WO2022254319A1 - Procédé de culture cellulaire pour la production du polypeptide sfgfr3 - Google Patents

Procédé de culture cellulaire pour la production du polypeptide sfgfr3 Download PDF

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WO2022254319A1
WO2022254319A1 PCT/IB2022/055062 IB2022055062W WO2022254319A1 WO 2022254319 A1 WO2022254319 A1 WO 2022254319A1 IB 2022055062 W IB2022055062 W IB 2022055062W WO 2022254319 A1 WO2022254319 A1 WO 2022254319A1
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cells
polypeptide
sfgfr3
seq
temperature
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PCT/IB2022/055062
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Timothy David Gryseels
Eugenia Adine HICKEY
Alexander Nicholas MILLER
Nichole Lea Wood
Erwin Yu
Ziaolu ZHENG
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Pfizer Inc.
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Publication of WO2022254319A1 publication Critical patent/WO2022254319A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/005—Protein-free medium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2523/00—Culture process characterised by temperature

Definitions

  • FGFR3 soluble fibroblast growth factor receptor 3
  • FGFs fibroblast growth factors
  • Fibroblast growth factor receptor 3 is a major negative regulator of bone growth and mutations in FGFR3 cause achondroplasia, or short- limbed dwarfism, in humans.
  • Soluble fibroblast growth factor receptor 3 (sFGFR3) polypeptides have been shown to be useful in the treatment of skeletal growth retardation disorders such as achondroplasia (see for example, WO 2014/111744, WO 2014/111467, WO 2016/110786, WO 2018/007597 or WO 2019/057820). It has also previously been shown that native FGFR3 is susceptible to proteolytic cleavage in the bovine rib growth plate (Pandit et al, 2002).
  • the invention relates to method for producing a sFGFR3 polypeptide in a cell culture.
  • the method comprises the steps of: (i) providing mammalian cells that contain a gene encoding a sFGFR3 polypeptide in a cell culture medium to start a cell culture, and, (ii) culturing the cells at a first temperature between about 36.0°C and 37.0°C, and, (iii) shifting the temperature to a second temperature between about 30.0°C and about 32.0°C.
  • the first temperature is about 36.5°C.
  • the second temperature is about 30.5°C or 31.5°C.
  • the temperature is shifted to a lower temperature between day 3 and day 7. In some embodiments, the temperature is shifted to a lower temperature on day 4.
  • the sFGFR3 polypeptide comprises 100-370 consecutive amino acids of an extracellular domain of the naturally occurring FGFR3 polypeptide; (ii) comprises 100, 200, 225, 250, 275, 300, 310, 320, 325, 330, 335, 336, 340, 345, or 350 consecutive amino acids of an ECD of a naturally occurring fibroblast growth factor receptor 3 (FGFR3); (iii) lacks a signal peptide and/or a transmembrane domain, such as the signal peptide and/or transmembrane domain of a naturally occurring FGFR3 polypeptide; (iv) comprises 175, 150, 125, 100, 75, 50, 40, 30, 20, 15, 13, or fewer consecutive amino acids of the intracellular domain of a naturally-occurring FGFR3 polypeptide; (v) comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 9
  • the sFGFR3 polypeptide comprises or consists of an amino acid sequence of SEQ ID NO: 5.
  • the percentage of intact sFGFR3 polypeptide is increased compared to the percentage that would be observed with an otherwise identical method that lacks a temperature shift or that comprises a temperature shift to 34°C.
  • the percentage of aggregated sFGFR3 polypeptide is reduced compared to the percentage that would be observed with an otherwise identical method that lacks a temperature shift or that comprises a temperature shift to 34°C.
  • Figure 1 shows the effect of a temperature shift to 34°C or 31.5°C on the titer (mg/mL) of sFGFR3 Del4-D3 polypeptide as measured by RP-HPLC in a small scale experiment.
  • Figure 2 shows the effect of a temperature shift to 34°C or 31.5°C on the percentage of intact species of sFGFR3_Del4-D3 polypeptide as measured by Capillary Gel Electrophoresis (CGE).
  • Figure 3 shows the effect of a temperature shift to 31.5°C on the titer (mg/mL) of sFGFR3_Del4-D3 polypeptide as measured by RP-HPLC in a 1L bioreactor experiment.
  • Figure 4 shows the effect of a temperature shift to 31.5°C on the percentage of intact species of sFGFR3_Del4-D3 polypeptide as measured by Capillary Gel Electrophoresis (CGE) in a 1L bioreactor experiment.
  • Figure 5 shows the effect of a temperature shift to 31.5°C on the percentage of HMMS as measured by Size Exclusion HPLC (SE-HPLC) in a 1L bioreactor experiment.
  • Figure 6 shows the effect of a temperature shift to 31.5°C, 30.5°C or 30.0°C on the titer (mg/mL) of sFGFR3_Del4-D3 polypeptide as measured by RP-HPLC in a 1L bioreactor experiment.
  • Figure 7 shows the effect of a temperature shift to 31.5°C, 30.5°C or 30.0°C on the percentage of intact species of sFGFR3_Del4-D3 polypeptide as measured by Capillary Gel Electrophoresis (CGE) in a 1L bioreactor experiment.
  • Figure 8 shows the effect of a temperature shift to 31.5°C, 30.5°C or 30.0°C on the percentage of HMMS as measured by Size Exclusion HPLC (SE-HPLC) in a 1L bioreactor experiment.
  • SE-HPLC Size Exclusion HPLC
  • the present disclosure is directed to a method for producing a sFGFR3 polypeptide.
  • the methods disclosed herein are particularly useful for producing a sFGFR3 polypeptide to be used as therapeutic agent.
  • the methods disclosed herein can be used for manufacturing sFGFR3 polypeptide at large scale, for example in cell culture medium volume of at least 100L, 500L or even at least 3000L.
  • the present application relates to a method for producing a sFGFR3 polypeptide in a cell culture, said method comprising the steps of: (i) providing mammalian cells that contain a gene encoding a sFGFR3 polypeptide in a cell culture medium to start a cell culture, and, (ii) culturing the cells at a first temperature between about 36.0°C and 37.0°C, and, (iii) shifting the temperature to a second temperature between about 30.0°C and about 32.0°C.
  • An advantage of the methods disclosed herein is to increase the percentage of intact (i.e. non fragmented) sFGFR3 polypeptide as compared to an identical method wherein the temperature is not shifted.
  • the methods disclosed herein also provide a good titer and reduce the percentage of aggregated sFGFR3 polypeptide.
  • the methods disclosed herein provide an increased percentage of intact sFGFR3 polypeptide (i.e less fragments) as compared to other methods such as for example methods conducted at a temperature higher or lower than the temperature or temperature ranges defined herein and/or methods without temperature shift.
  • the methods disclosed herein provide an increased percentage of intact sFGFR3 polypeptide (i.e less fragments) compared to an otherwise identical method that lacks a temperature shift or which comprises a temperature shift to 34°C.
  • the percentage of intact sFGFR3 polypeptide is calculated based on the amount of intact sFGFR3 relative to the total amount of sFGFR3 polypeptide (intact and fragmented) produced.
  • the percentage of intact sFGFR3 polypeptide can be determined by any method known to the skilled person. In one embodiment, the percentage of intact sFGFR3 polypeptide is determined by Capillary Gel Electrophoresis. In one embodiment, the percentage of intact sFGFR3 polypeptide is determined according to the method disclosed in the examples. In some embodiments, the methods disclosed herein provide an increased titer as compared to other methods such as for example methods conducted at a temperature higher or lower than the temperature or temperature ranges defined herein and/or methods without temperature shift.
  • the methods disclosed herein provides an increased titer compared to an otherwise identical method that lacks a temperature shift or that comprises a temperature shift to 34°C. Titer can be determined by any method known in the art. In one embodiment, titer is measured by reverse phase high-performance liquid chromatography (RP-HPLC). In some embodiments, the methods disclosed herein provide a reduced percentage of aggregated sFGFR3 polypeptide as compared to other methods such as for example methods conducted at a temperature higher or lower than the temperature or temperature ranges defined herein and/or methods without temperature shift.
  • RP-HPLC reverse phase high-performance liquid chromatography
  • the method disclosed herein provides a reduced percentage of aggregated sFGFR3 polypeptide compared to an otherwise identical method that lacks a temperature shift or that comprises a temperature shift to 34°C.
  • the amount of aggregated sFGFR3 polypeptide can be determined by any method known in the art. In one embodiment, the amount of aggregated sFGFR3 polypeptide is measured by Size Exclusion HPLC. In one embodiment, the amount of aggregated sFGFR3 polypeptide is determined according to the method disclosed in the examples. In some embodiments, the methods disclosed herein provides an increased percentage of intact sFGFR3 polypeptide and an increased titer compared to an otherwise identical method that lacks a temperature shift or that comprises a temperature shift to 34°C.
