino acids 1 to 323 of SEQ ID NO: 1, respectively), or sFGFR3 fusion polypeptides (e.g., sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 4 with or without amino acids 1 to 8 of SEQ ID NO: 4 or SEQ ID NO: 33)) include, e.g., 0.0002, 0.0005, 0.0010, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 2.5, 4, 5, 6, 7, 8, 9, 10, 15, or 20 mg/kg. For all dosages or ranges recited herein, the term about may be used to modify these dosages by 10% of the recited values or range endpoints. In particular, sFGFR3 compositions in accordance with the present disclosure can be administered to patients in doses ranging from about 0.0002 mg/kg/day to about 20 mg/kg/day, about 0.02 mg/kg/day to about 15 mg/kg/day, or about 0.2 mg/kg/day to about 10 mg/kg/day (e.g., 0.75 mg/kg/day). For example, the sFGFR3 compositions can be administered to patients in a weekly dosage ranging, e.g., from about 0.0014 mg/kg/week to about 140 mg/kg/week, e.g., about 0.14 mg/kg/week to about 105 mg/kg/week, or, e.g., about 1.4 mg/kg/week to about 70 mg/kg/week (e.g., 5 mg/kg/week). The dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease (e.g., achondroplasia) and different parameters from the patient (e.g., a patient with achondroplasia).
[0136] Dosages of compositions including sFGFR3 polypeptides, such as sFGFR3_Del2, sFGFR3_Del3, and sFGFR3_Del4 (e.g., amino acids 1 to 548 of SEQ ID NO: 1, amino acids 1 to 440 of SEQ ID NO: 1, and amino acids 1 to 323 of SEQ ID NO: 1, respectively), or sFGFR3 fusion polypeptides (e.g., sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 4 with or without amino acids 1 to 8 of SEQ ID NO: 4 or SEQ ID NO: 33)) may be provided in either a single or multiple dosage regimens. Doses can be administered, e.g., hourly, bihourly, daily, bidaily, twice a week, three times a week, four times a week, five times a week, six times a week, weekly, biweekly, monthly, bimonthly, or yearly. Alternatively, doses can be administered, e.g., twice, three times, four times, five times, six times, seven times, eight times, nine times, 10 times, 11 times, or 12 times per day. In particular, the dosing regimen is twice weekly. For example, the sFGFR3 polypeptides or sFGFR3 fusion polypeptides described herein can be administered at a dosage of 2.5 mg/kg twice weekly. The duration of the dosing regimen can be, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 day(s), week(s), or month(s), or even for the remaining lifespan of the patient. The amount, frequency, and duration of dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient.
Carriers/Vehicles
[0137] Preparations containing a sFGFR3 polypeptide (e.g., amino acids 1 to 548 of SEQ ID NO: 1, amino acids 1 to 440 of SEQ ID NO: 1, or amino acids 1 to 323 of SEQ ID NO: 1, respectively), or sFGFR3 fusion polypeptides (e.g., sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 4 with or without amino acids 1 to 8 of SEQ ID NO: 4 or SEQ ID NO: 33)) may be provided to patients in combination with pharmaceutically acceptable sterile aqueous or non-aqueous solvents, suspensions or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters. Aqueous carriers include water, water-alcohol solutions, emulsions or suspensions, including saline and buffered medical parenteral vehicles including sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, Ringer's solution containing lactose, or fixed oils.
[0138] Intravenous vehicles may include fluid and nutrient replenishers, electrolyte replenishers, such as those based upon Ringer's dextrose, and the like. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
[0139] Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1.000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day. To prepare pharmaceutical compositions, an effective amount of a polypeptide according to the invention may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
[0140] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
[0141] The polypeptides and nucleic acids according to the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and 5 the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
[0142] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution may be suitably buffered and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the subject.
[0143] Gene Therapy
[0144] The sFGFR3 polypeptides, such as sFGFR3_Del2, sFGFR3_Del3, and sFGFR3_Del4 (e.g., amino acids 1 to 548 of SEQ ID NO: 1, amino acids 1 to 440 of SEQ ID NO: 1, and amino acids 1 to 323 of SEQ ID NO: 1, respectively), or sFGFR3 fusion polypeptides such as sFGFR3_Del4-LK1-LK2 (e.g., SEQ ID NO: 4 with or without amino acid residues 1 to 8 of SEQ ID NO: 4 or SEQ ID NO: 33), could also be delivered through gene therapy, where an exogenous nucleic acid encoding the proteins is delivered to tissues of interest and expressed in vivo. Gene therapy methods are discussed, e.g., in Verme et al. (Nature 389:239-242, 1997), Yamamoto et al. (Molecular Therapy 17:S67-S68, 2009), and Yamamoto et al., (J. Bone Miner. Res. 26:135-142, 2011), each of which is hereby incorporated by reference. Both viral and non-viral vector systems can be used. The vectors may be, for example, plasmids, artificial chromosomes (e.g., bacterial, mammalian, or yeast artificial chromosomes), virus or phage vectors provided with an origin of replication, and optionally, a promoter for the expression of the nucleic acid encoding the viral polypeptide and optionally, a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example, an ampicillin or kanamycin resistance gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in in vitro, for example, for the production of DNA, RNA, or the viral polypeptide, or may be used to transfect or transform a host cell, for example, a mammalian host cell, e.g., for the production of the viral polypeptide encoded by the vector. The vectors may also be adapted to be used in vivo, for example, in a method of vaccination or gene therapy.
