NZ622174B2

NZ622174B2 - Glucagon-like peptide-2 compositions and methods of making and using same

Info

Publication number: NZ622174B2
Application number: NZ622174A
Authority: NZ
Inventors: Nathan Geething; Volker Schellenberger; Joshua Silverman; Benjamin Spink; Willem P Stemmer; Chiawei Wang; Chia Wei Wang
Original assignee: Amunix Operating Inc
Priority date: 2011-09-12
Filing date: 2012-09-12
Publication date: 2016-03-30

Abstract

Disclosed is a composition for use in achieving an intestinotrophic effect in a subject comprising a recombinant fusion protein comprising (i) a glucagon-like protein-2 (GLP-2) sequence selected from the group consisting of the sequences of SEQ ID NOS: 1 and 3-23, and (ii) an extended recombinant polypeptide (XTEN), wherein the XTEN is a sequence exhibiting at least 90% sequence identity to a sequence selected from the group consisting of the sequences in Table 4, and wherein the XTEN is further characterized in that: (a) the XTEN comprises at least 36 amino acid residues; (b) the sum of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues constitutes more than 80% of the total amino acid residues of the XTEN; (c) the XTEN is substantially non-repetitive such that (i) the XTEN contains no three contiguous amino acids that are identical unless the amino acids are serine; (ii) at least 80% of the XTEN sequence consists of non-overlapping sequence motifs, each of the sequence motifs comprising 9 to 14 amino acid residues consisting of four to six amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), wherein any two contiguous amino acid residues do not occur more than twice in each of the non-overlapping sequence motifs; or (iii) the XTEN sequence has a subsequence score of less than 10; (d) the XTEN has greater than 90% random coil formation as determined by GOR algorithm; (e) the XTEN has less than 2% alpha helices and 2% beta-sheets as determined by Chou-Fasman algorithm; and (f) the XTEN lacks a predicted T-cell epitope when analysed by TEPITOPE algorithm, wherein the TEPITOPE threshold score for the prediction by the algorithm has a threshold of –9, wherein the fusion protein exhibits an apparent molecular weight factor of at least 4 and is capable of achieving an intestinotrophic effect in a subject using a dosage of 2.5 nmol/kg to 6250 nmol/kg, or 25 nmol/kg to 3750 nmol/kg, or 75 nmol/kg/dose to 1250 nmol/kg/dose, or 125 nmol/kg/dose to 750 nmol/kg/dose. lypeptide (XTEN), wherein the XTEN is a sequence exhibiting at least 90% sequence identity to a sequence selected from the group consisting of the sequences in Table 4, and wherein the XTEN is further characterized in that: (a) the XTEN comprises at least 36 amino acid residues; (b) the sum of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues constitutes more than 80% of the total amino acid residues of the XTEN; (c) the XTEN is substantially non-repetitive such that (i) the XTEN contains no three contiguous amino acids that are identical unless the amino acids are serine; (ii) at least 80% of the XTEN sequence consists of non-overlapping sequence motifs, each of the sequence motifs comprising 9 to 14 amino acid residues consisting of four to six amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), wherein any two contiguous amino acid residues do not occur more than twice in each of the non-overlapping sequence motifs; or (iii) the XTEN sequence has a subsequence score of less than 10; (d) the XTEN has greater than 90% random coil formation as determined by GOR algorithm; (e) the XTEN has less than 2% alpha helices and 2% beta-sheets as determined by Chou-Fasman algorithm; and (f) the XTEN lacks a predicted T-cell epitope when analysed by TEPITOPE algorithm, wherein the TEPITOPE threshold score for the prediction by the algorithm has a threshold of –9, wherein the fusion protein exhibits an apparent molecular weight factor of at least 4 and is capable of achieving an intestinotrophic effect in a subject using a dosage of 2.5 nmol/kg to 6250 nmol/kg, or 25 nmol/kg to 3750 nmol/kg, or 75 nmol/kg/dose to 1250 nmol/kg/dose, or 125 nmol/kg/dose to 750 nmol/kg/dose.

Description

GLUCAGON-LIKE PEPTIDE-2 COMPOSITIONS AND METHODS OF MAKING AND USING SAME CROSS-REFERENCE TO RELATED APPLICATION This application claims priority beneﬁt to US. Provisional Application Serial No. 61/573,748 ﬁled September 12, 2011, and which application is incorporated herein by nce in its ty.

