CROSS REFERENCES TO RELATED APPLICATIONS The present application is a continuation of U.S. patent application Ser. No. 16/412,819, filed May 15, 2019, which is a divisional of U.S. patent application Ser. No. 15/586,463, filed on May 4, 2017, now issued U.S. Pat. No. 10,336,812, which claims priority to U.S. Provisional Patent Application No. 62/333,886, filed on May 10, 2016. Each disclosure is incorporated herein by reference in its entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 200310 401C1 SEQUENCE LISTING.txt. The text file is 403 KB, was created on Nov. 17, 2021, and is being submitted electronically via Patent Center.
FIELD OF THE INVENTION The invention relates to GDF15 fusion proteins. In particular, the invention relates to a fusion protein comprising a half-life extension protein, a linker and a GDF15 protein, nucleic acids and expression vectors encoding the fusion proteins, recombinant cells thereof, and pharmaceutical compositions comprising the fusion proteins. Methods of producing the fusion proteins and using the fusion proteins to treat metabolic disorders are also provided.
BACKGROUND OF THE INVENTION GDF15, a member of the TGFI3 family, is a secreted protein that circulates in plasma as a 25 kDa homodimer. Plasma levels of GDF15 range between 150 and 1150 pg/ml in most individuals (Tsai et al., J Cachexia Sarcopenia Muscle. 2012, 3: 239-243). Plasma levels of GDF15 are increased under conditions of injury, cardiovascular disease and certain types of cancer. This upregulation is thought to be a cytoprotective mechanism. High plasma levels of GDF15 are associated with weight loss due to anorexia and cachexia in cancer, and in renal and heart failure. In a clinical trial, GDF15 levels were an independent predictor of insulin resistance in obese, non-diabetic subjects (Kempf et al., Eur. J. Endo. 2012, 167: 671-678). A study in twins showed that the differences in levels of GDF15 within twin pairs correlated to the differences in BMI within that pair, suggesting that GDF15 serves as a long-term regulator of energy homeostasis (Tsai et al., PLoS One. 2015,10(7):e0133362).
While GDF15 has been extensively studied as a biomarker for several cardiovascular and other disease states, a protective role for GDF15 has also been described in myocardial hypertrophy and ischemic injury (Collinson, Curr. Opin. Cardiol. 2014, 29: 366-371; Kempf et al., Nat. Med. 2011, 17: 581-589; Xu et al., Circ Res. 2006, 98:342-50). GDF15 was shown to play an important role in protection from renal tubular and interstitial damage in mouse models of type 1 and type 2 diabetes (Mazagova et al., Am. J. Physiol. Renal Physiol. 2013; 305: F1249-F1264). GDF15 is proposed to have a protective effect against age-related sensory and motor neuron loss, and it improves recovery consequent to peripheral nerve damage (Strelau et al., Neurosci. 2009, 29: 13640-13648; Mensching et al., Cell Tissue Res. 2012, 350: 225-238). In fact, GDF15 transgenic mice were shown to have a longer lifespan than their littermate controls, which can indicate that this molecule serves as a long-term survival factor (Wang et al., Aging. 2014, 6: 690-700).
Numerous reports have demonstrated the improvement of glucose tolerance and insulin sensitivity in mouse models upon treatment with GDF15 protein. Two independent strains of transgenic mice overexpressing GDF15 have decreased body weight and fat mass, as well as improved glucose tolerance (Johnen et al., Nat. Med. 2007, 13:1333-1340; Macia et al., PLoS One. 2012, 7:e34868; Chrysovergis et al., Int. J. Obesity. 2014, 38: 1555-1564). Increases in whole-body energy expenditure and oxidative metabolism were reported in GDF15 transgenic mice (Chrysovergis et al., 2014, Id.). These were accompanied by an increase in thermogenic gene expression in brown adipose tissue and an increase in lipolytic gene expression in white adipose tissue. Mice lacking the GDF15 gene have increased body weight and fat mass (Tsai et al., PLoS One. 2013, 8(2):e55174). An Fc-fusion of GDF15 was shown to decrease body weight and improve glucose tolerance as well as insulin sensitivity in an obese cynomolgus monkey model when administered weekly over a period of six weeks (WO 2013/113008).
The effects of GDF15 on body weight are thought to be mediated via the reduction of food intake and increased energy expenditure. GDF15 may improve glycemic control via body weight-dependent and independent mechanisms.
Together, these observations suggest that increasing levels of GDF15 can be beneficial as a therapy for metabolic diseases. There is a need in the art for GDF15-based compositions that can be used to treat or prevent metabolic diseases, disorders, or conditions.
BRIEF SUMMARY OF THE INVENTION The invention satisfies this need by providing fusion proteins of GDF15 that demonstrate increased solubility/stability and exhibit features that indicate they can be used to treat or prevent metabolic diseases, disorders, or conditions. Such features include, for example, decreased body weight, increased glucose tolerance, and improved insulin sensitivity of a subject administered with a fusion protein according to an embodiment of the invention.
In one general aspect, the invention relates to a fusion protein comprising: (a) a half-life extension protein, (b) a linker, and (c) a GDF15 protein, wherein the fusion protein is arranged from N-terminus to C-terminus in the order (a)-(b)-(c).
In an embodiment of the invention, the GDF15 protein is a human GDF15 protein or a functional variant thereof In particular embodiments, the GDF15 protein comprises a mature GDF15 protein or functional variant thereof. In more particular embodiments, the GDF15 protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6-11. In other particular embodiments, the GDF15 protein comprises the amino acid sequence of SEQ ID NO:11, such as an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-11.
In an embodiment of the invention, the half life extension protein is human serum albumin (HSA) or a functional variant thereof. In particular embodiments, the half life extension protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 1. In other particular embodiments, the half life extension protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3.
In an embodiment of the invention, the linker is a flexible linker. In particular embodiments, the flexible linker contains the sequence (GGGGS)n wherein n is 2 to 20, preferably 4 to 10 (SEQ ID NO: 129). In another embodiment of the invention, the linker of the fusion protein is a structured linker. In particular embodiments, the structured linker contains the sequence (AP)n (SEQ ID NO: 144) or (EAAAK)n (SEQ ID NO: 130), wherein n is 2 to 20, preferably 4 to 10.
In embodiments of the invention, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 5, 25-30, 36-37, 40, 48, 55-60 or 64-75. In particular embodiments of the invention, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 25-30, 40, 55-60, and 70. In more particular embodiment of the invention, the fusion protein comprises the amino acid sequence of SEQ ID NO: 60.
In another general aspect, the invention relates to an isolated nucleic acid molecule encoding a fusion protein of the invention.
In another general aspect, the invention relates to an expression vector comprising a nucleic acid molecule encoding a fusion protein of the invention.
In another general aspect, the invention relates to a recombinant host cell comprising a nucleic acid molecule encoding a fusion protein of the invention.
In another general aspect, the invention relates to a method of obtaining a fusion protein of the invention. The method comprises: (1) culturing a host cell comprising a nucleic acid molecule encoding the fusion protein under a condition that the fusion protein is produced, and (2) recovering the fusion protein produced by the host cell.
In another general aspect, the invention relates to a pharmaceutical composition comprising a fusion protein of the invention and a pharmaceutically acceptable carrier.
In another general aspect, the invention relates to a pharmaceutical composition comprising a nucleic acid molecule encoding a fusion protein of the invention and a pharmaceutically acceptable carrier.
In another general aspect, the invention relates to a kit comprising a pharmaceutical composition of the invention.
In another general aspect, the invention relates to a method of treating or preventing a metabolic disease, disorder or condition, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a pharmaceutical composition of the invention. In a particular embodiment, the pharmaceutical composition is administered to the subject subcutaneously or intravenously.
According to embodiments of the invention, a method of treating a metabolic disorder selected from the group consisting of type 2 diabetes, elevated glucose levels, elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, or rheumatoid arthritis, in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 25-30, 40, 55-60, and 70 and a pharmaceutically acceptable carrier. In a particular embodiment, the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 60 and a pharmaceutically acceptable carrier.
Other aspects, features and advantages of the invention will be apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments and the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings.
In the drawings:
FIGS. 1A and 1B show the crystal structure of GDF15 (SEQ ID NO: 6), where the disulfide pairing of the first and second Cysteine residues (C1-C2) formed a loop at the N terminus of the protein, also shown is the crystal structure of TGF433 (SEQ ID NO: 139) for comparison.
FIG. 2 shows the effects of subcutaneous administration of fusion proteins according to embodiments of the invention, e.g., fusion proteins FP1 (SEQ ID NO: 60) and 6xHis-FP1 (SEQ ID NO: 26 with a 6xHis tag attached at the N-terminus), on food intake in C57BL/6 mice, the cumulative food intake at 24 hours post-administration is depicted. Values shown within the bars are % reduction compared to vehicle (PBS) group±SEM; N=8 animals per group for all groups, except N=9 in FP1 16 nmol/kg group. *-p<0.05, as compared to vehicle; p values were calculated using One-way ANOVA and Tukey's test for multiple comparisons.
FIG. 3 shows the effects of subcutaneous administration of FP1 and 6xHis-FP1 (SEQ ID NO: 26 with a 6xHis tag attached at the N-terminus) on food intake in Sprague-Dawley rats, the cumulative food intake at 48 hours post-administration is depicted. Values shown within the bars are % reduction compared to vehicle (PBS) group±SEM; N=8 animals per group. *-p<0.05, as compared to vehicle; p values were calculated using One-way ANOVA and Tukey's test for multiple comparisons.
FIG. 4 shows the change in body weight of diet induced obese (DIO) mice during treatment with FP1. Arrows indicate time (days) of subcutaneous administration post initial dose (Day 0); N=8 animals per group. *-p<0.05, for FP1 1 nmol/kg group as compared to vehicle; #-p<0.05, for FP1 10 nmol/kg group as compared to vehicle; p values were calculated using Two-way RM ANOVA and Tukey's test for multiple comparisons.
FIGS. 5A and 5B show the blood glucose levels in DIO mice during an oral glucose tolerance test (OGTT) after 14 days of dosing of FP1 every 3 days (q3d), the levels are expressed as the area under the curve. N=8 animals per group. *-p<0.05, for FP1 1 nmol/kg group as compared to vehicle; p values were calculated using One-way ANOVA and Tukey's test for multiple comparisons.
FIG. 6 shows the fed blood glucose levels in DIO mice during treatment with FP1. N=8 animals per group. *-p<0.05, as compared to vehicle; p values were calculated using Two-way RM ANOVA and Tukey's test for multiple comparisons.
FIG. 7 shows the 4 hour fasting homeostatic model assessment of insulin resistance (HOMA-IR) in DIO mice after 14 days of treatment with FP1. N=8 animals per group. *-p<0.05, as compared to vehicle; p values were calculated using One-way ANOVA and Tukey's test for multiple comparisons.
FIG. 8 shows the change in body weight in ob/ob mice during treatment with FP1 every 3 days (qd3). Arrows indicate time (days) of subcutaneous administration post initial dose (Day 0); N=9 animals per group. *-p<0.05, for FP1 10 nmol/kg group as compared to vehicle; #-p<0.05, for FP1 1 nmol/kg group as compared to vehicle; p values were calculated using Two-way RM ANOVA and Tukey's test for multiple comparisons.
FIG. 9 shows the blood glucose levels in ob/ob mice during treatment with FP1. Arrows indicate time (days) of subcutaneous administration post initial dose (Day 0); N=9 animals per group. *-p<0.05, for FP1 10 nmol/kg group as compared to vehicle; #-p<0.05, for FP1 1 nmol/kg group as compared to vehicle; p values were calculated using Two-way RM ANOVA and Tukey's test for multiple comparisons.
FIG. 10 shows the mean (±standard deviation, SD) of the serum drug concentration-time profile of FP1 following 2 mg/kg intravenous (IV) and subcutaneous (SC) administration in C57BI/6 mice.
FIG. 11 shows the mean (±SD) of the serum drug concentration-time profile of FP1 following 2 mg/kg IV and SC administration in Sprague-Dawley rats.
FIG. 12 shows the mean (±SD) of the serum drug concentration-time profile of FP1 following 1 mg/kg IV and SC administration in cynomolgus monkeys, as determined by immunoassays.
FIG. 13 shows the serum concentration (ng/mL) of FP1 as an intact dimer over time following a single IV administration in cynomolgus monkeys, as determined by immuno-affinity (IA) capture-LCMS analysis.
FIG. 14 shows the serum concentration (ng/mL) of FP1 as an intact dimer over time following a single SC administration in cynomolgus monkeys, as determined by immuno-affinity capture-LCMS analysis.
FIG. 15 shows the concentration of FP1, represented as a % of the starting concentration, after 0, 4, 24 and 48 hours of ex vivo incubation in plasma obtained from two human subjects (Sub), as determined by immunoassay.
FIG. 16 shows the average concentration of FP 1, represented as a % of time 0, as an intact dimer after 0, 4, 24 and 48 hours of ex vivo incubation in plasma obtained from two human subjects (Sub), as determined by intact mass immuno-affinity capture-LCMS analysis.
FIG. 17 shows acute food intake in lean C57BL6N male mice before and after the administration of various N-terminal deletion variants of GDF15. (SEQ ID NOs: 92, 111, and 112, compared to wild type fusion with no deletion (SEQ ID 26 with a 6xHis tag attached at the N-terminus). N=8 animals per group; *-p<0.05, as compared to vehicle; p values were calculated using Two-way RM ANOVA and Tukey's test for multiple comparisons.
FIG. 18 shows the effect of a single dose of FP2 on food intake in C57BL/6 mice; specifically, cumulative food intake at 24 hours post administration is shown. Values shown within the bars are % reduction compared to PBS group (mean+SEM); N=8 animals per group for all groups, except N=6 in 6xHis-FP1. **-p<0.01, ***-p<0.001, ****-p<0.0001; p values were calculated using Two-way ANOVA and Dunnetts's test for multiple comparisons.
FIG. 19 shows cumulative food intake measured in Sprague-Dawley rats at 24 hours post administration of a single dose of FP2. Values shown within the bars are % reduction compared to PBS group (mean+SEM); N=8 animals per group. **-p<0.01, p values were calculated using Two-way ANOVA and Tukey's test for multiple comparisons.
FIG. 20 shows the percent change in body weight during treatment with FP2 q3d in DIO mice. Arrows indicate the time of subcutaneous injections of FP2; N=6 animals per group; *-p,0.05, as compared with the vehicle, using Two Way ANOVA and Tukey's test for multiple comparisons;
FIGS. 21A and 21B show the area under the curve (AUC) for the blood glucose concentration levels during an OGTT test after 14 days of q3d dosing of FP2 in DIO mice. *-p<0.05, using One Way ANOVA and Tukey's multiple comparisons test, using n=8 animals per group.
FIG. 22A shows the plasma insulin levels during an OGTT after 8 days of q3d dosing of FP2 in DIO mice. *-p<0.05, Vehicle vs. FP2 (0.3 nmol/kg); FP2 (10 nmol/kg); and Rosiglitazone. #-p<0.05, as compared to Rosiglitazone (10 mg/kg), using Two Way RM ANOVA and Tukey's multiple comparisons test.
FIGS. 22B shows the AUC for the plasma insulin levels during an OGTT after 8 days of q3d dosing of FP2 in DIO mice. *-<0.05, as compared to Vehicle; #-p<0.05, as compared to Rosiglitazone.
FIG. 23 shows the fed blood glucose levels after 8 days of q3d dosing of FP2 in DIO mice. *-p<0.05, as compared to Vehicle, using Two Way RM ANOVA and Tukey's multiple comparisons test, n=8 animals per group.
FIG. 24 shows fasting HOMA-IR after 14 days of treatment with FP2 q3d, followed by 5-hour fast on Day 14, in DIO mice. *-p<0.05, as compared to Vehicle, using One Way ANOVA and Tukey's multiple comparisons test, for n=8 animals per group.
FIG. 25 shows serum concentrations of FP2 following 2 mg/kg intravenous (IV) and 2 mg/kg subcutaneous (SC) administration in C57B1/6 mice. Values represent mean±SD (n=5 samples per timepoint).
FIG. 26 shows serum concentrations of FP2 following 2 mg/kg intravenous (IV) and 2 mg/kg subcutaneous (SC) administration in Sprague Dawley rats. N=5 samples per time point.
FIG. 27 shows plasma concentrations of FP2 in cynomolgus monkeys analyzed by immunoassay. Values represent mean±SD of n=3, except n=2 for IV at day 22 (528 hr). IV—intravenous, SC—subcutaneous.
FIG. 28 shows plasma concentrations of FP2 as intact dimer in cynomolgus monkeys analyzed by LCMS. Values represent mean±SEM of n=3, except n=2 for subcutaneous (SC) at 168 hours, n=1 for SC at 120 hours and 432 hours, and n=1 for IV—intravenous at 168 hours and 432 hours.
FIG. 29 shows ex vivo stability of FP2 (Normalized Percent Recovery) over 48 hours in human plasma measured by immunoassay.
FIG. 30 shows ex vivo stability of FP2 (Normalized Percent Recovery) over 48 hours in human plasma measured by intact LC/MS.
FIG. 31 shows concentration response curves for FP2 and HSA-GDF15:GDF15 heterodimer using pAKT Assays in SK-N-AS cells expressing recombinant human GFRAL receptor (N=3).
FIG. 32 shows daily food intake (g) prior to and following a single dose of FP1 in cynomolgus monkeys. *-p<0.05 for 10mg/kg of FP1 as compared to vehicle.
FIG. 33 shows percent body weight change prior to and following a single dose of FP1 in cynomolgus monkeys. *-p<0.05 for 10mg/kg FP1 as compared to vehicle; #-p<0.05 for 3mg/kg as compared to vehicle using Two Way RM ANOVA and Tukey's multiple comparisons test, for n=8 animals per group.
FIG. 34 shows daily food intake (g) prior to and following a single dose of FP2 in cynomolgus monkeys. *-p<0.05, as compared to Vehicle, using Two Way RM ANOVA and Tukey's multiple comparisons test, for n=8 animals per group.
FIG. 35 shows percent body weight change prior to and following a single dose of FP2 in cynomolgus monkeys. *-p<0.05, for 10 nmol/kg of FP2 as compared to Vehicle, #-p<0.05, for 3 nmol/kg of FP2 as compared to Vehicle, & -p<0.05, for 1 nmol/kg of FP2 as compared to Vehicle, using Two Way RM ANOVA and Tukey's multiple comparisons test, for n=8 animals per group.
DETAILED DESCRIPTION OF THE INVENTION Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set in the specification. All patents, published patent applications and publications cited herein are incorporated by reference as if set forth fully herein. It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.
The invention relates to a fusion protein comprising (a) a half life-extension protein, (b) a linker, and (c) a GDF15 protein, wherein the fusion protein is arranged from N-terminus to C-terminus in the order (a)-(b)-(c).
It is found that a fusion protein according to an embodiment of the invention, comprising a half life-extension protein, a linker, and a GDF15 protein, results in an increased half life of the GDF15 protein, and fusion proteins of the invention exhibit metabolic effects that demonstrate their suitability as therapeutics for treating and preventing metabolic diseases, disorders or conditions. Such effects include, but are not limited to, decreasing body weight, increasing glucose tolerance, and improving insulin sensitivity of animals administered with the fusion proteins.
As used herein, the term “fusion protein” refers to a protein having two or more portions covalently linked together, where each of the portions is derived from different proteins.
Fusion proteins according to embodiments of the invention can include any GDF15 protein. As used herein, the term “GDF15 protein” refers to any naturally-occurring wild-type growth differentiation factor 15 protein or a functional variant thereof. The GDF15 protein can be from any mammal, such as a human or another suitable mammal, such as a mouse, rabbit, rat, pig, dog, or a primate. In particular embodiments, the GDF15 protein is a human GDF15 protein or a functional variant thereof. In preferred embodiments, the GDF15 protein is a mature GDF15 protein or a functional variant thereof.
As used herein, the term “mature GDF15 protein” refers to the portion of the pre-pro-protein of GDF15 that is released from the full-length protein following intracellular cleavage at the RXXR furin-like cleavage site. Mature GDF15 proteins are secreted as homodimers linked by disulfide bonds. In one embodiment of the invention, a mature GDF15 protein, shorthand GDF15(197-308) (SEQ ID NO: 6), contains amino acids 197-308 of a full-length human GDF15 protein.
As used herein, “functional variant” refers to a variant of a parent protein having substantial or significant sequence identity to the parent protein and retains at least one of the biological activities of the parent protein. A functional variant of a parent protein can be prepared by means known in the art in view of the present disclosure. A functional variant can include one or more modifications to the amino acid sequence of the parent protein. The modifications can change the physico-chemical properties of the polypeptide, for example, by improving the thermal stability of the polypeptide, altering the substrate specificity, changing the pH optimum, and the like. The modifications can also alter the biological activities of the parent protein, as long as they do not destroy or abolish all of the biological activities of the parent protein.
According to embodiments of the invention, a functional variant of a parent protein comprises a substitution, preferably a conservative amino acid substitution, to the parent protein that does not significantly affect the biological activity of the parent protein. Conservative substitutions include, but are not limited to, amino acid substitutions within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Non-standard or unnatural amino acids (such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline, and alpha-methyl serine) can also be used to substitute standard amino acid residues in a parent protein.
According to other embodiments of the invention, a functional variant of a parent protein comprises a deletion and/or insertion of one or more amino acids to the parent protein. For example, a functional variant of a mature GDF15 protein can include a deletion and/or insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids to the mature GDF15 protein, preferably, a deletion of 1 to 30 amino acids at the N-terminus of the mature GDF15 protein.
According to yet other embodiments of the invention, a functional variant of a parent protein comprises a substitution, preferably a conservative amino acid substitution, and a deletion and/or insertion, preferably a small deletion and/or insertion of amino acids, to the parent protein.
According to embodiments of the invention, a fusion protein of the invention comprises a GDF15 protein that has an amino acid sequence at least 90% identical to the amino acid sequence of a mature GDF15, such as GDF15(197-308) (SEQ ID NO: 6); or an amino acid sequence at least 90% identical to the amino acid sequence of a mature GDF15 truncated at the N-terminus, such as GDF15(200-308) (SEQ ID NO: 7), GDF15(201-308) (SEQ ID NO: 8), GDF15(202-308) (SEQ ID NO: 9), GDF15(203-308) (SEQ ID NO: 10), or GDF15(211-308) (SEQ ID NO: 11). The GDF15 protein can have at least one of substitutions, insertions and deletions to SEQ ID NO: 6, 7, 8, 9, 10 or 11, as long as it maintains at least one of the biological activities of the GDF15 protein, such as its effects on food intake, blood glucose levels, insulin resistance, and body weight, etc.
In particular embodiments, a fusion protein of the invention comprises a GDF15 protein having the amino acid sequence of SEQ ID NO: 11, including but not limited to, the amino acid sequence of SEQ ID NO: 6, 7, 8, 9, 10 or 11.
Any suitable half life extension protein can be used in fusion proteins according to embodiments of the invention. As used herein, the term “half life extension protein” can be any protein or fragment thereof that is known to extend the half life of proteins to which it is fused. Examples of such half life extension proteins include, but are not limited to, human serum albumin (HSA), the constant fragment domain (Fc) of an immunoglobulin (Ig), or transferrin (Tf). In embodiments of the invention, the half life extension protein comprises HSA or a functional variant thereof. In particular embodiments of the invention, the half life extension protein comprises an amino acid sequence that is at least 90% identity to SEQ ID NO: 1. In preferred embodiments of the invention, the half life extension protein comprises HSA or functional variant thereof wherein the cysteine residue at position 34 of the HSA has been replaced by serine or alanine.
In particular embodiments, a fusion protein of the invention comprises a half life extension protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3.
Any suitable linker can be used in fusion proteins according to embodiments of the invention. As used herein, the term “linker” refers to a linking moiety comprising a peptide linker. Preferably, the linker helps insure correct folding, minimizes steric hindrance and does not interfere significantly with the structure of each functional component within the fusion protein. In some embodiments of the invention, the peptide linker comprises 2 to 120 amino acids. For example, the peptide linker comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or 120 amino acids.
In embodiments of the invention, the linker increases the flexibility of the fusion protein components. In particular embodiments of the invention, the linker can be a flexible linker comprising the sequence (GGGGS)n (SEQ ID NO: 129), including but not limited to, GS-(GGGGS)n SEQ ID NO: 142 or AS-(GGGGS)n-GT SEQ ID NO: 143, wherein n is 2 to 20, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
In other embodiments of the invention, the linker is structured. In particular embodiments of the invention, the linker can be a structured linker comprising the sequence (AP)n (SEQ ID NO: 144) or (EAAAK)n (SEQ ID NO: 130), including but not limited to, AS-(AP)n-GT (SEQ ID NO: 145) or AS-(EAAAK)n-GT (SEQ ID NO: 140), wherein n is 2 to 20, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In other embodiments of the invention, the linker comprises the sequences (GGGGA)n (SEQ ID NO: 131), (PGGGS)n (SEQ ID NO: 132), (AGGGS)n (SEQ ID NO: 133) or GGS-(EGKSSGSGSESKST)n-GGS (SEQ ID NO: 134) wherein n is 2 to 20.
In embodiments of the invention, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 5, 25-30, 36-37, 40, 48, 55-56, 59-60 or 64-75. In particular embodiments of the invention, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 25-30, 36-37, 40, 48, 55-56, 59-60 and 64-75. In more particular embodiments of the invention, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 25-30, 40, 55-56, 55-56, 59-60, and 70. In further more particular embodiments of the invention, the fusion protein comprises the amino acid sequence of SEQ ID NO: 92, SEQ ID NO: 60 or SEQ ID NO: 26. The fusion protein can also include small extension(s) at the amino- or carboxyl-terminal end of the protein, such as a tag that facilitates purification, such as a poly-histidine tag, an antigenic epitope or a binding domain.
The fusion proteins disclosed herein can be characterized or assessed for GDF15 biological activities including, but not limited to effects on food intake, oral glucose tolerance tests, measurements of blood glucose levels, insulin resistance analysis, changes in body weight, pharmacokinetic analysis, toxicokinetic analysis, immunoassays and mass spec analysis of the level and stability of full-length fusion proteins, and human plasma ex vivo stability analysis.
The invention also provides an isolated nucleic acid molecule encoding a fusion protein of the invention. In embodiments of the invention, the isolated nucleic acid molecule encodes a fusion protein comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 5, 25-30, 36-37, 40, 48, 55-56, 59-60 or 64-75. In particular embodiments, the isolated nucleic acid molecule encodes a fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 25-31, 36-37, 40, 48, 55-56, 59-60 and 64-75. In more particular embodiments, the isolated nucleic acid molecule encodes a fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 25-30, 40, 55-56, 59-60, and 70. In further more particular embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NOs: 76-91.
According to other embodiments of the invention, the nucleic acid molecule encoding the fusion protein can be in an expression vector. Expression vectors include, but are not limited to, vectors for recombinant protein expression and vectors for delivery of nucleic acids into a subject for expression in a tissue of the subject, such as viral vectors. Examples of viral vectors suitable for use with the invention include, but are not limited to adenoviral vectors, adeno-associated virus vectors, lentiviral vectors, etc. The vector can also be a non-viral vector. Examples of non-viral vectors include, but are not limited to plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, etc. The vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, or an origin of replication.
According to other embodiments of the invention, the nucleic acid molecule encoding the fusion protein can be codon optimized for improved recombinant expression from a desired host cell, such as Human Embryonic Kidney (HEK) or Chinese hamster ovary (CHO) cells, using methods known in the art in view of the present disclosure.
The invention also provides a host cell comprising a nucleic acid molecule encoding a fusion protein of the invention. Host cells include, but are not limited to, host cells for recombinant protein expression and host cells for delivery of the nucleic acid into a subject for expression in a tissue of the subject. Examples of host cells suitable for use with the invention include, but are not limited to HEK or CHO cells.
In another general aspect, the invention relates to a method of obtaining a fusion protein of the invention. In a general aspect, the method comprises: (1) culturing a host cell comprising a nucleic acid molecule encoding a fusion protein under a condition that the fusion protein is produced, and (2) recovering the fusion protein produced by the host cell. The fusion protein can be purified further using methods known in the art.
In some embodiments, the fusion protein is expressed in host cells and purified therefrom using a combination of one or more standard purification techniques, including, but not limited to, affinity chromatography, size exclusion chromatography, ultrafiltration, and dialysis. Preferably, the fusion protein is purified to be free of any proteases.
The invention also provides a pharmaceutical composition comprising a fusion protein of the invention and a pharmaceutically acceptable carrier.
The invention further provides a composition comprising a nucleic acid molecule encoding a fusion protein of the invention and a pharmaceutically acceptable carrier. Compositions comprising a nucleic acid molecule encoding a fusion protein of the invention can comprise a delivery vehicle for introduction of the nucleic acid molecule into a cell for expression of the fusion protein. Examples of nucleic acid delivery vehicles include liposomes, biocompatible polymers, including natural polymers and synthetic polymers, lipoproteins, polypeptides, polysaccharides, lipopolysaccharides, artificial viral envelopes, metal particles, and bacteria, viruses, such as baculoviruses, adenoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic hosts.
The pharmaceutically acceptable carrier can include one or more of pharmaceutically acceptable excipient, buffer, stabilizer or other materials known to those skilled in the art. Examples of pharmaceutically acceptable carriers include, but are not limited to, one or more of water, saline, buffer, isotonic agents such as sugars, polyalcohols, auxiliary substances such as wetting or emulsifying agents, preservatives, as well as combinations thereof. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient at the dosages and concentrations employed. The precise nature of the carrier or other material can depend on the route of administration, e.g., intramuscular, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., gut), intranasal or intraperitoneal routes. For example, liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can also be included. Compositions for parenteral administration can be stored in lyophilized form or in a solution, and are generally placed into a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
According to embodiments of the invention, a pharmaceutical composition can comprise one or more additional components, such as another active ingredient.
The invention also relates to kits comprising a pharmaceutical composition of the invention. The kits can contain a first container having a dried fusion protein of the invention and a second container having an aqueous solution to be mixed with the dried fusion protein prior to administration to a subject, or a single container containing a liquid pharmaceutical composition of the invention. The kit can contain a single-dose administration unit or multiple dose administration units of a pharmaceutical composition of the invention. The kit can also include one or more pre-filled syringes (e.g., liquid syringes and lyosyringes). A kit can also comprise instructions for the use thereof. The instructions can describe the use and nature of the materials provided in the kit, and can be tailored to the precise metabolic disorder being treated.