[0145] Examples of suitable viral vectors include, retroviral, lentiviral, adenoviral, adeno-associated viral, herpes viral, including herpes simplex viral, alpha-viral, pox viral, such as Canarypox and vaccinia-viral based systems. Gene transfer techniques using these viruses are known in the art. Retrovirus vectors, for example, may be used to stably integrate the nucleic acids of the invention into the host genome. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression. Vectors capable of driving expression in insect cells (e.g., baculovirus vectors), in human cells, yeast, or in bacteria may be employed in order to produce quantities of the viral polypeptide(s) encoded by the nucleic acids of the invention, for example, for use in subunit vaccines or in immunoassays. In an additional example, a replication-deficient simian adenovirus vector may be used as a live vector. These viruses contain an E1 deletion and can be grown on cell lines that are transformed with an E1 gene. These vectors can be manipulated to insert a nucleic acid of the invention, such that the encoded viral polypeptide(s) may be expressed.
[0146] Promoters and other expression regulatory signals may be selected to be compatible with the host cell for which expression is designed. For example, mammalian promoters include the metallothionein promoter, which can be induced in response to heavy metals such as cadmium, and the -actin promoter. Viral promoters, such as the SV40 large T antigen promoter, human cytomegalovirus (CMV) immediate early (1 E) promoter, rous sarcoma virus LTR promoter, adenovirus promoter, or a HPV promoter, particularly the HPV upstream regulatory region (URR) may also be used. All these promoters, as well as additional promoters, are well-described in the art. The nucleic acid molecules described herein may also be administered using non-viral based systems. For example, these administration systems include microsphere encapsulation, poly(lactide-co-glycolide), nanoparticle, and liposome-based systems. Non-viral based systems also include techniques facilitating the delivery of naked polynucleotides (such as electroporation, gene gun delivery and various other techniques used for the introduction of polynucleotides).
[0147] The introduced polynucleotide can be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
[0148] The invention is described by way of the following examples which are to be construed as merely illustrative and not limitative of the scope of the invention.
EXAMPLES
Example 1
Decoy Design and Testing Procedures
[0149] Structures and Sequences of the Different Protein Variants.
[0150] A diagram of the different domains of FGFR3, HPLN1 and a soluble FGFR3 (sFGFR3) is shown in
[0151] SEQ ID NO: 1 provides the amino acid sequence of sFGFR3 of PCT/EP2014/050800, SEQ ID NO: 2 the amino acid sequence of the same sFGFR3 but with the full Ig like C2 type domain 3, SEQ ID NO: 3 the amino acid sequence of HPLN1, SEQ ID NO: 4 the amino acid sequence of FLAG-sFGFR3_Del4-LK1-LK2 (see
Cloning and PROTEIN PRODUCTION SYSTEM.
[0152] The Del plasmids were obtained by site directed mutagenesis of the sFGFR3-pFLAG-CMV3 plasmid. The cDNA sequence for LK1-LK2 was optimized for Homo Sapiens while encoding for the original protein sequence (GeneOptimizer process, GeneArt). The synthesized fragment was subcloned into sFGFR3-pFLAG-CMV3 (Garcia, S. et al. 5, 203ra124 (2013)) using Pmll and Kpnl cloning sites. Plasmid DNA was purified from transformed bacteria and concentration determined by UV spectroscopy. Final constructs were verified by sequencing.
[0153] Recombinant proteins were produced by transient transfection using CaCl2 transfection reagent in HEK 293 cells. Each transfection was performed in a cell factory (High flask T600, Merck Millipore) with 80% confluent HEK 293 in 100 ml DMEM without phenol red (Life Technologies) supplemented with 2 mM glutamine (Life Technologies) and antibiotics (Life Technologies). CaCl.sub.2 (690 l) and a pFLAG-sFGFR3 variant (240 g) were resuspended with 6.27 ml H.sub.2O in 7.2 ml HBS, incubated 30 min at room temperature, and then incubated for 16 h onto the cells at 37 C. in 5% CO2. Medium was then replaced by 120 ml supplemented DMEM without phenol red. After 72 h, production medium was filtrated using 0.22 m filters and concentrated on Amicon Ultra-15 60 kDa (Merck Millipore). Recombinant protein was then purified using an affinity column (ANTI-FLAG M2 Affinity Gel, Sigma Aldrich) according to the manufacturer's instructions. Protein amounts were measured by specific ELISA (R&D Systems) according to the manufacturer's instructions.
FGF Binding.
[0154] Fixed amounts of human FGF2, or human FGF9 (R&D Systems) were incubated for 2 h at 37 C. with increasing doses of each recombinant protein (0 to 250 ng/ml) in PBS 1% BSA. Specific commercial ELISA kits (R&D Systems; Clinisciences) were used to quantify remaining unbound FGFs. 1000 pg/ml or 1200 pg/ml were used for FGF2 and FGF9 respectively based on the sensitivity of the corresponding ELISA kits.
Aggrecan Binding.
[0155] ELISA plate were coated with 10 g/mL of Aggrecan (R&D systems #1220-PG-025) in a final volume of 50I per well and incubated overnight at room temperature. Plate were blocked with 300I of PBS 1% BSA per well during 1 h at room temperature. Different amount of protein (0 to 100 l ) were incubated for 1 h at room temperature. Specific detection antibody against FGFR3 from an R&D system ELISA kit (#DYC766E) was used to detect recombinant protein fixed to aggrecan as recommended by the manufacturer.
