NZ790135A - Soluble fibroblast growth factor receptor 3 (SFGFR3) polypeptides and uses thereof - Google Patents

Abstract

The invention features soluble fibroblast growth factor receptor 3 (sFGFR3) polypeptides. The invention also features methods of using sFGFR3 polypeptides to treat skeletal growth retardation disorders, such as achondroplasia.

Description

SOLUBLE FIBROBLAST GROWTH FACTOR RECEPTOR 3 (SFGFR3) POLYPEPTIDES AND USES THEREOF The entire disclosure in the complete specification of our Australian Patent Application No. 749910 is by this cross-reference incorporated into the present specification.

FIELD OF THE INVENTION The invention features soluble fibroblast growth factor receptor 3 (sFGFR3) polypeptides and compositions thereof. The invention also features methods to treat al growth retardation disorders, such as achondroplasia.

BACKGROUND OF THE INVENTION last growth factor receptor 3 (FGFR3) is a member of the fibroblast growth factor (FGFR) family, in which there is high amino acid sequence conservation between family members. Members of the FGFR family are entiated by both ligand binding ties and tissue distribution. A full-length FGFR polypeptide contains an extracellular domain (ECD), a hydrophobic transmembrane domain, and a asmic tyrosine kinase domain. The ECD of FGFR polypeptides interacts with fibroblast growth factors (FGFs) to mediate downstream signaling, which ultimately influences cellular differentiation. In particular, activation of the FGFR3 protein plays a role in bone development by inhibiting chondrocyte proliferation at the growth plate and limiting bone elongation.

Gain-of-function point mutations in FGFR3 are known to cause several types of human skeletal growth retardation disorders, such as roplasia, thanatophoric sia type I (TDI), ophoric dysplasia type II (TDII), severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN), hypochondroplasia, and synostosis syndromes (e.g., Muenke me, Crouzon syndrome, and Crouzonodermoskeletal syndrome). Loss-of-function point mutations in FGFR3 are also known to cause al growth retardation disorders, such as camptodactyly, tall stature, and hearing loss syndrome (CATSHL). Achondroplasia is the most common form of short-limb dwarfism and is characterized by disproportionate shortness of limbs and relative macrocephaly. Approximately 97% of achondroplasia is caused by a single point mutation in the gene encoding FGFR3, in which a glycine residue is substituted with an ne residue at on 380 of the FGFR3 amino acid sequence. Upon ligand binding, the mutation decreases the elimination of the receptor/ligand complex ing in prolonged intracellular signaling. This prolonged FGFR3 ing inhibits the proliferation and differentiation of the cartilage growth plate, consequently impairing endochondral bone growth.

There exists a need for improved therapeutics that target dysfunctional FGFR3 for treating skeletal growth retardation disorders, such achondroplasia.

SUMMARY OF THE INVENTION The invention features soluble fibroblast growth factor or 3 3) polypeptides and uses thereof, including the use of the sFGFR3 polypeptides for the treatment of skeletal growth ation 40 disorders (e.g., achondroplasia) in a patient (e.g., a human, particularly an infant, a child, or an adolescent).

In particular, the sFGFR3 polypeptides of the invention feature a deletion of, e.g., amino acids 289 to 400 of the amino acid sequence of the wildtype FGFR3 polypeptide (e.g., a polypeptide having the amino acid 18870863_1 (GHMatters) P110456.NZ.1 sequence of SEQ ID NO: 5 or 32), to provide the following sFGFR3 polypeptides: sFGFR3_Del4 including an amino acid substitution of a cysteine residue with a serine residue at position 253 (sFGFR3_Del4- C253S; SEQ ID NO: 2) and sFGFR3_Del4 including an extended Ig-like C2-type domain 3 (sFGFR3_Del4- D3; SEQ ID NO: 33) and variants thereof, such as a sFGFR3 polypeptide having the amino acid ce of SEQ ID NO: 4. Additionally, the sFGFR3 ptides of the invention may include a signal peptide, such as a sFGFR3 polypeptide having the amino acid sequence of SEQ ID NO: 18 or 34.

A first aspect of the invention es a soluble fibroblast growth factor or 3 (sFGFR3) polypeptide including a polypeptide sequence having at least 90% amino acid ce identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%) ce identity) to amino acid residues 23 to 357 of SEQ ID NO: 32. In particular, the polypeptide lacks a signal peptide (e.g., amino acids 1-22 of SEQ ID NO: 32) and a transmembrane domain of FGFR3 (e.g., amino acids of 367-399 of SEQ ID NO: 32) and (i) is less than 500 amino acids in length (e.g., less than 475, 450, 425, 400, 375, or 350 amino acids in length); (ii) includes 200 consecutive amino acids or fewer (e.g., 175, 150, 125, 100, 75, 50, 40, 30, 20, 15, or fewer consecutive amino acids) of an intracellular domain of FGFR3; and/or (iii) lacks a tyrosine kinase domain of FGFR3. The sFGFR3 polypeptide can also include an intracellular domain of FGFR3, such as amino acid residues 423 to 435 of SEQ ID NO: 32 or an amino acid sequence having at least 90%, 92%, 95%, 97%, or 99% sequence identity to amino acid residues 423 to 435 of SEQ ID NO: 32. In particular, the polypeptide includes an amino acid sequence having at least 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 33 (e.g., the polypeptide includes or consists of SEQ ID NO: 33). The sFGFR3 polypeptides can also e a signal peptide (e.g., the signal peptide can have the amino acid sequence of SEQ ID NO: 6 or 35 or an amino acid sequence having at least 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 6 or 35). For example, the sFGFR3 polypeptide may have an amino acid sequence with at least 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 34 (e.g., the sFGFR3 polypeptide includes or consists of SEQ ID NO: 34). The sFGFR3 polypeptide may also have a heterologous signal e (e.g., the polypeptide includes a heterologous signal peptide having the amino acid sequenceo of SEQ ID NO: 35).

A second aspect of the invention features an sFGFR3 polypeptide including an amino acid sequence having at least 85% ce identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the amino acid sequence of SEQ ID NO: 1, in which the sFGFR3 polypeptide r includes an amino acid substitution that s a cysteine residue at on 253 of SEQ ID NO: 1. For example, the cysteine residue at position 253 is substituted with a serine residue or, e.g., another conservative amino acid substitution, such as alanine, glycine, proline, or threonine. In particular, the sFGFR3 polypeptide includes or consists of the amino acid sequence of SEQ ID NO: 2. For instance, the sFGFR3 polypeptide can be an ed sFGFR3 polypeptide. The sFGFR3 polypeptides can also include a signal peptide (e.g., the signal peptide can have the amino acid ce of SEQ ID NO: 6 or 35 or an amino acid sequence having at least 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 6 or 35). For example, the sFGFR3 may have an amino acid sequence with at least 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 18 (e.g., the sFGFR3 polypeptide includes or consists of SEQ ID NO: 18). The sFGFR3 polypeptide may 40 also have a heterologous signal peptide (e.g., the polypeptide includes a logous signal peptide having the amino acid sequenceo of SEQ ID NO: 35).

A third aspect of the invention features a sFGFR3 polypeptide including an amino acid sequence 18870863_1 (GHMatters) P110456.NZ.1 having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the amino acid ce of SEQ ID NO: 1, in which the sFGFR3 polypeptide further includes a domain including an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to all or a nt of the amino acid sequence of SEQ ID NO: 3 (e.g., at least 10, 20, 30, 40, 45, or more consecutive amino acids of SEQ ID NO: 3), in which the domain is inserted between amino acid residues 288 and 289 of SEQ ID NO: 1. For example, the domain can include an amino acid sequence having at least 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 (e.g., the domain can include or consists of the amino acid sequence of SEQ ID NO: 3). Optionally, the sFGFR3 polypeptide includes an amino acid tution of a cysteine residue with a serine residue or, e.g., another conservative amino acid substitution, such as alanine, e, proline, or threonine, at position 253 of SEQ ID NO: 1 and/or position 28 of SEQ ID NO: 3. In ular, the sFGFR3 polypeptide includes or consists of the amino acid sequence of SEQ ID NO: 4. For example, the sFGFR3 polypeptide can be an isolated sFGFR3 polypeptide.

Also featured is a polynucleotide (e.g., an isolated polynucleotide) that encodes the sFGFR3 polypeptide of the first, , or third aspect of the invention ing a nucleic acid sequence having at least 85%, 90%, 92%, 95%, 97%, or 99% ce identity to the nucleic acid sequence of SEQ ID NO: 20, 21, 36, or 37 (e.g., the polynucleotide includes or consists of the nucleic acid of SEQ ID NO: 20, 21, 36, or 37). The invention also es a vector (e.g., an isolated vector) including the polynucleotide, such as a d, an artificial chromosome, a viral vector, or a phage vector. Additionally, the invention features a host cell (e.g., an isolated host cell) including the polynucleotide, such as a HEK 293 cell or CHO cell.

The invention features a ition including the sFGFR3 polypeptide of the first, second, or third aspects of the invention or the polynucleotide that encodes the sFGFR3 polypeptide of the first, second, or third aspects of the invention. In addition, the vector or host cell that includes the polynucleotide encoding the sFGFR3 polypeptide can be formulated in a composition. The ition can further include a pharmaceutically able excipient, carrier, or diluent. The ition including the sFGFR3 polypeptide, polynucleotide, or vector can be formulated for administration at a dose of about 0.002 mg/kg to about 30 mg/kg, such as about 0.001 mg/kg to about 10 mg/kg. The composition including the host cell can be formulated for administration at a dose of about 1 X 103 cells/mL to about 1 X 1012 cells/mL. The composition can be formulated for daily, weekly, or monthly administration, such as seven times a week, six times a week, five times a week, four times a week, three times a week, twice a week, weekly, every two weeks, or once a month. For e, the composition including the sFGFR3 polypeptide, polynucleotide, or vector is formulated for administration at a dose of about 0.25 mg/kg to about 10 mg/kg once or twice a week. The composition can be formulated for parenteral administration (e.g., subcutaneous administration, intravenous administration, intramuscular stration, intra-arterial administration, intrathecal administration, or intraperitoneal administration), enteral administration, or topical administration. ably, the composition is formulated for subcutaneous administration. The 40 invention also features a ment that includes one or more of the compositions described above.

The invention also features a method of delivering an sFGFR3 polypeptide to tissue (e.g., skeletal tissue) in a patient (e.g. a human) having a skeletal growth retardation disorder (e.g., achondroplasia) 18870863_1 (GHMatters) P110456.NZ.1 including stering to the patient an effective amount of the sFGFR3 polypeptide of the first, second, or third aspect of the ion, a polynucleotide encoding the sFGFR3 polypeptide, a vector containing the polynucleotide, a host cell containing the polynucleotide or vector, or a composition containing the polypeptide, polynucleotide, vector, or host cell.

A fourth aspect of the invention features a method of ng a al growth retardation disorder (e.g., a FGFR3-related skeletal disease) in a patient (e.g., a human) that includes administering the polypeptide of the first, second, or third aspect of the invention or a polynucleotide encoding the polypeptide, a vector containing the cleotide, a host cell containing the polynucleotide or vector, or a composition containing the polypeptide, polynucleotide, vector, or host cell. The FGFR3-related skeletal disease is selected from the group consisting of achondroplasia, thanatophoric dysplasia type I (TDI), thanatophoric dysplasia type II (TDII), severe achondroplasia with developmental delay and osis nigricans (SADDEN), hypochondroplasia, a craniosynostosis syndrome (e.g., Muenke syndrome, n syndrome, and Crouzonodermoskeletal syndrome), and camptodactyly, tall stature, and hearing loss syndrome (CATSHL). In ular, the skeletal growth retardation disorder is achondroplasia.

The FGFR3-related skeletal disease can be caused by expression in the patient of a constitutively active FGFR3, such as an amino acid substitution of a glycine e with an arginine residue at position 380 of SEQ ID NO: 5 or 32. In particul ar, the patient may be diagnosed with the skeletal growth retardation disorder (e.g., prior to treatment). For instance, the patient exhibits one or more symptoms of the skeletal growth retardation er selected from the group consisting of short limbs, short trunk, bowlegs, a waddling gait, skull malformations, cloverleaf skull, craniosynostosis, n bones, anomalies of the hands, anomalies of the feet, hitchhiker thumb, and chest ies, such that the patient exhibits an improvement in the one or more symptoms of the skeletal growth retardation disorder after administration of the sFGFR3 polypeptide (or a polynucleotide encoding the polypeptide, a vector containing the polynucleotide, a host cell containing the polynucleotide or vector, or a composition containing the polypeptide, polynucleotide, vector, or host cell). Additionally, the patient may have not been previously administered the sFGFR3 polypeptide. For e, the patient may be an infant, a child, an adolescent, or an adult.

For example, the polypeptide is administered to the patient at a dose of about 0.002 mg/kg to about 30 mg/kg (e.g., a dose of about 0.001 mg/kg to about 10 mg/kg). T he polypeptide may be administered to the patient one or more times daily, weekly (e.g., twice a week, three times a week, four times a week, five times a week, six times a week, or seven times a week), every two weeks, y, or . For e, the ptide is administered to the patient at a dose of about 0.25 mg/kg to about 30 mg/kg at least about once or twice a week or more (e.g., the ptide is administered to the patient at a dose of about 2.5 mg/kg or about 10 mg/kg once or twice weekly). The polypeptide can be administered to the patient in a composition including a ceutically acceptable excipient, carrier, or diluent. The polypeptide can be administered to the patient parenterally (e.g., subcutaneously, intravenously, intramuscularly, intra-arterially, intrathecally, or intraperitoneally), enterally, or topically. Preferably, the composition is administered to the patient by subcutaneous ion. Additionally, the polypeptide can 40 bind to fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (FGF9), last growth factor 18 (FGF18), fibroblast growth factor 19 (FGF19), or fibroblast growth factor 21 (FGF21). In particular, the binding can be characterized by an equilibrium dissociation constant 18870863_1 (GHMatters) P110456.NZ.1 (Kd) of about 0.2 nM to about 20 nM, such as the binding is characterized by a Kd of about 1 nM to about nM (e.g., 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). The polypeptide can exhibit greater binding affinity to FGF1, FGF2, FGF9, and FGF18 ve to the binding affinity of the polypeptide to FGF19 and FGF21.

The polypeptide can have an in vivo half-life of between about 2 hours to about 25 hours (e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or 25 hours). Preferably, administration of the polypeptide provides one or more, or all, of the ing: an increase in survival of the patient, an improvement in tion of the patient, an improvement in abdominal breathing in the patient, an increase in body and/or bone length of the patient, an improvement in the cranial ratio of the t, and/or restoration of the foramen magnum shape in the patient, e.g., relative to an ted patient (e.g., an untreated achondroplasia patient).

The invention also features a method of ing the sFGFR3 ptide of the first, second, or third aspect of the invention, which includes culturing the host cell bed above (e.g., a CHO cell or HEK 293 cell) in a culture medium under conditions suitable to effect expression of the sFGFR3 polypeptide and recovering the sFGFR3 polypeptide from the culture medium. In particular, the recovering includes chromatography, such as affinity chromatography (e.g., ion exchange tography or anti-FLAG chromatography, such as immunoprecipitation) or size exclusion chromatography.

A fifth aspect of the invention es the polypeptide of the first, second, or third aspect of the invention (or a polynucleotide encoding the polypeptide, a vector containing the polynucleotide, a host cell containing the cleotide or vector, or a composition containing the polypeptide, polynucleotide, vector, or host cell) for treating a al growth ation disorder in a patient. In particular, the sFGFR3 polypeptide can bind to fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), last growth factor 9 (FGF9), fibroblast growth factor 18 (FGF18), fibroblast growth factor 19 (FGF19), or fibroblast growth factor 21 (FGF21).

A sixth aspect of the invention features a sFGFR3 polypeptide (or a polynucleotide ng the polypeptide, a vector containing the polynucleotide, a host cell containing the polynucleotide or vector, or a composition containing the polypeptide, polynucleotide, vector, or host cell) including an amino acid sequence having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the amino acid sequence of SEQ ID NO: 1 for treating a skeletal growth retardation disorder in a patient (e.g., a human), in which the sFGFR3 polypeptide further includes an amino acid tution that removes a cysteine residue at position 253 of SEQ ID NO: 1. For e, the cysteine residue at position 253 is substituted with a serine e or, e.g., another conservative amino acid substitution, such as alanine, glycine, proline, or threonine. In ular, the sFGFR3 polypeptide includes or consists of the amino acid sequence of SEQ ID NO: 2. For example, the sFGFR3 polypeptide can be an isolated sFGFR3 polypeptide. Furthermore, the sFGFR3 polypeptide can bind to fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (FGF9), fibroblast growth factor 18 (FGF18), fibroblast growth factor 19 40 (FGF19), or fibroblast growth factor 21 (FGF21).

A seventh aspect of the invention features a sFGFR3 polypeptide (or a polynucleotide encoding the polypeptide, a vector ning the polynucleotide, a host cell containing the polynucleotide or vector, or 18870863_1 (GHMatters) P110456.NZ.1 a composition containing the polypeptide, polynucleotide, vector, or host cell) including an amino acid sequence having at least 85% ce identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the amino acid sequence of SEQ ID NO: 1 for treating a skeletal growth retardation disorder in a patient (e.g., a human), in which the sFGFR3 polypeptide further includes a domain including an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to all or a fragment of the amino acid sequence of SEQ ID NO: 3 (e.g., at least 10, 20, 30, 40, 45, or more utive amino acids of SEQ ID NO: 3), in which the domain is inserted between amino acid residues 288 and 289 of SEQ ID NO: 1. For example, the domain can include an amino acid sequence having at least 85%, 90%, 92%, 95%, 97%, or 99% ce identity to the amino acid sequence of SEQ ID NO: 3 (e.g., the domain can include or consists of the amino acid ce of SEQ ID NO: 3). Optionally, the sFGFR3 polypeptide includes an amino acid substitution of a cysteine residue with a serine residue or, e.g., another conservative amino acid substitution, such as alanine, glycine, proline, or threonine, at position 253 of SEQ ID NO: 1 and/or position 28 of SEQ ID NO: 3. In particular, the sFGFR3 polypeptide includes or consists of the amino acid sequence of SEQ ID NO: 4. For example, the sFGFR3 polypeptide can be an isolated sFGFR3 polypeptide. Furthermore, the sFGFR3 polypeptide can bind to fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (FGF9), fibroblast growth factor 18 (FGF18), fibroblast growth factor 19 ), or last growth factor 21 (FGF21).

The use of the fifth, sixth, or seventh aspect also features the administration of a polynucleotide, vector, host cell, or composition of the first, second, or third aspect of the invention. The sFGFR3 polypeptide of the sixth aspect of the invention can be encoded by a polynucleotide including a nucleic acid sequence having at least 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 20 or 36 (e.g., the polynucleotide includes or consists of the c acid of SEQ ID NO: 20 or 36). The sFGFR3 polypeptide of the fifth or seventh aspect of the invention can be encoded by a polynucleotide including a nucleic acid sequence having at least 85%, 90%, 92%, 95%, 97%, or 99% ce identity to the c acid sequence of SEQ ID NO: 21 or 37 (e.g., the polynucleotide es or consists of the nucleic acid of SEQ ID NO: 21 or 37).

The skeletal growth retardation disorder of the fifth, sixth, or seventh aspect of the invention can be any FGFR3-related skeletal disease, such as achondroplasia, TDI, TDII, severe achondroplasia with developmental delay and acanthosis nigricans (SADDEN), hypochondroplasia, a craniosynostosis syndrome (e.g., Muenke me, Crouzon syndrome, and Crouzonodermoskeletal syndrome), or CATSHL. In ular, the skeletal growth retardation disorder is achondroplasia. The FGFR3-related skeletal disease can be caused by expression in the patient of a constitutively active FGFR3, e.g., in which the constitutively active FGFR3 includes an amino acid substitution of a glycine residue with an arginine residue at position 380 of SEQ ID NO: 5.