BACKGROUND OF THE INVENTION Glucagon-like peptide-2 (GLP-2) is an endocrine peptide that, in humans, is generated as a 33 amino acid peptide by post-translational lytic cleavage of proglucagon; a process that also liberates the related glucagon-like peptide-1 (GLP-l). GLP-2 is produced and secreted in a nutrient-dependent fashion by the intestinal endocrine L cells. GLP-2 is trophic to the inal l epithelium via stimulation of crypt cell proliferation and reduction of enterocyte apoptosis. GLP—2 exerts its effects through speciﬁc GLP—2 receptors but the responses in the intestine are mediated by indirect pathways in that the receptor is not expressed on the epithelium but on enteric neurons (Redstone, HA, et al. The Effect of Glucagon-Like Peptide-2 Receptor Agonists on Colonic Anastomotic Wound Healing.

Gastroenterol Res Pract. (2010); 2010: Art. ID: ).

The effects of GLP-2 are multiple, including intestinaltrophic s resulting in an increase in intestinal absorption and nutrient assimilation (Lovshin, J. and DJ. Drucker, Synthesis, secretion and biological actions of the glucagon-like peptides. Ped. Diabetes (2000) 1(1):49-57); anti-inﬂammatory activities; mucosal healing and repair; sing intestinal permeability; and an increase in mesenteric blood ﬂow (Bremholm, L. et al. on-like e-2 increases mesenteric blood ﬂow in humans.

Scan. J. Gastro. (2009) 44(3):314-319). Exogenously administered GLP-2 produces a number of s in humans and rodents, including slowing gastric emptying, increasing intestinal blood ﬂow and intestinal /mucosal surface area, enhancement of intestinal function, reduction in bone breakdown and rotection. GLP-2 may act in an endocrine fashion to link intestinal growth and metabolism with nutrient intake. In inﬂamed , however, GLP-2 action is antiproliferative, decreasing the expression of proinﬂammatory nes while increasing the sion of IGF-1, promoting healing of inﬂamed mucosa.

Many patients e al removal of the small or large bowel for a wide range of conditions, including colorectal cancer, inﬂammatory bowel disease, irritable bowel syndrome, and trauma. Short bowel syndrome (SBS) patients with end jejunostomy and no colon have reduced release of GLP-2 in response to a meal due to the removal of secreting L cells. Patients with active Crohn’s Disease or ulcerative colitis have endogenous serum GLP-2 concentrations that are increased, suggesting the possibility of a normal adaptive response to mucosal injury (Buchman, A. L., et al. Teduglutide, a novel mucosally active analog of glucagon-like peptide-2 (GLP-2) for the ent ofmoderate to severe Crohn's disease. Inﬂammatory Bowel Diseases, (2010) 16:962—973).

Exogenously administered GLP-2 and GLP-2 analogues have been demonstrated in animal models to promote the growth and repair of the intestinal epithelium, including enhanced nutrient absorption following small bowel resection and alleviation of total parenteral nutrition-induced hypoplasia in rodents, as well as demonstration of sed mortality and ement of disease- related histopathology in animal models such as indomethacin-induced enteritis, dextran sulfate-induced colitis and Chemotherapy-induced mucositis. Accordingly, GLP-2 and related analogs may be treatments for short bowel syndrome, irritable bowel syndrome, Crohn's disease, and other diseases of the intestines (Moor, BA, et al. GLP-2 receptor m rates inﬂammation and gastrointestinal stasis in murine post-operative ileus. J Pharmacol Exp Ther. (2010) 333(2):574-583). However, native GLP-2 has a half- life of approximately seven minutes due to cleavage by dipeptidyl peptidase IV (DPP-IV) sen PB, et al., Teduglutide (ALX-0600), a dipeptidyl peptidase IV resistant glucagon-like peptide 2 analogue, improves intestinal function in short bowel syndrome patients. Gut. (2005) 54(9):]224-1231; Hartmann B, et al. (2000) Dipeptidyl peptidase IV inhibition enhances the intestinotrophic effect of glucagon-like peptide-2 in rats and mice. Endocrinology 141:4013—4020). It has been determined that modiﬁcation of the GLP-2 sequence by replacement of alanine with glycine in position 2 blocks degradation by DPP-IV, extending the half life of the analog called teduglutide to 0.9—2.3 hours (Marier JF, Population pharmacokinetics glutide ing repeated subcutaneous administrations in healthy participants and in patients with short bowel syndrome and Crohn's disease. J Clin Pharmacol. (2010) 50(1):36—49).