Efficacy Study in Mice and Evaluation of the Velocity of Growth.
[0156] The Principles of Laboratory Animal Care (NIH publication no. 85-23, revised 1985; grants1.nih.gov/grants/olaw/references/phspol.htm) and the European commission guidelines for the protection of animals used for scientific purposes (ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm) were followed at all times. All procedures were approved by the Institutional Ethic Committee for the use of Laboratory Animals (CIEPAL Azur) (approval #NCE-2012-52).
[0157] Experiments were performed on transgenic Fgfr3.sup.ach/+ animals in which expression of the mutant FGFR3 is driven by the Col2a1 promoter/enhancer (Naski et al., Development 125, 4977-4988, 1998). Mice were exposed to a 12 h light/dark cycle and had free access to standard laboratory food and water. Genotypes were verified by PCR of genomic DNA using the primers 5-AGGTGGCCTTTGACACCTACCAGG-3 (SEQ ID NO: 31) and 5-TCTGTTGTGTTTCCTCCCTGTTGG-3 (SEQ ID NO: 32), which amplify 360 bp of the FGFR3 transgene (Naski et al., supra).
[0158] Several doses of each recombinant protein were tested. At day 3, all newborn mice from a single litter received the same dose. Control litters received 10 l of PBS containing 50% glycerol (vehicle). Thereafter, subcutaneous injections were done twice a week for three weeks, alternatively on the left and right sides of the back. Mice were observed daily with particular attention to locomotion and urination alterations. Breeding was set up to theoretically generate litters with half wild type and half heterozygous Fgfr3.sup.ach/+ mice. To avoid bias due to variations of phenotype penetrance, experiments were performed on at least 2 litters (one treated and one control) arising from the same breeders. No statistical difference between males and females has been observed; they were thus considered as one group for all analyses (Garcia, S. et al. Sci. Transl. Med. 5, 203ra124, 2013).
[0159] At day 22, all animals were sacrificed by lethal injection of pentobarbital. Gender was determined. All subsequent measurements and analyses were performed without knowing mice genotype to avoid any experimental bias. Genotyping were performed at the end of the study to reveal the correspondence of data with a specific genotype. Because achondroplasia is a disease with an important phenotypic variability, all animals were included in the study, to improve the power of the study. Animals dead before day 22 were used for the study of the impact of treatment on premature death and animals reaching day 22 were used for all the analyses. All experiments and data measurements were performed in a blinded manner at all times.
[0160] Following sacrifice at day 22, body weights were measured. Blood (500 I) was harvested by cardiac puncture and 25 I were mixed with 25 l 0.5 M EDTA. Samples were analyzed without centrifugation for blood numeration (Hemavet 950FS, Mascot Hematology). Cadavers were carefully skinned and eviscerated and skeletal measurements (body and tail lengths) were obtained using an electronic digital calliper (Fisher Scientific). Total body length was measured from the nose to the end of the last caudal vertebra; tail was measured starting at the first caudal vertebra. Organs were harvested, weighed and stored in 10% formalin for further histological analysis using standard paraffin-embedded techniques. Organs were observed for macroscopic abnormalities such as modification of color or texture, presence of nodules.
PK/PD Analysis.
[0161] To determine the PK/PD parameters of FLAG-sFGFR3 and FLAG-sFGFR3_Del4-LK1-LK2, 8 week-old WT mice received an intravenous or subcutaneous bolus of 50 mg/kg and 100 mg/kg of protein, respectively. At 15 min, 1 h, 3 h, 8 h, 24 h, and 48 h blood was harvested by retro-orbital puncture using heparin catheter (n =4). Concentration of the FLAG tagged protein was measured by anti FLAG ELISA (Sigma).
Statistical Analysis.
[0162] Statistical analyses were performed with GraphPad Prism 6.0 software. To determine the statistical tests to be used, necessary assumptions were verified. To verify normality and equal variance, an Agostino and Pearson omnibus normality test (alpha=0.05) and a Brown-Forsythe test (p<0.05) were performed, respectively. Because all skeletal measurements data sets (body weight, body length, tail length, cranium length, width, length/width) fulfilled normality and equal variance requirements, two-tailed Student's t test for comparisons of two independent groups were used in the different statistical analyses. Comparison of mortality data between treated and control groups was done using a Kruskal-Wallis test (p<0.05) with a Dunn's test. Comparison of blood numeration was done using a one-way ANOVA with a Dunnett's multiple comparison test (95% CI). For organ weight correlation analyses, Pearson or Spearman tests were used when data sets followed or not normal distribution, respectively multiple comparison test (alpha 0.05). To compare correlations, a Fisher r-to-z transformation was performed. Comparison of decoys binding to human and murine FGFs was done by linear regression.
Example 2
In Vitro Testing of the Deletion Variants
[0163] Summary: All four sFGFR3_Del1, sFGFR3_Del2, sFGFR3_Del3 and sFGFR3_Del4 variants bind human FGF2 with similar affinity than the sFGFR3 full-length construct. sFGFR3_Del4 binds FGF9 with the same affinity as FLAG-sFGFR3.