The patient (e.g., a human) of the fifth, sixth, or seventh aspect of the invention can be one that has been diagnosed with the skeletal growth ation disorder (e.g., prior to treatment). The patient can exhibit one or more symptoms of the skeletal growth retardation disorder (e.g., achondroplasia) selected 40 from the group consisting of short limbs, short trunk, bowlegs, a ng gait, skull malformations, leaf skull, craniosynostosis, wormian bones, anomalies of the hands, anomalies of the feet, hitchhiker thumb, and chest anomalies. As a result of the s, the patient can exhibit an 18870863_1 ters) P110456.NZ.1 improvement in the one or more symptoms of the skeletal growth retardation disorder after administration of the sFGFR3 polypeptide. Moreover, administration of the sFGFR3 polypeptide can increase survival of the t and/or restore the shape of the foramen magnum of the patient. The t can be an infant, a child, an adolescent, or an adult. Additionally, the patient can be one that has not been previously administered the sFGFR3 polypeptide (or a polynucleotide encoding the polypeptide, a vector containing the polynucleotide, a host cell ning the polynucleotide or , or a composition containing the polypeptide, cleotide, vector, or host cell).

The sFGFR3 polypeptide, cleotide, or vector of the fifth, sixth, or seventh aspect of the ion can be administered to the patient (e.g., a human) at a dose of about 0.002 mg/kg to about 30 mg/kg, such as about 0.001 mg/kg to about 10 mg/kg. The composition including the host cell of the fourth or fifth aspect of the invention can be administered to the patient (e.g., a human) at a dose of about 1 X 103 cells/mL to about 1 X 1012 cells/mL. For example, the sFGFR3 polypeptide, polynucleotide, vector, or host cell is administered to the patient one or more times daily, weekly, monthly, or yearly (e.g., the sFGFR3 polypeptide is administered to the patient seven times a week, six times a week, five times a week, four times a week, three times a week, twice a week, , every two weeks, or once a month).

In particular, the sFGFR3 polypeptide is administered to the patient at a dose of about 0.25 mg/kg to about 10 mg/kg once or twice a week. The sFGFR3 polypeptide can be administered to the patient in a composition including a pharmaceutically acceptable ent, carrier, or diluent. For e, the composition is administered to the patient parenterally (e.g., subcutaneously, intravenously, intramuscularly, arterially, intrathecally, or intraperitoneally), enterally, or topically. In particular, the composition is administered to the patient by subcutaneous injection.

The invention features a method of cturing the sFGFR3 polypeptide of the first aspect of the invention by deleting the signal peptide, the transmembrane domain, and a portion of the intracellular domain from a FGFR3 polypeptide (e.g., to manufacture a polypeptide having the amino acid sequence of SEQ ID NO: 33). In particular, the intracellular domain consists of amino acid residues 436 to 806 of SEQ ID NO: 32. The invention also features a method of manufacturing the sFGFR3 polypeptide of the second aspect of the invention by introducing an amino acid substitution that s a cysteine e at position 253 of SEQ ID NO: 1 (e.g., to manufacture a polypeptide having the amino acid sequence of SEQ ID NO: 2). For example, the cysteine residue at position 253 is substituted with a serine residue or, e.g., another conservative amino acid substitution, such as alanine, glycine, e, or threonine.

The invention also features a kit including the sFGFR3 polypeptide of the first, second, or third aspect of the invention (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, or 33), the cleotide of the first, second, or third aspect of the invention (e.g., a cleotide having the nucleic acid sequence of SEQ ID NO: 20, 21, 36, or 37), the vector of the first, second, or third aspect of the invention (e.g., a plasmid, an artificial chromosome, a viral vector, or a phage vector), or the host cell of the first, second, or third aspect of the invention (e.g., a HEK 293 cell or a CHO cell), in which the kit optionally includes instructions for using the kit.

Definitions 40 As used herein, “a” and “an” means “at least one” or “one or more” unless otherwise indicated. In addition, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. 18870863_1 (GHMatters) P110456.NZ.1 As used herein, “about” refers to an amount that is ± 10% of the recited value and is preferably ±5% of the recited value, or more preferably ±2% of the recited value. For instance, the term “about” can be used to modify all dosages or ranges recited herein by ± 10% of the recited values or range endpoints.

The term “domain” refers to a conserved region of the amino acid ce of a polypeptide (e.g. a FGFR3 polypeptide) having an identifiable structure and/or function within the polypeptide. 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 FGFR3 (e.g., Ig-like e domain 1, Ig-like C2-type domain 2, and Ig-like C2-type domain 3).

The term “dosage” refers to a determined quantity of an active agent (e.g., an sFGFR3 polypeptide or variant thereof, such as a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, or 33) ated to produce a desired therapeutic effect (e.g., treatment of a skeletal growth retardation disorder, such as roplasia) when the active agent is administered to a t (e.g., a patient having a skeletal growth retardation disorder, such as achondroplasia). A dosage may be defined in terms of a defined amount of the active agent or a defined amount coupled with a particular frequency of administration. A dosage form can include an sFGFR3 polypeptide or fragment thereof in association with any suitable pharmaceutical excipient, r, or diluent.

The terms “effective amount,” “amount effective to,” and “therapeutically effective amount” refer to an amount of an sFGFR3 polypeptide, a vector encoding a sFGR3, and/or an sFGFR3 composition that is sufficient to e a desired result, for example, the treatment of a skeletal growth retardation er (e.g., achondroplasia).

The terms “extracellular domain” and “ECD” refer to the portion of a FGFR3 ptide 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). For instance, an ECD includes the e C2- type domains 1-3 of a FGFR3 polypeptide. In particular, the ECD includes the Ig-like C2-type domain 1 of a wildtype (wt) FGFR3 ptide (e.g., amino acids 36-88 of a wt FGFR3 polypeptide having the amino acid sequence of SEQ ID NO: 5 (a mature FGFR3 protein without a signal ce) or amino acids 57-110 of a wt FGFR3 polypeptide having the amino acid sequence of SEQ ID NO: 32 (a precursor FGFR3 protein with the signal sequence)), the Ig-like C2-type domain 2 of a wildtype (wt) FGFR3 polypeptide (e.g., amino acids 139-234 of a wt FGFR3 polypeptide having the amino acid sequence of SEQ ID NO: 5 or amino acids 161-245 of a wt FGFR3 polypeptide having the amino acid sequence of SEQ ID NO: 32), and the Ig-like C2-type domain 3 of a wt FGFR3 polypeptide (e.g., amino acids 5 of a wt FGFR3 polypeptide having the amino acid sequence of SEQ ID NO: 5 or amino acids 0 of a wt FGFR3 polypeptide having the amino acid sequence of SEQ ID NO: 32). An ECD of a FGFR3 can also include a nt of the wildtype FGFR3 Ig-like C2-type domain 3, for instance, aa 247-288 of SEQ ID NO: 1, which can further include, e.g., an amino acid substitution of a cysteine residue with a serine residue or another conservative amino acid substitution (e.g., alanine, e, proline, or threonine) at position 253 of SEQ ID NO: 1 (e.g., aa 247-288 of SEQ ID NO: 2). onally, an ECD can include an Ig-like C2-type domain 3 of, e.g., aa 247-335 of SEQ ID NO: 4. Thus, exemplary ECDs of FGFR3 polypeptides include, e.g., those polypeptides having the amino acid sequence of aa 1-288 of SEQ ID 40 NOs: 1 and 2 or aa 1-335 of SEQ ID NOs: 4 and 33. In particular, the ECD of a FGFR3 polypeptide includes aa 1-335 of SEQ ID NO: 33. 18870863_1 (GHMatters) P110456.NZ.1 The term “FGFR3-related skeletal e,” as used herein, refers to a skeletal disease that is caused by an al increase in the activation of FGFR3, such as by expression of a gain-of-function mutant of the FGFR3. The phrase “gain-of-function mutant of the FGFR3” refers to a mutant of the FGFR3 exhibiting a biological ty, 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 ce of SEQ ID NO: 5) in the ce of a FGF ligand. FGFR3-related skeletal diseases can e an inherited or a sporadic disease. Exemplary FGFR3-related skeletal diseases e, 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).

The terms blast growth factor” and “FGF” refer to a member of the FGF family, which es structurally related signaling molecules involved in various metabolic ses, 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, FGF18, FGF19, and FGF21, which have been shown to bind FGFR3. For instance, FGFs can include human FGF1 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 13), human FGF2 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 14), human FGF9 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 15), human FGF18 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 16), human FGF19 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 38), and human FGF21 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 39).

The terms “fibroblast growth factor receptor 3,” “FGFR3,” or “FGFR3 receptor,” as used herein, refer to a polypeptide that specifically binds one or more FGFs (e.g., FGF1, FGF2, FGF9, FGF18, FGF19, and/or FGF21). The human FGFR3 gene, which is located on the distal short arm of chromosome 4, s an 806 amino acid protein precursor (fibroblast growth factor receptor 3 m 1 precursor), which contains 19 exons, and includes a signal peptide (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 6 or 35). Mutations in the FGFR3 amino acid sequence that lead to skeletal growth disorders, (e.g., achondroplasia), include, e.g., the substitution of a glycine residue at position 380 with an arginine residue (i.e., G380R). The naturally occurring human FGFR3 gene has a nucleotide sequence as shown in Genbank Accession number 142.4 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: 5. The wildtype FGFR3 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 5) consists of an extracellular immunoglobulin-like membrane domain including Ig-like C2-type domains 1-3 (amino acid es 1 to 335), a transmembrane domain (amino acid es 345 to 377), and an intracellular domain (amino acid residues 378 to 784). 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, ype FGFR3 (e.g., a polypeptide having the amino acid sequence of 40 SEQ ID NO: 5).

The terms “fragment” and “portion” refer to a part of a whole, such as a polypeptide or nucleic acid molecule that contains, ably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 18870863_1 (GHMatters) P110456.NZ.1 90%, 95%, 96%, 97%, 98%, 99%, or more of the entire length of the reference nucleic acid molecule or polypeptide. A nt 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, 340, 350, 360, 370, 380, 390, 400, 500, 600, 700, or more amino acid residues, up to the entire length of the reference polypeptide (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 5 or 32). For example, a FGFR3 fragment can include any polypeptide having at least 200, 205, 210, 215, 220, 225, 235, 230, 240, 245, 250, 255, 260, 265, 275, 280, 285, 290, or 300 consecutive amino acids of SEQ ID NO: 1 or 2. Additionally, a FGFR3 fragment can include any polypeptide having at least 200, 205, 210, 215, 220, 225, 235, 230, 240, 245, 250, 255, 260, 265, 275, 280, 285, 290, 300, 305, 310, 315, 320, 325, 330, 335, 345, or 345 utive amino acids of SEQ ID NO: 4 or 33.

As used herein, the term “host cell” refers to a e that includes the necessary ar components, e.g., lles, needed to express an sFGFR3 polypeptide from a corresponding cleotide. The nucleic acid sequence of the polynucleotide is lly included in a nucleic acid vector (e.g., a plasmid, an artificial chromosome, a viral vector, or a phage ) that can be introduced into the host cell by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection). A host cell may be a prokaryotic cell, e.g., a bacterial or an archaeal cell, or a eukaryotic cell, e.g., a mammalian cell (e.g., a e Hamster Ovary (CHO) cell or a Human nic Kidney 293 (HEK 293)). Preferably, the host cell is a mammalian cell, such as a CHO cell.

By ted” is meant separated, recovered, or purified from its natural environment. For example, an ed sFGFR3 polypeptide (e.g., an sFGFR3 polypeptide or variant thereof, such as a polypeptide having the amino acid sequence of SEQ ID NO: 2 or 4) can be characterized by a certain degree of purity after isolating the sFGFR3 polypeptide from, e.g., cell culture media. An isolated sFGFR3 polype ptide can be at least 75% pure, such that the sFGFR3 polynucleotide constitutes at least 75% by weight of the total material (e.g., polypeptides, polynucleotides, cellular debris, and environmental contaminants) present in the preparation (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99%, or at least 99.5% by weight of the total material present in the preparation). se, an isolated polynucleotide encoding an sFGFR3 polypeptide (e.g., a polynucleotide having the nucleic acid sequence of SEQ ID NO: 20, 21, 36, or 37), or an isolated host cell (e.g., CHO cell, a HEK 293 cell, L cell, C127 cell, 3T3 cell, BHK cell, or COS-7 cell) can be at least 75% pure, such that the polynucleotide or host cell constitutes at least 75% by weight of the total material (e.g., polypeptides, polynucleotides, cellular debris, and environmental contaminants) present in the preparation (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99%, or at least 99.5% by weight of the total material present in the preparation).

“Polynucleotide” and “nucleic acid molecule,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or any substrate that can be incorporated into a r by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide can 40 include modified nucleotides, such as methylated nucleotides and analogs thereof. If present, cation to the nucleotide structure can be imparted before or after assembly of the polymer. The 18870863_1 ters) P110456.NZ.1 sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after synthesis, such as by conjugation with a label.

The terms “patient” and “subject” refer to a mammal, ing, but not limited to, a human (e.g., a human having a skeletal growth retardation disorder, such as roplasia) or a non-human mammal (e.g., a non-human mammal having a skeletal growth retardation disorder, such as roplasia), such as a bovine, equine, canine, ovine, or feline. Preferably, the t is a human having a skeletal growth retardation disorder (e.g., achondroplasia), particularly an infant, a child, or an adolescent having a skeletal growth retardation disorder (e.g., achondroplasia).

The terms “parenteral administration,” “administered parenterally,” and other grammatically equivalent phrases, as used herein, refer to a mode of administration of compositions including an sFGFR3 ptide (e.g., an sFGFR3 polypeptide or variant f, such as a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, or 33, or a sFGFR3 polypeptide including a signal peptide, such as a polypeptide having the amino acid sequence of SEQ ID NO: 18 or 34) other than enteral and topical administration, usually by injection, and include, t tion, subcutaneous, intradermal, intravenous, asal, intraocular, pulmonary, intramuscular, intra-arterial, hecal, intracapsular, intraorbital, ardiac, intradermal, ulmonary, intraperitoneal, racheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid, and intrasternal injection and infusion.

By “pharmaceutically acceptable t, excipient, carrier, or adjuvant” is meant a diluent, excipient, carrier, or nt, respectively that is physiologically acceptable to the subject (e.g., a human) while retaining the therapeutic properties of the pharmaceutical composition (e.g., an sFGFR3 polypeptide or variant thereof, such as a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, or 33, or a sFGFR3 polypeptide including a signal peptide, such as a polypeptide having the amino acid sequence of SEQ ID NO: 18 or 34) with which it is administered. One exemplary ceutically acceptable carrier is physiological saline. Other physiologically acceptable diluents, excipients, rs, or adjuvants and their formulations are known to one skilled in the art.

By “pharmaceutical composition” is meant a composition containing an active agent, such as an sFGFR3 (e.g., an sFGFR3 polypeptide or variant thereof, such as a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, or 33, or a sFGFR3 polypeptide including a signal peptide, such as a polypeptide having the amino acid sequence of SEQ ID NO: 18 or 34), formulated with at least one pharmaceutically acceptable excipient, carrier, or diluent. The pharmaceutical composition may be manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of a e or event (e.g., a skeletal growth retardation disorder, such achondroplasia) in a patient (e.g., a patient having a skeletal growth retardation disorder, such as a patient having roplasia). Pharmaceutical compositions can be formulated, e.g., for parenteral administration, such as for subcutaneous administration (e.g. by subcutaneous ion) or intravenous administration (e.g., as a sterile solution free of ulate emboli and in a solvent system suitable for intravenous use), or for oral administration (e.g., as a tablet, capsule, caplet, gelcap, or syrup).

As used herein, the term “sequence identity” refers to the percentage of amino acid (or nucleic 40 acid) es of a candidate sequence, e.g., an FGFR3 polypeptide, that are identical to the amino acid (or nucleic acid) residues of a nce sequence, e.g., a wild-type sFGFR3 polypeptide (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 5 or 32) or an sFGFR3 polypeptide (e.g., an 18870863_1 (GHMatters) P110456.NZ.1 sFGFR3 polypeptide or variant thereof, such as a polypeptide having the amino acid ce of SEQ ID NO: 2, 4, or 33, or a sFGFR3 polypeptide including a signal peptide, such as a polypeptide having the amino acid sequence of SEQ ID NO: 18 or 34), after aligning the sequences and introducing gaps, if ary, to achieve the maximum percent identity (e.g., gaps can be introduced in one or both of the ate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alignment for purposes of determining percent identity can be ed in various ways that are within the skill in the art, for ce, 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 ters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For instance, the percent amino acid (or nucleic acid) sequence identity of a given candidate ce to, with, or against a given nce ce (which can alternatively be phrased as a given candidate ce that has or includes a certain percent amino acid (or c acid) sequence identity to, with, or against a given nce sequence) is calculated as follows: 100 x (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. In particular, 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. When a position in the candidate sequence is occupied by the same amino acid (or nucleic acid) residue as the ponding position in the reference sequence, then the molecules are identical at that position.

By “signal e” is meant a short peptide (e.g., 5-30 amino acids in length, such as 22 amino acids in length) at the N-terminus of a polypeptide that directs a polypeptide towards the secretory pathway (e.g., the extracellular space). The signal peptide is typically cleaved during secretion of the polypeptide. The signal sequence may direct the polypeptide to an intracellular compartment or lle, e.g., the Golgi apparatus. A signal sequence may be identified by homology, or biological activity, to a peptide with the known function of targeting a polypeptide to a particular region of the cell.

One of ordinary skill in the art can identify a signal peptide by using readily available software (e.g., Sequence Analysis Software e of the Genetics Computer Group, University of Wisconsin hnology Center, 1710 sity Avenue, Madison, Wis. 53705, BLAST, or PILEUP/PRETTYBOX programs). A signal peptide can be one that is, for example, ntially identical to the amino acid sequence of SEQ ID NO: 6 or 35.

The term “skeletal growth retardation disorder,” as used , refers to a skeletal disease characterized by deformities and/or malformations of the bones. These disorders include, but are not 40 limiting to, skeletal growth retardation disorders caused by growth plate (physeal) fractures, idiopathic skeletal growth retardation ers, or FGFR3-related skeletal diseases. In particular, a patient having a skeletal growth retardation disorder (e.g., achondroplasia) may have bones that are shorter than the 18870863_1 ters) P110456.NZ.1 bones of a healthy patient. For example, the skeletal growth retardation disorder may include a skeletal dysplasia, e.g., achondroplasia, homozygous achondroplasia, heterozygous achondroplasia, achondrogenesis, acrodysostosis, acromesomelic sia, atelosteogenesis, camptomelic sia, chondrodysplasia punctata, rhizomelic type of chondrodysplasia punctata, cleidocranial dysostosis, congenital short femur, craniosynostosis (e.g., Muenke me, Crouzon syndrome, Apert syndrome, Jackson-Weiss me, Pfeiffer syndrome, or Crouzonodermoskeletal syndrome), dactyly, brachydactyly, camptodactyly, ctyly, syndactyly, diastrophic sia, dwarfism, dyssegmental dysplasia, enchondromatosis, fibrochondrogenesis, fibrous dysplasia, hereditary multiple exostoses, hypochondroplasia, hypophosphatasia, hypophosphatemic rickets, Lichtenstein syndrome, Kniest sia, 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, osteoarthritis, osteochondrodysplasia, osteogenesis imperfecta, tal lethal type of osteogenesis ecta, osteopetrosis, osteopoikilosis, peripheral dysostosis, Reinhardt syndrome, s syndrome, Robinow syndrome, short-rib polydactyly syndromes, short stature, spondyloepiphyseal dysplasia congenita, and spondyloepimetaphyseal dysplasia.

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 ptide. In particular, the transmembrane domain of FGFR3 extends from amino acid residues 345 to 377 of the wild-type FGFR3 sequence (e.g, a polypeptide having the amino acid sequence of SEQ ID NO: 5) or amino acid residues 367 to 399 of the wild-type FGFR3 sequence including a signal peptide (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 32). Thus, the sFGFR3 polypeptide can include a deletion of a portion or all of amino acid residues 345 to 377 of the wild-type FGFR3 polypeptide sequence (e.g., a polypeptide having the amino acid ce of SEQ ID NO: 5) or amino acid residues 367 to 399 of the wild-type FGFR3 sequence including a signal peptide (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 32). The sFGFR3 polypeptide can r include deletions of the cytoplasmic domain of the wild-type FGFR3 polypeptide sequence (amino acid residues 378 to 784 of SEQ ID NO: 5) or the wild-type FGFR3 polypeptide sequence including a signal peptide sequence (amino acid es 378 to 806 of SEQ ID NO: 32).