The invention also relates to use of the pharmaceutical compositions described herein to treat or prevent a metabolic disease, disorder or condition, such as type 2 diabetes, elevated glucose levels, elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, or rheumatoid arthritis. According to embodiments of the invention, a method of treating or preventing a metabolic disease, disorder or condition in a subject in need of the treatment comprises administering to the subject a therapeutically or prophylactically effective amount of a pharmaceutical composition of the invention. Any of the pharmaceutical compositions described herein can be used in a method of the invention, including pharmaceutical compositions comprising a fusion protein of the invention or pharmaceutical compositions comprising a nucleic acid encoding the fusion protein.
As used herein, “subject” means any animal, particularly a mammal, most particularly a human, who will be or has been treated by a method according to an embodiment of the invention. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans, etc., more particularly a human.
A “metabolic disease, disorder or condition” refers to any disorder related to abnormal metabolism. Examples of metabolic diseases, disorders or conditions that can be treated according to a method of the invention include, but are not limited to, type 2 diabetes, elevated glucose levels, elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, or rheumatoid arthritis.
The terms “treat,” “treating,” and “treatment” as used herein refer to administering a composition to a subject to achieve a desired therapeutic or clinical outcome in the subject. In one embodiment, the terms “treat,” “treating,” and “treatment” refer to administering a pharmaceutical composition of the invention to reduce, alleviate or slow the progression or development of a metabolic disorder, such as type 2 diabetes, elevated glucose levels, elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, or rheumatoid arthritis.
The term “therapeutically effective amount” means an amount of a therapeutically active compound needed to elicit the desired biological or clinical effect. According to embodiments of the invention, “a therapeutically effective amount” is an amount sufficient to effect beneficial or desired results, including clinical results. A therapeutically effective amount can be administered in one or more administrations. In terms of a disease state, an effective amount is an amount sufficient to ameliorate, stabilize, or delay development of a disease. According to specific embodiments of the invention, a therapeutically effective amount is an amount of a fusion protein needed to treat or prevent a metabolic disease, disorder or condition, such as type 2 diabetes, elevated glucose levels, elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, or rheumatoid arthritis.
According to embodiments of the invention, a pharmaceutical composition of the invention can be administered to a subject by any method known to those skilled in the art in view of the present disclosure, such as by intramuscular, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., gut), intranasal or intraperitoneal route of administration. In particular embodiments, a pharmaceutical composition of the invention is administered to a subject by intravenous injection or subcutaneous injection.
Parameters such as the dosage amount, frequency of administration, and duration of administration of a pharmaceutical composition to a subject according to an embodiment of the invention are not limited in any particular way. The optimum values of such parameters can depend on a variety of factors, such as the subject to be treated, the particular metabolic disease to be treated, the severity of the disease, the route of administration, etc., and one of ordinary skill in the art will be able to determine the optimum values for such parameters in order to achieve the desired therapeutic or clinical outcome. For example, a pharmaceutical composition can be administered once per day, or more than once per day, such as twice, three times, four times, etc. A typical dosage can range from about 0.1 μg/kg to up to about 100 mg/kg or more of the fusion protein, depending on the factors such as those mentioned above.
EMBODIMENTS Embodiment 1 is a fusion protein comprising: (a) a half-life extension protein, (b) a linker, and (c) a GDF15 protein; wherein the fusion protein is arranged from N-terminus to C-terminus in the order (a)-(b)-(c).
Embodiment 2 is a fusion protein according to Embodiment 1, wherein the GDF15 protein is a human GDF15 protein or a functional variant thereof
Embodiment 3 is a fusion protein according to Embodiment 1, wherein the GDF15 protein comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-11.
Embodiment 4 is a fusion protein according to Embodiment 1, wherein the GDF15 protein comprises the amino acid sequence of SEQ ID NO: 11.
Embodiment 5 is a fusion protein according to Embodiment 4, wherein the GDF15 protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-11.
Embodiment 6 is a fusion protein according to any of Embodiments 1 to 5, wherein the half-life extension protein comprises human serum albumin (HSA) or a functional variant thereof.
Embodiment 7 is a fusion protein according to Embodiment 6, wherein the half-life extension protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 1.
Embodiment 8 is a fusion protein according to Embodiment 7, wherein the half-life extension protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3.
Embodiment 9 is a fusion protein according to any of Embodiments 1 to 8, wherein the linker is a flexible linker.
Embodiment 10 is a fusion protein according to Embodiment 9, wherein the linker comprises the sequence (GGGGS)n, wherein n is 2 to 20 (SEQ ID NO: 129), such as GS-(GGGGS)x8 (SEQ ID NO: 12) or AS-(GGGGS)x8—GT (SEQ ID NO: 141).
Embodiment 11 is a fusion protein according to any of Embodiments 1 to 9, wherein the linker is a structured linker.
Embodiment 12 is a fusion protein according to Embodiment 11, wherein the linker comprises the sequence (AP)n (SEQ ID NO: 144) or (EAAAK)n (SEQ ID NO: 130), wherein n is 2 to 20, such as AS-(AP)n-GT (SEQ ID NO: 145) or AS-(EAAAK)n-GT (SEQ ID NO: 140).
Embodiment 13 is a fusion protein comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 5, 25-31, 36-37, 40, 48, 55-60 or 64-75.
Embodiment 14 is a fusion protein according to Embodiment 13, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 25-31, 36-37, 40, 48, 55-60 and 64-75.
Embodiment 15 is a fusion protein according to Embodiment 14, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 25-30, 40, 55-60, and 70.
Embodiment 16 is an isolated nucleic acid molecule encoding the fusion protein of any one of Embodiments 1 to 15.
Embodiment 17 is an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs: 76-91.
Embodiment 18 is an expression vector comprising the nucleic acid molecule of Embodiment 16 or 17.
Embodiment 19 is a host cell comprising the nucleic acid molecule of Embodiment 16 or 17.
Embodiment 20 is a method of producing the fusion protein of any one of Embodiments 1 to 15, comprising: (1) culturing a host cell comprising a nucleic acid molecule encoding the fusion protein under a condition that the fusion protein is produced; and (2) recovering the fusion protein produced by the host cell.
Embodiment 21 is a method according to Embodiment 20, wherein the recovering step comprises purifying the fusion protein to remove proteases.
Embodiment 22 is a pharmaceutical composition comprising a therapeutically effective amount of the fusion protein of any one of Embodiments 1 to 15 and a pharmaceutically acceptable carrier.
Embodiment 23 is a pharmaceutical composition comprising a therapeutically effective amount of a nucleic acid molecule encoding the fusion protein of any one of Embodiments 1 to 15 and a pharmaceutically acceptable carrier.
Embodiment 24 is a kit comprising a pharmaceutical composition according to Embodiment 22 or 23.
Embodiment 25 is a method of treating or preventing a metabolic disorder, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to any of Embodiments 22 and 23.
Embodiment 26 is a method according to Embodiment 25, wherein the metabolic disorder is selected from the group consisting of type 2 diabetes, elevated glucose levels, elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, or rheumatoid arthritis.
Embodiment 27 is a method according to Embodiment 25 or 26, wherein the pharmaceutical composition is administered to the subject subcutaneously or intravenously.
Embodiment 28 is a method of treating a metabolic disorder selected from the group consisting of type 2 diabetes, elevated glucose levels, elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, or rheumatoid arthritis, in a subject in need thereof, the method comprising subcutaneously or intravenously administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 25-30, 40, 55-60, and 70 and a pharmaceutically acceptable carrier.
Embodiment 29 is a method of treating a metabolic disorder selected from the group consisting of type 2 diabetes, elevated glucose levels, elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, or rheumatoid arthritis, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 60 and a pharmaceutically acceptable carrier.
Embodiment 30 is a method of any one of Embodiments 25 to 29, wherein the pharmaceutical composition is administered to the subject intravenously or subcutaneously.
Embodiment 31 is a fusion protein of any one of Embodiments 1 to 15 for use in treating or preventing a metabolic disorder selected from the group consisting of type 2 diabetes, elevated glucose levels, elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, or rheumatoid arthritis.
EXAMPLES The following examples of the invention are to further illustrate the nature of the invention. It should be understood that the following examples do not limit the invention and that the scope of the invention is to be determined by the appended claims.
Example 1: Design of Fusion Molecules Comprising GDF15-Effect of GDF15 Truncations Like other TGFβ family members, GDF15 is synthesized as a pre-pro-protein that forms a dimer in the endoplasmic reticulum and undergoes furin cleavage to produce secreted mature GDF15 (amino acids 197-308). The secreted mature GDF15 homodimer is about 25 k Daltons, and each monomer has the potential to form up to 4 intramolecular disulfide bonds with a single intermolecular disulfide linking the homodimer components.
The crystal structure of GDF15 was determined in the invention and is depicted in FIGS. 1A and 1B. The crystal structure shows that the C-terminus of the mature GDF15 is buried in the dimer interface, while the N-terminus is exposed. This exposed terminus allows for the linkage of fusion proteins, such as half life extension proteins, to the N-terminus of GDF15.
The crystal structure also depicts the novel disulfide paring pattern of GDF15 cysteine residues. While TGFβ1 has C1-C3 and C2-C7 pairing (i.e., pairing between its first and third cysteine residues as well as between its second and seventh cysteine residues), GDF15 has C1-C2 and C3-C7 pairing (see FIGS. 1A and 1B). This unique disulfide pairing results in a loop formed by the C1-C2 pairing that is located at the N-terminus of the protein and away from the cysteine knot that contains other disulfide bonds. The structure predicts that the N-terminus of GDF15 may not be critical for dimer formation or overall protein folding, and that GDF15 and N-terminal fusion molecules thereof may be tolerable to N-terminal deletions that delete C1 and C2, residues within the C1-C2 loop, or even residues C-terminal to C2.
Example 2: Design of Fusion Molecules Comprising GDF15 —Effect of the Linker Different linkers between the HSA molecule and the GDF15 molecule were evaluated. Both flexible linkers, containing the sequence (GGGGS)n (SEQ ID NO: 129), and structured linkers, containing the sequence (AP)n (SEQ ID NO: 144) or (EAAAK)n (SEQ ID NO: 130), wherein n is 2 to 20, were evaluated.
Fusion proteins comprising the different linkers were compared for their biophysical properties, their effect on the efficacy of food intake in lean mice, their mouse pharmacokinetic (PK) values, and their ex vivo stability in human blood. The results of tested linker variants are shown in Table 1. The molecule comprising SEQ ID NO: 31, which contained the (EAAAK)8 (SEQ ID NO: 138) linker, showed aggregation by HPLC. The remaining seven linker variants in Table 1 demonstrated no aggregation.
TABLE 1
Summary of linker variant analysis
SEQ Good Ex vivo
ID Aggre- Mouse stability in
NO* Linker of gation PK (WT) human blood
25 AS(GGGGS)2GT No Yes Yes
5 GS(GGGGS)4 No Yes Yes
26 AS(GGGGS)8GT No Yes Yes
27 AS(AP)5GT No Yes Yes
28 AS(AP)10GT No Yes Yes
29 AS(AP)20GT No Yes Yes
30 AS(EAAAK)4GT No Yes Yes
31 AS(EAAAK)8GT YES Not Not tested
tested
*-6xHis tag was attached at the N-terminus for purification purpose
Linker stability was also evaluated for these variants by in vivo studies in mice and by ex vivo stability studies in human whole blood and plasma samples. Two forms of detection were used to analyze the results from these studies. An immunoassay with anti-GDF15 capture and anti-HSA detection antibody pairs was used to evaluate how intact the linker was by measuring the presence of both molecules on either side of the linker. A broader picture of the whole-molecule integrity was analyzed by liquid chromatography-mass spectrometry (LC-MS) analysis using different surrogate peptide sequences from both HSA and GDF15 . The immunoassay demonstrated a stable PK profile for all of the linker variants and no loss of spiked plasma sample concentration for any of the linker variants observed over 48 hours. The LC-MS results were consistent with the immunoassay showing that the surrogate peptides from different parts of the HSA and GDF15 molecules were intact. The PK profile of the linker variants analyzed by LC-MS using surrogate peptides showed a similar trend for different linker variants, where they all had detectable levels at day 7. All the variants in Table 1 except for SEQ ID 31 had desirable biophysical properties and PK values.
The linker variants were evaluated for their in vivo activity by carrying out food intake studies in lean mice. Table 2 shows the influence of the linker variants on the efficacy of the fusion protein in decreasing food intake. There was a clear influence of the linker on the efficacy. With regard to the flexible (GGGGS)n (SEQ ID NO: 129) linkers, an increase in the linker length from 2 to 4 to 8 dramatically increased the fusion protein efficacy. For the more rigid (AP)n (SEQ ID NO: 144) linkers, the trend was less obvious, suggesting that the degree of freedom of the GDF15 molecule within the fusion protein plays a critical role in its efficacy.
TABLE 2
Effect of the linker on the in vivo efficacy of
HSA-GDF15 fusion proteins in lean mice
SEQ ID % Decrease in
NO* Linker food intake (mean)
25 AS(GGGGS)2GT 28.8
5 GS(GGGGS)4 40.5
26 AS(GGGGS)8GT 60.7
27 AS(AP)5GT 48.2
28 AS(AP)10GT 66.2
29 AS(AP)20GT 55.1
30 AS(EAAAK)4GT 51.9
*-6xHis tag was attached at the N-terminus for purification purpose
Example 3: Design of Fusion Molecules Comprising GDF15 —Effect of HSA Mutations Recombinant proteins with the half life extension protein human serum albumin fused to the N-terminus of GDF15 through a linker were designed. This design should allow for the GDF15 dimerization interface to remain unperturbed and allow for the formation of the native inter-chain disulfide linkages, resulting in a GDF15 homodimer with HSA fusion extended from each GDF15 arm. With this approach, only a single gene is required to generate the HSA-GDF15 homodimer.
Native human serum albumin protein contains 35 cysteine (Cys, C) residues that form 17 disulfide bonds, with the Cys-34 residue being the only free cysteine in the molecule. This free Cys-34 has been shown to function as a free radical scavenger, by trapping multiple reactive oxygen species (ROS) and reactive nitrogen species (RNS). This free Cys was thus mutated to minimize the risk of heterogeneity due to oxidation.
The free cysteine at position 34 of HSA was mutated to either serine or alanine, and the GDF15 fusion molecules with either a HSA(C34S) or a HSA(C34A) mutation were analyzed. Both of the molecules were purified using a three-step purification method: (i) ion-exchange chromatography, (ii) hydrophobic interaction chromatography, and (iii) size-exclusion chromatography. When they were first generated, HPLC analysis showed that both molecules were pure and aggregation-free (Table 3).
However, two weeks after its generation, the fusion protein containing the HSA(C34A) mutation (comprising SEQ ID NO: 48) showed aggregation by HPLC, while the fusion protein containing the HSA(C34S) mutation (SEQ ID NO: 40) remained aggregation-free after four weeks.
TABLE 3
The influence of mutating HSA C34 on fusion protein aggregation
% aggregation % aggregation
HSA when 2 weeks post
SEQ ID NO mutation purified purification
40 C34S 0 0
48 C34A 0 33.29
Example 4: Protease Cleavage Propensity on GDF15 It was observed by the inventors that the arginine residue at amino acid position 198 of GDF15 (R198) is susceptible to protease degradation within the HSA-GDF15 fusion molecules. Such degradation results in a heterogeneous population and is undesirable for therapeutic compositions. The cleavage can be prevented by a protease inhibitor cocktail. Purification methods were investigated for the removal of the protease. Table 4 lists the two types of HSA affinity columns that were tested for purification of HSA-GDF15 fusion proteins, as measured by HPLC. At the time of purification, the HSA-GDF15 fusion proteins purified by both methods were 100% pure and intact. At low concentrations (2-5 mg/ml), proteins purified by both methods remained intact for the entire test period of 4 weeks. However, at high concentrations (40-50 mg/ml), only the antibody-based HSA resin (CaptureSelect) produced protease-free proteins that remain intact for the entire 4 week test period. The HSA-ligand-based resin (Albupure) generated proteins that were intact initially but demonstrated degradation over time when stored at high concentrations. Adding a protease inhibitor cocktail (PI) and EDTA completely arrested the degradation of the high concentration HSA-GDF15 fusion protein batch purified using the Albupure resin. Thus, the purification method plays a critical role in generating a stable therapeutic composition. Corresponding degradation was not observed in vivo or ex vivo, suggesting that once the therapeutic composition has been made protease-free, degradation of the fusion proteins is not an issue in vivo. Therefore, purification methods that can effectively remove potential proteases during production, such as those using the CaptureSelect resin, are key to successfully manufacturing GDF15 therapeutics that are homogenous, intact and stable.
TABLE 4
Protease cleavage of the HSA-GDF15 fusion proteins can be eliminated
by sample purification methods
% degraded population of HSA-
Condition GDF15 (SEQ ID 60)
+PI/ WEEK WEEK WEEK WEEK
Concentration Resin EDTA 0 1 2 3 WEEK 4
Low Capture No 0 0 0 0 0
Select
Low Albupure No 0 0 0 0 0
High Capture No 0 0 0 0 0
Select
High Albupure No 0 3.04 9.47 11.57 13.89
High Capture Yes 0 0 0 0 0
Select
High Albupure Yes 0 0 0 0 0
Example 5: N-Terminal Deletion Variants of GDF15 The GDF15 crystal structure depicted in FIGS. 1A and 1B predicts that the N- terminus of GDF15 involved in the deletion variants is not critical for dimer formation and overall protein folding. It also predicts that such N-terminal deletions should not affect any potential receptor interaction. HSA-GDF15 fusion proteins comprising various deletions of the N terminal of GDF15 were tested for in vivo activity.
GDF15 N-terminal deletion variants were designed that removed the protease cleavage site at GDF15 (R198). Immediately following the R198 residue, there is a potential deamidation site at residues N199-G200, and substrate deamidation is also not favored in therapeutic compositions. GDF15 N-terminal deletions can remove both the proteolytic cleavage site and the deamidation sites simultaneously. The resulting GDF15 deletion variants that were incorporated into fusion proteins with HSA included GDF15 (201-308; SEQ ID NO: 8), GDF15 (202-308; SEQ ID NO: 9), and GDF15 (211-308; SEQ ID NO: 11). In vivo studies in mice showed that the N-terminal deletion variants of GDF15 are still active in reducing food intake (FIG. 17). The experimental results confirmed that such GDF15 N-terminal deletion variants express properly, form appropriate dimers, and are active in vivo.
Example 6: Inactive Mutants of GDF15 Table 5 lists twelve mutants of GDF15 that were made to eliminate GDF15 in vivo activity and identify the functional epitope of GDF15. The mutants include five single mutants, two double mutants, and five triple mutants. HSA-GDF15 fusion proteins comprising these mutations were characterized for their biophysical properties and activities (Table 5). Out of the 12 mutants, one did not express and four formed aggregates over time, indicating that the mutations interrupt protein folding and biophysical properties. Of the remaining seven mutants, four of them contained a single mutation of GDF15, and these mutants were tested in mice for food intake reduction compared to wild type. Three of the single mutants (I89R, I89W and W32A) lost in vivo activity, while the remaining mutant (Q60W) is as active as the wild type. These results indicated that the I89R, I89W or W32A mutation interrupts the interaction of the receptor/co-receptor with GDF15, suggesting that the functional epitopes of GDF15 are around residues I89 and W32. The numbering of the mutation is based on the mature GDF15 present in fusion protein, e.g., “1” refers to the 1st amino acid of the mature GDF15 (SEQ ID NO: 6) and “89” refers to the 89th amino acid of the mature GDF15 protein.
TABLE 5
Summary of the biophysical properties and activities of fusion proteins
comprising GDF15 mutants
SEQ ID Mutations
NO in GDF15 Biophysical properties Activities
5 Wild type Expresses well, stable Wild type
activity
64 I89R Expresses well, stable Complete loss of
activity
65 I89W Expresses well, stable Complete loss of
activity
66 L34A, S35A, R37A Expresses well, stable
67 V87A, I89A, L98A Expresses well, unstable
68 L34A, S35A, I89A Expresses well, stable
69 V87A, I89A Expresses well, stable
70 Q60W Expresses well, stable As active as
wild type
71 W32A Expresses well, stable Complete loss of
activity
72 W29A Expresses well, unstable
73 Q60A, S64A, R67A Expresses well, unstable
74 W29A, Q60A, I61A Does not express
75 W29A, W32A Expresses well, unstable
*6xHis tag was attached at the N-terminus for purification purpose
Example 7: Expression and Purification Methods Expression
For expression of 20 ml and greater, the expression was done using HEK Expi293™ cells grown in Expi293™ Expression media. The cells were grown at 37° C. while shaking at 125 RPM with 8% CO2. The cells were transfected at 2.5×106 cells per ml using the Expi293™ Expression Kit. For each liter of cells transfected, 1 mg of total DNA was diluted in 25 ml of Opti-MEM, and 2.6 ml of Expi293™ reagent was diluted in 25 ml of Opti-MEM and incubated for 5 minutes at room temperature. The diluted DNA and diluted Expi293 reagent were combined and incubated for 20 minutes at room temperature. The DNA complex was then added to the cells. The cells were placed in the shaking incubator overnight. The day after transfection, 5 ml of Enhancer 1 from the kit was diluted into 50 ml of Enhancer 2 from the kit, and the total volume of the two Enhancers was added to the cells. The transfected cells were placed back into the incubator for 4 days until they were harvested. The cells were concentrated by centrifugation at 6,000 g for 30 minutes and then filtered with a 0.2 μm filter before the purification step.
The expression was also done in CHO cells. The plasmid was purified and characterized. Prior to transfection, 1 aliquot of 200 μg of plasmid DNA containing the coding region of HSA-GDF15 was linearized by restriction enzyme digestion with Acl I. The digestion with this restriction endonuclease ensures the removal of the ampicillin resistance gene. Two linearized 15 μg DNA aliquots were transfected into two 1×107 CHO cells (designated transfection pool A and B) using the BTX ECM 830 Electro Cell Manipulator (Harvard Apparatus, Holliston, MA). Cells were electroporated 3 times at 250 volts with 15 millisecond pulse lengths and 5 second pulse intervals in a 4 mm gap cuvette. Transfected cells were transferred to MACH-1+L-glutamine in a shake flask and incubated for 1 day. Transfection pool A and transfection pool B were centrifuged, resuspended in MACH-1+MSX, and transferred to shake flasks to incubate for 6 days. Transfected HSA-protein fusion-producing cells from transfection pool A and transfection pool B were pooled and plated in methylcellulose on day 8 post-electroporation.
Purification
Two-step purification using CaptureSelect resin and size exclusion chromatography was used. Cell supernatants from transiently transfected Expi293™ cells were loaded onto a pre-equilibrated (PBS, pH 7.2) HSA CaptureSelect column (CaptureSelect Human Albumin Affinity Matrix from ThermoFisher Scientific) at an approximate capacity of 10 mg protein per ml of resin. After loading, unbound proteins were removed by washing the column with 10 column volumes (CV) of PBS pH7.2. The HSA-GDF15 that was bound to the column was eluted with 10 CV of 2 M MgCl2 in 20 mM Tris, pH 7.0. Peak fractions were pooled, filtered (0.2 μ), and dialyzed against PBS pH 7.2 at 4° C. After dialysis, the protein was filtered (0.2 μagain and concentrated to an appropriate volume before loading onto a 26/60 superdex 200 column (GE Healthcare). Protein fractions that eluted from the size exclusion chromatography (SEC) column with high purity (determined by SDS-PAGE) were pooled. The concentration of protein was determined by the absorbance at 280 nm on a BioTek Synergy HTTM spectrophotometer. The quality of the purified proteins was assessed by SDS-PAGE and analytical size exclusion HPLC (SE-HPLC, Dionex HPLC system). Endotoxin levels were measured using a LAL assay (Pyrotell®-T, Associates of Cape Cod).
Two-step purification using Albupure resin and SEC was also used. HSA-GDF15 fusion proteins were purified at room temperature using AlbuPure resin (ProMetic BioSciences Ltd) which utilizes an immobilized synthetic triazine ligand to selectively bind HSA. The expression supernatants were applied to the AlbuPure resin. The resin was then washed, first with 4 CV PBS pH 7.2 followed by 4 CV of 50 mM Tris pH 8.0, 150 mM NaCl buffer. The HSA-GDF15 that was bound to the column was eluted with 4 CV of PBS pH 7.2 buffer containing 100 mM Na Octanoate. The protein-containing fractions were concentrated to a 10 mL volume using a 30,000 kDa molecular weight cutoff spin concentrator (Amicon) and then applied to a 26/60 Superdex S200 pg column (GE) that was equilibrated in PBS pH 7.2 buffer. SEC fractions containing HSA-GDF15 homodimer were identified via SDS-PAGE and pooled for analysis. The protein purities were assessed by SDS-PAGE and SE-HPLC.
The Examples 8-14, and 19 involve characterization of an exemplary fusion protein of the invention, which has the amino acid sequence of SEQ ID NO: 60. This fusion protein is a fully recombinant protein that exists as a homodimer of a fusion of HSA with the mature human GDF15 through a 42-amino acid linker consisting of glycine and serine residues, GS-(GGGGS)8 (SEQ ID NO: 12). The predicted molecular weight of this fusion protein is 162,696 Daltons, and the single native free cysteine at position 34 of HSA has been mutated to serine. This particular HSA-GDF15 fusion protein will be referred to simply as “FP1” in the following examples, for simplicity. A 6xHis-tagged variant of FP1 (6xHis-FP1, SEQ ID NO: 26), containing an AS-(GGGGS)x 8-GT (SEQ ID NO: 141) linker, was used for comparison in some of the following examples.
Example 8: Effects of FP1 on the Food Intake of C57B1/6 Mice The purpose of this experiment was to demonstrate the dose-responsive effect of FP1 on the inhibition of food intake in C57B1/6 mice.
Male C57B1/6 mice were acclimated for a minimum of 72 hours in BioDAQ cages. Mice were then grouped based on food intake in the previous 24 hours into six groups of eight. Between 4:00 and 5:00 pm, animals were weighed and dosed with vehicle or a composition comprising FP1 via subcutaneous injection. The change in food weight for each cage was recorded continuously by the BioDAQ system for a period of 48 hours after the injections. 6xHis-FP1 was used for comparison in this study.
The results (FIG. 2 and Table 6) were expressed as an average of cumulative food intake for a given time interval. The results indicated that subcutaneous administration of FP1 to C57BL/6 mice significantly inhibited food intake relative to vehicle-treated animals at all doses and time points tested. 6xHis-FP1 reduced food intake at the 8 nmol/kg dose.
TABLE 6
Effects of subcutaneous administration of FP1 on food intake in
C57BL/6 mice; Cumulative food intake at 12, 24 and 48 hours
post administration is shown
Cumulative Food Intake (g)
Treatment 12 hours 24 hours 48 hours
PBS 3.7 ± 0.2 4.3 ± 0.1 8.4 ± 0.3
FP1, 1 nmol/kg 2.9 ± 0.1* 3.4 ± 0.2** 7.0 ± 0.3**
FP1, 4 nmol/kg 2.3 ± 0.2**** 3.1 ± 0.1**** 6.6 ± 0.3***
FP1, 8 nmol/kg 2.1 ± 0.2**** 3.2 ± 0.1*** 6.4 ± 0.2****
FP1, 16 nmol/kg 1.7 ± 0.2**** 2.7 ± 0.2**** 6.2 ± 0.3****
6xHis-FP1, 8 nmol/kg 1.8 ± 0.2**** 2.6 ± 0.2**** 6.0 ± 0.2****
Data are expressed as Mean ± SEM.
*p ≤ 0.05, versus PBS; **p ≤ 0.01, versus PBS; ***p ≤ 0.001, versus PBS; ****p ≤ 0.0001, versus PBS
One-Way ANOVA-Tukey's multiple comparisons test; n = 8/group
Example 9: Effects of FP1 on Food Intake in Sprague Dawley Rats The purpose of this experiment was to demonstrate the dose-responsive effect of FP1 on the inhibition of food intake in Sprague Dawley rats.
Male Sprague-Dawley rats were acclimated for a minimum of 72 hours in the BioDAQ cages. Rats were then grouped based on food intake in the previous 24 hours into six groups of eight. Between 4:00 and 5:00 pm, animals were weighed and dosed with vehicle or a composition comprising the fusion protein via subcutaneous injection. The change in food weight for each cage was recorded continuously by the BioDAQ system, for a period of 48 hours after the injections. 6xHis-FP1 was used for comparison in this study.
The results are shown in FIG. 3 and Table 7. Subcutaneous administration of FP1 inhibited food intake at doses of 2.5 nmol/kg and 10 nmol/kg compared to vehicle-treated animals. The inhibition reached statistical significance only with the highest dose tested (10 nmol/kg) at 24 and 48 hours post-administration. 6xHis-FP1 reduced food intake at the 8 nmol/kg dose, and the effect was significant at 24 and 48 hours.
TABLE 7
Effects of subcutaneous administration of FP1 on food intake in
Sprague-Dawley rats; cumulative food intake at 12, 24 and 48
hours post administration is shown
Cumulative Food Intake (g)
Treatment 12 hours 24 hours 48 hours
PBS 20.6 ± 1.3 25.3 ± 1.3 49.1 ± 2.1
FP1, 0.1 nmol/kg 23.3 ± 1.4 26.9 ± 0.8 52.8 ± 1.3
FP1, 0.5 nmol/kg 22.6 ± 1.7 25.1 ± 1.0 48.3 ± 1.8
FP1, 2.5 nmol/kg 20.0 ± 1.4 22.0 ± 1.0 44.6 ± 1.4
FP1, 10 nmol/kg 18.7 ± 0.9 19.9 ± 1.0* 39.9 ± 2.3*
6xHis-FP1, 8 nmol/kg 17.0 ± 1.5 18.8 ± 1.4** 38.4 ± 2.5**
Data are expressed as Mean ± SEM.
*p ≤ 0.05, versus PBS; **p ≤ 0.01, versus PBS
One-Way ANOVA-Tukey's multiple comparisons test; n = 8/group
Example 10: Effects of FP1 on Glucose Homeostasis and Body Weight in Diet-Induced Obese (DIO) Mice The purpose of this experiment was to evaluate the effects of FP1 on food intake, body weight, and glucose homeostasis throughout two weeks of treatment in DIO C57B1/6 mice.