[0164] All four variants were tested in vitro for their ability to bind human FGF2. Similar to the protocol used to validate the mechanism of action of the FLAG-sFGFR3 molecule; different amounts of FLAG-sFGFR3_Del were incubated with constant quantities of FGF2. All variants bind human FGF2 in a receptor-dose-dependent manner with a similar affinity than the initial FLAG-sFGFR3 protein (
Example 3
In Vitro Testing of the Del4 Deletion Variant
[0165] Summary: Del4 is effective at restoring bone growth in transgenic Fgfr3.sup.ach/+ mice.
[0166] To evaluate FLAG-sFGFR3_Del4 for its therapeutic efficacy, 3 day-old animals received 2.5 mg/kg of protein twice per week for 3 weeks. Control groups received vehicle. Experiments were performed blinded. A total of 108 animals were included.
[0167] The biological effects of FLAG-sFGFR3_Del4 were evaluated following a 3-week-long injection regimen to 3 day-old neonate mice. All newborn male and female mice from one litter received the treatment twice per week over the course of 3 weeks: 2.5 mg/kg FLAG-sFGFR3_Del4, (n=74) or vehicle for control groups (n=52). The first observation was the significant reduction in mortality with treatment: mortality for vehicle-treated Fgfr3.sup.ach/+ mice was 63% compared with 40% in the treated group.
[0168] The velocity of growth was evaluated during the three-week treatment by monitoring cranium length and body weight on growing animals. Results show an improvement in growth velocity with the FLAG-sFGFR3_Del4 treatment (
[0169] At day 22, all surviving animals were sacrificed and their growth was evaluated as measurements of body weight, body length, and tail length. FLAG-sFGFR3_Del4 treatment had a positive effect on overall skeletal growth in similar range than FLAG-sFGFR3 treatment (
Example 4
In Vitro Testing of a Targeted Decoy
[0170] Summary: FLAG-sFGFR3_Del4-LK1-LK2 effectively binds FGF2 and aggrecan.
[0171] Prior to initiating in vivo studies, FLAG-sFGFR3_Del4-LK1-LK2 was evaluated for its ability to bind FGF2 and aggrecan. For this, different amounts of recombinant protein were incubated with constant quantities of FGF2. Our results show that FLAG-sFGFR3_Del4-LK1-LK2 binds human FGF2 with the same affinity as FLAG-sFGFR3 (
[0172] To verify aggrecan binding, different amounts of recombinant protein were incubated with a fixed amount of aggrecan. As seen in
Example 5
In Vivo Testing of a Targeted Decoy
[0173] Summary: FLAG-sFGFR3_Del4-LK1-LK2 is effective at restoring bone growth in Fgfr3.sup.ach/+ mice.
[0174] Comparison of the pharmacokinetic parameters between the initial full-length protein and the fusion protein was performed by the injection of a bolus of protein either by the intravenous or subcutaneous route. Results show that the FLAG-sFGFR3_Del4-LK1-LK2 protein had similar PK parameters with a blood half-life within the same range than that of FLAG-sFGFR3 (see Table 1). However, it has to be kept in mind that the fusion protein targets the cartilage and is therefore retained in this tissue, such that the half-life measured using blood would be expected to be much lower in comparison to FLAG-sFGFR3 which keeps circulating. In fact, in view of the computed instability index (II) (37.41 for FLAG-sFGFR3_Del4-LK1-LK2 classifying it as stable vs. 44.04 for FLAG-sFGFR3, classifying it as unstable), surprisingly indicating that FLAG-sFGFR3_Del4-LK1-LK2 is more stable than FLAG-sFGFR3, and in view of the similar half-life determined in blood, it must be assumed that the actual time for elimination of FLAG-sFGFR3_Del4-LK1-LK2 from the body is significantly longer than for FLAG-sFGFR3, which is highly advantageous since it may allow reducing the effective dosage and/or the frequency of administration.
[0175] The therapeutic potential of FLAG-sFGFR3_Del4-LK1-LK2 was evaluated by injecting subcutaneously 0.3 mg/kg of protein twice per week for 3 weeks starting at age day 3. Control groups received vehicle and experiments were performed blinded. A total of 102 animals were included.
[0176] FLAG-sFGFR3_Del4-LK1-LK2 treatment resulted in a significant reduction in mortality of transgenic animals, from 63% in the control group to 37.5% in the treated group. Velocity of growth was significantly improved by the FLAG-sFGFR3_Del4-LK1-LK2 treatment as seen in
[0177] Potential side effects were evaluated in liver, lung, heart, spleen and kidneys at the time of sacrifice. None of the 102 animals that received chronic subcutaneous injections of FLAG-sFGFR3_Del4-LK1-LK2 or vehicle presented macroscopic abnormalities. In all groups, organ weight changes correlated with changes in total body weight (Table 2), suggesting no direct effect of FLAG-sFGFR3_Del4-LK1-LK2 treatment on organ growth. Blood counts were normal for all animals in this study (Table 3).