  • the methods disclosed herein provide an increased percentage of intact sFGFR3 polypeptide, a reduced percentage of aggregated sFGFR3 polypeptide and an increased titer compared to an otherwise identical method that lacks a temperature shift or that comprises a temperature shift to 34°C. In some embodiments, the methods disclosed herein provide a reduced percentage of aggregated sFGFR3 polypeptide and an increased titer compared to an otherwise identical method that lacks a temperature shift or that comprises a temperature shift to 34°C. In some embodiments of the methods disclosed herein, the first temperature is about 36.0, 36.1, 36.2, 36.3, 36.4, 36.5, 36.6, 36.7, 36.8, 36.9 or 37.0°C, preferably 36.5°C.
  • the second temperature is about 30.0, 30.1, 30.2, 30.3, 30.4, 30.5, 30.6, 30.7, 30.8, 30.9, 31.0, 31.1, 31.2, 31.3, 31.4, 31.5, 31.6, 31.7, 31.8, 31.9 or 32.0°C, preferably 31.5°C.
  • the first temperature is about 36.5°C and the second temperature is about 31.5°C. In some embodiments of the methods disclosed herein, the first temperature is about 36.5°C and the second temperature is about 30.5°C.
  • the temperature is shifted to a lower temperature between day 3 and day 7, i.e on day 3, day 4, day 5, day 6 or day 7.
  • the temperature is shifted on day 4.
  • the sFGFR3 polypeptide comprises 100-370 consecutive amino acids of an extracellular domain of the naturally occurring FGFR3 polypeptide; (ii) comprises 100, 200, 225, 250, 275, 300, 310, 320, 325, 330, 335, 336, 340, 345, or 350 consecutive amino acids of an ECD of a naturally occurring fibroblast growth factor receptor 3 (FGFR3); (iii) lacks a signal peptide and/or a transmembrane domain, such as the signal peptide and/or transmembrane domain of a naturally occurring FGFR3 polypeptide; (iv) comprises 175, 150, 125, 100, 75, 50, 40, 30, 20, 15, 13, or fewer consecutive amino acids
  • the sFGFR3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99% sequence identity to SEQ ID NO: 5.
  • the sFGFR3 polypeptide comprises or consists of an amino acid sequence of SEQ ID NO: 5.
  • the cell culture is a fed batch culture.
  • the disclosure also relates to a sFGFR3 polypeptide obtained by any of the methods disclosed herein.
  • the disclosure relates to a sFGFR3 polypeptide obtainable by any of the methods disclosed herein.
  • fibroblast growth factor and “FGF” refer to a member of the FGF family, which includes structurally related signaling molecules involved in various metabolic processes, including endocrine signaling pathways, development, wound healing, and angiogenesis. FGFs play key roles in the proliferation and differentiation of a wide range of cell and tissue types. The term preferably refers to FGF1, FGF2, FGF9, FGF 10, FGF18, FGF19, FGF21, and FGF23, which have been shown to bind FGFR3.
  • FGFs can include human FGF1 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 26), human FGF2 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 27), human FGF9 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 28), human FGF10 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 40), human FGF18 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 29), human FGF19 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 30), human FGF21 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 31), and human FGF23 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 41).
  • human FGF1 e.g., a polypeptide having the amino acid sequence of S
  • fibroblast growth factor receptor 3 refers to a polypeptide that specifically binds one or more FGFs (e.g., FGF1, FGF2, FGF9, FGF10, FGF18, FGF19, FGF 21, and/or FGF23).
  • FGFs e.g., FGF1, FGF2, FGF9, FGF10, FGF18, FGF19, FGF 21, and/or FGF23.
  • the human FGFR3 gene which is located on the distal short arm of chromosome 4, encodes an 806 amino acid protein precursor (fibroblast growth factor receptor 3 isoform 1 precursor), which contains 19 exons, and includes a signal peptide (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 21).
  • Mutations in the FGFR3 amino acid sequence that lead to skeletal growth disorders include, e.g., the substitution of a glycine residue at position 358 with an arginine residue (i.e., G358R; SEQ ID NO: 9).
  • the naturally occurring human FGFR3 gene has a nucleotide sequence as shown in Genbank Accession number NM_000142.4 and the naturally occurring human FGFR3 protein has an amino acid sequence as shown in Genbank Accession number NP_000133, herein represented by SEQ ID NO: 8.
  • the wildtype FGFR3 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 8) consists of an extracellular immunoglobulin-like membrane domain including Ig-like C2-type domains 1-3, a transmembrane domain, and an intracellular (i.e., cytoplasmic) domain.
  • FGFR3s can include fragments and/or variants (e.g., splice variants, such as splice variants utilizing alternate exon 8 rather than exon 9) of the full-length, wild-type FGFR3.
  • domain refers to a conserved region of the amino acid sequence of a polypeptide (e.g.
  • a domain can vary in length from, e.g., about 20 amino acids to about 600 amino acids.
  • Exemplary domains include the immunoglobulin domains of a FGFR3 (e.g., Ig- like C2-type domain 1, Ig-like C2-type domain 2, and Ig-like C2-type domain 3), the extracellular domain (ECD) of a FGFR3, the intracellular domain (ICD) of a FGFR3, or the transmembrane domain (TM) of a FGFR3, such as a FGFR3 having the sequence set forth in SEQ ID NO: 8).
  • ICD extracellular domain
  • ICD intracellular domain
  • TM transmembrane domain
  • extracellular domain and “ECD” refer to the portion of a FGFR3 polypeptide that extends beyond the transmembrane domain into the extracellular space.
  • the ECD mediates binding of a FGFR3 to one or more fibroblast growth factors (FGFs).
  • FGFs fibroblast growth factors
  • an ECD includes the Ig-like C2-type domains 1-3 of a FGFR3 polypeptide.
  • the ECD includes the Ig-like C2-type domain 1 of a wildtype (wt) FGFR3 polypeptide, the Ig-like C2-type domain 2 of a wildtype (wt) FGFR3 polypeptide, and/or the Ig-like C2-type domain 3 of a wt FGFR3 polypeptide.
  • An ECD of a FGFR3 can also include a fragment of the wildtype FGFR3 Ig-like 02-type domain for instance.
  • fragment and “portion” refer to a part of a whole, such as a polypeptide or nucleic acid molecule that contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the entire length of the reference nucleic acid molecule or polypeptide, or a domain thereof (e.g., the ECD, ICD, or TM of a sFGFR3 polypeptide).
  • a fragment or portion may contain, e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 336, 340, 348, 349, 350, 360, 370, 380, 390, 400, 500, 600, 700, or more consecutive amino acid residues, up to the entire length of the reference polypeptide.
  • a FGFR3 fragment can include any polypeptide having at least about 5 consecutive amino acids to about 350 consecutive amino acids, inclusive of the endpoints, e.g., at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 310, 320, 330, 335, 336, 340, or 350 consecutive amino acids of any one of SEQ ID Nos: 1-8.
  • a FGFR3 fragment includes a polypeptide having at least 336 consecutive amino acids of SEQ ID NO: 8.
  • the terms “soluble fibroblast growth factor receptor 3,” “soluble FGFR3,” and “sFGFR3” refer to a FGFR3 that is characterized by the absence or functional disruption of all or a substantial part of the transmembrane domain and any polypeptide portion that would anchor the FGFR3 polypeptide to a cell membrane (e.g., a tyrosine kinase domain).
  • An sFGFR3 polypeptide is a non-membrane bound form of an FGFR3 polypeptide.
  • an sFGFR3 polypeptide can include a deletion of a portion or all of the amino acid residues of the transmembrane domain of a wild-type FGFR3 polypeptide sequence (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 8).
  • the sFGFR3 polypeptide can further include deletions of the intracellular domain of the wild-type FGFR3 polypeptide.
  • Exemplary sFGFR3 polypeptides can include, but are not limited to, at least amino acids 1 to 100, 1 to 125, 1 to 150, 1 to 175, 1 to 200, 1 to 205, 1 to 210, 1 to 215, 1 to 220, 1 to 225, 1 to 230, 1 to 235, 1 to 240, 1 to 245, 1 to 250, 1 to 252, 1 to 255, 1 to 260, 1 to 265, 1 to 270, 1 to 275, 1 to 280, 1 to 285, 1 to 290, 1 to 295, or 1 to 300, or 1 to 301 of SEQ ID NOs: 1-8.
  • sFGFR3 polypeptides can include 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) sequence identity to any of these sFGFR3 polypeptides of SEQ ID NOs: 1-8.
