RU2794170C2 - TREATMENT OF ABNORMAL VISCERAL FAT DEPOSITION WITH FIBROBLAST GROWTH FACTOR 3 (sFGFR3) SOLUBLE POLYPEPTIDES - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for treating or reducing abnormal visceral fat deposition in a patient with an FGFR3-associated skeletal disorder.

EFFECT: control of abnormal visceral fat deposition and related conditions in patients with disorders of skeletal growth retardation, including after completion of bone growth.

17 cl, 7 dwg, 3 tbl, 2 ex

Description

BACKGROUND OF THE INVENTION

Achondroplasia, the most common form of short limb dwarfism, is a rare genetic disease for which there is no cure. In many patients, a G380R substitution in the transmembrane domain of the fibroblast growth factor receptor 3 (FGFR3) (Fgfr3ach) results in a gain-of-function mutation that prolongs intracellular MAPK signaling. In the growth plate, MAPK signaling is inhibitory and its subsequent constitutive activation results in inhibition of chondrocyte proliferation and differentiation. Cells expressing the mutant receptor do not mature and are not replaced by mineralized bone matrix, resulting in abnormally short bones.

Achondroplasia is also characterized by early obesity, which is a serious health problem for these patients, affecting approximately 50% of patients during childhood. Obesity increases the morbidity associated with lumbar lordosis as well as the physical impact of existing orthopedic complications, for example, increasing stress on already fragile knees. It can also increase the risk of serious complications such as cardiovascular risks, obstructive sleep apnea, or restrictive lung disease. The reasons for this increased susceptibility to obesity in patients with achondroplasia are not known, but do not appear to be related to hormonal or neurological dysfunction that may lead to decreased appetite regulation such as hyperphagia.

Obese patients undergoing achondroplasty may also suffer from associated metabolic complications such as dyslipidemia, low insulin levels, and dysregulation of glucose. It is not clear whether these complications are isolated and related to external factors such as excessive caloric intake and/or reduced physical activity, or whether they truly reflect an underlying defect in achondroplasia.

Abnormal fat deposition, and more specifically, abnormal visceral fat deposition, in the general population is also associated with the development of specific diseases, including cardiovascular, metabolic, pulmonary, reproductive, and neurological diseases. There is a need for medical preparations to treat or prevent the development of abnormal visceral fat deposition and related diseases.

SUMMARY OF THE INVENTION

In a first aspect, the application describes a method for treating or reducing abnormal fat deposition (e.g., visceral fat deposition) in a patient (e.g., human, such as a fetus, newborn, infant, child, adolescent, or adult) by administering a soluble growth factor receptor polypeptide to the patient. fibroblast 3 (sFGFR3), a polynucleotide encoding an sFGFR3 polypeptide, or a host cell that contains a polynucleotide encoding it. In some embodiments, the implementation of the abnormal deposition of visceral fat binds or surrounds one or more of the following organs: heart, liver, spleen, kidneys, pancreas, intestines, reproductive organs and gallbladder; or abnormal deposition of visceral fat causes disease in one or more of the following organs: heart, lungs, trachea, liver, pancreas, brain, reproductive organs, arteries, and gallbladder; or abnormal deposition of visceral fat is caused by dysfunction of an endocrine organ such as the adrenal gland, pituitary gland, or a reproductive organ such as the ovary. The method may reduce or eliminate or reduce the risk of developing one or more conditions associated with abnormal fat distribution, such as obstructive sleep apnea, lung disease, cardiovascular disease, metabolic disorder, neurological disease, dyslipidemia, hypertension, atherosclerosis, myocardial infarction, stroke, dementia, infertility, menstrual disorders, dysregulation of insulin secretion, and dysregulation of glucose. In particular, dyslipidemia is an abnormal level of one or more of triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and cholesterol; the cardiovascular disease is heart disease or stroke; lung disease is asthma and restrictive lung disease; the neurological disease is dementia or Alzheimer's disease; metabolic disorder is type 2 diabetes, glucose intolerance, non-alcoholic fatty liver disease and hepatotoxicity; dysregulation of insulin secretion is insulin resistance.

In one embodiment, the patient having abnormal fat deposition is not overweight, does not have significant subcutaneous fat deposition, and/or may not show significant abnormal extra-abdominal fat deposition. Abnormal fat storage can be determined using an anthropometric method (eg, body mass index (BMI) or android:gynoid fat ratio) or imaging (eg, computed tomography (CT), magnetic resonance imaging (MRI), and dual-energy X-ray absorptiometry (DXA). ), methods that can detect abnormal fat distribution in the absence of other fat phenotypes.

In other embodiments, the patient may have a skeletal growth retardation disorder, such as an FGFR3-associated skeletal disease (e.g., an FGFR3-associated skeletal disease caused by the patient's expression of a FGFR3 variant that exhibits ligand-dependent overactivation, such as a FGFR3 variant having an amino acid replacement of a glycine residue with an arginine residue at position 358 (G358R) as set forth in SEQ ID NO: 9). FGFR3 related skeletal disease may include, for example, achondroplasia, thanatophoric dysplasia type I (TDI), thanatophoric dysplasia type II (TDII), severe achondroplasia with developmental delay and acanthosis nigricans (SADDEN), hypochondroplasia, craniosynostosis syndrome (eg. , Muencke syndrome, Crouzon syndrome, and Crouzon syndrome with acanthosis nigricans), and camptodactyly, tall stature, and hearing loss syndrome (CATSHL). A patient suffering from skeletal growth retardation disorder may have one or more symptoms of skeletal stunting disorder selected from the group including short limbs, short torso, O-shaped curvature of the legs, duck gait, skull malformations, cloverleaf syndrome, craniosynostosis, wormian bones, hand anomalies, leg anomalies, an elongated thumb, and chest anomalies. The methods described herein may exclude a patient with a skeletal growth retardation disorder such as described above, such as achondroplasia, after bone growth has ceased in the patient (e.g., fetus, neonate, infant, child, and/or adolescent). ).

In an alternative embodiment, the patient does not have a skeletal growth retardation disorder, but can be characterized as being obese, polycystic ovary syndrome, or a form of hypercortisolism such as Cushing's disease.

In other embodiments, the sFGFR3 has at least 50 amino acid sequences of the extracellular domain of the natural fibroblast growth factor receptor 3 (FGFR3) polypeptide (e.g., 100-370 amino acid sequences of the extracellular domain of the natural fibroblast growth factor receptor 3 (FGFR3) polypeptide or less than 350 amino acids of the extracellular domain of the natural FGFR3 polypeptide). An sFGFR3 polypeptide may have an Ig-like domain of C2 type 1, 2 and/or 3 of a native FGFR3 polypeptide. An sFGFR3 polypeptide in particular lacks a signal peptide and/or a transmembrane domain, such as the signal peptide and/or transmembrane domain of a natural FGFR3 polypeptide. Alternatively, the sFGFR3 polypeptide is a mature polypeptide. A native FGFR3 polypeptide may have the amino acid sequence GenBank Accession No. NP_000133.

An sFGFR3 polypeptide may have 400 amino acid sequences or less of the extracellular domain of a native FGFR3 polypeptide (e.g., between 5 and 399 amino acid sequences, such as 175, 150, 125, 100, 75, 50, 40, 30, 20, 15 or fewer amino acids sequences of the extracellular domain of the native FGFR3 polypeptide). An sFGFR3 polypeptide may have an amino acid sequence having at least 90%, 92%, 95%, 97%, or 99% sequence identity with amino acids 401-413 of SEQ ID NO: 8 (for example, an sFGFR3 polypeptide may have amino acids 401- 413 sequences of SEQ ID NO: 8). In some embodiments, the sFGFR3 polypeptide lacks the tyrosine kinase domain of a native FGFR3 polypeptide, lacks the extracellular domain of a native FGFR3 polypeptide, or is less than 475, 450, 425, 400, 375, 350, 300, 250, 200, 150, or 100 amino acids in length. In other embodiments, the sFGFR3 polypeptide has an amino acid sequence having at least 85% sequence identity (e.g., 86%-100% sequence identity) with amino acid residues 1-280 of SEQ ID NO: 8. In still other embodiments, implementation of the sFGFR3 polypeptide has an amino acid sequence having at least 85% sequence identity (for example, 86%-100% sequence identity) with the sequence of SEQ ID NO: 1-7. The sFGFR3 polypeptide may also have a signal peptide, such as the signal peptide of a naturally occurring FGFR3 polypeptide (eg, a signal peptide having the amino acid sequence of SEQ ID NO: 21). The sFGFR3 polypeptide may also have a heterologous polypeptide (eg, a crystallizable immunoglobulin fragment (Fc region) or human serum albumin (HSA)).

In other embodiments, a polynucleotide encoding an sFGFR3 polypeptide may have a nucleic acid sequence having at least 85% and up to 100% sequence identity with the nucleic acid sequence of any of SEQ ID NOs: 10-18 (e.g., the polynucleotide may consist of nucleic acid sequences of any of SEQ ID NOs: 10-18). The polynucleotide may be an isolated polynucleotide and/or may be in a vector (eg, a vector selected from the group consisting of a plasmid, an artificial chromosome, a viral vector, and a phage vector). The vector may be in a host cell (eg, an isolated host cell, such as a host cell from a patient, or a HEK 293 cell or a CHO cell). The host cell can also be transformed with a polynucleotide encoding sFGFR3.

In other embodiments, the sFGFR3 polypeptide binds to fibroblast growth factor (FGF), wherein the FGF is selected from the group consisting of fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (FGF9), fibroblast growth factor 10 (FGF10), fibroblast growth factor 18 (FGF18), fibroblast growth factor 19 (FGF19), fibroblast growth factor 21 (FGF21), and fibroblast growth factor 23 (FGF23). Binding to FGF can be characterized by an equilibrium dissociation constant (K _d ) from about 0.2 nm to about 20 nm, or a K _d from about 1 nm to about 10 nm (e.g., K _d is about 1 nm, about 2 nm, about 3 nm, approximately 4 nm, approximately 5 nm, approximately 6 nm, approximately 7 nm, approximately 8 nm, approximately 9 nm or approximately 10 nm).

One aspect of treatment involves administering an sFGFR3 polypeptide, a polynucleotide encoding an sFGFR3 polypeptide, or a host cell containing a polynucleotide encoding an sFGFR3 polypeptide to a patient, e.g., a human such as a "naive" patient who has not yet been treated with an sFGFR3 polypeptide). The sFGFR3 polypeptide may be administered in compositions containing a pharmaceutically acceptable excipient, carrier, or diluent. Possible doses are from about 0.001 mg/kg to about 30 mg/kg (eg, from about 0.01 mg/kg to about 10 mg/kg). The administration may be daily, weekly or monthly. The dosing frequency can be 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. The dosing method may be subcutaneous, intravenous, intramuscular, intra-arterial, intrathecal, intraperitoneal, parenteral, enteral, or topical. It can also be re-introduced.

In other embodiments, the sFGFR3 polypeptide has an in vivo half-life ranging between about 2 hours to about 25 hours.

A second aspect of the invention is a composition comprising a soluble fibroblast growth factor receptor 3 (sFGFR3) polypeptide, a polynucleotide encoding an sFGFR3 polypeptide, or a host cell containing the polynucleotide, for the treatment or reduction of abnormal fat distribution in a patient in need thereof (e.g., a human ), for example, in accordance with the method of the first aspect of the invention.

A third aspect of the invention is the use of a soluble fibroblast growth factor receptor 3 (sFGFR3) polypeptide, a polynucleotide encoding an sFGFR3 polypeptide, or a host cell containing a polynucleotide encoding an sFGFR3 polypeptide in the manufacture of a medicament for the treatment or reduction of abnormal fat distribution in a patient in need thereof. (eg, in humans), for example, in accordance with the method of the first aspect of the invention.

DEFINITIONS

Within the scope of the invention, a reference in the singular means "at least one" or "one or more", unless otherwise indicated. In addition, singular forms include plural references unless the context clearly dictates otherwise.

The phrase "abnormal visceral fat deposition" refers to a level of fat deposition in the omentum, mesentery, retroperitoneum, and pericardium that exceeds the level of fat deposition seen in a normal patient, as determined by anthropometric or imaging techniques (see, e.g., Methods of Diagnosis ”, below, which lists the important values of each method for determining the threshold that distinguishes normal from abnormal visceral fat deposition). For example, patients with abnormal visceral fat deposits are those whose visceral fat deposits are equal to or greater than 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100 %, 200%, 500%, 750%, 1000% or more) limit value of visceral fat deposition compared to a normal patient. Typically, abnormal visceral fat deposition is associated with a subgroup of obese patients, such as patients with a BMI >30 kg/m ² . Abnormal deposition of visceral fat can be the result of various conditions. These include, for example, skeletal growth retardation syndromes (eg, achondroplasia), hypercortisolism (eg, Cushing's disease), and polycystic ovary syndrome.

Within the scope of the invention, the term "about" refers to an amount that is ±10% of the specified value, and preferably ±5% of the specified value, or more preferably ±2% of the specified value. For example, the term "approximately" can be used to change all dosages or ranges specified herein by ±10% of the given values or endpoints of the range.

The phrase "anthropometric methods" refers to measurements of body composition based on height, weight, waist and hip circumference, including body mass index (BMI), android:gynoid fat ratio, waist circumference, and sagittal diameter (SD); see, for example, Shuster et al., Br. J. Radiol. 85(1009):1-10, 2012).

The phrase "cardiovascular disease" refers to diseases of the heart and blood vessels, in particular atherosclerosis, myocardial infarction and hypertension.

The phrase "conditions associated with abnormal visceral fat deposition" refers to diseases observed in patients in whom visceral fat deposition is shown to be abnormal compared to normal patients. In particular, these conditions include cardiovascular disease, lung disease, metabolic disease, reproductive disease, and neurological disease.

The term "domain" refers to a conserved region of the amino acid sequence of a polypeptide (eg, an FGFR3 polypeptide) having an identifiable structure and/or function in the polypeptide. The length of a domain may vary from, for example, about 20 amino acids to about 600 amino acids. Exemplary domains include FGFR3 immunoglobulin domains (e.g., C2-type 1 Ig-like domain, C2-type 2 Ig-like domain, and C2-type 3 Ig-like domain), FGFR3 extracellular domain (ECD), FGFR3 extracellular domain (ICD) or a transmembrane domain (TM) of FGFR3, such as FGFR3 having the sequence shown as SEQ ID NO: 8).

The term "dosage" refers to a specific amount of an active agent (e.g., an sFGFR3 polypeptide or variant thereof, such as a polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-7, or a variant thereof having at least 85% to 100% sequence identity) calculated to produce the desired therapeutic effect (e.g., treatment of abnormal visceral fat deposition or a condition associated with visceral fat deposition) when the active agent is administered to a patient (e.g., a patient having abnormal visceral fat deposition or a condition associated with deposition of visceral fat). The dosage may be defined in terms of a specific amount of active agent, or a specific amount in combination with a specific frequency of administration. The dosage form may include the sFGFR3 polypeptide or fragment thereof in combination with any suitable pharmaceutical excipient, carrier or diluent.

The terms "effective amount", "amount effective for" and "therapeutically effective amount" refer to an amount of an sFGFR3 polypeptide, an sFGR3 encoding vector, and/or an sFGFR3 composition sufficient to produce the desired result, for example, to reduce abnormal fat deposition, abnormal deposition of visceral fat or reduction of symptoms associated with conditions associated with abnormal deposition of visceral fat.

The terms "extracellular domain" and "ECD" refer to the portion of the FGFR3 polypeptide that extends beyond the transmembrane domain into the extracellular space. The ECD mediates FGFR3 binding to one or more fibroblast growth factors (FGFs). For example, the ECD includes the C2-type 1-3 Ig-like domains of the FGFR3 polypeptide. In particular, the ECD includes the Ig-like domain C2 type 1 wild-type (wt) FGFR3 polypeptide, the Ig-like domain 2 C2 wild-type (wt) FGFR3 polypeptide, and/or the type 3 Ig-like domain 3 wt FGFR3 polypeptide. The FGFR3 ECD may also include, for example, a fragment of the IgG-like domain of the C2-type wild-type FGFR3.

The phrase "fat deposition" refers to deposition of visceral fat or deposition of subcutaneous fat.

The term "FGFR3-associated skeletal disease", as used herein, refers to a skeletal disease that is caused by an abnormal increase in FGFR3 activation, such as the expression of an FGFR3 up-function mutant. The phrase "FGFR3 up-function mutant" refers to an FGFR3 mutant exhibiting a biological activity, such as triggering downstream signaling, that is greater than that of a corresponding wild-type FGFR3 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 8) in the presence of FGF ligand. FGFR3-associated skeletal disorders may include hereditary or sporadic disease. Typical FGFR3-associated skeletal disorders include, but are not limited to, achondroplasia, thanatophoric dysplasia type I (TDI), thanatophoric dysplasia type II (TDII), severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN), hypochondroplasia, craniosynostosis syndrome (e.g. , Muencke syndrome, Crouzon syndrome and Crouzon syndrome with acanthosis nigricans) and camptodactyly, tall stature and hearing loss syndrome (CATSHL).

The terms "fibroblast growth factor" and "FGF" refer to a member of the FGF family, which includes structurally related signaling molecules involved in various metabolic processes, including endocrine signaling pathways, development, wound healing, and angiogenesis. FGFs play a key role in the proliferation and differentiation of a wide range of cell types and tissues. The term preferably refers to FGF1, FGF2, FGF9, FGF 10, FGF18, FGF19, FGF21 and FGF23 which exhibit binding to FGFR3. For example, FGFs may include human FGF1 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 26), human FGF2 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 27), human FGF9 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 28), human FGF10 (for example, a polypeptide having the amino acid sequence of SEQ ID NO: 40), human FGF18 (for example, a polypeptide having the amino acid sequence of SEQ ID NO: 29), human FGF19 (for example, a polypeptide having the amino acid sequence SEQ ID NO: 30), human FGF21 (eg, a polypeptide having the amino acid sequence of SEQ ID NO: 31), and human FGF23 (eg, a polypeptide having the amino acid sequence of SEQ ID NO: 41).

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, FGF10, FGF18, FGF19, FGF 21 and/or FGF23). The human FGFR3 gene, which is located on the distal short arm of chromosome 4, encodes a precursor of the amino acid protein 806 (precursor of fibroblast growth factor receptor isoform 1 3), which contains 19 exons and includes a signal peptide (for example, a polypeptide having the amino acid sequence of SEQ ID NO: 21) . Mutations in the amino acid sequence of FGFR3 that result in skeletal growth disorders (eg, achondroplasia) include, for example, the replacement of a glycine residue at position 358 with an arginine residue (ie G358R; SEQ ID NO: 9). The natural human FGFR3 gene has the nucleotide sequence shown in the genetic database, accession number NM_000142.4, and the natural human FGFR3 protein has the amino acid sequence shown in the genetic database, accession number NP_000133, represented in the present invention as SEQ ID NO: 8. Wild-type FGFR3 (eg, a polypeptide having the amino acid sequence of SEQ ID NO: 8) consists of an extracellular immunoglobulin-like membrane domain including C2-type Ig-like domains 1-3, a transmembrane domain, and an intracellular domain. FGFR3 may include fragments and/or variants (eg, splicing variants, such as splicing variants using an alternate exon 8 rather than exon 9) of full-length wild-type FGFR3.

The terms "fragment" and "part" refer to a part of a whole, such as a polypeptide or nucleic acid molecule, which preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the entire length of the reference nucleic acid molecule or polypeptide or domain thereof (e.g. ECD, ICD or TM of an sFGFR3 polypeptide) . The fragment or part may contain, for example, 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 sequences of amino acid residues, up to the entire length of the reference polypeptide. For example, an FGFR3 fragment may include any polypeptide having at least about 5 amino acid sequences to about 300 amino acid sequences, including endpoints, e.g., at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300 amino acid sequences of any of SEQ ID NOs: 1-8.

The phrase "glucose dysregulation" refers to a blood glucose level above or below an acceptable normal level.

Within the scope of the invention, the term "host cell" refers to a carrier medium that includes the necessary cellular components, for example, organelles, necessary for the expression of an sFGFR3 polypeptide from the corresponding polynucleotide. The nucleic acid sequence of a polynucleotide is typically incorporated into a nucleic acid vector (e.g., plasmid, artificial chromosome, viral vector, or phage vector) that can be introduced into the host cell by conventional methods known in the art (e.g., transformation, transfection, electroporation, precipitation). calcium phosphate and direct microinjection). The host cell can be a prokaryotic cell, such as a bacterial or archaeal cell, or a eukaryotic cell, such as a mammalian cell (eg, a Chinese Hamster Ovary (CHO) cell or human embryonic kidney 293 (HEK 293) cells). Preferably, the host cell is a mammalian cell, such as a CHO cell.

The phrase "imaging techniques" refers to techniques for creating visual representations of the inside of the body for the purpose of clinical analysis and medical intervention. Examples of imaging techniques include, for example, dual-energy X-ray absorptiometry (DXA) to give a fat mass index, cross-sectional imaging such as computed tomography (CT) and magnetic resonance imaging (MRI) to obtain the area of visceral fat in cm ² at a given level. lumbar spine (see, for example, Shuster et al., supra).

The phrase "dysregulation of insulin secretion" refers to a level of insulin in the blood that is above or below the acceptable normal range.

The term "isolated" means separated, extracted or purified from its natural environment. For example, an isolated sFGFR3 polypeptide (e.g., an sFGFR3 polypeptide or a variant thereof, such as a polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-7, or a variant thereof having at least about 85% to up to about 100% identity sequence) can be characterized by a certain degree of purity after isolation of the sFGFR3 polypeptide, for example, from a cell culture medium. An isolated sFGFR3 polypeptide may be at least 75% pure such that the sFGFR3 polynucleotide represents at least 75% by weight of all substances (e.g., polypeptides, polynucleotides, cellular debris, and environmental contaminants) present in the formulation ( 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 all substances present in the preparation). Similarly, an isolated polynucleotide encoding an sFGFR3 polypeptide (e.g., a polynucleotide having the nucleic acid sequence of any of SEQ ID NOs: 10-18, or a variant thereof having a sequence identity of at least about 85% to about 100%), or an isolated the host cell (e.g., CHO cell, HEK 293 cell, L cell, C127 cell, 3T3 cell, BHK cell, COS-7 cell, or patient cell) containing the polynucleotide may be at least 75% pure, such that the polynucleotide or the host cell makes up at least 75 wt.% of all substances (for example, polypeptides, polynucleotides, cellular debris and environmental contaminants) present in the preparation (for example, at least 80%, at least 85%, according to at least 90%, at least 95%, at least 96%, at least 97%, at least 99%, or at least 99.5% by weight of all substances present in the preparation).

The expression "metabolic disorder" refers to disorders of the chemical reactions that aid in energy processing, in particular dyslipidaemia, dysregulation of insulin secretion, dysregulation of glucose, non-alcoholic fatty liver disease, and hepatotoxicity.

The expression "neurological disease" refers to diseases of the brain or nerves, in particular dementia and stroke.

The terms "parenteral administration", "parenterally administered", and other grammatically equivalent phrases used in the present invention refer to a method of administering compositions comprising an sFGFR3 polypeptide (e.g., an sFGFR3 polypeptide or a variant thereof, such as a polypeptide having the amino acid sequence of any of SEQ ID NO: 1-7 (with or without signal peptide), other than enteral and topical administration, usually by injection, and include, without limitation, subcutaneous, intradermal, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid and intrasternal injection and infusion.

The terms "person" and "patient" refer to a mammal, including, but not limited to, a human (e.g., a human having abnormal fat deposition, abnormal visceral fat deposition, or conditions associated with abnormal visceral fat deposition), or a non-human mammal (e.g., a non-human mammal having abnormal fat deposition, abnormal visceral fat deposition, or conditions associated with abnormal visceral fat deposition, e.g., cattle, horses, dogs, sheep, or cats. Preferably, the patient is a human having abnormal fat deposition , abnormal visceral fat deposition, or conditions associated with abnormal visceral fat deposition), particularly a fetus, newborn, infant, child, adolescent, or adult having abnormal fat deposition, abnormal visceral fat deposition, or conditions associated with abnormal visceral fat deposition.

By "pharmaceutical composition" is meant a composition containing an active agent, such as sFGFR3, formulated with at least one pharmaceutically acceptable inert excipient, carrier or diluent. The pharmaceutical composition may be manufactured or marketed with government regulatory approval as part of a therapeutic regimen for the treatment of a disease or event (e.g., abnormal fat deposition, abnormal visceral fat deposition, or conditions associated with abnormal visceral fat deposition) in a patient (e.g., a patient having abnormal fat deposition, abnormal visceral fat deposition, or conditions associated with abnormal visceral fat deposition). Pharmaceutical compositions can be formulated, for example, for parenteral administration, such as subcutaneous administration (for example, subcutaneous injection) or intravenous administration (for example, in the form of a sterile solution that does not contain particulate emboli and in a system of solvents suitable for intravenous administration) , or oral administration (eg, as a tablet, capsule, capsule, gelatin capsule, or syrup).

By “pharmaceutically acceptable diluent, inert excipient, carrier, or adjuvant” is meant a diluent, excipient, carrier, or adjuvant, respectively, that is physiologically acceptable to a patient (e.g., human) while maintaining the therapeutic properties of the pharmaceutical composition (e.g., an sFGFR3 polypeptide or variant thereof). ) in which it is entered. One example of a pharmaceutically acceptable carrier is saline. Other physiologically acceptable diluents, inert excipients, carriers or adjuvants and formulations thereof are known to the person skilled in the art.

"Polynucleotide" and "nucleic acid molecule" used interchangeably in the present invention refer to polymers of nucleotides of any length and include DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or analogs thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. The polynucleotide may include modified nucleotides such as methylated nucleotides and their analogs. If present, the modification of the nucleotide structure may be given before or after the assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. The polynucleotide can be further modified after synthesis, for example by conjugation with a label.

The phrase "lung disease" refers to ventilation disorders such as obstructive sleep apnea, restrictive lung disease, and asthma.

The phrase "reproductive disease" refers to diseases of the reproductive system, in particular, infertility and menstrual irregularities.

In the context of the invention, the term "sequence identity" refers to the percentage of amino acid (or nucleic) residues of a candidate sequence, e.g., an FGFR3 polypeptide, that are identical to the amino acid (or nucleic) residues of a reference sequence, e.g., a wild-type sFGFR3 polypeptide (e.g., a polypeptide, having the amino acid sequence of SEQ ID NO: 8) or an sFGFR3 polypeptide (e.g., an sFGFR3 polypeptide or a variant thereof, such as a polypeptide having the amino acid sequence of any of SEQ ID NO: 1-7), after aligning the sequences and inserting gaps, if necessary, to achieving maximum percent identity (eg, gaps can be introduced in one or both candidates, and reference sequences for optimal alignment and non-homologous sequences can be ignored for comparison purposes). Alignment for purposes of determining percent identity can be achieved in a variety of ways that are known to those skilled in the art, e.g. using open source computer software such as BLAST, BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU -BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR). Suitable parameters for measuring alignment can be determined by those skilled in the art, including any algorithms necessary to achieve maximum alignment over the entire length of the compared sequences. For example, the percent amino acid (or nucleic acid) sequence identity of a given candidate sequence with or against a given reference sequence (which can alternatively be phrased as a given candidate sequence that has or includes a certain percentage of amino acid (or nucleic acid) sequence identity with the given reference sequence). or against it) is calculated as follows: 100×(fraction A/B) where A is the number of amino acid (or nucleic acid) residues judged to be identical when aligned with the candidate sequence and the reference sequence, and where B is the total number of amino acid residues ( or nucleic acids) in the reference sequence. In particular, a reference sequence aligned for comparison with a candidate sequence can show that the candidate sequence exhibits, for example, 50% to 100% identity over the entire length of the candidate sequence or a selected portion of non-essential amino acid (or nucleic acid) residues of the sequence. -candidate. The length of the candidate sequence aligned for comparison purposes is at least 30%, such as at least 40%, such as at least 50%, 60%, 70%, 80%, 90%, or 100% of the length of the reference sequence . When a position in a candidate sequence is occupied by the same amino acid (or nucleic) residue as the corresponding position in the reference sequence, then the molecules at that position are identical.