However, recent clinical trials utilizing teduglutide in patients with short bowel syndrome required daily administration of the GLP-2 analog to achieve a clinical beneﬁt (Jeppesen PB, Randomized placebo- controlled trial of teduglutide in reducing parenteral nutrition and/or intravenous ﬂuid requirements in patients with short bowel syndrome. Gut (2011) 902-9l4).

Chemical modiﬁcations to a therapeutic protein can modify its in vivo clearance rate and subsequent half-life. One example of a common ation is the addition of a polyethylene glycol (PEG) moiety, typically coupled to the protein via an aldehyde or oxysuccinimide (NHS) group on the PEG reacting with an amine group (e.g. lysine side chain or the N—terminus). However, the conjugation step can result in the formation of heterogeneous t mixtures that need to be separated, leading to cant product loss and complexity of manufacturing and does not result in a tely ally-uniform product. Also, the pharmacologic function of pharmacologically—active proteins may be hampered if amino acid side chains in the vicinity of its binding site become modiﬁed by the PEGylation process. Other ches include the genetic fusion of an EC domain to the eutic protein, which increases the size of the therapeutic protein, hence reducing the rate of clearance through the . Additionally, the Fc domain s the ability to bind to, and be recycled from lysosomes by, the FCRn receptor, which results in increased pharmacokinetic ife. A form of GLP-2 fused to PC has been evaluated in a murine model of gastrointestinal inﬂammation ated with postoperative ileus (Moor, BA, et al. GLP-2 receptor agonism ameliorates ation and gastrointestinal stasis in murine post-operative ileus. J Pharmacol Exp Ther. (2010) 333(2):574-583). Unfortunately, the Fc domain does not fold ntly during recombinant expression, and tends to form insoluble precipitates known as inclusion bodies. These inclusion bodies must be solubilized and onal protein must be renatured from the misfolded aggregate, a time-consuming, inefﬁcient, and expensive process.

SUMMARY OF THE INVENTION Accordingly, there remains a considerable need for GL—2 compositions and ations with increased half—life and retention of activity and bioavailability when administered as part of a preventive and/or therapeutic regimen for GLP-2 ated conditions and diseases that can be administered less frequently, and are safer and less complicated and costly to produce. The present ion addresses this need and provides related advantages as well. The present invention relates to novel GLP-2 itions and uses thereof. Speciﬁcally, the compositions provided herein are particularly used for the treatment or improvement of a gastrointestinal a condition. In one aspect, the present invention provides compositions of fusion proteins comprising a recombinant glucagon—like protein-2 (“GLP-2”) and one or more ed recombinant polypeptides (“XTEN”). A subject XTEN is typically a polypeptide with a non-repetitive sequence and unstructured conformation that is useful as a fusion partner to GLP-2 peptides in that it confers enhanced properties to the rsulting fusion protein. In one embodiment, one or more XTEN is linked to a GLP-2 or sequence variants thereof, ing in a GLP XTEN fusion n (“GLPZ-XTEN”). The present disclosure also provides pharmaceutical compositions comprising the fusion proteins and the uses thereof for treating GLPrelated ions.

In one aspect, the GLP2-XTEN compositions have enhanced pharmacokinetic and/or physicochemical ties compared to recombinant GLP-2 not linked to the XTEN, which permit more convenient dosing and result in improvement in one or more parameters ated with the gastrointestinal condition. The GLP2-XTEN fusion ns of the embodiments sed herein exhibit one or more or any combination of the improved properties and/or the embodiments as detailed herein. In some embodiments, the GLP2-XTEN compositions of the invention do not have a component selected the group consisting of: polyethylene glycol (PEG), n, antibody, and an antibody fragment.