  • exemplary sFGFR3 polypeptides can include, but are not limited to, at least amino acids 1 to 100, 1 to 125, 1 to 150, 1 to 175, 1 to 200, 1 to 205, 1 to 210, 1 to 215, 1 to 220, 1 to 225, 1 to 230, 1 to 235, 1 to 240, 1 to 245, 1 to 250, 1 to 255, 1 to 260, 1 to 265, 1 to 270, 1 to 275, 1 to 280, 1 to 285, 1 to 290, 1 to 295, 1 to 300, 1 to 305, 1 to 310, 1 to 315, 1 to 320, 1 to 325, 1 to 330, 1 to 335, 1 to 336, 1 to 340, 1 to 345, or 1 to 348 of SEQ ID NOs: 1-8.
  • the sFGFR3 polypeptide comprises 1-336 consecutive amino acids of SEQ ID NO: 8.
  • sFGFR3 polypeptides can include 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) sequence identity to any of these sFGFR3 polypeptides having the amino acid sequence of SEQ ID NOs: 1-8.
  • any of the above sFGFR3 polypeptides or variants thereof can optionally include a signal peptide at the N-terminal position, such as amino acids 1 to 22 of SEQ ID NO: 21 (MGAPACALALCVAVAIVAGASS) or amino acids 1 to 19 of SEQ ID NO: 43 (e.g., MMSFVSLLLVGILFHATQA).
  • a signal peptide at the N-terminal position such as amino acids 1 to 22 of SEQ ID NO: 21 (MGAPACALALCVAVAIVAGASS) or amino acids 1 to 19 of SEQ ID NO: 43 (e.g., MMSFVSLLLVGILFHATQA).
  • variant refers to a polypeptide (e.g., an sFGFR3 polypeptide or variant thereof, with or without a signal peptide) that differs by one or more changes in the amino acid sequence from the polypeptide from which the variant is derived (e.g., the reference polypeptide, such as, e.g., a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-7).
  • the reference polypeptide such as, e.g., a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-7.
  • variant refers to a polynucleotide that differs by one or more changes in the nucleic acid sequence from the polynucleotide from which the variant is derived (e.g., the reference polynucleotide, such as, e.g., a polynucleotide encoding a sFGFR3 polypeptide having the nucleic acid sequence of any one of SEQ ID NOs: 10-18).
  • the reference polynucleotide such as, e.g., a polynucleotide encoding a sFGFR3 polypeptide having the nucleic acid sequence of any one of SEQ ID NOs: 10-18.
  • the changes in the amino acid or nucleic acid sequence of the variant can be, e.g., amino acid or nucleic acid substitutions, insertions, deletions, N-term inal truncations, or C-terminal truncations, or any combination thereof.
  • the amino acid substitutions may be conservative and/or non-conservative substitutions.
  • a variant can be characterized by amino acid sequence identity or nucleic acid sequence identity to the reference polypeptide or parent polynucleotide, respectively.
  • a variant can include 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) sequence identity to the reference polypeptide or polynucleotide.
  • sequence identity refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence, e.g., an FGFR3 polypeptide, that are identical to the amino acid (or nucleic acid) residues of a reference sequence, e.g., a wild- type sFGFR3 polypeptide (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 8) or an sFGFR3 polypeptide (e.g., an sFGFR3 polypeptide or variant thereof, such as a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-7) after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (e.g., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • a wild- type sFGFR3 polypeptide e.g., a polypeptide having the amino acid sequence of SEQ
  • Alignment for purposes of determining percent 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, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • the percent amino acid (or nucleic acid) sequence identity of a given candidate sequence to, with, or against a given reference sequence is calculated as follows: 100 ⁇ (fraction of A/B) where A is the number of amino acid (or nucleic acid) residues scored as identical in the alignment of the candidate sequence and the reference sequence, and where B is the total number of amino acid (or nucleic acid) residues in the reference sequence.
  • a reference sequence aligned for comparison with a candidate sequence can show that the candidate sequence exhibits from, e.g., 50% to 100% identity across the full length of the candidate sequence or a selected portion of contiguous amino acid (or nucleic acid) residues of the candidate sequence.
  • the length of the candidate sequence aligned for comparison purpose is at least 30%, e.g., at least 40%, e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% of the length of the reference sequence.
  • a polypeptide that “preferentially binds” or “specifically binds” (used interchangeably herein) to a ligand is a term well understood in the art, and methods to determine such specific or preferential binding are also well known in the art.
  • a molecule is said to exhibit “specific binding” or “preferential binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances.
  • a polypeptide “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances.
  • a sFGFR3 polypeptide that specifically or preferentially binds to a fibroblast growth factor (FGF) is a polypeptide that binds the FGF with greater affinity, avidity, more readily, and/or with greater duration than it binds to other growth factors or other ligands. It is also understood by reading this definition that, for example, a polypeptide that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.
  • binding affinity is herein used as a measure of the strength of a non- covalent interaction between two molecules, e.g., a sFGFR3 polypeptide and its ligand, FGF.
  • binding affinity is used to describe monovalent interactions (intrinsic activity). Binding affinity between two molecules, e.g., a sFGFR3 polypeptide and its ligand, FGF or a sFGFR3 polypeptide and the receptor FGFR3, through a monovalent interaction may be quantified by determination of the dissociation constant (K D ).
  • K D can be determined by measurement of the kinetics of complex formation and dissociation using, e.g., the surface plasmon resonance (SPR) method (Biacore).
  • the rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constants ka (or kon) and dissociation rate constant kd (or koff), respectively.
  • the value of the dissociation constant can be determined directly by well-known methods, and can be computed even for complex mixtures by methods such as those, for example, set forth in Caceci et al.
  • the KD may be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432).
  • Other standard assays to evaluate the binding ability of ligands such as antibodies towards target antigens are known in the art, including for example, ELISAs, Western blots, RIAs, and flow cytometry analysis, and other assays exemplified elsewhere herein.
  • the binding kinetics and binding affinity of the polypeptide also can be assessed by standard assays known in the art, such as Surface Plasmon Resonance (SPR), e.g.
  • SPR Surface Plasmon Resonance
  • skeletal growth retardation disorder refers to a skeletal disease characterized by deformities and/or malformations of the bones. These disorders include, but are not limiting to, skeletal growth retardation disorders caused by growth plate (physeal) fractures, idiopathic skeletal growth retardation disorders, or FGFR3-related skeletal diseases.
  • a patient having a skeletal growth retardation disorder e.g., achondroplasia
  • the skeletal growth retardation disorder may include a skeletal dysplasia, e.g., achondroplasia, homozygous achondroplasia, heterozygous achondroplasia, achondrogenesis, acrodysostosis, acromesomelic dysplasia, atelosteogenesis, camptomelic dysplasia, chondrodysplasia punctata, rhizomelic type of chondrodysplasia punctata, cleidocranial dysostosis, congenital short femur, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, or Crouzonodermoskeletal syndrome), dactyly, brachydactyly, camptodactyly, polydactyly, syndactyly, diastrophic dysplasia, dwarfis
  • FGFR3-related skeletal disease refers to a skeletal disease that is caused by an abnormal increase in the activation of FGFR3, such as by expression of a gain-of-function mutant of the FGFR3.
  • gain-of-function mutant of the FGFR3 refers to a mutant of the FGFR3 exhibiting a biological activity, such as triggering downstream signaling, which is higher than the biological activity of the corresponding wild- type FGFR3 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 8) in the presence of a FGF ligand.
  • FGFR3-related skeletal diseases can include an inherited or a sporadic disease.
  • Exemplary FGFR3-related skeletal diseases 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, a craniosynostosis syndrome (e.g., Muenke syndrome, Crouzon syndrome, and Crouzonodermoskeletal syndrome), and camptodactyly, tall stature, and hearing loss syndrome (CATSHL).
  • TDI thanatophoric dysplasia type I
  • TDII thanatophoric dysplasia type II
  • SADDAN severe achondroplasia with developmental delay and Acanthosis nigricans
  • hypochondroplasia e.g., Muenke syndrome, Crouzon syndrome, and Crouzonodermoskeletal syndrome
  • CASHL tall stature, and hearing loss syndrome
  • the sFGFR3 produced according to the method disclosed herein is selected from sFGFR3 polypeptides disclosed in WO 2014/111744, WO 2014/111467, WO 2016/110786, WO 2018/007597, and WO 2019/057820, each of which is herein incorporated by reference in its entirety.
  • the sFGFR3 polypeptide is selected from the group consisting of SEQ ID NOs: 1-7.
  • the sFGFR3 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 5.
  • the sFGFR3 polypeptide comprises or consists of sFGFR3_Del4-D3 (SEQ ID NO: 5; also known as PF-07256472).