Exemplary sFGFR3 polypeptides can include, but are not d 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 or 2. sFGFR3 ptides 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 NO: 1 or 2. Additionally, exemplary sFGFR3 polypeptides can include, but are not d 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 40 210, 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 340, 1 to 345, or 1 to 348 of SEQ ID NO: 4 or 33. sFGFR3 18870863_1 (GHMatters) P110456.NZ.1 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 NO: 4 or 33.

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: 6 (MGAPACALALCVAVAIVAGASS) or amino acids 1 to 19 of SEQ ID NO: 35 (e.g., MMSFVSLLLVGILFHATQA).

By “treating” and “treatment” is meant a reduction (e.g., by at least about 10%, about 15%, about %, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or even 100%) in the progression or severity of a al growth retardation disorder (e.g., achondroplasia), or in the progression, severity, or ncy of one or more symptoms of a al growth ation disorder (e.g., achondroplasia) in a patient (e.g., a human, such as an infant, a child, or an adolescent). Treatment can occur for a ent period, in which an sFGFR3 polypeptide is administered for a period of time (e.g., days, months, years, or longer) to treat a patient (e.g., a human, such as an infant, a child, or an cent) having a skeletal growth retardation disorder, such as achondroplasia. ary symptoms of achondroplasia that can be treated with an sFGFR3 (e.g., an sFGFR3 polypeptide or variant thereof, such as a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, or 33, or a sFGFR3 polypeptide including a signal peptide, such as a polypeptide having the amino acid sequence of SEQ ID NO: 18 or 34) include, but are not limited to, short stature, a long trunk, shortened limbs, an adult height of n about 42 to about 56 inches, a relatively large head, a forehead that is prominent, underdeveloped portions of the face, genu valgum (e.g., “knock-knee”), a waddling gait, short and stubby fingers, short and stubby toes, limited ability to straighten the arm at the elbow, an excessive curve of the lower back, dental problems (e.g. from overcrowding of teeth), weight l problems, neurological problems, respiratory problems, and/or pain and numbness in the lower back and/or spine.

The term “variant,” with t to a polypeptide, refers to a polypeptide (e.g., an sFGFR3 ptide or variant thereof, such as a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, or 33, or a sFGFR3 polypeptide including a signal peptide, such as a polypeptide having the amino acid ce of SEQ ID NO: 18 or 34) that differs by one or more s in the amino acid sequence from the polypeptide from which the variant is derived (e.g., the parent polypeptide, such a polypeptide having the amino acid sequence of SEQ ID NO: 1 or 7). The term “variant,” with respect to a polynucleotide, refers to a cleotide (e.g., a polynucleotide encoding a sFGFR3 polypeptide, such as a polynucleotide having the nucleic acid ce of SEQ ID NO: 20, 21, 36, or 37) that differs by one or more changes in the nucleic acid sequence from the polynucleotide from which the variant is derived (e.g., the parent polynucleotide). The changes in the amino acid or nucleic acid sequence of the t can be, e.g., amino acid or nucleic acid substitutions, insertions, deletions, N-terminal truncations, or C- terminal truncations, or any ation thereof. In particular, 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 parent ptide (e.g., an sFGFR3 polypeptide or variant thereof, such as a polypeptide having the amino acid sequence of SEQ ID NO: 2, 40 4, or 33, or a sFGFR3 polypeptide including a signal peptide, such as a polypeptide having the amino acid sequence of SEQ ID NO: 18 or 34) or parent polynucleotide (e.g., a polynucleotide encoding a sFGFR3 polypeptide, such as a polynucleotide having the nucleic acid sequence of SEQ ID NO: 20, 21, 18870863_1 (GHMatters) P110456.NZ.1 36, or 37), respectively. For example, 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 a polypeptide having the amino acid sequence of SEQ ID NO: 1, 2, 4, or 33. A variant can also include any pol otide 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 a polynucleotide having the c acid ce of SEQ ID NO: 20, 21, 36, or 37.

By “vector” is meant a DNA uct that includes one or more polynucleotides, or fragments thereof, encoding an sFGFR3 polypeptide (e.g., an sFGFR3 ptide or variant f, such as a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, or 33, or a sFGFR3 polypeptide including a signal peptide, such as a polypeptide having the amino acid sequence of SEQ ID NO: 18 or 34). The vector can be used to infect a cell (e.g., a host cell or a cell of a patient having a human skeletal growth retardation disorder, such as achondroplasia), which results in the translation of the polynucleotides of the vector into a sFGFR3 polypeptide. One type of vector is a id,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Certain vectors are capable of mous replication in a host cell into which they are uced (e.g., bacterial s having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be ated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

The term “unit dosage form(s)” refers to physically discrete unit(s) suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired eutic effect, in association with any suitable pharmaceutical excipient, carrier, or diluent.

The recitation herein of numerical ranges by endpoints is intended to include all numbers ed within that range (e.g., a recitation of 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5).

Other features and advantages of the invention will be apparent from the following Detailed Description and from the .

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS FIGS. 1A-1D are graphs showing sensorgrams of the sFGFR3 polypeptides. Sensorgrams are shown for sFGFR3_Del1 (SEQ ID NO: 7), sFGFR3_Del4 (SEQ ID NO: 1), and sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 10; Fig. 1A); sFGFR3_Del1 (SEQ ID NO: 7) and _Del1-D3 (SEQ ID NO: 9; Fig. 1B); sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 10), sFGFR3_Del4-LK1-LK2-C253S (SEQ ID NO: 11), and sFGFR3_Del4-LK1-LK2-D3 (SEQ ID NO: 12; Fig. 1C); and sFGFR3_Del4 (SEQ ID NO: 1), sFGFR3_Del4-C253S (SEQ ID NO: 2), and sFGFR3_Del4-D3 (SEQ ID NO: 33; Fig. 1D).

FIGS. 2A-2C are images of Western blots of the sFGFR3 polypeptides. Western blots under reducing (R) and ducing (NR) conditions are shown for sFGFR3_Del1, sFGFR3_Del1-C253S (SEQ 40 ID NO: 8), and sFGFR3_Del1-D3 (Fig. 2A); sFGFR3_Del4-LK1-LK2, sFGFR3_Del4-LK1-LK2-C253S, and sFGFR3_Del4-LK1-LK2-D3 (Fig. 2B); and sFGFR3_Del4, sFGFR3_Del4-C253S, and sFGFR3_Del4-D3 (Fig. 2C). 18870863_1 (GHMatters) P110456.NZ.1 FIGS. 3A-3B are graphs showing a sensorgram (Fig. 3A) and proliferation assays of _Del4, sFGFR3_Del4-C253S, and _Del4-D3 (Fig. 3B) using Fgfr3ach/+ chondrocyte cells in the presence of FGF2. is a graph showing luciferase signaling in Serum Response Element-Luciferase uc) HEK cells expressing FGFR3G380R incubated with sFGFR3_Del4-D3 at 0 nM, 70 nM, and 280nM with or without 1 ng/mL of hFGF2 (* indicates p value < 0.05; *** indicates a p value < 0.001 compared to sFGFR3_Del4-D3 at 0 nM). is a graph showing the percentage of living animals (wild type (wt) and Fgfr3ach/+ mice) after 3 days of ent with a low dose (0.25 mg/kg) of sFGFR3_Del4-D3 ve to age (days). The percentage of living wt mice receiving vehicle (PBS) is also shown. is an image showing the amino acid residues corresponding to the Ig-like C2-type domains 1 (IgI), 2 (IgII), and 3 (IgIII) of wildtype FGFR3 polypeptide (SEQ ID NO: 5 or 32), sFGFR3_Del4-C253S (SEQ ID NO: 2), and a variant of sFGFR3_Del4-D3 (SEQ ID NO:33). sFGFR3_Del4-C253S includes an amino acid substitution of a cysteine residue with a serine residue at position 253 of SEQ ID NO: 1.

FIGS. 7A-7B are images of Western blots of the sFGFR3 polypeptides. Western blots under reducing (R) and non-reducing (NR) conditions are shown for 2.3 mg/ml and 23 mg/ml sFGFR3_Del1-D3 (Fig. 7A) and 1.5 mg/ml and 15 mg/ml sFGFR3_Del1-C253S (Fig. 7B).

FIGS. 8A-8B are graphs showing the melting temperature (Tm) of _Del4-C253S in 20 mM phosphate, 40mM NaCl, pH 7.5 buffer and 40 mM citrate, 40mM NaCl, pH 6.5 buffer (Fig. 8A) and the Tm of sFGFR3_Del4-D3 in 20 mM phosphate, 40mM NaCl, pH 7.5 buffer and 20 mM e, 40mM NaCl, pH 6.5 buffer (Fig. 8B).

FIGS. 9A-9C are graphs showing the fast protein liquid chromatography (FPLC) elution profiles of sFGFR3_Del4-D3. FPLC elution profiles are shown for Fig. 9A: sFGFR3_Del4-D3 at 0 minutes, 2 hours, and 24 hours in cpm/fraction (Fig. 9A); Fig. 9B: sFGFR3_Del4-D3 administered by intravenous bolus at 1 minute, 15 minutes, 30 minute, 2 hours, and 24 hours in cpm/fraction and as normalized to the highest peak (shown in Fig. 9B cont.); Fig 9C: sFGFR3_Del4-D3 administered by aneous injection at 30 minutes, 2 hours, 4 hours, and 24 hours in cpm/fraction and as normalized to the highest peak (shown in Fig. 9C cont.).

FIGS. B are graphs showing the tage (%) of proliferation of Fgfr3ach/+ chondrocyte cells in the presence of the sFGFR3 polypeptides. Fgfr3ach/+ chondrocyte proliferation is shown for 1 ug/ml, 10 ug/ml, and 50 ug/ml of _Del4-D3 (Fig. 10A) and for 1 ug/ml, 10 ug/ml, and 50 ug/ml of sFGFR3_Del4-C253S (Fig. 10B). is a graph showing the PK profiles of 2.5 mg/kg _Del4-D3 administered subcutaneously and 2.5 mg/kg sFGFR3_Del4-D3 administered intravenously. is a graph g the concentration of 125I- sFGFR3_Del4-D3 in kidney, liver, spleen, lung, and heart tissue at 30 minutes, 120 minutes, and 1440 minutes after intravenous administration.

The concentration is expressed as the percent of injected dose per gram (%ID/g). is a graph showing the concentration of 125I- sFGFR3_Del4-D3 in kidney, liver, spleen, lung, and heart tissue at 30 minutes, 120 minutes, 240 minutes, 480 minutes, and 1440 minutes after 40 subcutaneous administration. The concentration is expressed as %ID/g.

A-14B are graphs g the tration (c) and volume of distribution (Vd) of 125I- sFGFR3_Del4-D3 in brain tissue. Shown is the c of 125I-sFGFR3_Del4-D3 before and after correction for 18870863_1 (GHMatters) P110456.NZ.1 vascular content and degradation at 30 minutes, 2 hours, and 24 hours after intravenous bolus (Fig. 14A) and the Vd of 125I-sFGFR3_Del4-D3 and RSA at 30 minutes, 2 hours, and 24 hours after intravenous bolus (Fig. 14B). is a graph showing the percentage of surviving ch/+ mice administered sFGFR3_Del4- D3. Shown are the surviving wild type mice, Fgfr3ach/+ mice administered PBS as vehicle, Fgfr3ach/+ mice administered 2.5 mg/kg sFGFR3_Del4-D3 once weekly, Fgfr3ach/+ mice administered 2.5 mg/kg sFGFR3_Del4-D3 twice weekly, and Fgfr3ach/+ mice administered 10 mg/kg sFGFR3_Del4-D3 twice weekly over 22 days. is a graph showing the percentage (%) of tor and abdominal breathing complications in Fgfr3ach/+ mice administered PBS as vehicle, 2.5 mg/kg sFGFR3_Del4-D3 once weekly, 2.5 mg/kg _Del4-D3 twice weekly, and 10 mg/kg sFGFR3_Del4-D3 twice weekly.

FIGS. D are graphs and an x-ray radiograph showing the length of Fgfr3ach/+ mice administered sFGFR3_Del4-D3. Shown are the axial length (Fig. 17A), tail length (Fig. 17B), and tibia length (Fig. 17C) of wild type mice administered PBS as vehicle, and Fgfr3ach/+ mice administered PBS as vehicle, 2.5 mg/kg sFGFR3_Del4-D3 once , 2.5 mg/kg sFGFR3_Del4-D3 twice weekly, and 10 mg/kg sFGFR3_Del4-D3 twice weekly. Also shown is the x-ray radiograph (Fig. 17D) of wild type mice administered PBS as vehicle and Fgfr3ach/+ mice administered PBS as vehicle, 2.5 mg/kg sFGFR3_Del4- D3 twice weekly, and 10 mg/kg sFGFR3_Del4-D3 twice weekly. All measurements are in millimeters (mm).

FIGS. 18A-18B are a graph showing the cranium ratio and an x-ray radiograph g the skulls of ch/+ mice stered _Del4-D3, respectively. Shown in the graph (Fig. 18A) is the cranium ratio (L/W) of wild type mice administered PBS as vehicle and Fgfr3ach/+ mice administered PBS as vehicle, 2.5 mg/kg sFGFR3_Del4-D3 once weekly, 2.5 mg/kg sFGFR3_Del4-D3 twice weekly, and 10 mg/kg sFGFR3_Del4-D3 twice weekly. Shown in the x-ray radiograph (Fig. 18B) is the skulls of wild type mice stered PBS as vehicle, Fgfr3ach/+ mice administered PBS as vehicle, wild type mice administered 10 mg/kg sFGFR3_Del4-D3 twice weekly, and Fgfr3ach/+ mice administered 10 mg/kg sFGFR3_Del4-D3 twice weekly.

FIGS. E are graphs showing the kinetic profile for the binding of different concentrations of hFGF1, FGF2, hFGF9, hFGF18, hFGF19, and hFGF21 to immobilized SFGFR3_DEL4-D3 in real time.

Shown are the kinetic profiles for binding of hFGF1 at concentrations of 0.5 nM to 12 nM to immobilized SFGFR3_DEL4-D3 (A); hFGF2 at concentrations of 2 nM to 10 nM to immobilized SFGFR3_DEL4-D3 (B); hFGF9 at concentrations of 1 nM to 5 nM to lized _DEL4- D3 (C); hFGF18 at concentrations of 1 nM to 10 nM to immobilized SFGFR3_DEL4-D3 (D); hFGF19 at concentrations of 2 nM to 20 nM to immobilized SFGFR3_DEL4-D3 (E); and hFGF21 at concentrations of 100 nM to 10000 nM to lized SFGFR3_DEL4-D3 (F). The darker, overlapping lines represent the 2:1 binding model used to generate the Kd values. is an image of a n blot of non-induced wild type ATDC5 and retrovirally infected ATDC5 cells expressing FGFR3G380R.

FIG 21 is a graph showing the induction of proliferation of ATDC5 FGFR3G380R cells in the presence 40 of SFGFR3_DEL4-D3 for three experiments. Untreated ATDC5 380R cells were used as a control. 18870863_1 (GHMatters) P110456.NZ.1 DETAILED DESCRIPTION OF THE INVENTION We have discovered that soluble fibroblast growth factor receptor 3 3) polypeptides and variants thereof can be used to treat skeletal growth retardation disorders, such as achondroplasia, in a patient (e.g., a human, particularly an infant, a child, or an adolescent). In particular, sFGFR3 polypeptides of the invention feature a deletion of, e.g., amino acids 289 to 400 of SEQ ID NO: 5 or amino acids 311 to 422 of SEQ ID NO: 32, to provide the following exemplary sFGFR3 polypeptides: sFGFR3_Del4 ing an amino acid substitution of a cysteine residue with a serine residue at position 253 3_Del4-C253S; SEQ ID NO: 2) and sFGFR3_Del4 ing an extended Ig-like C2-type domain 3 (sFGFR3_Del4-D3; SEQ ID NO: 33) and variants thereof, such as a sFGFR3 ptide having the amino acid sequence of SEQ ID NO: 4. Additionally, the sFGFR3 polypeptides may include a signal peptide, such as a sFGFR3 polypeptide having the amino acid sequence of SEQ ID NO: 18 or 34.

See U.S. Provisional Application No. 62/276,222 and International Application No. PCT/US16/12553 for a ption of sFGFR3_Del4 (SEQ ID NO: 1), each of which is hereby incorporated herein by nce in their entirety.

For example, sFGFR3 polypeptides and variants thereof having at least 85% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the amino acid sequence of SEQ ID NO: 1 can include an amino acid substitution that removes a cysteine residue at position 253 of SEQ ID NO: 1 (e.g. sFGFR3_Del4-C253S; a polypeptide having the amino acid sequence of SEQ ID NO: 2). In particular, an sFGFR3 polypeptide of the invention can e a substitution of a cysteine e at position 253 of SEQ ID NO: 1 with, e.g., a serine residue. For example, the cysteine residue at position 253 is substituted with a serine residue or, e.g., r conservative amino acid substitution, such as alanine, glycine, proline, or threonine.

The sFGFR3 ptides can also include a polypeptide sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) amino acid sequence identity to amino acid residues 23 to 357 of SEQ ID NO: 32, in which the polypeptide lacks a signal e and a transmembrane domain of FGFR3 and (i) is less than 500 amino acids in length; (ii) comprises 200 consecutive amino acids or fewer of an intracellular domain of FGFR3; and/or (iii) lacks a tyrosine kinase domain of FGFR3 (e.g., sFGFR3_Del4-D3; a polypeptide having the amino acid sequence of SEQ ID NO: 33). Methods for administering the sFGFR3 polypeptides of the ion to treat skeletal growth retardation disorders (e.g., achondroplasia) in a patient (e.g., a human, particularly an infant, a child, or an adolescent) are also described.

The sFGFR3 polypeptides, methods of production, methods of treatment, compositions, and kits of the invention are described herein.

Soluble Fibroblast Growth Factor Receptor 3 (sFGFR3) Polypeptides The invention features sFGFR3 polypeptides and ts thereof formulated for the treatment of skeletal growth retardation disorders (e.g., achondroplasia). In ular, the sFGFR3 polypeptides can have 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 amino acid ce of SEQ ID NO: 1, in which the 40 sFGFR3 polypeptide includes an amino acid substitution that s a cysteine residue at position 253 of SEQ ID NO: 1 (e.g. sFGFR3_Del4-C253S; a polypeptide having the amino acid sequence of SEQ ID 18870863_1 (GHMatters) P110456.NZ.1 NO: 2). For example, the cysteine residue at position 253 of SEQ ID NO: 1 is substituted with a serine residue or a conservative amino acid substitution, such as alanine, e, proline, or threonine.

The sFGFR3 polypeptides and variants thereof can also include fragments of the amino acid sequence of SEQ ID NO: 2 (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, or 1 to 300, of SEQ ID NO: 2) having at least 50% sequence identity (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 ty) to SEQ ID NO: 2. Additionally, sFGFR3 polypeptides can include amino acids 1 to 301 of SEQ ID NO: 1, in which the sFGFR3 polypeptide includes an amino acid substitution of a ne residue with a serine residue at position 253 of SEQ ID NO: 1 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 2).

The sFGFR3 polypeptides and variants thereof can also include fragments of the amino acid sequence of SEQ ID NO: 33 (e.g., at least amino acids 1 to 200, 1 to 210, 1 to 220, 1 to 230, 1 to 240, 1 to 250, 1 to 260, 1 to 270, 1 to 280, 1 to 290, 1 to 300, 1 to 310, 1 to 320, 1 to 330, 1 to 340, 1 to 340, or 1 to 345 of SEQ ID NO: 33) having at least 50% sequence identity (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 SEQ ID NO: 33. In addition, the cysteine residue at position 253 of SEQ ID NO: 4 or 33 and/or position 316 of SEQ ID NO: 4, if present, can be substituted with a serine residue or a conservative amino acid substitution, such as alanine, glycine, proline, or threonine.

Given the results bed , the invention is not limited to a particular sFGFR3 polypeptide or variants f. In on to the exemplary sFGFR3 polypeptide s and ts thereof discussed above, any polypeptide that binds one or more FGFs (e.g., FGF1 (e.g., a ptide having the amino acid sequence of SEQ ID NO: 13), FGF2 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 14), FGF9 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 15), FGF18 (e.g., a polypeptide having the amino acid ce of SEQ ID NO: 16), FGF19 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 38), and/or FGF21 (e.g., a polypeptide having the amino acid ce of SEQ ID NO: 39)) with similar binding affinity as the sFGFR3 polypeptides of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 ptide including a signal peptide (SEQ ID NO: 18 or 34)) can be used in the methods, such as for treating a skeletal growth retardation disorder, e.g., achondroplasia. The sFGFR3 ptides can be, for example, fragments of FGFR3 isoform 2 lacking exons 8 and 9 encoding the C-terminal half of the Ig-like C2-type domain 3 and exon 10 including the transmembrane domain (e.g., fragments of the amino acid sequence of SEQ ID NO: 5 or 32), corresponding to fragments of FGFR3 transcript variant 2 (Accession No. NM_022965).

An sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4)) 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: 6 (e.g., MGAPACALALCVAVAIVAGASS) or amino acids 1 to 19 of SEQ ID NO: 35 (e.g., MMSFVSLLLVGILFHATQA). Accordingly, the sFGFR3 polypeptides include both secreted forms, 40 which lack the N-terminal signal peptide, and creted forms, which include the N-terminal signal peptide. For instance, a secreted sFGFR3 polypeptide can e the amino acid sequence of SEQ ID NOs: 2, 4, or 33. Alternatively , the sFGFR3 polypeptide does include a signal peptide, such the amino 18870863_1 (GHMatters) P110456.NZ.1 acid sequence of SEQ ID NOs: 18, 19, or 34. One skilled in the art will appreciate that the on of the N-terminal signal peptide will vary in different sFGFR3 polypeptides and can include, for example, the first , 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 ptide. One of skill in the art can predict the on of a signal sequence ge site, e.g., by an appropriate computer algorithm such as that described in en et al. (J.

Mol. Biol. 340(4):783-795, 2004) and available on the Web at u.dk/services/SignalP/.

Additionally, sFGFR3 polypeptides (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) of the invention can be glycosylated. In particular, a sFGFR3 polypeptide can be d to increase or decrease the extent to which the sFGFR3 polypeptide is glycosylated. on 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. For example, 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 sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 4 or 33), and variants thereof. One or more of these Asn residues can also be substituted to remove the glycosylation site. For instance, O-linked glycosylation, in which an oligosaccharide is ed 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 sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), ts thereof (SEQ ID NO: 4), and sFGFR3 polypeptides including a signal peptide (SEQ ID NO: 18 or 34). Additionally, O-linked glycosylation can occur at position Ser310 and/or Ser321 of _Del4-D3 (SEQ ID NO: 33) and variants thereof (SEQ ID NO: 4). One or more of these Ser or Thr residues can also be substituted to remove the glycosylation site. sFGFR3 Fusion Polypeptides sFGFR3 polypeptides of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4- D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) can be fused to a functional domain from a heterologous ptide (e.g., a fragment crystallizable region of an immunoglobulin (Fc ; such as a polypeptide having the amino acid sequence of SEQ ID NOs: 25 and 26) or human serum albumin (HSA; such as a polypeptide having the amino acid sequence of SEQ ID NO: 27)) to provide a sFGFR3 fusion polypeptide. Optionally, 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 polyglycine /serine linker, such as SEQ ID NOs: 28 and 29).

For example, the sFGFR3 polypeptides (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide ing a signal peptide (SEQ ID NO: 18 or 34)) can be a fusion polypeptide including, e.g., an Fc region of an immunoglobulin at the inal or C-terminal domain. In particular, useful Fc regions can include the Fc fragment of any immunoglobulin molecule, including IgG, IgM, IgA, IgD, or IgE and their 40 various subclasses (e.g., IgG-1, IgG-2, IgG-3, IgG-4, IgA-1, IgA-2) from any mammal (e.g., a human).

For instance, the Fc nt human IgG-1 (SEQ ID NO: 25) or a variant of human IgG-1, such as a variant including a substitution of gine at position 297 of SEQ ID NO: 25 with alanine (e.g., a 18870863_1 (GHMatters) P110456.NZ.1 polypeptide having the amino acid sequence of SEQ ID NO: 26). The Fc fragments of the invention can include, for example, the CH2 and CH3 domains of the heavy chain and any portion of the hinge region.

The sFGFR3 fusion polypeptides of the invention can also include, e.g., a monomeric Fc, such as a CH2 or CH3 domain. The Fc region may optionally be ylated 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 ons, deletions, or substitutions relative to any of the Fc fragments described herein.

Additionally, the sFGFR3 polypeptides (e.g. _Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4- D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) 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). For instance, the sFGFR3 ptides can be conjugated to human HSA (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 27) or a fragment thereof.

The sFGFR3 fusion polypeptides can e a peptide linker region between the sFGFR3 polypeptide (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants f (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) 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 ally hindered.

Exemplary linker lengths are between 1 and 200 amino acid residues, e.g., 1-5, 6-10, 11-15, 16-20, 21- , 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, 0, 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. red 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 t invention has an increased content of small amino acids, in particular of glycines, es, 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. In some cases, the amino acid ce of a linker consists only of e and serine residues. For example, the linker can be the amino acid sequence of GGGGAGGGG (SEQ ID NO: 28) or GGGGSGGGGSGGGGS (SEQ ID NO: 29). A linker can ally be glycosylated at any appropriate one or more amino acid residues. The linker can also be absent, in which the FGFR3 polypeptide and the heterologous polypeptide (e.g., an Fc region or HSA) are fused together directly, with no ening residues.

Polynucleotides encoding the sFGFR3 Polypeptides 40 The invention further includes polynucleotides encoding the sFGFR3 polypeptides (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) that can be used to 18870863_1 ters) P110456.NZ.1 treat skeletal growth retardation ers (e.g., achondroplasia) in a patient (e.g., a human, such as an infant, a child, or an adolescent), such as SEQ ID NOs: 20, 21, 36, or 37. For example, the cleotide can be the nucleic acid sequence of SEQ ID NO: 20 or 36, which encode sFGFR3_Del4- C253S (SEQ ID NO: 2), or a variant having at least 85% sequence ty (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: 20 or 36. Additionally, the polynucleotide can be the nucleic acid sequence of SEQ ID NO: 21 or 37, which encodes sFGFR3_Del4-D3 (SEQ ID NO: 33), having at least 85% sequence ty (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: 21 or 37. The invention also includes cleotides encoding sFGFR3 fusion polypeptides (e.g., a sFGFR3 ptide fused to a heterologous polypeptide, such as a Fc region or HSA) and polynucleotides encoding sFGFR3 polypeptides t a signal peptide (e.g., polypeptides having the amino acid sequence of SEQ ID NOs: 2, 4, and 33) or with a signal peptide (e.g., polypeptides having the amino acid sequence of SEQ ID NOs: 18, 19, and 34). Additionally, the ion includes polynucleotides include one or more mutations to alter any of the glycosylation sites bed herein. ally, the sFGFR3 polynucleotides of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) 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 cleotide. Codonoptimized polynucleotides (e.g., a polynucleotide having the nucleic acid sequence of SEQ ID NO: 20, 21, 36, or 37 ) can, e.g., facilitate genetic manipulations by decreasing the GC content and/or for sion in a host cell (e.g., a HEK 293 cell or a CHO cell). Codon-optimization can be performed by the d person, e.g. by using online tools such as the JAVA Codon Adaption Tool (www.jcat.de) or Integrated DNA Technologies Tool (www.eu.idtdna.com/CodonOpt) by simply ng the nucleic acid sequence of the polynucleotide and the host organism for which the codons are to be optimized. The codon usage of different organisms is available in online databases, for example, www.kazusa.or.jp/codon.

Host cells for expression of the sFGFR3 polypeptides Mammalian cells can be used as host cells for expression of the sFGFR3 polypeptides of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 ptide including a signal peptide (SEQ ID NO: 18 or 34)).

Exemplary mammalian cell types useful in the methods include, but are not limited to, human embryonic kidney (HEK; e.g., HEK 293) cells, Chinese Hamster Ovary (CHO) cells, L cells, C127 cells, 3T3 cells, BHK cells, COS-7 cells, HeLa cells, PC3 cells, Vero cells, MC3T3 cells, NS0 cells, Sp2/0 cells, VERY cells, BHK, MDCK cells, W138 cells, BT483 cells, Hs578T cells, HTB2 cells, BT20 cells, T47D cells, NS0 cells, CRL7O3O cells, and HsS78Bst cells, or any other suitable mammalian host cell known in the art.

Alternatively, E. coli cells can be used as host cells for expression of the sFGFR3 polypeptides.

Examples of E. coli strains include, but are not limited to, E. coli 294 (ATCC® 31,446), E. coli λ 1776 40 (ATCC® 31,537, E. coli BL21 (DE3) (ATCC® BAA-1025), E. coli RV308 (ATCC® 31,608), or any other suitable E. coli strain known in the art. 18870863_1 (GHMatters) P110456.NZ.1 Vectors including polynucleotides encoding the sFGFR3 polypeptides The invention also features recombinant vectors including any one or more of the polynucleotides described above. The vectors of the invention can be used to deliver a polynucleotide encoding a sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 ptide including a signal peptide (SEQ ID NO: 18 or 34)), which can e mammalian, viral, and bacterial expression vectors.

For example, the vectors can be plasmids, artificial chromosomes (e.g. BAG, PAC, and YAC), and virus or phage vectors, and may optionally include a promoter, enhancer, or regulator for the expression of the polynucleotide. The vectors can also contain one or more selectable marker genes, such as an ampicillin, in, and/or kanamycin resistance gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors can be used in vitro for the production of DNA or RNA or used to transfect or transform a host cell, such as a mammalian host cell for the production of a sFGFR3 polypeptide encoded by the vector. The vectors can also be adapted to be used in vivo in a method of gene therapy. ary viral vectors that can be used to deliver a polynucleotide encoding a sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 ptide including a signal peptide (SEQ ID NO: 18 or 34)) include a retrovirus, adenovirus (e.g., Ad2, Ad5, Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, and Pan9 (also known as AdC68)), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., nza virus), virus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as avirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes x virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), x and canarypox). Other viruses useful for ring polynucleotides encoding sFGFR3 polypeptides include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, and spumavirus n, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third n, B. N. , et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

Methods of Production Polynucleotides encoding sFGFR3 ptides of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) can be produced by any method known in the art. For instance, a polynucleotide is ted using molecular cloning s and is placed within a vector, such as a plasmid, an artificial some, a viral vector, or a phage vector. The vector is used to transform the polynucleotide into a host cell riate for the expression of the sFGFR3 polypeptide. 40 Nucleic acid vector construction and host cells The sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide 18870863_1 (GHMatters) P110456.NZ.1 including a signal peptide (SEQ ID NO: 18 or 34)) can be ed from a host cell. The polynucleotides (e.g., polynucleotides having the nucleic acid ce of SEQ ID NO: 20, 21, 36, or 37 and variants thereof) encoding sFGFR3 polypeptides can be included in vectors that can be introduced into the host cell by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, or infection). The choice of vector depends in part on the host cells to be used. Generally, host cells are of either prokaryotic (e.g., bacterial) or eukaryotic (e.g., ian) origin.

A polynucleotide encoding an sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) can be prepared by a variety of methods known in the art. These methods e, but are not d to, oligonucleotide-mediated (or sitedirected ) mutagenesis and PCR nesis. A polynucleotide encoding an sFGFR3 polypeptide can be obtained using standard techniques, e.g., gene synthesis. Alternatively, a polynucleotide encoding a wild-type sFGFR3 polypeptide (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 5 or 32) can be mutated to contain specific amino acid substitutions (e.g., an amino acid substitution of a cysteine residue with a serine residue or a conservative amino acid substitution, such as alanine, glycine, proline, or threonine, at position 253 of SEQ ID NO: 33 and/or position 316 of SEQ ID NO: 4) using rd techniques in the art, e.g., QuikChangeTM mutagenesis. Polynucleotides ng an sFGFR3 polypeptide can be synthesized using, e.g., a nucleotide synthesizer or PCR techniques. cleotides encoding sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) can be inserted into a vector capable of replicating and sing the polynucleotide in prokaryotic or eukaryotic host cells. Exemplary vectors useful in the methods can include, but are not limited to, a plasmid, an artificial chromosome, a viral vector, and a phage . For example, a viral vector can include the viral vectors described above, such as a retroviral vector, adenoviral vector, or poxviral vector (e.g., vaccinia viral , such as Modified Vaccinia Ankara (MVA)), adeno-associated viral , and alphaviral vector)) containing the nucleic acid sequence of a polynucleotide encoding the sFGFR3 polypeptide. Each vector can contain various components that may be adjusted and optimized for compatibility with the particular host cell. For example, the vector components may e, but are not limited to, an origin of ation, a selection marker gene, a promoter, a ribosome binding site, a signal sequence, the nucleic acid sequence of the cleotide encoding the sFGFR3 polypeptide, and/or a transcription termination sequence.

The above-described vectors may be uced into appropriate host cells (e.g., HEK 293 cells or CHO cells) using conventional techniques in the art, e.g., transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection. Once the vectors are introduced into host cells for the production of an sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)), host cells are cultured in conventional nutrient media ed as appropriate for ng promoters, selecting transformants, or amplifying the polynucleotides 40 (e.g. SEQ ID NOs: 20 and 21 and ts thereof) encoding the sFGFR3 polypeptide. Methods for expression of therapeutic proteins, such as sFGFR3 polypeptides, are known in the art, see, for example, Paulina Balbas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods 18870863_1 (GHMatters) P110456.NZ.1 in lar Biology), Humana Press; 2nd ed. 2004 (July 20, 2004) and Vladimir Voynov and Justin A.

Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd ed. 2012 (June 28, 2012), each of which is hereby orated by reference in its entirety. sFGFR3 polypeptide production, ry, and purification Host cells (e.g., HEK 293 cells or CHO cells) used to produce the sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) can be grown in media known in the art and suitable for culturing of the selected host cells. es of suitable media for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco’s Modified Eagle’s Medium (DMEM), Expi293™ Expression Medium, DMEM with supplemented fetal bovine serum (FBS), and RPMI-1640. Examples of suitable media for bacterial host cells include Luria broth (LB) plus necessary supplements, such as a selection agent, e.g., ampicillin. Host cells are cultured at suitable temperatures, such as from about 20 °C to about 39 °C, e.g., from 25 °C to about 37 °C, preferably 37 °C, and CO2 levels, such as 5 to 10% (preferably 8%). The pH of the medium is generally from about 6.8 to 7.4, e.g., 7.0, depending mainly on the host organism. If an inducible er is used in the expression vector, sFGFR3 ptide expression is induced under conditions suitable for the activation of the promoter.

An sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), _Del4-D3 (SEQ ID NO: 33), and variants f (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) can be recovered from the supernatant of the host cell.

Alternatively, the sFGFR3 polypeptide can be recovered by disrupting the host cell (e.g., using c shock, sonication, or , followed by centrifugation or filtration to remove the sFGFR3 polypeptide.

Upon recovery of the sFGFR3 polypeptide, the sFGFR3 polypeptide can then be further purified. An sFGFR3 polypeptide can be purified by any method known in the art of protein purification, such as protein A affinity, other chromatography (e.g., ion exchange, affinity, and size-exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins (see Process Scale Purification of dies, Uwe Gottschalk (ed.) John Wiley & Sons, Inc., 2009, hereby incorporated by reference in its entirety).

Optionally, the sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) can be ated to a detectable label for purification.

Examples of suitable labels for use in cation of the sFGFR3 ptides include, but are not limited to, a protein tag, a fluorophore, a phore, a radiolabel, a metal colloid, an enzyme, or a chemiluminescent, or inescent molecule. In particular, n tags that are useful for purification of the sFGFR3 polypeptides can include, but are not limited to, chromatography tags (e.g., peptide tags consisting of polyanionic amino acids, such as a FLAG-tag, or a hemagglutinin “HA” tag), affinity tags (e.g., a poly(His) tag, chitin binding protein (CBP), maltose binding protein (MBP), or glutathione-S- transferase (GST)), solubilization tags (e.g., thioredoxin (TRX) and poly(NANP)), epitope tags (e.g., V5- 40 tag, Myc-tag, and HA-tag), or fluorescence tags (e.g., GFP, GFP variants, RFP, and RFP variants). 18870863_1 (GHMatters) P110456.NZ.1 Methods of Treatment Provided herein are methods for treating a skeletal growth retardation disorder in a patient, such as a patient having roplasia (e.g., a human having achondroplasia). In particular, the patient is one that exhibits or is likely to develop one or more symptoms of a skeletal growth retardation disorder (e.g., achondroplasia). The method involves administering an sFGFR3 polypeptide of the invention (e.g. _Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) to the patient having a skeletal growth retardation er, such as a patient having achondroplasia (e.g., a human having achondroplasia). In particular, the method involves administering sFGFR3_Del4-C253S (SEQ ID NO: 2) or sFGFR3_Del4-D3 (SEQ ID NO: 33) to the patient having a skeletal growth retardation disorder, such as a t having roplasia (e.g., a human having achondroplasia). For example, the patient is an infant or child having a al growth retardation disorder, such as an infant, a child, or an cent having achondroplasia (e.g., a human having roplasia).

The patient (e.g., a human) can be treated before symptoms of a skeletal growth retardation disorder (e.g., achondroplasia) appear or after symptoms of a skeletal growth retardation disorder (e.g., achondroplasia) develop. In particular, patients that can be treated with a sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants f (SEQ ID NO: 4) or a sFGFR3 polypeptide ing a signal peptide (SEQ ID NO: 18 or 34)) are those exhibiting ms including, but not limited to, short limbs, short trunk, bowlegs, a ng gait, skull malformations, cloverleaf skull, craniosynostosis, wormian bones, anomalies of the hands, anomalies of the feet, hitchhiker thumb, and/or chest anomalies. Furthermore, treatment with an sFGFR3 polypeptide can result in an improvement in one or more of the entioned symptoms of a al growth ation disorder (e.g., relative to an untreated patient), such as achondroplasia.

The patient (e.g., a human) can be diagnosed with a skeletal growth retardation disorder, such as achondroplasia, before administration of an sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4- C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)). Additionally, the patient having a skeletal growth retardation disorder, such as achondroplasia, can be one that has not previously been treated with an sFGFR3 polypeptide.

Skeletal Growth Retardation Disorders al growth retardation disorders can be treated by administering an sFGFR3 polypeptide as described herein to a patient (e.g., a human) in need thereof. T he method involves administering to the patient (e.g., a human) having the skeletal growth retardation disorder an sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 ptide including a signal peptide (SEQ ID NO: 18 or 34)).

Skeletal growth retardation disorders that can be treated with the sFGFR3 polypeptides 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-C253S (SEQ ID NO: 2) or 40 sFGFR3_Del4-D3 (SEQ ID NO: 33).

Administration of an sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants f (SEQ ID NO: 4) or a sFGFR3 polypeptide 18870863_1 (GHMatters) P110456.NZ.1 including a signal peptide (SEQ ID NO: 18 or 34)) 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 osis, congenital short femur, Crouzon syndrome, Apert syndrome, n-Weiss syndrome, Pfeiffer syndrome, Crouzonodermoskeletal syndrome, dactyly, brachydactyly, camptodactyly, polydactyly, syndactyly, diastrophic dysplasia, dwarfism, dyssegmental dysplasia, enchondromatosis, fibrochondrogenesis, fibrous dysplasia, tary le exostoses, hypophosphatasia, hypophosphatemic s, Jaffe-Lichtenstein me, Kniest dysplasia, Kniest syndrome, Langer-type mesomelic dysplasia, Marfan syndrome, McCune-Albright syndrome, micromelia, metaphyseal dysplasia, -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, and spondyloepimetaphyseal For ce, the sFGFR3 polypeptides of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) can be used to treat symptoms associated with a skeletal growth ation disorder, including the disorders described above, such as achondroplasia.

Non-limiting examples of symptoms of skeletal growth retardation disorders that can be treated with the sFGFR3 ptides, include 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 al fingernails and toenails, and chest anomalies (e.g., pear-shaped chest or narrow thorax). Additional symptoms that can treated by administering sFGFR3 polypeptides can also include eletal abnormalities in patients having skeletal growth retardation disorders, such as anomalies of the eyes, mouth, and ears, such as congenital cataracts, myopia, cleft palate, or deafness; brain malformations, such as ephaly, porencephaly, encephaly, or agenesis of the corpus callosum; heart s, such as atrial septal defect, patent ductus arteriosus, or osition of the great vessels; pmental delays; or mental disabilities.