By "signal peptide" is meant a short peptide (eg, 5-30 amino acids in length, eg, 22 amino acids in length) at the N-terminus of a polypeptide that directs the polypeptide to a secretory pathway (eg, to the extracellular space). The signal peptide is usually cleaved during secretion of the polypeptide. The signal sequence may direct the polypeptide to an intracellular compartment or organelle, such as the Golgi apparatus. The signal sequence can be identified by homology or biological activity with a peptide with a known function of targeting the polypeptide to a specific region of the cell. One skilled in the art can identify the signal peptide using readily available software (e.g., sequence analysis software package Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, or PILEUP/PRETTYBOX programs) . The signal peptide may be, for example, substantially identical to the amino acid sequence of SEQ ID NO: 21.

The term "skeletal growth retardation disorder", within the scope of the invention, refers to a skeletal disease characterized by deformities and/or malformations of the bones. These disorders include, but are not limited to, skeletal growth retardation disorders caused by destruction of the growth epiphyseal cartilage, idiopathic skeletal growth retardation disorders, or FGFR3-related skeletal diseases. In particular, a patient having a skeletal growth retardation disorder (eg, achondroplasia) may have bones that are shorter than those of a healthy patient. For example, a disorder of skeletal growth retardation may include skeletal dysplasia, e.g., achondroplasia, homozygous achondroplasia, heterozygous achondroplasia, achondrogenesis, acrodysostosis, acromesomelic dysplasia, atelosteogenesis, campomelic dysplasia, punctate epiphyseal dysplasia, rhizomelic type of punctate epiphyseal dysplasia, short dyscongenital femoral bone , craniosynostosis (eg, Muencke syndrome, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, or Crouzon syndrome with acanthosis nigricans), dactyly, brachydactyly, camptodactyly, polydactyly, syndactyly, diastrophic dysplasia, dwarfism, dyssegmental dysplasia, enchondromatosis, fibrochondrogenesis , fibrous dysplasia, hereditary multiple exostoses, hypochondroplasia, hypophosphatasia, hypophosphatemic rickets, Jaffe-Liechtenstein syndrome, Knist dysplasia, Knist syndrome, mesomelic dysplasia of the Langer type, Marfan syndrome, McCune-Albright syndrome, micromelia, metaphyseal dysplasia, metaphyseal dysplasia of the Jansen type , metatropical dysplasia, Morquio's syndrome, Nivergelt's mesomelic dysplasia, neurofibromatosis, osteoarthritis, osteochondrodysplasia, osteogenesis imperfecta, perinatal lethal type of osteogenesis imperfecta, osteopetrosis, osteopoikilia, peripheral dysostosis, Reinhardt's syndrome, Roberts' syndrome, Robinov's syndrome, short rib-polydactyly syndrome, short stature, congenital spondyloepiphyseal dysplasia and spondyloepiphyseal dysplasia.

The terms "soluble fibroblast growth factor receptor 3", "soluble FGFR3", and "sFGFR3" refer to FGFR3 that is characterized by the absence or functional impairment of all or a significant portion of the transmembrane domain and any polypeptide portion that could bind the FGFR3 polypeptide to the cell membrane (e.g. , tyrosine kinase domain). The sFGFR3 polypeptide is a non-membrane-bound form of the FGFR3 polypeptide. Thus, an sFGFR3 polypeptide may include a deletion of part or all of the amino acid residues of the transmembrane domain of a wild-type FGFR3 polypeptide sequence (eg, a polypeptide having the amino acid sequence of SEQ ID NO: 8). The sFGFR3 polypeptide may further include deletions of the intracellular domain of the wild-type FGFR3 polypeptide.

Examples of sFGFR3 polypeptides may include, but are not limited to, at least amino acids 1-100, 1-125, 1-150, 1-175, 1-200, 1-205, 1-210, 1-215, 1-220 , 1-225, 1-230, 1-235, 1-240, 1-245, 1-250, 1-252, 1-255, 1-260, 1-265, 1-270, 1-275, 1 -280, 1-285, 1-290, 1-295 or 1-300 or 1-301 sequences of SEQ ID NOs: 1-8. sFGFR3 polypeptides may 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 with any of these sFGFR3 polypeptides of SEQ ID NOs: 1-8. In addition, examples of sFGFR3 polypeptides may include, but are not limited to, at least amino acids 1-100, 1-125, 1-150, 1-175, 1-200, 1-205, 1-210, 1-215, 1-220, 1-225, 1-230, 1-235, 1-240, 1-245, 1-250, 1-255, 1-260, 1-265, 1-270, 1-275, 1- 280, 1-285, 1-290, 1-295, 1-300, 1-305, 1-310, 1-315, 1-320, 1-325, 1-330, 1-335, 1-340, 1-345 or 1-348 sequences of SEQ ID NOs: 1-8. sFGFR3 polypeptides may 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 with any of these sFGFR3 polypeptides having the amino acid sequences of SEQ ID NO: 1 -8. Any of the above sFGFR3 polypeptides or variants thereof may optionally include a signal peptide at the N-terminus, such as amino acids 1-22 of SEQ ID NO: 21 (MGAPACALALCVAVAIVAGASS) or amino acids 1-19 of SEQ ID NO: 43 (e.g., MMSFVSLLLVGILFHATQA) .

The phrase "subcutaneous fat deposition" refers to the deposition of fat in the hypodermis.

By "treatment" and "cure" is meant a reduction (e.g., at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 95%, approximately 99%, or even 100%) of abnormal fat deposition or abnormal deposition of visceral fat, or progression, severity, or frequency of one or more conditions associated with abnormal deposition of visceral fat (eg, cardiovascular disease, lung disease, metabolic disorder, or neurological disease) in a patient (eg, a human such as a fetus, newborn, infant, child, adolescent, or adult). Treatment may occur over a period in which the sFGFR3 polypeptide is administered over a period of time (e.g., days, months, years, or longer) to treat a patient (e.g., a human such as a fetus, neonate, infant, child, adolescent, or adult), having abnormal fat deposition, abnormal visceral fat deposition, or a condition associated with abnormal visceral fat deposition. Typical symptoms associated with abnormal visceral fat deposition in achondroplasia that can be treated with sFGFR3 (e.g., an sFGFR3 polypeptide or a variant thereof, such as a polypeptide having the amino acid sequence of SEQ ID NOs: 1-7, or a variant thereof) include, but are not limited to , atherosclerosis, hypertension, lipid dysregulation, obstructive sleep apnea, glucose dysregulation, or dysregulation of insulin secretion (eg, insulin resistance). Examples of symptoms associated with abnormal visceral fat deposition in a patient with achondroplasia that can be treated with sFGFR3 (e.g., an sFGFR3 polypeptide or variants thereof, such as a polypeptide having the amino acid sequences of SEQ ID NOs: 1-7, or variants thereof) include but are not limited to atherosclerosis, hypertension, lipid dysregulation, obstructive sleep apnea, glucose dysregulation, or dysregulation of insulin secretion (eg, insulin resistance).

The term "unit dosage form(s)" refers to physically discrete unit(s) suitable as a single dose for humans and other mammals, each unit containing a predetermined amount of active substance calculated to produce the desired therapeutic effect, in combination with any suitable pharmaceutical inert excipient, carrier or diluent.

The term "variant" in relation to a polypeptide refers to a polypeptide (e.g., an sFGFR3 polypeptide or a variant thereof with or without a signal peptide) that differs by one or more amino acid sequence changes from the polypeptide from which the variant is derived (e.g., a reference polypeptide such as such as a polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-7). The term "variant" in relation to a polynucleotide refers to a polynucleotide that differs by one or more changes in the nucleic acid sequence from the polynucleotide from which the variant is derived (e.g., a reference polynucleotide, such as, for example, a polynucleotide encoding an sFGFR3 polypeptide having a nucleic acid sequence of any from SEQ ID NO: 10-18). Changes in the amino acid or nucleic acid sequence of a variant may be, for example, amino acid substitutions, nucleic acids, insertions, deletions, N-terminal truncations or C-terminal truncations, or any combination thereof. In particular, amino acid substitutions may be conservative and/or non-conservative substitutions. A variant may have amino acid sequence identity or nucleic acid sequence identity with a reference polypeptide or parent polynucleotide, respectively. For example, a variant may 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 with the reference polypeptide or polynucleotide.

By "vector" is meant a DNA construct that includes one or more polynucleotides or fragments thereof encoding an sFGFR3 polypeptide (e.g., an sFGFR3 polypeptide or a variant thereof, such as a polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-7, or variants thereof , with or without a signal peptide). The vector can be used to infect a cell (eg, a host cell or a patient cell with a human skeletal growth retardation disorder such as achondroplasia) resulting in translation of the vector polynucleotides into an sFGFR3 polypeptide. One type of vector is a "plasmid", which refers to a circular double stranded loop of DNA into which additional DNA segments can be ligated. Some vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of a host cell when introduced into the host cell and thus replicate along with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are operably linked. Typically, expression vectors used in recombinant DNA techniques are often in the form of plasmids.

The phrase "visceral fat deposition" refers to intra-abdominal fat depots, including mesenteric and omental, retroperitoneal fat deposits, and intrathoracic fat deposits, including pericardial.

The enumeration in the present invention of numeric endpoint ranges is intended to include all numbers related to that range (for example, an indication 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 become apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

Patent or application documents contain at least one figure in color. Copies of this patent or patent application publication with colored figures will be made available by the office upon request and payment of the required fee.

FIG. 1A-1D are tables, pictures, and graphs showing that children with achondroplasia develop abdominal obesity without an increase in blood glucose. FIG. 1A is a table showing weight, height, and BMI measurements and the corresponding height-for-age and BMI-for-age z-scores in three age groups (n=73 data points in the [0-3] year group, n=61 data points in the [4-8] years and n=36 data points in the [9-18] years group). Results of subsequent analysis: a: significantly different between [0-3] and [4-8] groups; b: significantly different between groups [0-3] and [9-18]; c: differ significantly between groups [4-8] and [9-18]. FIG. 1B is a depiction of various regions of interest (ROI) evaluated by DXA. FIG. 1C is a graph showing the measurement of android:gynoid fat ratio in three age groups (n=4 data points in the [0-3] year group, n=6 data points in the [4-8] year group, and n=9 data points in the group of [9-18] years). FIG. 1D is a graph showing fasting plasma glucose concentration in two age groups aged [4-8] and [9-18] years (n=16 data points in the [4-8] years group and n=12 data points in the [9-18 years). Horizontal lines represent normal values. Data are presented as mean±SD, **p<0.01, ***p<0.001.

FIG. 2A-2H are graphs and images showing that Fgfr3 ^ach/+ transgenic mice predominantly develop visceral obesity, which is prevented by sFGFR3 treatment (SEQ ID NO: 1). FIG. 2A is a graph showing the body weight of vehicle-treated WT and Fgfr3 ^{ach/+ mice} and sFGFR3-treated Fgfr3 ^ach/+ mice after 10 weeks of ND or HFD consumption. FIG. 2B is an image and graph showing the ratio of abdominal lean mass to fat. FIG. 2C is a graph showing epididymal adipose tissue (eAT) mass. FIG. 2D is a graph showing subcutaneous adipose tissue mass (scAT) per gram of body weight. FIG. 2E is a graph showing the area of scAT adipocytes. FIG. 2F is a graph showing the eAT area of adipocytes. FIG. 2G is a scatter plot of adipocytes versus their diameter. Data are presented as mean +/- standard deviation (n=8-10 mice per group). The data followed a normal distribution. *p<0.05, **p<0.01, ***p<0.001 depending on vehicle-treated WT, #p<0.05, ##p<0.01 versus Fgfr3 ^{ach/ +} , treated with a carrier medium; t -Student's criterion. FIG. 2H is a graph showing the eAT scatter of adipocytes according to their diameter. Data are presented as mean +/- standard deviation (n=8-10 mice per group). The data followed a normal distribution. *p<0.05, **p<0.01, ***p<0.001 depending on vehicle-treated WT, #p<0.05, ##p<0.01 versus Fgfr3 ^{ach/ +} , treated with a carrier medium; t -Student's criterion.

FIG. 3A-3B are graphs showing that MSCs isolated from untreated or sFGFR3-treated Fgfr3 ^ach/+ mice show prior participation in adipogenesis with no change in insulin response compared to WT mice. FIG. 3A is a graph showing the expression of genes involved in different stages of adipogenesis differentiation (the genes listed in Table 1). Expression was normalized to HPRT, RPL6 and RPL13a expression and expressed as percent change from WT. FIG. 3B is a graph showing the results of cells stimulated with 50 nM insulin for 0, 5, 15, or 30 minutes or 0, 1, 10, 50, or 100 nM insulin for 5 minutes. P-Erk1/2 expression normalized to total Erk1/2 expression was expressed as a normalized value to WT. Data are presented as mean ± standard deviation. The data followed a normal distribution. *p < 0.05, **p < 0.01, ***p < 0.001 compared to vehicle-treated WT, #p < 0.05, ##p < 0.01 compared to Fgfr3 ^{ach/ +} processed by the carrier medium. Two-way analysis of variance with Tukey's multiple test.

FIG. 4A-4E are graphs and microscopic images showing that glucose metabolism is altered in Fgfr3 ^ach/+ transgenic mice and restored by sFGFR3 treatment. FIG. 4A is a graph showing fasting glycemia and insulinemia in mice after 10 weeks ND. FIG. 4B is a graph showing fasting glycemia and insulinemia in mice after 10 weeks of HFD. FIG. 4C shows graphs of the results of the HFD glucose tolerance test; glucose levels were normalized to a time value of -15 min and area under the curve corresponding to each group of mice. FIG. 4D shows microscopy of mouse pancreatic insulin content for the first time (immunohistochemistry of paraffin sections, red: insulin; green: glucose; blue: DAPI staining). FIG. 4D also shows a graph of mouse pancreatic insulin levels, mean pancreatic islets normalized to total surface, and mean number of islets per group in HFD condition. FIG. 4E shows liver H&E microscopy and PAS staining under HFD conditions. FIG. 4F shows microscopy of H&E staining of hepatic nodules. Data are presented as mean±SD (n=8-10 mice for each group). The data followed a normal distribution. **p<0.01, ***p<0.001 depending on vehicle-treated WT, #p<0.05, ##p<0.01 versus vehicle-treated Fgfr3 ^ach/+ ; t -Student's criterion.

FIG. 5A-5D are graphs showing that untreated Fgfr3 ^ach/+ transgenic mice derive substantially all of their energy from lipids. FIG. 5A shows basal respiration rate (RQ=VCO2/VO2) during overnight or daytime fasting and feeding periods after 10 weeks of ND intake. FIG. 5B shows basic carbohydrate and lipid oxidation in WT and Fgfr3 ^ach/+ mice fed ND. FIG. 5C shows basal respiration rate (RQ=VCO2/VO2) during overnight or daytime fasting and feeding periods after 10 weeks of HFD intake. FIG. 5D shows basal carbohydrate and lipid oxidation in WT and Fgfr3 ^ach/+ mice fed HFD. Data are presented as mean±SD (n=8-10 mice for each group). The data followed a normal distribution. **p<0.01, ***p<0.001 depending on vehicle-treated WT, #p<0.05, ##p<0.01 versus vehicle-treated Fgfr3 ^ach/+ ; t -Student's criterion.

FIG. 6A-6B are graphs showing circulating adipokines studied in the sera of untreated or sFGFR3-treated Fgfr3 ^ach/+ mice. FIG. 6A is a graph showing the results for mice fed ND. FIG. 6B is a graph showing the results for mice fed with HFD. The results were expressed as percentage change from WT. AgRP, agutylated protein; ANGPT-L3, angiopoietin-3; CRP, C-reactive protein; DPPIV, dipeptidyl peptidase V; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; ICAM-1, intercellular adhesion molecule-1; IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein; MCP-1, monocytic chemotactic protein 1; M-CSF, macrophage colony stimulating factor; Pref-1, pre-adipocyte factor 1; RAGE, the end product of enhanced glycosylation; RANTES, receptor on activation, normal T cells expressed and secreted; RBP4, retinol binding protein; TIMP-1, tissue inhibitor of metalloproteinases; VEGF, vascular endothelial growth factor.

FIG. 7A-7H are graphs showing that achondroplasia transgenic mice had normal energy expenditure, cumulative activity, and cumulative growth during indirect calorimetry. FIG. 7A shows basal oxygen consumption during nighttime or daytime fasting and feeding periods in WT and Fgfr3 ^ach/+ mice fed ND. FIG. 7B shows basal carbon dioxide production during overnight or daytime fasting and feeding periods in WT and Fgfr3 ^ach/+ mice fed ND. FIG. 7C shows basal energy expenditure during overnight or daytime fasting and feeding periods in WT and Fgfr3 ^ach/+ mice fed ND. FIG. 7D shows basal cumulative activity and growth in WT and Fgfr3 ^ach/+ mice fed ND. FIG. 7E shows basal oxygen consumption during nighttime or daytime fasting and feeding periods in WT and Fgfr3 ^ach/+ mice fed HFD. FIG. 7F shows production of basic carbon dioxide during overnight or daytime fasting and feeding periods in WT and Fgfr3 ^ach/+ mice fed HFD. FIG. 7G shows basal energy expenditure during overnight or daytime fasting and feeding periods in WT and Fgfr3 ^ach/+ mice fed HFD. FIG. 7H shows basal cumulative activity and growth in WT and Fgfr3 ^ach/+ mice fed HFD. Data are presented as mean±SD (n=8-10 mice for each group). The data followed a normal distribution. **p<0.01, ***p<0.001 compared to vehicle-treated WT, ###p<0.001 compared to vehicle-treated Fgfr3 ^ach/+ ; t -Student's criterion.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have discovered that soluble fibroblast growth factor receptor 3 (sFGFR3) polypeptides and polynucleotides encoding sFGFR3 polypeptides, and variants thereof, can be used to treat abnormal visceral fat deposition in a patient (e.g., human, in particular, fetus, neonate, child , adolescent and adult) who need it. In particular, sFGFR3 polypeptides that can be used in the treatments described herein include those having the amino acid sequence of any of SEQ ID NOs: 1-7 and variants thereof having at least 85% sequence identity. Treatment methods may also administer polynucleotides encoding sFGFR polypeptides or cells containing the polynucleotides.

Methods of treatment

The invention relates to methods for treating a patient having abnormal visceral fat deposition and conditions associated with abnormal visceral fat deposition. In particular, the patient may have elevated body mass index, sagittal abdominal diameter, android:gynoid fat ratio, fat mass index, and visceral fat area. The patient may also have a skeletal growth retardation syndrome, such as achondroplasia, thanatophoric dysplasia type I (TDI), thanatophoric dysplasia type II (TDII), severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN), hypochondroplasia, and syndromes with craniosynostosis (eg, syndrome Münke, Crouzon syndrome, and Crouzon syndrome with acanthosis nigricans, camptodactyly, tall stature, and hearing loss syndrome (CATSHL) This may be the case when the patient has skeletal stunting syndrome and a condition associated with abnormal visceral fat deposition (eg, atherosclerosis, hypertension, myocardial infarction, dyslipidemia, sleep apnea, restrictive lung disease, asthma, dementia, dysregulation of insulin secretion (eg, insulin resistance), dysregulation of glucose metabolism, infertility, menstrual disorders, stroke, and dementia).

Alternatively, the patient may be one who does not have skeletal growth retardation syndrome but has a condition that results in abnormal visceral fat deposition (eg, obesity, polycystic ovary syndrome, and hypercortisolism (eg, Cushing's disease)). For example, a patient may have abnormal visceral fat storage and a condition associated with abnormal visceral fat storage (eg, atherosclerosis, hypertension, myocardial infarction, dyslipidaemia, sleep apnea, restrictive lung disease, asthma, dementia, dysregulation of insulin secretion (eg, resistance to to insulin), dysregulation of glucose metabolism, infertility, menstrual irregularities, stroke and dementia). The method may also include administering sFGFR3 to treat a patient with aberrant FGF10 signaling, such as a patient suffering from Cushing's disease caused by pituitary dysfunction.

The patient may also be characterized as having a deposit of visceral fat surrounding or associated with one or more of the following organs: heart, liver, spleen, kidneys, pancreas, intestines, reproductive organs, and gallbladder.

The method includes administering an sFGFR3 polypeptide of the invention, eg as described herein, to a patient having abnormal visceral fat deposition. The patient may be a fetus, newborn, infant, child, adolescent, or adult at risk of developing abnormal deposits of abdominal fat. The patient may also have skeletal growth retardation syndrome (eg, achondroplasia), obesity, hypercortisolism (eg, Cushing's disease), or polycystic ovary syndrome. The patient (eg, human) may be treated before signs and symptoms of abnormal visceral fat deposition appear. In particular, patients that may be treated with an sFGFR3 polypeptide of the invention described herein are those who exhibit symptoms such as, but not limited to, abnormal fat mass index, abnormal visceral fat area, increased BMI, increased waist circumference, increased sagittal diameter, and increased android:gynoid fat ratio. In addition, patients who can be treated with the sFGFR3 polypeptide have abnormal visceral fat distribution and conditions associated with abnormal visceral fat deposition (eg, metabolic, cardiovascular, pulmonary, reproductive, or neurological diseases). In addition, treatment with an sFGFR3 polypeptide may result in an improvement in one or more of the aforementioned symptoms associated with abnormal visceral fat deposition (eg, compared to an untreated patient). Prior to administration of the sFGFR3 polypeptide, a patient (e.g., human) may be diagnosed with abnormal visceral fat deposition, e.g., a patient with skeletal growth retardation syndrome (e.g., achondroplasia), obesity, hypercortisolism (e.g., Cushing's disease), and polycystic ovary syndrome. In addition, a patient having abnormal visceral fat deposition may be a patient who has not previously been treated with an sFGFR3 polypeptide.

A patient that can be treated with an sFGFR3 polypeptide can also be a patient with or at risk of developing diabetes, such as an obese patient. Treatment of this patient may include topical administration of soluble FGFR3 to the pancreas to treat or prevent the development of diabetes.

Soluble Fibroblast Growth Factor Receptor 3 (sFGFR3) Polypeptides

Soluble FGFR3 polypeptides and variants thereof can be used in methods of treating a patient having abnormal visceral fat deposition. An sFGFR3 polypeptide may comprise at least 50 amino acid sequences of the extracellular domain (ECD) of a naturally occurring fibroblast growth factor receptor 3 (FGFR3) polypeptide (e.g., an FGFR3 polypeptide having the sequence listed under GenBank Accession Number NP_000133; see also SEQ ID NO: 8 ). In particular, the sFGFR3 polypeptide may comprise 100-370 amino acid sequences (eg, less than 350 amino acid sequences) of the natural fibroblast growth factor receptor 3 (FGFR3) ECD polypeptide. An sFGFR3 polypeptide may also have the C2 type 1, 2 and/or 3 Ig-like domain of a natural FGFR3 polypeptide.

An sFGFR3 polypeptide may or may not have a signal peptide (e.g., the signal peptide of an FGFR3 polypeptide such as corresponding to SEQ ID NO: 21; in particular, sFGFR3 is a mature polypeptide without a signal peptide that is cleaved off during expression and secretion from the cell). The sFGFR3 polypeptide also lacks a transmembrane domain (TM) such as the TM of the native FGFR3 polypeptide.

sFGFR3 polypeptides may also contain all or part of the extracellular domain (ICD) of an FGFR3 polypeptide. For example, an sFGFR3 polypeptide may have 400 amino acid sequences or less (e.g., between 5 and 399 amino acid sequences, e.g., 175, 150, 125, 100, 75, 50, 40, 30, 20, 15 or fewer amino acid sequences) ICD natural FGFR3 polypeptide. An sFGFR3 polypeptide ICD may also lack the tyrosine kinase domain of a native FGFR3 polypeptide. Alternatively, an sFGFR3 polypeptide may lack any of the ICD amino acids of a native FGFR3 polypeptide (eg, an FGFR3 polypeptide of SEQ ID NO: 8).

The sFGFR3 polypeptide may also have an amino acid sequence having at least 90%, 92%, 95%, 97%, or 99% sequence identity, or have a sequence of amino acids 401-413 of SEQ ID NO: 8.

An sFGFR3 polypeptide for use in the methods described herein may be less than 475, 450, 425, 400, 375, 350, 300, 250, 200, 150, or 100 amino acids in length and/or may have an amino acid sequence having at least at least 85% sequence identity (e.g. 86%-100% sequence identity such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, or 99% sequence identity) with amino acid residues 1-280 of SEQ ID NO: 8. The sFGFR polypeptide can also be a polypeptide with an amino acid sequence having at least 85% sequence identity (e.g., 86%-100% sequence identity such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) with the amino acid sequence of any of SEQ ID NOs: 1-7. In particular, sFGFR3 has the amino acid sequence of SEQ ID NO: 5 or 6 (eg, the amino acid sequence of SEQ ID NO: 5). The sFGFR3 polypeptide may also have the sequence of SEQ ID NO: 6, except that the residue at position 253 is alanine, glycine, proline, or threonine.

Variants of the sFGFR3 polypeptide that can be administered by the methods also include fragments of the amino acid sequence of any of SEQ ID NOs: 1-8 (e.g., at least amino acids 1-200, 1-205, 1-210, 1-215, 1-220 , 1-225, 1-235, 1-230, 1-240, 1-245, 1-250, 1-253, 1-255, 1-260, 1-265, 1-275, 1-280, 1 -285, 1-290, or 1-300 sequences of SEQ ID NO: 8) or polypeptides having at least 50% sequence identity (e.g., 55%, 60%, 65%, 70%, 75%, 80% , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with any of the sequences of SEQ ID NO: 1-8 (and which, for example, lacks a signal peptide and TM domain).

The sFGFR3 polypeptides of the invention can also be characterized as being associated with fibroblast growth factor (FGF). In particular, FGF is selected from the group consisting of fibroblast growth factor 1 (FGF1; SEQ ID NO: 26), fibroblast growth factor 2 (FGF2; SEQ ID NO: 27), fibroblast growth factor 9 (FGF9; SEQ ID NO: 28) , fibroblast growth factor 10 (FGF10; SEQ ID NO: 40), fibroblast growth factor 18 (FGF18; SEQ ID NO: 29), fibroblast growth factor 19 (FGF19; SEQ ID NO: 30), fibroblast growth factor 21 (FGF21; SEQ ID NO: 31) and fibroblast growth factor 23 (FGF23; SEQ ID NO: 41). The binding is characterized by an equilibrium dissociation constant (K _d ) from about 0.2 nm to about 20 nm (for example, K _d from about 1 nm to about 10 nm, where optionally K _d is about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, or about 10 nm).