Male DIO mice were weighed, and FP1 was dosed subcutaneously at 2 mL/kg every three days (q3d) at Day 0, 3, 6, 9, and 12. The vehicle and rosiglitazone treatment groups were dosed with PBS on a similar regimen. The control rosiglitazone was provided in the diet at 0.015% ad libitum. Mouse and food weights were recorded daily. Glucose was measured using a glucometer (One Touch®Ultra®, Lifescan, Milpitas, Calif.). Fat and lean mass was quantitated in conscious mice by time-domain NMR (TD-NMR) using the Bruker Mini-Spec LF110. For an oral glucose tolerance test (OGTT), mice were fasted for 4 hours. Blood glucose was measured via tail snip at 0, 30, 60, 90, and 120 minutes post oral gavage administration of 2 g/kg glucose at 10 mL/kg. Insulin was measured at 0, 30, and 90 minutes post glucose administration.
At the end of the study, the mice were euthanized via CO2 inhalation, and a terminal blood sample was collected. Serum was placed into a 96 well plate on wet ice and then stored at −80° C. The liver was removed, and the fat content relative to the total mass of liver sections was assessed using TD-NMR with the Bruker Mini Spec mq60 according to the manufacturer's instructions.
The fasted homeostatic model assessment of insulin resistance (HOMA-IR) was calculated based on the product of fasted glucose (in mg/dL) and insulin (in mU/L) divided by a factor of 405.
Treatment of DIO mice with FP1 q3d at 1 nmol/kg and 10 nmol/kg reduced body weight (Table 8) and food intake (Table 9). The reductions reached statistical significance only at certain time points, as described below.
FP1 decreased body weight at doses of 1 (from day 2 to 14) and 10 nmol/kg (from day 1 to 14) in DIO mice (Table 8 and FIG. 4). A significant reduction in food intake was seen at days 1 and 2 of the study at the dose of 1 nmol/kg and at days 1, 8 and 9 at the 10 nmol/kg dose (Table 9).
TABLE 8
Body weight change (% of starting) during treatment with FP1
in DIO mice
Treat-
ment Vehicle FP1 (nmol/kg) Rosiglitazone
Day n/a 0.1 1 10 10 mpk/day
−2 0.1 ± 0.2 −0.1 ± 0.4 0.6 ± 0.4 0.2 ± 0.3 0.1 ± 0.2
−1 −0.8 ± 0.3 −0.2 ± 0.3 0.4 ± 0.3 −0.3 ± 0.4 −0.9 ± 0.2
0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
1 0.1 ± 0.2 −0.1 ± 0.4 −1.5 ± 0.5 −2.8 ± 0.4* 1.9 ± 0.2
2 0.0 ± 0.3 −0.8 ± 0.5 −3.2 ± 0.4* −3.1 ± 0.5* 2.2 ± 0.5
3 −0.2 ± 0.4 −0.4 ± 0.4 −3.2 ± 0.6* −4.0 ± 0.7* 2.6 ± 0.5
4 −0.4 ± 0.5 −0.7 ± 0.5 −3.8 ± 0.6* −4.4 ± 0.8* 3.0 ± 0.5*
5 −0.5 ± 0.4 −1.0 ± 0.3 −4.0 ± 0.5* −4.6 ± 0.9* 3.8 ± 0.6*
6 −0.4 ± 0.6 −0.5 ± 0.5 −3.8 ± 0.4* −5.6 ± 0.9* 4.0 ± 0.7*
7 −0.3 ± 0.5 −0.5 ± 0.5 −4.3 ± 0.6* −6.1 ± 0.9* 5.5 ± 0.8*
8 −0.6 ± 0.5 −0.5 ± 0.5 −4.2 ± 0.6* −6.7 ± 1.1* 6.1 ± 0.9*
9 −0.4 ± 0.5 −0.2 ± 0.5 −4.5 ± 0.6* −7.2 ± 1.2* 7.0 ± 1.0*
10 −0.3 ± 0.5 0.3 ± 0.5 −4.4 ± 0.8* −7.8 ± 1.2* 7.8 ± 1.1*
11 −0.5 ± 0.5 0.5 ± 0.4 −4.4 ± 0.8* −7.9 ± 1.4* 8.2 ± 1.2*
12 −0.8 ± 0.6 0.8 ± 0.5 −4.3 ± 1.0* −7.9 ± 1.5* 8.5 ± 1.2*
13 −0.8 ± 0.6 0.5 ± 0.5 −3.9 ± 0.9* −8.7 ± 1.6* 8.6 ± 1.3*
14 −1.7 ± 0.6 −0.4 ± 0.5 −4.6 ± 1.0* −9.0 ± 1.7* 7.6 ± 1.3*
Data are expressed as Mean ± SEM. n = 8 per group.
*= p < 0.05, compared to that of the vehicle treated group.
TABLE 9
Daily food intake (gm) during treatment with FP1 in DIO mice
Treatment
Vehicle FP1 (nmol/kg) Rosiglitazone
Day n/a 0.1 1 10 10 mpk/day
−2 2.9 ± 0.1 3.0 ± 0.2 2.9 ± 0.1 2.8 ± 0.1 2.8 ± 0.1
0 3.0 ± 0.1 3.0 ± 0.1 2.8 ± 0.1 2.8 ± 0.1 3.0 ± 0.1
1 2.9 ± 0.1 3.1 ± 0.1 2.1 ± 0.2* 1.5 ± 0.1* 3.6 ± 0.1*
2 3.0 ± 0.1 3.0 ± 0.1 2.3 ± 0.1* 2.6 ± 0.2 3.9 ± 0.2*
3 2.8 ± 0.1 3.0 ± 0.1 2.6 ± 0.1 2.4 ± 0.2 3.5 ± 0.1*
4 2.3 ± 0.1 2.6 ± 0.1 2.2 ± 0.1 2.3 ± 0.1 3.3 ± 0.1*
5 2.8 ± 0.1 3.0 ± 0.1 2.8 ± 0.1 2.6 ± 0.1 3.7 ± 0.2*
6 2.8 ± 0.1 3.1 ± 0.1 2.9 ± 0.1 2.4 ± 0.2 3.7 ± 0.2*
7 2.7 ± 0.1 2.9 ± 0.1 2.6 ± 0.1 2.4 ± 0.1 3.8 ± 0.2*
8 2.7 ± 0.1 2.9 ± 0.1 2.6 ± 0.1 2.1 ± 0.1* 3.5 ± 0.2*
9 3.0 ± 0.1 3.3 ± 0.1 2.8 ± 0.1 2.5 ± 0.2* 4.3 ± 0.2*
10 2.6 ± 0.1 3.0 ± 0.1 2.6 ± 0.1 2.2 ± 0.1 3.6 ± 0.1*
11 2.9 ± 0.1 3.1 ± 0.1 2.7 ± 0.2 2.6 ± 0.2 3.4 ± 0.1*
12 2.7 ± 0.1 3.1 ± 0.1 3.0 ± 0.1 3.0 ± 0.2 3.6 ± 0.2*
13 2.6 ± 0.1 2.8 ± 0.1 2.8 ± 0.1 2.2 ± 0.2 3.2 ± 0.1*
Data are expressed as Mean ± SEM.
n = 8 per group.
*= p < 0.05, compared to that of the vehicle treated group.
In an OGTT performed on day 14 of the study, FP1 significantly lowered glucose levels compared to vehicle-treated animals at all time points after time 0 at all three doses tested (Table 10). This was further quantitated as total area under the curve (AUC) and delta AUC, which were significantly lower compared to vehicle for all three doses tested (Table 10 and FIGS. 5A and 5B).
TABLE 10
Blood glucose (mg/dL) levels during an OGTT after fourteen days of
q3d dosing of FP1 in DIO mice
Time after Glucose Challenge Total AUC Delta AUC
Dose (min) (mg/dL/120 (mg/dL/120
Treatment (nmol/kg) 0 30 60 90 120 min) min)
Vehicle NA 209.6 ± 399.4 ± 338.5 ± 297.5 ± 269.3 ± 38244.4 ± 13089.4 ±
16.5 46.4 46.3 38.0 36.6 4308.2 3573.0
FP1 0.1 196.6 ± 319.0 ± 241.6 ± 196.0 ± 184.0 ± 28408.1 ± 4885.5 ±
7.6 21.5 14.4 12.8 6.3* 1236.1* 955.1*
1 164.0 ± 251.4 ± 227.6 ± 191.9 ± 180.6 ± 25295.6 ± 5615.6 ±
7.2 13.0 12.6 9.4* 13.1 1026.7* 647.6*
10 146.6 ± 195.6 ± 197.1 ± 172.1 ± 174.9 ± 21768.8 ± 4211.4 ±
5.9 14.5 6.9* 6.3* 13.8 603.0* 425.8*
Rosi- 10 130.8 ± 199.1 ± 204.4 ± 184.1 ± 169.1 ± 22115.6 ± 6425.6 ±
glitazone mpk/day 6.0 12.9 8.5* 10.9 5.7* 671.9* 599.6
Data are expressed as Mean ± SEM. n = 8 per group.
*= p < 0.05, compared to that of the vehicle treated group.
Fed blood glucose levels were measured at the start (day 0), at day 7 and at day 13 of the study (Table 11 and FIG. 6). FP1 decreased blood glucose in a statistically significant manner at doses of 1 nmol/kg and 10 nmol/kg on day 13 of the study.
TABLE 11
Fed blood glucose during treatment of DIO mice
with q3d treatment of FP1
Dose Time after start of treatment (days)
Treatment (nmol/kg) 0 7 13
Vehicle NA 177.6 ± 10.9 173.6 ± 10.6 225.3 ± 23.0
FP1 0.1 174.1 ± 8.5 171.8 ± 16.3 196.4 ± 8.1
1 167.5 ± 7.8 135.1 ± 11.2 165.3 ± 10.3*
10 165.3 ± 13.7 145.3 ± 4.9 153.8 ± 7.5*
Rosiglitazone 10 mpk/day 189.6 ± 17.3 122.6 ± 8.1* 154.8 ± 6.5*
Data are expressed as Mean ± SEM.
n = 8 per group.
*= p < 0.05, compared to that of the vehicle treated group.
Plasma insulin levels during the OGTT were significantly higher for FP1 than for the corresponding vehicle group for a 0.1 nmol/kg dose at 30 minutes, and lower at the 1 and 10 nmol/kg doses at the same time point (Table 12). The insulin excursion during the OGTT, as measured by total AUC, was higher than the vehicle group for the 0.1 nmol/kg dose of FP1 (Table 12), and lower at the 1 and 10 nmol/kg dose. In both cases, statistical significance was reached only at the lowest dose. At the 90 minute time point, mice treated with 1 and 10 nmol/kg of FP1 had lower insulin levels; however, this effect did not achieve statistical significance. HOMA-IR, used as a measure of insulin sensitivity, was measured on day 14 of the study. At this time point, FP1 decreased HOMA-IR, or improved insulin sensitivity, at 10 nmol/kg (Table 13 and FIG. 7).
TABLE 12
Plasma insulin (pg/mL) levels during an OGTT after fourteen days of
q3d dosing of FP1 in DIO mice
Dose Time after Glucose Challenge (min) Total AUC
Treatment (nmol/kg) 0 30 90 (pg/mL/90 min)
Vehicle NA 6096.0 ± 774.3 14660.0 ± 3031.2 5034.4 ± 405.1 902151.9 ± 143123.2
FP1 0.1 7861.0 ± 779.5 33825.8 ± 7902.0* 6494.4 ± 797.0 1834808.8 ± 381276.1*
1 4808.1 ± 795.8 13061.8 ± 2226.3 3443.5 ± 342.5 763218.1 ± 119766.8
10 3478.3 ± 634.6 7147.0 ± 823.8 2958.0 ± 414.0 462528.8 ± 44653.1
Rosiglitazone 10 mpk/day 2965.6 ± 524.2 2203.1 ± 193.5* 1180.0 ± 57.1 179025.0 ± 9970.7
Time after Glucose Challenge (min)
Data are expressed as Mean ± SEM.
n = 8 per group.
*= p < 0.05, compared to that of the vehicle treated group.
TABLE 13
Fasted HOMA-IR in DIO mice after fourteen
days of q3d treatment of FP1
Treatment Dose (nmol/kg) HOMA-IR
Vehicle NA 93.5 ± 15.3
FP1 0.1 112.4 ± 14.6
1 56.7 ± 9.7
10 37.7 ± 8.2*
Rosiglitazone 10 mpk/day 27.3 ± 4.7*
Data are expressed as Mean ± SEM.
n = 8 per group.
*= p < 0.05, compared to that of the vehicle treated group.
The magnitude of weight loss achieved by day 13 did not result in measurable changes in absolute fat mass or percent fat mass at any dose (Table 14). At the 10 nmol/kg dose, there was a significant decrease in absolute lean mass. This decrease was not observed when expressed as percent lean mass. Liver weights were measured during terminal necropsy on day 15 of the study (Table 15). FP1 decreased absolute liver weight and liver weight as a percentage of body weight at the 10 nmol/kg dose. A decrease was observed at the 1 nmol/kg dose, but this did not reach statistical significance for either parameter. Liver fat was measured on a biopsy by NMR (Table 16). FP1 fusion protein decreased hepatic fat content, expressed as a percentage of liver biopsy weight, at 1 and 10 nmol/kg doses. The reduction was significant at the higher dose.
TABLE 14
Body composition after thirteen days of treatment
with FP1 q3d in DIO mice
Fat Mass Lean Mass
Dose Fat Mass Lean Mass (% of (% of
Treatment (nmol/kg) (g) (g) body) body)
Vehicle NA 11.6 ± 0.5 29.6 ± 0.4 23.4 ± 0.8 59.6 ± 0.9
FP1 0.1 12.2 ± 0.4 29.7 ± 0.4 24.1 ± 0.6 58.5 ± 0.6
1 12.1 ± 0.3 28.0 ± 0.4 25.1 ± 0.3 58.3 ± 0.6
10 10.6 ± 0.3 27.7 ± 0.4* 23.1 ± 0.3 60.6 ± 0.6
Rosiglita- 10 mpk/day 14.9 ± 0.6* 30.0 ± 0.5 27.2 ± 0.4* 55.0 ± 0.6*
zone
Data are expressed as Mean ± SEM.
n = 8 per group.
*= p < 0.05, compared to that of the vehicle treated group.
TABLE 15
Liver weight after fifteen days of treatment with FP1 q3d in DIO mice
Liver Weight
Treatment Dose (nmol/kg) Liver weight (g) (% of body)
Vehicle NA 2.6 ± 0.1 5.4 ± 0.2
FP1 0.1 2.9 ± 0.2 5.8 ± 0.2
1 2.2 ± 0.1 4.6 ± 0.2
10 1.9 ± 0.1* 4.2 ± 0.1*
Rosiglitazone 10 mpk/day 2.5 ± 0.2 4.6 ± 0.2
Data are expressed as Mean ± SEM.
n = 8 per group.
*= p < 0.05, compared to that of the vehicle treated group.
TABLE 16
Liver fat content measured after fifteen days of
treatment with FP1 q3d in DIO mice
Treatment Dose (nmol/kg) Fat (%)
Vehicle NA 27.3 ± 2.1
FP1 0.1 26.0 ± 1.4
1 22.5 ± 1.4
10 17.8 ± 1.6*
Rosiglitazone 10 mpk/day 25.9 ± 0.8
Data are expressed as Mean ± SEM.
n = 8 per group.
*= p < 0.05, compared to that of the vehicle treated group.
Example 11: Effects of FP1 on Blood Glucose Levels and Body Weight in ob/ob Mice The purpose of this experiment was to evaluate the effects of FP1 on body weight and blood glucose levels over eight days of treatment in obese, hyperglycemic, leptin-deficient ob/ob mice.
Male ob/ob mice were weighed and FP1 was administered subcutaneously at 2mL/kg every three days (q3d) at Day 0, 3 and 6. Mouse and food weights were recorded daily. Glucose was measured daily using a glucometer. At the end of the study, mice were euthanized, and a terminal blood sample was collected.
FP1, at the 1 nmol/kg dose, significantly decreased body weight (expressed as a percentage of starting body weight) in ob/ob mice starting at day 2 until day 8, relative to vehicle-treated mice. FP1, at the 10 nmol/kg dose, decreased body weight (expressed as a percentage of starting body weight) in ob/ob mice starting at day 1 until day 8 relative to vehicle-treated mice (Table 17 and FIG. 8).
TABLE 17
Body weight change (% of starting) during treatment
with FP1 q3d in ob/ob mice
Treatment
Vehicle FP1
Day n/a 1 nmol/kg 10 nmol/kg
−6 −2.5 ± 0.3 −2.8 ± 0.4 −3.6 ± 0.5
−5 −2.4 ± 0.3 −3.0 ± 0.5 −3.5 ± 0.5
−4 −1.9 ± 0.2 −2.5 ± 0.4 −2.8 ± 0.3
−1 −0.3 ± 0.3 0.1 ± 0.2 −0.3 ± 0.2
0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
1 0.5 ± 0.2 −1.6 ± 0.2 −2.3 ± 0.5*
2 1.1 ± 0.2 −1.8 ± 0.4* −2.0 ± 0.9*
3 1.3 ± 0.3 −2.5 ± 0.4* −3.1 ± 1.1*
4 1.8 ± 0.3 −3.3 ± 0.5* −4.2 ± 1.3*
5 1.8 ± 0.4 −3.5 ± 0.5* −4.7 ± 1.5*
6 2.0 ± 0.5 −4.0 ± 0.7* −5.7 ± 1.6*
7 2.8 ± 0.6 −4.8 ± 0.8* −6.3 ± 1.7*
8 3.5 ± 0.8 −4.5 ± 1.0* −6.1 ± 1.9*
Data are expressed as Mean ± SEM.
n = 8 per group.
*= p < 0.05, compared to that of the vehicle treated group.
FP1, at the 10 nmol/kg dose, decreased fed blood glucose values in ob/ob mice on study day 1 and 2 and from day 4 until day 8 relative to vehicle-treated mice. A reduction in in blood glucose was observed at 1 nmol/kg; however, this effect did not reach statistical significance (Table 18 and FIG. 9).
TABLE 18
Fed blood glucose during treatment of ob/ob mice with FP1 q3d
Treatment
Vehicle FP1
Day n/a 1 nmol/kg 10 nmol/kg
−6 463.6 ± 31.2 395.7 ± 52.0 463.6 ± 41.0
−5 554.9 ± 57.5 609.6 ± 53.6 552.9 ± 53.0
−4 502.9 ± 41.6 517.9 ± 71.6 490.3 ± 54.4
−1 552.1 ± 45.1 567.2 ± 51.2 586.0 ± 42.6
0 468.8 ± 57.3 479.7 ± 61.8 437.0 ± 42.7
1 537.0 ± 42.8 439.0 ± 80.4 336.2 ± 51.0*
2 511.7 ± 43.5 440.0 ± 74.3 293.7 ± 37.6*
3 447.8 ± 54.2 369.6 ± 75.9 279.4 ± 38.8
4 516.3 ± 47.0 384.6 ± 84.0 261.2 ± 43.7*
5 531.0 ± 47.1 410.9 ± 92.2 286.9 ± 55.8*
6 596.1 ± 45.1 451.1 ± 82.6 301.9 ± 49.3*
7 566.7 ± 44.3 425.3 ± 86.9 246.7 ± 43.5*
8 509.9 ± 37.6 337.8 ± 75.1 223.4 ± 34.1*
Data are expressed as Mean ± SEM.
n = 8 per group.
Example 12: Multispecies Pharmacokinetics Mouse Pharmacokinetics
FP1 was administered to female C57B1/6 mice at a dose of 2 mg/kg IV and SC in PBS, pH 7. Blood samples were collected, serum was processed and drug concentrations were measured up to 7 days following both routes of administration. The concentration of FP1 was determined using an immunoassay method. The serum drug concentration-time profile is summarized in Tables 19 and 20 and illustrated in FIG. 10.
TABLE 19
Serum concentration (nM) of FP1 over time following a single SC
administration in C57Bl/6 female mice
FPl-SC Dose
Animal Animal Animal Animal Animal Average
56 Result 57 Result 58 Result 60 Result 63 Result Result Std
Timepoint (nM) (nM) (nM) (nM) (nM) (nM) Dev
4 hr 32.299 50.735 42.766 32.407 23.018 36.245 10.698
24 hr 88.822 106.418 88.648 103.841 80.346 93.615 11.093
72 hr 33.563 38.473 32.473 33.625 32.769 34.181 2.451
96 hr 20.639 24.988 21.247 19.356 20.771 21.400 2.124
Day 7 5.399 6.919 7.234 5.994 5.637 6.237 0.803
TABLE 20
Serum concentration (nM) of FP1 over time following a single IV
administration in C57Bl/6 female mice
FP1-IV Dose
Animal Animal Animal Animal Animal Average
52 Result 53 Result 65 Result 66 Result 70 Result Result Std
Timepoint (nM) (nM) (nM) (nM) (nM) (nM) Dev
1 hr 240.419 233.318 232.484 276.913 272.727 251.172 21.857
24 hr 86.823 95.774 80.201 93.153 88.853 88.961 6.027
72 hr 33.634 37.447 33.108 41.680 34.034 35.981 3.612
96 hr 22.666 20.588 19.458 33.718 20.361 23.358 5.909
Day 7 7.401 5.606 4.896 8.556 4.205 6.133 1.803
Pharmacokinetic analysis revealed a terminal half-life of 1.67 and 1.57 days for FP1 in C57B1/6 mice following SC and IV administration, respectively (Table 21). FP1 demonstrated a mean bioavailability of ˜71% following SC administration.
TABLE 21
Mean (±SD) pharmacokinetic parameters of FP1 following 2 mg/kg IV
and SC administration in female C57Bl/6 mice
CL or
t1/2 CL/F Vss Cmax Tmax* AUC0-last AUC0-inf
Route (day) (ml/day/kg) (ml/kg) (ng/ml) (day) (day*ng/ml) (day*ng/ml)
SC Mean 1.67 49.48 14994 1 38315 40734
(SD) (0.14) (4.791) (1776) (3851) (4072)
IV Mean 1.57 35.00 69.03 40231 0.04 55263 57531
(SD) (0.19) (3.13) (5.8) (3500) (4853) (5416)
Note:
*Tmax (median)
Rat Pharmacokinetics
FP1 was administered to female Sprague Dawley rats at a dose of 2 mg/kg IV and SC in PBS, pH 7. Blood samples were collected, serum was processed and drug concentrations were measured up to 7 days following both routes of administration. The concentration of FP1 was determined using an immunoassay method. The serum drug concentration-time profile is summarized in Tables 22 and 23 and illustrated in FIG. 11.
TABLE 22
Serum concentration (nM) of FP1 over time following a single SC
administration in female Sprague Dawley rats.
FP1-Group 3 (SC Dose)
Animal Animal Animal Animal Animal Average
53 Result 55 Result 67 Result 68 Result 69 Result Result
Timepoint (nM) (nM) (nM) (nM) (nM) (nM) Std Dev
4 hr 4.766 3.500 3.932 3.546 3.250 3.799 0.593
24 hr 45.118 53.192 39.196 39.823 40.804 43.627 5.826
72 hr 18.900 24.102 23.124 23.718 18.933 21.755 2.615
96 hr 12.193 14.333 14.185 14.669 11.256 13.327 1.511
Day 7 2.805 2.821 2.447 3.438 2.358 2.774 0.426
TABLE 23
Serum concentration (nM) of FP1 over time following a single IV
administration in female Sprague Dawley rats
FP1-Group 4 (IV Dose)
Animal Animal Animal Animal Animal
51 52 57 64 66 Average
Result Result Result Result Result Result
Timepoint (nM) (nM) (nM) (nM) (nM) (nM) Std Dev
1 hr 43.620* 382.676 403.255 443.080 510.105 356.547 181.560
24 hr 102.665* 142.661 139.066 124.528 126.425 127.069 15.728
72 hr 46.720 60.105 67.090 59.257 70.423 60.719 9.127
96 hr 29.409 39.897 41.225 42.258 48.074 40.173 6.779
Day 7 6.976 11.251 8.913 9.540 13.006 9.937 2.298
*Repeat analysis confirmed results
Pharmacokinetic analysis revealed a terminal half-life of 1.34 and 1.51 days for FP1 in Sprague Dawley rats following SC and IV administration, respectively (Table 24). FP1 demonstrated a mean bioavailability of ˜23% following SC administration.
TABLE 24
Mean (±SD) pharmacokinetic parameters of FP1 following 2 mg/kg
IV and SC administration in Sprague Dawley rats
CL or
t1/2 CL/F Vss Cmax Tmax* AUC0-last AUC0-inf
Route (day) (ml/day/kg) (ml/kg) (ng/ml) (day) (day*ng/ml) (day*ng/ml)
SC Mean 1.34 100.09 6987 1 19250 20112
(SD) (0.04) (3.97) (417) (794) (820)
IV Mean 1.51 24.75 53.41 59000 0.04 83028 86525
(SD) (0.12) (8.53) (17.15) (25031) (20126) (20881)
Note:
*Tmax (median)
Monkey Pharmacokinetics
FP1 was administered to naive male cynomolgus monkeys (Macaca fascicularis) at a dose of 1 mg/kg IV and SC in PBS, pH 7. Blood samples were collected, serum was processed and drug concentrations were measured up to 21 days following both routes of administration, using immunoassay bioanalysis. The serum drug concentration-time profile is summarized in Tables 25 and 26 and illustrated in FIG. 12.
TABLE 25
Serum concentration (nM) of FP1 over time following a single
SC administration in cynomolgus monkeys as
determined by immunoassay
FP1 (SC Dose)
Animal Animal Animal Average Std
Timepoint 110 (nM) 111 (nM) 112 (nM) Result (nM) Dev
Predose <LLOQ <LLOQ <LLOQ <LLOQ N/A
6 hr 49.304 43.784 72.110 55.066 15.017
24 hr 93.368 71.958 96.863 87.396 13.483
48 hr 107.689 97.509 115.144 106.781 8.853
72 hr 113.601 104.190 104.449 107.414 5.360
120 hr 101.490 95.049 91.717 96.085 4.968
168 hr 82.167 75.435 81.569 79.724 3.726
240 hr 71.033 56.732 59.266 62.344 7.631
336 hr 44.380 42.758 42.571 43.236 0.995
432 hr 30.911 29.445 32.839 31.065 1.702
528 hr 21.277 20.404 26.427 22.703 3.255
TABLE 26
Serum concentration (nM) of FP1 over time following a single
IV administration in cynomolgus monkeys as
determined by immunoassay
FP1 (IV Dose)
Animal Animal Animal Average Std
Timepoint 104 (nM) 105 (nM) 106 (nM) Result (nM) Dev
Predose <LLOQ <LLOQ <LLOQ <LLOQ N/A
1 hr 212.661 235.168 189.000 212.276 23.087
6 hr 190.315 185.331 183.575 186.407 3.497
24 hr 141.743 155.943 146.487 148.058 7.229
48 hr 111.765 126.105 120.744 119.538 7.246
72 hr 105.076 106.955 106.441 106.157 0.971
120 hr 92.591 103.882 103.757 100.077 6.483
168 hr 71.368 93.055 87.706 84.043 11.298
240 hr 71.554 65.093 65.685 67.444 3.572
336 hr 46.184 38.961 41.696 42.280 3.647
432 hr 34.589 31.266 19.492 28.449 7.933
528 hr 26.885 24.154 22.422 24.487 2.250
Pharmacokinetic analysis revealed a terminal half-life between 8.5 and 9.2 days for FP1 in cynomolgus monkeys following SC and IV administration, respectively with a mean bioavailability of ˜88% following SC administration (Table 27).
TABLE 27
Mean (±SD) pharmacokinetic parameters of FP1 following 1 mg/kg IV
and SC administration in cynomolgus monkeys.
CL or
t1/2 CL/F Vss Cmax Tmax* AUC0-last AUC0-inf
Route (day) (ml/day/kg) (ml/kg) (ng/ml) (day) (day*ng/ml) (day*ng/ml)
SC Mean 8.5 3.9 17776 3 211030 256202
(SD) (1.5) (0.3) (950) (11332) (20192)
IV Mean 9.2 3.4 45.6 34001 0.04 239758 292206
(SD) (0.5) (0.1) (1.6) (3698) (6095) (11516)
Note:
*Tmax (median)
Immuno-affinity capture-LCMS analysis was used to quantitate the concentration of intact dimer present in the serum of cynomolgus monkeys after IV and SC administration (Tables 28 and 29 and FIGS. 13 and 14). Concentrations determined by this method were similar to concentrations determined by the immunoassay (IA), demonstrating that FP1 circulates as an intact dimer, with no detectable metabolic liability in cynomolgus monkeys.
TABLE 28
Serum concentration (ng/mL) of FP1 as an intact dimer over time
following a single IV administration in cynomolgus monkeys as
determined by immuno-affinity capture-LCMS analysis.
Dimer Intact MS Data
Day (Pooled Samples) IA Data (Average)
0.00 0 0
0.04 33347 34537
0.25 29686 30328
1.00 28787 24089
2.00 17249 19449
3.00 16827 17272
5.00 16159 16282
7.00 11124 13674
10.00 8746 10973
14.00 5328 6879
18.00 3857 4629
22.00 2252 3984
TABLE 29
Serum concentration (ng/mL) of FP1 as an intact dimer over time
following a single SC administration in cynomolgus monkeys as
determined by immuno-affinity capture-LCMS analysis.
Dimer Intact MS Data IA Data
Day (Pooled Samples) (Average)
0.00 0 0
0.25 9625 8959
1.00 15799 14219
2.00 17671 17373
3.00 19130 17476
5.00 12284 15633
7.00 10808 12971
10.00 8910 10143
14.00 5814 7034
18.00 4074 5054
22.00 2967 3694
The concentration of analytes in cynomolgus monkey serum after IV and SC administration was also measured by immuno-affinity capture-trypsin digestion-LC-MS/MS analysis (Tables 30 and 31). Selected tryptic peptides, namely, ALV (ALVLIAFAQYLQQSPFEDHVK) (SEQ ID NO: 135), ASL (ASLEDLGWADWVLSPR) (SEQ ID NO: 136), and TDT (TDTGVSLQTYDDLLAK) (SEQ ID NO: 137), which are located within FP1 near the N-terminus of the HSA region, the N-terminus of GDF15, and the C-terminal of GDF15, respectively. The peptides were monitored as surrogate peptides of FP1. The concentrations of all of the surrogate peptides were similar to each other and the concentrations measured by immunoassay, demonstrating that the GDF15 sequence in FP1 remains intact and linked to the full HSA sequence in vivo.
TABLE 30
Serum concentration (ng/mL) of surrogate peptides representing various
regions of FP1 over time following a single IV administration in
cynomolgus monkeys as determined by immuno-affinity capture-
trypsin digestion-LC-MS/MS analysis.