TABLE-US-00001 TABLE 1 Evaluation of the PK/PD parameters of FLAG-sFGFR3_Del4-LK1- LK2. Comparison with the a full-length FLAG-sFGFR3 protein. Distribution half-life Elimination half-life (t.sub.1/2) (t.sub.1/2) Ka FLAG-SFGFR3 0.3 h 4.5 h 3.05 FLAG- 0.4 h 3.7 h 2.59 sFGFR3_Del4- LK1-LK2
TABLE-US-00002 TABLE 2 Coefficient correlation (r) between organ and body weight in the different treatment groups (FLAG-sFGFR3_Del4-KL1-KL2 (FP) or vehicle). Pearson or Spearman tests were used for statistical analysis of organ/body weights correlations in each treatment group. No statistical difference was found (Fisher r-to-z transformation) (n = 19-38). Treatment pancreas spleen kidney L kidney R liver heart lung Correlation coefficient WT Vehicle 0.5612 0.8016 0.7636 0.7651 0.821 0.7771 0.7442 0.3 mg/kg FP 0.749 0.6826 0.7411 0.749 0.9136 0.747 0.7168 Fgfr3.sup.ach/+ Vehicle 0.8103 0.7109 0.7641 0.7467 0.7715 0.6757 0.6174 0.3 mg/kg FP 0.7001 0.9038 0.824 0.8569 0.9276 0.8179 0.8351 Fisher r to z transformation WT control vs treated 1.11 0.892 0.172 0.184 1.28 0.188 0.195 Fgfr3.sup.ach/+ control vs treated 0.644 1.493 0.404 0.782 1.526 0.814 1.19 p value WT control vs treated 0.132 0.186 0.431 0.427 0.197 0.425 0.422 Fgfr3.sup.ach/+ control vs treated 0.259 0.067 0.343 0.217 0.063 0.207 0.115
TABLE-US-00003 TABLE 3 Blood counts were not modified by sFGFR3 treatment. Plasma from the vehicle and 0.3 mg/kg FLAG- sFGFR3_Del4-LK1-LK2 (FP) groups were analyzed at time of sacrifice (n = 19-38). Data are means SD. Statistical comparisons with vehicle-treated WT mice were performed using a one way ANOVA. WBCs Neutrophils Lymphocytes Monocytes Eosinophils Basophils (K/l) (%) (%) (%) (%) (%) WT Vehicle 1.82 0.59 0.17 0.17 1.49 0.41 0.08 0.04 0.05 0.06 0.02 0.03 0.3 mg/kg FP 2.7 1.09 0.25 0.31 2.2 0.75 0.17 0.09 0.06 0.07 0.02 0.03 Fgfr3.sup.ach/+ Vehicle 1.79 0.9 0.2 0.19 1.41 0.64 0.1 0.1 0.05 0.05 0.02 0.02 0.3 mg/kg FP 2.36 0.81 0.18 0.12 1.97 0.65 0.14 0.07 0.05 0.05 0.02 0.02 RBCs Hemoglobin Hematocrit Platelets (K/l) (g/dl) (%) MCV MCH MCHC (K/ l) WT Vehicle 3.25 0.39 4.72 0.57 17.59 2.23 54.17 1.76 14.53 0.31 26.87 0.98 307.56 84.71 0.3 mg/kg FP 3.49 0.82 5.12 1.29 19.31 4.82 55.26 1.27 14.65 0.54 26.51 0.69 312.11 80.99 Fgfr3.sup.ach/+ Vehicle 3.13 0.32 4.54 0.42 17.19 2.12 54.83 1.65 14.55 0.74 26.57 1.88 466.17 45.69 0.3 mg/kg FP 3.43 0.45 5 2.54 18.68 2.54 54.51 1.93 14.6 0.68 26.78 0.88 456.9 117.23
Conclusion
[0178] This study shows that a fusion protein, containing portions of sFGFR3 and of HPLN1, can be used to restore endochondral bone growth in a murine model of achondroplasia.
[0179] We first validated that a decoy variant containing the extracellular portion and a very short portion of the intracellular domain of sFGFR3 was sufficient to cause effective FGF binding and engender in vivo efficacy restoring bone growth in mice carrying the G380R mutation.
[0180] The fusion protein has been designed to specifically target cartilage, thus increasing exposure of the target tissue, allowing decreasing a potential effective dose. Biodistribution studies suggest cartilage trapping with similar diffusion through body (PK/PD analysis). FLAG-sFGFR3_Del4-LK1-LK2 treatment is effective at restoring bone growth in the transgenic murine model. Similar to FLAG-sFGFR3, no apparent toxicity was associated with FLAG-sFGFR3_Del4-LK1-LK2 treatment, suggesting that this protein can be used as a therapeutic for achondroplasia and related disorders.
[0181] We have also validated the use of body weight and skull length and/or width monitoring to evaluate velocity of growth. While both approaches give similar results, it is technically easier to use body weight as an index of velocity as skull measurement on newborn animals may created unwanted side effects if not done properly.
[0182] In conclusion, it has been demonstrated that FLAG-sFGFR3_Del4-LK1-LK2 is a viable treatment option for achondroplasia and related disorders.