  • the sFGFR3 polypeptide may include at least 50 consecutive amino acids of an extracellular domain (ECD) of a naturally occurring fibroblast growth factor receptor 3 (FGFR3) polypeptide (e.g., the FGFR3 polypeptide having the sequence set forth in GenBank Accession No. NP_000133; see also SEQ ID NO: 8).
  • the sFGFR3 polypeptide may include 100-370 consecutive amino acids (e.g., fewer than 350 consecutive amino acids) of an ECD of a naturally occurring fibroblast growth factor receptor 3 (FGFR3) polypeptide.
  • the sFGFR3 polypeptide includes 100, 200, 225, 250, 275, 300, 310, 320, 325, 330, 335, 336, 340, 345, or 350 consecutive amino acids of an ECD of a naturally occurring fibroblast growth factor receptor 3 (FGFR3) (e.g., the FGFR3 polypeptide having the sequence set forth in GenBank Accession No. NP_000133; see also SEQ ID NO: 8).
  • the sFGFR3 polypeptide includes 336 consecutive amino acids of an ECD of a naturally occurring fibroblast growth factor receptor 3 (FGFR3) (e.g., the FGFR3 polypeptide having the sequence set forth in GenBank Accession No. NP_000133; see also SEQ ID NO: 8).
  • the sFGFR3 polypeptide may also have an Ig-like C2-type domain 1, 2, and/or 3 of a naturally occurring FGFR3 polypeptide.
  • the sFGFR3 polypeptide have an Ig-like C2-type domain 1, 2, and 3 of a naturally occurring FGFR3 polypeptide.
  • Exemplary sFGFR3 polypeptides can include, but are not limited to, at least amino acids 1 to 100, 1 to 125, 1 to 150, 1 to 175, 1 to 200, 1 to 205, 1 to 210, 1 to 215, 1 to 220, 1 to 225, 1 to 230, 1 to 235, 1 to 240, 1 to 245, 1 to 250, 1 to 252, 1 to 255, 1 to 260, 1 to 265, 1 to 270, 1 to 275, 1 to 280, 1 to 285, 1 to 290, 1 to 295, or 1 to 300, or 1 to 301 of SEQ ID NOs: 1-8.
  • sFGFR3 polypeptides can include 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) sequence identity to any of these sFGFR3 polypeptides of SEQ ID NOs: 1-8.
  • exemplary sFGFR3 polypeptides can include, but are not limited to, at least amino acids 1 to 100, 1 to 125, 1 to 150, 1 to 175, 1 to 200, 1 to 205, 1 to 210, 1 to 215, 1 to 220, 1 to 225, 1 to 230, 1 to 235, 1 to 240, 1 to 245, 1 to 250, 1 to 255, 1 to 260, 1 to 265, 1 to 270, 1 to 275, 1 to 280, 1 to 285, 1 to 290, 1 to 295, 1 to 300, 1 to 305, 1 to 310, 1 to 315, 1 to 320, 1 to 325, 1 to 330, 1 to 335, 1 to 336, 1 to 340, 1 to 345, or 1 to 348 of SEQ ID NOs: 1-8.
  • the sFGFR3 polypeptide comprises 1-336 consecutive amino acids of SEQ ID NO: 8.
  • sFGFR3 polypeptides can include 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) sequence identity to any of these sFGFR3 polypeptides having the amino acid sequence of SEQ ID NOs: 1-8.
  • any of the above sFGFR3 polypeptides or variants thereof can optionally include a signal peptide at the N-terminal position, such as amino acids 1 to 22 of SEQ ID NO: 21 (MGAPACALALCVAVAIVAGASS) or amino acids 1 to 19 of SEQ ID NO: 43 (e.g., MMSFVSLLLVGILFHATQA).
  • the sFGFR3 is a mature polypeptide lacking the signal peptide, which is cleaved during expression and secretion from the cell.
  • the sFGFR3 polypeptide may also lack a transmembrane domain (TM), such as the TM of a naturally occurring FGFR3 polypeptide.
  • TM transmembrane domain
  • the sFGFR3 polypeptides may also contain all or a portion of an intracellular domain (ICD) of an FGFR3 polypeptide.
  • the sFGFR3 polypeptide may have 400 consecutive amino acids or fewer (e.g., between 5 and 399 consecutive amino acids, such as 175, 150, 125, 100, 75, 50, 40, 30, 20, 15, 13, 10, 5 or fewer consecutive amino acids) of an ICD of a naturally-occurring FGFR3 polypeptide.
  • the sFGFR3 polypeptide comprises 13 consecutive amino acids of an ICD of a naturally-occurring FGFR3 polypeptide (e.g., SEQ ID NO: 8).
  • the ICD of the sFGFR3 polypeptide may also lack a tyrosine kinase domain of a naturally-occurring FGFR3 polypeptide.
  • the sFGFR3 polypeptide may lack any amino acids of an ICD of a naturally- occurring FGFR3 polypeptide (e.g., the FGFR3 polypeptide of SEQ ID NO: 8).
  • the sFGFR3 polypeptide may also have an amino acid sequence with at least 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to, or the sequence of, amino acids 401 to 413 of SEQ ID NO: 8.
  • the sFGFR3 polypeptide comprises amino acids 401 to 413 of SEQ ID NO: 8.
  • An sFGFR3 polypeptide produced by the method described herein may be fewer than 475, 450, 425, 400, 375, 350, 300, 250, 200, 150, or 100 amino acids in length and/or may have an amino acid sequence with at least 85% sequence identity (e.g., 86%-100% sequence identity, such as 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to amino acids residues 1 to 280 of SEQ ID NO: 8.
  • sequence identity e.g., 86%-100% sequence identity, such as 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity
  • the sFGFR polypeptide may also be one with an amino acid sequence having at least 85% sequence identity (e.g., 86%-100% sequence identity, such as 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of any one of SEQ ID NOs: 1 -7 (e.g., SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7).
  • the sFGFR3 has the amino acid sequence of SEQ ID NO: 5 or 6 (e.g., the amino acid sequence of SEQ ID NO: 5).
  • the sFGFR3 polypeptide may also have the sequence of SEQ ID NO: 6, except that the residue at position 253 is an alanine, glycine, proline, or threonine.
  • sFGFR3 polypeptide variants that may be produced according to the method disclosed herein also include fragments of the amino acid sequence of any one of SEQ ID NOs: 1 -8 (e.g., at least amino acids 1 to 200, 1 to 205, 1 to 210, 1 to 215, 1 to 220, 1 to 225, 1 to 235, 1 to 230, 1 to 240, 1 to 245, 1 to 250, 1 to 253, 1 to 255, 1 to 260, 1 to 265, 1 to 275, 1 to 280, 1 to 285, 1 to 290, 1 to 300, of SEQ ID NO: 8) or polypeptides having at least 50% sequence identity (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,
  • exemplary sFGFR3 polypeptides can include, but are not limited to, at least amino acids 1 to 100, 1 to 125, 1 to 150, 1 to 175, 1 to 200, 1 to 205, 1 to 210, 1 to 215, 1 to 220, 1 to 225, 1 to 230, 1 to 235, 1 to 240, 1 to 245, 1 to 250, 1 to 255, 1 to 260, 1 to 265, 1 to 270, 1 to 275, 1 to 280, 1 to 285, 1 to 290, 1 to 295, 1 to 300, 1 to 305, 1 to 310, 1 to 315, 1 to 320, 1 to 325, 1 to 330, 1 to 335, 1 to 336, 1 to 340, 1 to 345, or 1 to 348 of SEQ ID NOs: 1-8.
  • the sFGFR3 polypeptide comprises 1-336 consecutive amino acids of SEQ ID NO: 8.
  • sFGFR3 polypeptides can include 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) sequence identity to any of these sFGFR3 polypeptides having the amino acid sequence of SEQ ID NOs: 1-8.
  • the sFGFR3 polypeptides produced according to the method disclosed herein can also be characterized as binding to a fibroblast growth factor (FGF).
  • FGF fibroblast growth factor
  • the FGF is selected from the group consisting of fibroblast growth factor 1 (FGF1 ; SEQ ID NO: 26), fibroblast growth factor 2 (FGF2; SEQ ID NO: 27), fibroblast growth factor 9 (FGF9; SEQ ID NO: 28), fibroblast growth fact 10 (FGF10; SEQ ID NO: 40), fibroblast growth factor 18 (FGF18; SEQ ID NO: 29), fibroblast growth factor 19 (FGF19; SEQ ID NO: 30), fibroblast growth factor 21 (FGF21 ; SEQ ID NO: 31), and fibroblast growth factor 23 (FGF23; SEQ ID NO: 41 ).