Time point FP1 Average (ng/mL) Std Dev IA Data
Day hour ALV TDT ASL ALV TDT ASL (ng/mL)
0.00 0 <LLOQ <LLOQ <LLOQ N/A N/A N/A 0.0
0.04 1 32400.0 39766.7 33333.3 2623.0 3162.8 2722.7 34536.9
0.25 6 29100.0 27166.7 30600.0 3439.5 1006.6 2095.2 30328.1
1.00 24 24366.7 23300.0 23800.0 4215.8 3996.2 4100.0 24088.7
2.00 48 19433.3 17733.3 18700.0 1457.2 1193.0 854.4 19448.6
3.00 72 18100.0 17200.0 17166.7 360.6 871.8 1001.7 17271.6
5.00 120 15966.7 14033.3 14233.3 1001.7 642.9 1011.6 16282.3
7.00 168 13733.3 11800.0 12100.0 1115.0 600.0 300.0 13673.6
10.00 240 9303.3 8570.0 8570.0 2290.1 1682.2 1685.9 10973.0
14.00 336 5860.0 5890.0 6056.7 415.8 312.2 388.0 6878.9
18.00 432 4143.3 4400.0 4226.7 374.3 52.9 571.2 4628.6
22.00 528 2830.0 3256.7 2753.3 355.4 420.0 319.0 3984.0
TABLE 31
Serum concentration (ng/mL) of surrogate peptides representing
various regions of FP1 over time following a single SC administration
in cynomolgus monkeys as determined by immuno-affinity capture-
trypsin digestion-LC-MS/MS analysis.
Time point FPI Average (ng/mL) Std Dev IA Data
Day hour ALV TDT ASL ALV TDT ASL (ng/mL)
0.00 0 <LLOQ <LLOQ <LLOQ N/A N/A N/A 0.0
0.25 6 9323.3 7430.0 8123.3 1900.3 2471.6 1954.1 8959.1
1.00 24 15233.3 13390.0 14533.3 2926.3 3222.0 2683.9 14219.2
2.00 48 15366.7 14000.0 14166.7 2579.4 1646.2 2311.6 17373.0
3.00 72 17300.0 16033.3 15333.3 1571.6 1001.7 986.6 17476.0
5.00 120 15333.3 13366.7 13666.7 1222.0 1692.1 1501.1 15632.9
7.00 168 13333.3 11633.3 11700.0 709.5 472.6 781.0 12970.9
10.00 240 8496.7 7376.7 8343.3 1475.5 189.0 1039.1 10143.2
14.00 336 6046.7 6116.7 6253.3 90.2 118.5 110.6 7034.5
18.00 432 4593.3 5250.0 4636.7 802.6 1157.5 621.7 5054.2
22.00 528 3056.7 3490.0 3116.7 424.5 687.7 220.5 3693.7
Human plasma stability assay
The purpose of this study was to analyze the ex vivo stability of FP1 in human plasma. Fresh, non-frozen human plasma was generated from heparinized blood from two subjects (one male and one female) by centrifugation. FP1 was incubated in this matrix at 37° C. with gentle mixing, for 0, 4, 24 and 48 hours. The concentration of FP1 was determined using an immunoassay method. The average percent difference from the starting concentration (0 hours) ranged from −4.1 to −12.9 and did not increase over time, demonstrating that FP1 is stable in human plasma for up to 48 hours ex vivo (Table 32 and FIG. 15).
TABLE 32
FP1 concentration (μg/mL) after 0, 4, 24, and
48 hours (hr) of ex vivo incubation in plasma obtained from two
human subjects (Sub) as determined by immunoassay
Sub 1 Sub 2
Male % Female % Average %
Conc. Diff Conc. Diff Conc. Diff
Ex Vivo Sample (μg/mL) T0 (μg/mL) T0 (μg/mL) T0
Plasma-T0 hr_FP1 11.503 N/A 12.649 N/A 12.076 N/A
Plasma-T4 hr_FP1 10.524 −8.5 10.521 −16.8 10.523 −12.9
Plasma-T24 hr_ 9.934 −13.6 12.402 −2.0 11.168 −7.5
FP1
Plasma-T48 hr_ 10.582 −8.0 12.575 −0.6 11.578 −4.1
FP1
Immuno-affinity capture-LCMS was used to quantitate the concentration of intact dimer present after incubation in human plasma. Concentrations determined by this method were stable over time (0, 4, 24, and 48 hours), demonstrating that FP1 remains an intact dimer in human plasma ex vivo up to 48 hours (Table 33 and FIG. 16).
TABLE 33
Average FP1 concentration (μg/mL) and % difference from starting
concentration as an intact dimer after 0, 4, 24, and 48 hours (hr) of
ex vivo incubation in plasma obtained from two human subjects
as determined by immuno-affinity capture-LCMS analysis
Dimer Conc. (ug/mL) % Difference
0 hr 15.8 100.0
4 hr 15.8 100.1
24 hr 15.9 100.9
48 hr 15.2 96.0
Example 13: IR Strategy Immune response (IR) assays will be developed for anti-drug antibody (ADA) detection in animals and clinical samples. The IR assay will identify ADA-positive samples for comparison of ADA status with pharmacokinetic/toxicokinetic (PK/TK) results, enabling assessment of FP1 exposure and pharmacokinetics. The clinical IR assay will be used to screen serum samples, confirm specificity of ADA-positive samples, and determine the ADA titer for confirmed positive samples. Neutralizing antibody (NAb) assay development will follow for use in confirmed positive samples from ADA-positive subjects in Phase 1 of the program. Additionally, determination of ADA cross-reactivity to endogenous GDF15 will follow for use in Phase 2 of the program. An immunogenicity risk assessment will be conducted prior to the first in human (FIH) study, and additional immune response characterization assays can be implemented if they are warranted.
Example 14: Toxicology Plan Since the endogenous target receptor for GDF15 has not been identified, there is a lack of in vitro binding and functional data for FP1. However, single and multiple dose pharmacology and efficacy studies in rats, mice and cynomolgus monkeys have demonstrated activity of FP1 in these species, showing its effect of reducing food intake, decreasing body weight and modulating oral glucose tolerance. The rat and monkey will be the rodent and non-rodent toxicology testing species, respectively, based on the efficacy results, with the understanding that the intrinsic potency of FP I on the receptor in these species (versus that of humans) has not been fully characterized.
The Examples 15-19 involve characterization of another exemplary fusion protein of the invention, described in Example 5, which has the amino acid sequence of SEQ ID NO: 92 (encoded by nucleotide sequences SEQ ID NOs: 95 (codon optimization 1) or 110 (codon optimization 2)). This fusion protein is a fully recombinant protein that exists as a homodimer of a fusion of HSA (C34S) with the deletion variant of the mature human GDF15 (201-308; SEQ ID NO: 8) through a 42-amino acid linker consisting of glycine and serine residues, GS-(GGGGS)8 (SEQ ID NO: 12). The single native free cysteine at position 34 of HSA has been mutated to serine. This particular HSA-GDF15 fusion protein will be referred to as “FP2” in the following examples, for simplicity.
Example 15: Effects of FP2 on the Food Intake of C57B1/6 Mice FP2 was evaluated for its ability to reduce food intake in male C57B1/6 mice after a single dose. Male C57B1/6N mice (age 10-12 weeks) obtained from Taconic Biosciences (Hudson, N.Y.) were used in the study. Mice were singly housed in a temperature-controlled room with 12-hour light/dark cycle (6 am/6 pm) and allowed ad libitum access to water and chow. Male C57B1/6 mice were acclimated for a minimum of 72 hours in the BioDAQ cages; mice were then grouped based on food intake in the last 24 hours into six groups of eight each. Between 4:00-5:00 pm, animals were weighed and dosed with vehicle or compounds via subcutaneous injection. Change in food weight for each cage was recorded continuously by the BioDAQ system, for a period of 48 hours after compound administration. 6xHis-FP1 was used as a comparator in this study.
FP2 had significant effects on reducing food intake at 12, 24 and 48 hours after administration at all dose levels tested (Table 34). There was a reduction in percent change in food intake relative to PBS at all time points and all dose levels (Table 35) in mice.
TABLE 34
Effect of a Single Dose of FP2 on Food Intake
over 48 hours in C57Bl/6 Mice
Cumulative Food Intake (g)
Treatment 12 hours 24 hours 48 hours
PBS 3.7 ± 0.2 4.4 ± 0.2 8.8 ± 0.4
FP2, 1 nmol/kg 2.8 ± 0.4* 3.0 ± 0.4** 6.9 ± 0.7*
FP2, 4 nmol/kg 2.4 ± 0.2*** 3.1 ± 0.2** 7.0 ± 0.3*
FP2, 8 nmol/kg 1.9 ± 0.2**** 2.4 ± 0.1**** 6.2 ± 0.2***
FP2, 16 nmol/kg 1 8 ± 0.1**** 2.7 ± 0.1*** 6.3 ± 0.4**
6xHis-FP1, 8 nmol/kg 2.3 ± 0.3*** 2.8 ± 0.4** 6.6 ± 0.7**
Data are expressed as Mean ± SEM.
*p ≤ 0.05, versus PBS
**p ≤ 0.01, versus PBS
***p ≤ 0.001, versus PBS
****p ≤ 0.0001, versus PBS, respectively
Statistical analyses used: ANOVA and Dunnett's multiple comparisons test.
n = 8/group, except for 6xHis-FP1 8 nmol/kg (n = 6).
TABLE 35
Effect of a Single Dose of FP2 on Percent Reduction in Food
Intake (Relative to Vehicle) over 48 hours in C57Bl/6 Mice
Percentage of inhibition relative to PBS
Treatment 12 hours 24 hours 48 hours
PBS 0.0 ± 7.9 0.0 ± 7.7 0.0 ± 5.9
FP2, 1 nmol/kg 22.4 ± 12.5* 31.8 ± 10.5** 21.8 ± 8.1*
FP2, 4 nmol/kg 36.6 ± 7.2*** 30.4 ± 5.8** 20.2+4.7*
FP2, 8 nmol/kg 47.0 ± 6.2**** 45.7 ± 3.8**** 29 7-3 9***
FP2, 16 nmol/kg 49 2 ± 4.1**** 38.1 ± 4.1*** 27.8 ± 5.1**
6 × His-FP1, 8 nmol/kg 36.9 ± 9.9*** 36.5 ± 8.8** 24.6 ± 8.2**
The anorectic effect of FP2 is expressed as the relative reduction in food intake compared with the respective PBS controls.
Data are expressed as Mean ± SEM.
*p ≤ 0.05, versus PBS
**p ≤ 0.01, versus PBS
***p ≤ 0.001, versus PBS
****p ≤ 0.0001, versus PBS, respectively
Statistical analyses used: ANOVA and Dunnett's multiple comparisons test.
n = 8/group, except for 6 × His-FP1 8nmol/kg (n = 6).
Example 16: Effects of FP2 on Food Intake in Sprague Dawley Rats FP2 was evaluated for its ability to reduce food intake and body weight gain in male Sprague-Dawley rats after a single dose. The animals were obtained from Charles River (Wilmington, Mass.) at 200-225 g body weight and used within one week of delivery. They were housed one per cage on alpha dry bedding and a plastic tube for enrichment in a temperature-controlled room with 12-hour light/dark cycle. They were allowed ad libitum access to water and were fed laboratory rodent diet; Irradiated Certified PicoLab® Rodent Diet 20, 5K75* (supplied from Purina Mills, St. Louis, Mo. via ASAP Quakertown, Pa.). Animal weights were taken and recorded for each rat prior to dosing.
Animals were acclimated for a minimum of 72 hours in the BioDAQ cages; rats were then grouped based on food intake in the last 24 hours into six groups of eight each. Between 4:00-5:00 pm, animals were weighed and dosed with vehicle or compounds via subcutaneous injection. Change in food weight for each cage was recorded continuously by the BioDAQ system, for a period of 48 hours after compound administration. 6XHis-FP1 was used as a comparator in this study.
Dose-dependent reductions of food intake were tested after a single dose of FP2. No significant differences in food intake were observed at the dose of 0.3 nmol/kg. Significant effects in reduction of food intake were observed 12 hours but not 24 or 48 hours at 1 nmol/kg. Significant reductions in food intake were observed at all time points for the 3 and 10 nmol/kg dose levels (Table 36, FIG. 19). There was a reduction in percent change in food intake relative to PBS at all time points and all dose levels (Table 37).
TABLE 36
Effect of a single dose of FP2 on food intake over 48 hours
in Sprague Dawley rats
Cumulative Food Intake (g)
Treatment 12 hours 24 hours 48 hours
PBS 21.7 ± 0.6 25.0 ± 0.8 53.1 ± 1.6
FP2, 0.3 nmol/Kg 19.5 ± 0.9 22.9 ± 0.7 48.4 ± 1.5
FP2, 1 nmol/Kg 17.5 ± 0.9* 19.8 ± 0.8 47.0 ± 2.6
FP2, 3 nmol/Kg 16.1 ± 1.2** 17.0 ± 1.0** 39.2 ± 3.2**
FP2, 10 nmol/Kg 15.8 ± 0.9*** 16.5 ± 0.9** 36.5 ± 3.2**
6 × His-FP1 8 nmol/Kg 15.0 ± 1.4*** 15.7 ± 1.2** 37.2 ± 4.5**
Data are expressed as Mean ± SEM.
*p ≤ 0.05, versus PBS
**p ≤ 0.01, versus PBS
***p ≤ 0.001, versus PBS, respectively
Statistical analyses used: ANOVA and Dunnett's multiple comparisons test.
n = 8/group
TABLE 37
Effect of a single dose of FP2 on percent reduction in food intake
(relative to vehicle)over 48 hours in Sprague Dawley rats.
Percentage of inhibition relative to PBS
Treatment 12 hours 24 hours 48 hours
PBS 0.0 ± 4.0 0.0 ± 4.6 0.0 ± 4.3
FP2 0.3 nmol/kg 10.0 ± 4.7 8.5 ± 4.1 8.8 ± 3.9
FP2 1 nmol/kg 19.3 ± 4.7* 20.8 ± 4.1 11.5 ± 5.5
FP2 3 nmol/kg 25.9 ± 6.1** 32.0 ± 4.7** 26.2 ± 6.5**
FP2 10 nmol/kg 27.4 ± 4.8*** 33 8 ± 4.1** 31.2 ± 6.4**
6 × His-FP1 8 nmol/kg 30.8 ± 6.6*** 37.0 ± 5.4** 29.9 ± 8.8**
The anorectic effect of FP2 is expressed as the relative reduction in food intake compared with the respective PBS controls.
Data are expressed as Mean ± SEM.
*p ≤ 0.05, versus PBS
**p ≤ 0.01, versus PBS
***p ≤ 0.001, versus PBS, versus PBS, respectively
Statistical analyses used: ANOVA and Dunnett's multiple comparisons test.
n = 8/group
Example 17: Effects of FP2 on Food Intake, Body Weight and Glucose Homeostasis in Diet-induced Obese (DIO) C57B1/6 Mice FP2 was evaluated for its ability to reduce food intake and body weight and improve glucose homeostasis on repeat dosing in male DIO C57B1/6 mice over a period of 8 days. Male DIO C57B1/6 mice (age 21 weeks, high fat-fed for 15 weeks) obtained from Taconic Biosciences (Hudson, NY) were used in the study. Mice were singly housed in a temperature-controlled room with 12-hour light/dark cycle (6 am/6 pm) and allowed ad libitum access to water and fed with Research Diet D12492 (Research Diets, New Brunswick, N.J.). Mice were acclimated >1 week in the mouse housing room prior to the experiment. The endpoints of the study were measurements of food intake, body weight, body composition and glycemic endpoints (OGTT, blood glucose). One day prior to dosing, animals were weighed and grouped by body weight (BW). Mice were dosed by subcutaneous injection. Animals dosed with FP2 received this compound on Day 0, Day 3, and Day 6, Day 9 and Day 12. The vehicle group and rosiglitazone group received sterile PBS s.c. on these days as well. Rosliglitazone was provided in the diet at 0.015% w/w ad libitum. BW and food intake were recorded daily, over a period of fifteen days. Blood glucose was measured on Days 0, 7 and 13. An oral glucose tolerance test (OGTT) was performed on Day 14. Insulin levels were measured at selected time points during the OGTT. Mice were euthanized with CO2 and terminal blood samples were collected for exposure via cardiac puncture on day 15. A separate PK arm was run with three mice per dose group with a total of 15 mice.
Exposure-response (E-R) Analysis for FP2 in DIO Mouse
Most animals in the pharmacodynamics (PD) (efficacy) arms had undetectable drug concentrations on the last study day when the pharmacokinetics (PK) samples were obtained, potentially due to immunogenicity. Therefore, the mean PK profiles from the PK arms, instead of individual PK from the PD arms, were used to conduct exposure-response (from day 3, 6 and 9, respectively) for the % weight change from baseline in the PD arms at the corresponding dose level. This method assumes that the PK arms behave similarly to the PD arms in terms of drug exposure.
The Emax model (GraphPad Prism 6, log(agonist) vs. response) was used to correlate exposure with response data (log transformed drug concentrations). Hill Slope was set to be 1. Note that the model fitted EC10 to EC50 values were within two fold amongst day 3, 6 and 9, despite that the Emax estimates were different (Emax=−4.26%, −8.18% and −9.85%, respectively). Some animals on day 9 also showed the loss of drug exposure, due to potential ADA formation and therefore, the E-R parameter estimates based on day 9 data should be interpreted with caution.
The effects of two weeks of exposure of FP2 on food intake, body weight, glucose homeostasis, and liver fat content was assessed in diet induced obese male C57B1/6 mice. Trough exposure between 1.7 and 3.3 nM FP2 for the 0.3 nmol/kg treatment group, between 7.1 and 14 nM for the 1.0 nmol/kg treatment group, between 20.8 and 41.6 nM for the 3.0 nmol/kg treatment group, and between 28.5 and 112.9 nM FP2 for the 10 nmol/kg treatment group was maintained until day 9 in the PK arm of the study (n=2 or 3, Table 49). After day 9, a decrease in circulating levels was observed in the majority of animals despite continued q3d dosing (Table 49). Consistent with this accelerated clearance, the majority of animals in the PD arm of the study had undetectable circulating levels of FP2 on day 15 (Table 50).
Treatment of DIO mice with FP2 q3d reduced food intake (Table 38), body weight (Table 39, 40 and FIG. 20) and fed blood glucose compared to vehicle treatment (Table 43 and FIG. 23). A significant reduction in food intake was seen on day 2, day 5, and day 8 for 0.3 nmol/kg, from day 1 through day 7 for 1.0 nmol/kg, on day 1, day 2, day 4 through day 6, and day 8 for 3.0 nmol/kg, and on day 1, day 3 through day 6, day 8 and day 9 for 10.0 nmol/kg. Percent body weight changes were significant from day 5 through day 13 for 0.3 nmol/kg, from day 3 through day 13 for 1.0 nmol/kg and 10.0 nmol/kg, and from day 4 through day 13 for 3.0 nmol/kg. Changes in grams of body weight were significant from day 8 for 0.3 nmol/kg, from day 6 for 1.0 nmol/kg, from day 7 for 3.0 nmol/kg and from day 5 for 10.0 nmol/kg. Decreases in fed blood glucose levels were significant on day 7 for the animals in the 3.0 nmol/kg dose level and were significant on day 13 for the animals in the 3.0 and 10.0 nmol/kg dose levels.
DIO mice treated with FP2 q3d had improved glucose tolerance on day 14 compared to vehicle treatment during an oral glucose challenge (Table 41; FIG. 21A and 21B). Glucose was significantly lower at 30 minutes for the 0.3 nmol/kg group, at 60 minutes and 120 minutes for the 1.0 nmol/kg group, at 120 minutes for the 3.0 nmol/kg group, and at 30, 90, and 120 minutes for the 10.0 nmol/kg group. Total area under the curve was significant for all dose groups. Insulin levels during the glucose challenge were significantly lower for 0.3 and 10.0 nmol/kg groups at 30 minutes (Table 42; FIGS. 22A and 22B). In addition, compared to vehicle treated animals, there was a significant reduction in the calculated fasted HOMA-IR in DIO mice after 14 days of treatment with FP2 q3d at 10.0 nmol/kg indicative of improved insulin sensitivity (Table 44 and FIG. 24).
Body composition was measured by MM on day -1 before the start of the study and on day 13 (Table 47 and Table 48). DIO mice treated with FP2 at 1.0 nmol/kg and 10.0 nmol/kg had significant reductions in fat mass on day 13; whereas there were no changes in lean mass for any treatment groups. On day 13, the 10.0 nmol/kg treatment group had a significant increase in percent lean mass and a significant reduction in percent fat mass compared to the vehicle treated group. Changes from day -1 to day 13 were significant for lean mass in the 0.3 nmol/kg, 1.0 nmol/kg, and 10.0 nmol/kg treatment groups and were significant for percent lean mass in the 1.0, 3.0, and 10.0 nmol/kg treatment groups. Changes from day -1 to day 13 were significant for fat mass and percent lean mass in all treatment groups compared to vehicle.
There was no significant difference in endogenous mouse GDF15 serum levels between vehicle treated animals and mice treated with FP2 q3d for 15 days (Table 46).
Conclusion: the results suggest that the higher drug exposure is generally associated with greater % weight change from baseline on a population level across the studied dose groups on day 3, 6 and 9.
Exposure to FP2 over two weeks led to reduced food intake, decreased body weight, decreased blood glucose, improved glucose tolerance and insulin sensitivity in DIO mice. Significant decreases in food intake over multiple days were achieved at 1.0, 3.0, and 10.0 nmol/kg q3d. Body weight was decreased significantly starting three to five days after the initiation of the study. Fed blood glucose on day 13 was significantly decreased after q3d administration of FP2 at 3.0 and 10.0 nmol/kg. Insulin sensitivity represented by significantly decreased fasting HOMA-IR was achieved 14 days after 10.0 nmol/kg FP2 administered q3d. On day 13, a significant increase in percent lean mass and a significant reduction in percent fat mass was observed in DIO mice treated q3d with 10.0 nmol/kg FP2.
TABLE 38
Effect of FP2 on daily food intake (g) over 13 days of treatment.