In view of the results described in the present invention, the invention is not limited to a particular sFGFR3 polypeptide or variant thereof. In addition to the exemplary sFGFR3 polypeptides and variants thereof discussed above, any sFGFR3 polypeptide that binds one or more FGFs with a similar binding affinity as sFGFR3 polypeptides having the amino acid sequence of SEQ ID NOs: 1-7 is also contemplated as one that can be used to treat abnormal deposition of visceral fat in a patient. sFGFR3 polypeptides can be, for example, fragments of FGFR3 isoform 2 without exons 8 and 9 encoding the C-terminal half of the IgG3 domain and exon 10 including the transmembrane domain (for example, fragments of the amino acid sequence of SEQ ID NO: 8) corresponding to fragments of the FGFR3 transcript option 2 (access number NM_022965).

As indicated above, an sFGFR3 polypeptide for use in the methods of the invention may include a signal peptide at the N-terminal position. An example of a signal peptide may include, but is not limited to, amino acids 1-22 of SEQ ID NO: 21 (eg, MGAPACALALCVAVAIVAGASS). Accordingly, sFGFR3 polypeptides include both selected forms that lack an N-terminal signal peptide and non-secreted forms that include an N-terminal signal peptide. For example, a secreted sFGFR3 polypeptide may include the amino acid sequence of any of SEQ ID NOs: 1-7, but without the N-terminal signal peptide (eg, SEQ ID NO: 21). Alternatively, an sFGFR3 polypeptide (e.g., a polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-7) includes a signal peptide, such as the amino acid sequence of SEQ ID NO: 21. One skilled in the art will appreciate that the position of the N-terminal signal peptide will vary across sFGFR3 polypeptides and may include, for example, the first 5, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27 , 30 or more amino acid residues at the N-terminus of the polypeptide. One skilled in the art can predict the position of a signal sequence cleavage site, for example, using an appropriate computer algorithm such as that described by Bendtsen et al. ( J. Mol. Biol. 340(4):783-795, 2004) and available online at cbs.dtu.dk/services/SignalP/.

In addition, the sFGFR3 polypeptides of the invention may be glycosylated. In particular, the sFGFR3 polypeptide can be modified to increase or decrease the degree of glycosylation of the sFGFR3 polypeptide. The addition or removal of glycosylation sites in an sFGFR3 polypeptide can be accomplished by changing the amino acid sequence such that one or more glycosylation sites are 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 SEQ ID NO: 5 or 6 and variants thereof. . One or more of these Asn residues may also be substituted to remove the glycosylation site. For example, O-linked glycosylation, in which an oligosaccharide is attached to the oxygen atom of an amino acid residue, may occur at position Ser109, Thr126, Ser199, Ser274, Thr281, Ser298, Ser299 and/or Thr301 of the amino acid sequence of SEQ ID NO: 5 or 6 and variants thereof. . In addition, O-linked glycosylation can occur at the serine residue in sFGFR3. One or more of these Ser or Thr residues may also be substituted to remove the glycosylation site.

sFGFR3 fusion polypeptides

sFGFR3 polypeptides of the invention (e.g., sFGFR3 polypeptides having the amino acid sequence of any of SEQ ID NOs: 1-7 or a variant thereof, having at least 85% sequence identity (e.g., 86%-100% sequence identity, e.g. , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) can be fused to a functional domain of a heterologous polypeptide (e.g., a fragment of a crystallizable region (Fc region; e.g., a polypeptide having the amino acid sequences of SEQ ID NOs: 35 and 36) or human serum albumin (HSA; e.g., a polypeptide having the amino acid sequence of SEQ ID NOs: 37 )) to produce an sFGFR3 fusion polypeptide Optionally, a flexible linker, such as a serine or glycine rich sequence (for example, a polyglycine or polyglycine/serine linker such as as SEQ ID NOs: 38 and 39).

For example, sFGFR3 polypeptides and variants thereof can be a fusion polypeptide, including, for example, an immunoglobulin Fc region of the N-terminal or C-terminal domain. In particular, useful Fc regions may include the Fc fragment of any immunoglobulin molecule, including IgG, IgM, IgA, IgD, or IgE and their various subclasses (e.g., IgG-1, IgG-2, IgG-3, IgG-4, IgA-1 , IgA-2) of any mammal (eg human). For example, a human IgG-1 Fc fragment (SEQ ID NO: 35) or a human IgG-1 variant, such as a variant comprising replacing asparagine at position 297 of SEQ ID NO: 35 with alanine (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: : 36). Fc fragments of the invention may include, for example, the CH2 and CH3 domains of the heavy chain and any part of the hinge region. The sFGFR3 fusion polypeptides of the invention may also include, for example, a monomeric Fc such as a CH2 or CH3 domain. The Fc region may optionally be glycosylated at any suitable one or more amino acid residues known to those skilled in the art. The Fc fragment described in the present invention may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50 or more additions, deletions or substitutions relative to any of the Fc fragments described in the present invention.

In addition, sFGFR3 polypeptides can be conjugated to other molecules at the N-terminal or C-terminal domain to improve 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 example, sFGFR3 polypeptides can be conjugated to a human HSA (eg, a polypeptide having the amino acid sequence of SEQ ID NO: 37) or a fragment thereof.

Fusion SFGFR3 polypeptides may include a peptide linker region between an sFGFR3 polypeptide and a heterologous polypeptide (eg, an Fc or HSA region). The linker region can be of any sequence and length that allows the sFGFR3 to remain biologically active, eg, be free of steric hindrance. Approximate linker lengths are from 1 to 200 amino acid residues, e.g. -50, 51-55, 56-60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-90, 91-95, 96-100, 101-110, 111-120 , 121-130, 131-140, 141-150, 151-160, 161-170, 171-180, 181-190 or 191-200 amino acid residues. For example, linkers include or consist of flexible portions, such as regions without significant fixed secondary or tertiary structure. Preferred ranges are 5 to 25 and 10 to 20 amino acids in length. This flexibility is usually increased if the amino acids are small and do not have bulky side chains that prevent rotation or bending of the amino acid chain. Thus, preferably, the peptide linker of the present invention has an increased content of small amino acids, in particular glycines, alanines, serines, threonines, leucines and isoleucines.

Examples of flexible linkers are glycine-rich linkers, for example, containing at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or even 100% glycine residues. Linkers may also include, for example, serine-rich linkers, for example, containing at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or even 100% serine residues. In some cases, the amino acid sequence of the linker consists of only glycine and serine residues. For example, the linker may be the amino acid sequence GGGGAGGGG (SEQ ID NO: 38) or GGGGSGGGGSGGGGS (SEQ ID NO: 39). The linker may optionally be glycosylated at any suitable one or more amino acid residues. The linker may also be absent when an sFGFR3 polypeptide and a heterologous polypeptide (eg, Fc or HSA region) are fused to each other directly without intermediate residues.

Polynucleotide encoding sFGFR3 polypeptides

Polynucleotides encoding sFGFR3 polypeptides may be used to treat a patient having abnormal visceral fat deposition (eg, a human such as a fetus, neonate, infant, child, adolescent, or adult). For example, a polynucleotide may have a nucleic acid sequence of any of SEQ ID NOs: 10-18 or variants thereof having at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with the nucleic acid sequence of any of SEQ ID NO: 10-18. In addition, the polynucleotide may have a nucleic acid sequence of SEQ ID NO: 14 or 15, or a variant thereof having at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with the nucleic acid sequence of SEQ ID NO: 14 or 15.

Also characterized are polynucleotides encoding sFGFR3 fusion polypeptides (e.g., an sFGFR3 polypeptide fused to a heterologous polypeptide such as an Fc or HSA region) and polynucleotides encoding sFGFR3 polypeptides without a signal peptide (e.g., polypeptides having the amino acid sequence of any of SEQ ID NO: 1 -7), or with a signal peptide (eg, polypeptides having the amino acid sequence of any of SEQ ID NOs: 1-7). In addition, polynucleotides may have one or more mutations to alter any of the glycosylation sites described herein or known to be present in the polypeptide.

Optionally, the polynucleotides of the invention can be codon-optimized to change the codons in the nucleic acid, in particular to reflect the typical codon usage of the host organism (e.g., human) without changing the sFGFR3 polypeptide encoded by the nucleic acid sequence of the polynucleotide. Codon-optimized polynucleotides (e.g., a polynucleotide having the nucleic acid sequence of SEQ ID NO: 14 or 16) can, for example, facilitate genetic manipulation by reducing GC content and/or for expression in a host cell (e.g., a HEK 293 cell or a CHO). A person skilled in the art can perform codon optimization, for example using online tools such as the JAVA Codon Adaption Tool (www.jcat.de) or the Integrated DNA Technologies Tool (www.eu.idtdna.com/CodonOpt), simply by entering the nucleic acid sequence acids of the polynucleotide and the host organism for which the codons are to be optimized. The use of codons by various organisms is available in online databases such as www.kazusa.or.jp/codon.

Host Cells for the Expression of sFGFR3 Polypeptides

Mammalian cells can be used as host cells to express an sFGFR3 polypeptide (eg, a polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-7 and variants thereof). Exemplary mammalian cell types useful in the methods include, but are not limited to, human embryonic kidney (HEK; e.g., HEK 293), Chinese hamster ovary (CHO) cells, L cells, C127 cell line, 3T3 cell line, BHK cells, COS-7 cells, HeLa cells, PC3 cells, Vero cells, MC3T3 cells, NS0 cells, Sp2/0 cells, VERY cells, BHK cells, MDCK cells, W138 cells, cells BT483 cell line, Hs578T cell line, HTB2 cell line, BT20 cell line, T47D cell line, NS0 cell line, CRL7O3O cell line and HsS78Bst cell line, or any other suitable mammalian host cell known in the art. Alternatively, cellsE. coli can be used as host cells for the expression of sFGFR3 polypeptides. Strain examplesE. coli include, but are not limited to,E. coli294 (ATCC^®31.446),E. coliλ 1776 (ATCC^®31.537,E. coliBL21 (DE3) (ATCC^®BAA-1025),E. coliRV308 (ATCC^®31.608) or any other suitable strainE. coliknown in the art.

Vectors Including Polynucleotides Encoding sFGFR3 Polypeptides

Also provided are recombinant vectors comprising any one or more of the polynucleotides described above (eg, a polynucleotide encoding a polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-7 and variants thereof). The vectors of the invention may be used to deliver a polynucleotide encoding an sFGFR3 polypeptide of the invention and variants thereof, which may include mammalian, viral, and bacterial expression vectors. For example, the vectors may be plasmids, artificial chromosomes (eg BAG, PAC and YAC), and viral or phage vectors, and may optionally include a promoter, enhancer or regulator for polynucleotide expression. The vectors may also contain one or more selectable marker genes, such as an ampicillin, neomycin and/or kanamycin resistance gene for a bacterial plasmid, or a resistance gene for a fungal vector. Vectors can be used in vitro to produce DNA or RNA, or to transfect or transform a host cell, such as a mammalian host cell, to produce the sFGFR3 polypeptide encoded by the vector. The vectors can also be adapted for use in vivo in a gene therapy method.

Examples of viral vectors that can be used to deliver a polynucleotide encoding an sFGFR3 polypeptide of the invention (e.g., a polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-7 and variants thereof) include 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. , influenza virus), rhabdovirus (eg, rabies and vesicular stomatitis virus), paramyxovirus (eg, measles and Sendai), positive-strand RNA viruses such as picornavirus and alpha virus, and double-stranded DNA viruses, including adenovirus, virus herpes (eg, herpes simplex viruses types 1 and 2, Epstein-Barr virus, cytomegalovirus); and poxvirus (eg, vaccinia, modified Ankara vaccinia (MVA), avian pox, and canary pox). Other viruses used to deliver polynucleotides encoding sFGFR3 polypeptides include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukemia-sarcoma, C-type mammals, B-type viruses, D-type viruses, the HTLV-BLV group, lentivirus, and spumavirus (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology , Third Edition , BN Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

How to get

Polynucleotides encoding the sFGFR3 polypeptides of the invention (eg, a polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-7 and variants thereof) can be prepared by any method known in the art. For example, a polynucleotide is generated using molecular cloning techniques and placed in a vector such as a plasmid, artificial chromosome, viral vector, or phage vector. The vector is used to transform the polynucleotide into a host cell suitable for expression of the sFGFR3 polypeptide.

Nucleic acid vector and host cell construction

The sFGFR3 polypeptides of the invention (eg, a polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-7 and variants thereof) can be obtained from a host cell. Polynucleotides (e.g., polynucleotides having the nucleic acid sequence of SEQ ID NO: 14 or 16 and variants thereof) encoding sFGFR3 polypeptides can be included in vectors that can be introduced into the host cell by conventional methods 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 used. Typically, host cells are of prokaryotic (eg, bacterial) or eukaryotic (eg, mammalian) origin.

A polynucleotide encoding an sFGFR3 polypeptide of the invention (eg, a polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-7 and variants thereof) can be obtained by various methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. A polynucleotide encoding an sFGFR3 polypeptide can be obtained using standard techniques such as gene synthesis. Alternatively, a polynucleotide encoding a wild-type sFGFR3 polypeptide (eg, a polypeptide having the amino acid sequence of SEQ ID NO: 8) can be mutated to contain specific amino acid substitutions using standard techniques in the art, eg, QuikChange™ mutagenesis. Polynucleotides encoding an sFGFR3 polypeptide can be synthesized using, for example, nucleotide synthesis or PCR methods.

Polynucleotides encoding an sFGFR3 polypeptide of the invention (e.g., a polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-7 and variants thereof) can be inserted into a vector capable of replicating and expressing the polynucleotide in prokaryotic or eukaryotic host cells. Examples of vectors used in the methods may include, but are not limited to, plasmid, artificial chromosome, viral vector, and phage vector. For example, a viral vector may include the viral vectors described above, such as a retroviral vector, an adenoviral vector, or a poxvirus vector (e.g., a vaccinia virus-based expression vector such as a modified vaccinia virus (MVA)), an adeno-associated viral vector, and an alpha viral vector. )) containing the nucleic acid sequence of the polynucleotide encoding the sFGFR3 polypeptide. Each vector may contain various components that can be tuned and optimized for compatibility with a particular host cell. For example, vector components may include, but are not limited to, an origin of replication, a selection marker gene, a promoter, a ribosome binding site, a signal sequence, a nucleic acid sequence of a polynucleotide encoding an sFGFR3 polypeptide, and/or a transcription termination sequence.

The above vectors can be introduced into appropriate host cells (e.g., HEK 293 cells or CHO cells, or into a patient host cell) using methods conventional in the art, such as transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection. When vectors are introduced into host cells to produce an sFGFR3 polypeptide of the invention, the host cells are cultured in conventional nutrient media modified as necessary to induce promoters, select transformants, or amplify polynucleotides encoding the sFGFR3 polypeptide. Methods for expressing 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 in Molecular 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), which are hereby incorporated by reference.

sFGFR3 polypeptide preparation, isolation and purification

Host cells (e.g., HEK 293 cells or CHO cells) used to produce an sFGFR3 polypeptide of the invention (e.g., a polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-7 and variants thereof) can be grown in a medium known in the art, and suitable for culturing selected host cells. Examples of suitable media for mammalian host cells include minimal maintenance medium (MEM), Dulbecco's modified Eagle's medium (DMEM), Expi293™ expression medium, DMEM supplemented with 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 selective agent such as ampicillin. Host cells are cultured at suitable temperatures, such as from about 20°C to about 39°C, for example, from 25°C to about 37°C, preferably 37°C, and CO ₂ levels, such as 5-10% (preferably 8%). The pH of the medium is typically from about 6.8 to 7.4, eg 7.0, depending primarily on the host organism. If an inducible promoter is used in the expression vector, expression of the sFGFR3 polypeptide is induced under conditions suitable for promoter activation.

An sFGFR3 polypeptide of the invention (eg, a polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-7 and variants thereof) can be isolated from host cell supernatant. Alternatively, the sFGFR3 polypeptide can be isolated by disruption of the host cell (eg, by osmotic shock, sonication, or lysis) followed by centrifugation or filtration to recover the sFGFR3 polypeptide. After the sFGFR3 polypeptide has been recovered, the sFGFR3 polypeptide can then be further purified. The 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 any other standard protein purification technique. (See Process Scale Purification of Antibodies , Uwe Gottschalk (ed.) John Wiley & Sons, Inc., 2009, incorporated herein by reference in its entirety).

Optionally, an sFGFR3 polypeptide of the invention (eg, a polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-7 and variants thereof) can be conjugated to a detectable label for purification. Examples of suitable labels for use in the purification of sFGFR3 polypeptides include, but are not limited to, a protein label, a fluorophore, a chromophore, a radioactive label, a metal colloid, an enzyme, or a chemiluminescent or bioluminescent molecule. In particular, protein tags that can be used to purify sFGFR3 polypeptides may include, but are not limited to, chromatographic tags (e.g., polyanionic amino acid peptide tags such as the FLAG tag or the hemagglutinin "HA" tag), affinity tags (eg, poly(His) tag, chitin binding protein (CBP), maltose binding protein (MBP), or glutathione S-transferase (GST)), solubilization tags (eg, thioredoxin (TRX) and poly(NANP)) , epitope tags (eg, V5 tag, Myc tag, and HA tag), or fluorescence tags (eg, GFP variants, GFP, RFP and RFP variants).

Diagnostic methods

A patient with abnormal visceral fat deposits may be identified as in need of treatment by one of the many methods known in the art. There are two categories of methods: anthropometric methods and imaging. Anthropometric methods are based on measurements based on tape measure and scale. Imaging techniques are based on the attenuation of X-rays as they pass through the patient, or on the magnetic properties of hydrogen nuclei in the patient.

A commonly used anthropometric method that classifies patients as normal weight, overweight, or obese is the body mass index (BMI). BMI is calculated by dividing the patient's weight (in kg) by the patient's height squared (in m). The threshold for obesity in adults differs from the thresholds for children. For adults, obesity is considered to be present when the BMI is 30 kg/m ² or greater (see, for example, Nuttall, Nutr. Today 50(3):117-28, 2015); for children, the threshold for obesity varies with age and height (see, for example, van der Sluis et al., Arch. Dis. Child . 87(4):341-347, 2002). Standard tables for BMI in children have limitations when achondroplasty children are considered (see, for example, Hoover-Fong et al., Am. J. Clin. Nutr. 88, 364-371, 2008).

Among anthropometric methods, BMI does not allow distinguishing between subcutaneous and visceral fat deposition. As a result, abnormal abdominal visceral fat is better identified using waist circumference (measured just above the palpable upper iliac crest bone), sagittal diameter (anteroposterior distance from the lower back to the anterior abdomen), and android:gynoid fat ratio (waist circumference divided by to the circumference of the hips).

The cutoff value for abnormal waist circumference for adult women is greater than 83 cm and for adult men greater than 90 cm (see, for example, Zhu et al., Am. J. Clin. Nutr. 76(4):743-749, 2002 ). For sagittal diameters, the limit value for women is more than 20.1 cm, and for men more than 23.1 cm; see, for example, Pimentel et al, Nutr. hosp. 25(4):656-61, 2010. Finally, for an android:gynoid fat ratio in women over 0.85 and in men over 0.9; see, for example, Price et al, Am. J.Clin. Nutr. 84(2):449-460, 2006).

One imaging technique that relies on X-rays is dual-energy X-ray absorptiometry (DXA), a technique in which two X-ray beams of different energies are used to determine fat mass (as opposed to lean mass) and from this determination a measure is obtained called fat mass index (measured fat mass divided by height squared). The limit value for abnormal fat deposition in women is greater than 13, and the limit value for abnormal fat deposition in men is greater than 9; see, for example, Kelly et al, PLOS One, 4(9): 2009).

Both CT (using X-rays) and MRI (depending on the magnetic properties of the body) can visualize the abdomen in cross section and thus distinguish between visceral and subcutaneous fat deposits. With the help of CT or MRI, the boundary value of abnormal visceral fat deposition in women is 110 cm ² and in men 132 cm ² ; see, for example, Wajchenberg, Endocr. Rev. 21(6):697-738, 2000.

Ways to monitor therapy

To evaluate the effect of therapy, a patient treated with an sFGFR3 polypeptide can be screened using various disease state biomarkers. For a patient being treated to reduce or prevent abnormal visceral fat deposition, monitoring methods include, for example, determination of improvement in BMI, sagittal diameter, android:gynoid ratio, waist circumference, fat mass index, and visceral fat area on a standardized cross section. The effect of therapy can also be assessed using one or more biomarkers found, for example, in a patient sample (eg, in a blood, urine or sputum sample). In the case of a patient receiving treatment to prevent or improve metabolic diseases such as diabetes or fatty liver, blood tests to monitor the effect of therapy include, for example, evaluation of a patient sample for changes in glucose and/or insulin levels, improvement in glucose tolerance testing and reducing elevated levels of alanine transaminase, aspartate aminotransferase and/or alkaline phosphatase.

If a patient is receiving sFGFR3 polypeptide therapy to treat or reduce the risk of cardiovascular disease, a blood test may be performed to show improvement in triglycerides, high-density lipoprotein, low-density lipoprotein, and/or cholesterol levels. Alternatively, radioisotope stress testing may be performed to show improvement in the patient's exercise tolerance and/or myocardial perfusion.

In patients being treated to improve or reduce the risk of dementia, a serial neuropsychology study may be performed.

In cases where the sFGFR3 polypeptide is administered to treat or reduce the risk of infertility or menstrual irregularities, blood levels of a biomarker such as follicle stimulating hormone, luteinizing hormone, estradiol and prolactin can be measured.

If a patient suffers from or is at risk of sleep apnea, a study that measures, for example, oxygen, respiratory rate, heart rate, snoring, and/or body movements of the sleeping patient, may be performed after sFGFR3 polypeptide therapy to evaluate the effect of therapy.

In a patient for whom an sFGFR3 polypeptide is indicated for the treatment or prevention of restrictive lung disease or asthma, lung function testing using e.g. spirometry or plethysmography can be used to show improvement in lung parameters such as tidal volume, vital capacity, residual volume and/or or forced vital capacity.

In the event that monitoring methods indicate that a patient may not respond to treatment with an sFGFR3 polypeptide or for whom a greater effect of treatment may be desirable, administration of the sFGFR3 polypeptide may be repeated one or more times, or the frequency of administration may be increased.

Introduction of sFGFR3 polypeptides

The sFGFR3 polypeptide may be administered to treat a patient who has or is at risk of abnormal visceral fat deposits. Examples of sFGFR3 polypeptides include, for example, sFGFR3 polypeptides having the amino acid sequence of any of SEQ ID NOs: 1-7, or variants thereof having at least 85% sequence identity (e.g., 86%-100% sequence identity, e.g. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity). The sFGFR3 polypeptide may be administered by any route known in the art, such as parenteral, enteral, or topical administration. In particular, the sFGFR3 polypeptide can be administered to a patient having increased visceral fat deposition) subcutaneously (eg, by subcutaneous injection), intravenously, intramuscularly, intraarterially, intrathecally, or intraperitoneally.

The sFGFR3 polypeptide may be administered to a patient (eg, human) at a predetermined dosage, eg, an effective amount, to treat increased visceral fat deposition without causing significant toxicity. For example, sFGFR3 polypeptides may be administered to a patient with increased visceral fat deposition in discrete doses ranging from about 0.002 mg/kg to about 20 mg/kg (e.g., 0.002 mg/kg to 20 mg/kg, 0.01 mg/kg up 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 up to 3 mg/kg). In particular, the sFGFR3 polypeptide may be administered at individual doses, for example, from 0.001 mg/kg to 7 mg/kg, such as from 0.3 mg/kg to about 2.5 mg/kg.

Exemplary dosages of the sFGFR3 polypeptide of the invention for administration to a patient (e.g. human) having increased visceral fat deposition) include, for example, 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 or 20 mg/kg. These doses may be administered one or more times (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more times) per day, week, month, or year. For example, the sFGFR3 polypeptide may be administered to patients at a weekly dose in the range, for example, from about 0.0014 mg/kg/week to about 140 mg/kg/week, for example, from about 0.14 mg/kg/week to about 105 mg/week. kg/week, or, for example, from about 1.4 mg/kg/week to about 70 mg/kg/week (eg, 5 mg/kg/week).

Gene therapy

The sFGFR3 polypeptide may be administered to a patient as a nucleic acid molecule. Examples of nucleic acid molecules that can be administered include those that encode sFGFR3 polypeptides having the amino acid sequence of any of SEQ ID NOs: 1-7, or variants thereof having at least 85% sequence identity (e.g., 86% 100% sequence identity, e.g. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% -th sequence identity). For example, nucleic acid molecules may have the sequence of any of SEQ ID NOs: 10-18, or variants thereof, having at least 85% sequence identity (e.g., 86%-100% sequence identity, e.g., 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity).

Nucleic acid molecules encoding an sFGFR3 polypeptide can be delivered by gene therapy, in which a polynucleotide encoding an sFGFR3 polypeptide is delivered to tissues of interest and expressed in vivo . Gene therapy methods are discussed, for example, Verme et al. ( Nature 389:239-242, 1997), Yamamoto et al. ( Molecular Therapy 17:S67-S68, 2009), and Yamamoto et al., ( J. Bone Miner. Res. 26: 135-142, 2011), are incorporated herein by reference.

The sFGFR3 polypeptide of the invention can be produced in cells of a patient (eg, human) having increased visceral fat deposition by introducing a vector (eg, plasmid, artificial chromosome (eg, BAG, PAC, and YAC), or viral vector) containing the nucleic acid sequence of the polynucleotide encoding the sFGFR3 polypeptide. For example, the viral vector can be a retroviral vector, an adenoviral vector, or a poxvirus vector (eg, a vaccinia virus-based expression vector such as a modified vaccinia virus (MVA)), an adeno-associated viral vector, or an alpha viral vector. The vector, when placed inside a cell of a patient (e.g., human) having a skeletal growth retardation disorder (e.g., achondroplasia), for example, by transformation, transfection, electroporation, calcium phosphate precipitation, or direct microinjection, will stimulate the expression of the sFGFR3 polypeptide, which is then secreted from the cell. . The invention further includes a cellular therapy in which a cell expressing an sFGFR3 polypeptide is administered to a patient (eg, a human).

Pharmaceutical compositions

Pharmaceutical compositions that can be administered to treat a patient having abnormal visceral fat deposition as discussed herein comprise an sFGFR3 polypeptide, a polynucleotide encoding an sFGFR3 polypeptide, or a host cell that contains an sFGFR3 polynucleotide. An sFGFR3 polypeptide may have an amino acid sequence of any of SEQ ID NOs: 1-7, or variants thereof, having at least 85% sequence identity (e.g., 86%-100% sequence identity, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity). Nucleic acid molecules can have the sequence of any of SEQ ID NOs: 10-18 or variants thereof having at least 85% sequence identity (e.g. 86%-100% sequence identity, e.g. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity). Compositions comprising an sFGFR3 polypeptide, a polynucleotide encoding an sFGFR3 polypeptide, or a host cell that contains an sFGFR3 polynucleotide can be formulated at various dosages, in various formulations and in combination with pharmaceutically acceptable inert excipients, carriers or diluents.

A pharmaceutical composition comprising an sFGFR3 polypeptide, a polynucleotide encoding an sFGFR3 polypeptide, or a host cell that contains an sFGFR3 polynucleotide may be formulated at a specific dosage, such as a dosage that is effective for treating a patient (e.g., human) with increased visceral fat deposition without significant toxicity. For example, compositions may be formulated to include from about 1 mg/ml to about 500 mg/ml of an sFGFR3 polypeptide or polynucleotide (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 sFGFR3 polypeptide or polynucleotide).