  • the binding is characterized by an equilibrium dissociation constant (K d ) of about 0.2 nM to about 20 nM (e.g., a K d of about 1 nM to about 10 nM, wherein optionally the K d is about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, or about 10 nm).
  • K d equilibrium dissociation constant
  • the methods described herein are not limited to the production of a particular sFGFR3 polypeptide or variant thereof.
  • any sFGFR3 polypeptide that binds one or more FGFs with a similar binding affinity as the sFGFR3 polypeptides having the amino acids sequence of SEQ ID NOs: 1 -7 are also envisioned as being produced by the methods disclosed herein.
  • the sFGFR3 polypeptides can be, for example, fragments of FGFR3 isoform 2 lacking exons 8 and 9 encoding the C-terminal half of the lgG3 domain and exon 10 including the transmembrane domain (e.g., fragments of the amino acid sequence of SEQ ID NO: 8), corresponding to fragments of FGFR3 transcript variant 2 (Accession No.
  • an sFGFR3 polypeptide can include a signal peptide at the N- terminal position.
  • An exemplary signal peptide can include, but is not limited to, amino acids 1 to 22 of SEQ ID NO: 21 (e.g., MGAPACALALCVAVAIVAGASS). Accordingly, the sFGFR3 polypeptides include both secreted forms, which lack the N-terminal signal peptide, and non- secreted forms, which include the N-terminal signal peptide.
  • a secreted sFGFR3 polypeptide can include the amino acid sequence of any one of SEQ ID NOs: 1 -7, but without an N-terminal signal peptide (e.g., the sequence of SEQ ID NO: 21).
  • the sFGFR3 polypeptide e.g., a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1 -7) does include a signal peptide, such as the amino acid sequence of SEQ ID NO: 21.
  • the position of the N- terminal signal peptide will vary in different sFGFR3 polypeptides and can 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.
  • 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 cbs.dtu.dk/services/SignalP/.
  • sFGFR3 polypeptides can be glycosylated.
  • a sFGFR3 polypeptide can be altered to increase or decrease the extent to which the sFGFR3 polypeptide is glycosylated.
  • Addition or deletion of glycosylation sites to an sFGFR3 polypeptide can be accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
  • N-linked glycosylation in which an oligosaccharide is attached to the amide nitrogen of an asparagine residue, can occur at position Asn76, Asn148, Asn169, Asn 203, Asn240, Asn272, and/or Asn 294 of the amino acid sequence of SEQ ID NO: 5 or 6 and variants thereof.
  • One or more of these Asn residues can also be substituted to remove the glycosylation site.
  • O-linked glycosylation in which an oligosaccharide is attached to an oxygen atom of an amino acid residue, can occur at position Ser109, Thr126, Ser199, Ser274, Thr281 , Ser298, Ser299, and/or Thr301 of the amino acid sequence of SEQ ID NO: 5 or 6 and variants thereof.
  • O-linked glycosylation can occur at a serine residue within the sFGFR3.
  • Ser or Thr residues can also be substituted to remove the glycosylation site.
  • sFGFR3 Fusion Polypeptides sFGFR3 polypeptides produced by the method described herein (e.g., sFGFR3 polypeptides having the amino acid sequence of any one of SEQ ID NOs: 1 -7 (e.g., SEQ ID NO: 1, 2, 3, 4, 5, 6, 7) or a variant thereof having at least 85% sequence identity thereto (e.g., 86%-100% sequence identity, such as 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto) can be fused to a functional domain from a heterologous polypeptide (e.g., a fragment crystallizable region (Fc region; such as a polypeptide having the amino acid sequence of SEQ ID NOs: 35 and 36) or human serum albumin (HSA; such as a polypeptide having the amino acid sequence of SEQ ID NO: 37)) to provide a sFGFR3
  • a flexible linker can be included between the sFGFR3 polypeptide and the heterologous polypeptide (e.g., an Fc region or HSA), such as a serine or glycine-rich sequence (e.g., a poly-glycine or a poly-glycine/serine linker, such as SEQ ID NOs: 38 and 39).
  • the sFGFR3 polypeptides and variants thereof can be a fusion polypeptide including, e.g., an Fc region of an immunoglobulin at the N-terminal or C-terminal domain.
  • useful Fc regions can include the Fc fragment of any immunoglobulin molecule, including IgG, IgM, IgA, IgD, or IgE and their various subclasses (e.g., lgG-1 , lgG- 2, lgG-3, lgG-4, lgA-1 , lgA-2) from any mammal (e.g., a human).
  • immunoglobulin molecule including IgG, IgM, IgA, IgD, or IgE and their various subclasses (e.g., lgG-1 , lgG- 2, lgG-3, lgG-4, lgA-1 , lgA-2) from any mammal (e.g., a human).
  • the Fc fragment human lgG-1 (SEQ ID NO: 35) or a variant of human lgG-1 , such as a variant including a substitution of asparagine at position 297 of SEQ ID NO: 35 with alanine (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 36).
  • the Fc fragments can include, for example, the CH2 and CH3 domains of the heavy chain and any portion of the hinge region.
  • the sFGFR3 fusion polypeptides can also include, e.g., a monomeric Fc, such as a CH2 or CH3 domain.
  • 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.
  • the sFGFR3 polypeptides can be conjugated to other molecules at the N-terminal or C-terminal domain for the purpose of improving the solubility and stability of the protein in aqueous solution. Examples of such molecules include human serum albumin (HSA), PEG, PSA, and bovine serum albumin (BSA).
  • the sFGFR3 polypeptides can be conjugated to human HSA (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 37) or a fragment thereof.
  • the sFGFR3 fusion polypeptides can include a peptide linker region between the sFGFR3 polypeptide and the heterologous polypeptide (e.g., an Fc region or HSA).
  • the linker region may be of any sequence and length that allows the sFGFR3 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 -1 10, 111 -120, 121 -130, 131 -140, 141 -150, 151 -160, 161 -170, 171 -180, 181 -190, or 191- 200 amino acid residues.
  • 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.
  • 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.
  • 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.
  • the amino acid sequence of a linker consists only of glycine and serine residues.
  • the linker can be the amino acid sequence of GGGGAGGGG (SEQ ID NO: 38) or GGGGSGGGGSGGGGS (SEQ ID NO: 39).
  • a linker can optionally be glycosylated at any appropriate one or more amino acid residues.
  • the linker can also be absent, in which the sFGFR3 polypeptide and the heterologous polypeptide (e.g., an Fc region or HSA) are fused together directly, with no intervening residues.
  • Polynucleotides encoding the sFGFR3 Polypeptides Polynucleotides encoding the sFGFR3 polypeptides can be used to treat a patient having abnormal visceral fat deposition in a patient (e.g., a human, such as a fetus, a neonate, an infant, a child, an adolescent, or an adult).
  • the polynucleotide can have the nucleic acid sequence of any one of SEQ ID NOs: 10-18 or a variant thereof having at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the nucleic acid sequence of any one of SEQ ID NOs: 10-18.
  • sequence identity e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity
  • polynucleotide can have the nucleic acid sequence of SEQ ID NO: 14 or 15 or a variant having at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the nucleic acid sequence of SEQ ID NO: 14 or 15.
  • sequence identity e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity
  • polynucleotides encoding sFGFR3 fusion polypeptides e.g., a sFGFR3 polypeptide fused to a heterologous polypeptide, such as a Fc region or HSA
  • polynucleotides encoding sFGFR3 polypeptides without a signal peptide e.g., polypeptides having the amino acid sequence of any one of SEQ ID NOs: 1 -7) or with a signal peptide (e.g., polypeptides having the amino acid sequence of any one of SEQ ID NOs: 1 -7.
  • polynucleotides can have one or more mutations to alter any of the glycosylation sites described herein or known to be present in the polypeptide.
  • the polynucleotides of the invention can be codon optimized to alter the codons in the nucleic acid, in particular to reflect the typical codon usage of the host organism (e.g., a human) without altering the sFGFR3 polypeptide encoded by the nucleic acid sequence of the polynucleotide.
  • Codon- optimized polynucleotides can, e.g., facilitate genetic manipulations by decreasing the GC content and/or for expression in a host cell (e.g., a HEK 293 cell or a CHO cell). Codon-optimization can be performed by the skilled person, e.g.
  • the medium may comprise Ala, Arg, Asn, Asp, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val and Cystine and/or Cys.
  • Such a solution may also contain supplementary components that enhance growth and/or survival above the minimal rate, including, but not limited to, hormones and/or other growth factors, particular ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds usually present at very low final concentrations), inorganic compounds present at high final concentrations (e.g., iron), amino acids, lipids, and/or glucose or other energy source.
  • a medium is advantageously formulated to a pH and salt concentration optimal for cell survival and proliferation.