Treatment with the sFGFR3 polypeptides of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 ptide including a signal peptide (SEQ ID NO: 18 or 34)) can also increase survival of patients (e.g., humans) with skeletal growth retardation disorders (e.g., achondroplasia). For example, the survival rate of ts treated with the sFGFR3 polypeptides can increase by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more relative to, e.g., an untreated patient with a skeletal growth retardation disorder (e.g., achondroplasia), over a treatment period of days, months, years, or . In particular, administration of sFGFR3_Del4-D3 can increase survival of patients (e.g., humans) with skeletal growth 40 retardation disorders (e.g., relative to an untreated patient), such as achondroplasia.

Any skeletal growth retardation disorder that is a FGFR3-related skeletal disease (e.g., caused by or ated with overactivation of FGFR3 as result of a gain-of-function FGFR3 mutation) can be 18870863_1 (GHMatters) P110456.NZ.1 treated by administering an sFGFR3 polypeptide of the invention ((e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), _Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 ptide including a signal peptide (SEQ ID NO: 18 or 34)) to a patient (e.g., a human). For example, 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 pmental delay and acanthosis nigricans (SADDAN), hypochondroplasia, and synostosis (e.g., Muenke syndrome, Crouzon syndrome, and Crouzonodermoskeletal syndrome).

Patients (e.g., humans) with mutations in the FGFR3 gene associated with different related skeletal disorders, such as achondroplasia, hypochondroplasia, SADDAN, TDI, and TDII, can be d with sFGFR3 ptides of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4- D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 ptide including a signal e (SEQ ID NO: 18 or 34)). For example, the sFGFR3 polypeptides can be administered to treat achondroplasia resulting from the G380R, G375C, G346E or S279C mutations of the FGFR3 gene.

Administration of the sFGFR3 polypeptides can be used to treat the following ary 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, X807S, X807W and K650M mutations of the FGFR3 gene; TDII resulting from the K650E mutation of the FGFR3 gene; and SADDAN ing from the K650M mutation of the FGFR3 gene.

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 with an sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)). Additionally, the parents of the patient and/or fetal samples (e.g., fetal nucleic acid ed from maternal blood, placental, or fetal samples) can be tested by methods known in the art for the mutation in the FGFR3 gene to determine their need for treatment. roplasia Achondroplasia is the most common cause of dwarfism in humans and can be treated by administering sFGFR3 polypeptides as described herein. In particular, achondroplasia can be treated by administering an sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)). Accordingly, administration of the sFGFR3 polypeptides can result in an ement 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 is (e.g., leg and lower back pain), hydrocephalus (e.g., requiring cerebral shunt surgery), g loss due to chronic otitis, cardiovascular disease, neurological disease, respiratory ms, fatigue, pain, numbness in the lower back and/or spine, and/or obesity.

Patients treated using the sFGFR3 polypeptides of the invention (e.g. sFGFR3_Del4-C253S (SEQ 40 ID NO: 2), _Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) can include infants, children, and adults with achondroplasia. In particular, infants are often diagnosed with achondroplasia at birth, and thus, 18870863_1 (GHMatters) P110456.NZ.1 ent with the sFGFR3 polypeptides can begin as early as possible in the patient’s life, e.g., shortly after birth, or prior to birth (in utero).

Symptoms of achondroplasia in patients (e.g., humans) can also be monitored prior to or after a t is treated with an sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)). For instance, symptoms of achondroplasia can be monitored prior to treatment to assess the severity of achondroplasia and condition of the patient prior to performing the methods.

The methods can include diagnosis of achondroplasia in a patient and monitoring the patient for changes in the symptoms of achondroplasia, such as s in body weight and skull size (e.g., skull length and/or skull width) of the patient. s in body weight and skull size can be 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 of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants f (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)). Body weight and/or skull size of the patient having achondroplasia can also be determined at treatment specific events, such as before and/or after administration of the sFGFR3 polypeptide.

For example, body weight and/or skull size can be measured in response to administration of the sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal e (SEQ ID NO: 18 or 34)). Body weight can be measured by weighing the patient having roplasia on a scale, preferably in a standardized manner, such as with the same or no clothes or at a certain time of the day, preferably in a g state (e.g., in the morning before breakfast or after at least 1, 2, 3, 4, 5 or more hours of fasting). Skull size can be represented by length, , width, and/or circumference of the skull. Measurements can be performed using any known or self-devised standardized method. For a human subject, the measurement of skull ference is preferred, which can be measured using a flexible and non-stretchable material, such as a tape, d around the widest possible circumference of the head (e.g. from the most prominent part of the ad around to the widest part of the back of the head). The height of the skull of the subject (e.g., human) can also be determined from the underside of the chin to the uppermost point of the head. Preferably, any measurement is performed more than once, e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times.

Administration of sFGFR3 Polypeptides An sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) can be administered by any route known in the art, such as by parenteral administration, enteral administration, or topical administration. In particular, the sFGFR3 polypeptide can be stered to the patient having a skeletal growth retardation disorder (e.g., achondroplasia) subcutaneously (e.g., by subcutaneous injection), intravenously, intramuscularly, 40 intra-arterially, intrathecally, or intraperitoneally.

An sFGFR3 polypeptide of the ion (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and ts thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide 18870863_1 (GHMatters) P110456.NZ.1 ing a signal peptide (SEQ ID NO: 18 or 34)) can be administered to a patient (e.g., a human) at a predetermined , such as in an effective amount to treat a skeletal growth retardation disorder (e.g., achondroplasia), without inducing significant toxicity. For example, sFGFR3 polypeptides can be administered to a patient having skeletal growth retardation disorders (e.g., roplasia) in individual doses ranging from about 0.002 mg/kg to about 50 mg/kg (e.g., from 2.5 mg/kg to 30 mgkg, 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 can be administered in individual doses of, e.g., 0.001 mg/kg to 50 mg/kg, such as 2.5 mg/kg to about 10 mg/kg.

Exemplary doses of an sFGFR3 polypeptide of the ion (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide ing a signal peptide (SEQ ID NO: 18 or 34)) for stration to a patient (e.g., a human) having a skeletal growth ation disorder (e.g., achondroplasia) include, e.g., 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 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. For example, an sFGFR3 polypeptide can be administered to patients in a weekly dosage ranging, e.g., from about 0.0014 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., 2.5 mg/kg/week, 5 mg/kg/week, 10 mg/kg/week, 20 mg/kg/week, 30 mg/kg/week, 40 week, or 50 mg/kg/week).

Gene Therapy An sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal e (SEQ ID NO: 18 or 34)) can also be delivered through gene therapy, where a polynucleotide encoding the sFGFR3 polypeptide is delivered to tissues of interest and expressed in vivo.

Gene therapy s 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: 2, 2011), each of which is hereby incorporated by reference.

An sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) can be produced by the cells of a patient (e.g., a human) having a skeletal growth retardation disorder (e.g., achondroplasia) by administrating a vector (e.g., a plasmid, an artificial chromosome (e.g. BAG, PAC, and YAC), or a viral vector) containing the nucleic acid sequence of a polynucleotide encoding the sFGFR3 polypeptide. For example, a viral vector can be a retroviral vector, adenoviral vector, or poxviral vector (e.g., vaccinia viral vector, such as Modified Vaccinia Ankara (MVA)), adeno-associated viral vector, or alphaviral vector. The vector, once inside a cell of the patient (e.g., a human) having a skeletal growth retardation disorder (e.g., achondroplasia), by, e.g., ormation, transfection, oporation, calcium phosphate precipitation, 40 or direct microinjection, will promote expression of the sFGFR3 polypeptide, which is then secreted from the cell. The invention further includes cell-based therapies, in which the patient (e.g., a human) is administered a cell expressing the sFGFR3 polypeptide. 63_1 ters) P110456.NZ.1 Pharmaceutical Compositions Pharmaceutical compositions of the invention can include an sFGFR3 ptide (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)), polynucleotide, vector, and/or host cell of the invention. Compositions including an sFGFR3 ptide, polynucleotide, vector, and/or host cell can be formulated at a range of dosages, in a variety of formulations, and in combination with pharmaceutically acceptable excipients, rs, or diluents.

A ceutical composition including an sFGFR3 polypeptide (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 ptide including a signal peptide (SEQ ID NO: 18 or 34)), polynucleotide, vector, and/or host cell of the invention can be formulated at a specific dosage, such as a dosage that is effective for treating a t (e.g., a human) skeletal growth retardation er (e.g., achondroplasia), t inducing significant toxicity. For example, the compositions can be formulated to include between about 1 mg/mL and about 500 mg/mL of the sFGFR3 polypeptide (e.g., between 10 mg/mL and 300 mg/mL, 20 mg/mL and 120 mg/mL, 40 mg/mL and 200 mg/mL, 30 mg/mL and 150 mg/mL, 40 mg/mL and 100 mg/mL, 50 mg/mL and 80 mg/mL, or 60 mg/mL and 70 mg/mL of the sFGFR3 polypeptide).

The pharmaceutical compositions including an sFGFR3 polypeptide (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 ptide including a signal peptide (SEQ ID NO: 18 or 34)), polynucleotide, vector, and/or host cell of the invention can be prepared in a variety of forms, such as a liquid solution, dispersion or suspension, powder, or other ordered structure suitable for stable storage. For example, compositions including an sFGFR3 polypeptide intended for systemic or local delivery can be in the form of injectable or infusible solutions, such as for parenteral administration (e.g., aneous, intravenous, intramuscular, intraarterial , intrathecal, or intraperitoneal administration). sFGFR3 compositions for injection (e.g., subcutaneous or intravenous injection) can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically able vehicles include, but are not limited to, e water, physiological saline, and cell culture media (e.g., Dulbecco’s Modified Eagle Medium (DMEM), α- Modified Eagles Medium (α-MEM), F-12 medium). ation methods are known in the art, see e.g., Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (2nd ed.) Taylor & Francis Group, CRC Press (2006), which is hereby incorporated by reference in its entirety.

Compositions including an sFGFR3 polypeptide (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)), polynucleotide, vector, and/or host cell of the invention can be provided to patients (e.g., humans) having al growth retardation disorders (e.g. achondroplasia) in ation with pharmaceutically acceptable excipients, carriers, or diluents.

Acceptable excipients, carriers, or diluents can include buffers, antioxidants, preservatives, polymers, amino acids, and carbohydrates. Aqueous ents, rs, or diluents can e water, wateralcohol solutions, emulsions or suspensions including saline, buffered medical parenteral vehicles including sodium de solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, 40 Ringer's solution containing lactose, and fixed oils. Examples of non-aqueous excipients, carriers, or ts are propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters. 18870863_1 (GHMatters) P110456.NZ.1 Pharmaceutically acceptable salts can also be included in the compositions including an sFGFR3 polypeptide (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)), polynucleotide, vector, and/or host cell of the invention. Exemplary pharmaceutically acceptable salts can include mineral acid salts (e.g., hydrochlorides, hydrobromides, ates, and sulfates) and salts of organic acids (e.g., acetates, nates, malonates, and benzoates). Additionally, auxiliary substances, such as g or emulsifying agents and pH ing substances, can be t. A thorough discussion of pharmaceutically acceptable excipients, carriers, and diluents is available in Remington: The e and Practice of Pharmacy, 22nd Ed., Allen (2012), which is hereby incorporated by reference in its ty.

Pharmaceutical compositions including an sFGFR3 polypeptide (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)), polynucleotide, vector, and/or host cell of the invention can also be formulated with a carrier that will protect the sFGFR3 polypeptide against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. For example, the sFGFR3 composition can be entrapped in microcapsules prepared by vation techniques or by interfacial polymerization, such as hydroxymethylcellulose, n, or poly- (methylmethacylate) microcapsules; colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nano-particles, or nanocapsules); or macroemulsions. Additionally, an sFGFR3 ition can be formulated as a sustained-release composition. For example, sustainedrelease compositions can include semi-permeable matrices of solid hydrophobic polymers containing the sFGFR3 polypeptides, polynucleotides, vectors, or host cells of the invention, in which the matrices are in the form of shaped articles, such as films or microcapsules.

Kits Kits of the ion can include one or more sFGFR3 polypeptides (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)), polynucleotides, vectors, and/or cells of the invention as described herein. For example, the sFGFR3 polypeptide, polynucleotide, vector, and/or cell can be present in a container (e.g., a glass vial) in liquid form (e.g., in water or a ed salt solution, such as, 2 mM to 20 mM of sodium phosphate, pH 6.5 or 7.0, and 25 mM to 250 mM sodium chloride). Alternatively, the sFGFR3 polypeptide, polynucleotide, and/or vector is t in a ner (e.g., a glass vial) in lyophilized form, which can optionally include a diluent (e.g., water or a buffered salt solution) for reconstitution of the lyophilized sFGFR3 polypeptide, polynucleotide, vector, and/or cell into liquid form prior to administration. The sFGFR3 polypeptide, polynucleotide, vector, and/or cell can also be present in a kit in r formulation as described herein. The kit components can be ed in dosage form to facilitate administration, and optionally, can include materials required for administration and/or ctions for patient treatment consistent with the methods. For example, the kit can include instructions for use, which guides the user (e.g., the physician) with respect to the administration of the 40 sFGFR3 ptide, cleotide, vector, and/or cell. 18870863_1 (GHMatters) P110456.NZ.1 EXAMPLES The ing examples are intended to illustrate, rather than limit, the disclosure. These studies feature the administration of the sFGFR3 polypeptides of sFGFR3_Del4-C253S (SEQ ID NO: 2) and _Del4-D3 (SEQ ID NO: 33) to patients (e.g., humans) having achondroplasia, to treat achondroplasia and symptoms associated ith.

Example 1: Production of sFGFR3 Polypeptides sFGFR3_Del4-C253S (SEQ ID NO: 2) and sFGFR3_Del4-D3 (SEQ ID NO: 33) were produced by transient transfection in three different suspension cell types: HEK 293 freestyle, CHO-S freestyle cells and Expi CHO-S cells. For tion in HEK 293 freestyle and CHO-S freestyle cells, ection was performed using polyethylenimine (PEIpro® - us-transfection), according to the manufacturer’s directions. Proteins were harvested after three days. For sFGFR3 polyp eptide production in Expi CHO-S cells, transfection was performed using Expifectamine as described by the manufacturer using the High Titer production ol. A time course was performed and sFGFR3 polypeptides were optimally harvested after 12 days. Western blots were then performed using 50 ng of sFGFR3 polypeptide.

Classical western blot protocols were used with B9 as a primary antibody (anti FGFR3, 21, Santa Cruz) diluted 1:2000 in blocking buffer and anti-mouse IgG secondary antibody (Anti-mouse IgG, #7076, Cell signaling) diluted 1:5000 in blocking buffer.

Example 2: Purification of sFGFR3 Polypeptides sFGFR3_Del4-C253S and sFGFR3_Del4-D3 were each purified using a two-step purification process ing ion exchange chromatography and size exclusion chromatography.

For ion exchange chromatography, 300 mL of culture supernatant was purified by cross flow filtration (ÄKTA™ flux, GE Healthcare) using 5 µm and 0.2 µm capsules (KGF-A0504 TT and KMP-HEC 9204 TT, GE care, respectively). The purified sample including sFGFR3_Del4-C253S or sFGFR3_Del4-D3 was then loaded on an equilibrated column at 20 mL/min, after adjusting the sample’s conductivity to 14 mS/cm (ÄKTA™ pure 25 (GE Healthcare)). Columns used were HiPrep Q FF 26/10 (GE Healthcare) with a bed volume of 53 mL. The binding buffer was 1X PBS and the elution buffer was PBS 1X + 1 M NaCl. The column was washed with four column volumes of 1X PBS. Elution of sFGFR3_Del4-C253S and sFGFR3_Del4-D3 was performed by two steps of 5% NaCl and 10% NaCl using four column volumes of each. Both 5% NaCl and 10% NaCl were pooled and concentrated by cross flow tion (ÄKTA™ flux, GE Healthcare). The remaining volume was then concentrated on a 30 kDa filter by centrifugation at 4°C, 3,900 g for 10 min (MILLLIPORE® UFC903024 AMICON® Ultra-15 Centrifugal Filter Concentrator). For size exclusion chromatography, t he ing volume was loaded on a HiLoad 26/600 SUPERDEX™ 200 prep grade (2836, GE care) with a bed volume of 320 mL. Loading volume did not exceed 12.8 mL. Elution was performed in 1X PBS.

Example 3: Kinetic Assays and Dissociation Constant (Kd) Measurements of sFGFR3 ptides Calibration Free Concentration Analysis and kinetic assays of sFGFR3_Del4-C253S and 40 sFGFR3_Del4-D3 were performed with a Sensor Chip CM5 (GE Healthcare). Human FGF2 (hFGF2) was ntly immobilized to the Sensor Chip CM5 at a level of about 5000 RU by amine coupling. To achieve 5000 RU, hFGF2 was lized for 420 seconds at a flow rate 10 µl/min and a concentration 18870863_1 (GHMatters) P110456.NZ.1 µg/ml. g buffer was HBS-EP+ Buffer (GE Healthcare). Regeneration buffer was 100mM sodium acetate with 2M sodium chloride pH 4.5. FGF g, dissociation constant (Kd) measurements, and kinetic ters were determined by Surface Plasmon nce using a BIACORE™ T200 (GE Healthcare). The model used for kinetic assays and Kd determination was a 1:1 binding algorithm.

Example 4: Proliferation Assays of sFGFR3 Polypeptides Both ATDC5 and ATDC5 FGFR3G380R cell lines were seeded at a density of 25,000 cells/cm2 in NUNC™ MICROWELL™ 96-Well Optical-Bottom Plates with Polymer Base (ThermoFisher Scientific, Catalog No. 165305). After a 24 hour incubation period, cells were depleted for 48 hour in 0.5 % BSA and then stimulated for 72 hour with _Del4-C253S or sFGFR3_Del4-D3 with and t hFGF2 (Peprotech). Cell proliferation was then ed using the CyQUANT® Direct Cell Proliferation Assay (Molecular Probes, Catalog No. C35012). After stimulation, 10µL of CyQUANT® Direct Cell Proliferation (Invitrogen; 1mL 1X PBS, 250µL background suppressor, and 50µL nuclear stain) was added per well.

ATDC5 and ATDC5 FGFR3G380R cells were then incubated at room temperature in the dark for 2 hours.

Fluorescence was read using the VARIOSKAN™ LUX ode microplate reader (ThermoFisher Scientific). e 5: Luciferase Assays of sFGFR3 ptides Serum Response Element-Luciferase (SRE-Luc) HEK cells expressing FGFR3G380R were seeded at a density of 100,000 cells/cm2 in a standard culture 96 well plate. Cells were then depleted for 24 hours with 0.5% heat inactivated Fetal Bovine Serum (hiFBS), before being treated with sFGFR3_Del4- D3 at concentrations of 0 nm, 70 nm, and 280 nm with or without 1 ng/ml of hFGF2 for 24h. The culture plate was equilibrated to room temperature for 15 minutes prior to adding 100µL per well of Firefly Luc One-Step Glow Assay Working Solution (ThermoFisher Scientific, Catalog No. 16197), then shaken at 600 rpm for 3 minutes. The plate was incubated at room ature for 10 s and each cell lysate was transferred to a white opaque 96 well plate to increase luminescence signal and decrease cross contamination. The luminescence signal was read using the VARIOSKAN™ LUX multimode microplate reader (ThermoFisher Scientific).

Example 6: In vivo Efficacy Study of sFGFR3 Polypeptides ments were performed on transgenic Fgfr3ach/+ animals in which expression of the mutant FGFR3 is driven by the Col2a1 er/enhancer. Mice were exposed to a 12 hour 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: 30) and 5’- TCTGTTGTGTTTCCTCCCTGTTGG-3’ (SEQ ID NO: 31), which amplify 360 bp of the FGFR3 transgene. sFGFR3_Del4-D3 produced using CHO cells was evaluated at a subcutaneous dose of 0.25 mg/kg twice weekly. At day 3, all newborn mice from a single litter received the same dose. Control s received 10 µl of PBS (vehicle). Thereafter, subcutaneous injections of sFGFR3_Del4-D3 (0.25 mg/kg) were administered twice a week for three weeks, alternatively on the left and right sides of the back. Mice 40 were observed daily with particular attention to locomotion and urination tions. Breeding was med to generate litters with half wild type and half heterozygous Fgfr3ach/+ mice. To avoid bias due to phenotype penetrance variations, experiments were performed on at least two litters (one treated and 18870863_1 (GHMatters) P110456.NZ.1 one l) from the same breeders. Previous data indicated there was no statistical difference between males and females, and thus, males and females were considered one group for all analyses.