Pharmaceutical compositions comprising an sFGFR3 polypeptide or polynucleotide can be prepared in various forms such as liquid solution, dispersion or suspension, powder, or other ordered structure suitable for stable storage. For example, compositions comprising an sFGFR3 polypeptide or polynucleotide intended for systemic or local delivery may be in the form of injectable or infusion solutions, such as for parenteral administration (eg, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, or intraperitoneal administration). Compositions of sFGFR3 for injection (eg, subcutaneous or intravenous injection) can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a carrier medium. Pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, and cell culture media (eg, Dulbecco's modified Eagle's medium (DMEM), α-modified Eagle's medium (α-MEM), F-12 medium). Methods of preparation are known in the art, see for example, Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (2nd ed.) Taylor & Francis Group, CRC Press (2006), incorporated herein by reference in in all its fullness.

Compositions comprising an sFGFR3 polypeptide or polynucleotide may be provided to patients (eg, humans) having increased visceral fat deposition in combination with pharmaceutically acceptable inert excipients, carriers, or diluents. Acceptable inert excipients, carriers or diluents may include buffers, antioxidants, preservatives, polymers, amino acids and carbohydrates. Aqueous vehicles, carriers or diluents may include water, hydroalcoholic solutions, emulsions or suspensions, including saline, buffered medical parenteral vehicles, including sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, Ringer's solution containing lactose and fixed oils. Examples of non-aqueous inert excipients, carriers or diluents are propylene glycol, polyethylene glycol, vegetable oil, fish oil and injectable organic esters.

Pharmaceutically acceptable salts may also be included in sFGFR3 compositions. Typical pharmaceutically acceptable salts may include mineral acid salts (eg hydrochlorides, hydrobromides, phosphates and sulfates) and organic acid salts (eg acetates, propionates, malonates and benzoates). In addition, adjuvants such as wetting or emulsifying agents and pH buffering agents may be present. A detailed discussion of pharmaceutically acceptable excipients, carriers, and diluents is available in Remington: The Science and Practice of Pharmacy , ^22nd Ed., Allen (2012), which is incorporated herein by reference in its entirety.

Pharmaceutical compositions comprising an sFGFR3 polypeptide or polynucleotide can also be formulated with a carrier that will protect the sFGFR3 polypeptide or polynucleotide from rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. For example, the sFGFR3 composition can be enclosed in microcapsules prepared by coacervation or interfacial polymerization methods, such as hydroxymethylcellulose, gelatin, or poly(methyl methacylate) microcapsules; colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles, or nanocapsules); or macroemulsions. In addition, the sFGFR3 composition may be formulated as a sustained release composition. For example, sustained release compositions may include semi-permeable matrices of solid hydrophobic polymers containing an sFGFR3 polypeptide or polynucleotide, in which the matrices are in the form of shaped articles such as films or microcapsules.

EXAMPLES

The following examples are intended to illustrate and not limit the invention.

Example 1 :

Study plan

To evaluate the potential of sFGFR3 therapy (e.g., sFGFR3 having the sequence of SEQ ID NO: 1) to prevent the development of abdominal obesity in achondroplasia, the inventors first characterized the development of obesity in children using a retrospective chart review, and then conducted a study in Fgfr3 ^ach/+ mice , in mouse models reproducing most human symptoms. For a chart review in the Department of Endocrinology, Bone Diseases, Genetic and Medical Gynecology of the Purpan Children's Hospital in Toulouse, France, 11 patients (5 girls and 6 boys) with achondroplasia were observed from birth to 18 years of age. This retrospective review study was conducted in accordance with the Declaration of Helsinki and the French Biomedical Research Regulations, which do not require ethics committee approval for non-interventional research. All patients had a G380R FGFR3 mutation (G380R refers to the full-length FGFR3 numbering including the signal peptide; the residue numbering without the signal peptide is G358R (see SEQ ID NO: 9) confirmed by molecular testing. Patients receiving any growth-related treatment were excluded.On average, children visited a referral center every 4 months.During each visit, data determined included height, weight, and subsequent body mass index (BMI=kg/ ^m2 ).Blood tests were performed only once a year, and dual-energy X-ray absorptiometry (DXA) was performed once.A mouse study was performed on Fgfr3 ^ach/+ transgenic mice or their littermates (WT) by blind treatment twice a week with subcutaneous injections of 2.5 μg/kg sFGFR3 or vehicle, starting from day 3 to day 21. Mice were weaned from mother's milk at the age of 3 weeks.After one week of acclimatization, mice were transferred to a normal (ND) or high fat (HFD) diet for 10 weeks. The development of obesity was assessed using measurements of body composition, indirect calorimetry and classical assessment of the glucose and lipid profile, as well as assessment of liver and pancreas function. The number per group is presented in figure captions.

Clinical Analysis

Anthropometric calculations, including height and age z-score and BMI-age z-score, were performed using the WHO AnthroPlus software (WHO AnthroPlus Personal Computer Manual: Software for Estimating the Growth of Children and Adolescents in the World. Geneva: WHO, 2009 (www.who.int/growthref/tools/en/) z-scores are based on WHO standards (birth to 60 months) and WHO reference material 2007 (61 months to 19 years) Body composition was assessed by DXA using a Lunar Prodigy device (GE Healthcare).Regions of Interest (ROI) for regional body composition were determined using the manufacturer's instructions (Fig. 1B).Briefly, ROI of the torso was measured from the pelvic incision (lower border) to the neck incision (upper border), The android ROI was measured from the pelvic incision (lower limit) to the pelvic incision at 20% of the distance between the neck and pelvic incisions (upper limit), the navel ROI was measured from the lower border from android at 150% of the android distance, and the gynoid ROI was from the lower limit of the ROI navel to a line equal to twice the height of the android ROI (lower bound). Blood samples were taken after at least a 12 hour fasting period and analyzed at the Federal Institute of Biology (IFB) at Purpana Hospital. Fasting glucose and insulin, total cholesterol, HDL and LDL cholesterol, triglyceride concentrations, TGO, TGP, gGT, and total calcium, sodium, potassium, bicarbonate, phosphate, chloride, and alkaline phosphatase in plasma were measured using standard colorimetric or colorimetric enzymatic methods on the Cobas 8000 series modular analyzer using the C701 module from Roche Diagnostics. Serum total 25OH vitamin D concentration was measured by chemiluminescent immunoassay on a Cobas 8000 series modular analyzer using an E602 module from Roche Diagnostics. All values were compared with reference values established for each age group in a children's hospital and using published references (Mellerio et al., Pediatrics 129: e1020-1029 (2012); Fischer et al., Ann. Clin. Biochem. 49:546 -553, 2012; and Haine et al., J. Bone Miner Res. 30:1369-1376, 2015). After a 12-hour overnight fast, patients underwent OGTT according to established guidelines (Alberti et al., Diabet. Med. 15:539-553, 1998). After oral administration of 1.75 g/kg glucose, blood samples were taken at baseline and 30 and 120 minutes later to measure glucose concentration using the hexokinase method. Glucose regulation was assessed according to the American Diabetes Association guidelines: normal glucose regulation was defined as fasting glucose <5.6 mmol/L and 120 min glucose <7.8 mmol/L, and fasting glucose as fasting glucose 5.6 -6.9 mmol/l, glucose level impaired tolerance as 120 min glucose 7.8-11.0 mmol/l.

Animals and treatment

Experiments were performed on Fgfr3 ^ach/+ transgenic mice (Naski et al., Development 125: 4977-4988, 1998). At weaning from mother's milk, ear biopsy was used to confirm the genotype of mice by genomic DNA PCR, as previously described (Garcia et al., Science Translational Medicine 5:203ra124, 2013).

During the experiment, the mice were kept under standard laboratory conditions and allowed access to food and water ad libitum. The study was approved by the local Institutional Ethical Committee for the Use of Laboratory Animals (CIEPAL Azur) (approvals # NCE-2012-52 and NCE-2015-225). On day 3, neonatal mice were injected with 2.5 mg/kg of FLAG-labeled sFGFR3 as previously described (Garcia et al., Science Translational Medicine 5:203ra124, 2013). Control litters received 10 μl of PBS containing 50% glycerol (carrier medium). From day 3 to day 22, Fgfr3 ^ach/+ mice received 6 subcutaneous injections of sFGFR3 or vehicle. One week after weaning at 4 weeks of age, treated and untreated mice were divided into two groups and exposed for 10 weeks to a normal (ND, A03, SAFE) or high fat (HFD, 52% kcal as cooked fat) diet. to order, containing 54% lipids, SAFE), respectively. After 6 hours of fasting, blood was taken from the tail vein. Glycemia was measured using a glucometer (Abbot) and serum insulin was determined using ELISA (Mercodia). Glucose tolerance tests (GTT) were performed on mice after 10 weeks of ND or HFD intake. After 6 hours, fasting mice were injected with intraperitoneal glucose solution (1 g/kg). Blood was taken from the tail vein and glucose levels were monitored over time using a glucometer or EnzyChrom glucose assay kit (BioAssay Systems). Glucose levels were normalized to a time value of -15 min for each mouse.

Indirect calorimetry was studied in mice fed a normal (ND) diet or a high fat diet (HFD) for 10 weeks. As indicated above, mice were treated during the rearing period with 2.5 mg/kg sFGFR3 or vehicle, dieted for 2 weeks, and then placed in metabolic chambers. After 24 h of acclimatization in separate metabolic chambers, O2 consumption (VO2) and CO2 production (VCO2) (Oxylet; Panlab-Bioseb) were measured in individual mice at 32 min intervals over a 24 h period with unrestricted access to food, followed by one night without food. The respiration coefficient was calculated and analyzed as follows: RQ=VCO2/VO2, RQ=1 correspond to carbohydrate oxidation, and RQ~0.7 corresponds to fat oxidation. Energy expenditure (kcal/day/weight 0.75=1.44×VO2x [3.815+1.232×RQ]), carbohydrates (g/min/kg 0.75=[4.55×VCO2]-[3.21× VO2]) and lipids (g/min/kg 0.75=[1.67×VO2]-[1.67×VCO2]). Outpatient activities and rearing of mice were monitored using a weight sensor or an infrared photocell interruption method (Oxylet; Panlab-Bioseb).

Body composition was determined using a SkyScan 1178 X-ray micro-CT system. Four and 10 week old mice were anesthetized and scanned using the same parameters: 104 µm pixel size, 49 kV, 0.5 mm thick aluminum filter, 0.9° rotation pitch. The total volume of adipose tissue was determined between the tip of the nose and the end of the tail, and the volume of the abdominal cavity was determined between the lumbar L1 and the sacral S1. The adipose tissue was then quantified more accurately. Body composition analysis is based on the determination of the boundaries of the region of interest after a three-dimensional reconstruction of scanned images. Analysis of 3D reconstructions was performed using NRecon and CTAn (Skyscan) software.

After sacrifice, the animals were weighed and several tissues and organs (subcutaneous, epididymal adipose tissue, liver, pancreas) were harvested for further analysis by histochemistry or qPCR. In some groups, bone marrow-derived mesenchymal stem cells were collected by washing the femurs (Zhu et al., Nat Protoc 5:550-560, 2010).

The lipid profile was assessed by taking intracardiac blood. Total cholesterol, triglycerides (TG), high density lipoprotein (HDL), and low density lipoprotein (LDL) were measured in serum using a Beckman AU 2700 analyzer. Serum was analyzed for protein levels in selected adipokines associated with inflammation, obesity, by insulin or FGF , using an array of mouse adipokines (#ARY013, R&D Systems) according to the manufacturer's instructions for nitrocellulose membranes. Following streptavidin-HRP and chemiluminescent detection, the proteins associated with each captured antibody were quantified using densitometry and levels compared to the percentage change in WT mice.

Mesenchymal stem cell research

Mesenchymal stem cells were isolated from femoral marrow from untreated or sFGFR3-treated mice aged 6 to 8 weeks by washing the femurs (Zhu et al., Nat. Protoc. 5:550-560, 2010) and cultured up to 80 % confluence in medium supplemented with 10% serum. The medium was then changed to adipogenic medium (DMEM F12 supplemented with 2% serum, 1% antibiotics, 66 mM insulin, 1 nM triiodothyronine, 100 mM cortisol, 10 μg/ml transferrin and 3 μM rosiglitazone in DMEM-F12). Total RNAs were extracted using Trizol reagent (Life Technologies) and chloroform (Sigma). One microgram of total RNA was reverse transcribed into cDNA using random hexamers and a Superscript II reverse transcriptase kit (Invitrogen). Real-time qPCR was performed on the StepOne Plus Real-Time PCR System (Applied Biosystems) with Fast SYBR Green Master Mix (Sigma) using a dedicated RT2 Profiler PCR Array (CAPM13080). The genes studied are listed in Table 1. Gene expression was normalized for the housekeeping gene HPRT, RPL13a and RPL6. FGFR expression was performed using the following primers: FGFR1, For-5' CAGATGCACTCCCATCCTCG 3'Rev-5' TCT GGGGATGTCCAGTAGGG 3'; FGFR2 For-5' TGGCAGTGAAGATGTTGAAAG 3'Rev-5' ATCATCTTCATCATCTCCATCTCTTG 3'; FGFR3 For-5' TTATCCTTGGCTCCTGGGTG 3'Rev-5' CTGGAAGGTAGCAGTGGGAA 3'; FGFR4 For-5' GCTCGGAGGTAGAGGTCTTGT 3'Rev-5' CCACGCTGACTGGTAGGAA 3'; HSP90 For-5' TTTGGTGGACACAGGCATTG 3'Rev-5' CAAACTGCCCGATCATGGAG 3'.

Table 1. List of genes examined using the RT2 Profiler PCR Array in mesenchymal stem cells

Gene designation Full official name No. standard. sequences Adipoq Adiponectin containing C1Q and collagen domains NM_009605 Cebpa CCAAT/enhancer-binding protein (C/EBP), alpha NM_007678 Cebpb CCAAT/enhancer-binding protein (C/EBP), beta NM_009883 Cebpd CCAAT/enhancer-binding protein (C/EBP), delta NM_007679 Dlk 1 Delta-like 1 homolog (Drosophila) NM_010052 Fabp4 Fatty acid binding protein 4, adipocyte NM_024406 Fasn fatty acid synthase NM_007988 Gata3 GATA binding protein 3 NM_008509 Hprt Hypoxanthine guanine phosphoribosyltransferase NM_013556 Lep Leptin NM_008493 Lipe Lipase, hormone-sensitive NM_010719 Lpl Lipoprotein lipase NM_008509 Pparg Peroxisome proliferator-activated gamma receptor NM_011146 Ppargc1a Peroxisome proliferator-activated receptor gamma, coactivator 1 alpha NM_008904 Rpl13a Ribosomal protein L13a NM_009438 Rpl6 Ribosomal protein L6 NM_011290 Scd1 Stearoyl-coenzyme-A-desaturase 1 NM_009204 Slc2a4 Solute Carrier Family 2 (Lightweight Glucose Transporter), Member 4 NM_011480 Srebf1 Transcription factor binding sterol regulatory element 1 NM_021 Ucp1 Uncoupling protein 1 (mitochondrial, proton transporter) NM_009463 Wnt1 Wingless Type 1 MMTV Integration Site NM_021279

In insulin signaling experiments 24 h after isolation, cells were depleted for 6 h and then stimulated with 50 nM insulin for 0, 5, 15, 30 min or 0, 1, 10, 50, 100 nM for 5 min. Cell extracts were processed for Western blot analysis. For this, cells were lysed in RIPA lysis buffer (Millipore) and homogenized for 30 minutes using vortex mixing. Proteins were precipitated by centrifugation at 1400 g for 15 minutes, and the total protein content was assessed using a BCA protein assay kit (ThermoScientific). Samples were diluted in Sample Reduction Buffer (Life Technology), boiled, and processed for immunoblotting using a standard procedure. Monoclonal antibodies were used as follows: anti-p44/42 MAPK (Erk1/2) (4695S, Cell Signaling), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (4370S, Cell Signaling). The results were normalized to HSP90 expression (4877S, Cell Signaling).

Histology and immunohistochemistry

Histological analysis was performed on adipose tissue, liver and pancreas from mice aged 12 weeks. Organs were fixed in 4% formalin for 24 hours, added dropwise to paraffin, and 5 µm sections were stained with hematoxylin and eosin. Adipocyte diameters were measured in one or two different sections in each sample (100 to 300 adipocytes were counted in each section).

The number and area of islets of the pancreas were measured in one section using the Fiji Image J system (the area of the islets was normalized to the total area of the pancreas). Liver glycogen content was assessed by Periodic Acid Schiff (PAS) staining. Immunohistochemistry was performed on 5 μm sections of the pancreas. Sections were blocked for 45 minutes with PBS with 1% BSA, incubated overnight in a humid chamber with anti-insulin monoclonal primary antibody (4 μg/ml) (Santa Cruz, sc-9168), polyclonal anti-glucagon antibody (4 μg/ml) (Santa Cruz, sc-7779) and 1 h with Alexa Fluor 594 Secondary Antibody (2 µg/mL) (Life Technologies, A-21442) and Alexa Fluor 488 Secondary Antibody (4 µg/mL) (Life Technologies, A-21467 ). Sections were stained with DAPI solution (Santa Cruz), treated with anti-fluorescence reagent and visualized under fluorescence microscopy. Staining without secondary antibodies was used as a negative control.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 6.0 software. Data were expressed as mean ± standard deviation. To test for normality and equality of variance, the Agostino and Pearson normality test (a=0.05), the Shapiro-Wilk normality test (a=0.05), and the KS normality test (a=0.05) were performed. Significance was determined using unpaired two-tailed Student's t-test or Tukey's multiple comparison test. The raw height and BMI results of the patients were converted to age z-score from the mean of the control population using control data. The expected mean of these values in a healthy population is 0. P values <0.05 were considered significant (*P<0.05; **P<0.01; ***P<0.001).

Example 2:

Development of excess abdominal adipose tissue in patients with achondroplasia without classic complications

The patients were children and adolescents with achondroplasia. They were included in a longitudinal retrospective study conducted by the same observer for a mean period of 8.6 ± 5.6 years. Anthropometric measures and body composition were recorded from birth at follow-up visits and compared between the three age groups aged [0-3], [4-8] and [9-18] years. Several metabolic blood parameters were measured and compared across different age groups. Blood values at visits under 3 years of age were not considered due to the difficulty of restricting food intake in children and therefore controlling metabolic parameters at this early age.

It is known that patients with achondroplasia not only exhibit growth disorders, but also tend to become overweight, resulting in overweight or even obesity (Hoover-Fong et al., Am. J. Med. Genet. A. 143A:2227- 2235, 2007). As seen in FIG. 1A, BMI of patients with achondroplasia increased significantly during childhood, reaching a value of approximately 30 kg/m ² in the [9-18] years group (p=0.2407 between groups [0-3] and [4-8], P<0 .0001 comparing group [9-18] with both other groups, Tukey's multiple comparison test). A negative correlation was observed between BMI and height (Pearson's r=-0.5660, p=0.0021), with the youngest children having the highest BMI. To study their metabolic status and its evolution, densitometric analyzes were first performed to determine the distribution of body fat and lean mass in different age groups (Fig. 1B). The inventors observed that under the age of 8 years, the ratio of total fat:lean mass was relatively constant and that it increased significantly during adolescence, from 0.21±0.02 and 0.20±0.05 in age groups [0 -3] and [4-8] to 0.84±0.29 in the age group [9-18], respectively (P=0.9954 between groups [0-3] and [4-8], P=0 .0053 and P<0.0001 when comparing age groups [9-18] with [0-3] and [4-8], respectively, Tukey's multiple comparison test (Table 2).

Table 2. The results of densitometry of achondroplasia patients in three age groups. Values are mean±SD (range). The p-values represent the significance of the difference between the three groups (one-way ANOVA followed by Tukey's multiple comparison tests, ns=not significant). Results of subsequent analysis: significant difference between [0-3] and [4-8] groups; b significant difference between groups [0-3] and [9-18]; with a significant difference between groups [4-8] and [9-18].

Age (years) [0-3] [4-8] Parameter Average+SD Interval (min-max) Average+SD Interval (min-max) Redistribution of fat and lean mass Total body fat mass 1.52+0.32 (1.29-1.76) 2.64+0.73 (1.98-4.06) Total lean body mass 0.88+0.04 (0.29-0.91) 0.84+0.09 (0.75-0.90) Torso weight 3.67+0.45 (3.38-4.02) 6.93+1.46 (4.83-8.68) Fat mass of the torso 0.41+0.12 (0.32-0.49) 0.83+0.44 (0.23-1.64) Lean mass of the torso 3.29+0.33 (3.06-3.53) 6.11+1.15 (4.35-7.08) Leg mass 1.77+0.28 (1.57-1.97) 4.61+1.29 (3.12-7.21) Fat mass of the leg 0.65+0.12 (0.57-0.74) 1.22+0.31 (0.86-1.69) Lean mass of the leg 1.11+0.16 (0.99-1.23) 3.39+1.04 (2.26-5.52) Hand mass 0.65+0.04 (0.62-0.68) 1.21+0.23 (0.99-1.53) Fat mass of the arm 0.09+0.06 (0.08-0.10) 0.21+0.09 (0.12-0.35) Lean mass of the arm 0.56+0.04 (0.53-0.58) 0.99+0.18 (0.66-1.18) Redistribution of fat and lean mass
(% of total body weight) Total body fat mass 19.04+0.74 (18.51-19.57) 16.08+3.10 (11.86-21.23) Total lean body mass 88.29+3.51 (85.81-90.77) 83.89+9.25 (74.75-90.23) Torso weight 46.47+2.57 (44.64-48.29) 47.26+4.08 (28.42-50.23) Fat mass of the torso 5.01+0.64 (4.56-5.46) 5.60+1.74 (1.37-8.23) Lean mass of the torso 41.47+3.22 (39.19-43.74) 41.66+5.82 (27.05-45.23) Leg mass 22.14+0.37 (21.88-22.40) 25.22+0.28 (25.03-42.23) Fat mass of the leg 8.17+0.02 (8.16-8.19) 6.94+0.87 (5.95-9.23) Lean mass of the leg 13.97+0.40 (13.69-14.26) 18.28+0.60 (17.64-32.23) Hand mass 8.21+0.88 (7.59-8.83) 7.33+0.21 (6.66-8.23) Fat mass of the arm <0.001 (10 ^-6 -10 ^-6 ) 1.08+0.42 (0.78-1.23) Lean mass of the arm 7.05+0.76 (6.51-7.59) 6.25+0.21 (5.44+6.23) Gynoid body fat 43.75+0.07 (43.70-43.80) 33.97+3.00 (29.30-38.40) 52.91+6.37 (40.60-58.80) 0.0007 ^s android body fat 13.45+3.47 (11.00-15.90) 14.73+4.02 (8.90-19.50) 44.79+11.39 (29.90-55.90) 0.0001b ^{, s} Gynoid lean body mass 56.25+0.07 (56.20-56.30) 67.52+4.70 (60.90-74.91) 47.10+6.38 (40.50-58.70) ns Android lean body mass 86.6+3.40 (84.20-89.00) 85.27+3.97 (80.52-91.07) 55.21+11.39 (44.10-69.20) ns

Interestingly, as seen in Fig. 1C, the increase in fat mass was not uniform, and patients predominantly developed abdominal (android) fat mass (+204%) compared to femoral (gynoid) fat mass (+55%) (P = 0.0974 between groups [0– 3] and [4-8], P=0.0002 and P=0.001, when comparing age groups [9-18] with [0-3] and [4-8], respectively, Tukey's multiple comparison test). Simultaneously, the masses of both androids and gynoids did not change during this period (Table 1). Therefore, the ratio of fat:lean mass increased significantly in the abdominal region throughout childhood (P=0.3187 between groups [0-3] and [4-8], P=0.0924 and P<0.0001 when comparing age groups [9-18] with [0-3] and [4-8], respectively, Tukey's multiple comparison test). The trunk, legs, and arms followed a very similar trend, with an increase in percentage fat mass and a decrease in percentage lean mass from infancy to adulthood (Table 1). Spinal mineral density (BMD) was determined between L1 and L4 after 3 years. In both age groups, BMD was found to be below the respective normal age range (van der Sluis et al., Arch. Dis. Child. 87:341-347, 2002) (0.511±0.065 g/ ^cm2 and 0.898±0.223 g/cm2). cm ² in the age groups [4-8] and [9-18], respectively, compared with 0.645±0.071 g/cm ² and 0.913±0.199 g/cm ² in the same age groups).

A comparison was made of various blood parameters between age groups [4-8] and [9-18]. Surprisingly, in both age groups and regardless of their BMI, children with achondroplasia tended to have low plasma total cholesterol (3.38 ± 0.36 mmol/L and 3.73 ± 0.44 mmol/L in age groups [4-8] and [9-18]), respectively, with normal values ranging from 3.90 to 5.70 mmol/l in children), and low triglycerides (0.56±0.14 mmol/l and 0.63±0 .13 mmol/l in the age groups [4-8] and [9-18], respectively, with normal values from 0.60 to 1.70 mmol/l in children). Similarly, fasting blood glucose levels (Fig. 1D) and insulin levels did not increase with age and remained within the normal range (7.3 ± 5.4 mU/L and 13.4 ± 3.4 mU/L in age groups). [4-8] and [9-18]), respectively, with normal values ranging from 2.6 to 16 mIU/l in children). These results were confirmed by glucose levels obtained during the oral glucose tolerance test (OGTT), which showed normal glucose levels at 0, 30 and 120 minutes after oral administration (4.53±0.22 mmol/l at 0 minutes, 7.97±1.72 mmol/l at 30 minutes and 5.17 mmol/l at 120 minutes). No statistical differences were found between age groups. Due to the high level of hemolysis during OGTT, unfortunately, there were no data on insulin levels.

All other blood counts were within normal limits (data not shown).

Metabolic changes seen in lean and obese Fgfr3 mice ^ach/+ and corrected by sFGFR3 treatment

To determine the role of FGFR3ach in this predominant development of visceral obesity in achondroplasia, Fgfr3 ^ach/+ transgenic mice carrying the G380R mutation or their wild-type (WT) littermates were treated with sFGFR3 or vehicle for 3 weeks starting on day 3. Mice were then maintained on a normal (ND) or high fat (HFD) diet starting at 4 weeks of age for 10 weeks to evaluate the development of obesity.

After weaning at 4 weeks of age, as expected, untreated Fgfr3 ^ach/+ mice lost 20.4% body weight compared to their WT littermates. This was associated with a decrease in lean and adipose tissue (50% and 33.9%, respectively). Treated animals showed a 14.1% reduction in body weight compared to WT mice (P < 0.0001).

After 10 weeks of diet, all HFD groups showed a significant increase in body weight compared to ND (Fig. 2A). Interestingly, however, body composition was different for both genotypes. Untreated Fgfr3 ^ach/+ mice had a higher lean to belly fat ratio measured between L1 and S1 compared to WT mice, regardless of diet (FIG. 2B). When fed ND, untreated Fgfr3 ^ach/+ mice had less epididymal (visceral) and subcutaneous adipose tissue than WT animals (FIGS. 2C and 2D). However, after 10 weeks of HFD consumption, untreated Fgfr3 ^ach/+ mice developed more epididymal adipose tissue compared to WT animals, which predominantly developed subcutaneous fat (FIGS. 2C and 2D). These data showed that, like patients with achondroplasia, Fgfr3 ^ach/+ mice were prone to developing visceral adipose tissue.