Treatment Vehicle FP2 (nmol/kg) Rosiglitazone
Day N/A 0.3 1.0 3.0 10.0 10 mpk/day
0 2.0 ± 0.1 1.9 ± 0.1 2.2 ± 0.2 1.9 ± 0.2 1.9 ± 0.2 2.2 ± 0.1
1 2.4 ± 0.2 2.1 ± 0.1 1.6 ± 0.1* 1.5 ± 0.1* 1.3 ± 0.1* 2.7 ± 0.1
2 2.5 ± 0.1 1.8 ± 0.1* 1.7 ± 0.2* 1.9 ± 0.1* 2.0 ± 0.1 3.0 ± 0.2
3 2.5 ± 0.1 2.1 ± 0.1 1.7 ± 0.1* 1.9 ± 0.1 1.8 ± 0.1* 2.8 ± 0.2
4 2.6 ± 0.1 2.0 ± 0.1 1.8 ± 0.1* 1.9 ± 0.1* 1.9 ± 0.1* 2.9 ± 0.2
5 2.8 ± 0.1 2.1 ± 0.2* 2.2 ± 0.1* 2.2 ± 0.0* 1.9 ± 0.1* 2.7 ± 0.2
6 2.8 ± 0.1 2.3 ± 0.2 2.1 ± 0.1* 2.2 ± 0.1* 2.0 ± 0.1* 2.8 ± 0.2
7 2.6 ± 0.2 2.3 ± 0.1 2.0 ± 0.1* 2.0 ± 0.2 2.1 ± 0.1 2.9 ± 0.2
8 2.7 ± 0.2 2.1 ± 0.1* 2.2 ± 0.1 2.0 ± 0.1* 2.0 ± 0.1* 3.1 ± 0.2
9 2.8 ± 0.1 2.3 ± 0.1 2.3 ± 0.2 2.3 ± 0.2{circumflex over ( )} 2.1 ± 0.2* 3.2 ± 0.1
10 2.6 ± 0.1 2.5 ± 0.1 2.4 ± 0.2 2.3 ± 0.2{circumflex over ( )} 2.1 ± 0.2 3.0 ± 0.2
11 2.9 ± 0.1 2.5 ± 0.1 2.8 ± 0.2 2.6 ± 0.4 2.6 ± 0.1 3.3 ± 0.1
12 2.8 ± 0.1 2.7 ± 0.2 2.8 ± 0.1 2.8 ± 0.2 2.9 ± 0.1 3.1 ± 0.2
13 2.7 ± 0.1 2.5 ± 0.2 2.6 ± 0.1 2.6 ± 0.1 2.2 ± 0.1 3.1 ± 0.2
Values represent mean ± SEM or data from 8 animals per time per group, except n = 7 when noted by {circumflex over ( )}
*p < 0.05, versus vehicle
Statistical analyses used: Two-Way ANOVA RM, Tukey's multiple comparison test
TABLE 39
Effect of FP2 on Percent Body Weight Change Over 13 days of Treatment
Treatment Vehicle FP2 (nmol/kg) Rosiglitazone
Day N/A 0.3 1.0 3.0 10.0 10 mpk/day
−1 −0.1 ± 0.4 0.5 ± 0.2 −0.1 ± 0.1 −0.4 ± 0.2 0.0 ± 0.6 0.1 ± 0.2
0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
1 −0.4 ± 0.5 −1.0 ± 0.3 −2.2 ± 0.4 −1.8 ± 0.6 −2.4 ± 0.5 1.3 ± 0.4
2 −0.4 ± 0.3 −2.2 ± 0.4 −3.4 ± 0.6 −3.0 ± 0.6 −3.1 ± 0.4 1.3 ± 0.5
3 −0.3 ± 0.4 −2.3 ± 0.4 −4.2 ± 0.6* −3.5 ± 0.6 −4.3 ± 0.5* 1.9 ± 0.5
4 −0.3 ± 0.5 −2.9 ± 0.5 −5.3 ± 0.6* −4.9 ± 0.7* −5.8 ± 0.7* 1.8 ± 0.7
5 −0.2 ± 0.4 −4.2 ± 0.7* −6.2 ± 0.6* −5.6 ± 0.7* −6.8 ± 0.7* 1.7 ± 0.8
6 −0.4 ± 0.6 −5.1 ± 0.7* −7.4 ± 0.8* −6.9 ± 0.7* −8.5 ± 0.9* 1.1 ± 0.9
7 0.2 ± 0.8 −5.1 ± 0.7* −7.7 ± 0.9* −7.4 ± 0.7* −8.7 ± 0.9* 2.0 ± 1.0
8 0.2 ± 1.0 −6.1 ± 0.8* −7.9 ± 0.9* −8.0 ± 0.8* −9.7 ± 0.8* 2.4 ± 1.1
9 0.5 ± 1.1 −6.0 ± 0.9* −8.4 ± 0.9* −8.8 ± 0.9* −10.1 ± 1.0* 3.1 ± 1.0
10 1.1 ± 1.2 −5.7 ± 0.8* −8.1 ± 0.9* −8.9 ± 0.9* −10.7 ± 1.2* 3.5 ± 1.2
11 1.2 ± 1.3 −6.1 ± 0.9* −8.2 ± 0.8* −8.6 ± 1.3* −11.1 ± 1.4* 3.7 ± 1.2
12 1.4 ± 1.3 −6.3 ± 1.2* −7.7 ± 0.8* −8.2 ± 1.4* −10.9 ± 1.4* 4.1 ± 1.3
13 1.7 ± 0.9 −5.8 ± 1.4* −7.1 ± 1.0* −7.2 ± 1.4* −10.9 ± 1.4* 4.7 ± 1.3
Values represent mean ± SEM for data from 8 animals per time per group
*p < 0.05, versus Vehicle
Statistical analyses used: Two-Way ANOVA RM, Tukey's multiple comparison test
TABLE 40
Effect of FP2 on body weight change (g) over 13 days of treatment
Treatment Vehicle FP2 (nmol/kg) Rosiglitazone
Day N/A 0.3 1.0 3.0 10.0 10 mpk/day
−1 44.6 ± 0.6 44.5 ± 0.6 44.5 ± 0.6 44.5 ± 0.6 44.5 ± 0.6 44.5 ± 0.6
0 44.6 ± 0.6 44.3 ± 0.6 44.6 ± 0.6 44.7 ± 0.6 44.6 ± 0.7 44.4 ± 0.6
1 44.5 ± 0.7 43.8 ± 0.6 43.6 ± 0.6 43.9 ± 0.7 43.5 ± 0.7 45.0 ± 0.7
2 44.5 ± 0.7 43.3 ± 0.7 43.1 ± 0.6 43.3 ± 0.7 43.2 ± 0.7 45.0 ± 0.7
3 44.5 ± 0.7 43.2 ± 0.7 42.7 ± 0.6 43.1 ± 0.7 42.6 ± 0.6 45.3 ± 0.7
4 44.5 ± 0.7 43.0 ± 0.7 42.2 ± 0.6 42.5 ± 0.7 42.0 ± 0.7 45.2 ± 0.7
5 44.5 ± 0.7 42.4 ± 0.7 41.8 ± 0.5 42.2 ± 0.7 41.5 ± 0.6* 45.2 ± 0.7
6 44.5 ± 0.7 42.0 ± 0.7 41.3 ± 0.6* 41.6 ± 0.7 40.7 ± 0.5* 44.9 ± 0.8
7 44.7 ± 0.7 42.0 ± 0.7 41.1 ± 0.6* 41.4 ± 0.7* 40.6 ± 0.5* 45.3 ± 0.8
8 44.7 ± 0.7 41.6 ± 0.7* 41.1 ± 0.7* 41.1 ± 0.7* 40.2 ± 0.6* 45.5 ± 0.8
9 44.8 ± 0.7 41.6 ± 0.8* 40.8 ± 0.7* 40.8 ± 0.8* 40.0 ± 0.8* 45.8 ± 0.7
10 45.1 ± 0.7 41.8 ± 0.8* 41.0 ± 0.8* 40.8 ± 0.8* 39.8 ± 0.8* 46.0 ± 0.8
11 45.2 ± 0.8 41.6 ± 0.8* 40.9 ± 0.7* 40.8 ± 0.9* 39.6 ± 0.9* 46.1 ± 0.8
12 45.3 ± 0.7 41.5 ± 0.9* 41.2 ± 0.7* 41.0 ± 0.9* 39.7 ± 0.9* 46.3 ± 0.9
13 45.4 ± 0.7 41.7 ± 0.9* 41.4 ± 0.8* 41.5 ± 0.9* 39.7 ± 0.9* 46.5 ± 0.9
Values represent mean ± SEM for data from 8 animals per time per group
*p < 0.05, versus Vehicle
Statistical analyses: Two-Way ANOVA RM, Tukey's multiple comparison test
TABLE 41
Effect of FP2 on blood glucose (mg/dL) levels during an OGTT after 14 days of
treatment
Dose Total AUC Δ AUC
(nmol/ Time after Glucose Challenge (min) 120 min) 120 min)
Treatment kg) 0 30 60 90 120 (mg/dL/ (mg/dL/
Vehicle NA 161 ± 10 225 ± 17 228 ± 14 208 ± 18 213 ± 19 25429 ± 1228 6094 ± 1430
FP2 0.3 144 ± 4 163 ± 14* 209 ± 11 180 ± 12 171 ± 7 21270 ± 399* 3985 ± 521
1.0 151 ± 9 179 ± 18 188 ± 7 179 ± 13 163 ± 8* 21096 ± 754* 2972 ± 907
3.0 149 ± 8 180 ± 18 179 ± 11* 164 ± 6 163 ± 9* 20338 ± 876* 2426 ± 1058
10.0 134 ± 6 163 ± 7* 190 ± 13 152 ± 7* 163 ± 8* 19599 ± 614* 3539 ± 1198
Rosiglitazone 10 mpk/ 132 ± 9 152 ± 10* 162 ± 13* 138 ± 9* 159 ± 9* 17904 ± 632* 2139 ± 1179
day
Values represent mean ± SEM for data from 8 animals per time per group
*p < 0.05, versus Vehicle
Statistical analyses:
Two-Way ANOVA RM, Tukey's multiple comparison test for Glucose Values;
One-Way ANOVA, Tukey's multiple comparison test for AUC
TABLE 42
Effect of FP2 on insulin (ng/mL) levels during an OGTT after
14 days of treatment
Dose Time after Glucose Total AUC
(nmol/ Challenge (min) (ng/mL/
Treatment kg) 0 30 90 90 min)
Vehicle NA 4.2 ± 0.7 12.2 ± 2.3 3.6 ± 0.5 716.1 ± 122.0
FP2 0.3 2.9 ± 0.6 5.8 ± 1.8* 2.4 ± 0.4 377.1 ± 97.3
1.0 2.9 ± 0.4 8.2 ± 2.6 2.6 ± 0.4 491.8 ± 118.2
3.0 3.0 ± 0.3 8.4 ± 1.5 2.5 ± 0.3 498.6 ± 77.0
10.0 2.4 ± 0.4 4.3 ± 0.5* 2.1 ± 0.4 295.5 ± 32.0*
Rosiglita- 10 mpk/ 0.9 ± 0.1 1.8 ± 0.2* 0.8 ± 0.1 118.5 ± 9.6*
zone day
Values represent mean ± SEM for data from 8 animals per time per group
*—p < 0.05, versus Vehicle
Statistical analyses: Two-Way ANOVA RM, Tukey's multiple comparison test for insulin Values; One-Way ANOVA, Tukey's multiple comparison test for AUC
TABLE 43
Effect of FP2 on fed blood glucose (mg/dL) levels
Time after start of treatment
Dose (days)
Treatment (nmol/kg) 0 7 13
Vehicle NA 162 ± 5 159 ± 6 180 ± 9
FP2 0.3 143 ± 5 150 ± 9 168 ± 9
1.0 153 ± 12 135 ± 7 159 ± 7
3.0 148 ± 3 127 ± 6* 151 ± 8*
10.0 146 ± 6 136 ± 4 148 ± 3*
Rosiglitazone 10 mpk/day 137 ± 7 117 ± 4* 134 ± 6*
Values represent mean ± SEM for data from 8 animals per time per group
*—p < 0.05, versus Vehicle
Statistical analyses: Two-Way ANOVA RM, Tukey's multiple comparison test
TABLE 44
Fasted HOMA-IR in DIO mice after 14 days of
q3d treatement with FP2
Dose HOMA-
Treatment (nmol/kg) IR
Vehicle NA 48.7 ± 9.2
FP2 0.1 30.1 ± 6.6
1.0 30.5 ± 3.8
3.0 31.3 ± 3.2
10.0 22.7 ± 3.1*
Rosiglitazone 10 mpk/day 8.7 ± 1.0*
Values represent mean ± SEM for data from 8 animals per time per group
*—p < 0.05, versus Vehicle
Statistical analyses: One-Way ANOVA, Tukey's multiple comparison test
TABLE 45
Liver weight after 15 days of treatment with FP2 q3d in DIO mice
Liver
Dose Liver Weight (%
Treatment (nmol/kg) Weight, (g) of body)
Vehicle NA 1.9 ± 0.1 4.3 ± 0.3
FP2 0.1 1.7 ± 0.1 4.0 ± 0.1
1.0 1.7 ± 0.0 4.2 ± 0.1
3.0 1.8 ± 0.1 4.2 ± 0.1
10.0 1.7 ± 0.1 4.2 ± 0.1
Rosiglitazone 10 mpk/day 1.9 ± 0.1 4.1 ± 0.2
Values represent mean ± SEM or data from 8 animals per time per group
*—p < 0.05, versus Vehicle
Statistical analyses: One-Way ANOVA, Tukey's multiple comparison test
TABLE 46
Serum mouse GDF15 (pg/mL) levels after 15 days of treatement
with FP2 q3d in DIO mice
Dose
Treatment (nmol/kg) mGDF15
Vehicle NA 258.3 ± 21.4
FP2 0.1 214.1 ± 10.3
1.0 191.6 ± 12.7
3.0 254.5 ± 28.6
10.0 202.0 ± 10.0
Rosiglitazone 10 mpk/day 509.6 ± 26.2*
Values represent mean ± SEM or data from 8 animals per time per group
*—p < 0.05, versus Vehicle
Statistical analyses: One-Way ANOVA, Tukey's multiple comparison test
TABLE 47
Effect of JNJ-64739090 q3d in DIO mice on body composition (g) measured by MRI
Dose
Treat- (nmol/ Fat (g) Fat (g) Lean (g) Lean (g) Δ Lean
ment kg) Day −1 Day 13 Δ Fat (g) Day −1 Day 13 (g)
Vehicle NA 17.4 ± 0.6 18.2 ± 0.6 0.9 ± 0.3 25.4 ± 0.3 25.1 ± 0.3 −0.4 ± 0.2
FP2 0.3 17.0 ± 0.6 15.2 ± 0.8 −1.9 ± 0.6* 25.8 ± 0.7 24.5 ± 0.5 −1.2 ± 0.2*
1.0 17.0 ± 0.6 14.9 ± 0.6* −2.1 ± 0.4* 25.8 ± 0.5 24.6 ± 0.4 −1.2 ± 0.2*
3.0 17.4 ± 0.7 15.1 ± 1.1 −2.3 ± 0.5* 25.3 ± 0.4 24.2 ± 0.4 −1.1 ± 0.1
10.0 16.7 ± 0.6 13.2 ± 0.8* −3.6 ± 0.5* 26.1 ± 0.6 24.5 ± 0.6 −1.7 ± 0.2*
Rosi- 10 mpk/ 17.2 ± 0.6 19.1 ± 0.5 1.9 ± 0.6 25.5 ± 0.7 25.1 ± 0.6 −0.4 ± 0.2
glitazone day
Values represent mean ± SEM for data from 8 animals per time per group
*p < 0.05, versus Vehicle
Statistical analyses: One-Way ANOVA, Tukey's multiple comparison test
TABLE 48
Effect of FP2 q3d in DIO mice on body composition (%) measured by MRI
Dose
Treat- (nmol/ Fat (%) Fat (%) Lean (%) Lean (%) Δ Lean
ment kg) Day −1 Day 13 Δ Fat (%) Day −1 Day 13 (%)
Vehicle NA 38.1 ± 1.0 40.1 ± 1.0 2.1 ± 0.3 55.9 ± 0.9 55.3 ± 0.9 −0.6 ± 0.3
FP2 0.3 37.6 ± 1.2 36.3 ± 1.3 −1.3 ± 0.9* 56.8 ± 1.3 59.0 ± 1.4 2.2 ± 0.8
1.0 37.2 ± 1.1 35.8 ± 0.9 −1.4 ± 0.5* 56.7 ± 1.1 59.5 ± 1.0 2.8 ± 0.6*
3.0 38.3 ± 1.4 36.1 ± 2.0 −2.3 ± 0.7* 55.9 ± 1.2 58.7 ± 2.0 2.8 ± 0.8*
10.0 36.9 ± 1.1 33.1 ± 1.5* −3.8 ± 0.8* 57.5 ± 1.0 61.7 ± 1.4* 4.2 ± 0.7*
Rosi- 10 mpk/ 38.0 ± 1.2 41.1 ± 0.9 3.1 ± 0.9 56.2 ± 1.2 53.9 ± 0.8 −2.3 ± 0.8
glitazone day
Values represent mean ± SEM for data from 8 animals per time per group
*p < 0.05, versus Vehicle
Statistical analyses: One-Way ANOVA, Tukey's multiple comparison test
TABLE 49
FP2 serum exposures (nM) of the PK arm
during q3d treatment in DIO mice
Time Post Initial Dose
(Time post last dose)
Treatment Subject Day 14 Day 15 Day 17
Group ID Day 3 Day 6 Day 9 Day 12 (48 hr) (72 hr) (120 hr)
0.1 9 1.930 2.993 <LOQ <LOQ <LOQ <LOQ <LOQ
nmol/ 10 1.853 3.369 2.817 <LOQ <LOQ <LOQ <LOQ
kg 11 1.715 2.637 2.568 2.709 3.441 1.313 <LOQ
1.0 9 8.198 9.660 9.645 <LOQ <LOQ <LOQ <LOQ
nmol/ 10 7.073 10.595 8.966 <LOQ <LOQ <LOQ <LOQ
kg 11 6.689 13.967 10.802 <LOQ <LOQ <LOQ <LOQ
3.0 9 20.802 26.329 27.863 <LOQ <LOQ <LOQ <LOQ
nmol/ 10 23.563 32.020 41.576 1.168 <LOQ <LOQ <LOQ
kg 11 21.704 30.101 <LOQ <LOQ <LOQ <LOQ <LOQ
10.0 9 28.495 33.050 <LOQ <LOQ <LOQ <LOQ <LOQ
nmol/ 10 70.779 112.898 <LOQ <LOQ <LOQ <LOQ <LOQ
kg 11 70.404 111.767 55.117 <LOQ <LOQ <LOQ <LOQ
Data are expressed as concentration for each animal.
<LOQ = below limit of quantitation; LOQ is 0.494 nM
** Values at Day 3, 6, 9 and 12 are immediately prior to next dose
TABLE 50
Terminal serum exposures (nM) of FP2 after 15 days of q3d
treatment in DIO mice
Treatment Group (nmol/kg)
Animal
ID 0.1 1.0 3.0 10.0
1 2.567 <LOQ <LOQ <LOQ
2 <LOQ <LOQ <LOQ 44.149
3 <LOQ <LOQ <LOQ <LOQ
4 1.144 0.678 <LOQ <LOQ
5 <LOQ <LOQ <LOQ <LOQ
6 3.440 <LOQ <LOQ <LOQ
7 2.727 <LOQ <LOQ <LOQ
8 <LOQ <LOQ <LOQ <LOQ
Data are expressed as concentration for each animal.
<LOQ = below limit of quantitation; LOQ is 0.494 nM
Example 18: FP2 Multispecies Pharmacokinetics and Immune Response Mouse pharmacokinetics
The pharmacokinetic properties of FP2 were evaluated when administered subcutaneously to female C57B1/6 mice. FP2 was administered subcutaneously (n=5 samples per time point) and intravenously (n=5 samples per time point) to female C57B1/6 mice (Sage Laboratories, St Louis, Mo.) at a dose level of 2.0 mg/kg in PBS, (pH 7.3-7.5). The collection of sample at the last time point was via a terminal bleed. Blood samples were collected, serum processed and drug concentrations were measured up to 168 hours. The levels of FP2 were measured using an immunoassay method. The drug concentration profiles in plasma are summarized in Table 51 and 52 and illustrated in FIG. 25.
Pharmacokinetic analysis of FP2 in C57B1/6 mice demonstrated a terminal half-life of ˜1.51 and ˜1.76 days following IV and SC dosing respectively, with a mean bioavailability of ˜61% following SC administration.
TABLE 51
Serum concentration (ng/mL) of FP2 following a single subcutaneous
(SC) dose in C57Bl/6 mice
FP2-SC Dose
Animal Animal Animal Animal Animal
31 32 33 35 36 Average
Result Result Result Result Result Result Std Dev
Timepoint (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL)
4 hr 11310.11 6323.44 23489.21 4357.74 4835.54 10063.21 7995.0
24 hr 12378.58 10896.80 17769.03 15928.14 15404.08 14475.33 2785.0
72 hr 5127.95 5569.27 6727.37 7909.66 7550.32 6576.91 1210.5
96 hr 3059.83 3350.25 4042.04 4095.15 4431.94 3795.84 569.0
168 hr 784.61 1202.66 1364.68 1557.60 1678.02 1317.51 348.9
TABLE 52
Serum concentration (ng/mL) of FP2 following a single intravenous (IV)
dose in C57Bl/6 mice
FP2-IV Dose
Animal Animal Animal Animal Animal
37 40 41 45 48 Average
Result Result Result Result Result Result Std Dev
Timepoint (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL)
1 hr 49573.59 42382.59 40001.18 43085.75 46443.59 44297.34 3742.9
24 hr 22173.85 19120.39 19393.78 19798.29 17459.96 19589.25 1696.7
72 hr 8334.33 7989.10 8573.44 9086.37 8186.68 8433.98 422.5
96 hr 4478.83 4699.80 4749.93 5331.39 4579.08 4767.81 332.3
168 hr 1044.24 1419.64 1393.44 1835.63 979.49 1334.49 343.6
TABLE 53
Pharmacokinetic parameters of FP2 following 2 mg/kg IV and
2 mg/kg SC administration in C57B1/6 mice.
CL or Vz or AUC0-last AUC0-inf
t1/2 CL/F Vz/F Cmax Tmax* (day* (day*
Route (day) (ml/day/kg) (ml/kg) (ng/ml) (day) ng/ml) ng/ml)
SC Mean 1.76 43 108 15619 1 44972 48379
SD 0.16 8 20 4870 8563 9147
IV Mean 1.51 25 55 44297 0.042 76269 79253
SD 0.18 1 6 3743 3788 4051
Rat Pharmacokinetics
FP2 was administered subcutaneously (n=5 samples per time point) and intravenously (n=5 samples per time point) to female Sprague-Dawley rats (Sage Laboratories, St. Louis, MO) at a dose level of 2.0 mg/kg in PBS, (pH 7.3-7.5). The collection of sample at the last time point was via a terminal bleed. Blood samples were collected, serum processed and drug concentrations were measured up to 168 hours. The levels of FP2 were measured using an immunoassay method. The drug concentration profiles in plasma are summarized in Table 54 and 55, and illustrated in FIG. 26. Pharmacokinetic parameters calculated from these data are summarized in Table 56.
Pharmacokinetic analysis of FP2 in Sprague Dawley rats demonstrated a terminal half-life of ˜1.46 and ˜1.37 days following IV and SC dosing respectively, with a mean bioavailability of ˜28% following SC administration.
TABLE 54
Serum concentration (ng/mL) of FP2 following a single subcutaneous
(SC) dose in Sprague-Dawley rats.
FP2-SC Dose
Animal Animal Animal Animal Animal
01 02 03 04 05 Average Std
Result Result Result Result Result Result Dev
Timepoint (ng/ml) (ng/ml) (ng/ml) (ng/ml) (ng/ml) (ng/ml) (ng/ml)
4 hr 730.95 257.94 469.95 496.34 400.65 471.16 172.2
24 hr 9236.27 6658.79 7728.92 7449.56 6502.60 7515.23 1092.1
72 hr 4559.04 4464.72 5619.82 4287.25 3844.15 4555.00 655.6
96 hr 2791.97 2452.35 3387.55 2316.06 2176.00 2624.79 483.8
168 hr 622.32 606.36 <80.00 514.19 476.49 554.84 70.7
TABLE 55
Serum concentration (ng/mL) of FP2 following a single intravenous (IV) dose in
Sprague-Dawley rats.
FP2-IV Dose
Animal Animal Animal Animal Animal
06 07 08 09 10 Average Std
Result Result Result Result Result Result Dev
Timepoint (ng/ml) (ng/ml) (ng/ml) (ng/ml) (ng/ml) (ng/ml) (ng/ml)
1 hr 60785.80 47392.80 43046.54 46014.25 44547.65 48357.41 7134.5
24 hr 25470.36 24729.88 21157.29 20497.32 21459.71 22662.91 2267.1
72 hr 8208.46 9262.13 8866.76 8635.93 8843.83 8763.42 384.1
96 hr 4433.18 4833.22 4995.63 4630.60 4604.13 4699.35 218.1
168 hr 1083.14 1469.35 1614.61 1394.17 1053.89 1323.03 245.7
TABLE 56
Pharmacokinetic parameters of FP2 following 2 mg/kg IV and
2 mg/kg SC administration in Sprague-Dawley Rats.
CL or Vz or AUC0-last AUC0-inf
t1/2 CL/F Vz/F Cmax Tmax* (day* (day*
Route (day) (ml/day/kg) (ml/kg) (ng/ml) (day) ng/ml) ng/ml)
SC Mean 1.37 84 165 7515 1 22614 24036
SD 0.04 10 17 1092 2509 2893
IV Mean 1.46 23 49 48357 0.042 83271 86089
SD 0.12 2 6 7135 6081 5820
Monkey Pharmacokinetics
FP2 was administered subcutaneously at 1 mg/kg and intravenously at 1 mg/kg to three male cynomolgus monkeys each in PBS, (pH 7.0-7.6). Blood samples were collected, plasma processed and drug concentrations were measured up to 21 days.
The pharmacokinetics (PK) of FP2 was characterized following administration of a single dose IV (1.0 mg/kg) and SC (1.0 mg/kg) in cynomolgus monkeys. The plasma drug concentration-time profile after SC administration is summarized in Tables 57 and 58 for immunoassay and LCMS analyses respectively and after IV administration in Tables 59 and 60 for immunoassay and LCMS analyses respectively. The immunoassay data is graphed in FIG. 27, and the LCMS data is represented in FIG. 28.
Using results from the immunoassay analysis, the mean NCA-based terminal half-life (t1/2) for FP2 was ˜7.05 and ˜8.51 days following IV and SC dosing, respectively. The mean PK parameters following IV and SC administration are summarized in Table 61. Using results from the immunoassay bioanalysis, the mean non-compartment model estimated terminal half-life (t1/2) for FP2 was 7.05 and 8.51 days following IV and SC dosing, respectively. The mean bioavailability (F%) of FP2 was estimated to be ˜98.5% based on AUC0-last and estimated to be ˜109.2% based on AUC0-Inf in cynomolgus monkeys following SC administration.
TABLE 57
Plasma concentration (ng/mL) of FP2 measured by immunoassay
following a single SC dose in cynomolgus monkeys.
Immunoassay
Animal Animal Animal
Time (hr) 704 705 706 SC Ave Std Dev
0 <80.0 <80.0 <80.0 <80.0 N/A
6 7365.3 6128.5 6056.9 6516.9 735.6
24 19716.8 10903.8 10554.7 13725.1 5191.9
48 18191.7 14353.7 14464.7 15670.0 2184.5
72 17813.9 14207.5 12684.0 14901.8 2634.5
120 13823.9 11445.8 10590.8 11953.5 1675.3
168 12359.6 10103.8 9467.8 10643.8 1519.7
240 9457.9 7642.6 8109.1 8403.2 942.7
336 6796.8 5679.8 5235.7 5904.1 804.3
432 5581.3 3830.6 3746.5 4386.1 1035.9
528 4126.2 2613.4 2879.3 3206.3 807.7
N/A = not applicable
TABLE 58
Plasma concentration (ng/mL) of FP2 measured by LCMS
following a single SC dose in cynomolgus monkeys.
LC/MS
Animal Animal Animal
Time (hr) 704 705 706 SC Ave SEM
0 <1000 <1000 <1000 <1000 N/A
6 6110.0 5910 6200.0 6073.3 85.7
24 # 10420.0 11300.0 10860.0 359.3
48 16560.0 13510.0 14960.0 15010.0 880.8
72 13690.0 13450.0 13380.0 13506.7 93.9
120 11040.0 # # 11040.0 N/A
168 # 8680.0 9400.0 9040.0 293.9
240 6940.0 7070.0 7140.0 7050.0 58.6
336 4400.0 4580.0 4430.0 4470.0 55.7
432 — 2910.0 — 2910.0 N/A
528 <1000 <1000 <1000 <1000 N/A
— = initial run failed, not enough sample for repeat analysis
# = mislabeled tube, sample excluded from the analysis
N/A = not applicable
TABLE 59
Plasma concentration (ng/mL) of FP2 measured by immunoassay
following a single IV dose in cynomolgus monkeys.
Immunoassay
Time (hr) Animal 701 Animal 702 Animal 703 IV Ave Std Dev
0 <80.0 <80.0 <80.0 <80.0 N/A
1 29938.3 31139.0 23545.3 28207.6 4082.0
6 24711.9 21173.0 20434.4 22106.4 2286.5
24 19648.4 21420.9 8662.7 16577.3 6911.3
48 17790.1 17107.0 12983.1 15960.0 2600.7
72 17889.4 13871.1 13692.4 15151.0 2373.3
120 15298.6 11982.5 11361.5 12880.9 2116.7
168 11752.0 10982.8 9933.8 10889.5 912.7
240 8921.1 7465.0 6733.4 7706.5 1113.6
336 6572.8 5750.5 4687.6 5670.3 945.1
432 1099.6 3425.0 3458.0 2660.9 1352.2
528 <80.0 2069.0 2416.2 2242.6 N/A
N/A = not applicable
TABLE 60
Plasma concentration (ng/mL) of FP2 measured by LCMS following
a single IV dose in cynomolgus monkeys
LC/MS
Animal Animal Animal
Time (hr) 701 702 703 IV Ave SEM
0 <1000 <1000 <1000 <1000 N/A
1 25340.0 28820 26220.0 26793.3 1044.7
6 24610.0 26340.0 23810.0 24920.0 746.6
24 18410.0 18680.0 9810.0 15633.3 2912.7
48 16290.0 17370.0 13840.0 15833.3 1044.3
72 15430.0 14920.0 14280.0 14876.7 332.7
120 11180.0 11480.0 11440.0 11366.7 94.0
168 9170.0 # # 9170.0 N/A
240 6800.0 7380.0 7600.0 7260.0 238.6
336 2870.0 3860.0 3480.0 3403.3 288.3
432 — — 3030.0 3030.0 N/A
528 1150.0 1680.0 1400.0 1410.0 153.1
— = initial run failed, not enough sample for repeat analysis
N/A = not applicable
# = mislabeled tube, sample excluded from the analysis
TABLE 61
Mean (±SD) pharmacokinetic parameters of FP2 following 1 mg/kg
IV and SC administration in cynomolgus monkey.
CL or Vz or
CL/F Vz/F AUC0-last AUC0-inf
t1/2 (ml/day/ (ml/ Cmax Tmax* (day* (day*
Route (day) kg) kg) (ng/ml) (day) ng/ml) ng/ml)
SC Mean 8.51 4.6 56 16178 1.67 180792 221032
SD 1.28 0.8 7 3065 29990 44931
IV Mean 7.05 4.9 51 28208 0.042 183468 202380
SD 1.45 0.2 12 4082 18268 9617
PK parameters are mean values baser on NCA of immunoassay PK data.
*Tmax (median)
Human Plasma Stability Assay
The ex vivo stability of FP2 was examined in fresh heparinized plasma at 37° C. for up to 48 hours. Fresh, non-frozen human plasma was generated from heparinized blood from two subjects (one male and one female) by centrifugation. FP2 was incubated in this matrix at 37° C. wih gentle mixing or 0. 4. 24 and 48 hours. The concentration of FP2 was determine using an immunoassay method. An independent immunoaffinity capture followed by LCMS method was used to quantitate the concentration of the intact dimer present in this matrix under the assay conditions.
In the immunoassay method, the percent recovery from the starting concentration ranged from 104.8 to 94.1 and did not decrease over time, demonstrating that FP2 is stable in human plasma up to 48 hours ex vivo (FIG. 29 and Table 62). The LCMS method showed that concentrations were stable over time demonstrating that JNJ-64739090 remains an intact dimer in human plasma up to 48 hours ex vivo (FIG. 30 and Table 63).
TABLE 62
Ex vivo stability of FP2 (Normalized Percent Recovery) over 48
hours in human plasma (ng/ml) measured by immunoassay.
Concentration Normalized %
Compound Gender Time (hr) (ng/mL) Recovery
FP2 female 0 9104 100.0
4 9056 99.5
24 9332 102.5
48 9374 103.0
male 0 9473 100.0
4 9929 104.8
24 9081 95.9
48 8912 94.1
TABLE 63
Ex vivo stability of FP2 (Normalized Percent Recovery) over 48
hours in human plasma (ng/ml) measured by intact LC/MS.
Concentration Normalized %
Compound Gender Time (hr) (ng/mL) Recovery
FP2 female 0 11090 100.0
4 10830 97.7
24 10500 94.7
48 10030 90.4
male 0 10760 100.0
4 9640 89.6
24 10190 94.7
48 8500 79.0
Example 19: Efficacy of FP1 and FP2 in Cynomolgus Monkeys The effects of FP1 and FP2 on food intake and body weight after a single dose in naive cynomolgus monkeys were evaluated.
FP1 was administered subcutaneously to a cohort of naive cynomolgus monkeys at three dose levels; 1, 3 and 10 nmol/kg. A vehicle treated group was also included. The animals were treated in a blinded manner. The study lasted a total of 6 weeks: 2 weeks of baseline food intake measurement and data collection, 4 weeks of data collection after single administration of compound. Plasma drug exposures were measured on days 1, 7, 14, 21, and 28 following dosing.
Treatment of cynomolgus monkeys with a single dose of FP1 reduced food intake and body weight compared to vehicle treatment (FIGS. 32-33). A significant reduction in daily food intake was seen on days 4, 5, 6, and 8 through 12 for the 10 nmol/kg dose level (FIG. 32). The weekly average of daily food intake was significantly reduced for during week 2 post administration for the 10 nmol/kg dose level. The 3 nmol/kg dose level had a significant percent reduction from the average weekly food intake prior to dosing on week 2 post administration and the 10 nmol/kg dose level had a significant percent reduction from the average weekly food intake prior to dosing in weeks 1 and 2 post administration. A significant reduction in percent body weight change from day 0 was seen at day 28 for the 3 nmol/kg dose level, and on day 14, 21, and 28 for the 10 nmol/kg dose level (FIG. 33).
FP2 was administered subcutaneously to a cohort of naïve cynomolgus monkeys at three dose levels; 1, 3 and 10 nmol/kg. A vehicle treated group was also included. The animals were treated in a blinded manner. The study lasted a total of 11 weeks: 5 weeks of baseline food intake measurement and data collection, 1 week of treatment and 5 weeks of wash-out phase data collection. Plasma drug exposures were measured on days 1, 7, 14, 21, 28, 35, and 42 following dosing.
Treatment of cynomolgus monkeys with a single dose of FP2 reduced food intake and body weight compared to vehicle treatment (FIGS. 34-35). A significant reduction in daily food intake was seen on days 3, 5 through 8, 10 and 12 for the 3 nmol/kg dose level and from days 3 through 38 and day 40 for the 10 nmol/kg dose level (FIG. 34). The weekly average of daily food intake was significantly reduced for week 1 post administration for the 3 nmol/kg dose level and significantly reduced for weeks 1 through 6 for the 10 nmol/kg dose level. The 3 nmol/kg dose level had a significant percent reduction from the week prior to dosing in weekly average daily food intake on week 2 post administration and the 10 nmol/kg dose level had a significant percent reduction from the week prior to dosing in weekly average daily food intake on weeks 1 through 6 post administration. A significant reduction in percent body weight change from day 0 was seen from days 21 through 42 for the 1 nmol/kg dose level, from days 14 through 42 for the 3 nmol/kg dose level and from days 7 through 42 for the 10 nmol/kg dose level (FIG. 34).
Example 20: HSA-GDF15:GDF15 Heterodimer Bioactivity of HSA-GDF15:GDF15 heterodimer was investigated.
To generate an HSA-GDF15:GDF15 heterodimer two constructs were designed. The first construct contained HSA fused to the N-terminus of mature GDF15 (AA 203-308) via a glycine-serine linker (SEQ ID NO: 93). The second construct contained a 6x histidine tagged HSA fused to the N-terminus of mature GDF15 (AA 197-308) via a glycine-serine linker and an HRV3C protease cleavage site (SEQ ID NO: 94). The plasmids were co-transfected at a 1:1 ratio using the Expi293™ Expression System (Thermo Fisher Scientific) according to the manufacturer's protocol. Peptides were secreted as HSA-GDF15 proteins, including both heterodimer and homodimer forms, wherein monomers were linked by disulfide bonds.
Cell supernatants from transiently transfected Expi293™ cells were harvested 5 days after transfection, clarified by centrifugation and sterile filtered (0.2 μPES membrane, Corning). Clarified supernatants were loaded onto a Histrap HP column (GE Healthcare) equilibrated with 20 mM sodium phosphate, 500 mM NaCl, pH 7.4. After loading, unbound protein was removed by washing the column with equilibration buffer. HSA-GDF15 proteins, including both heterodimer and homodimer forms, bound to the column and were eluted with 20 mM sodium phosphate, 150 mM imidazole, pH 7.4. Eluate fractions were pooled and incubated overnight at 4° C. in the presence of 6x histidine tagged HRV3C enzyme (Janssen) to generate the HSA-GDF15:GDF15 heterodimer. Following incubation, the protein solution was dialyzed into equilibration buffer to remove imidazole before being applied to a HisTrap HP column one more time. The HSA-GDF15:GDF15 heterodimer eluted in the 20 mM sodium phosphate, 50 mM imidazole, pH 7.4 wash step, while histidine tagged proteins were retained. The heterodimer was further polished by size exclusion chromatography (SEC) using a HiLoad 26/60 Superdex 200 pg column (GE Healthcare) equilibrated in lx DPBS, pH 7.2. Eluate fractions from the SEC with high purity (determined by SDS-PAGE) of the HSA-GDF15:GDF15 heterodimer were pooled and filtered. The protein concentration was determined by absorbance at 280 nm on a BioTek SynergyHTTM spectrophotometer. The quality of the purified protein was assessed by SDS-PAGE and analytical size exclusion HPLC (Ultimate3000 HPLC system). Endotoxin levels were measured using an LAL assay (Pyrotell®-T, Associates of Cape Cod). The purified protein was stored at 4° C.