Example 6
Decoy Design and Purification of sFGFR3 Polypeptides and Fusion Polypeptides
[0183] Further experiments were performed to generate sFGFR3 polypeptides (e.g., sFGFR3_Del4) and a sFGFR3 fusion polypeptide (e.g., sFGFR3_Del4-LK1-LK2) with a FLAG-tag in the C-terminal position. Recombinant proteins were produced by transient transfection using CaCl2 transfection reagent in FreeStyle 293FT cells (TheroFisher Scientific). The plasmid used was pBApo-EF1 -sFGFR3-FLAG plasmid for all variants (sFGFR3_Del1, sFGFR3_Del4, and sFGFR3_Del4-LK1-LK2). An Erlenmeyer flask (2000 ml) was seeded with 293FT cells at a concentration of 1.810.sup.6 cells/ml. Cells were centrifuged for 5 minutes at room temperature prior to transfection, and then resuspended in 300 ml final volume of medium (FreeStyle 293 Expression Medium Life technologies). A solution including sFGFR3-FLAG (600 g plasmid DNA) and 1.73 m of 2M CaCl2 was resuspended in 18 ml of 2 Hepes Buffered Saline (HBS) and then incubated for 30 min at room temperature. The mixture was then added to an erlenmeyer flask and 600 ml of fresh media was added after 4 hours. After 72 hours, the supernatant was purified using the Akta Flux 6 cross-flow filtration system with a combination of two different capsules: 1) the ULTA Prime GF capsule featuring a pleated glass microfibre depth filter with a 5 micron pore size rating and a filter surface area of 0.095 m.sup.2, and 2) the ULTA Pure HC capsule featuring a pleated polyethersulfone sterilizing grade membrane layer with a polyethersulfone prefilter with 0.6/0.2 micron pore size rating and a filter surface area of 0.1 m.sup.2 (GE Healthercare Life Sciences).
[0184] Stable cells lines were then generated by stable transfection of the GP2-293 packaging cell line using CaCl.sub.2 transfection (Clontech). The Del vectors were designed based on the transient transfection results obtained in 293FT cells to create stable cell lines that produce sFGFR3_Del1, sFGFR3_Del4, and sFGFR3_Del4-LK1-LK2. In particular, the plasmids included the signal peptide (MGAPACALALCVAVAIVAGASS; SEQ ID NO: 36) in the same as the previously designed N-terminal FLAG-sFGFR3 variants, the FLAG tag in the C terminal position, an EF1 promoter instead of a CMV promoter, and a pBAbe backbone. Co-transfection of pVSVg and pBABE-EF1 Puro sFGFR3_Del1 Cter FLAG, pBABE-EF1 Puro sFGFR3_Del4-LK1-LK2 Cter FLAG, or pBABE-EF1 a Puro sFGFR3_Del4 Cter FLAG were performed according to the manufacturer's instructions.
[0185] The recombinant sFGFR3 polypeptides and fusion polypeptides were then purified using cross flow filtration system. The advantages of this technique are the elimination of contaminants without aggregation and the purified recombinant sFGFR3 polypeptide are produced under optimized conditions for IEX chromatography. The cross-flow filtration was performed using an Ultrafiltration Hollow Fiber Cartridge (Fiber: UFP-750C from GE Healthcare) at 10 concentration, followed by 3 times volume exchange in Tris 20 mM pH 7 for sFGFR3_Del1 and Tris 20 mM pH 8.5 for sFGFR3_Del4 and sFGFR3_Del4-LK1-LK2. Ion Exchange Chromatography (IEX) was then performed at pH 7 for sFGFR3_Del1 and at pH 8.5 for sFGFR3_Del4 and sFGFR3_Del4-LK1-LK2 using a Hi Prep Q FF 20 ml column (GE Healthcare Life Sciences). Aspiration and elution was performed at a rate of 5 ml/min. A volume fraction of 8 ml was added to the column, then the unbound sFGFR3 polypeptide was washed with a buffer including Tris 20 mM followed by a wash with Tris 20 mM, 1 M NaCl. Finally, size exclusion chromatography was performed using a HiLoad Superdex 200 prep grad (GE Healthcare), with 13 ml of the supernatant at a flow rate of 1 ml/min and an elution buffer of 20 mM Tris, 150 mM NaCl pH 7.4 for all recombinant sFGFR3 polypeptides and fusion polypeptides.
Example 7
Role of Signal Sequence in sFGFR3 Polypeptides and Fusion Polypeptides
[0186] Western blots of the sFGFR3_Del1, sFGFR3_Del4, and sFGFR3_Del4-LK1-LK2 were performed using TBOLTTM 4-12% Bis-Tris Plus Gels (Life technologies) and an IBLOT 2 Gel Transfer Device (Life Technologies) with a PVDF membrane stack. Sample preparation and migration were performed using the manufacturer's instructions. The transfer procedure was the PO program of the iBlot 2 Gel Transfer Device (i.e., 20V for 1 min, 23V for 4 min, and 25V for 2min). After 30 minutes of blocking, incubation with anti-FLAG monoclonal M2 antibody (Sigma Aldrich) was performed for 1 hour. A C-DiGit Chemiluminescence Western Blot Scanner was then used for analysis with a standard ECL substrate (Licor).