Treatment with sFGFR3 did not affect weight gain (Figure 2A). However, body composition was significantly affected by sFGFR3 treatment with a significant reduction in the ratio of abdominal lean:fat mass in ND-treated mice (FIG. 2B) induced by a decrease in lean mass and an increase in fat mass, respectively. Very interestingly, sFGFR3-treated Fgfr3 ^ach/+ mice showed body fat distributions that were the same as WT animals whether they received ND or HFD (FIGS. 2C and 2D).

After HFD consumption, histological analysis showed fewer adipocytes in the subcutaneous region and an increased proportion of these small adipocytes in untreated Fgfr3 ^ach/+ mice compared to WT (FIGS. 2E and 2G). In epididymal adipocytes, no difference in size or variance was found between both genotypes (Fig. 2F and 2H). Treatment with sFGFR3 restored the size and spread of subcutaneous adipocytes and induced a slight increase in the size of epididymal adipocytes (Fig. 2E-2H). Since an increase in the proportion of small fat cells has been associated with inflammation (Kursawe et al., Diabetes 59: 2288-2296, 2010; and Lafontan, Diabetes Metab. 40:16-28, 2014), several circulating adipokines have been measured to assess the degree of systemic inflammation in these animals (FIGS. 6A and 6B). Adipokines were divided into four categories - pro-inflammatory, obesity-related, insulin pathway, and FGF - all of which were increased in transgenic mice compared to WT littermates (Table 3). Untreated Fgfr3 ^ach/+ mice had a low baseline inflammation compared to WT animals, which was exacerbated by HFD (Table 3). Fgfr3 ^ach/+ treated animals in ND or HFD had a systemic profile that resembled that of their WT littermates. In vitro mesenchymal stem cells (MSCs) isolated from Fgfr3 ^ach/+ mice showed that early and intermediate genes for the differentiation process such as Srebf-1, CEBP/d, CEPB/a, and PPARg were already expressed (Fig. 3A ). Very interestingly, MSCs isolated from Fgfr3 ^ach/+ mice treated with sFGFR3 showed a significant increase in anti-adipogenic markers and activation markers in brown adipose tissue, as well as a decrease in the expression of genes involved in mature adipocyte functions (Fig. 3A). Together with in vivo data, this suggests a predisposition to adipogenesis in Fgfr3 ^ach/+ mice, which can be prevented by treatment with sFGFR3.

Table 3. Raw miceFgfr3 ^ach/+ showed increased baseline inflammation prevented by sFGFR3 treatment. Pro-inflammation, obesity, the insulin pathway, and expression of circulating FGF adipokines were performed in mice. WT andFgfr3 ^ach/+ treated with carrier medium and in miceFgfr3 ^ach/+ treated with sFGFR3 after 10 weeks of receiving ND or HFD. "-"=<2 "+"=10-30 A.U., "++"=30-100 A.U., "+++"> 100 A.U.

ND HFD wt Fgfr3 ^ach/+ wt Fgfr3 ^ach/+ Markers (n) carrier medium carrier medium 2.5 mg/kg sFGFR3 carrier medium carrier medium 2.5 mg/kg sFGFR3 Pro-inflammation (14) + ++ + + ++ + Obesity (15) ++ +++ + ++ +++ ++ Insulin pathway (7) ++ ++ ++ ++ +++ ++ FGF(2) + + - - ++ -

Compared to their WT littermates, mice carrying the FGFR3 mutation had low fasting glycemia and very low baseline insulin levels (Fig. 4A). When consuming the HFD diet (FIG. 4B), glycemic levels increased but remained lower than in WT animals. Insulin levels remained extremely low. In sFGFR3-treated Fgfr3 ^ach/+ animals, glycemia was restored and insulin levels increased significantly compared to untreated Fgfr3 ^ach/+ mice (FIGS. 4A and 4B).

To evaluate the development of glucose intolerance, after 10 weeks of diet, glucose tolerance tests (GTT) were performed. Raw miceFgfr3 ^ach/+ showed higher glucose and AUC levels compared to their WT littermates (Cmax 320.9±32.0 mg/dl and 273.3±23.9 mg/dl, AUC 1.2×104±0.7×104 and 1.7×104±0.4×104, respectively), showing some basal glucose intolerance even in ND. This was further exacerbated with HFD (Fig. 4C). When the miceFgfr3 ^ach/+ treated with 2.5 mg/kg sFGFR3, normal GTT responses were restored (Fig. 4C). The inventors attempted to perform an insulin tolerance test in transgenic mice fed ND or HFD, but due to the mouse's lower baseline glycemiaFgfr3 ^ach/+ could not withstand the injection of insulin and quickly died. Insulin sensitivity analysis of mesenchymal stem cells isolated from miceFgfr3 ^ach/+ or WT showed no difference in Erk1/2 phosphorylation levels, indicating a similar response to insulin stimulation in both types of mice (FIG. 3B). These results show that miceFgfr3 ^ach/+ do not appear to be more sensitive to insulin regulation, but that insulin injection during ITT probably caused lethal hypoglycemia due to their low baseline glycemia. Pancreatic analyzes showed fewer and more islets of Langerhans with lower levels of insulin and glucagon in untreated miceFgfr3 ^ach/+ (Fig. 4D), which indicates a change in the production and/or accumulation of insulin. This was partially restored in treated animals (FIGS. 3B and 4A-4C). Glucose accumulation also appeared to be impaired in the liver of untreated animals.Fgfr3 ^ach/+ , as evidenced by the decrease in glycogen in liver sections (Fig. 4E). As expected, after 10 weeks of HFD consumption, WT mice developed grade III macrovesicular steatosis with over 75% of hepatocytes that had lipid vacuoles that were larger than the nucleus (Fig. 4E). In contrast, after 10 weeks of HFD in untreated miceFgfr3 ^ach/+ reversible benign hepatic nodules developed (Fig. 4F) and grade II microvesicular steatosis: less than 50% of hepatocytes showed small vacuoles (Fig. 4E). In contrast, after 10 weeks of HFD in untreated miceFgfr3 ^ach/+ reversible benign hepatic nodules developed (Fig. 4F) and grade II microvesicular steatosis: less than 50% of hepatocytes showed small vacuoles (Fig. 4E). Interestingly, sFGFR3 treatment restored normal hepatic response in treated mice.Fgfr3 ^ach/+ (FIG. 4E) and no nodules were observed.

The metabolic rate of basal energy in Fgfr3 ^ach/+ mice was assessed using indirect calorimetry. The inventors found that while lean WT animals that consumed a normal diet (ND) obtained energy from carbohydrate sources (respiratory RQ of approximately 1), mice with transgenic achondroplasia obtained their energy primarily from lipid sources ( RQ approximately 0.7) (FIG. 5A). In the fasting episode, as expected, both types of animals drew their energy from lipid sources. This preferential lipid utilization was confirmed by calculating carbohydrate and lipid oxidation, which were respectively lower and higher in Fgfr3 ^ach/+ mice compared to WT animals (FIG. 5B). During a 24-hour period, Fgfr3 ^ach/+ mice tend to feed constantly, not only during the night period, however, energy expenditure and food intake did not differ significantly between both genotypes (Fig. 7A-D). As expected in HFD, all animals derived their energy from lipid sources, resulting in similar rates of carbohydrate and lipid oxidation (Fig. 5C-D, Fig. 7E-H). Very interestingly, Fgfr3 ^ach/+ animals that received sFGFR3 treatment during the growth period behaved like untreated WT mice after weaning, regardless of whether they consumed ND or HFD, suggesting restoration of glucose metabolism capacity during the period. treatment (Fig. 5 and Fig. 7A-H).

Differences in the deposition of adipose tissue in different areas of the body can be assessed to assess the severity of obesity in achondroplasia. Compared to BMI z-scores and skinfold measurements, the android:gynoid ratio is strongly associated with all risk factors in overweight and obese children. In achondroplasia, children have a higher android:gynoid ratio that develops very early in childhood. The results obtained by the inventors in mice Fgfr3 ^ach/+ indicate a predisposition to predominant visceral obesity in these patients. Indeed, cells derived from the mesenchymal lineage appeared to be more prone to adipogenesis than cells isolated from WT animals and, apparently, are preliminarily involved in the process of differentiation towards adipocytes. Moreover, a different distribution of adipocytes was observed with a higher proportion of small adipocytes in the subcutaneous adipose tissue of Fgfr3 ^ach/+ mice compared to WT animals. The number of fat cells is set during childhood and adolescence and remains constant in adulthood. Obesity in patients with achondroplasia can be identified very early in childhood, and monitoring at 4-8 years of age, for example by DXA scanning, may be warranted.

The development of abdominal obesity is generally considered the most harmful type of obesity (Smith, J. Clin. Invest. 125:1790-1792, 2015). The results obtained by the inventors show that abdominal obesity is probably the result of a mutation affecting FGFR3. Currently, three members of the FGF family are associated with obesity (Nies et al., Front. Endocrinol. (Lausanne) 6:193, 2015): FGF1, FGF15/19 and FGF21. FGF1 is regulated by PPARg and is markedly elevated in WAT (28). FGF1 is known to promote proliferation and differentiation of preadipocytes through Erk1/2 signaling. It also causes an acute blood-lowering effect that appears to be dependent on FGFR2 signaling in the WAT. FGF15/19 is considered to be a regulator of feeding responses. It binds to the FGFR4/bklotho receptor complex on the hepatocyte cell membrane, which ultimately leads to repression of gluconeogenesis (Tomlinson et al., Endocrinology 143:1741-1747, 2002). FGF21 mainly binds to FGFR1 and regulates the adaptive response to starvation through PPARa (Kharitonenkov et al., J. Clin. Invest. 115:1627-1635, 2005). In Fgfr3 ^ach/+ mice, overexpression of FGFR3ach during embryogenesis can modify FGF signaling in different cell types. Accordingly, the inventors have found that mesenchymal stem cells from Fgfr3 ^ach/+ mice express high levels of FGFR3 compared to their WT littermates. Similarly, neonatal Fgfr3 ^ach/+ mice express elevated levels of FGFR2 and FGFR4 in AT and liver. Overall, these data may explain the low glycemia associated with abdominal obesity in Fgfr3 ^ach/+ mice. Along this line, we also observed that patients even tended to have decreased fasting glycemia and insulinemia, and no patient had glucose intolerance, suggesting that similar mechanisms may apply in humans.

Treatment with sFGFR3 was administered immediately after birth and prevented most metabolic complications, including the development of abdominal obesity. Processed miceFgfr3 ^ach/+ were not protected from obesity per se, but behave essentially like WT animals with the development of homogeneous obesity and restoration of glucose metabolism leading to glucose resistance in HFD. This suggests that, if applied at an early age, treatment could reverse the effect of the Fgfr3ach mutation on these atypical metabolic tissues.

In conclusion, the inventors' data confirm that the development of inappropriate obesity is predominantly abdominal and appears to be triggered by FGFR3 mutation. The data of the inventors also show that the treatment of achondroplastic patients with abnormal visceral fat deposition can be applied to other groups of patients with abnormal visceral fat deposition and the resulting conditions. Patients with skeletal growth retardation disorders can be treated to control abnormal visceral fat deposition and related conditions, eg, after bone growth has been completed, when sFGFR3 polypeptides would not otherwise be considered an appropriate therapy. Patients may also benefit from monitoring comorbidities such as diabetes and cardiovascular disease risk.

OTHER IMPLEMENTATION OPTIONS

All publications, patents and patent applications referred to in the foregoing description are hereby incorporated by reference to the same extent as if each individual publication, patent or patent application were expressly and separately indicated for inclusion by reference in their entirety.

Various modifications and variations of the described methods, pharmaceutical compositions and kits according to 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 should be understood that further modifications are possible and that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described ways of carrying out the invention, which are obvious to experts in the art, should be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention generally consistent with the principles of the invention and including such departures from the present invention, within known common practice in the art to which the invention pertains, and may be applicable to the essential features herein. before expounding.

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SEQUENCE LIST

<110> THERACHON

INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE

(INSERM)

<120> TREATMENT OF ABNORMAL VISCERAL FAT USING

SOLUBLE POLYPEPTIDE RECEPTOR 3 FIBROBLAST GROWTH FACTORS

(SFGFR3)

<130> 51088-011WO2

<150> US 61/561140

<151> 2017-09-20

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<170>PatentIn version 3.5

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Val Leu Val Gly Pro Gln Arg Leu Gln Val Leu Asn Ala Ser His Glu

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Ser Asp Ala Gln Pro His Ile Gln Trp Leu Lys His Val Glu Val Asn

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Gly Ser Lys Val Gly Pro Asp Gly Thr Pro Tyr Val Thr Val Leu Lys

275 280 285

Thr Ala Gly Ala Asn Thr Thr Asp Lys Glu Leu Glu Val Leu Ser Leu

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His Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala Gly

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Asn Ser Ile Gly Phe Ser His His Ser Ala Trp Leu Val Val Leu Pro

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Asp Ala Val Glu Leu Ser Cys Pro Pro Pro Gly Gly Gly Pro Met Gly

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Pro Thr Val Trp Val Lys Asp Gly Thr Gly Leu Val Pro Ser Glu Arg

50 55 60

Val Leu Val Gly Pro Gln Arg Leu Gln Val Leu Asn Ala Ser His Glu

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Asp Ser Gly Ala Tyr Ser Cys Arg Gln Arg Leu Thr Gln Arg Val Leu

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Cys His Phe Ser Val Arg Val Thr Asp Ala Pro Ser Ser Gly Asp Asp

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Glu Asp Gly Glu Asp Glu Ala Glu Asp Thr Gly Val Asp Thr Gly Ala

115 120 125

Pro Tyr Trp Thr Arg Pro Glu Arg Met Asp Lys Lys Leu Leu Ala Val

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Ser Asp Ala Gln Pro His Ile Gln Trp Leu Lys His Val Glu Val Asn

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Pro Thr Val Trp Val Lys Asp Gly Thr Gly Leu Val Pro Ser Glu Arg