In one ment, the invention provides a recombinant GLP-2 fusion protein comprising an XTEN, wherein the XTEN is characterized in that a) the XTEN comprises at least 36, or at least 72, or at least 96, or at least 120, or at least 144, or at least 288, or at least 576, or at least 864, or at least 1000, or at least 2000, or at least 3000 amino acid residues; b) the sum of glycine (G), alanine (A), serine (S), ine (T), glutamate (E) and proline (P) residues constitutes at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, of the total amino acid residues of the XTEN; c) the XTEN is substantially non-repetitive such that (i) the XTEN contains no three contiguous amino acids that are identical unless the amino acids are serine; (ii) at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, of the XTEN sequence consists of non-overlapping sequence motifs, each of the sequence motifs comprising about 9 to about 14, or about 12 amino acid residues consisting of three, four, ﬁve or six types of amino acids selected from e (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), n any two contiguous amino acid residues do not occur more than twice in each of the non-overlapping sequence motifs; or (iii) the XTEN sequence has a subsequence score of less than 10; d) the XTEN has greater than 90%, or greater than 95%, or greater than 99%, random coil formation as determined by GOR algorithm; e) the XTEN has less than 2% alpha helices and 2% beta-sheets as determined by Chou-Fasman thm; f) the XTEN lacks a predicted T- cell epitope when analyzed by TEPITOPE thm, wherein the TEPITOPE threshold score for said prediction by said algorithm has a threshold of —9; wherein said ﬁJsion protein exhibits an apparent molecular weight factor of at least about 4, or at least about 5, or at least about 6, or at least about 7, or at least about 8, or at least about 9, or at least about 10, or at least about 11, or at least about 12, or at least about 15, or at least about 20 When measured by size exclusion chromatography or comparable method and exhibits an intestinotrophic effect when administered to a t using a therapeutically effective amount. In the foregoing embodiment, the XTEN can have any one of elements (a)-(d) or any combination of (a)—(d). In another embodiment of the foregoing, the fusion protein exhibits an nt molecular weight of at least about 200 kDa, or at least about 400 kDa, or at least about 500 kDa, or at least about 700 kDa, or at least about 1000 kDa, or at least about 1400 kDa, or at least about 1600 kDa, or at least about 1800kDa, or at least about 2000 kDa, or at least about 3000 kDa. In another embodiment of the foregoing, the fusion protein exhibits a al half—life that is longer than about 24, or about 30, or about 48, or about 72, or about 96, or about 120, or about 144 hours when stered to a subject, wherein the subject is selected from mouse, rat, monkey and man. In one embodiment, the XTEN ofthe fusion protein is characterized in that at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the XTEN sequence consists of non-overlapping sequence motifs wherein the motifs are selected from Table 3. In some ments, the XTEN of the fusion proteins are further characterized in that the sum of asparagine and glutamine residues is less than 10%, or less than 5%, or less than 2% of the total amino acid sequence of the XTEN. In other embodiments, the XTEN of the fusion proteins are further characterized in that the sum of methionine and tryptophan residues is less than 2% of the total amino acid sequence of the XTEN. In still other ments, the XTEN of the fusion proteins are further characterized in that the XTEN has less than 5% amino acid residues with a positive charge. In one embodiment, the intestinotrophic effect of the administered fusion protein is at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100% or at least about 120% or at least about 150% or at least about 200% of the inotrophic effect compared to the corresponding GLP-2 not linked to XTEN and administered to a subject using a comparable dose. In one ment, the intestinotrophic effect is manifest in a subject selected from the group consisting of mouse, rat, , and human. In the foregoing embodiments, said administration is subcutaneous, intramuscular, or intravenous. In another embodiment, the inotrophic effect is determined after administration of 1 dose, or 3 doses, or 6 doses, or 10 doses, or 12 or more doses of the fusion protein. In another embodiment, the intestinotrophic effect is selected from the group consisting of intestinal growth, increased hyperplasia of the villus epithelium, increased crypt cell proliferation, increased height of the crypt and villus axis, increased healing after intestinal anastomosis, increased small bowel weight, increased small bowel length, decreased small bowel epithelium apoptosis, reduced ulceration, d intestinal adhesions, and enhancement of intestinal function.

In one embodiment, the administration of the GLP2-XTEN fusion protein results in an increase in small intestine weight of at least about 10%, or at least about 20%, or at least about 30%. In another embodiment, the stration results in an increase in small intestine length of at least about 5%, or at least about 6%, or at least about 7%, or at least about 8%, or at least about 9%, or at least about 10%, or at least about 20%, or at least about 30%.

In one embodiment, the GLP-2 ce of the fusion n has at least 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity to a sequence selected from the group consisting of the sequences in Table 1, when optimally aligned. In r embodiment, the GLP-2 of the fusion protein comprises human GLP-2. In another embodiment, the GLP—2 of the fusion protein comprises a GLP—2 of a species origin other than human, such as bovine GLP—2, pig GLP—2, sheep GLP—2, chicken GLP—2, and canine GLP—2. In some embodiments, the GLP-2 of the fusion proteins has an amino acid substitution in place of Alaz, n the substitution is glycine. In yet another embodiment, the GLP-2 of the fusion protein has the sequence HGDGSFSDEMNTILDNLAARDFINWLIQTKITD.