SK-N-AS cells (ATCC) stably expressing GDF15 receptor (GFRAL) were seeded in growth medium (10% FBS) in a 96-well plate 24 hours prior to the assay. After 24 hrs the cells were starved by replacing the culture medium with 200 μl of DMEM medium supplemented with 1% HI horse serum for 3 hours in a 37° C. incubator. The 1% HI horse serum supplemented medium was then replaced with 200 μl of AB1 and incubated for an additional 2 hours in a 37° C. incubator. To perform the assay, the AB1 was aspirated from all wells and 100 μl of variable concentrations of the testing compound in AB2 was added and the plate was incubated for 15 min in a 37° C. incubator. After 15 minutes, the testing solution was removed and 30 μl of lysis buffer (provided in the detection kit) was added and the plate was shaken on a plate shaker at room temperature for 30 min. For detection, 16 μl of the lysed sample was transferred to a 384-well assay plate and 4 μl of HTRF pAKT detection antibodies were added. The plate was incubated overnight at room temperature and then the HTRF signal read on the Envision (Perkin Elmer).
EC50 values were calculated using GraphPad Prism® Nonlinear Regression (Curve fit). Data are expressed as the Mean± Standard Error (SE) from three separate experiments with three replicates per data point. Molecular identity of the HSA-GDF15:GDF15 heterodimer was confirmed by mass spectrometry. The left-shift of the heterodimer curve suggested that the HSA-GDF15:GDF15 heterodimer is more potent in inducing pAKT than the relevant homodimer molecule with an additional albumin.
Example 21: Linker Thermal Stability Thermal stability was investigated for various linkers that connect HSA and GDF15. To evaluate the potential to fragment and aggregate, HSA-GDF15 fusion proteins with various linkers were diluted to I Omg/ml. After addition of EDTA and Methionine, the samples were incubated under 40° C. for 14 days. Then samples were diluted to the concentration of 1 mg/ml and evaluated under size-exclusion high-performance liquid chromatography (SE-HPLC). Percent of intact protein as well as aggregate and fragment were quantified for these proteins. Table 64 shows that the HSA-GDF15 proteins with linkers that consist of AP repeats are most stable against fragment under thermal stress.
To evaluate the whether these linker affects GDF15 interaction with its receptor, an immunoassay with GFRAL-Fc fusion protein coated on plate and anti-GDF15 or anti-HSA detection was performed, using monoclonal antibodies for GDF15 (Janssen) and HSA (Kerafast, Inc., Boston, Mass.). The assay showed all these linker variants in Table 66 has similar binding to receptor.
TABLE 64
SE-HPLC results after thermal stress for 14 days.
SEQ aggre- frag-
ID gate intact ment
NO Linker (%) (%) (%)
113 GS(GGGGS)8 3.33 84.44 12.22
115 GA(GGGGA)8 3.51 87.98 8.5
117 (AP)10 1.64 98.36 0
119 (AP)12 2.36 97.64 0
121 GGS-(EGKSSGSGSESKST)3-GGS 1.67 85.24 13.09
123 GS(PGGGS)8 2.96 88.12 8.91
125 GS(AGGGS)8 3.44 86.22 10.34
127 GGS-(EGKSSGSGSESKST)2-GGS 1.71 91.17 7.12
While the invention has been described in detail, and with reference to specific embodiments thereof, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention.
The sequences referenced in this application are provided in the table below: WT—wild type
WT-wild type
SEQ
ID
NO Description Sequence
1 Human Serum DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNE
Albumin, WT VTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADC
(HSA) CAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFL
KKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPK
LDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKA
EFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSIS
SKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKN
YAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAA
ADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLV
RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLS
VVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPK
EFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKA
VMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL
2 HSA variant, DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
C34S TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL
3 HSA variant, DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNE
C34A VTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADC
CAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFL
KKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPK
LDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKA
EFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSIS
SKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKN
YAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAA
ADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLV
RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLS
VVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPK
EFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKA
VMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL
5 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GS(GGGGS)4- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 (WT) AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
Fusion KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGW
ADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVP
APCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI
6 Mature GDF15 ARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCI
(197-308) GACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQK
TDTGVSLQTYDDLLAKDCHCI
7 Truncated GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGAC
mature GDF15 PSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDT
(200-308) GVSLQTYDDLLAKDCHCI
8 Truncated DHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACP
mature GDF15 SQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTG
(201-308) VSLQTYDDLLAKDCHCI
9 Truncated HCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPS
mature GDF15 QFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGV
(202-308) SLQTYDDLLAKDCHCI
10 Truncated CPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQ
mature GDF15 FRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVS
(203-308) LQTYDDLLAKDCHCI
11 Truncated CRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMH
mature GDF15 AQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDL
(211-308) LAKDCHCI
25 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
AS(GGGGS)2GT- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
-GDF15 Fusion AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLASGGGG
SGGGGSGTARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPR
EVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASY
NPMVLIQKTDTGVSLQTYDDLLAKDCHCI
26 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
AS(GGGGS)8GT- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 (WT) AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLASGGGG
SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGTARNGDH
CPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQ
FRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVS
LQTYDDLLAKDCHCI
27 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
AS(AP)5GT- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 Fusion AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLASAPAPA
PAPAPGTARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPRE
VQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYN
PMVLIQKTDTGVSLQTYDDLLAKDCHCI
28 HSA (C34S) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
AS(AP)10GT- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 Fusion AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLASAPAPA
PAPAPAPAPAPAPAPGTARNGDHCPLGPGRCCRLHTVRASLEDLGW
ADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVP
APCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI
29 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
AS(AP)20GT- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 Fusion AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLASAPAPA
PAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPGTARNGDHCPLG
PGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAA
NMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTY
DDLLAKDCHCI
30 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
AS(EAAAK)4GT- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 Fusion AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLASEAAA
KEAAAKEAAAKEAAAKGTARNGDHCPLGPGRCCRLHTVRASLEDL
GWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDT
VPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI
31 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
AS(EAAAK)8GT- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 Fusion AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLASEAAA
KEAAAKEAAAKEAAAKEAAAKEAAAKEAAAKEAAAKGTARNGD
HCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPS
QFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGV
SLQTYDDLLAKDCHCI
36 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GS(GGGGS)4- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 Fusion AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
(deletion 13 KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
mutant) DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSCSRLHTVRASLEDLGWADWVLSPREVQVT
MCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVL
IQKTDTGVSLQTYDDLLAKDCHCI
37 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GS(GGGGS)4- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 Fusion AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
(deletion 14 KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
mutant) DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSCRLHTVRASLEDLGWADWVLSPREVQVT
MCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVL
IQKTDTGVSLQTYDDLLAKDCHCI
40 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GS(GGGGS)4- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 Fusion AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGW
ADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVP
APCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI
48 HSA (C34A)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNE
GS(GGGGS)4- VTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADC
GDF15 Fusion CAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFL
KKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPK
LDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKA
EFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSIS
SKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKN
YAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAA
ADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLV
RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLS
VVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPK
EFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKA
VMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGG
GSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLG
WADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTV
PAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI
55 HSA(C34A)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNE
GS(GGGGS)8- VTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADC
GDF15 CAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFL
KKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPK
LDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKA
EFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSIS
SKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKN
YAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAA
ADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLV
RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLS
VVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPK
EFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKA
VMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGG
GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSARNGDHC
PLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQF
RAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSL
QTYDDLLAKDCHCI
56 HSA (C34A)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNE
(AP)10- VTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADC
GDF15 CAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFL
KKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPK
LDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKA
EFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSIS
SKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKN
YAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAA
ADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLV
RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLS
VVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPK
EFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKA
VMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLAPAPAP
APAPAPAPAPAPAPARNGDHCPLGPGRCCRLHTVRASLEDLGWAD
WVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPC
CVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI
59 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
(AP)10- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLAPAPAPA
PAPAPAPAPAPAPARNGDHCPLGPGRCCRLHTVRASLEDLGWADW
VLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCC
VPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI
60 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GS-(GGGGS)8- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15(WT) AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSARNGDHCP
LGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFR
AANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQ
TYDDLLAKDCHCI
64 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GS(GGGGS)4- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 (I89R) AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGW
ADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVP
APCCVPASYNPMVLRQKTDTGVSLQTYDDLLAKDCHCI
65 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GS(GGGGS)4- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 (I89W) AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGW
ADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVP
APCCVPASYNPMVLWQKTDTGVSLQTYDDLLAKDCHCI
66 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GS(GGGGS)4- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 (L34A, AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
S35A, R37A) KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGW
ADWVAAPAEVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVP
APCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI
67 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GS(GGGGS)4- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 (V87A, AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
I89A, L98A) KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGW
ADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVP
APCCVPASYNPMALAQKTDTGVSAQTYDDLLAKDCHCI
68 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GS(GGGGS)4- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 (L34A, AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
S35A, I89A) KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGW
ADWVAAPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVP
APCCVPASYNPMVLAQKTDTGVSLQTYDDLLAKDCHCI
69 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GS(GGGGS)4- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 (V87A, AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
I89A) KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGW
ADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVP
APCCVPASYNPMALAQKTDTGVSLQTYDDLLAKDCHCI
70 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GS(GGGGS)4- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 (Q60W) AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGW
ADWVLSPREVQVTMCIGACPSQFRAANMHAWIKTSLHRLKPDTVP
APCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI
71 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GS(GGGGS)4- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 (W32A) AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGW
ADAVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPA
PCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI
72 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GS(GGGGS)4- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 (W29A) AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGA
ADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVP
APCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI
73 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GS(GGGGS)4- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 (Q60A, AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
S64A, R67A) KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGW
ADWVLSPREVQVTMCIGACPSQFRAANMHAAIKTALHALKPDTVP
APCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI
74 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GS(GGGGS)4- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 (W29A, AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
Q60A, I61A) KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGA
ADWVLSPREVQVTMCIGACPSQFRAANMHAAAKTSLHRLKPDTVP
APCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI
75 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GS(GGGGS)4- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 (W29A, AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
W32A) KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGA
ADAVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPA
PCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI
76 nucleic acid gacgcccacaagagcgaggtggcccaccggttcaaggacctgggcgaggagaacttcaaggc
encoding cctggtgctgatcgccttcgcccagtacctgcagcagtccccatcgaggaccacgtgaagctgg
SEQ ID NO: 5 tgaacgaggtgaccgagttcgccaagacctgcgtggccgacgagagcgccgagaactgcgac
aagagcctgcacaccctgttcggcgacaagctgtgcaccgtggccaccctgcgggagacctac
ggcgagatggccgactgctgcgccaagcaggagcccgagcggaacgagtgatcctgcagca
caaggacgacaaccccaacctgccccggctggtgcggcccgaggtggacgtgatgtgcaccg
ccttccacgacaacgaggagaccttcctgaagaagtacctgtacgagatcgcccggcggcaccc
ctacttctacgcccccgagctgctgttcttcgccaagcggtacaaggccgccttcaccgagtgctg
ccaggccgccgacaaggccgcctgcctgctgcccaagctggacgagctgcgggacgagggc
aaggccagcagcgccaagcagcggctgaagtgcgccagcctgcagaagttcggcgagcggg
ccttcaaggcctgggccgtggcccggctgagccagcggttccccaaggccgagttcgccgagg
tgagcaagctggtgaccgacctgaccaaggtgcacaccgagtgctgccacggcgacctgctgg
agtgcgccgacgaccgggccgacctggccaagtacatctgcgagaaccaggacagcatcagc
agcaagctgaaggagtgctgcgagaagcccctgctggagaagagccactgcatcgccgaggtg
gagaacgacgagatgcccgccgacctgcccagcctggccgccgacttcgtggagagcaagga
cgtgtgcaagaactacgccgaggccaaggacgtgttcctgggcatgttcctgtacgagtacgccc
ggcggcaccccgactacagcgtggtgctgctgctgcggctggccaagacctacgagaccaccc
tggagaagtgctgcgccgccgccgacccccacgagtgctacgccaaggtgttcgacgagttcaa
gcccctggtggaggagccccagaacctgatcaagcagaactgcgagctgttcgagcagctggg
cgagtacaagttccagaacgccctgctggtgcggtacaccaagaaggtgccccaggtgagcac
ccccaccctggtggaggtgagccggaacctgggcaaggtgggcagcaagtgctgcaagcacc
ccgaggccaagcggatgccctgcgccgaggactacctgagcgtggtgctgaaccagctgtgcg
tgctgcacgagaagacccccgtgagcgaccgggtgaccaagtgctgcaccgagagcctggtga
accggcggccctgatcagcgccctggaggtggacgagacctacgtgcccaaggagttcaacg
ccgagaccttcaccttccacgccgacatctgcaccctgagcgagaaggagcggcagatcaaga
agcagaccgccctggtggagctggtgaagcacaagcccaaggccaccaaggagcagctgaag
gccgtgatggacgacttcgccgccttcgtggagaagtgctgcaaggccgacgacaaggagacc
tgcttcgccgaggagggcaagaagctggtggccgccagccaggccgccctgggcctgggcag
cggcggcggcggcagcggcggcggcggatctggtggaggtggcagtggaggagggggatc
cgctcgcaacggtgaccactgccctctgggtcctggtcgctgctgccgcctgcacaccgttcgcg
cttctctggaagacctgggttgggctgactgggttctgtctcctcgcgaagttcaggttaccatgtgc
atcggtgatgcccttctcagttccgcgctgctaacatgcacgctcagatcaaaacctctctgcacc
gcctgaaacctgacaccgttcctgctccttgctgcgttcctgcttcttacaaccctatggttctgatcc
agaaaaccgacaccggtgtttctctgcagacctacgacgacctgctggctaaagactgccactgc
atc
77 nucleic acid gatgctcataagtccgaagtcgcccacagattcaaggacctcggagaagaaaattttaaggccct
encoding cgtgcttatcgccttcgcccaatacctccagcagtccccgttcgaggaccacgtgaagctcgtgaa
SEQ ID NO: 25 cgaagtgaccgagtttgccaagacttgtgtggcggatgaatccgccgagaactgcgacaagagc
ctccacacgctgttcggcgacaagctgtgcaccgtcgccacgctgagagaaacttacggagaga
tggccgactgctgcgcaaagcaggagccggaacggaacgaatgcttcctgcaacataaggacg
ataaccctaacttgcctcgcctggtccgccctgaggtcgacgtgatgtgcaccgcgttccacgaca
acgaggaaacctttcttaagaagtacctgtacgagattgcgcggaggcacccttatttctacgcccc
cgaactgttgttcttcgccaagcggtacaaggctgcctttaccgaatgctgccaggccgccgataa
ggcggcttgcctgctgccgaagctcgacgagttgcgcgatgaggggaaggcgtcctccgctaa
gcagcggctgaaatgtgcgagcctccagaagttcggggagcgcgccttcaaggcctgggccgt
ggcgcgcctgtctcaacggttcccgaaggccgagttcgccgaagtgtcgaagctggtcaccgac
ctgacgaaagtgcacaccgaatgttgtcacggcgatctgctggaatgcgccgatgacagagccg
atttggccaagtacatctgcgaaaaccaggacagcatttcgtcaaagctgaaggaatgctgcgaa
aagcccttgctggaaaagtcccactgcatcgcggaagtggagaacgacgagatgcccgccgac
ctcccgtccctggccgccgatttcgtggagtcgaaggatgtgtgcaagaactacgcagaagccaa
ggacgtgttcctgggaatgtttctgtatgagtacgcccgccgccacccggactactcggtcgtgct
cctgctgcgactggcaaagacctacgaaaccactctggagaagtgctgcgccgccgcggaccc
gcacgagtgctacgcaaaggtgttcgacgagttcaagccacttgtcgaggagcctcagaacctga
tcaagcagaactgcgaactgttcgagcagctgggagagtacaaattccagaacgcgcttctcgtg
cgctacaccaagaaggtcccccaggtgtccactccgaccctggtggaagtgtccaggaacctgg
gaaaggtcggctccaagtgttgcaagcatcccgaggctaagcgcatgccctgcgccgaggacta
cttgtccgtggtgctgaatcagctgtgcgtgctccatgaaaagaccccagtgtccgacagagtgac
caagtgctgtaccgaatcgctcgtgaaccggcggccgtgcttttccgcactggaggtggacgaaa
cctacgtgccgaaggagttcaacgcagaaaccttcactttccacgccgacatctgcactctgtccg
agaaggagcggcagattaagaagcagactgccctggtggagcttgtgaaacacaagcctaagg
ccaccaaagagcagctgaaggccgtcatggatgatttcgcggccttcgtggaaaagtgttgtaaa
gcggacgacaaggagacttgcttcgccgaagaaggaaagaagctcgtggcagcgtcacaggc
cgctctgggcctcgctagcggtggagggggcagcggtggtggaggatccggtaccgcgcgca
acggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgct
ggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatc
ggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgca
ccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtg
ctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgc
cactgcata
78 nucleic acid gatgctcataagtccgaagtcgcccacagattcaaggacctcggagaagaaaattttaaggccct
encoding cgtgcttatcgccttcgcccaatacctccagcagtccccgttcgaggaccacgtgaagctcgtgaa
SEQ ID NO: 26 cgaagtgaccgagtttgccaagacttgtgtggcggatgaatccgccgagaactgcgacaagagc
ctccacacgctgttcggcgacaagctgtgcaccgtcgccacgctgagagaaacttacggagaga
tggccgactgctgcgcaaagcaggagccggaacggaacgaatgcttcctgcaacataaggacg
ataaccctaacttgcctcgcctggtccgccctgaggtcgacgtgatgtgcaccgcgttccacgaca
acgaggaaacctttcttaagaagtacctgtacgagattgcgcggaggcacccttatttctacgcccc
cgaactgttgttcttcgccaagcggtacaaggctgcctttaccgaatgctgccaggccgccgataa
ggcggcttgcctgctgccgaagctcgacgagttgcgcgatgaggggaaggcgtcctccgctaa
gcagcggctgaaatgtgcgagcctccagaagttcggggagcgcgccttcaaggcctgggccgt
ggcgcgcctgtctcaacggttcccgaaggccgagttcgccgaagtgtcgaagctggtcaccgac
ctgacgaaagtgcacaccgaatgttgtcacggcgatctgctggaatgcgccgatgacagagccg
atttggccaagtacatctgcgaaaaccaggacagcatttcgtcaaagctgaaggaatgctgcgaa
aagcccttgctggaaaagtcccactgcatcgcggaagtggagaacgacgagatgcccgccgac
ctcccgtccctggccgccgatttcgtggagtcgaaggatgtgtgcaagaactacgcagaagccaa
ggacgtgttcctgggaatgtttctgtatgagtacgcccgccgccacccggactactcggtcgtgct
cctgctgcgactggcaaagacctacgaaaccactctggagaagtgctgcgccgccgcggaccc
gcacgagtgctacgcaaaggtgttcgacgagttcaagccacttgtcgaggagcctcagaacctga
tcaagcagaactgcgaactgttcgagcagctgggagagtacaaattccagaacgcgcttctcgtg
cgctacaccaagaaggtcccccaggtgtccactccgaccctggtggaagtgtccaggaacctgg
gaaaggtcggctccaagtgttgcaagcatcccgaggctaagcgcatgccctgcgccgaggacta
cttgtccgtggtgctgaatcagctgtgcgtgctccatgaaaagaccccagtgtccgacagagtgac
caagtgctgtaccgaatcgctcgtgaaccggcggccgtgcttttccgcactggaggtggacgaaa
cctacgtgccgaaggagttcaacgcagaaaccttcactttccacgccgacatctgcactctgtccg
agaaggagcggcagattaagaagcagactgccctggtggagcttgtgaaacacaagcctaagg
ccaccaaagagcagctgaaggccgtcatggatgatttcgcggccttcgtggaaaagtgttgtaaa
gcggacgacaaggagacttgatcgccgaagaaggaaagaagctcgtggcagcgtcacaggc
cgctctgggcctcgctagcggaggtggcggatcaggtggcggaggtagcggtggaggcggct
ctggcggaggtggatcaggcggaggaggttccggtggaggaggctcaggaggaggaggaag
tggaggagggggatccggtaccgcgcgcaacggggaccactgtccgctcgggcccgggcgtt
gctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtc
gccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaa
acatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccct
gctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctc
cagacctatgatgacttgttagccaaagactgccactgcata
79 nucleic acid gatgctcataagtccgaagtcgcccacagattcaaggacctcggagaagaaaattttaaggccct
encoding cgtgcttatcgccttcgcccaatacctccagcagtccccgttcgaggaccacgtgaagctcgtgaa
SEQ ID NO: 27 cgaagtgaccgagtttgccaagacttgtgtggcggatgaatccgccgagaactgcgacaagagc
ctccacacgctgttcggcgacaagctgtgcaccgtcgccacgctgagagaaacttacggagaga
tggccgactgctgcgcaaagcaggagccggaacggaacgaatgcttcctgcaacataaggacg
ataaccctaacttgcctcgcctggtccgccctgaggtcgacgtgatgtgcaccgcgttccacgaca
acgaggaaacctttcttaagaagtacctgtacgagattgcgcggaggcacccttatttctacgcccc
cgaactgttgttcttcgccaagcggtacaaggctgcctttaccgaatgctgccaggccgccgataa
ggcggcttgcctgctgccgaagctcgacgagttgcgcgatgaggggaaggcgtcctccgctaa
gcagcggctgaaatgtgcgagcctccagaagttcggggagcgcgccttcaaggcctgggccgt
ggcgcgcctgtctcaacggttcccgaaggccgagttcgccgaagtgtcgaagctggtcaccgac
ctgacgaaagtgcacaccgaatgttgtcacggcgatctgctggaatgcgccgatgacagagccg
atttggccaagtacatctgcgaaaaccaggacagcatttcgtcaaagctgaaggaatgctgcgaa
aagcccttgctggaaaagtcccactgcatcgcggaagtggagaacgacgagatgcccgccgac
ctcccgtccctggccgccgatttcgtggagtcgaaggatgtgtgcaagaactacgcagaagccaa
ggacgtgttcctgggaatgtttctgtatgagtacgcccgccgccacccggactactcggtcgtgct
cctgctgcgactggcaaagacctacgaaaccactctggagaagtgctgcgccgccgcggaccc
gcacgagtgctacgcaaaggtgttcgacgagttcaagccacttgtcgaggagcctcagaacctga
tcaagcagaactgcgaactgttcgagcagctgggagagtacaaattccagaacgcgcttctcgtg
cgctacaccaagaaggtcccccaggtgtccactccgaccctggtggaagtgtccaggaacctgg
gaaaggtcggctccaagtgttgcaagcatcccgaggctaagcgcatgccctgcgccgaggacta
cttgtccgtggtgctgaatcagctgtgcgtgctccatgaaaagaccccagtgtccgacagagtgac
caagtgctgtaccgaatcgctcgtgaaccggcggccgtgcttttccgcactggaggtggacgaaa
cctacgtgccgaaggagttcaacgcagaaaccttcactttccacgccgacatctgcactctgtccg
agaaggagcggcagattaagaagcagactgccctggtggagcttgtgaaacacaagcctaagg
ccaccaaagagcagctgaaggccgtcatggatgatttcgcggccttcgtggaaaagtgttgtaaa
gcggacgacaaggagacttgatcgccgaagaaggaaagaagctcgtggcagcgtcacaggc
cgctctgggcctcgctagcgcacctgcccccgctccagctcctgcaccaggtaccgcgcgcaac
ggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctg
gaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcg
gcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcac
cgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgc
tcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgcc
actgcata
80 nucleic acid gatgctcataagtccgaagtcgcccacagattcaaggacctcggagaagaaaattttaaggccct
encoding cgtgcttatcgccttcgcccaatacctccagcagtccccgttcgaggaccacgtgaagctcgtgaa
SEQ ID NO: 28 cgaagtgaccgagtttgccaagacttgtgtggcggatgaatccgccgagaactgcgacaagagc
ctccacacgctgttcggcgacaagctgtgcaccgtcgccacgctgagagaaacttacggagaga
tggccgactgctgcgcaaagcaggagccggaacggaacgaatgcttcctgcaacataaggacg
ataaccctaacttgcctcgcctggtccgccctgaggtcgacgtgatgtgcaccgcgttccacgaca
acgaggaaacctttcttaagaagtacctgtacgagattgcgcggaggcacccttatttctacgcccc
cgaactgttgttcttcgccaagcggtacaaggctgcctttaccgaatgctgccaggccgccgataa
ggcggcttgcctgctgccgaagctcgacgagttgcgcgatgaggggaaggcgtcctccgctaa
gcagcggctgaaatgtgcgagcctccagaagttcggggagcgcgccttcaaggcctgggccgt
ggcgcgcctgtctcaacggttcccgaaggccgagttcgccgaagtgtcgaagctggtcaccgac
ctgacgaaagtgcacaccgaatgttgtcacggcgatctgctggaatgcgccgatgacagagccg
atttggccaagtacatctgcgaaaaccaggacagcatttcgtcaaagctgaaggaatgctgcgaa
aagcccttgctggaaaagtcccactgcatcgcggaagtggagaacgacgagatgcccgccgac
ctcccgtccctggccgccgatttcgtggagtcgaaggatgtgtgcaagaactacgcagaagccaa
ggacgtgttcctgggaatgtttctgtatgagtacgcccgccgccacccggactactcggtcgtgct
cctgctgcgactggcaaagacctacgaaaccactctggagaagtgctgcgccgccgcggaccc
gcacgagtgctacgcaaaggtgttcgacgagttcaagccacttgtcgaggagcctcagaacctga
tcaagcagaactgcgaactgttcgagcagctgggagagtacaaattccagaacgcgcttctcgtg
cgctacaccaagaaggtcccccaggtgtccactccgaccctggtggaagtgtccaggaacctgg
gaaaggtcggctccaagtgttgcaagcatcccgaggctaagcgcatgccctgcgccgaggacta
cttgtccgtggtgctgaatcagctgtgcgtgctccatgaaaagaccccagtgtccgacagagtgac
caagtgctgtaccgaatcgctcgtgaaccggcggccgtgcttttccgcactggaggtggacgaaa
cctacgtgccgaaggagttcaacgcagaaaccttcactttccacgccgacatctgcactctgtccg
agaaggagcggcagattaagaagcagactgccctggtggagcttgtgaaacacaagcctaagg
ccaccaaagagcagctgaaggccgtcatggatgatttcgcggccttcgtggaaaagtgttgtaaa
gcggacgacaaggagacttgatcgccgaagaaggaaagaagctcgtggcagcgtcacaggc
cgctctgggcctcgctagcgcacctgcccccgctccagcacccgccccagcccctgctcccgca
ccagctcctgcaccaggtaccgctcgcaacggtgaccactgccctctgggtcctggtcgctgctg
ccgcctgcacaccgttcgcgcttctctggaagacctgggttgggctgactgggttctgtctcctcgc
gaagttcaggttaccatgtgcatcggtgcttgcccttctcagttccgcgctgctaacatgcacgctca
gatcaaaacctctctgcaccgcctgaaacctgacaccgttcctgctccttgctgcgttcctgcttctta
caaccctatggttctgatccagaaaaccgacaccggtgtttctctgcagacctacgacgacctgct
ggctaaagactgccactgcatc
81 nucleic acid gatgctcataagtccgaagtcgcccacagattcaaggacctcggagaagaaaattttaaggccct
encoding cgtgcttatcgccttcgcccaatacctccagcagtccccgttcgaggaccacgtgaagctcgtgaa
SEQ ID NO: 29 cgaagtgaccgagtttgccaagacttgtgtggcggatgaatccgccgagaactgcgacaagagc
ctccacacgctgttcggcgacaagctgtgcaccgtcgccacgctgagagaaacttacggagaga
tggccgactgctgcgcaaagcaggagccggaacggaacgaatgcttcctgcaacataaggacg
ataaccctaacttgcctcgcctggtccgccctgaggtcgacgtgatgtgcaccgcgttccacgaca
acgaggaaacctttcttaagaagtacctgtacgagattgcgcggaggcacccttatttctacgcccc
cgaactgttgttcttcgccaagcggtacaaggctgcctttaccgaatgctgccaggccgccgataa
ggcggcttgcctgctgccgaagctcgacgagttgcgcgatgaggggaaggcgtcctccgctaa
gcagcggctgaaatgtgcgagcctccagaagttcggggagcgcgccttcaaggcctgggccgt
ggcgcgcctgtctcaacggttcccgaaggccgagttcgccgaagtgtcgaagctggtcaccgac
ctgacgaaagtgcacaccgaatgttgtcacggcgatctgctggaatgcgccgatgacagagccg
atttggccaagtacatctgcgaaaaccaggacagcatttcgtcaaagctgaaggaatgctgcgaa
aagcccttgctggaaaagtcccactgcatcgcggaagtggagaacgacgagatgcccgccgac
ctcccgtccctggccgccgatttcgtggagtcgaaggatgtgtgcaagaactacgcagaagccaa
ggacgtgttcctgggaatgtttctgtatgagtacgcccgccgccacccggactactcggtcgtgct
cctgctgcgactggcaaagacctacgaaaccactctggagaagtgctgcgccgccgcggaccc
gcacgagtgctacgcaaaggtgttcgacgagttcaagccacttgtcgaggagcctcagaacctga
tcaagcagaactgcgaactgttcgagcagctgggagagtacaaattccagaacgcgcttctcgtg
cgctacaccaagaaggtcccccaggtgtccactccgaccctggtggaagtgtccaggaacctgg
gaaaggtcggctccaagtgttgcaagcatcccgaggctaagcgcatgccctgcgccgaggacta
cttgtccgtggtgctgaatcagctgtgcgtgctccatgaaaagaccccagtgtccgacagagtgac
caagtgctgtaccgaatcgctcgtgaaccggcggccgtgcttttccgcactggaggtggacgaaa
cctacgtgccgaaggagttcaacgcagaaaccttcactttccacgccgacatctgcactctgtccg
agaaggagcggcagattaagaagcagactgccctggtggagcttgtgaaacacaagcctaagg
ccaccaaagagcagctgaaggccgtcatggatgatttcgcggccttcgtggaaaagtgttgtaaa
gcggacgacaaggagacttgatcgccgaagaaggaaagaagctcgtggcagcgtcacaggc
cgctctgggcctcgctagcgcacctgcccccgctccagccccagctcctgcacctgctccagca
ccagctcctgcaccagctccagcccctgcacctgcacccgctccagccccagctcctgcacctg
ctccagcaccaggtaccgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgcc
gtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacg
ggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgca
cgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgt
gcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacct
atgatgacttgttagccaaagactgccactgcata
82 nucleic acid gatgctcataagtccgaagtcgcccacagattcaaggacctcggagaagaaaattttaaggccct
encoding cgtgcttatcgccttcgcccaatacctccagcagtccccgttcgaggaccacgtgaagctcgtgaa
SEQ ID NO: 30 cgaagtgaccgagtttgccaagacttgtgtggcggatgaatccgccgagaactgcgacaagagc
ctccacacgctgttcggcgacaagctgtgcaccgtcgccacgctgagagaaacttacggagaga
tggccgactgctgcgcaaagcaggagccggaacggaacgaatgcttcctgcaacataaggacg
ataaccctaacttgcctcgcctggtccgccctgaggtcgacgtgatgtgcaccgcgttccacgaca
acgaggaaacctttcttaagaagtacctgtacgagattgcgcggaggcacccttatttctacgcccc