50 55 60

Val Leu Val Gly Pro Gln Arg Leu Gln Val Leu Asn Ala Ser His Glu

65 70 75 80

Asp Ser Gly Ala Tyr Ser Cys Arg Gln Arg Leu Thr Gln Arg Val Leu

85 90 95

Cys His Phe Ser Val Arg Val Thr Asp Ala Pro Ser Ser Gly Asp Asp

100 105 110

Glu Asp Gly Glu Asp Glu Ala Glu Asp Thr Gly Val Asp Thr Gly Ala

115 120 125

Pro Tyr Trp Thr Arg Pro Glu Arg Met Asp Lys Lys Leu Leu Ala Val

130 135 140

Pro Ala Ala Asn Thr Val Arg Phe Arg Cys Pro Ala Ala Gly Asn Pro

145 150 155 160

Thr Pro Ser Ile Ser Trp Leu Lys Asn Gly Arg Glu Phe Arg Gly Glu

165 170 175

His Arg Ile Gly Gly Ile Lys Leu Arg His Gln Gln Trp Ser Leu Val

180 185 190

Met Glu Ser Val Val Pro Ser Asp Arg Gly Asn Tyr Thr Cys Val Val

195 200 205

Glu Asn Lys Phe Gly Ser Ile Arg Gln Thr Tyr Thr Leu Asp Val Leu

210 215 220

Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala Asn

225 230 235 240

Gln Thr Ala Val Leu Gly Ser Asp Val Glu Phe His Cys Lys Val Tyr

245 250 255

Ser Asp Ala Gln Pro His Ile Gln Trp Leu Lys His Val Glu Val Asn

260 265 270

Gly Ser Lys Val Gly Pro Asp Gly Thr Pro Tyr Val Thr Val Leu Lys

275 280 285

Val Ser Leu Glu Ser Asn Ala Ser Met Ser Ser Asn Thr Ser Gly Ser

290 295 300

Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Val Val Phe Pro

305 310 315 320

Tyr Phe Pro Arg Leu Gly Arg Tyr Asn Leu Asn Phe His Glu Ala Gln

325 330 335

Gln Ala Cys Leu Asp Gln Asp Ala Val Ile Ala Ser Phe Asp Gln Leu

340 345 350

Tyr Asp Ala Trp Arg Gly Gly Leu Asp Trp Cys Asn Ala Gly Trp Leu

355 360 365

Ser Asp Gly Ser Val Gln Tyr Pro Ile Thr Lys Pro Arg Glu Pro Cys

370 375 380

Gly Gly Gln Asn Thr Val Pro Gly Val Arg Asn Tyr Gly Phe Trp Asp

385 390 395 400

Lys Asp Lys Ser Arg Tyr Asp Val Phe Cys Phe Thr Ser Asn Phe Asn

405 410 415

Gly Arg Phe Tyr Tyr Leu Ile His Pro Thr Lys Leu Thr Tyr Asp Glu

420 425 430

Ala Val Gln Ala Cys Leu Asn Asp Gly Ala Gln Ile Ala Lys Val Gly

435 440 445

Gln Ile Phe Ala Ala Trp Lys Ile Leu Gly Tyr Asp Arg Cys Asp Ala

450 455 460

Gly Trp Leu Ala Asp Gly Ser Val Arg Tyr Pro Ile Ser Arg Pro Arg

465 470 475 480

Arg Arg Cys Ser Pro Thr Glu Ala Ala Val Arg Phe Val Gly Phe Pro

485 490 495

Asp Lys Lys His Lys Leu Tyr Gly Val Tyr Cys Phe Arg Ala Tyr Asn

500 505 510

<210> 8

<211> 784

<212> protein

<213> Homo sapiens

<400> 8

Glu Ser Leu Gly Thr Glu Gln Arg Val Val Gly Arg Ala Ala Glu Val

1 5 10 15

Pro Gly Pro Glu Pro Gly Gln Gln Glu Gln Leu Val Phe Gly Ser Gly

20 25 30

Asp Ala Val Glu Leu Ser Cys Pro Pro Pro Gly Gly Gly Pro Met Gly

35 40 45

Pro Thr Val Trp Val Lys Asp Gly Thr Gly Leu Val Pro Ser Glu Arg

50 55 60

Val Leu Val Gly Pro Gln Arg Leu Gln Val Leu Asn Ala Ser His Glu

65 70 75 80

Asp Ser Gly Ala Tyr Ser Cys Arg Gln Arg Leu Thr Gln Arg Val Leu

85 90 95

Cys His Phe Ser Val Arg Val Thr Asp Ala Pro Ser Ser Gly Asp Asp

100 105 110

Glu Asp Gly Glu Asp Glu Ala Glu Asp Thr Gly Val Asp Thr Gly Ala

115 120 125

Pro Tyr Trp Thr Arg Pro Glu Arg Met Asp Lys Lys Leu Leu Ala Val

130 135 140

Pro Ala Ala Asn Thr Val Arg Phe Arg Cys Pro Ala Ala Gly Asn Pro

145 150 155 160

Thr Pro Ser Ile Ser Trp Leu Lys Asn Gly Arg Glu Phe Arg Gly Glu

165 170 175

His Arg Ile Gly Gly Ile Lys Leu Arg His Gln Gln Trp Ser Leu Val

180 185 190

Met Glu Ser Val Val Pro Ser Asp Arg Gly Asn Tyr Thr Cys Val Val

195 200 205

Glu Asn Lys Phe Gly Ser Ile Arg Gln Thr Tyr Thr Leu Asp Val Leu

210 215 220

Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala Asn

225 230 235 240

Gln Thr Ala Val Leu Gly Ser Asp Val Glu Phe His Cys Lys Val Tyr

245 250 255

Ser Asp Ala Gln Pro His Ile Gln Trp Leu Lys His Val Glu Val Asn

260 265 270

Gly Ser Lys Val Gly Pro Asp Gly Thr Pro Tyr Val Thr Val Leu Lys

275 280 285

Thr Ala Gly Ala Asn Thr Thr Asp Lys Glu Leu Glu Val Leu Ser Leu

290 295 300

His Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala Gly

305 310 315 320

Asn Ser Ile Gly Phe Ser His His Ser Ala Trp Leu Val Val Leu Pro

325 330 335

Ala Glu Glu Glu Glu Leu Val Glu Ala Asp Glu Ala Gly Ser Val Tyr Ala

340 345 350

Gly Ile Leu Ser Tyr Gly Val Gly Phe Phe Leu Phe Ile Leu Val Val

355 360 365

Ala Ala Val Thr Leu Cys Arg Leu Arg Ser Pro Pro Lys Lys Gly Leu

370 375 380

Gly Ser Pro Thr Val His Lys Ile Ser Arg Phe Pro Leu Lys Arg Gln

385 390 395 400

Val Ser Leu Glu Ser Asn Ala Ser Met Ser Ser Asn Thr Pro Leu Val

405 410 415

Arg Ile Ala Arg Leu Ser Ser Gly Glu Gly Pro Thr Leu Ala Asn Val

420 425 430

Ser Glu Leu Glu Leu Pro Ala Asp Pro Lys Trp Glu Leu Ser Arg Ala

435 440 445

Arg Leu Thr Leu Gly Lys Pro Leu Gly Glu Gly Cys Phe Gly Gln Val

450 455 460

Val Met Ala Glu Ala Ile Gly Ile Asp Lys Asp Arg Ala Ala Lys Pro

465 470 475 480

Val Thr Val Ala Val Lys Met Leu Lys Asp Asp Ala Thr Asp Lys Asp

485 490 495

Leu Ser Asp Leu Val Ser Glu Met Glu Met Met Lys Met Ile Gly Lys

500 505 510

His Lys Asn Ile Ile Asn Leu Leu Gly Ala Cys Thr Gln Gly Gly Pro

515 520 525

Leu Tyr Val Leu Val Glu Tyr Ala Ala Lys Gly Asn Leu Arg Glu Phe

530 535 540

Leu Arg Ala Arg Arg Pro Pro Gly Leu Asp Tyr Ser Phe Asp Thr Cys

545 550 555 560

Lys Pro Pro Glu Glu Gln Leu Thr Phe Lys Asp Leu Val Ser Cys Ala

565 570 575

Tyr Gln Val Ala Arg Gly Met Glu Tyr Leu Ala Ser Gln Lys Cys Ile

580 585 590

His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Thr Glu Asp Asn Val

595 600 605

Met Lys Ile Ala Asp Phe Gly Leu Ala Arg Asp Val His Asn Leu Asp

610 615 620

Tyr Tyr Lys Lys Thr Thr Asn Gly Arg Leu Pro Val Lys Trp Met Ala

625 630 635 640

Pro Glu Ala Leu Phe Asp Arg Val Tyr Thr His Gln Ser Asp Val Trp

645 650 655

Ser Phe Gly Val Leu Leu Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro

660 665 670

Tyr Pro Gly Ile Pro Val Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly

675 680 685

His Arg Met Asp Lys Pro Ala Asn Cys Thr His Asp Leu Tyr Met Ile

690 695 700

Met Arg Glu Cys Trp His Ala Ala Pro Ser Gln Arg Pro Thr Phe Lys

705 710 715 720

Gln Leu Val Glu Asp Leu Asp Arg Val Leu Thr Val Thr Ser Thr Asp

725 730 735

Glu Tyr Leu Asp Leu Ser Ala Pro Phe Glu Gln Tyr Ser Pro Gly Gly

740 745 750

Gln Asp Thr Pro Ser Ser Ser Ser Ser Gly Asp Asp Ser Val Phe Ala

755 760 765

His Asp Leu Leu Pro Pro Ala Pro Pro Ser Ser Gly Gly Ser Arg Thr

770 775 780

<210> 9

<211> 784

<212> protein

<213> Homo sapiens

<400> 9

Glu Ser Leu Gly Thr Glu Gln Arg Val Val Gly Arg Ala Ala Glu Val

1 5 10 15

Pro Gly Pro Glu Pro Gly Gln Gln Glu Gln Leu Val Phe Gly Ser Gly

20 25 30

Asp Ala Val Glu Leu Ser Cys Pro Pro Pro Gly Gly Gly Pro Met Gly

35 40 45

Pro Thr Val Trp Val Lys Asp Gly Thr Gly Leu Val Pro Ser Glu Arg

50 55 60

Val Leu Val Gly Pro Gln Arg Leu Gln Val Leu Asn Ala Ser His Glu

65 70 75 80

Asp Ser Gly Ala Tyr Ser Cys Arg Gln Arg Leu Thr Gln Arg Val Leu

85 90 95

Cys His Phe Ser Val Arg Val Thr Asp Ala Pro Ser Ser Gly Asp Asp

100 105 110

Glu Asp Gly Glu Asp Glu Ala Glu Asp Thr Gly Val Asp Thr Gly Ala

115 120 125

Pro Tyr Trp Thr Arg Pro Glu Arg Met Asp Lys Lys Leu Leu Ala Val

130 135 140

Pro Ala Ala Asn Thr Val Arg Phe Arg Cys Pro Ala Ala Gly Asn Pro

145 150 155 160

Thr Pro Ser Ile Ser Trp Leu Lys Asn Gly Arg Glu Phe Arg Gly Glu

165 170 175

His Arg Ile Gly Gly Ile Lys Leu Arg His Gln Gln Trp Ser Leu Val

180 185 190

Met Glu Ser Val Val Pro Ser Asp Arg Gly Asn Tyr Thr Cys Val Val

195 200 205

Glu Asn Lys Phe Gly Ser Ile Arg Gln Thr Tyr Thr Leu Asp Val Leu

210 215 220

Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala Asn

225 230 235 240

Gln Thr Ala Val Leu Gly Ser Asp Val Glu Phe His Cys Lys Val Tyr

245 250 255

Ser Asp Ala Gln Pro His Ile Gln Trp Leu Lys His Val Glu Val Asn

260 265 270

Gly Ser Lys Val Gly Pro Asp Gly Thr Pro Tyr Val Thr Val Leu Lys

275 280 285

Thr Ala Gly Ala Asn Thr Thr Asp Lys Glu Leu Glu Val Leu Ser Leu

290 295 300

His Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala Gly

305 310 315 320

Asn Ser Ile Gly Phe Ser His His Ser Ala Trp Leu Val Val Leu Pro

325 330 335

Ala Glu Glu Glu Glu Leu Val Glu Ala Asp Glu Ala Gly Ser Val Tyr Ala

340 345 350

Gly Ile Leu Ser Tyr Arg Val Gly Phe Phe Leu Phe Ile Leu Val Val

355 360 365

Ala Ala Val Thr Leu Cys Arg Leu Arg Ser Pro Pro Lys Lys Gly Leu

370 375 380

Gly Ser Pro Thr Val His Lys Ile Ser Arg Phe Pro Leu Lys Arg Gln

385 390 395 400

Val Ser Leu Glu Ser Asn Ala Ser Met Ser Ser Asn Thr Pro Leu Val

405 410 415

Arg Ile Ala Arg Leu Ser Ser Gly Glu Gly Pro Thr Leu Ala Asn Val

420 425 430

Ser Glu Leu Glu Leu Pro Ala Asp Pro Lys Trp Glu Leu Ser Arg Ala

435 440 445

Arg Leu Thr Leu Gly Lys Pro Leu Gly Glu Gly Cys Phe Gly Gln Val

450 455 460

Val Met Ala Glu Ala Ile Gly Ile Asp Lys Asp Arg Ala Ala Lys Pro

465 470 475 480

Val Thr Val Ala Val Lys Met Leu Lys Asp Asp Ala Thr Asp Lys Asp

485 490 495

Leu Ser Asp Leu Val Ser Glu Met Glu Met Met Lys Met Ile Gly Lys

500 505 510

His Lys Asn Ile Ile Asn Leu Leu Gly Ala Cys Thr Gln Gly Gly Pro

515 520 525

Leu Tyr Val Leu Val Glu Tyr Ala Ala Lys Gly Asn Leu Arg Glu Phe

530 535 540

Leu Arg Ala Arg Arg Pro Pro Gly Leu Asp Tyr Ser Phe Asp Thr Cys

545 550 555 560

Lys Pro Pro Glu Glu Gln Leu Thr Phe Lys Asp Leu Val Ser Cys Ala

565 570 575

Tyr Gln Val Ala Arg Gly Met Glu Tyr Leu Ala Ser Gln Lys Cys Ile

580 585 590

His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Thr Glu Asp Asn Val

595 600 605

Met Lys Ile Ala Asp Phe Gly Leu Ala Arg Asp Val His Asn Leu Asp

610 615 620

Tyr Tyr Lys Lys Thr Thr Asn Gly Arg Leu Pro Val Lys Trp Met Ala

625 630 635 640

Pro Glu Ala Leu Phe Asp Arg Val Tyr Thr His Gln Ser Asp Val Trp

645 650 655

Ser Phe Gly Val Leu Leu Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro

660 665 670

Tyr Pro Gly Ile Pro Val Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly

675 680 685

His Arg Met Asp Lys Pro Ala Asn Cys Thr His Asp Leu Tyr Met Ile

690 695 700

Met Arg Glu Cys Trp His Ala Ala Pro Ser Gln Arg Pro Thr Phe Lys

705 710 715 720

Gln Leu Val Glu Asp Leu Asp Arg Val Leu Thr Val Thr Ser Thr Asp

725 730 735

Glu Tyr Leu Asp Leu Ser Ala Pro Phe Glu Gln Tyr Ser Pro Gly Gly

740 745 750

Gln Asp Thr Pro Ser Ser Ser Ser Ser Gly Asp Asp Ser Val Phe Ala

755 760 765

His Asp Leu Leu Pro Pro Ala Pro Pro Ser Ser Gly Gly Ser Arg Thr

770 775 780

<210> 10

<211> 2016

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 10

gagtccttgg ggacggagca gcgcgtcgtg gggcgagcgg cagaagtccc gggcccagag 60

cccggccagc aggagcagtt ggtcttcggc agcggggatg ctgtggagct gagctgtccc 120

ccgcccgggg gtggtcccat ggggcccact gtctggggtca aggatggcac agggctggtg 180

ccctcggagc gtgtcctggt ggggccccag cggctgcagg tgctgaatgc ctccccgag 240

gactccgggg ccctacagctg ccggcagcgg ctcacgcagc gcgtactgtg ccacttcagt 300

gtgcgggtga cagacgctcc atcctcggga gatgacgaag acggggagga cgaggctgag 360

gacacaggtg tggacacagg ggccccttac tggacacggc ccgagcggat ggacaagaag 420

ctgctggccg tgccggccgc caacaccgtc cgcttccgct gccagccgc tggcaacccc 480

actccctcca tctcctggct gaagaacggc agggagttcc gcggcgagca ccgcattgga 540

ggcatcaagc tgcggcatca gcagtggagc ctggtcatgg aaagcgtggt gccctcggac 600

cgcggcaact acacctgcgt cgtggagaac aagtttggca gcatccggca gacgtacacg 660

ctggacgtgc tggagcgctc cccgcaccgg cccatcctgc aggcggggct gccggccaac 720

cagacggcgg tgctgggcag cgacgtggag ttccactgca aggtgtacag tgacgcacag 780

ccccacatcc agtggctcaa gcacgtggag gtgaatggca gcaaggtggg cccggacggc 840

acaccctacg ttaccgtgct caaggtgtcc ctggagtcca acgcgtccat gagctccaac 900

acaccactgg tgcgcatcgc aaggctgtcc tcaggggagg gccccacgct ggccaatgtc 960

tccgagctcg agctgcctgc cgaccccaaa tgggagctgt ctcgggcccg gctgaccctg 1020

ggcaagcccc ttggggaggg ctgcttcggc caggtggtca tggcgggaggc catcggcatt 1080

gacaaggacc gggccgccaa gcctgtcacc gtagccgtga agatgctgaa agacgatgcc 1140

actgacaagg acctgtcgga cctggtgtct gagatggaga tgatgaagat gatcgggaaa 1200

cacaaaaaca tcatcaacct gctgggcgcc tgcacgcagg gcgggcccct gtacgtgctg 1260

gtggagtacg cggccaaggg taacctgcgg gagtttctgc gggcgcggcg gcccccgggc 1320

ctggactact ccttcgacac ctgcaagccg cccgaggagc agctcacctt caaggacctg 1380

gtgtcctgtg cctaccaggt ggcccggggc atggagtact tggcctccca gaagtgcatc 1440

cacagggacc tggctgcccg caatgtgctg gtgaccgagg acaacgtgat gaagatcgca 1500

gacttcgggc tggcccggga cgtgcacaac ctcgactact acaagaagac aaccaacggc 1560

cggctgcccg tgaagtggat ggcgcctgag gccttgtttg accgagtcta cactcaccag 1620

agtgacgtct ggtcctttgg ggtcctgctc tgggagatct tcacgctggg gggctccccg 1680

taccccggca tccctgtgga ggagctcttc aagctgctga aggagggcca ccgcatggac 1740

aagcccgcca actgcacaca cgacctgtac atgatcatgc gggagtgctg gcatgccgcg 1800

ccctcccaga ggcccacctt caagcagctg gtggaggacc tggaccgtgt ccttaccgtg 1860

acgtccaccg acgagtacct ggacctgtcg gcgcctttcg agcagtactc cccgggtggc 1920

caggacaccc ccagctccag ctcctcaggg gacgactccg tgtttgccca cgacctgctg 1980

cccccggccc cacccagcag tgggggctcg cggacg 2016

<210> 11

<211> 1579

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 11

gagtccttgg ggacggagca gcgcgtcgtg gggcgagcgg cagaagtccc gggcccagag 60

cccggccagc aggagcagtt ggtcttcggc agcggggatg ctgtggagct gagctgtccc 120

ccgcccgggg gtggtcccat ggggcccact gtctggggtca aggatggcac agggctggtg 180

ccctcggagc gtgtcctggt ggggccccag cggctgcagg tgctgaatgc ctccccgag 240

gactccgggg ccctacagctg ccggcagcgg ctcacgcagc gcgtactgtg ccacttcagt 300

gtgcgggtga cagacgctcc atcctcggga gatgacgaag acggggagga cgaggctgag 360

gacacaggtg tggacacagg ggccccttac tggacacggc ccgagcggat ggacaagaag 420

ctgctggccg tgccggccgc caacaccgtc cgcttccgct gccagccgc tggcaacccc 480

actccctcca tctcctggct gaagaacggc agggagttcc gcggcgagca ccgcattgga 540

ggcatcaagc tgcggcatca gcagtggagc ctggtcatgg aaagcgtggt gccctcggac 600

cgcggcaact acacctgcgt cgtggagaac aagtttggca gcatccggca gacgtacacg 660

ctggacgtgc tggagcgctc cccgcaccgg cccatcctgc aggcggggct gccggccaac 720

cagacggcgg tgctgggcag cgacgtggag ttccactgca aggtgtacag tgacgcacag 780

ccccacatcc agtggctcaa gcacgtggag gtgaatggca gcaaggtggg cccggacggc 840

acaccctacg ttaccgtgct caaggtgtcc ctggagtcca acgcgtccat gagctccaac 900

acaccactgg tgcgcatcgc aaggctgtcc tcaggggagg gccccacgct ggccaatgtc 960

tccgagctcg agctgcctgc cgaccccaaa tgggagctgt ctcgggcccg gctgaccctg 1020

ggcaagcccc ttggggaggg ctgcttcggc caggtggtca tggcgggaggc catcggcatt 1080

gacaaggacc gggccgccaa gcctgtcacc gtagccgtga agatgctgaa agacgatgcc 1140

actgacaagg acctgtcgga cctggtgtct gagatggaga tgatgaagat gatcgggaaa 1200

cacaaaaaca tcatcaacct gctgggcgcc tgcacgcagg gcgggcccct gtacgtgctg 1260

gtggagtacg cggccaaggg taacctgcgg gagtttctgc gggcgcggcg gcccccgggc 1320

ctggactact ccttcgacac ctgcaagccg cccgaggagc agctcacctt caaggacctg 1380

gtgtcctgtg cctaccaggt ggcccggggc atggagtact tggcctccca gaagtgcatc 1440

cacagggacc tggctgcccg caatgtgctg gtgaccgagg acaacgtgat gaagatcgca 1500

gacttcgggc tggcccggga cgtgcacaac ctcgactact acaagaagac aaccaacggc 1560

cggctgcccg tgaagtgga 1579

<210> 12

<211> 1254

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 12

gagtccttgg ggacggagca gcgcgtcgtg gggcgagcgg cagaagtccc gggcccagag 60

cccggccagc aggagcagtt ggtcttcggc agcggggatg ctgtggagct gagctgtccc 120

ccgcccgggg gtggtcccat ggggcccact gtctggggtca aggatggcac agggctggtg 180

ccctcggagc gtgtcctggt ggggccccag cggctgcagg tgctgaatgc ctccccgag 240

gactccgggg ccctacagctg ccggcagcgg ctcacgcagc gcgtactgtg ccacttcagt 300

gtgcgggtga cagacgctcc atcctcggga gatgacgaag acggggagga cgaggctgag 360

gacacaggtg tggacacagg ggccccttac tggacacggc ccgagcggat ggacaagaag 420

ctgctggccg tgccggccgc caacaccgtc cgcttccgct gccagccgc tggcaacccc 480

actccctcca tctcctggct gaagaacggc agggagttcc gcggcgagca ccgcattgga 540

ggcatcaagc tgcggcatca gcagtggagc ctggtcatgg aaagcgtggt gccctcggac 600

cgcggcaact acacctgcgt cgtggagaac aagtttggca gcatccggca gacgtacacg 660

ctggacgtgc tggagcgctc cccgcaccgg cccatcctgc aggcggggct gccggccaac 720

cagacggcgg tgctgggcag cgacgtggag ttccactgca aggtgtacag tgacgcacag 780

ccccacatcc agtggctcaa gcacgtggag gtgaatggca gcaaggtggg cccggacggc 840

acaccctacg ttaccgtgct caaggtgtcc ctggagtcca acgcgtccat gagctccaac 900

acaccactgg tgcgcatcgc aaggctgtcc tcaggggagg gccccacgct ggccaatgtc 960

tccgagctcg agctgcctgc cgaccccaaa tgggagctgt ctcgggcccg gctgaccctg 1020

ggcaagcccc ttggggaggg ctgcttcggc caggtggtca tggcgggaggc catcggcatt 1080

gacaaggacc gggccgccaa gcctgtcacc gtagccgtga agatgctgaa agacgatgcc 1140

actgacaagg acctgtcgga cctggtgtct gagatggaga tgatgaagat gatcgggaaa 1200

gtac 1254

<210> 13

<211> 903

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 13

gagtccttgg ggacggagca gcgcgtcgtg gggcgagcgg cagaagtccc gggcccagag 60

cccggccagc aggagcagtt ggtcttcggc agcggggatg ctgtggagct gagctgtccc 120

ccgcccgggg gtggtcccat ggggcccact gtctggggtca aggatggcac agggctggtg 180

ccctcggagc gtgtcctggt ggggccccag cggctgcagg tgctgaatgc ctccccgag 240

gactccgggg ccctacagctg ccggcagcgg ctcacgcagc gcgtactgtg ccacttcagt 300

gtgcgggtga cagacgctcc atcctcggga gatgacgaag acggggagga cgaggctgag 360

gacacaggtg tggacacagg ggccccttac tggacacggc ccgagcggat ggacaagaag 420

ctgctggccg tgccggccgc caacaccgtc cgcttccgct gccagccgc tggcaacccc 480

actccctcca tctcctggct gaagaacggc agggagttcc gcggcgagca ccgcattgga 540

ggcatcaagc tgcggcatca gcagtggagc ctggtcatgg aaagcgtggt gccctcggac 600

cgcggcaact acacctgcgt cgtggagaac aagtttggca gcatccggca gacgtacacg 660

ctggacgtgc tggagcgctc cccgcaccgg cccatcctgc aggcggggct gccggccaac 720

cagacggcgg tgctgggcag cgacgtggag ttccactgca aggtgtacag tgacgcacag 780

ccccacatcc agtggctcaa gcacgtggag gtgaatggca gcaaggtggg cccggacggc 840

acaccctacg ttaccgtgct caaggtgtcc ctggagtcca acgcgtccat gagctccaac 900

aca 903

<210> 14

<211> 1131

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 14

atgggagtga aggtgctgtt cgccctgatc tgtatcgccg tggccgaggc cgagtctctg 60

ggcacagaac agagagtcgt gggcagagcc gccgaagtgc ctggacctga acctggccag 120

caggaacagc tggtgtttgg cagcggcgac gccgtggaac tgagctgtcc tccacctggc 180

ggaggccta tgggacctac cgtgtgggtc aaggatggca ccggactggt gcctagcgag 240

agggtgctcg tgggacctca gagactgcag gtgctgaacg ccagccacga ggatagcggc 300

gcctacagct gcagacagag actgacagg cgggtgctgt gccacttctc cgtcagagtg 360

accgacgccc ctagctccgg cgacgatgag gatggcgaag atgaggccga ggacaccggc 420

gtggacacag gcgctccata ctggaccaga cccgagcgga tggacaagaa actgctggcc 480

gtgcctgccg ccaacaccgt gcggtttaga tgtcctgccg cgggaaaccc cacccccagc 540

atcagctggc tgaagaacgg cagagagttc cggggcgagc acagaatcgg cggcatcaag 600

660

tacacctgtg tggtggaaaa caagttcggc agcatccggc agacctacac cctggacgtg 720

ctggaaagaa gcccccacag acccatcctg caggccggac tgcctgccaa tcagacagcc 780

gtgctgggca gcgacgtgga atttcactgc aaggtgtaca gcgacgccca gccccacatc 840

cagtggctga aacacgtgga agtgaacggc agcaaagtgg gccccgacgg caccccttat 900

gtgaccgtgc tgaaaaccgc tggcgccaat accaccgaca aagaactgga agtgctgagc 960

ctgcacaacg tgaccttcga ggatgccggc gagtacacct gtctggccgg caacagcatc 1020

ggcttcagcc accattctgc ctggctggtg gtgctgcccg tgtccctgga aagcaacgcc 1080

agcatgagca gcaacaccga ctacaaggac gacgacgaca agtgaaagct t 1131

<210> 15

<211> 1107

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 15

atgatgtcct ttgtctctct gctcctggtt ggcatcctat tccatgccac ccaggccgag 60

tccttgggga cggagcagcg cgtcgtgggg cgagcggcag aagtcccgggg cccagagccc 120

ggccagcagg agcagttggt cttcggcagc ggggatgctg tggagctgag ctgtcccccg 180

cccggggggtg gtcccatggg gcccactgtc tgggtcaagg atggcacagg gctggtgccc 240

tcggagcgtg tcctggtggg gccccagcgg ctgcaggtgc tgaatgcctc ccacgaggac 300

tccggggcct acagctgccg gcagcggctc acgcagcgcg tactgtgcca cttcagtgtg 360

cgggtgacag acgctccatc ctcgggagat gacgaagacg gggaggacga ggctgaggac 420

acaggtgtgg acacaggggc cccttactgg acacggcccg agcggatgga caagaagctg 480

ctggccgtgc cggccgccaa caccgtccgc ttccgctgcc cagccgctgg caaccccact 540

ccctccatct cctggctgaa gaacggcagg gagttccgcg gcgagcaccg cattggaggc 600

atcaagctgc ggcatcagca gtggagcctg gtcatggaaa gcgtggtgcc ctcggaccgc 660

ggcaactaca cctgcgtcgt ggagaacaag tttggcagca tccggcagac gtacacgctg 720

gacgtgctgg agcgctcccc gcaccggccc atcctgcagg ctgggctgcc tgctaaccag 780

acagcggtgc tgggcagcga cgtggagttc cactgcaagg tgtacagtga cgcacagccc 840

cacatccagt ggctcaagca cgtggaggtg aatggcagca aggtgggccc ggacggcaca 900

ccctacgtta ccgtgctcaa gacggcgggc gctaacacca ccgacaagga gctagaggtt 960

ctctccttgc acaacgtcac ctttgaggac gccggggagt acacctgcct gggggcaat 1020

tctattgggt tttctcatca ctctgcgtgg ctggtggtgc tgccagtgtc cctggagtcc 1080

aacgcgtcca tgagctccaa cacatga 1107

<210> 16

<211> 987

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 16

atgggagtga aggtgctgtt cgccctgatc tgtatcgccg tggccgaggc cgagtctctg 60

ggcacagaac agagagtcgt gggcagagcc gccgaagtgc ctggacctga acctggccag 120

caggaacagc tggtgtttgg cagcggcgac gccgtggaac tgagctgtcc tccacctggc 180

ggaggccta tgggacctac cgtgtgggtc aaggatggca ccggactggt gcctagcgag 240

agggtgctcg tgggacctca gagactgcag gtgctgaacg ccagccacga ggatagcggc 300

gcctacagct gcagacagag actgacagg cgggtgctgt gccacttctc cgtcagagtg 360

accgacgccc ctagctccgg cgacgatgag gatggcgaag atgaggccga ggacaccggc 420

gtggacacag gcgctccata ctggaccaga cccgagcgga tggacaagaa actgctggcc 480

gtgcctgccg ccaacaccgt gcggtttaga tgtcctgccg cgggaaaccc cacccccagc 540

atcagctggc tgaagaacgg cagagagttc cggggcgagc acagaatcgg cggcatcaag 600

660

tacacctgtg tggtggaaaa caagttcggc agcatccggc agacctacac cctggacgtg 720

ctggaaagaa gcccccacag acccatcctg caggccggac tgcctgccaa tcagacagcc 780

gtgctgggca gcgacgtgga atttcacagc aaggtgtaca gcgacgccca gccccacatc 840

cagtggctga aacacgtgga agtgaacggc agcaaagtgg gccccgacgg caccccttat 900

gtgaccgtgc tgaaggtgtc cctggaaagc aacgccagca tgagcagcaa caccgactac 960

aaggacgacg acgacaagtg aaagctt 987

<210> 17

<211> 1107

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 17

atgatgtcct ttgtctctct gctcctggtt ggcatcctat tccatgccac ccaggccgag 60

tccttgggga cggagcagcg cgtcgtgggg cgagcggcag aagtcccgggg cccagagccc 120

ggccagcagg agcagttggt cttcggcagc ggggatgctg tggagctgag ctgtcccccg 180

cccggggggtg gtcccatggg gcccactgtc tgggtcaagg atggcacagg gctggtgccc 240

tcggagcgtg tcctggtggg gccccagcgg ctgcaggtgc tgaatgcctc ccacgaggac 300

tccggggcct acagctgccg gcagcggctc acgcagcgcg tactgtgcca cttcagtgtg 360

cgggtgacag acgctccatc ctcgggagat gacgaagacg gggaggacga ggctgaggac 420

acaggtgtgg acacaggggc cccttactgg acacggcccg agcggatgga caagaagctg 480

ctggccgtgc cggccgccaa caccgtccgc ttccgctgcc cagccgctgg caaccccact 540

ccctccatct cctggctgaa gaacggcagg gagttccgcg gcgagcaccg cattggaggc 600

atcaagctgc ggcatcagca gtggagcctg gtcatggaaa gcgtggtgcc ctcggaccgc 660

ggcaactaca cctgcgtcgt ggagaacaag tttggcagca tccggcagac gtacacgctg 720

gacgtgctgg agcgctcccc gcaccggccc atcctgcagg ctgggctgcc tgctaaccag 780

acagcggtgc tgggcagcga cgtggagttc cactgcaagg tgtacagtga cgcacagccc 840

cacatccagt ggctcaagca cgtggaggtg aatggcagca aggtgggccc ggacggcaca 900

ccctacgtta ccgtgctcaa gacggcgggc gctaacacca ccgacaagga gctagaggtt 960

ctctccttgc acaacgtcac ctttgaggac gccggggagt acacctgcct gggggcaat 1020

tctattgggt tttctcatca ctctgcgtgg ctggtggtgc tgccagtgtc cctggagtcc 1080

aacgcgtcca tgagctccaa cacatga 1107

<210> 18

<211> 1548

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 18

gagagcctgg gcacagagca gcgggtggtg ggaagggccg ctgaagtgcc tggccctgag 60

cctggccagc aggaacagct ggtctttggc agcggcgacg ccgtggaact gagctgtcct 120

ccacccggcg gaggccctat gggccctacc gtgtgggtga aagacggcac cggcctggtg 180

cccagcgaga gggtgctggt gggaccccag cggctgcagg tcctgaacgc cagccacgag 240

gacagcggcg cctacagctg cagacagaga ctgacccagc gggtgctgtg ccacttcagc 300

gtgcgcgtga ccgacgcccc tagctctggc gacgacgagg acggcgagga cgaggccgag 360

gataccggcg tggacacagg cgccccttac tggaccagac ccgagcggat ggacaagaaa 420

ctgctggccg tgcctgccgc caacaccgtg cggttcagat gtcccgccgc tggcaacccc 480

acccccagca tcagctggct gaagaacggc agagagttcc ggggcgagca ccggatcggc 540

ggcatcaagc tgcggcacca gcagtggtcc ctggtcatgg aaagcgtggt gccctccgac 600

cggggcaact acacctgtgt ggtggaaaac aagttcggca gcatccggca gacctacacc 660

ctggacgtgc tggaaagaag cccccaccgg cccatcctgc aggctggact gcccgccaat 720

cagacagccg tgctgggcag cgacgtggaa tttcactgca aggtgtacag cgacgcccag 780

ccccacatcc agtggctcaa gcacgtggaa gtgaacggca gcaaagtggg ccccgacggc 840

accccttacg tgaccgtgct gaaagtgtcc ctggaaagca acgccagcat gagcagcaac 900

acctgaggta ccagcggcag cggctctggc tctggaagcg gaagcggatc tgggagcgtg 960

gtgttcccct acttcccccg gctgggccgg tacaacctga acttccacga ggcccagcag 1020

gcttgcctcg accaggatgc cgtgatcgcc agcttcgacc agctgtacga tgcctggcgg 1080

ggaggcctgg actggtgcaa tgccggctgg ctgtccgacg gcagcgtcca gtaccccatc 1140

accaagccca gagagccctg cggcggacag aacacagtgc ccggcgtgcg gaactacggc 1200

ttctgggaca aggacaagag cagatacgac gtgttctgct tcaccagcaa cttcaacggc 1260

cggttctact acctgatcca ccccaccaag ctgacctacg acgaagccgt ccaggcctgt 1320

ctgaacgacg gcgccgat cgccaaagtg ggacagatct tcgccgcctg gaagatcctg 1380

ggctacgaca gatgcgacgc cggatggctg gccgatggct ccgtgcgcta ccctatcagc 1440

agaccccgca gaagatgcag ccctaccgag gccgccgtgc gcttcgtggg cttccccgac 1500

aagaagcaca agctgtacgg cgtctactgc ttccgggcct acaactga 1548

<210> 19

<211> 2355

<212> DNA

<213> Homo sapiens

<400> 19

gagtccttgg ggacggagca gcgcgtcgtg gggcgagcgg cagaagtccc gggcccagag 60

cccggccagc aggagcagtt ggtcttcggc agcggggatg ctgtggagct gagctgtccc 120

ccgcccgggg gtggtcccat ggggcccact gtctggggtca aggatggcac agggctggtg 180

ccctcggagc gtgtcctggt ggggccccag cggctgcagg tgctgaatgc ctccccgag 240

gactccgggg ccctacagctg ccggcagcgg ctcacgcagc gcgtactgtg ccacttcagt 300

gtgcgggtga cagacgctcc atcctcggga gatgacgaag acggggagga cgaggctgag 360

gacacaggtg tggacacagg ggccccttac tggacacggc ccgagcggat ggacaagaag 420

ctgctggccg tgccggccgc caacaccgtc cgcttccgct gccagccgc tggcaacccc 480

actccctcca tctcctggct gaagaacggc agggagttcc gcggcgagca ccgcattgga 540

ggcatcaagc tgcggcatca gcagtggagc ctggtcatgg aaagcgtggt gccctcggac 600

cgcggcaact acacctgcgt cgtggagaac aagtttggca gcatccggca gacgtacacg 660

ctggacgtgc tggagcgctc cccgcaccgg cccatcctgc aggcggggct gccggccaac 720

cagacggcgg tgctgggcag cgacgtggag ttccactgca aggtgtacag tgacgcacag 780

ccccacatcc agtggctcaa gcacgtggag gtgaatggca gcaaggtggg cccggacggc 840

acaccctacg ttaccgtgct caagacggcg ggcgctaaca ccaccgacaa ggagctagag 900

gttctctcct tgcacaacgt cacctttgag gacgccgggg agtacacctg cctggcgggc 960

aattctattg ggttttctca tcactctgcg tggctggtgg tgctgccagc cgaggagggag 1020

ctggtggagg ctgacgaggc gggcagtgtg tatgcaggca tcctcagcta cggggtgggc 1080

ttcttcctgt tcatcctggt ggtggcggct gtgacgctct gccgcctgcg cagcccccccc 1140

aagaaaggcc tgggctcccc caccgtgcac aagatctccc gcttcccgct caagcgacag 1200

gtgtccctgg agtccaacgc gtccatgagc tccaacacac cactggtgcg catcgcaagg 1260

ctgtcctcag gggagggccc cacgctggcc aatgtctccg agctcgagct gcctgccgac 1320

cccaaatggg agctgtctcg ggcccggctg accctgggca agccccttgg ggagggctgc 1380

ttcggccagg tggtcatggc ggaggccatc ggcattgaca aggaccgggc cgccaagcct 1440

gtcaccgtag ccgtgaagat gctgaaagac gatgccactg acaaggacct gtcggacctg 1500

gtgtctgaga tggagatgat gaagatgatc gggaaacaca aaaacatcat caacctgctg 1560

ggcgcctgca cgcagggcgg gcccctgtac gtgctggtgg agtacgcggc caagggtaac 1620

ctgcgggagt ttctgcgggc gcggcggccc ccgggcctgg actactcctt cgacacctgc 1680

aagccgcccg aggagcagct caccttcaag gacctggtgt cctgtgccta ccaggtggcc 1740

cggggcatgg agtacttggc ctcccagaag tgcatccaca gggacctggc tgcccgcaat 1800

1860

cacaacctcg actactacaa gaagacaacc aacggccggc tgcccgtgaa gtggatggcg 1920

cctgaggcct tgtttgaccg agtctacact caccagagtg acgtctggtc ctttggggtc 1980

ctgctctggg agatcttcac gctggggggc tccccgtacc ccggcatccc tgtggaggag 2040

ctcttcaagc tgctgaagga gggccaccgc atggacaagc ccgccaactg cacacacgac 2100

ctgtacatga tcatgcggga gtgctggcat gccgcgccct cccagaggcc caccttcaag 2160

cagctggtgg aggacctgga ccgtgtcctt accgtgacgt ccaccgacga gtacctggac 2220

ctgtcggcgc ctttcgagca gtactccccg ggtggccagg acacccccag ctccagctcc 2280

tcaggggacg actccgtgtt tgccccgac ctgctgcccc cggccccacc cagcagtggg 2340

ggctcgcgga cgtga 2355

<210> 20

<211> 2355

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1073)..(1073)