In one ment of the GLP2-XTEN fusion protein, the XTEN is linked to the C-terminus of the GLP-2. In another embodiment of the GLP2-XTEN fusion n wherein the XTEN is linked to the C-terminus of the GLP-2, the fusion protein further comprises a spacer sequence of 1 to about 50 amino acid residues linking the GLP-2 and XTEN components. In one embodiment, the spacer sequence is a single glycine residue.

In one embodiment of the GLP2-XTEN fusion protein, the XTEN is characterized in that: (a) the total XTEN amino acid es is at least 36 to about 3000, or about 144 to about 2000, or about 288 to about 1000 amino acid residues; and (b) the sum of glycine (G), e (A), serine (S), threonine (T), glutamate (E) and proline (P) residues constitutes at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, of the total amino acid residues of the XTEN.

In one embodiment of the GLP2-XTEN fusion protein, the fusion protein comprises one or more XTEN having at least 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or sequence identity compared to a ce of able length selected from any one of Table 4, Table 8, Table 9, Table 10, Table 11, and Table 12, when optimally aligned. In r embodiment, the fusion protein comprises an XTEN wherein the sequence is AE864 of Table 4. In r embodiment, the fusion protein sequence has a sequence with at least 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence ty to the sequence set forth in .

In one embodiment, the ﬁision protein comprising a GLP-2 and XTEN binds to a GLP-2 receptor with an ECso of less than about 30 nM, or about 100 nM, or about 200 nM, or about 300 nM, or about 370 nM, or about 400 nM, or about 500 nM, or about 600 nM, or about 700 nM, or about 800 nM, or about 1000 nM, or about 1200 nM, or about 1400 nM when assayed using an in vitro GLP2R cell assay.

In another embodiment, the fusion protein retains at least about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 10%, or about 20%, or about 30% 0f the potency of the corresponding GLP-2 not linked to XTEN when d using an in vitro GLP2R cell assay. In the foregoing embodiments of the paragraph, the GLP2R cell can be a human recombinant GLP-2 glucagon family or calcium- optimized cell or another cell comprising GLP2R known in the art.

Non-limiting examples of fusion proteins with a single GLP-2 linked to one or two XTEN are ted in Tables 13 and 32. In one embodiment, the ion provides a fusion protein composition has at least about 80% sequence identity compared to a sequence from Table 13 or Table 33, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity as compared to a sequence from Table 13 or Table 33. However, the invention also provides substitution of any of the GLP-2 sequences of Table 1 for a GLP-2 in a sequence of Table 33, and substitution of any XTEN ce of Table 4 for an XTEN in a sequence of Table 33. In some embodiments, the GLP-2 and the XTEN further comprise a spacer sequence of 1 to about 50 amino acid residues linking the GLP-2 and XTEN components, wherein the spacer sequence optionally comprises a cleavage sequence that is cleavable by a protease, including endogenous mammalian proteases. Examples of such se include, but are not limited to, FXIa, FXIIa, kallikrein, FVIIIa, FVIIIa, FXa, thrombin, se-2, granzyme B, MMP-12, MMP-13, MMP-17 or MMP-ZO, TEV, enterokinase, rhinovirus 3C protease, and e A, or a ce selected from Table 6. In one embodiment, a fusion protein composition with a cleavage sequence has a sequence having at least about 80% sequence identity compared to a sequence from Table 34, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity as compared to a sequence from Table 34. However, the invention also provides substitution of any of the GLP-2 sequences of Table 1 for a GLP-2 in a sequence of Table 34, and substitution of any XTEN ce of Table 4 for an XTEN in a sequence of Table 34, and substitution of any cleavage sequence of Table 6 for a cleavage sequence in a sequence of Table 34.

In ments having the subject cleavage sequences linked to the XTEN, cleavage of the cleavage sequence by the protease releases the XTEN from the ﬁlsion protein. In some embodiments of the fusion proteins comprising cleavage sequences that link XTEN to GLP-2, the GLP-2 component becomes WO 40093 biologically active or has an increase in the ty to bind to GLP-2 receptor upon its release from the XTEN by cleavage of the cleavage sequence, wherein the ing activity of the cleaved protein is at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to the ponding GLP-2 not linked to XTEN. In one embodiment of the foregoing, the cleavage sequence is cleavable by a protease of Table 6.

In another embodiment, the fusion protein comprises XTEN linked to the GLP-2 by two heterologous ge sequences that are cleavable by different proteases, which can be sequences of Table 6. In one ment of the foregoing, the cleaved GLPZ-XTEN has increased ty to bind the GLP-2 receptor.