cgaactgttgttcttcgccaagcggtacaaggctgcctttaccgaatgctgccaggccgccgataa
ggcggcttgcctgctgccgaagctcgacgagttgcgcgatgaggggaaggcgtcctccgctaa
gcagcggctgaaatgtgcgagcctccagaagttcggggagcgcgccttcaaggcctgggccgt
ggcgcgcctgtctcaacggttcccgaaggccgagttcgccgaagtgtcgaagctggtcaccgac
ctgacgaaagtgcacaccgaatgttgtcacggcgatctgctggaatgcgccgatgacagagccg
atttggccaagtacatctgcgaaaaccaggacagcatttcgtcaaagctgaaggaatgctgcgaa
aagcccttgctggaaaagtcccactgcatcgcggaagtggagaacgacgagatgcccgccgac
ctcccgtccctggccgccgatttcgtggagtcgaaggatgtgtgcaagaactacgcagaagccaa
ggacgtgttcctgggaatgtttctgtatgagtacgcccgccgccacccggactactcggtcgtgct
cctgctgcgactggcaaagacctacgaaaccactctggagaagtgctgcgccgccgcggaccc
gcacgagtgctacgcaaaggtgttcgacgagttcaagccacttgtcgaggagcctcagaacctga
tcaagcagaactgcgaactgttcgagcagctgggagagtacaaattccagaacgcgcttctcgtg
cgctacaccaagaaggtcccccaggtgtccactccgaccctggtggaagtgtccaggaacctgg
gaaaggtcggctccaagtgttgcaagcatcccgaggctaagcgcatgccctgcgccgaggacta
cttgtccgtggtgctgaatcagctgtgcgtgctccatgaaaagaccccagtgtccgacagagtgac
caagtgctgtaccgaatcgctcgtgaaccggcggccgtgcttttccgcactggaggtggacgaaa
cctacgtgccgaaggagttcaacgcagaaaccttcactttccacgccgacatctgcactctgtccg
agaaggagcggcagattaagaagcagactgccctggtggagcttgtgaaacacaagcctaagg
ccaccaaagagcagctgaaggccgtcatggatgatttcgcggccttcgtggaaaagtgttgtaaa
gcggacgacaaggagacttgatcgccgaagaaggaaagaagctcgtggcagcgtcacaggc
cgctctgggcctcgctagcgaagcagcagccaaagaagcagccgcaaaagaagcagccgcta
aggaggccgcagcaaagggtaccgcgcgcaacggggaccactgtccgctcgggcccgggcg
ttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgt
cgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggca
aacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgcc
ctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgc
tccagacctatgatgacttgttagccaaagactgccactgcata
83 nucleic acid gacgcccacaagagcgaggtggcccaccggttcaaggacctgggcgaggagaacttcaaggc
encoding cctggtgctgatcgccttcgcccagtacctgcagcagtccccatcgaggaccacgtgaagctgg
SEQ ID NO: 40 tgaacgaggtgaccgagttcgccaagacctgcgtggccgacgagagcgccgagaactgcgac
aagagcctgcacaccctgttcggcgacaagctgtgcaccgtggccaccctgcgggagacctac
ggcgagatggccgactgctgcgccaagcaggagcccgagcggaacgagtgatcctgcagca
caaggacgacaaccccaacctgccccggctggtgcggcccgaggtggacgtgatgtgcaccg
ccttccacgacaacgaggagaccttcctgaagaagtacctgtacgagatcgcccggcggcaccc
ctacttctacgcccccgagctgctgttcttcgccaagcggtacaaggccgccttcaccgagtgctg
ccaggccgccgacaaggccgcctgcctgctgcccaagctggacgagctgcgggacgagggc
aaggccagcagcgccaagcagcggctgaagtgcgccagcctgcagaagttcggcgagcggg
ccttcaaggcctgggccgtggcccggctgagccagcggttccccaaggccgagttcgccgagg
tgagcaagctggtgaccgacctgaccaaggtgcacaccgagtgctgccacggcgacctgctgg
agtgcgccgacgaccgggccgacctggccaagtacatctgcgagaaccaggacagcatcagc
agcaagctgaaggagtgctgcgagaagcccctgctggagaagagccactgcatcgccgaggtg
gagaacgacgagatgcccgccgacctgcccagcctggccgccgacttcgtggagagcaagga
cgtgtgcaagaactacgccgaggccaaggacgtgttcctgggcatgttcctgtacgagtacgccc
ggcggcaccccgactacagcgtggtgctgctgctgcggctggccaagacctacgagaccaccc
tggagaagtgctgcgccgccgccgacccccacgagtgctacgccaaggtgttcgacgagttcaa
gcccctggtggaggagccccagaacctgatcaagcagaactgcgagctgttcgagcagctggg
cgagtacaagttccagaacgccctgctggtgcggtacaccaagaaggtgccccaggtgagcac
ccccaccctggtggaggtgagccggaacctgggcaaggtgggcagcaagtgctgcaagcacc
ccgaggccaagcggatgccctgcgccgaggactacctgagcgtggtgctgaaccagctgtgcg
tgctgcacgagaagacccccgtgagcgaccgggtgaccaagtgctgcaccgagagcctggtga
accggcggccctgatcagcgccctggaggtggacgagacctacgtgcccaaggagttcaacg
ccgagaccttcaccttccacgccgacatctgcaccctgagcgagaaggagcggcagatcaaga
agcagaccgccctggtggagctggtgaagcacaagcccaaggccaccaaggagcagctgaag
gccgtgatggacgacttcgccgccttcgtggagaagtgctgcaaggccgacgacaaggagacc
tgcttcgccgaggagggcaagaagctggtggccgccagccaggccgccctgggcctgggcag
cggcggcggcggcagcggcggcggcggatctggtggaggtggcagtggaggagggggatc
cgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccg
cgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgac
catgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagac
gagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaa
tcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagc
caaagactgccactgcata
84 nucleic acid gatgcacacaagagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttg
encoding gtgttgattgcctttgctcagtatcttcagcaggccccatttgaagatcatgtaaaattagtgaatgaa
SEQ ID NO: 55 gtaactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcatac
cctttttggagacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgct
gtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctc
ccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacattttt
gaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgct
aaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaa
agctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagt
ctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttccc
aaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgcc
atggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaag
attcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgc
cgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaagg
atgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaa
ggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaa
gtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctatgtgg
aagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagcttggagagtacaaattcca
gaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtct
caagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgt
gcagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtg
acagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaag
tcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcaca
ctttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcc
caaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagatttgtagagaagtgctg
caaggctgacgataaggagacctgattgccgaggagggtaaaaaacttgttgctgcaagtcaag
ctgccttaggcttaggcagcggcggcggcggcagcggcggcggcggatctggtggaggtggc
agtggaggagggggatccggcggcggcggcagcggcggcggcggatctggtggaggtggc
agtggaggagggggatccgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctg
ccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgcca
cgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatg
cacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctg
cgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccaga
cctatgatgacttgttagccaaagactgccactgcata
85 nucleic acid gatgcacacaagagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttg
encoding gtgttgattgcctttgctcagtatcttcagcaggccccatttgaagatcatgtaaaattagtgaatgaa
SEQ ID NO: 56 gtaactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcatac
cctttttggagacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgct
gtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctc
ccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacattttt
gaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgct
aaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaa
agctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagt
ctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttccc
aaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgcc
atggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaag
attcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgc
cgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaagg
atgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaa
ggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaa
gtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtgg
aagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagcttggagagtacaaattcca
gaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtct
caagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgt
gcagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtg
acagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaag
tcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcaca
ctttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcc
caaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctg
caaggctgacgataaggagacctgattgccgaggagggtaaaaaacttgttgctgcaagtcaag
ctgccttaggcttagcacctgcccccgctccagcacccgccccagcccctgctcccgcaccagct
cctgcaccagcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcac
acggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtg
caagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcag
atcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgcc
agctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatga
cttgttagccaaagactgccactgcata
86 nucleic acid gatgcacacaagagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttg
encoding gtgttgattgcctttgctcagtatcttcagcagtccccatttgaagatcatgtaaaattagtgaatgaag
SEQ ID NO: 40 taactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcatacc
(Codon ctttttggagacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgctgt
optimization 1) gcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctccc
ccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttga
aaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaa
aaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaag
ctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtct
ccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttccca
aagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgcca
tggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaagat
tcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccg
aagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggat
gtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaag
gcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaag
tgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtgga
agagcctcagaatttaatcaaacaaaattgtgagattttgagcagatggagagtacaaattccag
aatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactatgtagaggtctc
aagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtg
cagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtga
cagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagt
cgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcacac
tttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagccc
aaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgc
aaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgctgcaagtcaagc
tgccttaggcttaggcagcggcggcggcggcagcggcggcggcggatctggtggaggtggca
gtggaggagggggatccgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgc
cgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccac
gggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgc
acgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgc
gtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagac
ctatgatgacttgttagccaaagactgccactgcata
87 nucleic acid gacgcccacaagagcgaggtggcccacagattcaaggacctgggcgaggaaaacttcaaggc
encoding cctggtgctgatcgccttcgcccagtacctgcagcagagcccatcgaggaccacgtgaagctgg
SEQ ID NO: 40 tcaacgaagtgaccgagttcgccaagacctgcgtggccgacgagagcgccgagaactgcgaca
(Codon agagcctgcacaccctgttcggcgacaagctgtgcaccgtggccaccctgcgggaaacctacg
optimization 2) gcgagatggccgactgctgcgccaagcaggaacccgagcggaacgagtgcttcctgcagcac
aaggacgacaaccccaacctgcccagactcgtgcggcccgaggtggacgtgatgtgcaccgcc
ttccacgacaacgaggaaaccttcctgaagaagtacctgtacgagatcgccagacggcacccct
acttctacgcccccgagctgctgttcttcgccaagcggtacaaggccgccttcaccgagtgctgcc
aggccgccgataaggccgcctgcctgctgcccaagctggacgagctgagagatgagggcaag
gccagctccgccaagcagcggctgaagtgcgccagcctgcagaagttcggcgagcgggccttt
aaggcttgggctgtggcccggctgagccagagattccccaaggccgagtttgccgaggtgtcca
agctggtcaccgacctgaccaaggtgcacaccgagtgttgtcacggcgacctgctggaatgcgc
cgacgacagagccgacctggccaagtacatctgcgagaaccaggacagcatcagcagcaagct
gaaagagtgctgcgagaagcccctgctggaaaagagccactgtatcgccgaggtggaaaacga
cgagatgcccgctgacctgcccagcctggccgccgacttcgtggaaagcaaggacgtgtgcaa
gaactacgccgaggccaaggatgtgttcctgggcatgttcctgtatgagtacgcccgcagacacc
ccgactacagcgtggtgctgctgctgcggctggccaagacctacgagacaaccctggaaaagtg
ctgcgccgctgccgacccccacgagtgctacgccaaggtgttcgacgagttcaagcctctggtg
gaagaaccccagaacctgatcaagcagaactgcgagctgttcgagcagctgggcgagtacaag
ttccagaacgccctgctcgtgcggtacaccaagaaagtgccccaggtgtccacccccaccctggt
cgaagtgtcccggaacctgggcaaagtgggcagcaagtgctgcaagcaccctgaggccaagc
ggatgccctgcgccgaggactacctgtccgtggtgctgaaccagctgtgcgtgctgcacgagaa
aacccccgtgtccgacagagtgaccaagtgctgtaccgagagcctggtcaacagacggccctgc
ttcagcgccctggaagtggacgagacatacgtgcccaaagagttcaacgccgagacattcacctt
ccacgccgacatctgcaccctgagcgagaaagagcggcagatcaagaagcagaccgccctgg
tcgagctggtcaagcacaagcccaaggccaccaaagaacagctgaaggccgtgatggacgact
tcgccgccttcgtcgagaagtgttgcaaggccgacgacaaagagacatgcttcgccgaagaggg
caagaaactggtggccgcctctcaggccgccctgggactgggatctggcggcggaggaagcg
gaggcggaggatctgggggaggcggctctggcggagggggatccgccagaaatggcgacca
ctgtcccctgggccctggccggtgttgcagactgcacacagtgcgggccagcctggaagatctg
ggctgggccgattgggtgctgagccccagagaagtgcaggtcacaatgtgcatcggcgcctgcc
ccagccagttcagagccgccaacatgcacgcccagatcaagaccagcctgcaccggctgaagc
ccgacaccgtgcctgccccttgttgcgtgcccgccagctacaaccccatggtgctgattcagaaa
accgacaccggcgtgtccctgcagacctacgacgatctgctggccaaggactgccactgcatc
88 nucleic acid gatgcacacaagagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttg
encoding gtgttgattgcctttgctcagtatcttcagcaggccccatttgaagatcatgtaaaattagtgaatgaa
SEQ ID NO: 48 gtaactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcatac
cctttttggagacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgct
gtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctc
ccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacattttt
gaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgct
aaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaa
agctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagt
ctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttccc
aaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgcc
atggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaag
attcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgc
cgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaagg
atgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaa
ggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaa
gtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtgg
aagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagcttggagagtacaaattcca
gaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtct
caagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgt
gcagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtg
acagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaag
tcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcaca
ctttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcc
caaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctg
caaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgctgcaagtcaag
ctgccttaggcttaggcagcggcggcggcggcagcggcggcggcggatctggtggaggtggc
agtggaggagggggatccgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctg
ccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgcca
cgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatg
cacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctg
cgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccaga
cctatgatgacttgttagccaaagactgccactgcata
89 nucleic acid gatgcacacaagagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttg
encoding gtgttgattgcctttgctcagtatcttcagcagtccccatttgaagatcatgtaaaattagtgaatgaag
SEQ ID NO: 59 taactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcatacc
attttggagacaaattatgcacagttgcaactatcgtgaaacctatggtgaaatggctgactgctgt
gcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctccc
ccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttga
aaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaa
aaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaag
ctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtct
ccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttccca
aagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgcca
tggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaagat
tcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccg
aagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggat
gtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaag
gcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaag
tgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtgga
agagcctcagaatttaatcaaacaaaattgtgagattttgagcagcttggagagtacaaattccag
aatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtctc
aagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtg
cagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtga
cagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagt
cgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcacac
tttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagccc
aaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgc
aaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgctgcaagtcaagc
tgccttaggcttagcacctgcccccgctccagcacccgccccagcccctgctcccgcaccagctc
ctgcaccagcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcaca
cggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgc
aagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcaga
tcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgcca
gctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgact
tgttagccaaagactgccactgcata
90 nucleic acid gatgcacacaagagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttg
encoding gtgttgattgcctttgctcagtatcttcagcagtccccatttgaagatcatgtaaaattagtgaatgaag
SEQ ID NO: 60 taactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcatacc
(Codon ctttttggagacaaattatgcacagttgcaactatcgtgaaacctatggtgaaatggctgactgctgt
optimization 1) gcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctccc
ccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttga
aaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaa
aaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaag
ctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtct
ccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttccca
aagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgcca
tggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaagat
tcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccg
aagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggat
gtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaag
gcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaag
tgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtgga
agagcctcagaatttaatcaaacaaaattgtgagattttgagcagatggagagtacaaattccag
aatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtctc
aagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtg
cagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtga
cagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagt
cgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcacac
tttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagccc
aaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgc
aaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgctgcaagtcaagc
tgccttaggcttaggcagcggcggcggcggcagcggcggcggcggatctggtggaggtggca
gtggaggagggggatccggcggcggcggcagcggcggcggcggatctggtggaggtggca
gtggaggagggggatccgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgc
cgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccac
gggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgc
acgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgc
gtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagac
ctatgatgacttgttagccaaagactgccactgcata
91 nucleic acid gacgcccacaagagcgaggtggcccaccggttcaaggacctgggcgaggagaacttcaaggc
encoding cctggtgctgatcgccttcgcccagtacctgcagcagtccccatcgaggaccacgtgaagctgg
SEQ ID NO: 70 tgaacgaggtgaccgagttcgccaagacctgcgtggccgacgagagcgccgagaactgcgac
aagagcctgcacaccctgttcggcgacaagctgtgcaccgtggccaccctgcgggagacctac
ggcgagatggccgactgctgcgccaagcaggagcccgagcggaacgagtgatcctgcagca
caaggacgacaaccccaacctgccccggctggtgcggcccgaggtggacgtgatgtgcaccg
ccttccacgacaacgaggagaccttcctgaagaagtacctgtacgagatcgcccggcggcaccc
ctacttctacgcccccgagctgctgttcttcgccaagcggtacaaggccgccttcaccgagtgctg
ccaggccgccgacaaggccgcctgcctgctgcccaagctggacgagctgcgggacgagggc
aaggccagcagcgccaagcagcggctgaagtgcgccagcctgcagaagttcggcgagcggg
ccttcaaggcctgggccgtggcccggctgagccagcggttccccaaggccgagttcgccgagg
tgagcaagctggtgaccgacctgaccaaggtgcacaccgagtgctgccacggcgacctgctgg
agtgcgccgacgaccgggccgacctggccaagtacatctgcgagaaccaggacagcatcagc
agcaagctgaaggagtgctgcgagaagcccctgctggagaagagccactgcatcgccgaggtg
gagaacgacgagatgcccgccgacctgcccagcctggccgccgacttcgtggagagcaagga
cgtgtgcaagaactacgccgaggccaaggacgtgttcctgggcatgttcctgtacgagtacgccc
ggcggcaccccgactacagcgtggtgctgctgctgcggctggccaagacctacgagaccaccc
tggagaagtgctgcgccgccgccgacccccacgagtgctacgccaaggtgttcgacgagttcaa
gcccctggtggaggagccccagaacctgatcaagcagaactgcgagctgttcgagcagctggg
cgagtacaagttccagaacgccctgctggtgcggtacaccaagaaggtgccccaggtgagcac
ccccaccctggtggaggtgagccggaacctgggcaaggtgggcagcaagtgctgcaagcacc
ccgaggccaagcggatgccctgcgccgaggactacctgagcgtggtgctgaaccagctgtgcg
tgctgcacgagaagacccccgtgagcgaccgggtgaccaagtgctgcaccgagagcctggtga
accggcggccctgatcagcgccctggaggtggacgagacctacgtgcccaaggagttcaacg
ccgagaccttcaccttccacgccgacatctgcaccctgagcgagaaggagcggcagatcaaga
agcagaccgccctggtggagctggtgaagcacaagcccaaggccaccaaggagcagctgaag
gccgtgatggacgacttcgccgccttcgtggagaagtgctgcaaggccgacgacaaggagacc
tgcttcgccgaggagggcaagaagctggtggccgccagccaggccgccctgggcctgggcag
cggcggcggcggcagcggcggcggcggatctggtggaggtggcagtggaggagggggatc
cgctcgcaacggtgaccactgccctctgggtcctggtcgctgctgccgcctgcacaccgttcgcg
cttctctggaagacctgggttgggctgactgggttctgtctcctcgcgaagttcaggttaccatgtgc
atcggtgcttgcccttctcagttccgcgctgctaacatgcacgcttggatcaaaacctctctgcacc
gcctgaaacctgacaccgttcctgctccttgctgcgttcctgcttcttacaaccctatggttctgatcc
agaaaaccgacaccggtgtttctctgcagacctacgacgacctgctggctaaagactgccactgc
atc
92 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GS-(GGGGS)8- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
(deletion 4) KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDHCPLGPG
RCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANM
HAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDD
LLAKDCHCI
93 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GS-(GGGGS)8- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
GDF15 AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
(deletion 5) KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSHCPLGPGR
CCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANM
HAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDD
LLAKDCHCI
94 6x histidine tag- EFHHHHHHDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFED
HSA (C34S)- HVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRET
GS-(GGGGS)8- YGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFH
HRV3C site DNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADK
GDF15 AACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARL
SQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYIC
ENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVES
KDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTL
EKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKF
QNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPC
AEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVD
ETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATK
EQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL
GSGGGGSGGGGSGGGGSGGGGSLEVLFQGPARNGDHCPLGPGRCC
RLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHA
QIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLL
AKDCHCI
95 nucleic acid GATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGG
encoding AGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTA
SEQ ID NO: 92 TCTTCAGCAGTCCCCATTTGAAGATCATGTAAAATTAGTGAATGA
(Codon AGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTG
optimization 1) AAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTAT
GCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGAC
TGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCA
ACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAG
AGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACA
TTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTAC
TTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCT
GCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTG
TTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTC
TGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAG
AAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGA
TTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGAT
CTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGA
ATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAA
ATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAA
CCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGA
TGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGA
AAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCT
TCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATT
ACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACC
ACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTA
TGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTC
AGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGA
GAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAA
AGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAA
ACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCA
AAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAA
CCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAG
TCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGC
TTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTT
AATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCT
GAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGC
TCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGC
TGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGC
TGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTG
TTGCTGCAAGTCAAGCTGCCTTAGGGCTTGGAAGCGGCGGAGGG
GGGAGTGGCGGCGGTGGCTCCGGGGGGGGCGGATCCGGCGGAG
GGGGCAGCGGGGGTGGAGGGAGTGGCGGGGGAGGATCAGGGGG
AGGAGGATCAGGAGGGGGCGGAAGTGATCATTGCCCTCTCGGGC
CCGGACGGTGTTGCCGCCTCCACACTGTGAGGGCTTCACTTGAA
GACCTTGGATGGGCCGACTGGGTGCTGTCCCCAAGAGAGGTACA
AGTCACAATGTGTATTGGCGCCTGCCCCAGCCAGTTTCGCGCCGC
TAACATGCACGCCCAGATAAAAACCAGCCTGCACCGCCTGAAGC
CCGACACGGTGCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAAT
CCCATGGTGCTCATTCAAAAGACCGACACCGGGGTGTCGCTCCA
GACCTATGATGACTTGTTAGCCAAAGACTGCCACTGCATA
96 nucleic acid GGGGACCACTGTCCGCTCGGGCCCGGGCGTTGCTGCCGTCTGCA
encoding CACGGTCCGCGCGTCGCTGGAAGACCTGGGCTGGGCCGATTGGG
SEQ ID NO: 7 TGCTGTCGCCACGGGAGGTGCAAGTGACCATGTGCATCGGCGCG
TGCCCGAGCCAGTTCCGGGCGGCAAACATGCACGCGCAGATCAA
GACGAGCCTGCACCGCCTGAAGCCCGACACGGTGCCAGCGCCCT
GCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAG
ACCGACACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGC
CAAAGACTGCCACTGCATA
97 nucleic acid GACCACTGTCCGCTCGGGCCCGGGCGTTGCTGCCGTCTGCACAC
encoding GGTCCGCGCGTCGCTGGAAGACCTGGGCTGGGCCGATTGGGTGC
SEQ ID NO: 8 TGTCGCCACGGGAGGTGCAAGTGACCATGTGCATCGGCGCGTGC
(Codon CCGAGCCAGTTCCGGGCGGCAAACATGCACGCGCAGATCAAGAC
optimization 1) GAGCCTGCACCGCCTGAAGCCCGACACGGTGCCAGCGCCCTGCT
GCGTGCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACC
GACACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAA
AGACTGCCACTGCATA
98 nucleic acid GATCATTGCCCTCTCGGGCCCGGACGGTGTTGCCGCCTCCACACT
encoding GTGAGGGCTTCACTTGAAGACCTTGGATGGGCCGACTGGGTGCT
SEQ ID NO: 8 GTCCCCAAGAGAGGTACAAGTCACAATGTGTATTGGCGCCTGCC
(Codon CCAGCCAGTTTCGCGCCGCTAACATGCACGCCCAGATAAAAACC
optimization 2) AGCCTGCACCGCCTGAAGCCCGACACGGTGCCAGCGCCCTGCTG
CGTGCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCG
ACACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAAA
GACTGCCACTGCATA
99 nucleic acid GATCATTGTCCCCTTGGACCGGGTAGATGCTGTCGCCTGCACACT
encoding GTGCGGGCTTCACTGGAGGACCTCGGCTGGGCTGACTGGGTGCT
SEQ ID NO: 8 GTCCCCACGGGAGGTGCAAGTGACCATGTGCATCGGCGCCTGTC
(Codon CTTCGCAATTCCGGGCCGCGAATATGCACGCCCAGATCAAGACC
optimization 3) TCCCTGCATCGCCTCAAGCCCGACACTGTGCCTGCTCCATGCTGT
GTGCCGGCCTCCTATAACCCCATGGTGCTGATCCAGAAAACCGA
TACCGGCGTCAGCCTGCAGACGTATGATGATCTGCTGGCCAAGG
ACTGCCATTGCATC
100 nucleic acid CACTGTCCGCTCGGGCCCGGGCGTTGCTGCCGTCTGCACACGGTC
encoding CGCGCGTCGCTGGAAGACCTGGGCTGGGCCGATTGGGTGCTGTC
SEQ ID NO: 9 GCCACGGGAGGTGCAAGTGACCATGTGCATCGGCGCGTGCCCGA
(Codon GCCAGTTCCGGGCGGCAAACATGCACGCGCAGATCAAGACGAG
optimization 1) CCTGCACCGCCTGAAGCCCGACACGGTGCCAGCGCCCTGCTGCG
TGCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCGAC
ACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAAAGA
CTGCCACTGCATA
101 nucleic acid CATTGCCCTCTCGGGCCCGGACGGTGTTGCCGCCTCCACACTGTG
encoding AGGGCTTCACTTGAAGACCTTGGATGGGCCGACTGGGTGCTGTC
SEQ ID NO: 9 CCCAAGAGAGGTACAAGTCACAATGTGTATTGGCGCCTGCCCCA
(Codon GCCAGTTTCGCGCCGCTAACATGCACGCCCAGATAAAAACCAGC
optimization 2) CTGCACCGCCTGAAGCCCGACACGGTGCCAGCGCCCTGCTGCGT
GCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCGACA
CCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAAAGAC
TGCCACTGCATA
102 nucleic acid CATTGTCCCCTTGGACCGGGTAGATGCTGTCGCCTGCACACTGTG
encoding CGGGCTTCACTGGAGGACCTCGGCTGGGCTGACTGGGTGCTGTC
SEQ ID NO: 9 CCCACGGGAGGTGCAAGTGACCATGTGCATCGGCGCCTGTCCTT
(Codon CGCAATTCCGGGCCGCGAATATGCACGCCCAGATCAAGACCTCC
optimization 3) CTGCATCGCCTCAAGCCCGACACTGTGCCTGCTCCATGCTGTGTG
CCGGCCTCCTATAACCCCATGGTGCTGATCCAGAAAACCGATAC
CGGCGTCAGCCTGCAGACGTATGATGATCTGCTGGCCAAGGACT
GCCATTGCATC
103 nucleic acid TGTCCGCTCGGGCCCGGGCGTTGCTGCCGTCTGCACACGGTCCGC
encoding GCGTCGCTGGAAGACCTGGGCTGGGCCGATTGGGTGCTGTCGCC
SEQ ID NO: 10 ACGGGAGGTGCAAGTGACCATGTGCATCGGCGCGTGCCCGAGCC
(Codon AGTTCCGGGCGGCAAACATGCACGCGCAGATCAAGACGAGCCTG
optimization 1) CACCGCCTGAAGCCCGACACGGTGCCAGCGCCCTGCTGCGTGCC
CGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCGACACCG
GGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAAAGACTGC
CACTGCATA
104 nucleic acid TGCCCTCTCGGGCCCGGACGGTGTTGCCGCCTCCACACTGTGAG
encoding GGCTTCACTTGAAGACCTTGGATGGGCCGACTGGGTGCTGTCCC
SEQ ID NO: 10 CAAGAGAGGTACAAGTCACAATGTGTATTGGCGCCTGCCCCAGC
(Codon CAGTTTCGCGCCGCTAACATGCACGCCCAGATAAAAACCAGCCT
optimization 2) GCACCGCCTGAAGCCCGACACGGTGCCAGCGCCCTGCTGCGTGC
CCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCGACACC
GGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAAAGACTG
CCACTGCATA
105 nucleic acid TGTCCCCTTGGACCGGGTAGATGCTGTCGCCTGCACACTGTGCGG
encoding GCTTCACTGGAGGACCTCGGCTGGGCTGACTGGGTGCTGTCCCC
SEQ ID NO: 10 ACGGGAGGTGCAAGTGACCATGTGCATCGGCGCCTGTCCTTCGC
(Codon AATTCCGGGCCGCGAATATGCACGCCCAGATCAAGACCTCCCTG
optimization 3) CATCGCCTCAAGCCCGACACTGTGCCTGCTCCATGCTGTGTGCCG
GCCTCCTATAACCCCATGGTGCTGATCCAGAAAACCGATACCGG
CGTCAGCCTGCAGACGTATGATGATCTGCTGGCCAAGGACTGCC
ATTGCATC
106 nucleic acid TGCCGTCTGCACACGGTCCGCGCGTCGCTGGAAGACCTGGGCTG
encoding GGCCGATTGGGTGCTGTCGCCACGGGAGGTGCAAGTGACCATGT
SEQ ID NO: 11 GCATCGGCGCGTGCCCGAGCCAGTTCCGGGCGGCAAACATGCAC
(Codon GCGCAGATCAAGACGAGCCTGCACCGCCTGAAGCCCGACACGGT
optimization 1) GCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGC
TCATTCAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGAT
GACTTGTTAGCCAAAGACTGCCACTGCATA
107 nucleic acid TGCCGCCTCCACACTGTGAGGGCTTCACTTGAAGACCTTGGATG
encoding GGCCGACTGGGTGCTGTCCCCAAGAGAGGTACAAGTCACAATGT
SEQ ID NO: 11 GTATTGGCGCCTGCCCCAGCCAGTTTCGCGCCGCTAACATGCAC
(Codon GCCCAGATAAAAACCAGCCTGCACCGCCTGAAGCCCGACACGGT
optimization 2) GCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGC
TCATTCAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGAT
GACTTGTTAGCCAAAGACTGCCACTGCATA
108 nucleic acid TGTCGCCTGCACACTGTGCGGGCTTCACTGGAGGACCTCGGCTG
encoding GGCTGACTGGGTGCTGTCCCCACGGGAGGTGCAAGTGACCATGT
SEQ ID NO: 11 GCATCGGCGCCTGTCCTTCGCAATTCCGGGCCGCGAATATGCAC
(Codon GCCCAGATCAAGACCTCCCTGCATCGCCTCAAGCCCGACACTGT
optimization 3) GCCTGCTCCATGCTGTGTGCCGGCCTCCTATAACCCCATGGTGCT
GATCCAGAAAACCGATACCGGCGTCAGCCTGCAGACGTATGATG
ATCTGCTGGCCAAGGACTGCCATTGCATC
109 nucleic acid GATGCGCACAAGTCGGAAGTGGCCCATCGCTTTAAGGACCTGGG
encoding AGAAGAGAACTTCAAGGCCCTGGTCCTGATCGCGTTCGCCCAGT
SEQ ID NO: 60 ACCTCCAGCAGTCCCCGTTTGAGGACCACGTCAAGCTTGTGAAC
(Codon GAAGTGACCGAGTTCGCAAAGACTTGTGTGGCCGATGAGTCCGC
optimization 2) CGAAAACTGCGACAAGTCCCTGCACACCTTGTTCGGAGACAAGC
TGTGCACCGTCGCGACTTTGCGGGAGACTTACGGCGAAATGGCG
GACTGCTGCGCAAAGCAGGAGCCCGAAAGGAACGAGTGCTTCCT
GCAACACAAGGACGACAACCCGAACCTTCCGAGACTCGTGCGGC
CTGAGGTCGACGTGATGTGCACTGCATTCCATGATAACGAAGAA
ACATTCCTGAAGAAGTACCTGTATGAAATTGCCAGACGCCACCC
GTACTTCTACGCCCCCGAACTGCTGTTCTTCGCCAAGAGATACAA
GGCCGCCTTTACCGAATGTTGTCAAGCCGCCGATAAGGCAGCGT
GCCTGCTGCCGAAGTTGGACGAGCTCAGGGACGAAGGAAAGGC