<223> N is a or c

<400> 20

gagtccttgg ggacggagca gcgcgtcgtg gggcgagcgg cagaagtccc gggcccagag 60

cccggccagc aggagcagtt ggtcttcggc agcggggatg ctgtggagct gagctgtccc 120

ccgcccgggg gtggtcccat ggggcccact gtctggggtca aggatggcac agggctggtg 180

ccctcggagc gtgtcctggt ggggccccag cggctgcagg tgctgaatgc ctccccgag 240

gactccgggg ccctacagctg ccggcagcgg ctcacgcagc gcgtactgtg ccacttcagt 300

gtgcgggtga cagacgctcc atcctcggga gatgacgaag acggggagga cgaggctgag 360

gacacaggtg tggacacagg ggccccttac tggacacggc ccgagcggat ggacaagaag 420

ctgctggccg tgccggccgc caacaccgtc cgcttccgct gccagccgc tggcaacccc 480

actccctcca tctcctggct gaagaacggc agggagttcc gcggcgagca ccgcattgga 540

ggcatcaagc tgcggcatca gcagtggagc ctggtcatgg aaagcgtggt gccctcggac 600

cgcggcaact acacctgcgt cgtggagaac aagtttggca gcatccggca gacgtacacg 660

ctggacgtgc tggagcgctc cccgcaccgg cccatcctgc aggcggggct gccggccaac 720

cagacggcgg tgctgggcag cgacgtggag ttccactgca aggtgtacag tgacgcacag 780

ccccacatcc agtggctcaa gcacgtggag gtgaatggca gcaaggtggg cccggacggc 840

acaccctacg ttaccgtgct caagacggcg ggcgctaaca ccaccgacaa ggagctagag 900

gttctctcct tgcacaacgt cacctttgag gacgccgggg agtacacctg cctggcgggc 960

aattctattg ggttttctca tcactctgcg tggctggtgg tgctgccagc cgaggagggag 1020

ctggtggagg ctgacgaggc gggcagtgtg tatgcaggca tcctcagcta cgnggtgggc 1080

ttcttcctgt tcatcctggt ggtggcggct gtgacgctct gccgcctgcg cagcccccccc 1140

aagaaaggcc tgggctcccc caccgtgcac aagatctccc gcttcccgct caagcgacag 1200

gtgtccctgg agtccaacgc gtccatgagc tccaacacac cactggtgcg catcgcaagg 1260

ctgtcctcag gggagggccc cacgctggcc aatgtctccg agctcgagct gcctgccgac 1320

cccaaatggg agctgtctcg ggcccggctg accctgggca agccccttgg ggagggctgc 1380

ttcggccagg tggtcatggc ggaggccatc ggcattgaca aggaccgggc cgccaagcct 1440

gtcaccgtag ccgtgaagat gctgaaagac gatgccactg acaaggacct gtcggacctg 1500

gtgtctgaga tggagatgat gaagatgatc gggaaacaca aaaacatcat caacctgctg 1560

ggcgcctgca cgcagggcgg gcccctgtac gtgctggtgg agtacgcggc caagggtaac 1620

ctgcgggagt ttctgcgggc gcggcggccc ccgggcctgg actactcctt cgacacctgc 1680

aagccgcccg aggagcagct caccttcaag gacctggtgt cctgtgccta ccaggtggcc 1740

cggggcatgg agtacttggc ctcccagaag tgcatccaca gggacctggc tgcccgcaat 1800

1860

cacaacctcg actactacaa gaagacaacc aacggccggc tgcccgtgaa gtggatggcg 1920

cctgaggcct tgtttgaccg agtctacact caccagagtg acgtctggtc ctttggggtc 1980

ctgctctggg agatcttcac gctggggggc tccccgtacc ccggcatccc tgtggaggag 2040

ctcttcaagc tgctgaagga gggccaccgc atggacaagc ccgccaactg cacacacgac 2100

ctgtacatga tcatgcggga gtgctggcat gccgcgccct cccagaggcc caccttcaag 2160

cagctggtgg aggacctgga ccgtgtcctt accgtgacgt ccaccgacga gtacctggac 2220

ctgtcggcgc ctttcgagca gtactccccg ggtggccagg acacccccag ctccagctcc 2280

tcaggggacg actccgtgtt tgccccgac ctgctgcccc cggccccacc cagcagtggg 2340

ggctcgcgga cgtga 2355

<210> 21

<211> 22

<212> protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 21

Met Gly Ala Pro Ala Cys Ala Leu Ala Leu Cys Val Ala Val Ala Ile

1 5 10 15

Val Ala Gly Ala Ser Ser

20

<210> 22

<211> 672

<212> protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 22

Glu Ser Leu Gly Thr Glu Gln Arg Val Val Gly Arg Ala Ala Glu Val

1 5 10 15

Pro Gly Pro Glu Pro Gly Gln Gln Glu Gln Leu Val Phe Gly Ser Gly

20 25 30

Asp Ala Val Glu Leu Ser Cys Pro Pro Pro Gly Gly Gly Pro Met Gly

35 40 45

Pro Thr Val Trp Val Lys Asp Gly Thr Gly Leu Val Pro Ser Glu Arg

50 55 60

Val Leu Val Gly Pro Gln Arg Leu Gln Val Leu Asn Ala Ser His Glu

65 70 75 80

Asp Ser Gly Ala Tyr Ser Cys Arg Gln Arg Leu Thr Gln Arg Val Leu

85 90 95

Cys His Phe Ser Val Arg Val Thr Asp Ala Pro Ser Ser Gly Asp Asp

100 105 110

Glu Asp Gly Glu Asp Glu Ala Glu Asp Thr Gly Val Asp Thr Gly Ala

115 120 125

Pro Tyr Trp Thr Arg Pro Glu Arg Met Asp Lys Lys Leu Leu Ala Val

130 135 140

Pro Ala Ala Asn Thr Val Arg Phe Arg Cys Pro Ala Ala Gly Asn Pro

145 150 155 160

Thr Pro Ser Ile Ser Trp Leu Lys Asn Gly Arg Glu Phe Arg Gly Glu

165 170 175

His Arg Ile Gly Gly Ile Lys Leu Arg His Gln Gln Trp Ser Leu Val

180 185 190

Met Glu Ser Val Val Pro Ser Asp Arg Gly Asn Tyr Thr Cys Val Val

195 200 205

Glu Asn Lys Phe Gly Ser Ile Arg Gln Thr Tyr Thr Leu Asp Val Leu

210 215 220

Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala Asn

225 230 235 240

Gln Thr Ala Val Leu Gly Ser Asp Val Glu Phe His Ser Lys Val Tyr

245 250 255

Ser Asp Ala Gln Pro His Ile Gln Trp Leu Lys His Val Glu Val Asn

260 265 270

Gly Ser Lys Val Gly Pro Asp Gly Thr Pro Tyr Val Thr Val Leu Lys

275 280 285

Val Ser Leu Glu Ser Asn Ala Ser Met Ser Ser Asn Thr Pro Leu Val

290 295 300

Arg Ile Ala Arg Leu Ser Ser Gly Glu Gly Pro Thr Leu Ala Asn Val

305 310 315 320

Ser Glu Leu Glu Leu Pro Ala Asp Pro Lys Trp Glu Leu Ser Arg Ala

325 330 335

Arg Leu Thr Leu Gly Lys Pro Leu Gly Glu Gly Cys Phe Gly Gln Val

340 345 350

Val Met Ala Glu Ala Ile Gly Ile Asp Lys Asp Arg Ala Ala Lys Pro

355 360 365

Val Thr Val Ala Val Lys Met Leu Lys Asp Asp Ala Thr Asp Lys Asp

370 375 380

Leu Ser Asp Leu Val Ser Glu Met Glu Met Met Lys Met Ile Gly Lys

385 390 395 400

His Lys Asn Ile Ile Asn Leu Leu Gly Ala Cys Thr Gln Gly Gly Pro

405 410 415

Leu Tyr Val Leu Val Glu Tyr Ala Ala Lys Gly Asn Leu Arg Glu Phe

420 425 430

Leu Arg Ala Arg Arg Pro Pro Gly Leu Asp Tyr Ser Phe Asp Thr Cys

435 440 445

Lys Pro Pro Glu Glu Gln Leu Thr Phe Lys Asp Leu Val Ser Cys Ala

450 455 460

Tyr Gln Val Ala Arg Gly Met Glu Tyr Leu Ala Ser Gln Lys Cys Ile

465 470 475 480

His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Thr Glu Asp Asn Val

485 490 495

Met Lys Ile Ala Asp Phe Gly Leu Ala Arg Asp Val His Asn Leu Asp

500 505 510

Tyr Tyr Lys Lys Thr Thr Asn Gly Arg Leu Pro Val Lys Trp Met Ala

515 520 525

Pro Glu Ala Leu Phe Asp Arg Val Tyr Thr His Gln Ser Asp Val Trp

530 535 540

Ser Phe Gly Val Leu Leu Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro

545 550 555 560

Tyr Pro Gly Ile Pro Val Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly

565 570 575

His Arg Met Asp Lys Pro Ala Asn Cys Thr His Asp Leu Tyr Met Ile

580 585 590

Met Arg Glu Cys Trp His Ala Ala Pro Ser Gln Arg Pro Thr Phe Lys

595 600 605

Gln Leu Val Glu Asp Leu Asp Arg Val Leu Thr Val Thr Ser Thr Asp

610 615 620

Glu Tyr Leu Asp Leu Ser Ala Pro Phe Glu Gln Tyr Ser Pro Gly Gly

625 630 635 640

Gln Asp Thr Pro Ser Ser Ser Ser Ser Gly Asp Asp Ser Val Phe Ala

645 650 655

His Asp Leu Leu Pro Pro Ala Pro Pro Ser Ser Gly Gly Ser Arg Thr

660 665 670

<210> 23

<211> 720

<212> protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 23

Glu Ser Leu Gly Thr Glu Gln Arg Val Val Gly Arg Ala Ala Glu Val

1 5 10 15

Pro Gly Pro Glu Pro Gly Gln Gln Glu Gln Leu Val Phe Gly Ser Gly

20 25 30

Asp Ala Val Glu Leu Ser Cys Pro Pro Pro Gly Gly Gly Pro Met Gly

35 40 45

Pro Thr Val Trp Val Lys Asp Gly Thr Gly Leu Val Pro Ser Glu Arg

50 55 60

Val Leu Val Gly Pro Gln Arg Leu Gln Val Leu Asn Ala Ser His Glu

65 70 75 80

Asp Ser Gly Ala Tyr Ser Cys Arg Gln Arg Leu Thr Gln Arg Val Leu

85 90 95

Cys His Phe Ser Val Arg Val Thr Asp Ala Pro Ser Ser Gly Asp Asp

100 105 110

Glu Asp Gly Glu Asp Glu Ala Glu Asp Thr Gly Val Asp Thr Gly Ala

115 120 125

Pro Tyr Trp Thr Arg Pro Glu Arg Met Asp Lys Lys Leu Leu Ala Val

130 135 140

Pro Ala Ala Asn Thr Val Arg Phe Arg Cys Pro Ala Ala Gly Asn Pro

145 150 155 160

Thr Pro Ser Ile Ser Trp Leu Lys Asn Gly Arg Glu Phe Arg Gly Glu

165 170 175

His Arg Ile Gly Gly Ile Lys Leu Arg His Gln Gln Trp Ser Leu Val

180 185 190

Met Glu Ser Val Val Pro Ser Asp Arg Gly Asn Tyr Thr Cys Val Val

195 200 205

Glu Asn Lys Phe Gly Ser Ile Arg Gln Thr Tyr Thr Leu Asp Val Leu

210 215 220

Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala Asn

225 230 235 240

Gln Thr Ala Val Leu Gly Ser Asp Val Glu Phe His Cys Lys Val Tyr

245 250 255

Ser Asp Ala Gln Pro His Ile Gln Trp Leu Lys His Val Glu Val Asn

260 265 270

Gly Ser Lys Val Gly Pro Asp Gly Thr Pro Tyr Val Thr Val Leu Lys

275 280 285

Thr Ala Gly Ala Asn Thr Thr Asp Lys Glu Leu Glu Val Leu Ser Leu

290 295 300

His Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala Gly

305 310 315 320

Asn Ser Ile Gly Phe Ser His His Ser Ala Trp Leu Val Val Leu Pro

325 330 335

Val Ser Leu Glu Ser Asn Ala Ser Met Ser Ser Asn Thr Pro Leu Val

340 345 350

Arg Ile Ala Arg Leu Ser Ser Gly Glu Gly Pro Thr Leu Ala Asn Val

355 360 365

Ser Glu Leu Glu Leu Pro Ala Asp Pro Lys Trp Glu Leu Ser Arg Ala

370 375 380

Arg Leu Thr Leu Gly Lys Pro Leu Gly Glu Gly Cys Phe Gly Gln Val

385 390 395 400

Val Met Ala Glu Ala Ile Gly Ile Asp Lys Asp Arg Ala Ala Lys Pro

405 410 415

Val Thr Val Ala Val Lys Met Leu Lys Asp Asp Ala Thr Asp Lys Asp

420 425 430

Leu Ser Asp Leu Val Ser Glu Met Glu Met Met Lys Met Ile Gly Lys

435 440 445

His Lys Asn Ile Ile Asn Leu Leu Gly Ala Cys Thr Gln Gly Gly Pro

450 455 460

Leu Tyr Val Leu Val Glu Tyr Ala Ala Lys Gly Asn Leu Arg Glu Phe

465 470 475 480

Leu Arg Ala Arg Arg Pro Pro Gly Leu Asp Tyr Ser Phe Asp Thr Cys

485 490 495

Lys Pro Pro Glu Glu Gln Leu Thr Phe Lys Asp Leu Val Ser Cys Ala

500 505 510

Tyr Gln Val Ala Arg Gly Met Glu Tyr Leu Ala Ser Gln Lys Cys Ile

515 520 525

His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Thr Glu Asp Asn Val

530 535 540

Met Lys Ile Ala Asp Phe Gly Leu Ala Arg Asp Val His Asn Leu Asp

545 550 555 560

Tyr Tyr Lys Lys Thr Thr Asn Gly Arg Leu Pro Val Lys Trp Met Ala

565 570 575

Pro Glu Ala Leu Phe Asp Arg Val Tyr Thr His Gln Ser Asp Val Trp

580 585 590

Ser Phe Gly Val Leu Leu Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro

595 600 605

Tyr Pro Gly Ile Pro Val Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly

610 615 620

His Arg Met Asp Lys Pro Ala Asn Cys Thr His Asp Leu Tyr Met Ile

625 630 635 640

Met Arg Glu Cys Trp His Ala Ala Pro Ser Gln Arg Pro Thr Phe Lys

645 650 655

Gln Leu Val Glu Asp Leu Asp Arg Val Leu Thr Val Thr Ser Thr Asp

660 665 670

Glu Tyr Leu Asp Leu Ser Ala Pro Phe Glu Gln Tyr Ser Pro Gly Gly

675 680 685

Gln Asp Thr Pro Ser Ser Ser Ser Ser Gly Asp Asp Ser Val Phe Ala

690 695 700

His Asp Leu Leu Pro Pro Ala Pro Pro Ser Ser Gly Gly Ser Arg Thr

705 710 715 720

<210> 24

<211> 512

<212> protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 24

Glu Ser Leu Gly Thr Glu Gln Arg Val Val Gly Arg Ala Ala Glu Val

1 5 10 15

Pro Gly Pro Glu Pro Gly Gln Gln Glu Gln Leu Val Phe Gly Ser Gly

20 25 30

Asp Ala Val Glu Leu Ser Cys Pro Pro Pro Gly Gly Gly Pro Met Gly

35 40 45

Pro Thr Val Trp Val Lys Asp Gly Thr Gly Leu Val Pro Ser Glu Arg

50 55 60

Val Leu Val Gly Pro Gln Arg Leu Gln Val Leu Asn Ala Ser His Glu

65 70 75 80

Asp Ser Gly Ala Tyr Ser Cys Arg Gln Arg Leu Thr Gln Arg Val Leu

85 90 95

Cys His Phe Ser Val Arg Val Thr Asp Ala Pro Ser Ser Gly Asp Asp

100 105 110

Glu Asp Gly Glu Asp Glu Ala Glu Asp Thr Gly Val Asp Thr Gly Ala

115 120 125

Pro Tyr Trp Thr Arg Pro Glu Arg Met Asp Lys Lys Leu Leu Ala Val

130 135 140

Pro Ala Ala Asn Thr Val Arg Phe Arg Cys Pro Ala Ala Gly Asn Pro

145 150 155 160

Thr Pro Ser Ile Ser Trp Leu Lys Asn Gly Arg Glu Phe Arg Gly Glu

165 170 175

His Arg Ile Gly Gly Ile Lys Leu Arg His Gln Gln Trp Ser Leu Val

180 185 190

Met Glu Ser Val Val Pro Ser Asp Arg Gly Asn Tyr Thr Cys Val Val

195 200 205

Glu Asn Lys Phe Gly Ser Ile Arg Gln Thr Tyr Thr Leu Asp Val Leu

210 215 220

Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala Asn

225 230 235 240

Gln Thr Ala Val Leu Gly Ser Asp Val Glu Phe His Ser Lys Val Tyr

245 250 255

Ser Asp Ala Gln Pro His Ile Gln Trp Leu Lys His Val Glu Val Asn

260 265 270

Gly Ser Lys Val Gly Pro Asp Gly Thr Pro Tyr Val Thr Val Leu Lys

275 280 285

Val Ser Leu Glu Ser Asn Ala Ser Met Ser Ser Asn Thr Ser Gly Ser

290 295 300

Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Val Val Phe Pro

305 310 315 320

Tyr Phe Pro Arg Leu Gly Arg Tyr Asn Leu Asn Phe His Glu Ala Gln

325 330 335

Gln Ala Cys Leu Asp Gln Asp Ala Val Ile Ala Ser Phe Asp Gln Leu

340 345 350

Tyr Asp Ala Trp Arg Gly Gly Leu Asp Trp Cys Asn Ala Gly Trp Leu

355 360 365

Ser Asp Gly Ser Val Gln Tyr Pro Ile Thr Lys Pro Arg Glu Pro Cys

370 375 380

Gly Gly Gln Asn Thr Val Pro Gly Val Arg Asn Tyr Gly Phe Trp Asp

385 390 395 400

Lys Asp Lys Ser Arg Tyr Asp Val Phe Cys Phe Thr Ser Asn Phe Asn

405 410 415

Gly Arg Phe Tyr Tyr Leu Ile His Pro Thr Lys Leu Thr Tyr Asp Glu

420 425 430

Ala Val Gln Ala Cys Leu Asn Asp Gly Ala Gln Ile Ala Lys Val Gly

435 440 445

Gln Ile Phe Ala Ala Trp Lys Ile Leu Gly Tyr Asp Arg Cys Asp Ala

450 455 460

Gly Trp Leu Ala Asp Gly Ser Val Arg Tyr Pro Ile Ser Arg Pro Arg

465 470 475 480

Arg Arg Cys Ser Pro Thr Glu Ala Ala Val Arg Phe Val Gly Phe Pro

485 490 495

Asp Lys Lys His Lys Leu Tyr Gly Val Tyr Cys Phe Arg Ala Tyr Asn

500 505 510

<210> 25

<211> 560

<212> protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 25

Glu Ser Leu Gly Thr Glu Gln Arg Val Val Gly Arg Ala Ala Glu Val

1 5 10 15

Pro Gly Pro Glu Pro Gly Gln Gln Glu Gln Leu Val Phe Gly Ser Gly

20 25 30

Asp Ala Val Glu Leu Ser Cys Pro Pro Pro Gly Gly Gly Pro Met Gly

35 40 45

Pro Thr Val Trp Val Lys Asp Gly Thr Gly Leu Val Pro Ser Glu Arg

50 55 60

Val Leu Val Gly Pro Gln Arg Leu Gln Val Leu Asn Ala Ser His Glu

65 70 75 80

Asp Ser Gly Ala Tyr Ser Cys Arg Gln Arg Leu Thr Gln Arg Val Leu

85 90 95

Cys His Phe Ser Val Arg Val Thr Asp Ala Pro Ser Ser Gly Asp Asp

100 105 110

Glu Asp Gly Glu Asp Glu Ala Glu Asp Thr Gly Val Asp Thr Gly Ala

115 120 125

Pro Tyr Trp Thr Arg Pro Glu Arg Met Asp Lys Lys Leu Leu Ala Val

130 135 140

Pro Ala Ala Asn Thr Val Arg Phe Arg Cys Pro Ala Ala Gly Asn Pro

145 150 155 160

Thr Pro Ser Ile Ser Trp Leu Lys Asn Gly Arg Glu Phe Arg Gly Glu

165 170 175

His Arg Ile Gly Gly Ile Lys Leu Arg His Gln Gln Trp Ser Leu Val

180 185 190

Met Glu Ser Val Val Pro Ser Asp Arg Gly Asn Tyr Thr Cys Val Val

195 200 205

Glu Asn Lys Phe Gly Ser Ile Arg Gln Thr Tyr Thr Leu Asp Val Leu

210 215 220

Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala Asn

225 230 235 240

Gln Thr Ala Val Leu Gly Ser Asp Val Glu Phe His Cys Lys Val Tyr

245 250 255

Ser Asp Ala Gln Pro His Ile Gln Trp Leu Lys His Val Glu Val Asn

260 265 270

Gly Ser Lys Val Gly Pro Asp Gly Thr Pro Tyr Val Thr Val Leu Lys

275 280 285

Thr Ala Gly Ala Asn Thr Thr Asp Lys Glu Leu Glu Val Leu Ser Leu

290 295 300

His Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala Gly

305 310 315 320

Asn Ser Ile Gly Phe Ser His His Ser Ala Trp Leu Val Val Leu Pro

325 330 335

Val Ser Leu Glu Ser Asn Ala Ser Met Ser Ser Asn Thr Ser Gly Ser

340 345 350

Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Val Val Phe Pro

355 360 365

Tyr Phe Pro Arg Leu Gly Arg Tyr Asn Leu Asn Phe His Glu Ala Gln

370 375 380

Gln Ala Cys Leu Asp Gln Asp Ala Val Ile Ala Ser Phe Asp Gln Leu

385 390 395 400

Tyr Asp Ala Trp Arg Gly Gly Leu Asp Trp Cys Asn Ala Gly Trp Leu

405 410 415

Ser Asp Gly Ser Val Gln Tyr Pro Ile Thr Lys Pro Arg Glu Pro Cys

420 425 430

Gly Gly Gln Asn Thr Val Pro Gly Val Arg Asn Tyr Gly Phe Trp Asp

435 440 445

Lys Asp Lys Ser Arg Tyr Asp Val Phe Cys Phe Thr Ser Asn Phe Asn

450 455 460

Gly Arg Phe Tyr Tyr Leu Ile His Pro Thr Lys Leu Thr Tyr Asp Glu

465 470 475 480

Ala Val Gln Ala Cys Leu Asn Asp Gly Ala Gln Ile Ala Lys Val Gly

485 490 495

Gln Ile Phe Ala Ala Trp Lys Ile Leu Gly Tyr Asp Arg Cys Asp Ala

500 505 510

Gly Trp Leu Ala Asp Gly Ser Val Arg Tyr Pro Ile Ser Arg Pro Arg

515 520 525

Arg Arg Cys Ser Pro Thr Glu Ala Ala Val Arg Phe Val Gly Phe Pro

530 535 540

Asp Lys Lys His Lys Leu Tyr Gly Val Tyr Cys Phe Arg Ala Tyr Asn

545 550 555 560

<210> 26

<211> 155

<212> protein

<213> Homo sapiens

<400> 26

Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe

1 5 10 15

Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser

20 25 30

Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly

35 40 45

Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu

50 55 60

Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu

65 70 75 80

Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu

85 90 95

Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr

100 105 110

Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys

115 120 125

Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala

130 135 140

Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp

145 150 155

<210> 27

<211> 288

<212> protein

<213> Homo sapiens

<400> 27

Met Val Gly Val Gly Gly Gly Asp Val Glu Asp Val Thr Pro Arg Pro

1 5 10 15

Gly Gly Cys Gln Ile Ser Gly Arg Gly Ala Arg Gly Cys Asn Gly Ile

20 25 30

Pro Gly Ala Ala Ala Trp Glu Ala Ala Leu Pro Arg Arg Arg Pro Arg

35 40 45

Arg His Pro Ser Val Asn Pro Arg Ser Arg Ala Ala Gly Ser Pro Arg

50 55 60

Thr Arg Gly Arg Arg Thr Glu Glu Arg Pro Ser Gly Ser Arg Leu Gly

65 70 75 80

Asp Arg Gly Arg Gly Arg Ala Leu Pro Gly Gly Arg Leu Gly Gly Arg

85 90 95

Gly Arg Gly Arg Ala Pro Glu Arg Val Gly Gly Arg Gly Arg Gly Arg

100 105 110

Gly Thr Ala Ala Pro Arg Ala Ala Pro Ala Ala Arg Gly Ser Arg Pro

115 120 125

Gly Pro Ala Gly Thr Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala

130 135 140

Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys

145 150 155 160

Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile

165 170 175

His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro His

180 185 190

Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys

195 200 205

Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu

210 215 220

Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu

225 230 235 240

Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp

245 250 255

Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr

260 265 270

Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser

275 280 285

<210> 28

<211> 208

<212> protein

<213> Homo sapiens

<400> 28

Met Ala Pro Leu Gly Glu Val Gly Asn Tyr Phe Gly Val Gln Asp Ala

1 5 10 15

Val Pro Phe Gly Asn Val Pro Val Leu Pro Val Asp Ser Pro Val Leu

20 25 30

Leu Ser Asp His Leu Gly Gln Ser Glu Ala Gly Gly Leu Pro Arg Gly

35 40 45

Pro Ala Val Thr Asp Leu Asp His Leu Lys Gly Ile Leu Arg Arg Arg

50 55 60

Gln Leu Tyr Cys Arg Thr Gly Phe His Leu Glu Ile Phe Pro Asn Gly

65 70 75 80

Thr Ile Gln Gly Thr Arg Lys Asp His Ser Arg Phe Gly Ile Leu Glu

85 90 95

Phe Ile Ser Ile Ala Val Gly Leu Val Ser Ile Arg Gly Val Asp Ser

100 105 110

Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr Gly Ser Glu

115 120 125

Lys Leu Thr Gln Glu Cys Val Phe Arg Glu Gln Phe Glu Glu Asn Trp

130 135 140

Tyr Asn Thr Tyr Ser Ser Asn Leu Tyr Lys His Val Asp Thr Gly Arg

145 150 155 160

Arg Tyr Tyr Val Ala Leu Asn Lys Asp Gly Thr Pro Arg Glu Gly Thr

165 170 175

Arg Thr Lys Arg His Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val

180 185 190

Asp Pro Asp Lys Val Pro Glu Leu Tyr Lys Asp Ile Leu Ser Gln Ser

195 200 205

<210> 29

<211> 207

<212> protein

<213> Homo sapiens

<400> 29

Met Tyr Ser Ala Pro Ser Ala Cys Thr Cys Leu Cys Leu His Phe Leu

1 5 10 15

Leu Leu Cys Phe Gln Val Gln Val Leu Val Ala Glu Glu Asn Val Asp

20 25 30

Phe Arg Ile His Val Glu Asn Gln Thr Arg Ala Arg Asp Asp Val Ser

35 40 45

Arg Lys Gln Leu Arg Leu Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys

50 55 60

His Ile Gln Val Leu Gly Arg Arg Ile Ser Ala Arg Gly Glu Asp Gly

65 70 75 80

Asp Lys Tyr Ala Gln Leu Leu Val Glu Thr Asp Thr Phe Gly Ser Gln

85 90 95

Val Arg Ile Lys Gly Lys Glu Thr Glu Phe Tyr Leu Cys Met Asn Arg

100 105 110

Lys Gly Lys Leu Val Gly Lys Pro Asp Gly Thr Ser Lys Glu Cys Val

115 120 125

Phe Ile Glu Lys Val Leu Glu Asn Asn Tyr Thr Ala Leu Met Ser Ala

130 135 140

Lys Tyr Ser Gly Trp Tyr Val Gly Phe Thr Lys Lys Gly Arg Pro Arg

145 150 155 160

Lys Gly Pro Lys Thr Arg Glu Asn Gln Gln Asp Val His Phe Met Lys

165 170 175

Arg Tyr Pro Lys Gly Gln Pro Glu Leu Gln Lys Pro Phe Lys Tyr Thr

180 185 190

Thr Val Thr Lys Arg Ser Arg Arg Ile Arg Pro Thr His Pro Ala

195 200 205

<210> 30

<211> 216

<212> protein

<213> Homo sapiens

<400> 30

Met Arg Ser Gly Cys Val Val Val His Val Trp Ile Leu Ala Gly Leu

1 5 10 15

Trp Leu Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro

20 25 30

His Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr

35 40 45

Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala

50 55 60

Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu Leu

65 70 75 80

Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly Val His

85 90 95

Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met Gln Gly Leu

100 105 110

Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro

115 120 125

Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser

130 135 140

Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu

145 150 155 160

Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro

165 170 175

Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu

180 185 190

Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala

195 200 205

Val Arg Ser Pro Ser Phe Glu Lys

210 215

<210> 31

<211> 209

<212> protein

<213> Homo sapiens

<400> 31

Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser

1 5 10 15

Val Leu Ala Gly Leu Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro

20 25 30

Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr

35 40 45

Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg

50 55 60

Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu

65 70 75 80

Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val

85 90 95

Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly

100 105 110

Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu

115 120 125

Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu

130 135 140

His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly

145 150 155 160

Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu

165 170 175

Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp

180 185 190

Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala

195 200 205

Ser

<210> 32

<211> 2019

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 32

atggagtctc tgggcacaga gcagagagtc gtgggcagag ccgctgaagt gcctggacct 60

gagcctggcc agcaggaaca gctggtcttt ggctctggcg acgccgtgga actgagctgt 120

cctccacctg gcggaggccc tatgggacct accgtgtggg tcaaggatgg caccggactg 180

gtgcctagcg aggggtgct cgtgggacct cagagactgc aggtcctgaa cgccagccac 240

gaggatagcg gcgcctacag ctgcagacag agactgaccc agcgggtgct gtgccacttc 300

agcgtcagag tgaccgatgc ccccagcagc ggagatgacg aggatggcga ggatgaggcc 360