The invention provides that the fusion proteins compositions of the embodiments comprising GLP-2 and XTEN characterized as described above, can be in different N— to C-terminus conﬁgurations.

In one ment of the GLPZ-XTEN composition, the ion provides a fusion protein of formula (GLP-2)-(XTEN) I wherein independently for each occurrence, GLP-2 is a GLP-2 protein or analog as defined herein, including sequences of Table l, and XTEN is an extended recombinant polypeptide as defined herein, including sequences exhibiting at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% sequence identity to a sequence of comparable length from any one of of Table 4, Table 8, Table 9, Table 10, Table 11, and Table 12, when optimally aligned. In one embodiment, the XTEN is AE864.

In another embodiment of the GLPZ-XTEN composition, the invention provides a fusion protein of formula II: (XTEN)-(GLP-2) 11 wherein independently for each occurrence, GLP-2 is a GLP-2 protein or analog as d herein, including sequences of Table l, and XTEN is an extended inant polypeptide as defined , including sequences exhibiting at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% sequence identity to a sequence of comparable length from any one of of Table 4, Table 8, Table 9, Table 10, Table 11, and Table 12, when optimally aligned. In one embodiment, the XTEN is AE864.

In another embodiment of the GLP2-XTEN composition, the invention provides an isolated fusion protein, wherein the fusion protein is of formula III: (XTEN)-(GLP-2)-(XTEN) 111 wherein independently for each occurrence, GLP-2 is a GLP-2 protein or analog as defined herein (e.g., including sequences of Table l), and XTEN is an extended recombinant polypeptide as defined herein, including sequences exhibiting at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% sequence identity to a sequence of comparable length from any one of of Table 4, Table 8, Table 9, Table 10, Table 11, and Table 12, when optimally aligned. In one embodiment, the XTEN is AE864.

In r embodiment of the GLPZ-XTEN composition, the invention provides an isolated fusion protein, wherein the ﬁJsion protein is of formula IV: (GLP-2)-(XTEN)-(GLP-2) IV n independently for each occurrence, GLP-2 is a GLP-2 protein or analog as d herein (e.g., including sequences of Table 1), and XTEN is an extended recombinant polypeptide as deﬁned herein e. g., including sequences exhibiting at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% sequence identity to a sequence of comparable length from any one of of Table 4, Table 8, Table 9, Table 10, Table 11, and Table 12, when lly aligned. In one embodiment, the XTEN is AE864.

In another embodiment of the GLPZ-XTEN composition, the invention provides an isolated ﬁJsion protein, wherein the fusion protein is of formula V: (GLP-2)-(S)x-(XTEN)y V wherein independently for each ence, GLP-2 is a GLP-2 n or analog as d herein, including sequences of Table 1; S is a spacer ce having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; X is either 0 or 1; and XTEN is an extended recombinant polypeptide as deﬁned herein, including ces ting at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% ce identity to a sequence of comparable length from any one of of Table 4, Table 8, Table 9, Table , Table 11, and Table 12, when lly aligned. In one embodiment, the XTEN is AE864. In the embodiments of formula V, the spacer sequence comprising a cleavage sequence is a sequence that is cleavable by a mammalian protease selected from the group consisting of factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin), elastase-2, MMP-12, MMP13, MMP- 17 and MMP-ZO. In one embodiment of the fusion protein of formula V, the GLP-2 comprises human GLP-Z. In another embodiment of the fusion protein of formula V, the GLP-Z comprises a GLP-Z of a s origin other than human, e.g., bovine GLP-Z, pig GLP-Z, sheep GLP-Z, chicken GLP-2, and canine GLP-Z. In another embodiment of the fusion protein of formula V, the GLP-2 has an amino acid substitution in place of Alaz, and wherein the substitution is glycine. In another embodiment, of the ﬁJsion protein of formula V, the GLP-2 has the sequence HGDGSFSDEMNTILDNLAARDFINWLIQTKITD. In another ment of the fusion protein of formula V, the fusion protein comprises a spacer sequence wherein the spacer sequence is a glycine residue.