  • the medium may be formulated to a pH between around 6.9 and 7.3 and a final osmolality between around 1000 and 1300mOsm.
  • basal and/or feed cell culture media which can be used in the methods disclosed herein include those disclosed in WO2006/026445, WO2008/109410, WO2008/063892, EP2243827, WO2002/066603, WO2015/140708 and WO2006/050050.
  • the feed medium used in the method of the invention comprises 4 to 10mM Ala, 30 to 60mM Arg, 50 to 90mM Asn, 10 to 30mM Asp, 2 to 40mM Glu, 2 to 15mM Gly, 8 to 20mM His, 25 to 32mM Ile, 35 to 60mM Leu, 28 to 60mM Lys, 9 to 25mM Met, 10 to 30mM Phe, 15 to 40mM Pro, 44 to 80mM Ser, 20 to 45mM Thr, 2 to 10mM Trp and 20 to 50mM Val.
  • the medium is a serum free medium.
  • the medium is a protein free medium.
  • the medium is a chemically defined medium, wherein the components of the medium are both known and controlled.
  • the medium is a complex medium, in which not all components of the medium are known and/or controlled.
  • Chemically defined growth media for mammalian cell culture have been extensively developed and published over the last several decades. All components of defined media are well characterized, and so defined media do not contain complex additives such as serum or hydrolysates. Early media formulations were developed to permit cell growth and maintenance of viability with little or no concern for protein production. More recently, media formulations have been developed with the express purpose of supporting highly productive recombinant protein producing cell cultures. Such media are preferred for use in the methods disclosed herein.
  • Such media generally comprises high amounts of nutrients and in particular of amino acids to support the growth and/or the maintenance of cells at high density. If necessary, these media can be modified by the skilled person for use in the methods disclosed herein. Not all components of complex media are well characterized, and so complex media may contain additives such as simple and/or complex carbon sources, simple and/or complex nitrogen sources, and serum, among other things.
  • complex media suitable for the present invention contains additives such as hydrolysates in addition to other components of defined medium as described herein.
  • defined media typically includes roughly fifty chemical entities at known concentrations in water.
  • Some of them also contain one or more well-characterized proteins such as insulin, IGF-1, transferrin or BSA, but others require no protein components and so are referred to as protein-free defined media.
  • Typical chemical components of the media fall into five broad categories: amino acids, vitamins, inorganic salts, trace elements, and a miscellaneous category that defies neat categorization.
  • Cell culture medium may be optionally supplemented with supplementary components.
  • supplementary components refers to components that enhance growth and/or survival above the minimal rate, including, but not limited to, hormones and/or other growth factors, particular ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds usually present at very low final concentrations), amino acids, lipids, and/or glucose or other energy source.
  • supplementary components may be added to the initial cell culture.
  • supplementary components may be added after the beginning of the cell culture.
  • trace elements refer to a variety of inorganic salts included at micromolar or lower levels. For example, commonly included trace elements are zinc, selenium, copper, and others.
  • iron can be included as a trace element in the initial cell culture medium at micromolar concentrations.
  • Manganese is also frequently included among the trace elements as a divalent cation (MnCl 2 or MnSO 4 ) in a range of nanomolar to micromolar concentrations. Numerous less common trace elements are usually added at nanomolar concentrations.
  • the medium used in the method of the invention is a medium suitable for supporting high viable cell density, such as for example 1 x 10 6 cells/mL, 5 x 10 6 cells/mL, 1 x 10 7 cells /mL, 5 x 10 7 cells/mL, 1 x 10 8 cells/mL or 5 x 10 8 cells/mL, in a cell culture.
  • the cell culture is a CHO cell fed-batch culture.
  • the cells are grown to a viable cell density greater than 1 x 10 6 cells/mL, 5 x 10 6 cells/mL, 1 x 10 7 cells /mL, 5 x 10 7 cells/mL, 1 x 10 8 cells/mL or 5 x 10 8 cells/mL.
  • viable cell density refers to the number of cells present in a given volume of medium. Viable cell density can be measured by any method known to the skilled person. Preferably, viable cell density is measured using an automated cell counter such as Bioprofile Flex®.
  • the term maximum cell density as used herein refers to the maximum cell density achieved during the cell culture.
  • cell viability refers to the ability of cells in culture to survive under a given set of culture conditions or experimental variations. Those of ordinary skill in the art will appreciate that one of many methods for determining cell viability are encompassed in this invention. For example, one may use a dye (e.g., trypan blue) that does not pass through the membrane of a living cell, but can pass through the disrupted membrane of a dead or dying cell in order to determine cell viability.
  • a dye e.g., trypan blue
  • culture and “cell culture” as used herein refer to a cell population that is suspended in a medium under conditions suitable to survival and/or growth of the cell population.
  • fed-batch culture or “fed-batch cell culture” as used herein refers to a method of culturing cells in which additional components are provided to the culture at a time or times subsequent to the beginning of the culture process. Such provided components typically comprise nutritional components for the cells which have been depleted during the culturing process.
  • a fed-batch culture is typically stopped at some point and the cells and/or components in the medium are harvested and optionally purified.
  • the fed-batch culture comprises a basal medium supplemented with a feed medium.
  • Cells may be grown in any convenient volume chosen by the practitioner. For example, cells may be grown in small scale reaction vessels ranging in volume from a few milliliters to several liters. Alternatively, cells may be grown in large scale bioreactors ranging in volume from at least 500, 1000, 2500, 5000, 8000, 10,000, 12,000, 15000, 20000 or 25000 liters or more, or any volume in between. In some embodiments, the volume of the cell culture is at least 100L, 300L or 500L. In some embodiments, the volume of the cell culture is at least 3000L. In some embodiments, the cells may be grown during the initial growth phase (or growth phase) for a greater or lesser amount of time, depending on the needs of the practitioner and the requirement of the cells themselves.
  • the cells are grown for a period of time sufficient to achieve a predefined cell density. In some embodiments, the cells are grown for a period of time sufficient to achieve a predefined cell density of about 1 x 10 6 cells/mL, about 5 x 10 6 cells/mL, about 1 x 10 7 cells /mL, about 5 x 10 7 cells/mL, about 1 x 10 8 cells/mL or about 5 x 10 8 cells/mL. In some embodiments, the cells are grown for a period of time sufficient to achieve a cell density that is a given percentage of the maximal cell density that the cells would eventually reach if allowed to grow undisturbed.
  • the cells may be grown for a period of time sufficient to achieve a desired viable cell density of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent of maximal cell density.
  • the cells are grown until the cell density does not increase by more than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% per day of culture.
  • the cells are grown until the cell density does not increase by more than 5% per day of culture.
  • the cells are allowed to grow for a defined period of time.
  • the cells may be grown for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days, preferably for 4 to 10 days.
  • the practitioner of the methods disclosed herein will be able to choose the duration of the initial growth phase depending on protein production requirements and the needs of the cells themselves.
  • the cell culture may be agitated or shaken during the initial culture phase in order to increase oxygenation and dispersion of nutrients to the cells.
  • it can be beneficial to control or regulate certain internal conditions of the bioreactor during the initial growth phase, including but not limited to pH, temperature, oxygenation, etc.
  • a temperature shift to a lower temperature is used in the methods disclosed herein.
  • the temperature shift may be relatively gradual. For example, it may take several hours or days to complete the temperature change.
  • the temperature shift may be relatively abrupt.
  • the temperature change may be complete in less than several hours.
  • the appropriate production and control equipment such as is standard in the commercial large-scale production of polypeptides or proteins, the temperature change may even be complete within less than an hour.
  • the cell culture is maintained for a subsequent production phase under conditions conducive to the survival and viability of the cell culture and appropriate for expression of the desired polypeptide or protein at commercially adequate levels.
  • the cells may be maintained in the subsequent production phase until a desired cell density or production titer is reached.
  • the duration of the production phase is comprised between 2 and 10 days, i.e 2, 3 ,4, 5, 6, 7, 8, 9 or 10 days, preferably between 4 to 8 days, preferably 6, 7 or 8 days.
  • the duration of the growth phase is about 4 days and the duration of the production phase is about 8 days.
  • the cell culture may be agitated or shaken during the subsequent production phase in order to increase oxygenation and dispersion of nutrients to the cells.
  • Non-limiting examples of mammalian cells include BALB/c mouse myeloma line (NSO/l, ECACC No: 85110503); human retinoblasts (PER.C6, CruCell, Leiden, The Netherlands); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59,1977); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells +/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci.