At day 22, all animals were sacrificed by lethal injection of pentobarbital, and gender was determined. All subsequent ements and analyses were performed without dge of mice genotype to avoid investigator bias. Genotyping was performed at the end of the study to reveal the correspondence of data with a specific pe. Since achondroplasia is a disease with phenotypic variability, all animals were included in the study. Animals dead before day 22 were used to investigate the impact of treatment on premature death. Surviving animals at day 22 were used for all analyses. All experiments and data measurements were performed by blinded experimenters at all time points.

Following sacrifice at day 22, body s were measured. Cadavers were carefully d, eviscerated, and skeletal measurements were performed based on X-rays. Organs were harvested, d, and stored in 10% formalin for further histological analysis using standard paraffin-embedded techniques. Organs were then observed for macroscopic abnormalities, such as modification of color or texture and presence of nodules. The Principles of Laboratory Animal Care (NIH publication no. 85–23, revised 1985; http://grants1.nih.gov/grants/olaw/references/phspol.htm) and the an commission ines for the protection of animals used for scientific purposes (http://ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm) were followed during all animal experiments. All procedures were approved by the Institutional Ethic tee for the use of Laboratory Animals (CIEPAL Azur) (approval # NCE52).

Example 7: The Cell Line used to produce sFGFR3 Polypeptides did not impact Activity The FGF2 g activity, Kd, and effect on cellular signaling of sFGFR3_Del1 (SEQ ID NO: 7), sFGFR3_Del4 (SEQ ID NO: 1), and sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 10) produced in suspension HEK 293 cells or CHO cells were compared. HEK 293 cells or CHO cells differ in post-translation modification of proteins. Expression of the sFGFR3 polypeptides in ent cell lines did not impact Kd, g activity, or the effect of the sFGFR3 polypeptides on intracellular signaling inhibition (FIGS. 1A- Example 8: Improved Production of sFGFR3_Del4-C253S and sFGFR3_Del4-D3 The sFGFR3 polypeptides of sFGFR3_Del1 (SEQ ID NO: 7), sFGFR3_Del4 (SEQ ID NO: 1), and sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 10) were each modified to include either an amino acid substitution of a cysteine residue with a serine e at position 253 or an extended Ig-like C2-type domain 3 (SEQ ID NO: 33). These modifications of sFGFR3_Del1 and sFGFR3_Del4-LK1-LK2 had no or minimal effect on production of the sFGFR3 polypeptides, since aggregation was still visible (FIGS. 2A and 2B, respectively). Surprisingly, cation of sFGFR3_Del4 to include either an amino acid substitution of a cysteine residue with a serine e at position 253 (sFGFR3_Del4-C253S) or an extended Ig-like C2- type domain 3 (SEQ ID NO: 33)) improved production of the sFGFR3 ptides. In particular, there was minimal ation of sFGFR3_Del4-C253S and sFGFR3_Del4-D3 under both reducing and nonreducing ions (). The inclusion of C253S or D3 also resulted in a relative increase in 40 production compared to sFGFR3_Del4, a two-fold increase in sFGFR3_Del4-C253S production and a 3- fold increase in sFGFR3_Del4-D3 production. 18870863_1 (GHMatters) P110456.NZ.1 Additionally, sFGFR3_Del4, _Del4-C253S, and sFGFR3_Del4-D3 exhibited similar Kd and were not affected by cell type specific changes in post translational modifications. In Expi CHO cells, the Kd of sFGFR3_Del4 was 0.8 nM, the Kd of sFGFR3_Del4-C253S was 0.6 nM, and the Kd of sFGFR3_Del4-D3 was 0.7 nM ( and Table 1).

Table 1. Dissociation constant (Kd) of sFGFR3 polypeptides. sFGFR3 Polypeptide Kd (nM) sFGFR3_Del4 0.8 sFGFR3_Del4-C253S 0.6 sFGFR3_Del4-D3 0.7 Example 9: sFGFR3_Del4-C253S and sFGFR3_Del4-D3 are Equally Active In Vitro sFGFR3_Del4, sFGFR3_Del4-C253S, and sFGFR3_Del4-D3 restored proliferation of ATDC5 cells genetically modified to overexpress the FGFR3ach mutation (ATDC5 380R cell lines). At a dose of 36 nM, sFGFR3_Del4 produced using HEK 293 cells increased proliferation to 115.5%, sFGFR3_Del4 produced using CHO-S cells sed proliferation to 116%, _Del4-C253S produced using CHO-S cells increased proliferation to 114.4%, and sFGFR3_Del4-D3 using CHO-S cells increased proliferation to 120.1% (). sFGFR3_Del4-D3 was also tested in the 380R expressing SRE(-Luc) HEK cell line at doses of 0 nM, 70 nM, and 280nM with or without 1 ng/ml of hFGF2 ( n=8). Data shown in are the mean +/- standard error of the mean (SEM). These data followed a normal law and have equal variance based on the D'Agostino- Pearson omnibus normality test. Statistical comparisons with and without sFGFR3_Del4-D3 were med using a student t-test. As shown in sFGFR3_Del4-D3 decreases luciferase signalling in the SRE cell line.

Example 10: sFGFR3_Del4-D3 restores Bone Growth, prevents Mortality, and restores Foramen Magnum Shape in Mice with Achondroplasia An in vivo efficacy study was performed as in e 6 using a low dose (0.25 mg/kg) of _Del4-D3. A total of 60 mice were included in the vehicle group, with 32 wild type (wt) mice and 28 Fgfr3ach/+ mice. The treated group included 40 mice, with 19 wt mice and 21 Fgfr3ach/+ mice.

Surprisingly, the low dose of sFGFR3_Del4-D3 almost completely ted the premature death of mice with achondroplasia (. In the l group, 53.6% of the Fgfr3ach/+ mice died before weaning, whereas only 4.8% of mice in the treated group died before day 22 and 20% of mice died following treatment with sFGFR3_Del1 at 0.25 mg/kg (Table 2; see also Garcia et al. Sci. Transl. Med. 5:203ra124, 2013, hereby incorporated by reference in its entirety). sFGFR3_Del4-D3 also partially ed bone growth with correction of the initial discrepancy between wt and Fgfr3ach/+ mice on the axial and appendicular on (Table 2). In contrast to prior results of treatment with a low dose of sFGFR3_Del1, treatment with low dose of sFGFR3_Del4-D3 restored normal foramen magnum shape. 18870863_1 (GHMatters) P110456.NZ.1 Table 2. In vivo results of stering a high dose of _Del1, a low dose of sFGFR3_Del1, and a low dose of sFGFR3_Del4-D3 to mice with roplasia 2.5 mg/kg 0.25 mg/kg 0.25 mg/kg sFGFR3_Del1 sFGFR3_Del1 sFGFR3_Del4-D3 (Garcia et al.) a et al.) Mortality 12% 20% 4.8% Axial correction 77% 24% 10% Appendicular correction 150-215% 18-42% 11-42% Foramen shape Not determined Not ined 111% correction (ratio W/H) Example 11: Treatment of Achondroplasia by Administration of sFGFR3_Del4-C253S A human patient (e.g., an infant, child, adolescent, or adult) suffering from achondroplasia can be treated by administering sFGFR3_Del4-C253S ( SEQ ID NO: 2) by an appropriate route (e.g., by subcutaneous injection) at a particular dosage (e.g., between 0.0002 mg/kg/day to about 20 mg/kg/day, such as 0.001 mg/kg/day to 7 mg/kg/day) over a course of days, weeks, months, or years. The progression of roplasia that is treated with sFGFR3_Del4-C253S can be monitored by one or more of several established methods. A physician can monitor the patient by direct observation in order to evaluate how the symptoms of achondroplasia ted by the patient have changed in response to treatment. For instance, a physician may monitor changes in body weight, skull length, and/or skull width of the patient 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 sFGFR3_Del4-C253S. Body weight and/or skull size of the patient or changes thereof can also be determined at treatment specific , e.g. before and/or after administration of sFGFR3_Del4-C253S. For example, body weight and/or skull siz e are measured in response to administration of sFGFR3_Del4-C253S.

Example 12: Treatment of Achondroplasia by Administration of sFGFR3_Del4-D3 Additionally, a human patient (e.g., an infant, child, adolescent, or adult) suffering from achondroplasia can be treated by administering the sFGFR3 polypeptide of sFGFR3_Del4-D3 (SEQ ID NO: 33) by an appropriate route (e.g., by subcutaneous ion) at a particular dosage (e.g., between 0.0002 mg/kg/day to about 20 mg/kg/day, such as 0.001 mg/kg/day to 7 mg/kg/day) over a course of days, weeks, months, or years. The progression of achondroplasia that is treated with sFGFR3_Del4-D3 can be monitored by one or more of several established methods. A physician can monitor the patient by direct observation in order to evaluate how the symptoms of achondroplasia exhibited by the patient have changed in response to treatment. For instance, a physician may monitor changes in body , skull length, and/or skull width of the patient 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 sFGFR3_Del4-D3. Body weight and/or skull size of the patient or s thereof can also be determined at treatment specific events, e.g. before and/or after administration of sFGFR3_Del4-D3. For e, body weight and/or skull size are measured in response to administration of sFGFR3_Del4-D3.

Example 13: Production of _Del4-D3 and sFGFR3_Del4-C253S The _Del4-D3 and sFGFR3_Del4-C253S polypeptides were purified as described in Example 2. Modification of sFGFR3_Del4 to include either an ed Ig-like C2-type domain 3 18870863_1 (GHMatters) P110456.NZ.1 (FGFR3_Del4-D3) or an amino acid substitution of a cysteine residue with a serine residue at position 253 (sFGFR3_Del4-C253S) improved production of the sFGFR3 polypeptides. In particular, there was less than about 2% aggregation of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S (as observed upon loading using a tration of 2.3 mg/ml or 23 mg/ml for FGFR3_Del4-D3 and 1.5 mg/ml and 15 mg/ml of sFGFR3_Del4-C253S) under both reducing and non-reducing conditions using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; FIGS. 7A and 7B, respectively). Following production of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S in fed-batch cultures, the top five clones were separated using capillary electrophoresis to yield 0.93 to 1.0 g/L and 0.98 to 1.1 g/L of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S, respectively. Viral filtration using ion-exchange chromatography ed in a yield of greater than 60% for both sFGFR3_Del4-D3 and sFGFR3_Del4-C253S.

Example 14: Pharmacokinetics and Tissue bution of _Del4-D3 in vivo In vivo studies were med to investigate the pharmacokinetic parameters of sFGFR3_Del4- D3, the uptake of sFGFR3_Del4-D3 across the blood brain barrier, and the tissue distribution of _Del4-D3 in kidney, liver, spleen, lung, and heart. The studies described herein included four arms with five groups of C57BL/6J mice per arm and a total of four mice (n=4) per group (Table 3). Mice were male and weighed 25 to 30 grams.

Table 3. Overview of mice used in s of sFGFR3_Del4-D3.

Arm sFGFR3_Del4- Route PK BBB Tissue D3 (mg/kg) distribution 1 0.25 SC yes no no 2 2.5 SC yes no yes 3 2.5 IV yes Yes yes 4 10 SC yes no no Group 1 was sampled at 1 minute, 15 minutes, and 30 minutes; group 2 was sampled at 4 hours; group 3 was sampled at 24 hours; group 4 was d at 36 hours; and group 5 was sampled at 48 hours. For Group 1, an indwelling intra-arterial catheter (PE-10) was inserted into one common carotid artery under isoflurane anesthesia and used for repeated blood ng at the 30 minute final sampling time point. For intravenous injection, 125I- sFGFR3_Del4-D3 was injected intravenously into the jugular vein, which was exposed by skin incision under isoflurane anesthesia. Group 1 mice remained anesthetized throughout the experiments. Repeated blood s (2 x ~50µL) were drawn from the arterial er at 1 minute and 15 s after enous injection. For groups 2 to 5, after injection of 125I- sFGFR3_Del4-D3, the skin was closed with a surgical clip, and the mice were allowed to wake up and returned to the cage. At 5 minutes before termination time for group 3, mice were re-anesthetized and received an intravenous bolus of 3H-albumin into the jugular vein. The 3H tracer dose was targeted to yield a ratio of 125I to 3H in blood, which is suitable for double isotope labeling with a lower dose at later sampling times. At the terminal sampling time (2 hours, 3 hours, 24 hours, 36 hours, and 48 hours), a blood sample was collected, and the animal was euthanized. The brain was sampled for homogenization and determination of tissue concentration of tracers. Endpoints of the studies included pharmacokinetic parameters for sFGFR3_Del4-D3 (terminal half life), uptake of sFGFR3_Del4-D3 across the blood brain barrier, and the tissue distribution of _Del4-D3 in kidney, liver, spleen, lung, and heart. 18870863_1 (GHMatters) P110456.NZ.1 Example 15: Thermal and Plasma Stability of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S The thermal stability of sFGFR3_Del4-D3 and _Del4-C253S in mouse plasma was investigated using differential scanning colorimetry. For sFGFR3_Del4-D3, two buffers (20 mM phosphate, 40mM NaCl, pH 7.5, and 20 mM citrate, 40mM NaCl, pH 6.5) were added to polypeptide samples. For _Del4-C253S, two buffers (20 mM phosphate, 40mM NaCl, pH 7.5, and 40 mM e, 40mM NaCl, pH 6.5) were added to polypeptide samples. The melting temperature (Tm) for sFGFR3_Del4-C253S in the 20 mM phosphate, 40mM NaCl, pH 7.5 buffer was 52°C and 56°C, and the Tm for sFGFR3_Del4-C253S in the 40 mM citrate, 40mM NaCl, pH 6.5 buffer was 55°C and 60°C ( 8A). For sFGFR3_Del4-D3, two buffers (20 mM phosphate, 40mM NaCl, pH 7.5, and 20 mM citrate, 40mM NaCl, pH 6.5) were added to polypeptide samples. The Tm for sFGFR3_Del4-D3 in the 20 mM phosphate, 40mM NaCl, pH 7.5 buffer was 50°C and 54°C, and the Tm for sFGFR3_Del4-D3 in the 20 mM citrate, 40mM NaCl, pH 6.5 buffer was 53°C and 58°C (). These s indicate that both sFGFR3_Del4-D3 and sFGFR3_Del4-C253S show two domains of polypeptide stability and unfolding.

The ex vivo plasma stability of sFGFR3_Del4-D3 with a Histidine tag was determined by labeling purified sFGFR3_Del4-D3 with 125I-tracer using the Bolton-Hunter method, followed by purification on PD- (Sephadex® G-25) columns. The trichloroacetic acid (TCA) precipitability of peak fractions was also determined to confirm stability of the racer. Mouse plasma (n = 4) pre-warmed to 37°C was spiked with the 125I-sFGFR3_Del4-D3 to a concentration of ~10 cpm/mL and then vortexed. The plasma samples were incubated with the FGFR3_Del4-D3 in an Eppendorf ThermoMixer® under gentle rotation (300 rpm). Aliquots were then collected for TCA precipitation (10 µl sample and 100 µl 2% BSA) and for injection onto an Fast Performance Liquid Chromatography (FPLC) column (20 µl sample and 150 µl 10 mM PBS, pH 7.4) at intervals of 0, 30, 60, 120, 180, and 360 minutes. Aliquots were stored on ice until TCA precipitation or FPLC injection was performed.

For TCA precipitation, 1 mL ice cold 10% TCA was added to plasma samples, incubated for 10 minutes on ice, centrifuged at 4,000g for 5 minutes, and then the atant and pellet were separated and both were counted in a gamma counter. For evaluation of the ex vivo plasma stability, 100 µl of the sample was injected on an FPLC column (Superdex® 200 10/300 GL) and eluted at a rate of 0.75 ml/min for 1.5 column volumes. ons of 1 ml were ted from the column and then ed in a gamma counter. The plasma ity of sFGFR3_Del4-D3 at 37°C was determined to be 95% at 0 s, 95% at 2 hours, and ~92% at 24 hours with only minor aggregation ().

The in vivo stability of sFGFR3_Del4-D3 in plasma after administration by intravenous and aneous injection was also determined. sFGFR3_Del4-D3 was d with 125I-tracer using the Bolton-Hunter method, followed by purification on PD-10 (Sephadex® G-25) columns. The 125I-labeled sFGFR3_Del4-D3 (10 µCi in ~50 µL PBS) was administered by intravenous or subcutaneous injection into anesthetized C57Bl/6 mice. The 125I-tracer protein dose (approximately 0.1 mg/kg) was complemented with unlabeled n to a total dose of 2.5 mg/kg. Rat serum albumin used as a ar marker was labeled with [3H]-NSP (N-succininidyl[2,3-3H]Propionate; Perkin Elmer) and purified on PD-10 (Sephadex® G25) columns. 40 For the stability of sFGFR3_Del4-D3 in plasma after intravenous bolus injection, FPLC elution profiles showed no degradation products in plasma up to 15 s (). At 30 minutes after administration of sFGFR3_Del4-D3, a small amount of low molecular weight degradation products 18870863_1 (GHMatters) P110456.NZ.1 ed, which increased by 2 hours, but largely disappeared by 24 hours. For the stability of sFGFR3_Del4-D3 in plasma after subcutaneous injection, FPLC elution profiles showed some degradation products in plasma at 30 s, with increased degradation by 2 hours and 4 hours (). The low amount of tracer left in plasma after 24 hours appears largely as the intact sFGFR3_Del4- D3 polypeptide. The lower panel chromatograms for FIGS. 9B and 9C are presented as normalized to the highest peak in each individual run for easier comparison of the elution patterns.

Example 16: Ligand Binding Activity of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S Experiments were performed to characterize the binding affinity of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S for human FGF2. The dissociation constant (Kd) of sFGFR3_Del4-D3 and Kd of sFGFR3_Del4-C253S for FGF2 were determined as described in Example 3 with a regeneration buffer of 20mM phosphate, 40mM NaCl, pH 7.5. Concentrations of 13 nM, 6.5 nM, 3.25 nM, and 1.75 nM were tested for both _Del4-D3 and sFGFR3_Del4-C253S. The Kd of sFGFR3_Del4-D3 was determined to be ~3.6 nm, and the Kd of sFGFR3_Del4-C253S was determined to be ~6.9 nm. These results indicate that sFGFR3_Del4-D3 and sFGFR3_Del4-C253S have binding activity for FGF2 in the low nM range.

Example 17: sFGFR3_Del4-D3 and _Del4-C253S Exhibit Functional ty in vitro Functional activity of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S was tested using a proliferation assay. Proliferation assays using ATDC5 cells cally modified to overexpress the FGFR3ach mutation (ATDC5 FGFR3G380R cell lines) were performed as described in Example 4 with trations of 1 ug/ml, 10 ug/ml, and 50 ug/ml for sFGFR3_Del4-D3 and sFGFR3_Del4-C253S. At each of these concentrations, sFGFR3_Del4-C253S and sFGFR3_Del4-D3 ed proliferation of the FGFR3G380R cells (A and 10B). The EC50 was determined to be about 10 nM for both sFGFR3_Del4-D3 and sFGFR3_Del4-C253S based on a concentration of 1 ug/ml. These results indicate that sFGFR3_Del4-D3 and sFGFR3_Del4-C253S are biologically active in the low nM range.

Example 18: Pharmacokinetic Profile of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S The pharmacokinetic (PK) e of sFGFR3_Del4-D3 stered subcutaneously or intravenously at a dose of 2.5 mg/kg was used to determine the terminal ation ife of sFGFR3_Del4-D3 (). Samples were collected at 30 minutes, 2 hours, 4 hours, 8 hours, 24 hours, 36 hours, and 48 hours for mice administered sFGFR3_Del4-D3 subcutaneously. Samples were collected at 1 minute, 15 minutes, 30 minutes, 2 hours, 24 hours, and 36 hours for mice administered sFGFR3_Del4-D3 intravenously. The subcutaneous terminal elimination half-life of 2.5 mg/kg sFGFR3_Del4-D3 was ~20 hours, while the intravenous terminal elimination half-life of 2.5 mg/kg sFGFR3_Del4-D3 was ~7 hours. From the PK e, the Tmax was ~8 hours, the Cmax was ~ 4.5 nM, and the estimated bioavailability was ~30% for 2.5 mg/kg sFGFR3_Del4-D3 administered subcutaneously.