] In another embodiment of the GLPZ-XTEN composition, the invention provides an isolated ﬁJsion protein, wherein the fusion protein is of formula VI: (XTEN)X-(S)X-(GLP-2)-(S)y-(XTEN)y v1 wherein independently for each occurrence, GLP-2 is a GLP-2 protein or analog as deﬁned herein (e.g., ing sequences of Table 1); S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; x is either 0 or 1 and y is either 0 or 1 wherein x+y 31; and XTEN is an extended recombinant polypeptide as deﬁned herein, e. g., including exhibiting at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% sequence identity to a sequence of comparable length from any one of of Table 4, Table 8, Table 9, Table 10, Table 11, and Table 12, when optimally aligned. In one embodiment, the XTEN is AE864. In the ments of formula VI, the spacer sequence comprising a cleavage ce is a sequence that is ble by a mammalian protease not limited to , includingbut factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, factor Ila (thrombin), se-2, , MMP13, MMP-17 and MMP-20.

In some embodiments, administration of a therapeutically effective dose of a fusion protein of one of formulae I-VI to a subject in need thereof can result in a gain in time of at least two-fold, or at least three-fold, or at least four-fold, or at least ﬁve-fold, or at least 10-fold or more spent within a therapeutic window for the fusion protein compared to the corresponding GLP-2 not linked to the XTEN and stered at a comparable dose to a subject. In other cases, administration of a therapeutically effective dose of a fusion protein of an ment of formulae I-VI to a subject in need thereof can result in a gain in time between consecutive doses necessary to maintain a therapeutically effective dose regimen of at least 48 h, or at least 72 h, or at least about 96 h, or at least about 120 h, or at least about 7 days, or at least about 14 days, or at least about 21 days between consecutive doses compared to administration of a corresponding GLP-2 not linked to XTEN at a able dose.

The ﬁlsion protein compositions ofthe embodiments bed herein can be evaluated for retention of activity (including after cleavage of any incorporated XTEN-releasing cleavage sites) using any appropriate in vitro assay disclosed herein (e.g., the assays of Table 32 or the assays described in the Examples), to determine the suitability of the conﬁguration for use as a therapeutic agent in the treatment of a GLPfactor related condition. In one embodiment, the fusion protein ts at least about 2%, or at least about 5%, or at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the activity ed to the ponding GLP-2 not linked to XTEN. In another embodiment, the GLP-2 component released from the ﬁlsion protein by enzymatic cleavage of the incorporated cleavage sequence g the GLP-2 and XTEN components exhibits at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the biological activity compared to the corresponding GLP-2 not linked to XTEN.

In some embodiments, ﬁlsion proteins comprising GLP-2 and one or more XTEN, wherein the ﬁasion proteins exhibit enhanced pharmacokinetic properties when administered to a subject ed to a GLP-2 not linked to the XTEN, wherein the enhanced properties include but are not limited to longer terminal half-life, larger area under the curve, increased time in which the blood concentration remains within the therapeutic window, increased time between consecutive doses resulting in blood concentrations within the therapeutic window, increased time between Cmax and Cmin blood concentrations when consecutive doses are administered, and decreased cumulative dose over time required to be administered compared to a GLP-2 not linked to the XTEN, yet still result in a blood concentration within the therapeutic window. A subject to which a XTEN composition is administered can include but is not limited to mouse, rat, monkey and human. In some embodiments, the terminal ife of the fusion protein administered to a subject is increased at least about three-fold, or at least about four-fold, or at least about ﬁve-fold, or at least about ld, or at least about eight-fold, or at least about ten-fold, or at least about 20-fold, or at least about d, or at least about 60-fold, or at least about ld, or even longer as ed to the corresponding recombinant GLP-2 not linked to the XTEN when the corresponding GLP-2 is administered to a t at a able dose. In other embodiments, the terminal half-life of the fusion protein administered to a subject is at least about 12 h, or at least about 24 h, or at least about 48 h, or at least about 72 h, or at least about 96 h, or at least about 120 h, or at least about 144 h, or at least about 21 days or greater. In other embodiments, the enhanced pharmacokinetic property is reﬂected by the fact that the blood concentrations remain within the therapeutic window for the fusion protein for a period that is at least about two-fold, or at least about three-fold, or at least about four-fold, or at least about ﬁve-fold, or at least about six-fold, or at least about eight—fold, or at least about ten—fold longer, or at least about 20—fold, or at least about 40—fold, or at least about 60—fold, or at least about 100—fold greater compared to the corresponding GLP—2 not linked to the XTEN when thee corresponding GLP-2 is administered to a subject at a comparable dose. The se in half-life and time spent within the therapeutic window permits less nt dosing and decreased amounts of the fusion protein (in nmoles/kg equivalent) that are administered to a subject, compared to the corresponding GLP-2 not linked to the XTEN. In one embodiment, administration of three or more doses of a GLP2-XTEN fusion protein to a subject in need thereof using a therapeutically—effective dose regimen results in a gain in time of at least two-fold, or at least three-fold, or at least four-fold, or at least ﬁve-fold, or at least ld, or at least fold, or at least 10-fold, or at least about 20-fold, or at least about 40-fold, or at least about 60-fold, or at least about 100-fold or higher between at least two consecutive Cmax peaks and/or Cm troughs for blood levels of the fusion protein compared to the corresponding GLP-2 not linked to the XTEN and administered using a comparable dose n to a subject. In one embodiment, the GLP2-XTEN administered using a therapeutically effective amount to a subject in need f results in blood concentrations of the GLPZ-XTEN fusion protein that remain above at least about 500 ng/ml, at least about 1000 ng/ml, or at least about 2000 ng/ml, or at least about 3000 ng/ml, or at least about 4000 ng/ml, or at least about 5000 ng/ml, or at least about 10000 ng/ml, or at least about 15000 ng/ml, or at least about 20000 ng/ml, or at least about 30000 ng/ml, or at least about 40000 ng/ml for at least about 24 hours, or at least about 48 hours, or at least about 72 hours, or at least about 96 hours, or at least about 120 hours, or at least about 144 hours. In another embodiment, the GLP2-XTEN administered at an appropriate dose to a subject results in area under the curve concentrations of the GLP2-XTEN fusion protein of at least 100000 hr*ng/mL, or at least about 200000 hr*ng/mL, or at least about 400000 hr*ng/mL, or at least about 600000 hr*ng/mL, or at least about 800000 hr*ng/mL, or at least about 1000000 hr*ng/mL, or at least about 2000000 hr*ng/mL after a single dose. In one ment, the GLP2-XTEN ﬁJsion protein has a terminal half-life that results in a gain in time between consecutive doses ary to maintain a therapeutically effective dose regimen of at least 48 h, or at least 72 h, or at least about 96 h, or at least about 120 h, or at least about 7 days, or at least about 14 days, or at least about 21 days between utive doses compared to the regimen of a GLP-2 not linked to XTEN and administered at a comparable close.