[0187] When the FLAG tag of sFGFR3_Del1, sFGFR3_Del4, and sFGFR3_Del4-LK1-LK2 is in the N-terminal position followed by the signal peptide sequence, the sFGFR3 polypeptides and fusion polypeptide were not detectable via Western blot using the anti-FLAG monoclonal M2 antibody (
TABLE-US-00004 TABLE 4 Measurement of body lengthy, tail lenght, skull height, and skull length of wild-type mice and Fgfr3.sup.ach/+ mice after 5 days of treatment with sFGFR3_Del1 and sFGFR3_Del4-LK1-LK2. Body Tail Skull Skull Skull Time Polypeptide GENOTYPE length Length height 1 height 2 length D 3 + 2 sFGFR3_Del1 wt 57.42 27.42 4.42 2.99 13.02 ach 53.45 25.66 4.61 3.01 12.53 SFGFR3 Del4- wt 62.77 30.71 4.89 2.96 14.10 LK1-LK2 ach 60.06 29.07 4.22 2.51 13.28
Example 8
Biodistribution
[0188] Treatment with sFGFR3_Del1 or sFGFR3_Del4-LK1-LK2 was performed in Fgfr3.sup.ach/+ mice as described in Garcia et al. (supra), which is incorporated herein by reference in its entirety. Mice were sacrificed at day 5 and day at day 22 after birth. The biobiodistribution of sFGFR3_Del1 and sFGFR3_Del4-LK1-LK2 in the growth plate of the mice and the effect of sFGFR3_Del1 and sFGFR3_Del4-LK1-LK2 on all bones, growth plate thickness, and the hypertrophic chondrocyte zone were determined over a three week period. The presence of the FLAG tagged polypeptides was determined in the growth plate. These results demonstrate that sFGFR3_Del4-LK1-LK2 is trapped faster within the growth plate (e.g., at day 5), which may be due to an aggrecan defect in Fgfr3.sup.ach/+ mice (
[0189] Skull measurements and x-ray radiography were also performed on wild-type mice and Fgfr3.sup.ach/+ mice after 5 days of treatment with sFGFR3_Del1 and sFGFR3_Del4-LK1-LK2 (Table 4;
TABLE-US-00005 TABLE 5 Measurements of skull parameters in wild-type mice and Fgfr3.sup.ach/+ mice (n = 12). Dorsal measurements include skull length, skull width, palatine length, foramen magnum height, and foramen magnum weidth. Agostino and Pearson omnibus normality test (a = 0.05) and Brown-Forsythe test (P < 0.05) followed by two-tailed Student's t test were performed to determine statistical significance. wt ach p value Skull length 20.75 0.19 18.61 0.40 <0.0001 *** Skull width 10.55 0.06 10.61 0.17 0.7568 ns Skull L/W 1.96 0.02 1.75 0.03 <0.0001 *** palatine 3.163 0.09 2.722 0.11 0.0097 ** foramen height 3.499 0.08 3.396 0.07 0.3871 ns foramen width 4.762 0.02 4.599 0.05 0.0122 *
TABLE-US-00006 TABLE 6 Measurements of axial/appendicular and lateral skeletal parameters in wild-type mice and Fgfr3.sup.ach/+ mice (n = 12). The axial/appendicular measurements include CTL (cervico-thoraco-lumbar length) and femur length (lateral). The lateral measurements include skull length and height. Agostino and Pearson omnibus normality test (a = 0.05) and Brown-Forsythe test (P < 0.05) followed by two-tailed Student's t test were performed to determine statistical significance. wt ach p value CTL 35.11 0.58 31.17 1.05 0.0030 ** Femur length 10.23 0.30 8.83 0.32 0.0053 ** Skull length 20.75 0.19 18.61 0.40 <0.0001 *** Skull height 4.76 0.17 5.107 0.28 0.3076 ns.sup.
Example 9
Potency Assays
[0190] Potency assays were performed to determine the effect of sFGFR3_Del1 (SEQ ID NO: 1) on Erk/P-Erk Intracellular signaling in ATDCS chondrocyte cells. ATDCS cells were plated at a density of 7.510.sup.3 in 96-well plates and cultured for 24 hours in DMEM-F12/0.5% BSA (Life Technologies). Cells were then challenged for 24 hours with hFGF2 (100 pg/ml) in the presence of sFGFR3_Del1 (0 or 20 ng/ml). Intracellular signaling was evaluated with the ICW kit PhosphoPlus p44/42 MAPK (Erk1/2)(Thr202/Tyr204) In-Cell Duet (ICW Compatible)-(cell signaling). After a 24 hour incubation, there was a decrease of Erk phosphorylation in ATDCS cells incubated with sFGFR3_Del1 (SEQ ID
[0191] NO: 1) and FGF decreased relative to ATDCS cells incubated with sFGFR3_Del1 (SEQ ID NO: 1) or FGF alone (
Example 10
Cellular Proliferation
[0192] The effect of different fractions of purified sFGFR3 polypeptide or sFGFR3 fusion polypeptide on cellular proliferation was determined in ATDCS chondrocyte cells. Size-exclusion chromatography followed by western blot analysis was performed as described above to purify and identify fractions of sFGFR3_Del1 (
Example 11
Behavioral Studies
[0193] Behavioral studies were performed to characterize the long-term effects of sFGFR3_Del1 and sFGFR3 Del4-LK1-LK2 in the C57BL6/J mouse. sFGFR3 Del1 and sFGFR3 Del4-LK1-LK2 were each administered subcutaneously twice per week during the first 8 postnatal weeks, starting from postnatal day 3. There was three different groups (16 male mice per group) treated with sFGFR3_Del1, sFGFR3_Del4-LK1-LK2, or a vehicle at 2.5 mg/kg. Behavioral evaluation was conducted between postnatal weeks 10 and 14. The effects of sFGFR3_Del1 and sFGFR3_Del4-LK1-LK2 on behavior, sensory capacities, motor capacities, psychological characteristics, and cognitive function were investigated.