There was rapid clearance of sFGFR3_Del4-D3 administered intravenously during the α phase followed by a slower β phase clearance, with a similar intravenous PK profile for sFGFR3_Del4-C253S. 18870863_1 (GHMatters) P110456.NZ.1 Example 19: The Kidney and Liver are the Main Clearance Routes of sFGFR3_Del4-D3 Clearance of sFGFR3_Del4-D3 was evaluated in kidney, liver, spleen, lung, and heart tissue after minutes, 120 minutes, and 1440 minutes following intravenous administration of 2.5 mg/kg sFGFR3_Del4-D3 and after 30 minutes, 120 minutes, 240 minutes, 480 minutes, and 1440 minutes following subcutaneous administration of 2.5 mg/kg sFGFR3_Del4-D3. The liver and kidney were the major route of sFGFR3_Del4-D3 clearance for enous administration (). The kidney was the major route of sFGFR3_Del4-D3 clearance for subcutaneous administration ().

Example 20: sFGFR3_Del4-D3 does not Cross the Blood Brain Barrier cokinetic studies were also performed to determine the uptake of sFGFR3_Del4-D3 across the blood brain barrier in ype mice. After intravenous bolus injection, brain tissue uptake of sFGFR3_Del4-D3 was measured at three time points (30 minutes, 2 hours, and 24 hours). sFGFR3_Del4-D3 was injected as radiolabeled tracer (125I- sFGFR3_Del4-D3) with 2.5 mg/kg unlabeled sFGFR3_Del4-D3. The injected dose of 125I- sFGFR3_Del4-D3 was about 10 µCi per animal, which corresponds to less than 0.1 mg/kg. After euthanizing the mice at 30 minutes, 2 hours, and 24 hours, the concentration of 125I- sFGFR3_Del4-D3 in organs and plasma was measured by liquid scintillation The 125I- sFGFR3_Del4-D3 concentration was ted for lism 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 ing samples on a size exclusion fast protein liquid chromatography (FPLC) column. The organ concentration of 125I- _Del4-D3 was corrected for intravascular content (V0) by injecting radiolabeled albumin (3H-RSA) shortly before sacrificing the animal. The apparent organ volume of distribution of RSA represents V0. The dose of albumin was negligible (on the order of 1% of the physiological concentration). For all organs other than the 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.

The brain concentrations were calculated by the following formula: (corr.) = [Vd(sFGFR3_Del4- D3) – V0] X Cplasma (terminal), in which Vd(sFGFR3_Del4-D3) is the volume of distribution of sFGFR3_Del4- D3 in brain (calculated as Cbrain / Cplasma), V0 is the volume of albumin distributed in the brain, and Cplasma(terminal) is the plasma concentration of sFGFR3_Del4-D3 at the terminal sampling time. All concentrations were sed as the t of injected dose per gram or ml (%ID/g or %ID/mL), respectively, and the dose of the intravenous bolus equals 100%. These values can be converted to [mg/g] or [mg/mL] by multiplication with the injected dose: (body weight in g /1000 g) x 2.5 mg. All body weights were in the range of 25 g – 30 g.

There was no detectable brain uptake of 125I- sFGFR3_Del4-D3, as indicated by corrected brain trations (after correction for ar content and degradation (TCA precipitability)) at at any of the measured time points (A). Additionally, the Vd of RSA (=V0) and 125I- sFGFR3_Del4-D3 was not significantly different at any of the measured time points (30 minutes, 2 hours, and 24 hours) as ined by a paired t-test (B). In conclusion, there is no able uptake of sFGFR3_Del4- 40 D3 into brain tissue of mice at 30 s, 2 hours, and 24 hours at a dose of 2.5 mg/kg injected as an intravenous bolus. 18870863_1 ters) P110456.NZ.1 Example 21: In Vivo Efficacy of sFGFR3_Del4-D3 for the Treatment of Achondroplasia sFGFR3_Del4-D3 and sFGFR3_Del4-C253S were each evaluated at a subcutaneous dose of 2.5 mg/kg once or twice weekly or 10 mg/kg twice weekly. Breeding was performed to te 30 litters with half wild type and half heterozygous Fgfr3ach/+ mice (Table 4).

Table 4. Subcutaneous administration of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S to wild type (WT) and ch/+ mice.

PBS 2.5mg 2.5mg 10mg (pooled) 1X week 2X week 2X week sFGFR3_Del4-D3 WT 65 26 22 23 Fgfr3ach/+ 43 26 25 30 total N= sFGFR3_Del4-C253S WT 65 26 22 23 Fgfr3ach/+ 27 22 18 28 total N= % survival 62.8 84.6 72.0 93.3 % mortality 37.2 15.4 28.0 6.7 At day 3, all newborn mice from a single litter received the same dose. l litters received 10 µl of PBS le). Thereafter, subcutaneous ions of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S were administered at doses of 2.5 mg/kg once or twice weekly or 10 mg/kg 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 ion alterations and weighed on days of injection. Mice with complications were observed twice a day for surveillance. Previous data indicated there was no statistical difference between males and females, and thus, males and s were considered one group for all analyses.

At day 22, all animals were sacrificed by lethal injection of pentobarbital, and gender was ined. All subsequent measurements and analyses were performed t knowledge of mice genotype to avoid igator bias. Genotyping was performed at the end of the study to reveal the correspondence of data with a specific genotype. Since roplasia is a disease with ypic variability, all animals were included in the study. Animals dead before day 22 were used to investigate the impact of treatment on premature death. Surviving animals at day 22 were used for all analyses. All experiments and data measurements were performed by blinded experimenters at all time points.

Subcutaneous administration of sFGFR3_Del4-D3 at 2.5 mg/kg once or twice weekly or 10 mg/kg twice weekly increased survival of Fgfr3ach/+ mice relative to Fgfr3ach/+ mice receiving PBS ( and Table 4). In particular, administration of 10 mg/kg sFGFR3_Del4-D3 twice weekly resulted in 93% survival of Fgfr3ach/+ mice, administration of 2.5 mg/kg sFGFR3_Del4-D3 once weekly resulted in 84% survival in Fgfr3ach/+ mice, and administration of 2.5 mg/kg sFGFR3_Del4-D3 twice weekly resulted in 72% survival in Fgfr3ach/+ mice, while the survival of Fgfr3ach/+ mice receiving PBS was 62.8%. The 18870863_1 (GHMatters) P110456.NZ.1 mortality of Fgfr3ach/+ mice administered 10 mg/kg sFGFR3_Del4-D3 twice weekly was 6.7%, the mortality of Fgfr3ach/+ mice administered 2.5 mg/kg sFGFR3_Del4-D3 once weekly was 15.4%, the mortality of Fgfr3ach/+ mice administered 2.5 mg/kg sFGFR3_Del4-D3 twice weekly was 28.0%, and the mortality of Fgfr3ach/+ mice administered PBS was 37.2%. Statistical analysis of Fgfr3ach/+ mice survival following treatment with sFGFR3_Del4-D3 was performed using the Agostino and Pearson omnibus normality test following by a t-test. All investigated groups passed the ity tests. T he P-values from these es are shown below, in which * represent a P-value of <0.05 and *** represents a P-value of <0.001 (Table 5).

Table 5. P-values for subcutaneous administration of sFGFR3_Del4-D3 to wild type (WT) and Fgfr3ach/+ mice.

Group Comparison P Value Wt vs ach *** Fgfr3ach/+ PBS vs ch/+ 2.5 mg/kg, 1x *** ch/+ PBS vs ch/+ 2.5 mg/kg, 2x * Fgfr3ach/+ PBS vs Fgfr3ach/+ 10 mg/kg, 2x *** Wt PBS vs ch/+ 10 mg/kg, 2x ns Subcutaneous stration of sFGFR3_Del4-D3 at 2.5 mg/kg once or twice weekly or 10 mg/kg twice weekly also decreased the severity and frequency of locomotor problems and complications in abdominal breathing in ch/+ mice relative to Fgfr3ach/+ mice receiving PBS (). In particular, locomotor problems decreased the most in Fgfr3ach/+ mice stered subcutaneously 10 mg/kg sFGFR3_Del4-D3 twice weekly followed by mice administered sFGFR3_Del4-D3 2.5 mg/kg twice weekly and mice administered sFGFR3_Del4-D3 2.5 mg/kg once weekly. Complications in abdominal ing decreased the most in Fgfr3ach/+ mice administered subcutaneously 10 mg/kg sFGFR3_Del4-D3 twice weekly followed by mice administered sFGFR3_Del4-D3 2.5 mg/kg once weekly and then mice administered sFGFR3_Del4-D3 2.5 mg/kg twice weekly. These results show that _Del4-D3 reduces symptoms of achondroplasia in Fgfr3ach/+ mice.

Subcutaneous administration of sFGFR3_Del4-D3 also significantly increased total body length, including axial length and tail length, and long bones (p = 0.07) in Fgfr3ach/+ mice receiving 2.5 mg/kg sFGFR3_Del4-D3 once or twice weekly or 10 mg/kg sFGFR3_Del4-D3 twice weekly relative to Fgfr3ach/+ mice receiving PBS (FIGS. 17A-17C). Tail and body length (axial length) were measured using the same digital r on whole skeletons. Tibia length was measured on digital X-rays. Administration of 10 mg/kg sFGFR3_Del4-D3 twice weekly resulted in 51% axial correction (body and tail length) of Fgfr3ach/+ mice, followed by 43% axial correction in Fgfr3ach/+ receiving 2.5 mg/kg sFGFR3_Del4-D3 twice weekly, and 39% axial tion in Fgfr3ach/+ mice receiving 2.5 mg/kg sFGFR3_Del4-D3 once . Increases in bone and body length were also evident from x-ray radiographs of Fgfr3ach/+ mice administered 2.5 mg/kg or 10 mg/kg sFGFR3_Del4-D3 twice weekly relative to Fgfr3ach/+ mice receiving PBS (D).

Administration of 10 mg/kg sFGFR3_Del4-D3 twice weekly resulted in 86% appendicular correction (tibia and femur length) of Fgfr3ach/+ mice, ed by 68% appendicular correction in ch/+ receiving 2.5 18870863_1 (GHMatters) P110456.NZ.1 mg/kg sFGFR3_Del4-D3 twice weekly and 54% appendicular correction in Fgfr3ach/+ mice receiving 2.5 mg/kg sFGFR3_Del4-D3 once weekly.

Subcutaneous stration of sFGFR3_Del4-D3 also resulted in a dose-dependent improvement in l ratio (length/width (L/W)) in Fgfr3ach/+ mice relative to Fgfr3ach/+ mice receiving PBS (A).

Fgfr3ach/+ mice subcutaneously administered 10 mg/kg _Del4-D3 twice weekly exhibited the greatest improvement in the cranium ratio (L/W), followed by Fgfr3ach/+ mice administered 2 mg/kg _Del4-D3 twice weekly and Fgfr3ach/+ mice administered 2 mg/kg sFGFR3_Del4-D3 once weekly.

In particular, administration of 10 mg/kg sFGFR3_Del4-D3 twice weekly ed in 37% skull shape correction (L/W ratio) of Fgfr3ach/+ mice, followed by 29% skull shape correction in Fgfr3ach/+ receiving 2.5 mg/kg sFGFR3_Del4-D3 twice weekly and 19% skull shape correction in Fgfr3ach/+ mice receiving 2.5 mg/kg sFGFR3_Del4-D3 once weekly. Improvements in the cranial ratio were also evident from x-ray radiographs of ch/+ mice administered 10 mg/kg _Del4-D3 relative to Fgfr3ach/+ mice receiving PBS (B). Bone measurements (presented in mm and mean ± SEM) for body length, tail, femur, tibia, and cranial ratio are shown below (Table 6). These results indicate the dose-dependent in vivo efficacy of sFGFR3_Del4-D3 as trated by increased al, reduced number of complications, increased bone growth, and improvements in skeletal proportions of Fgfr3ach/+ mice.

Table 6. Bone measurements (presented in mm and mean ± SEM) for body length, tail, femur, tibia, and cranial ratio of WT and Fgfr3ach/+ mice administered subcutaneously sFGFR3_Del4-D3. cy of sFGFR3_Del4-D3 WT PBS in 2.5 mg/kg 2.5 mg/kg 10 mg/kg Fgfr3ach/+ once weekly twice weekly twice weekly Body length 144.8 ± 0.53 129.2 ± 1.98 135 ± 1.48 135.5 ± 1.75 135.2 ± 1.58 Tail 77.65 ± 0.39 70.25 ± 1.1 73.37 ± 1.66 73.69 ± 1.5 74.95 ± 0.91 Femur 10.94 ± 0.05 10.14 ± 0.13 10.47 ± 0.08 10.58 ± 0.09 10.63 ± 0.10 Tibia 14.19 ± 0.05 13.67 ± 0.14 14.02 ± 0.10 14.09 ± 0.12 14.25 ± 0.12 Cranial ratio 1.99 ± 0.01 1.79 ± 0.01 1.83 ± 0.02 1.85 ± 0.01 1.86 ± 0.02 Additionally, comparison of the bone measurements for Fgfr3ach/+ mice administered sFGFR3_Del1 at a dosage of 2.5 mg/kg twice weekly show that administration sFGFR3_Del4-D3 at a dosage of 2.5 mg/kg twice weekly was comparable to or more ive in increasing the bone, tail, femur, and tibia length and improving the cranial ratio of Fgfr3ach/+ mice (Table 7). In particular, the body length of Fgfr3ach/+ mice administered sFGFR3_Del4-D3 improved to 135.5 ± 1.75 mm relative to 134.4 ± 1.17 mm for Fgfr3ach/+ mice administered sFGFR3_Del1; the tail length of Fgfr3ach/+ mice administered sFGFR3_Del4-D3 improved to 73.69 ± 1.5 mm relative to 71.58 ± 0.86 mm for Fgfr3ach/+ mice administered sFGFR3_Del1; the femur length of Fgfr3ach/+ mice administered sFGFR3_Del4-D3 improved to 10.58 ± 0.09 mm relative to 10.01 ± 0.06 mm for Fgfr3ach/+ mice administered sFGFR3_Del1; the tibia length of Fgfr3ach/+ mice administered sFGFR3_Del4-D3 improved to 14.09 ± 0.12 mm relative to 13.27 ± 0.31 mm for ch/+ mice administered _Del1; and the cranial ratio of Fgfr3ach/+ mice 18870863_1 (GHMatters) P110456.NZ.1 administered sFGFR3_Del4-D3 improved to 1.85 ± 0.01 mm relative to 1.81 ± 0.02 mm for Fgfr3ach/+ mice administered sFGFR3_Del1.

Table 7. Bone measurements (presented in mm and mean ± SEM) for body length, tail, femur, tibia, and cranial ratio of WT and ch/+ mice administered subcutaneously sFGFR3_Del1 (data described in Garcia et al. Sci. . Med. 5:203ra124, 2013).

Efficacy of sFGFR3_Del1 WT PBS in Fgfr3ach/+ 0.25 mg/kg 2.5 mg/kg mice twice weekly twice weekly body length 133.9 ± 0.8 118.5 ± 1.76 132.4 ± 1.26 134.4 ± 1.17 tail 71.9 ± 0.49 64.48 ± 1.1 71.05 ± 0.99 71.58 ± 0.86 femur 10.05 ± 0.17 9.67 ± 0.16 9.85 ± 0.10 10.01 ± 0.06 tibia 13.43 ± 0.19 12.62 ± 0.18 12.87 ± 0.14 13.27 ± 0.31 cranial ratio 1.94 ± 0.01 1.75 ± 0.01 1.77 ± 0.02 1.81 ± 0.02 Example 22: No Organ Toxicity Associated with Administration of sFGFR3_Del4-D3 Histopathological studies were performed to characterize organ ty associated with sFGFR3_Del4-D3 administration. Wild type mice (6 males and 6 females per dose) were administered PBS, 2.5 mg/kg sFGFR3_Del4-D3 once weekly, 2.5 mg/kg sFGFR3_Del4-D3 twice weekly, or 10 mg/kg sFGFR3_Del4-D3 twice weekly. Organs igated included the kidney, skin, salivary glands, mandibular lymph nodes, gall bladder, spleen, pancreas, lungs, heart, aorta, jejunum, colon, and liver. There were no histopathological results indicating organ toxicity in wild-type mice stered any of the doses of _Del4-D3. These results indicate that there was no toxicity associated with administration of sFGFR3_Del4-D3 up 10 mg/kg twice weekly.

Example 23: Determination of g Affinity of sFGFR3_Del4-D3 to Fibroblast Growth Factors We determined that sFGFR3_Del4-D3 binds to Fibroblast Growth s (FGF) ligands and acts as a decoy to prevent the binding of FGFs to the membrane bound FGFR3. Surface Plasmon Resonance was performed using a BIACORE™ T200 (GE Healthcare) to ine the Kd values for different human FGFs (hFGFs) binding to immobilized sFGFR3_Del4-D3. In particular, Kd values for the paracrine hFGFs of hFGF1 (A), hFGF2 (B), hFGF9 (C), and hFGF18 (D) and the endocrine hFGFs of hFGF19 (E) and hFGF21 (F) were determined. All four paracrine FGF ligands bound sFGFR3_Del4-D3 with lar (nM) affinity (Table 8).

Table 8. Summary of Kd determination and values for human, paracrine FGFs (hFGF1, hFGF2, hFGF9, and hFGF18) and human, endocrine FGFs (hFGF19 and hFGF21). 18870863_1 (GHMatters) P110456.NZ.1 Paracrine Binding ka1 ka2 kd1 kd2 KD (M) Chi2 KD (M) Chi2 FGFs (1/Ms) (1/Ms) (1/s) (1/s) Kinetic (RU2) Steady (RU2) average state average FGF1 binding 2.0* 1.2* 1610 6.4 * 2.6* 10-9 0.138 5.7* 10-9 0.247 & 10+11 10-3 10-4 (+/- 1.9*10- (+/-2.1*10-9, steady 9, n = 3) n=3) state 1:1 6.1* 10-10 FGF2 binding 9.0* 4.75* (+/- 1.7*10- 13.6 +5 10-4 10, n = 3) FGF9 binding 2.3* 3.0* 2.6* 3.6* 1.8* 10-9 0.14 3.6* 10-9 0.25 & 10+6 10-2 10-2 10-3 (+/- (n = 1) steady 0.25*10-9, state n = 3) FGF18 binding 2.0* 9.1* 4.5* 10-9 9.7 6.4*10-9 11.8 & 10+5 10-3 (+/- 2.5*10- (+/- steady 9, n = 3) 0.89*10-9, state n=4) Endocrine 2:1 4.8* 10-7 FGF19 g 5.4* 7.3* 1.5* 3.6* (+/- 3.2*10- 0.05 +4 10-3 10-1 10-3 7, n = 3) 2:1 2.8* 10-5 FGF21 binding 258 1.8* 5.5* 1.4* (n = 2) 0.56 -2 10-3 10-3 For FGF2 and FGF18, a good fit was achieved with a 1:1 binding model, which is the most direct model of binding affinity. This model describes a 1:1 binding interaction at the surface of the chip with immobilized SFGFR3_DEL4-D3 binding different FGFs: A + B = AB with single on- and off rate. The 2:1 model also describes a 1:1 interaction of FGF binding to _DEL4-D3, but also assumes a conformational change that stabilizes the complex: A + B = AB = AB* and represents two on- and offrates.

This model assumes that the mationally changed complex (SFGFR3_DEL4-D3 bound to FGF) can only dissociate by reversing the conformational change. The experimental data for hFGF1, hFGF9, hFGF19, and hFGF21 were determined to fit the 2:1 model very well, and thus, Kd for hFGF1, hFGF9, hFGF19, and hFGF21 were derived from the 2:1 model.

Despite hFGF1, hFGF9, hFGF19, and hFGF21 all having a Kd in the low nM range, the kinetic profiles of these hFGFs differed significantly. For example, FGF1 binds sFGFR3_Del4-D3 with a very fast on-rate and off-rate, while FGF2 does not bind sFGFR3_Del4-D3 with as fast of an on-rate or off-rate as FGF1, resulting in an overall smaller Kd for FGF2 compared to FGF1 (Table 8). A significantly lower affinity was ed between sFGFR3_Del4-D3 and hFGF19 or , which are members of the endocrine FGF15/FGF19 subfamily, relative to the paracrine hFGFs (Table 8 and FIGS. 19D and 19E).