  • BALB/c mouse myeloma line NSO/l, ECACC No: 85110503
  • human retinoblasts PER.C6, CruCell, Leiden, The Netherlands
  • monkey kidney CV1 line transformed by SV40 COS-7, ATCC CRL 1651
  • mice sertoli cells TM4, Mather, Biol. Reprod., 23:243-251, 1980
  • monkey kidney cells CV1 ATCC CCL 70
  • African green monkey kidney cells VOD-76, ATCC CRL-1587
  • human cervical carcinoma cells HeLa, ATCC CCL 2
  • canine kidney cells MDCK, ATCC CCL 34
  • buffalo rat liver cells BRL 3A, ATCC CRL 1442
  • human lung cells W138, ATCC CCL 75
  • human liver cells Hep G2, HB 8065
  • mouse mammary tumor MMT 060562, ATCC CCL51
  • TRI cells Mather et al., Annals N.Y. Acad.
  • the cells are CHO cells. In some preferred embodiments, the cells are GS-CHO cells. Expression of Proteins As noted above, in many instances the cells will be selected or engineered to produce high levels of desired products. Often, cells will be manipulated by the hand of man to produce high levels of recombinant protein, for example by introduction of a gene encoding the protein of interest and/or by introduction of genetic control elements that regulate expression of that gene (whether endogenous or introduced).
  • a cell line is empirically selected by the practitioner for robust growth under the particular conditions chosen for culturing the cells.
  • individual cells engineered to express a particular protein are chosen for large-scale production based on cell growth, final cell density, percent cell viability, titer of the expressed protein or any combination of these or any other conditions deemed important by the practitioner.
  • the term “host cell” as used herein refers to a cell that is manipulated to produce a protein of interest as described herein.
  • a protein may be expressed from a gene that is endogenous to the cell, or from a heterologous gene that is introduced into the cell.
  • a protein may be one that occurs in nature, or may alternatively have a sequence that was engineered or selected by the hand of man. Isolation of the Expressed Protein In general, it will typically be desirable to isolate and/or purify proteins expressed according to the present invention. In certain embodiments, the expressed protein is secreted into the medium and thus cells and other solids may be removed, as by centrifugation or filtering for example, as a first step in the purification process.
  • the expressed protein may be isolated and purified by standard methods including, but not limited to, chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), gel filtration, centrifugation, or differential solubility, ethanol precipitation and/or by any other available technique for the purification of proteins (See, e.g., Scopes, Protein Purification Principles and Practice 2nd Edition, Springer-Verlag, New York, 1987; Higgins, S.J. and Hames, B.D. (eds.), Protein Expression : A Practical Approach, Oxford Univ Press, 1999; and Deutscher, M.P., Simon, M.I., Abelson, J.N.
  • the protein may be isolated by binding it to an affinity column comprising antibodies that were raised against that protein and were affixed to a stationary support.
  • affinity tags such as an influenza coat sequence, poly-histidine, or glutathione-S-transferase can be attached to the protein by standard recombinant techniques to allow for easy purification by passage over the appropriate affinity column.
  • Protease inhibitors such as phenyl methyl sulfonyl fluoride (PMSF), leupeptin, pepstatin or aprotinin may be added at any or all stages in order to reduce or eliminate degradation of the protein during the purification process. Protease inhibitors are particularly advantageous when cells must be lysed in order to isolate and purify the expressed protein.
  • PMSF phenyl methyl sulfonyl fluoride
  • leupeptin phenyl methyl sulfonyl fluoride
  • pepstatin or aprotinin
  • aprotinin may be added at any or all stages in order to reduce or eliminate degradation of the protein during the purification process.
  • Protease inhibitors are particularly advantageous when cells must be lysed in order to isolate and purify the expressed protein.
  • the exact purification technique will vary depending on the character of the protein to be purified, the character of the cells from which the protein is expressed, and/or the composition of the medium in which the cells were
  • a nucleic acid to be introduced is in the form of a naked nucleic acid molecule.
  • the nucleic acid molecule introduced into a cell may consist only of the nucleic acid encoding the protein and the necessary genetic control elements.
  • a nucleic acid encoding the protein may be contained within a plasmid vector.
  • Non-limiting representative examples of suitable vectors for expression of proteins in mammalian cells include pCDNA1; pCD, see Okayama, et al. Mol. Cell Biol.5:1136-1142, 1985; pMClneo Poly-A, see Thomas, et al. Cell 51:503-512, 1987; a baculovirus vector such as pAC 373 or pAC 610; CDM8 , see Seed, B. Nature 329:840, 1987; and pMT2PC, see Kaufman, et al. EMBO J. 6:187-195, 1987, each of which is incorporated herein by reference in its entirety.
  • a nucleic acid molecule to be introduced into a cell is contained within a viral vector.
  • a nucleic acid encoding the protein may be inserted into the viral genome (or a partial viral genome).
  • Regulatory elements directing the expression of the protein may be included with the nucleic acid inserted into the viral genome (i.e., linked to the gene inserted into the viral genome) or can be provided by the viral genome itself.
  • naked DNA can be introduced into cells by forming a precipitate containing the DNA and calcium phosphate.
  • naked DNA can also be introduced into cells by forming a mixture of the DNA and DEAE-dextran and incubating the mixture with the cells or by incubating the cells and the DNA together in an appropriate buffer and subjecting the cells to a high-voltage electric pulse (e.g., by electroporation).
  • a further method for introducing naked DNA cells is by mixing the DNA with a liposome suspension containing cationic lipids. The DNA/liposome complex is then incubated with cells. Naked DNA can also be directly injected into cells by, for example, microinjection.
  • naked DNA can also be introduced into cells by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G. and Wu, C.H. J. Biol. Chem.263:14621, 1988; Wilson et al. J. Biol. Chem. 267:963-967, 1992; and U.S. Patent No.5,166,320, each of which is hereby incorporated by reference in its entirety). Binding of the DNA-ligand complex to the receptor facilitates uptake of the DNA by receptor-mediated endocytosis.
  • a cation such as polylysine
  • viral vectors containing particular nucleic acid sequences e.g., a cDNA encoding a protein
  • Infection of cells with a viral vector has the advantage that a large proportion of cells receive the nucleic acid, which can obviate the need for selection of cells which have received the nucleic acid.
  • molecules encoded within the viral vector e.g., by a cDNA contained in the viral vector, are generally expressed efficiently in cells that have taken up viral vector nucleic acid.
  • Defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. Blood 76:271, 1990).
  • a recombinant retrovirus can be constructed having a nucleic acid encoding a protein of interest inserted into the retroviral genome. Additionally, portions of the retroviral genome can be removed to render the retrovirus replication defective. Such a replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques.
  • the genome of an adenovirus can be manipulated such that it encodes and expresses a protein of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See, for example, Berkner et al. BioTechniques 6:616, 1988; Rosenfeld et al. Science 252:431-434, 1991; and Rosenfeld et al.
  • adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus are known to those skilled in the art.
  • Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al., 1992, cited supra), endothelial cells (Lemarchand et al., Proc. Natl. Acad. Sci.
  • adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA).
  • Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle.
  • the modified population of cells may be used without further isolation or subcloning of individual cells within the population. That is, there may be sufficient production of the protein by the population of cells such that no further cell isolation is needed and the population can immediately be used to seed a cell culture for the production of the protein. Alternatively, it may be desirable to isolate and expand a homogenous population of cells from a few cells or a single cell that efficiently produce(s) the protein.
  • a gene encoding a protein of interest may optionally be linked to one or more regulatory genetic control elements. In certain embodiments, a genetic control element directs constitutive expression of the protein.
  • a genetic control element that provides inducible expression of a gene encoding the protein of interest can be used.
  • an inducible genetic control element e.g., an inducible promoter
  • potentially useful inducible genetic control elements for use in eukaryotic cells include hormone- regulated elements (e.g., see Mader, S. and White, J.H., Proc. Natl. Acad. Sci. USA 90:5603-5607, 1993), synthetic ligand-regulated elements (see, e.g. Spencer, D.M.
  • compositions The sFGFR3 produced according to the method described herein can be included in pharmaceutical compositions.
  • sFGFR3 produced according to the methods described herein may be included in formulations described in U.S. Provisional Application No. 63/115,170, which is incorporated by reference herein in its entirety.
  • Such produced polypeptide may be administered to a subject or may first be formulated for delivery by any available route including, but not limited to parenteral (e.g., intravenous), intradermal, subcutaneous, oral, nasal, bronchial, ophthalmic, transdermal (topical), transmucosal, rectal, and vaginal routes.
  • compositions typically include a purified polypeptide expressed from a mammalian cell line, a delivery agent (i.e., a cationic polymer, peptide molecular transporter, surfactant, etc., as described above) in combination with a pharmaceutically acceptable carrier.
  • a delivery agent i.e., a cationic polymer, peptide molecular transporter, surfactant, etc., as described above
  • pharmaceutically acceptable carrier includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants
  • compositions suitable for injectable use typically include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition should be sterile and should be fluid to the extent that easy syringability exists.