[0194] These studies showed no indications of long term toxicity of treatment with sFGFR3_Del1 and sFGFR3_Del4-LK1-LK2. For the Irwin test, activity meter test, elevated plus-maze test, and forced swimming test, no changes in any group were observed. For odor discrimination, I observed a minor decrease with sFGFR3_Del4-LK1-LK2. Administration of sFGFR3_Del4-LK1-LK2 also appeared to result in a minor decrease of motor coordination during the accelerating rotarod test and a minor improvement of motor coordination and spatial memory during the Morris water maze test, which were not statistically different.
Example 12
FGFR3_Del1 and sFGFR3_Del4-LK1-LK2 Do Not Cross the Blood Brain Barrier
[0195] Pharmacokinetic studies were performed to determine the uptake of FGFR3_Del1 and sFGFR3_Del4-LK1-LK2 across the blood brain barrier in C57BL/6 mice. After intravenous (i.v.) bolus injection, brain tissue uptake of FGFR3_Del1 and sFGFR3_Del4-LK1-LK2 was measured at 4 time points (1 hour (h), 3 hours (h), 6 hours (h), and 24 hours (h)). FGFR3_Del1 and sFGFR3_Del4-LK1-LK2 were injected as radiolabeled tracer (.sup.125I-FGFR3_Del1 or .sup.125I-sFGFR3_Del4-LK1-LK2) with 2.5 mg/kg unlabeled FGFR3_Del1 and sFGFR3_Del4-LK1-LK2. The injected dose of .sup.125I-FGFR3_Del1 or .sup.125I-sFGFR3_Del4-LK1-LK2 was about 10 Ci per animal, which corresponds to less than 0.1 mg/kg. After euthanizing the mice at 1 h, 3 h, 6 h or 24 h, the concentration of .sup.125I-FGFR3_Del1 or .sup.125I-sFGFR3_Del4-LK1-LK2 in organs and plasma was measured by liquid scintillation counting.
[0196] .sup.125I-FGFR3 Del1 or .sup.125I-sFGFR3 Del4-LK1-LK2 concentrations were corrected for metabolism in plasma and in brain samples by measuring the fraction of trichloroacetic acid (TCA) precipitable material (e.g., intact tracer). The validity of the TCA correction was also confirmed by injecting samples on a size exclusion fast protein liquid chromatography (FPLC) column. The organ concentration of .sup.125I-FGFR3 Del1 or .sup.125I-sFGFR3 Del4-LK1-LK2 was corrected for intravascular content (V.sub.0) by injecting radiolabeled albumin (.sup.3H-RSA) shortly before sacrificing the animal. The apparent organ volume of distribution of RSA represents V.sub.0. The dose of albumin was negligible (on the order of 1% of the physiological concentration). For all organs other than brain, the concentrations were calculated by subtracting the vascular content and taking into account the TCA precipitable fraction in plasma. However, no correction was made for the uptake of degraded material into these organs other than the brain because no TCA precipitation was performed.
[0197] The brain concentrations were calculated by the following formula: C.sub.brain(corr,)=[Vd(FGFR3_Del1)V.sub.0]C.sub.plasma (terminal), in which Vd(FGFR3_Del1) is the volume of distribution of FGFR3_Del1 in brain (calculated as C.sub.brain/C.sub.plasma), V.sub.0 is the volume of albumin distributed in the brain, and C.sub.plasma(terminal) is the plasma concentration of FGFR3_Del1 at the terminal sampling time. Calculations for sFGFR3_Del4-LK1-LK2 were performed identically, and all concentrations were expressed as the percent of injected dose per gram or ml (%ID/g or %ID/mL), respectively, and the dose of the i.v. bolus equals 100%. If desired, these values may be converted to [mg/g] or [mg/mL] by multiplication with the injected dose: (body weight in g/1000 g)2.5 mg. All body weights were in the range of 25 g-30 g.
[0198] There was no detectable brain uptake of .sup.125I-FGFR3_Del2, as indicated by corrected brain concentrations (after correction for vascular content and degradation (TCA precipitability)), at any of the measured time points (1 h, 3 h, 6 h, or 24 h) (
[0199] Similar results were obtained for brain tissue uptake of .sup.125I-sFGFR3_Del4-LK1-LK2. There was no detectable brain uptake of .sup.125I-sFGFR3_Del4-LK1-LK2, as indicated by corrected brain concentrations (after correction for vascular content and degradation (TCA precipitability)), at any of the measured time points (1 h, 3 h, 6 h, or 24 h) (
[0200] In a non-compartmental analysis (NCA) of .sup.125I-FGFR3_Del1 and .sup.125I-sFGFR3_Del4-LK1-LK2 plasma concentrations with Phoenix WinNonlin, the area under the curve (AUC) of .sup.125I-sFGFR3_Del4-LK1-LK2 was lower than the AUC of .sup.125I-FGFR3_Del1 by a factor of 0.71 (
[0201] In conclusion, there is no measurable uptake of either FGFR3_Del1 or sFGFR3_Del4-LK1-LK2 into brain tissue of mice at any of the time points analyzed in this study, at a dose of 2.5 mg/kg injected as an intravenous bolus. These results are based on the assumption that the .sup.125I-FGFR3_Del1 and .sup.125I-sFGFR3_Del4-LK1-LK2 (tracer-labeled proteins) behave similarly to unlabeled protein in brain tissue and plasma.