The FGF15/FGF19 subfamily uses Klotho d of proteoglycans as a co-factor and has evolved into endocrine-acting growth factors, which are important for the ic regulation of metabolic parameters, such as phosphate, bile acid, carbohydrate, and lipid metabolism.

These results demonstrate that there was a high affinity ction of sFGFR3_Del4-D3 with hFGF1, hFGF2, hFGF9, and hFGF18, while there was a low ty interaction of _Del4-D3 with FGF19 and FGF21. The low affinity of _Del4-D3 for FGF19 and FGF21 is advantageous as sFGFR3_Del4-D3 will have a low probability of interfering with the function of these FGFs in vivo. 18870863_1 (GHMatters) P110456.NZ.1 Example 24: In Vitro Proliferation Assay of sFGFR3_Del4-D3 Following binding of FGFs, FGFR3 dimerizes to initiate ing cascades. Several downstream signaling pathways are associated with FGF signaling. In chondrocytes, dimerized FGFR3 results in an anti-proliferative signal/early differentiation signal into the ocyte, which eventually leads to inhibition of bone growth. For e, the RAS/MAPK pathway propagates signals to negatively affect proliferation, terminal differentiation, and post-mitotic matrix sis, and the STAT1 pathway mediates the inhibition of chondrocyte proliferation in concert with the cell cycle regulators p107 and 130 and cell cycle inhibitor p21Waf/Cip1. Gene expression studies suggest a number of other ys are also ed in down-regulation of growth-promoting molecules or induction of roliferative functions.

To study FGFR3-decoy induced inhibition of FGFR3G380R in a chondrocytic cell model, studies were performed to determine the effect of sFGFR3_Del4-D3 on the proliferation of ATDC5 cells genetically modified to overexpress the FGFR3ach mutation (ATDC5 FGFR3G380R . The chondrocytic cell line ATDC5 cell, which was first isolated from the differentiating teratocarcinoma stem cell line AT805, is commonly used as a model for in vitro chondrocyte research. ATDC5 cells were first ed with a retroviral expression vector and a stable cell line expressing 380R was generated. The expression of FGFR3G380R in the ATDC5 cell line was ined via n blot (). Extracts of ATDC5 cells expressing FGFR3G380R at passage one (G380R #1) and two (G380R #2) after resistant cell selection and extracts of l ATDC5 cells were blotted and detected with antibodies for total phosphorylation of FGFR3 (pFGFR3), the specific phosphotyrosine 724 in FGFR3 (pFGFR3 Y724), and total FGFR3 expression (FGFR3). Total extracellular signal-related kinase expression was used as loading control (ERK). Addition of SFGFR3_DEL4-D3 to the ATDC5 FGFR3G380R cells dose-dependently increased the proliferation index of the ATDC5 FGFR3G380R cells by two-fold with an EC50 of 1.25 +/- 0.27 nM ().

These results demonstrate that on of SFGFR3_DEL4-D3 to ATDC5 FGFR3G380R cells overcomes the negative growth signal mediated by FGFR3G380R in a cellular model of achondroplasia and are in line with the roliferative signal mediated by FGFR3 in chondrocytes, which is more pronounced when the chondrocytes s a FGFR3 including the G380R on.

OTHER EMBODIMENTS All publications, patents, and patent applications mentioned in the above ication are hereby incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are s to those skilled in the art are intended to be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present 40 sure come within known customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth. 18870863_1 ters) P110456.NZ.1

Claims (98)

1. A soluble fibroblast growth factor receptor 3 (sFGFR3) polypeptide comprising a polypeptide sequence having at least 90% amino acid sequence identity to amino acid residues 23 to 357 of SEQ ID 5 NO: 32, wherein the polypeptide lacks a signal peptide and a transmembrane domain of FGFR3 and (i) is less than 500 amino acids in length; (ii) comprises 200 consecutive amino acids or fewer of an intracellular domain of FGFR3; or (iii) lacks a ne kinase domain of FGFR3.

2. The polypeptide of claim 1, wherein the polypeptide is less than 475, 450, 425, 400, 375, or 350 10 amino acids in length.

3. The polypeptide of claim 1, wherein the ptide comprises 175, 150, 125, 100, 75, 50, 40, 30, 20, 15, or fewer consecutive amino acids of the intracellular domain of FGFR3. 15

4. The polypeptide of any one of claims 1 to 3, wherein the polypeptide further comprises an amino acid sequence having at least 90%, 92%, 95%, 97%, or 99% sequence identity to amino acid residues 423 to 435 of SEQ ID NO: 32.

5. The polypeptide of any one of claims 1 to 3, wherein the polypeptide further comprises amino acid 20 residues 423 to 435 of SEQ ID NO: 32.

6. The polypeptide of any one of claims 1 to 5, wherein the polypeptide ses an amino acid ce having at least 92%, 95%, 97%, 99%, or 100% sequence identity to amino acid residues 23 to 357 of SEQ ID NO: 32.

7. The polypeptide of any one of claims 1 to 5, wherein the polypeptide ses an amino acid sequence having at least 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 33.

8. The polypeptide of any one of claims 1 to 5, wherein the polypeptide comprises or consists of SEQ ID 30 NO: 33.

9. A soluble last growth factor or 3 (sFGFR3) polypeptide comprising an amino acid sequence having at least 85% sequence ty to the amino acid ce of SEQ ID NO: 1, wherein the polypeptide further comprises an amino acid tution that removes a cysteine residue at position 35 253 of SEQ ID NO: 1.

10. The polypeptide of claim 9, wherein the cysteine residue at position 253 is substituted with a serine residue. 40

11. The polypeptide of claim 9 or 10, wherein the polypeptide comprises an amino acid sequence having at least 87%, 90%, 92%, 95%, 97%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. 18870863_1 (GHMatters) P110456.NZ.1

12. The polypeptide of any one of claims 9 to 11, wherein the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 2. 5

13. The polypeptide of any one of claims 1 to 12, wherein the polypeptide is an ed sFGFR3 polypeptide.

14. The polypeptide of any one of claims 1 to 13, wherein the polypeptide binds to fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (FGF9), fibroblast growth 10 factor 18 (FGF18), fibroblast growth factor 19 (FGF19), or fibroblast growth factor 21 (FGF21).

15. The polypeptide of claim 14, wherein the binding is characterized by an equilibrium dissociation constant (Kd) of about 0.2 nM to about 20 nM. 15

16. The polypeptide of claim 15, wherein the g is characterized by a Kd of about 1 nM to about 10 nM, wherein ally the Kd 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.

17. The polypeptide of any one of claims 1 to 16, wherein the polypeptide is encoded by a 20 polynucleotide comprising a nucleic acid ce having at least 85%, 87%, 90%, 92%, 95%, 97%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NOs: 20, 21, 36, or 37.

18. The polypeptide of claim 17, n the polynucleotide comprises or consists of the nucleic acid ce of SEQ ID NOs: 20, 21, 36, or 37.

19. The polypeptide of claim 17 or 18, wherein the polynucleotide is an isolated polynucleotide.

20. A vector comprising the polynucleotide of any one of claims 17 to 19. 30

21. The vector of claim 20, wherein the vector is selected from the group ting of a plasmid, an artificial chromosome, a viral vector, and a phage vector.

22. The vector of claim 21, wherein the vector is an isolated polynucleotide. 35

23. A host cell comprising the polynucleotide of any one of claims 17 to 19 or the vector of any one of claims 20 to 22.

24. The host cell of claim 23, wherein the host cell is an isolated host cell. 40

25. The host cell of claim 23 or 24, wherein the host cell is a HEK 293 cell or CHO cell.

26. A composition sing the polypeptide of any one of claims 1 to 16, the polynucleotide of any 18870863_1 (GHMatters) P110456.NZ.1 one of claims 17 to 19, the vector of any one of claims 20 to 22, or the host cell of any one of claims 23 to

27. The composition of claim 26, further comprising a pharmaceutically acceptable excipient, carrier, or 5 diluent.

28. The composition of claim 26 or 27, wherein the composition is formulated for administration at a dose of about 0.002 mg/kg to about 30 mg/kg. 10

29. The composition of claim 28, wherein the composition is formulated for stration at a dose of about 0.001 mg/kg to about 10 mg/kg.

30. The composition of any one of claims 26 to 29, wherein the composition is formulated for daily, weekly, or monthly administration.

31. The composition of any one of claims 26 to 30, wherein the composition is formulated for stration seven times a week, six times a week, five times a week, four times a week, three times a week, twice a week, , every two weeks, or once a month. 20

32. The composition of claim 31, n the composition is formulated for administration at a dose of about 2.5 mg/kg to about 10 mg/kg once or twice a week.

33. The composition of any one of claims 26 to 32, wherein the composition is formulated for eral administration, enteral administration, or topical administration.

34. The composition of claim 33, wherein the composition is formulated for subcutaneous administration, intravenous administration, intramuscular administration, intra-arterial administration, intrathecal administration, or intraperitoneal administration. 30

35. The composition of claim 34, wherein the composition is ated for subcutaneous administration.

36. A medicament comprising the composition of any one of claims 26 to 35. 35

37. A method of treating a al growth retardation disorder in a patient comprising stering the polypeptide of any one of claims 1 to 16, the polynucleotide of any one of claims 17 to 19, the vector of any one of claims 20 to 22, the host cell of any one of claims 23 to 25, the composition of any one of claims 26 to 35, or the ment of claim 36. 40

38. A method of delivering a polypeptide to tissue in a patient having a skeletal growth retardation disorder comprising administering to the patient an effective amount of the polypeptide of any one of claims 1 to 16, the polynucleotide of any one of claims 17 to 19, the vector of any one of claims 20 to 22, 18870863_1 (GHMatters) P110456.NZ.1 the host cell of any one of claims 23 to 25, the composition of any one of claims 26 to 35, or the medicament of claim 36.

39. The method of claim 38, wherein the tissue is skeletal tissue.

40. The method of any one of claims 37 to 39, wherein the skeletal growth retardation er is a FGFR3-related skeletal disease.

41. The method of claim 40, wherein the FGFR3-related skeletal e is selected from the group 10 consisting of achondroplasia, thanatophoric dysplasia type I (TDI), thanatophoric sia type II (TDII), severe achondroplasia with developmental delay and acanthosis nigricans (SADDEN), hypochondroplasia, a craniosynostosis me, and camptodactyly, tall e, and hearing loss syndrome (CATSHL). 15

42. The method of claim 41, wherein the skeletal growth retardation disorder is achondroplasia.

43. The method of claim 41, n the craniosynostosis syndrome is selected from the group consisting of Muenke syndrome, Crouzon syndrome, and Crouzonodermoskeletal syndrome. 20

44. The method of any one of claims 40 to 43, wherein the FGFR3-related skeletal disease is caused by expression in the patient of a constitutively active FGFR3.

45. The method of claim 44, n the constitutively active FGFR3 comprises an amino acid substitution of a glycine residue with an arginine residue at position 380 of SEQ ID NO: 5.

46. The method of any one of claims 37 to 45, wherein the patient has been diagnosed with the al growth retardation disorder.

47. The method of any one of claims 37 to 46, wherein the patient exhibits one or more symptoms of 30 the skeletal growth retardation disorder selected from the group consisting of short limbs, short trunk, bowlegs, a waddling gait, skull malformations, cloverleaf skull, craniosynostosis, wormian bones, anomalies of the hands, anomalies of the feet, hitchhiker thumb, and chest anomalies.

48. The method of claim 47, wherein the patient exhibits an improvement in the one or more symptoms 35 of the skeletal growth retardation disorder after administration of the ptide.

49. The method of any one of claims 37 to 48, wherein the t has not been previously administered the polypeptide. 40

50. The method of any one of claims 37 to 49, wherein the patient is selected from the group consisting of an infant, a child, an cent, and an adult. 18870863_1 (GHMatters) P110456.NZ.1

51. The method of any one of claims 37 to 50, wherein the patient is a human.

52. The method of any one of claims 37 to 51, wherein the polypeptide is stered to the patient at a dose of about 0.002 mg/kg to about 30 mg/kg.

53. The method of claim 52, wherein the polypeptide is administered to the t at a dose of about 0.001 mg/kg to about 10 mg/kg.

54. The method of any one of claims 37 to 53, wherein the ptide is administered to the t 10 one or more times daily, weekly, y, or yearly.

55. The method of any one of claims 37 to 54, n the polypeptide is administered to the patient seven times a week, six times a week, five times a week, four times a week, three times a week, twice a week, weekly, every two weeks, or once a month.

56. The method of claim 55, wherein the polypeptide is administered to the patient at a dose of about 0.25 mg/kg to about 20 mg/kg at least about once or twice a week or more.

57. The method of claim 56, wherein the polypeptide is administered to the patient at a dose of about 20 2.5 mg/kg or about 10 mg/kg once or twice weekly.

58. The method of any one of claims 37 to 57, n the polypeptide is administered to the patient in a composition comprising a pharmaceutically able excipient, carrier, or diluent. 25

59. The method of claim 58, wherein the composition is administered to the patient subcutaneously, intravenously, intramuscularly, intra-arterially, intrathecally, or intraperitoneally.

60. The method of claim 59, wherein the composition is administered to the patient by subcutaneous injection.

61. The method of claim 59 or 60, wherein the polypeptide has an in vivo half-life of between about 2 hours to about 25 hours.

62. The method of any one of claims 37 to 61, wherein administration of the polypeptide increases 35 survival of the patient.

63. The method of any one of claims 37 to 62, wherein administration of the polypeptide improves locomotion of the patient. 40

64. The method of any one of claims 37 to 63, wherein administration of the polypeptide improves abdominal breathing in the patient. 18870863_1 (GHMatters) P110456.NZ.1

65. The method of any one of claims 37 to 64, wherein administration of the polypeptide increases body and/or bone length of the patient.

66. The method of any one of claims 37 to 65, wherein administration of the polypeptide improves the 5 cranial ratio and/or restores foramen magnum shape in the patient.

67. A method of producing the polypeptide of any one of claims 1 to 16, comprising culturing the host cell of any one of claims 23 to 25 in a culture medium under conditions suitable to effect expression of the polypeptide and recovering the polypeptide from the culture medium.

68. The method of claim 67, wherein the ring comprises chromatography.

69. The method of claim 68, wherein the chromatography comprises affinity chromatography or size exclusion chromatography.

70. The method of claim 69, wherein the affinity chromatography comprises ion exchange chromatography or anti-FLAG tography.

71. The method of claim 70, wherein the anti-FLAG chromatography comprises immunoprecipitation.

72. The polypeptide of any one of claims 1 to 16 for treating a skeletal growth retardation er in a

73. The polypeptide of claim 72, wherein the skeletal growth retardation disorder is a related 25 skeletal disease.

74. The polypeptide of claim 73, wherein the FGFR3-related skeletal disease is selected from the group consisting of achondroplasia, TDI, TDII, SADDEN, ondroplasia, a craniosynostosis syndrome, and CATSHL.

75. The polypeptide of claim 74, wherein the FGFR3-related skeletal disease is achondroplasia.

76. The polypeptide of claim 75, wherein the craniosynostosis syndrome is selected from the group consisting of Muenke me, n syndrome, and Crouzonodermoskeletal me.

77. The ptide of any one of claims 73 to 76, wherein the FGFR3-related skeletal disease is caused by sion in the patient of a constitutively active FGFR3.

78. The polypeptide of claim 77, wherein the constitutively active FGFR3 comprises an amino acid 40 substitution of a glycine residue with an arginine residue at position 380 of SEQ ID NO: 5.

79. The polypeptide of any one of claims 72 to 78, wherein the patient has been diagnosed with the 18870863_1 (GHMatters) P110456.NZ.1 skeletal growth retardation disorder.

80. The polypeptide of any one of claims 72 to 79, wherein the patient exhibits one or more ms of the skeletal growth retardation disorder selected from the group consisting of short limbs, short trunk, 5 bowlegs, a waddling gait, skull malformations, cloverleaf skull, craniosynostosis, wormian bones, anomalies of the hands, anomalies of the feet, hitchhiker thumb, and chest anomalies.

81. The polypeptide of claim 80, wherein the patient exhibits an improvement in the one or more symptoms of the skeletal growth retardation disorder after stration of the sFGFR3 polypeptide.

82. The polypeptide of any one of claims 72 to 81, wherein the t has not been previously administered the polypeptide.

83. The ptide of any one of claims 72 to 82, wherein the patient is selected from the group 15 consisting of an infant, a child, an adolescent, and an adult.

84. The polypeptide of any one of claims 72 to 83, wherein the patient is a human.

85. The polypeptide of any one of claims 72 to 84, wherein the polypeptide is administered to the 20 patient at a dose of about 0.002 mg/kg to about 30 mg/kg.

86. The polypeptide of claim 85, n the ptide is administered to the patient at a dose of about 0.001 mg/kg to about 10 mg/kg. 25

87. The polypeptide of any one of claims 72 to 86, wherein the polypeptide is administered to the patient one or more times daily, weekly, monthly, or yearly.

88. The polypeptide of any one of claims 72 to 87, wherein the polypeptide is administered to the t seven times a week, six times a week, five times a week, four times a week, three times a week, 30 twice a week, weekly, every two weeks, or once a month.

89. The polypeptide of claim 88, wherein the polypeptide is administered to the patient at a dose of about 2.5 mg/kg to about 10 mg/kg twice a week. 35

90. The polypeptide of any one of claims 72 to 89, wherein the polypeptide is administered to the patient in a ition comprising a pharmaceutically acceptable excipient, carrier, or diluent.

91. The polypeptide of claim 90, wherein the composition is stered to the patient parenterally, enterally, or topically.

92. The polypeptide of claim 91, wherein the composition is administered to the patient subcutaneously, intravenously, uscularly, intra-arterially, intrathecally, or intraperitoneally. 63_1 (GHMatters) P110456.NZ.1

93. The polypeptide of claim 92, wherein the ition is administered to the patient by subcutaneous injection. 5

94. The polypeptide of any one of claims 72 to 93, wherein the polypeptide binds to fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (FGF9), fibroblast growth factor 18 (FGF18), fibroblast growth factor 19 (FGF19), or fibroblast growth factor 21 (FGF21).

95. A method of manufacturing the polypeptide of any one of 1 to 8, said method comprising deleting 10 the signal peptide, the embrane domain, and a portion of the intracellular domain from a FGFR3 polypeptide.

96. The method of claim 95, n the portion of the intracellular domain consists of amino acid residues 436 to 806 of SEQ ID NO: 32.

97. A method of manufacturing the polypeptide of any one of claims 9 to 12, said method comprising introducing an amino acid substitution that removes a cysteine residue at on 253 of SEQ ID NO: 1.

98. A kit comprising the polypeptide of any one of claims 1 to 16, the polynucleotide of any one of 20 claims 17 to 19, the vector of any one of claims 20 to 22, or the host cell of any one of claims 23 to 25, wherein the kit optionally ses instructions for using the kit. 18870863_1 (GHMatters) P110456.NZ.1 sFGFR3_Del4 _Del1 sFGFR3_Del4-LK1-LK2 (secs) _Del1 sFGFR3_Del1-D3 (secs) _Del4-LK1-LK2-C253S sFGFR3_Del4-LK1-LK2 sFGFR3_Del4-LK1-LK2-D3 (secs) bilit y) sta sFGFR3_Del4-C253S (100 = 60 _Del4-D3 asel in b (0 = se Respon sFGFR3_Del4 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Time (secs) sFGFR3_Del1 _Del1-D3 sFGFR3_Del1-C253S NR NR NR kDa sFGFR3_Del1 sFGFR3_Del1-D3 Sfgfr3_DeL1-c253S R R R sFGFR3_Del4-LK1-LK2-C253S sFGFR3_Del4-LK1-LK2 NR _Del4-LK1-LK2-D3 NR NR sFGFR3_Del4-LK1-LK2 sFGFR3_Del4-LK1-LK2-C253S sFGFR3_Del4-LK1-LK2-D3 R R kDa R sFGFR3_Del4-C253S sFGFR3_Del4-D3 _Del4 NR NR sFGFR3_Del4 NR sFGFR3_Del4-C253S sFGFR3_Del4-D3 R kDa R kDa R _Del4-C253S sFGFR3_Del4-D3 ) e 300 in l ase sFGFR3_Del4 0 = b ( e 200 s Res pon 100 hFGFR2a Embrel -500 0 500 1000 1500 2000 Time (secs) 80 100