gaggatacag gcgtggacac aggcgcccct tactggacca gacccgagcg gatggacaag 420

aaactgctgg ccgtgcctgc cgccaacacc gtgcggttta gatgccctgc cgccggaaac 480

cccaccccca gcatctcttg gctgaagaac ggcagagagt tccggggcga gcaccggatc 540

ggcggcatta agctgagaca ccagcagtgg tccctggtca tggaaagcgt ggtgcccagc 600

gaccggggca actacacctg tgtggtggaa aacaagttcg gcagcatccg gcagacctac 660

accctggacg tgctggaaag aagcccccac agacctatcc tgcaggccgg actgcctgcc 720

aatcagacag ccgtgctggg cagcgacgtg gaatttcaca gcaaggtgta cagcgacgcc 780

cagccccaca tccagtggct gaagcacgtg gaagtgaacg gcagcaaagt gggccccgac 840

ggcacccctt acgtgaccgt gctgaaagtg tccctggaaa gcaacgccag catgagcagc 900

aacaccccccc tcgtgcggat cgccagactg tctagcggag agggccctac cctggccaac 960

gtgtccgaac tggaactgcc cgccgacccc aagtgggagc tgagcagagc tagactgacc 1020

ctgggcaagc ctctggggcga gggctgtttt ggacaggtgg tcatggccga ggccatcggc 1080

atcgacaagg acagagccgc caagcctgtg accgtggccg tgaagatgct gaaggacgac 1140

gccaccgaca aggacctgag cgacctggtg tccgagatgg aaatgatgaa gatgatcggc 1200

aagcacaaga acatcatcaa cctgctgggc gcctgcaccc agggcggacc tctgtacgtg 1260

ctggtggaat acgccgccaa gggcaacctg agagagttcc tgagagccag aaggccccct 1320

ggcctggact acagcttcga tacctgcaag ccccccgaag aacagctgac cttcaaggat 1380

ctggtgtcct gcgcctatca ggtggccaga ggcatggaat acctggccag ccagaagtgc 1440

atccaccggg atctggccgc cagaaacgtg ctggtcaccg aggacaacgt gatgaagatc 1500

gccgacttcg gcctggcccg ggacgtgcac aacctggact actacaagaa aaccaccaac 1560

ggccggctgc ccgtgaagtg gatggcccct gaggccctgt tcgacagagt gtacacccac 1620

cagagcgacg tgtggtcctt cggcgtgctg ctgtgggaga tctttaccct gggcggcagc 1680

ccttaccccg gcatccctgt ggaagaactg ttcaagctgc tgaaagaggg ccacagaatg 1740

gacaagcccg ccaactgcac ccacgacctg tacatgatca tgagagagtg ctggcacgcc 1800

gctcccagcc agaggcctac ctttaagcag ctggtggaag atctggaccg ggtgctgacc 1860

gtgaccagca ccgacgagta cctggatctg agcgccccct tcgagcagta ctctcctggc 1920

ggccaggata cccctagcag cagctctagc ggcgacgaca gcgtgttcgc ccacgatctg 1980

ctgcctccag ccctcctag ctctggcggc tctagaacc 2019

<210> 33

<211> 1491

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 33

atggagagcc tgggcacaga acagagagtc gtgggcagag ccgccgaagt gcctggacct 60

gaacctggcc agcaggaaca gctggtcttt ggctctggcg acgccgtgga actgagctgt 120

cctccacctg gcggaggccc tatgggacct accgtgtggg tcaaggatgg caccggactg 180

gtgcctagcg aggggtgct cgtgggacct cagagactgc aggtcctgaa cgccagccac 240

gaggatagcg gcgcctacag ctgcagacag agactgaccc agcgggtgct gtgccacttc 300

agcgtcagag tgaccgatgc ccccagcagc ggagatgacg aggatggcga ggatgaggcc 360

gaggatacag gcgtggacac aggcgcccct tactggacca gacccgagcg gatggacaag 420

aaactgctgg ccgtgcctgc cgccaacacc gtgcggttta gatgccctgc cgccggaaac 480

cccaccccca gcatctcttg gctgaagaac ggcagagagt tccggggcga gcaccggatc 540

ggcggcatta agctgagaca ccagcagtgg tccctggtca tggaaagcgt ggtgcccagc 600

gaccggggca actacacctg tgtggtggaa aacaagttcg gcagcatccg gcagacctac 660

accctggacg tgctggaaag aagcccccac agacctatcc tgcaggccgg actgcctgcc 720

aatcagacag ccgtgctggg cagcgacgtg gaatttcact gcaaggtgta cagcgacgcc 780

cagccccaca tccagtggct gaagcacgtg gaagtgaacg gcagcaaagt gggccccgac 840

ggcacccctt acgtgaccgt gctgaaagtg tccctggaaa gcaacgccag catgagcagc 900

aacaccagcg gcagcggctc tggcagcgga tctggttctg gctccggcag cgtggtgttc 960

ccctacttcc cccggctggg ccggtacaac ctgaactttc atgaggccca gcaggcctgc 1020

ctggaccagg atgccgtgat cgccagcttc gaccagctgt acgatgcttg gagaggcggc 1080

ctggactggt gcaatgccgg ctggctgtct gacggcagcg tgcagtaccc catcaccaag 1140

cccagagagc cctgcggcgg acagaatacc gtgcccggcg tgcggaacta cggcttctgg 1200

gacaaggaca agagcagata cgacgtgttc tgcttcacca gcaacttcaa cggccggttc 1260

tactacctga tccaccccac caagctgacc tacgacgagg ccgtgcaggc ctgtctgaac 1320

1380

gacagatgtg acgccggatg gctggccgac ggctccgtgc ggtatcccat cagccggcct 1440

agaagaagat gcagccctac cgaggccgcc gtcagattcg tgggcttccc c 1491

<210> 34

<211> 1710

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 34

atgggagcac cagcttgtgc cctggctctg tgtgtggccg tggctattgt ggctggcgcc 60

tctagcgaga gcctgggcac agaacagaga gtcgtgggca gagccgccga agtgcctgga 120

cctgaacctg gccagcagga acagctggtc tttggctctg gcgacgccgt ggaactgagc 180

tgtcctccac ctggcgggagg ccctatggga cctaccgtgt gggtcaagga tggcaccgga 240

ctggtgccta gcgagagggt gctcgtggga cctcagagac tgcaggtcct gaacgccagc 300

cacgaggata gcggcgccta cagctgcaga cagagactga cccagcgggt gctgtgccac 360

ttcagcgtca gagtgaccga tgcccccagc agcggagatg acgaggatgg cgaggatgag 420

gccgaggata caggcgtgga cacaggcgcc ccttactgga ccagacccga gcggatggac 480

aagaaactgc tggccgtgcc tgccgccaac accgtgcggt ttagatgccc tgccgccgga 540

aaccccaccc ccagcatctc ttggctgaag aacggcagag agttccgggg cgagcaccgg 600

atcggcggca ttaagctgag acaccagcag tggtccctgg tcatggaaag cgtggtgccc 660

agcgaccggg gcaactacac ctgtgtggtg gaaaacaagt tcggcagcat ccggcagacc 720

tacaccctgg acgtgctgga aagaagcccc cacagaccta tcctgcaggc cggactgcct 780

gccaatcaga cagccgtgct gggcagcgac gtggaatttc actgcaaggt gtacagcgac 840

gcccagcccc acatccagtg gctgaagcac gtggaagtga acggcagcaa agtgggcccc 900

gacggcaccc cttacgtgac cgtgctgaaa accgctggcg ccaacaccac cgacaaagaa 960

ctggaagtgc tgagcctgca caacgtgacc ttcgaggacg ccggcgagta cacctgtctg 1020

gccggcaata gcatcggctt cagccaccac tctgcctggc tggtggtgct gccaggcgga 1080

ggctctgtgt ccctggaaag caacgccagc atgagcagca acaccagcgg cagcggctct 1140

ggcagcggat ctggttctgg ctccggcagc gtggtgttcc cctacttccc ccggctgggc 1200

cggtacaacc tgaactttca tgaggcccag caggcctgcc tggaccagga tgccgtgatc 1260

gccagcttcg accagctgta cgatgcttgg agaggcggcc tggactggtg caatgccggc 1320

tggctgtctg acggcagcgt gcagtacccc atcaccaagc ccagagagcc ctgcggcgga 1380

cagaataccg tgcccggcgt gcggaactac ggcttctggg acaaggacaa gagcagatac 1440

gacgtgttct gcttcaccag caacttcaac ggccggttct actacctgat ccaccccacc 1500

aagctgacct acgacgaggc cgtgcaggcc tgtctgaacg atggcgccca gatcgccaaa 1560

1620

ctggccgacg gctccgtgcg gtatcccatc agccggccta gaagaagatg cagccctacc 1680

gaggccgccg tcagattcgt gggcttcccc 1710

<210> 35

<211> 236

<212> protein

<213> Homo sapiens

<400> 35

Gly Gly Gly Gly Ala Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro

1 5 10 15

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

20 25 30

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

35 40 45

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

50 55 60

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

65 70 75 80

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

85 90 95

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

100 105 110

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

115 120 125

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

130 135 140

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

145 150 155 160

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

165 170 175

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

180 185 190

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

195 200 205

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

210 215 220

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

225 230 235

<210> 36

<211> 236

<212> protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 36

Gly Gly Gly Gly Ala Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro

1 5 10 15

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

20 25 30

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

35 40 45

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

50 55 60

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

65 70 75 80

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

85 90 95

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

100 105 110

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

115 120 125

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

130 135 140

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

145 150 155 160

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

165 170 175

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

180 185 190

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

195 200 205

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

210 215 220

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

225 230 235

<210> 37

<211> 585

<212> protein

<213> Homo sapiens

<400> 37

Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu

1 5 10 15

Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln

20 25 30

Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu

35 40 45

Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys

50 55 60

Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu

65 70 75 80

Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro

85 90 95

Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu

100 105 110

Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His

115 120 125

Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg

130 135 140

Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg

145 150 155 160

Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala

165 170 175

Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser

180 185 190

Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu

195 200 205

Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro

210 215 220

Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys

225 230 235 240

Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp

245 250 255

Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser

260 265 270

Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His

275 280 285

Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser

290 295 300

Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala

305 310 315 320

Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg

325 330 335

Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr

340 345 350

Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu

355 360 365

Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro

370 375 380

Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu

385 390 395 400

Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro

405 410 415

Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys

420 425 430

Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys

435 440 445

Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His

450 455 460

Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser

465 470 475 480

Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr

485 490 495

Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp

500 505 510

Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala

515 520 525

Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu

530 535 540

Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys

545 550 555 560

Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val

565 570 575

Ala Ala Ser Gln Ala Ala Leu Gly Leu

580 585

<210> 38

<211> 9

<212> protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 38

Gly Gly Gly Gly Ala Gly Gly Gly Gly

15

<210> 39

<211> 15

<212> protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 39

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 40

<211> 208

<212> protein

<213> Homo sapiens

<400> 40

Met Trp Lys Trp Ile Leu Thr His Cys Ala Ser Ala Phe Pro His Leu

1 5 10 15

Pro Gly Cys Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser

20 25 30

Val Pro Val Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser Pro Glu

35 40 45

Ala Thr Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly

50 55 60

Arg His Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg

65 70 75 80

Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly

85 90 95

Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu

100 105 110

Ile Thr Ser Val Glu Ile Gly Val Val Ala Val Lys Ala Ile Asn Ser

115 120 125

Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys

130 135 140

Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly

145 150 155 160

Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met

165 170 175

Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr

180 185 190

Arg Arg Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser

195 200 205

<210> 41

<211> 251

<212> protein

<213> Homo sapiens

<400> 41

Met Leu Gly Ala Arg Leu Arg Leu Trp Val Cys Ala Leu Cys Ser Val

1 5 10 15

Cys Ser Met Ser Val Leu Arg Ala Tyr Pro Asn Ala Ser Pro Leu Leu

20 25 30

Gly Ser Ser Trp Gly Gly Leu Ile His Leu Tyr Thr Ala Thr Ala Arg

35 40 45

Asn Ser Tyr His Leu Gln Ile His Lys Asn Gly His Val Asp Gly Ala

50 55 60

Pro His Gln Thr Ile Tyr Ser Ala Leu Met Ile Arg Ser Glu Asp Ala

65 70 75 80

Gly Phe Val Val Ile Thr Gly Val Met Ser Arg Arg Tyr Leu Cys Met

85 90 95

Asp Phe Arg Gly Asn Ile Phe Gly Ser His Tyr Phe Asp Pro Glu Asn

100 105 110

Cys Arg Phe Gln His Gln Thr Leu Glu Asn Gly Tyr Asp Val Tyr His

115 120 125

Ser Pro Gln Tyr His Phe Leu Val Ser Leu Gly Arg Ala Lys Arg Ala

130 135 140

Phe Leu Pro Gly Met Asn Pro Pro Tyr Ser Gln Phe Leu Ser Arg

145 150 155 160

Arg Asn Glu Ile Pro Leu Ile His Phe Asn Thr Pro Ile Pro Arg Arg

165 170 175

His Thr Arg Ser Ala Glu Asp Asp Ser Glu Arg Asp Pro Leu Asn Val

180 185 190

Leu Lys Pro Arg Ala Arg Met Thr Pro Ala Pro Ala Ser Cys Ser Gln

195 200 205

Glu Leu Pro Ser Ala Glu Asp Asn Ser Pro Met Ala Ser Asp Pro Leu

210 215 220

Gly Val Val Arg Gly Gly Arg Val Asn Thr His Ala Gly Gly Thr Gly

225 230 235 240

Pro Glu Gly Cys Arg Pro Phe Ala Lys Phe Ile

245 250

<210> 42

<211> 806

<212> protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 42

Met Gly Ala Pro Ala Cys Ala Leu Ala Leu Cys Val Ala Val Ala Ile

1 5 10 15

Val Ala Gly Ala Ser Ser Glu Ser Leu Gly Thr Glu Gln Arg Val Val

20 25 30

Gly Arg Ala Ala Glu Val Pro Gly Pro Glu Pro Gly Gln Gln Glu Gln

35 40 45

Leu Val Phe Gly Ser Gly Asp Ala Val Glu Leu Ser Cys Pro Pro Pro

50 55 60

Gly Gly Gly Pro Met Gly Pro Thr Val Trp Val Lys Asp Gly Thr Gly

65 70 75 80

Leu Val Pro Ser Glu Arg Val Leu Val Gly Pro Gln Arg Leu Gln Val

85 90 95

Leu Asn Ala Ser His Glu Asp Ser Gly Ala Tyr Ser Cys Arg Gln Arg

100 105 110

Leu Thr Gln Arg Val Leu Cys His Phe Ser Val Arg Val Thr Asp Ala

115 120 125

Pro Ser Ser Gly Asp Asp Glu Asp Gly Glu Asp Glu Ala Glu Asp Thr

130 135 140

Gly Val Asp Thr Gly Ala Pro Tyr Trp Thr Arg Pro Glu Arg Met Asp

145 150 155 160

Lys Lys Leu Leu Ala Val Pro Ala Ala Asn Thr Val Arg Phe Arg Cys

165 170 175

Pro Ala Ala Gly Asn Pro Thr Pro Ser Ile Ser Trp Leu Lys Asn Gly

180 185 190

Arg Glu Phe Arg Gly Glu His Arg Ile Gly Gly Ile Lys Leu Arg His

195 200 205

Gln Gln Trp Ser Leu Val Met Glu Ser Val Val Pro Ser Asp Arg Gly

210 215 220

Asn Tyr Thr Cys Val Val Glu Asn Lys Phe Gly Ser Ile Arg Gln Thr

225 230 235 240

Tyr Thr Leu Asp Val Leu Glu Arg Ser Pro His Arg Pro Ile Leu Gln

245 250 255

Ala Gly Leu Pro Ala Asn Gln Thr Ala Val Leu Gly Ser Asp Val Glu

260 265 270

Phe His Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Leu

275 280 285

Lys His Val Glu Val Asn Gly Ser Lys Val Gly Pro Asp Gly Thr Pro

290 295 300

Tyr Val Thr Val Leu Lys Thr Ala Gly Ala Asn Thr Thr Asp Lys Glu

305 310 315 320

Leu Glu Val Leu Ser Leu His Asn Val Thr Phe Glu Asp Ala Gly Glu

325 330 335

Tyr Thr Cys Leu Ala Gly Asn Ser Ile Gly Phe Ser His His Ser Ala

340 345 350

Trp Leu Val Val Leu Pro Ala Glu Glu Glu Leu Val Glu Ala Asp Glu

355 360 365

Ala Gly Ser Val Tyr Ala Gly Ile Leu Ser Tyr Gly Val Gly Phe Phe

370 375 380

Leu Phe Ile Leu Val Val Ala Ala Val Thr Leu Cys Arg Leu Arg Ser

385 390 395 400

Pro Pro Lys Lys Gly Leu Gly Ser Pro Thr Val His Lys Ile Ser Arg

405 410 415

Phe Pro Leu Lys Arg Gln Val Ser Leu Glu Ser Asn Ala Ser Met Ser

420 425 430

Ser Asn Thr Pro Leu Val Arg Ile Ala Arg Leu Ser Ser Ser Gly Glu Gly

435 440 445

Pro Thr Leu Ala Asn Val Ser Glu Leu Glu Leu Pro Ala Asp Pro Lys

450 455 460

Trp Glu Leu Ser Arg Ala Arg Leu Thr Leu Gly Lys Pro Leu Gly Glu

465 470 475 480

Gly Cys Phe Gly Gln Val Val Met Ala Glu Ala Ile Gly Ile Asp Lys

485 490 495

Asp Arg Ala Ala Lys Pro Val Thr Val Ala Val Lys Met Leu Lys Asp

500 505 510

Asp Ala Thr Asp Lys Asp Leu Ser Asp Leu Val Ser Glu Met Glu Met

515 520 525

Met Lys Met Ile Gly Lys His Lys Asn Ile Ile Asn Leu Leu Gly Ala

530 535 540

Cys Thr Gln Gly Gly Pro Leu Tyr Val Leu Val Glu Tyr Ala Ala Lys

545 550 555 560

Gly Asn Leu Arg Glu Phe Leu Arg Ala Arg Arg Pro Pro Gly Leu Asp

565 570 575

Tyr Ser Phe Asp Thr Cys Lys Pro Glu Glu Gln Leu Thr Phe Lys

580 585 590

Asp Leu Val Ser Cys Ala Tyr Gln Val Ala Arg Gly Met Glu Tyr Leu

595 600 605

Ala Ser Gln Lys Cys Ile His Arg Asp Leu Ala Ala Arg Asn Val Leu

610 615 620

Val Thr Glu Asp Asn Val Met Lys Ile Ala Asp Phe Gly Leu Ala Arg

625 630 635 640

Asp Val His Asn Leu Asp Tyr Tyr Lys Lys Thr Thr Asn Gly Arg Leu

645 650 655

Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp Arg Val Tyr Thr

660 665 670

His Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Phe

675 680 685

Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro Val Glu Glu Leu Phe

690 695 700

Lys Leu Leu Lys Glu Gly His Arg Met Asp Lys Pro Ala Asn Cys Thr

705 710 715 720

His Asp Leu Tyr Met Ile Met Arg Glu Cys Trp His Ala Ala Pro Ser

725 730 735

Gln Arg Pro Thr Phe Lys Gln Leu Val Glu Asp Leu Asp Arg Val Leu

740 745 750

Thr Val Thr Ser Thr Asp Glu Tyr Leu Asp Leu Ser Ala Pro Phe Glu

755 760 765

Gln Tyr Ser Pro Gly Gly Gln Asp Thr Pro Ser Ser Ser Ser Ser Ser Gly

770 775 780

Asp Asp Ser Val Phe Ala His Asp Leu Leu Pro Pro Ala Pro Pro Ser

785 790 795 800

Ser Gly Gly Ser Arg Thr

805

<210> 43

<211> 19

<212> protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 43

Met Met Ser Phe Val Ser Leu Leu Leu Val Gly Ile Leu Phe His Ala

1 5 10 15

Thr Gln Ala

<210> 44

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 44

cagatgcact cccatcctcg 20

<210> 45

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 45

tctggggatg tccagtaggg 20

<210> 46

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 46

tggcagtgaa gatgttgaaa g 21

<210> 47

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 47

atcatcttca tcatctccat ctcttg 26

<210> 48

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 48

ttatccttgg ctcctggggtg 20

<210> 49

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 49

ctggaaggta gcagtgggaa 20

<210> 50

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 50

gctcgggaggt agaggtcttg t 21

<210> 51

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 51

ccacgctgac tggtaggaa 19

<210> 52

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 52

tttggtggac acaggcattg 20

<210> 53

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 53

caaactgccc gatcatggag 20

<---

Claims (18)

1. A method of treating or reducing abnormal visceral fat deposition in a patient having an FGFR3-related skeletal disease, comprising administering to the patient an effective amount of a soluble fibroblast growth factor receptor 3 (sFGFR3) polypeptide, wherein the sFGFR3 polypeptide consists of the amino acid sequence of SEQ ID NO: 5, and where the sFGFR3 polypeptide reduces insulin dysregulation or glucose dysregulation. 2. The method of claim. 1, in which the abnormal deposition of visceral fat is associated with one or more of the following organs or around them: heart, liver, spleen, kidneys, pancreas, intestines, reproductive organs and gallbladder. 3. The method according to claim 1 or 2, wherein the patient is not overweight or the patient does not have significant deposits of subcutaneous fat. 4. The method according to any one of paragraphs. 1-3, in which the patient has no significant abnormal fat deposition outside the abdomen. 5. The method according to any one of paragraphs. 1-4, wherein the patient is a fetus, neonate, infant, child, adolescent, or adult. 6. The method according to any one of paragraphs. 1-5, in which the patient is a human. 7. The method of claim 6, wherein the FGFR3-associated skeletal disease is selected from achondroplasia, thanatophoric dysplasia type I (TDI), thanatophoric dysplasia type II (TDM), severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN), hypochondroplasia, syndrome craniosynostosis and camptodactyly, tall stature and hearing loss syndrome (CATSHL). 8. The method of claim 6 or 7, wherein the patient exhibits one or more symptoms of an FGFR3-associated skeletal disease selected from short limbs, short torso, crooked legs, waddling gait, malformations of the skull, cloverleaf skull, craniosynostosis, vermiform bones, anomalies of the hands, anomalies of the feet, an elongated thumb, and anomalies of the chest. 9. The method according to any one of paragraphs. 1-8, wherein the sFGFR3 polypeptide binds to a fibroblast growth factor (FGF) selected from fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (FGF9), fibroblast growth factor 10 (FGF10) , fibroblast growth factor 18 (FGF18) and combinations thereof. 10. The method according to any one of paragraphs. 1-9, wherein the sFGFR3 polypeptide has an in vivo half-life of about 2 hours to about 25 hours. 11. The method according to any one of paragraphs. 1-10, in which the amino acid sequence of the sFGFR3 polypeptide is encoded by a polynucleotide containing the nucleic acid sequence of SEQ ID NO: 15. 12. The method of claim 11, wherein the polynucleotide encoding the sFGFR3 polypeptide is in a vector, the vector being selected from a plasmid, an artificial chromosome, a viral vector, and a phage vector. 13. The method according to any one of paragraphs. 1-12, wherein the sFGFR3 polypeptide is administered to the patient at a dose of about 0.001 mg/kg to about 30 mg/kg of the sFGFR3 polypeptide. 14. The method according to any one of paragraphs. 1-13, wherein the sFGFR3 polypeptide drug is administered to the patient at a dose of about 0.01 mg/kg to about 10 mg/kg of the sFGFR3 polypeptide. 15. The method according to any one of paragraphs. 1-14, wherein the sFGFR3 polypeptide is administered to the patient at a dose of about 0.2 mg/kg to about 3 mg/kg of the sFGFR3 polypeptide. 16. The method according to any one of paragraphs. 1-15, wherein the sFGFR3 polypeptide is administered to the patient daily, weekly, or monthly. 17. The method according to any one of paragraphs. 1-16, wherein 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, weekly, every two weeks, or once a month. 18. The method according to any one of paragraphs. 1-17, wherein the sFGFR3 polypeptide is administered to a patient by subcutaneous, intravenous, intramuscular, intra-arterial, intrathecal, or intraperitoneal administration.

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