CTCGTCCGCCAAGCAGAGGCTGAAGTGCGCGTCGCTCCAGAAGT
TTGGAGAGCGGGCTTTTAAGGCCTGGGCAGTGGCTAGGTTGAGC
CAGAGGTTCCCCAAGGCGGAGTTTGCCGAAGTGTCCAAGCTCGT
GACTGACCTGACTAAAGTCCATACCGAATGCTGCCACGGCGATC
TGCTCGAATGCGCAGATGACCGGGCGGATTTGGCCAAGTACATT
TGCGAAAACCAAGACTCCATAAGCTCCAAGCTGAAGGAGTGCTG
TGAAAAGCCTCTGCTCGAGAAGTCCCACTGTATCGCCGAGGTGG
AGAACGACGAAATGCCGGCAGACCTCCCTAGCCTGGCAGCCGAC
TTCGTCGAATCCAAGGACGTGTGCAAGAACTACGCCGAAGCGAA
GGACGTGTTCCTGGGAATGTTCCTGTACGAGTACGCCAGACGGC
ATCCAGACTACTCCGTGGTGCTTCTCTTGCGGCTGGCCAAGACTT
ATGAAACGACCCTGGAGAAATGTTGCGCTGCTGCTGACCCACAC
GAGTGCTACGCCAAAGTGTTCGACGAGTTTAAGCCTCTCGTGGA
GGAACCCCAGAACCTCATCAAGCAGAACTGCGAACTTTTCGAGC
AGCTCGGGGAGTACAAGTTCCAAAACGCGCTGCTTGTCCGCTAC
ACCAAGAAAGTGCCGCAAGTGTCCACACCGACCCTCGTGGAAGT
GTCCAGGAACCTGGGCAAAGTCGGAAGCAAATGTTGCAAGCACC
CCGAAGCCAAGCGCATGCCGTGCGCAGAGGACTACCTTTCGGTG
GTGTTGAACCAGCTCTGCGTCCTGCACGAAAAGACCCCGGTGTC
AGACCGCGTGACCAAGTGCTGTACCGAAAGCCTCGTGAATCGGC
GCCCCTGCTTCTCGGCCCTGGAGGTGGACGAAACTTACGTGCCG
AAAGAGTTCAACGCGGAAACCTTCACCTTTCATGCCGATATCTG
CACCCTGTCCGAGAAGGAGCGGCAGATCAAGAAGCAGACCGCC
CTGGTGGAGCTTGTGAAACACAAGCCGAAGGCCACTAAGGAAC
AGCTGAAGGCCGTCATGGACGATTTCGCTGCCTTCGTCGAGAAG
TGCTGCAAGGCCGACGACAAGGAGACTTGCTTCGCTGAAGAAGG
GAAGAAGCTTGTGGCCGCTAGCCAGGCTGCACTGGGACTGGGTA
GCGGTGGAGGGGGATCAGGGGGTGGTGGATCGGGAGGAGGAGG
ATCAGGAGGTGGCGGCTCAGGAGGAGGCGGATCAGGCGGTGGA
GGATCCGGAGGCGGAGGATCGGGTGGAGGAGGCTCAGCGAGGA
ACGGGGATCATTGTCCCCTTGGACCGGGTAGATGCTGTCGCCTG
CACACTGTGCGGGCTTCACTGGAGGACCTCGGCTGGGCTGACTG
GGTGCTGTCCCCACGGGAGGTGCAAGTGACCATGTGCATCGGCG
CCTGTCCTTCGCAATTCCGGGCCGCGAATATGCACGCCCAGATC
AAGACCTCCCTGCATCGCCTCAAGCCCGACACTGTGCCTGCTCCA
TGCTGTGTGCCGGCCTCCTATAACCCCATGGTGCTGATCCAGAAA
ACCGATACCGGCGTCAGCCTGCAGACGTATGATGATCTGCTGGC
CAAGGACTGCCATTGCATC
110 nucleic acid GATGCGCACAAGTCGGAAGTGGCCCATCGCTTTAAGGACCTGGG
encoding AGAAGAGAACTTCAAGGCCCTGGTCCTGATCGCGTTCGCCCAGT
SEQ ID NO: 92 ACCTCCAGCAGTCCCCGTTTGAGGACCACGTCAAGCTTGTGAAC
(Codon GAAGTGACCGAGTTCGCAAAGACTTGTGTGGCCGATGAGTCCGC
optimization 2) CGAAAACTGCGACAAGTCCCTGCACACCTTGTTCGGAGACAAGC
TGTGCACCGTCGCGACTTTGCGGGAGACTTACGGCGAAATGGCG
GACTGCTGCGCAAAGCAGGAGCCCGAAAGGAACGAGTGCTTCCT
GCAACACAAGGACGACAACCCGAACCTTCCGAGACTCGTGCGGC
CTGAGGTCGACGTGATGTGCACTGCATTCCATGATAACGAAGAA
ACATTCCTGAAGAAGTACCTGTATGAAATTGCCAGACGCCACCC
GTACTTCTACGCCCCCGAACTGCTGTTCTTCGCCAAGAGATACAA
GGCCGCCTTTACCGAATGTTGTCAAGCCGCCGATAAGGCAGCGT
GCCTGCTGCCGAAGTTGGACGAGCTCAGGGACGAAGGAAAGGC
CTCGTCCGCCAAGCAGAGGCTGAAGTGCGCGTCGCTCCAGAAGT
TTGGAGAGCGGGCTTTTAAGGCCTGGGCAGTGGCTAGGTTGAGC
CAGAGGTTCCCCAAGGCGGAGTTTGCCGAAGTGTCCAAGCTCGT
GACTGACCTGACTAAAGTCCATACCGAATGCTGCCACGGCGATC
TGCTCGAATGCGCAGATGACCGGGCGGATTTGGCCAAGTACATT
TGCGAAAACCAAGACTCCATAAGCTCCAAGCTGAAGGAGTGCTG
TGAAAAGCCTCTGCTCGAGAAGTCCCACTGTATCGCCGAGGTGG
AGAACGACGAAATGCCGGCAGACCTCCCTAGCCTGGCAGCCGAC
TTCGTCGAATCCAAGGACGTGTGCAAGAACTACGCCGAAGCGAA
GGACGTGTTCCTGGGAATGTTCCTGTACGAGTACGCCAGACGGC
ATCCAGACTACTCCGTGGTGCTTCTCTTGCGGCTGGCCAAGACTT
ATGAAACGACCCTGGAGAAATGTTGCGCTGCTGCTGACCCACAC
GAGTGCTACGCCAAAGTGTTCGACGAGTTTAAGCCTCTCGTGGA
GGAACCCCAGAACCTCATCAAGCAGAACTGCGAACTTTTCGAGC
AGCTCGGGGAGTACAAGTTCCAAAACGCGCTGCTTGTCCGCTAC
ACCAAGAAAGTGCCGCAAGTGTCCACACCGACCCTCGTGGAAGT
GTCCAGGAACCTGGGCAAAGTCGGAAGCAAATGTTGCAAGCACC
CCGAAGCCAAGCGCATGCCGTGCGCAGAGGACTACCTTTCGGTG
GTGTTGAACCAGCTCTGCGTCCTGCACGAAAAGACCCCGGTGTC
AGACCGCGTGACCAAGTGCTGTACCGAAAGCCTCGTGAATCGGC
GCCCCTGCTTCTCGGCCCTGGAGGTGGACGAAACTTACGTGCCG
AAAGAGTTCAACGCGGAAACCTTCACCTTTCATGCCGATATCTG
CACCCTGTCCGAGAAGGAGCGGCAGATCAAGAAGCAGACCGCC
CTGGTGGAGCTTGTGAAACACAAGCCGAAGGCCACTAAGGAAC
AGCTGAAGGCCGTCATGGACGATTTCGCTGCCTTCGTCGAGAAG
TGCTGCAAGGCCGACGACAAGGAGACTTGCTTCGCTGAAGAAGG
GAAGAAGCTTGTGGCCGCTAGCCAGGCTGCACTGGGACTGGGTA
GCGGTGGAGGGGGATCAGGGGGTGGTGGATCGGGAGGAGGAGG
ATCAGGAGGTGGCGGCTCAGGAGGAGGCGGATCAGGCGGTGGA
GGATCCGGAGGCGGAGGATCGGGTGGAGGAGGCTCAGATCATT
GTCCCCTTGGACCGGGTAGATGCTGTCGCCTGCACACTGTGCGG
GCTTCACTGGAGGACCTCGGCTGGGCTGACTGGGTGCTGTCCCC
ACGGGAGGTGCAAGTGACCATGTGCATCGGCGCCTGTCCTTCGC
AATTCCGGGCCGCGAATATGCACGCCCAGATCAAGACCTCCCTG
CATCGCCTCAAGCCCGACACTGTGCCTGCTCCATGCTGTGTGCCG
GCCTCCTATAACCCCATGGTGCTGATCCAGAAAACCGATACCGG
CGTCAGCCTGCAGACGTATGATGATCTGCTGGCCAAGGACTGCC
ATTGCATC
111 HSA (C34S)-GS- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
(GGGGS)8- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
(Deletion 5) AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
GDF15 KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSHCPLGPGR
CCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANM
HAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDD
LLAKDCHCI
112 HSA (C34S)-GS- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
(GGGGS)8- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
(deletion 14) AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
GDF15 KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGG
SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCRLHTVRA
SLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHR
LKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI
113 (Deletion 2) HKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTE
HSA (C34S)-GS- FAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAK
(GGGGS)8- QEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYL
(deletion 4) YEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDEL
GDF15 RDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAE
VSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLK
ECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEA
KDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPH
ECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTK
KVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLN
QLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA
ETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDD
FAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGG
GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDHCPLGPGRCCR
LHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQI
KTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAK
DCHCI
114 DNA for SEQ CACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGA
ID NO: 113 AAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTCA
(Deletion 2) GCAGTCCCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAA
HSA (C34S)-GS- CTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAAT
(GGGGS)8- TGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACA
(deletion 4) GTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGT
GDF15 GCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAA
AGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTG
ATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGA
AAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATG
CCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTA
CAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCA
AAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAA
ACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAG
CTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCC
AAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTAC
CAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTG
CTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAA
GATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCT
GTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGA
TGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTA
AGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTG
GGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCT
GTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCT
AGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCA
AAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAAT
TTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTA
CAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTAC
CCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTA
GGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAA
GAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAG
TTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCAC
CAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTT
CAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAAT
GCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAG
AAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTCGT
GAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTT
ATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGA
CGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTG
CTGCAAGTCAAGCTGCCTTAGGGCTTGGAAGCGGCGGAGGGGGG
AGTGGCGGCGGTGGCTCCGGGGGGGGCGGATCCGGCGGAGGGG
GCAGCGGGGGTGGAGGGAGTGGCGGGGGAGGATCAGGGGGAGG
AGGATCAGGAGGGGGCGGAAGTGATCATTGCCCTCTCGGGCCCG
GACGGTGTTGCCGCCTCCACACTGTGAGGGCTTCACTTGAAGAC
CTTGGATGGGCCGACTGGGTGCTGTCCCCAAGAGAGGTACAAGT
CACAATGTGTATTGGCGCCTGCCCCAGCCAGTTTCGCGCCGCTAA
CATGCACGCCCAGATAAAAACCAGCCTGCACCGCCTGAAGCCCG
ACACGGTGCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCC
ATGGTGCTCATTCAAAAGACCGACACCGGGGTGTCGCTCCAGAC
CTATGATGACTTGTTAGCCAAAGACTGCCACTGCATA
115 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GA-(GGGGA)8- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
(deletion 4) AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
GDF15 KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGAGGGG
AGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGADHCPLG
PGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAA
NMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTY
DDLLAKDCHCI
116 DNA for SEQ GATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGG
ID NO: 115 AGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTA
HSA (C34S)- TCTTCAGCAGTCCCCATTTGAAGATCATGTAAAATTAGTGAATGA
GA-(GGGGA)8- AGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTG
(deletion 4) AAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTAT
GDF15 GCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGAC
TGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCA
ACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAG
AGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACA
TTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTAC
TTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCT
GCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTG
TTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTC
TGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAG
AAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGA
TTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGAT
CTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGA
ATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAA
ATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAA
CCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGA
TGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGA
AAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCT
TCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATT
ACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACC
ACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTA
TGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTC
AGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGA
GAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAA
AGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAA
ACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCA
AAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAA
CCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAG
TCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGC
TTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTT
AATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCT
GAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGC
TCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGC
TGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGC
TGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTG
TTGCTGCAAGTCAAGCTGCCTTAGGGCTTGGTGCTGGAGGAGGC
GGGGCGGGCGGCGGGGGTGCCGGTGGGGGTGGCGCAGGGGGAG
GTGGTGCGGGTGGTGGTGGGGCTGGTGGGGGAGGTGCAGGCGG
TGGCGGTGCCGGGGGGGGTGGCGCGGATCATTGCCCTCTCGGGC
CCGGACGGTGTTGCCGCCTCCACACTGTGAGGGCTTCACTTGAA
GACCTTGGATGGGCCGACTGGGTGCTGTCCCCAAGAGAGGTACA
AGTCACAATGTGTATTGGCGCCTGCCCCAGCCAGTTTCGCGCCGC
TAACATGCACGCCCAGATAAAAACCAGCCTGCACCGCCTGAAGC
CCGACACGGTGCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAAT
CCCATGGTGCTCATTCAAAAGACCGACACCGGGGTGTCGCTCCA
GACCTATGATGACTTGTTAGCCAAAGACTGCCACTGCATA
117 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
(AP)10- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
(deletion 4) AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
GDF15 KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLAPAPAPA
PAPAPAPAPAPAPDHCPLGPGRCCRLHTVRASLEDLGWADWVLSP
REVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPAS
YNPMVLIQKTDTGVSLQTYDDLLAKDCHCI
118 DNA for SEQ GATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGG
ID NO: 117 AGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTA
HSA (C34S)- TCTTCAGCAGTCCCCATTTGAAGATCATGTAAAATTAGTGAATGA
(AP)10- AGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTG
(deletion 4) AAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTAT
GDF15 GCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGAC
TGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCA
ACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAG
AGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACA
TTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTAC
TTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCT
GCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTG
TTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTC
TGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAG
AAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGA
TTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGAT
CTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGA
ATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAA
ATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAA
CCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGA
TGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGA
AAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCT
TCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATT
ACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACC
ACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTA
TGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTC
AGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGA
GAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAA
AGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAA
ACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCA
AAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAA
CCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAG
TCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGC
TTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTT
AATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCT
GAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGC
TCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGC
TGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGC
TGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTG
TTGCTGCAAGTCAAGCTGCCTTAGGGCTTGCACCAGCCCCTGCCC
CTGCACCTGCACCTGCTCCCGCACCGGCTCCAGCCCCAGCTCCG
GATCATTGCCCTCTCGGGCCCGGACGGTGTTGCCGCCTCCACACT
GTGAGGGCTTCACTTGAAGACCTTGGATGGGCCGACTGGGTGCT
GTCCCCAAGAGAGGTACAAGTCACAATGTGTATTGGCGCCTGCC
CCAGCCAGTTTCGCGCCGCTAACATGCACGCCCAGATAAAAACC
AGCCTGCACCGCCTGAAGCCCGACACGGTGCCAGCGCCCTGCTG
CGTGCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCG
ACACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAAA
GACTGCCACTGCATA
119 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
(AP)12- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
(deletion 4) AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
GDF15 KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLAPAPAPA
PAPAPAPAPAPAPAPAPDHCPLGPGRCCRLHTVRASLEDLGWADW
VLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCC
VPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI
120 DNA for SEQ GATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGG
ID NO: 119 AGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTA
HSA (C34S)- TCTTCAGCAGTCCCCATTTGAAGATCATGTAAAATTAGTGAATGA
(AP)12- AGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTG
(deletion 4) AAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTAT
GDF15 GCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGAC
TGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCA
ACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAG
AGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACA
TTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTAC
TTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCT
GCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTG
TTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTC
TGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAG
AAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGA
TTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGAT
CTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGA
ATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAA
ATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAA
CCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGA
TGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGA
AAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCT
TCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATT
ACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACC
ACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTA
TGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTC
AGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGA
GAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAA
AGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAA
ACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCA
AAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAA
CCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAG
TCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGC
TTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTT
AATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCT
GAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGC
TCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGC
TGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGC
TGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTG
TTGCTGCAAGTCAAGCTGCCTTAGGGCTTGCACCAGCCCCTGCCC
CTGCACCTGCACCTGCTCCCGCACCGGCTCCAGCCCCAGCTCCG
GCTCCAGCTCCTGATCATTGCCCTCTCGGGCCCGGACGGTGTTGC
CGCCTCCACACTGTGAGGGCTTCACTTGAAGACCTTGGATGGGC
CGACTGGGTGCTGTCCCCAAGAGAGGTACAAGTCACAATGTGTA
TTGGCGCCTGCCCCAGCCAGTTTCGCGCCGCTAACATGCACGCCC
AGATAAAAACCAGCCTGCACCGCCTGAAGCCCGACACGGTGCCA
GCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCATT
CAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGATGACTT
GTTAGCCAAAGACTGCCACTGCATA
121 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GGS- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
(EGKSSGSGSESKST)3- AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
GGS- KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
(deletion4) DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
GDF15 FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGSEGKS
SGSGSESKSTEGKSSGSGSESKSTEGKSSGSGSESKSTGGSDHCPLGP
GRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAAN
MHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYD
DLLAKDCHCI
122 DNA for SEQ GATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGG
ID NO: 121 AGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTA
HSA (C34S)- TCTTCAGCAGTCCCCATTTGAAGATCATGTAAAATTAGTGAATGA
GGS- AGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTG
(EGKSSGSGSESKST)3- AAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTAT
GGS- GCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGAC
(deletion4) TGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCA
GDF15 ACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAG
AGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACA
TTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTAC
TTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCT
GCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTG
TTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTC
TGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAG
AAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGA
TTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGAT
CTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGA
ATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAA
ATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAA
CCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGA
TGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGA
AAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCT
TCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATT
ACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACC
ACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTA
TGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTC
AGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGA
GAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAA
AGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAA
ACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCA
AAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAA
CCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAG
TCACAAAATGCTGTACTGAGAGCTTGGTCAACAGGCGGCCGTGC
TTCAGCGCCCTCGAGGTGGATGAGACTTATGTCCCAAAGGAGTT
TAATGCGGAAACTTTTACTTTCCACGCAGACATTTGCACCTTGTC
TGAAAAGGAAAGACAGATTAAGAAACAGACTGCTCTTGTGGAA
CTGGTAAAACATAAACCAAAAGCTACGAAGGAGCAGCTTAAGG
CTGTTATGGATGATTTCGCCGCGTTTGTCGAGAAGTGCTGCAAAG
CGGACGATAAGGAAACTTGCTTTGCAGAGGAAGGTAAGAAACTC
GTAGCGGCAAGTCAGGCTGCGCTTGGCCTTGGAGGCAGTGAAGG
CAAATCCTCTGGGAGTGGCTCTGAAAGTAAATCCACCGAGGGCA
AATCCAGTGGATCTGGGTCTGAATCTAAGTCTACCGAGGGGAAG
TCTTCTGGCAGTGGGTCAGAATCTAAATCTACAGGCGGCTCTGA
CCATTGCCCGTTGGGACCAGGACGCTGCTGTCGCCTTCATACAGT
GCGAGCGAGTTTGGAAGACCTGGGCTGGGCTGACTGGGTGCTTA
GCCCTCGGGAGGTCCAGGTCACAATGTGCATTGGCGCGTGTCCC
AGTCAATTTAGAGCAGCAAATATGCACGCCCAAATAAAAACCTC
CCTGCATAGGCTTAAGCCAGATACTGTCCCCGCACCATGCTGTGT
GCCTGCTTCTTACAATCCTATGGTACTCATCCAGAAGACCGACAC
GGGAGTTAGCCTCCAGACTTATGACGACCTCTTGGCTAAAGATT
GCCATTGTATT
123 HSA (C34S)-GS- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
(PGGGS)8- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
(deletion4) AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
GDF15 KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSPGGGS
PGGGSPGGGSPGGGSPGGGSPGGGSPGGGSPGGGSDHCPLGPGRCC
RLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHA
QIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLL
AKDCHCI
124 DNA for SEQ GATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGG
ID NO: 123 AGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTA
HSA (C34S)-GS- TCTTCAGCAGTCCCCATTTGAAGATCATGTAAAATTAGTGAATGA
(PGGGS)8- AGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTG
(deletion4) AAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTAT
GDF15 GCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGAC
TGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCA
ACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAG
AGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACA
TTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTAC
TTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCT
GCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTG
TTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTC
TGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAG
AAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGA
TTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGAT
CTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGA
ATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAA
ATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAA
CCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGA
TGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGA
AAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCT
TCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATT
ACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACC
ACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTA
TGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTC
AGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGA
GAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAA
AGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAA
ACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCA
AAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAA
CCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAG
TCACGAAATGTTGCACAGAGTCACTGGTCAACAGGAGACCTTGC
TTCTCCGCTCTTGAGGTTGACGAAACGTATGTCCCAAAAGAGTTC
AACGCCGAAACGTTTACGTTTCATGCGGACATATGCACTCTCAGT
GAGAAGGAGCGACAAATCAAAAAACAGACTGCTCTTGTAGAGTT
GGTAAAACACAAACCTAAAGCAACAAAAGAGCAATTGAAAGCT
GTGATGGACGATTTTGCAGCTTTCGTAGAAAAGTGCTGCAAGGC
CGACGATAAAGAAACCTGTTTCGCTGAAGAAGGCAAAAAACTTG
TTGCGGCATCTCAGGCCGCTCTTGGACTTGGGAGCCCGGGTGGC
GGGTCTCCAGGCGGAGGCTCTCCGGGCGGAGGTAGTCCCGGAGG
GGGTAGTCCGGGCGGCGGTTCTCCAGGTGGAGGTTCTCCTGGTG
GTGGCAGTCCTGGCGGAGGATCTGATCACTGTCCCCTTGGGCCC
GGGAGGTGCTGCCGACTTCATACAGTTCGCGCCAGCCTTGAAGA
TTTGGGGTGGGCCGACTGGGTGTTGAGCCCGAGAGAGGTCCAAG
TCACGATGTGTATTGGAGCCTGTCCCTCTCAATTCCGAGCCGCAA
ATATGCATGCGCAAATAAAGACGAGTCTCCATCGGTTGAAGCCT
GATACTGTCCCAGCTCCGTGCTGCGTCCCCGCGAGTTATAATCCC
ATGGTCCTTATACAGAAAACAGACACTGGTGTCAGCCTTCAGAC
GTATGACGATTTGCTTGCTAAAGACTGTCATTGTATT
125 HSA (C34S)-GS- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
(AGGGS)8- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
(deletion 4) AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
GDF15 KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSAGGG
SAGGGSAGGGSAGGGSAGGGSAGGGSAGGGSAGGGSDHCPLGPG
RCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANM
HAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDD
LLAKDCHCI
126 DNA for SEQ GATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGG
ID NO: 125 AGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTA
TCTTCAGCAGTCCCCATTTGAAGATCATGTAAAATTAGTGAATGA
AGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTG
AAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTAT
GCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGAC
TGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCA
ACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAG
AGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACA
TTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTAC
TTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCT
GCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTG
TTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTC
TGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAG
AAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGA
TTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGAT
CTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGA
ATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAA
ATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAA
CCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGA
TGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGA
AAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCT
TCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATT
ACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACC
ACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTA
TGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTC
AGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGA
GAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAA
AGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAA
ACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCA
AAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAA
CCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAG
TCACTAAATGTTGTACCGAGTCTCTTGTTAATAGGCGGCCATGCT
TCAGTGCATTGGAAGTCGACGAAACCTATGTACCAAAGGAGTTC
AACGCAGAAACATTTACATTCCATGCTGATATCTGCACATTGAG
CGAGAAAGAGAGACAGATTAAGAAACAGACAGCGCTTGTTGAA
CTGGTTAAACACAAACCAAAAGCTACCAAGGAGCAGCTTAAGGC
AGTAATGGATGACTTCGCGGCCTTTGTCGAGAAATGTTGTAAAG
CGGATGATAAAGAGACATGCTTCGCCGAAGAGGGCAAAAAACT
TGTAGCGGCAAGCCAGGCCGCACTGGGTCTCGGTAGTGCGGGCG
GTGGTTCAGCGGGGGGAGGATCTGCAGGTGGTGGCTCAGCGGGT
GGCGGTAGCGCTGGGGGGGGCTCCGCAGGTGGGGGATCAGCAG
GCGGCGGATCAGCCGGCGGTGGATCCGACCACTGTCCTCTCGGG
CCTGGTCGGTGTTGCCGCCTCCATACTGTGCGCGCGTCTCTTGAG
GATCTGGGGTGGGCTGATTGGGTTCTCTCTCCCCGCGAAGTGCA
GGTGACCATGTGTATTGGTGCTTGCCCAAGTCAATTCCGAGCAG
CTAACATGCACGCCCAGATCAAGACTAGCCTGCATCGGCTTAAG
CCCGACACTGTTCCTGCCCCTTGCTGTGTTCCTGCATCTTATAATC
CAATGGTCCTGATCCAGAAAACCGATACGGGTGTATCATTGCAA
ACATACGACGACTTGCTTGCCAAAGATTGCCATTGCATT
127 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV
GGS- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC
(EGKSSGSGSESKST)2- AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
GGS- KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL
(deletion4) DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE
GDF15 FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS
KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNY
AEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR
YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE
FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGSEGKS
SGSGSESKSTEGKSSGSGSESKSTGGSDHCPLGPGRCCRLHTVRASL
EDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLK
PDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI
128 DNA for SEQ GATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGG
ID NO: 127 AGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTA
TCTTCAGCAGTCCCCATTTGAAGATCATGTAAAATTAGTGAATGA
AGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTG
AAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTAT
GCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGAC
TGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCA
ACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAG
AGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACA
TTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTAC
TTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCT
GCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTG
TTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTC
TGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAG
AAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGA
TTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGAT
CTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGA
ATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAA
ATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAA
CCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGA
TGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGA
AAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCT
TCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATT
ACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACC
ACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTA
TGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTC
AGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGA
GAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAA
AGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAA
ACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCA
AAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAA
CCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAG
TCACGAAATGCTGTACAGAATCCCTCGTGAATAGAAGGCCCTGC
TTCTCTGCCCTTGAGGTGGACGAGACTTACGTCCCTAAGGAGTTT
AACGCCGAGACCTTTACTTTTCATGCTGATATTTGCACCCTTTCC
GAAAAGGAGCGGCAGATCAAGAAACAAACAGCCTTGGTGGAAC
TCGTAAAACATAAACCCAAAGCCACCAAGGAACAACTTAAAGCT
GTTATGGATGACTTCGCAGCCTTCGTCGAGAAATGTTGCAAGGC
GGATGATAAGGAAACGTGTTTTGCTGAGGAAGGGAAGAAGTTG
GTTGCTGCCTCTCAAGCGGCTCTGGGGCTTGGCGGATCAGAGGG
GAAGTCCTCCGGGTCCGGTAGCGAGTCCAAATCTACGGAAGGGA
AGTCATCCGGTTCTGGGTCAGAGTCCAAATCCACAGGAGGATCA
GACCATTGCCCATTGGGACCAGGACGATGTTGTCGCCTGCATAC
GGTAAGAGCGTCTCTGGAGGATCTCGGCTGGGCCGATTGGGTTC
TCTCACCACGAGAAGTACAGGTCACAATGTGCATAGGAGCTTGT
CCGAGCCAATTCCGGGCGGCTAATATGCACGCACAGATCAAGAC
CTCTTTGCACCGCTTGAAGCCCGATACCGTGCCAGCACCGTGTTG
CGTCCCAGCATCTTACAACCCTATGGTTTTGATACAGAAAACTGA
CACAGGTGTGAGCCTCCAGACATATGATGATTTGCTGGCTAAGG
ATTGCCACTGTATA