  • the relevant carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • 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.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the purified polypeptide or protein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the purified polypeptide or protein expressed from a mammalian cell line into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • the purified polypeptide can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • compositions comprising a purified polypeptide expressed from a mammalian cell line and a delivery agent are preferably delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • the present disclosure particularly contemplates delivery of the compositions using a nasal spray, inhaler, or other direct delivery to the upper and/or lower airway.
  • Intranasal administration of DNA vaccines directed against influenza viruses has been shown to induce CD8 T cell responses, indicating that at least some cells in the respiratory tract can take up DNA when delivered by this route, and the delivery agents of the invention will enhance cellular uptake.
  • compositions comprising a purified polypeptide expressed from a mammalian cell line and a delivery agent are formulated as large porous particles for aerosol administration.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the purified polypeptide and delivery agents are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compositions can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the compositions are prepared with carriers that will protect the polypeptide or protein against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • compositions for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially for example from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No.4,522,811. It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active polypeptide or protein calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the polypeptide expressed according to the present method can be administered at various intervals and over different periods of time as required, e.g., one time per week for between about 1 to 10 weeks, between 2 to 8 weeks, between about 3 to 7 weeks, about 4, 5, or 6 weeks, etc.
  • certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
  • treatment of a subject with a polypeptide or protein as described herein can include a single treatment or, in many cases, can include a series of treatments. It is furthermore understood that appropriate doses may depend upon the potency of the polypeptide or protein and may optionally be tailored to the particular recipient, for example, through administration of increasing doses until a preselected desired response is achieved. It is understood that the specific dose level for any particular animal subject may depend upon a variety of factors including the activity of the specific polypeptide or protein employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • All publications, patents, and patent applications cited herein are hereby incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication, patent, and patent application were specifically and individually indicated to be so incorporated by reference.
  • this application controls.
  • Examples sFGFR3_Del4-D3 was developed as a recombinant soluble form of FGFR3 expressed in CHO cells comprising a glutamine synthetase expression system (hereafter GS-CHO).
  • sFGFR3_Del4-D3 is useful for example for treatment of achondroplasia.
  • sFGFR3_Del4-D3 underwent increased fragmentation and aggregation and that the produced polypeptides comprised substantial amount of undesirable fragments and aggregates. Characterization of the sFGFR3_Del4-D3 fragments suggested cathepsin-L mediated proteolytic cleavage.
  • studies were performed to develop a sFGFR3_Del4-D3 cell culture production process that would provide suitable expression level while limiting fragmentation and aggregation of the sFGFR3 polypeptide.
  • Example 1 Small scale experiment to determine conditions to improve the titer while decreasing fragment levels of sFGFR3_Del4-D3 Experimental Goals This experiment was carried out to identify culture conditions yielding improved productivity while lowering fragmentation of the sFGFR3_Del4-D3 protein.
  • the experiment was a high throughput DOE utilizing the AMBR (Sartorius) automated bioreactor system. Varying levels of pH setpoint, temperature shift temperature, feed rate, and dissolved oxygen (DO) were evaluated.
  • Materials and Methods Cells and Medium CHO cells comprising a glutamine synthetase expression system (hereafter GS-CHO, Cell line A) and expressing the recombinant polypeptide sFGFR3_Del4- D3 were used in this experiment.
  • GS-CHO glutamine synthetase expression system
  • AMBR15 Micro Bioreactor Setup The AMBR system is a small-scale bioreactor setup. The vessels have a marine type impellar and are sparged oxygen or CO2 based on the needs of the culture. The dissolved oxygen and pH are monitored and controlled throughout the run.
  • the experiment was designed to look at optimal conditions with respect to titer and product quality of sFGFR3_Del4-D3.
  • the primary variable highlighted in this example was the temperature shift temperature (see Table 1). Multiple reactors were run for each temperature shift condition. Other variables (feed rate, pH, DO) included in this experiment had minimal effect on the titer or fragmentation levels.
  • the pH deadband was ⁇ 0.15 units from the setpoint
  • the RPM was 1300
  • the feeds were all started on day 3.
  • all conditions were started at a temperature of 36.5°C. All reactor conditions were shifted to their respective temperatures on day 5 of the culture. Glucose was also added periodically to maintain levels above 2 g/L in the culture.
  • Samples were denatured with Rapigest®, deglycosylated with peptide-N-glycosidase F, then heated in the presence of a reducing agent. After reduction, the fragmented species that are smaller than the main species are electrophoretically separated in a capillary containing sieving medium containing a fluorescent dye and detected using fluorescence. The separation allows quantitation of the resolved species. The results are expressed as the percentage of intact species (percent main peak) which represent the percentage of non-fragmented sFGFR3 polypeptide relative to the total amount of sFGFR3 produced (intact + fragment).
  • Example 2 Effects of a temperature shift in fed batch culture on titer, fragmentation, and aggregation of sFGFR3_Del4-D3 in a 1L bioreactor.
  • Experimental Goals A further experiment was conducted in 1L bioreactor to confirm the effect observed in example 1 at a larger scale. The experiment compared a fed batch process with and without shift to a lower temperature. Overall culture performance was evaluated based on cell growth, viability, cell culture metabolites (lactate and ammonia), and titer. Also, the two conditions were compared with respect to product quality to see if a temperature shift improved certain quality attributes, namely fragmentation and aggregation of the polypeptide sFGFR3_Del4-D3.
  • the advantageous temperature shift condition (31.5°C) identified in example 1 was also used in this experiment.
  • Materials and Methods Cells and Medium CHO cells comprising a glutamine synthetase expression system and expressing the recombinant polypeptide sFGFR3_Del4-D3 (cell line B) were used in this experiment.
  • Cell line B was thawed, cultured, and expanded in medium in shake flasks as well as a 1L stirred tank bioreactor. Seventeen days from thaw, the Cell line B was used to inoculate 2x1L stirred tank bioreactors.
  • Bioreactor Setup 2x1L bioreactors (1L working volume) were inoculated and a 12-day fed batch process was run.
  • Bioreactor Temperature Conditions for Cell Line B Conditioned media samples were taken on days 7, 10-12 of the reactors to determine protein titer. In addition, harvest samples (day 12) were prepared for further product quality analysis using a bench scale affinity purification method. The samples were then buffer exchanged into 20mM Histidine, pH 6.2, and concentrated to 5-10 mg/mL. Protein titers and percentage of intact polypeptide were determined as disclosed in example 1. Size Exclusion HPLC (SE-HPLC) was used to determine the presence of high molecular mass species (aggregated polypeptide). The test samples were diluted and injected onto a size-exclusion column. The content of high molecular mass species (HMMS) and monomer were reported as the percent of the total area for all protein-related peaks.
  • SE-HPLC Size Exclusion HPLC
  • Example 3 expanding the temperature shift range and its effects on titer, fragmentation, and aggregation of sFGFR3_Del4-D3 Experimental Goals This experiment was carried out to further evaluate the effect of temperature shift temperatures on fragmentation and aggregation of the sFGFR3 polypeptide .
  • the experiment outlined in Example 1 evaluated 36.5°C, 34°C, and 31.5°C.
  • This experiment expanded on the temperature shift ranges to include 30.5°C and 29.5°C, compared with 31.5°C.
  • Overall culture performance was evaluated based on cell growth, viability, cell culture metabolites (lactate and ammonia), and titer.
  • Cell line B was thawed, cultured, and expanded in medium in shake flasks as well as a 1L stirred tank bioreactor. Seventeen days from thaw, the Cell line B was used to inoculate 3x1L stirred tank bioreactors. Bioreactor Setup: 3x1L bioreactors (1L working volume) were inoculated and a 12-day fed batch process was run.
  • Example 4 Effects of a temperature shift to 31.5°C on titer, fragmentation, and aggregation of sFGFR3_Del4-D3 in a 500L bioreactor.
  • An optimized process based on a temperature shift to 31.5°C was run in a 500L reactor (350 L -480 L culture volume) to evaluate the reduced levels of fragmentation and aggregates at large scale.
  • the scaled-up bioreactor was run following the outlined parameters in Example 2, condition R13. The only difference was that the temperature was shifted to 31.5°C on day 4 rather than day 5.
  • Conditioned media samples were taken as well as harvest samples purified for further product quality analysis as disclosed in examples 2 and 3.

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Abstract

L'invention concerne des procédés de culture cellulaire pour produire un polypeptide sFGFR3 dans des cellules de mammifères.
PCT/IB2022/055062 2021-06-01 2022-05-30 Procédé de culture cellulaire pour la production du polypeptide sfgfr3 WO2022254319A1 (fr)

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