In one embodiment, the GLP2-XTEN fusion protein is characterized in that when an equivalent amount, in nmoles/kg of the fusion n and the corresponding GLP-2 that lacks the XTEN are each administered to comparable subjects, the fusion protein achieves a terminal half-life in the subject that is at least about 3-fold, or at least , or at least 5-fold, or at least 10-fold, or at least 15-fold, or at least -fold longer compared to the corresponding GLP-2 that lacks the XTEN. In another embodiment, the GLP2-XTEN fusion protein is characterized in that when a 2-fold, or 3-fold, or 4-fold, or 5-fold, or 6- fold smaller amount, in nmoles/kg, of the fusion protein than the corresponding GLP-2 that lacks the XTEN are each administered to comparable subjects with a gastrointestinal condition, the fusion protein achieves a comparable therapeutic effect in the subject as the corresponding GLP-2 that lacks the XTEN.

In another embodiment, the GLP2—XTEN fusion protein is characterized in that when the fusion protein is administered to a subject in utive doses to a subject using a dose interval that is at least about 2— fold, or at least 3-fold, or at least 4-fold, or at least 5-fold, or at least 10-fold, or at least 15-fold, or at least 20-fold longer as ed to a dose interval for the corresponding GLP-2 that lacks the XTEN and is administered to a comparable subject using an otherwise equivalent nmoles/kg amount, the fusion protein achieves a similar blood concentration in the t as compared to the ponding GLP-2 that lacks the XTEN. In another embodiment, the GLP2-XTEN fusion protein is characterized in that when the fusion protein is administered to a subject in consecutive doses to a subject using a dose interval that is at least about 3-fold, or at least 4-fold, or at least 5-fold, or at least d, or at least 15- fold, or at least 20-fold longer as compared to a dose interval for the corresponding GLP-2 that lacks the XTEN and is administered to a comparable subject using an otherwise equivalent /kg amount, the fusion protein achieves a comparable therapeutic effect in the subject as the corresponding GLP-2 that lacks the XTEN. In another embodiment, the GLP2-XTEN fusion protein ts any combination of, or all of the ing characterisitics of this paragraph. In the embodiments of this paragraph, the subject to which the subject composition is administered can e but is not, limited to mouse, rat, , and human. In one embodiment, the subject is rat. In another embodiment, the subject is human.

In one ment, the stration of a GLP2-XTEN fusion protein to a subject results in a greater therapeutic effect compared to the effect seen with the corresponding GLP-2 not linked to XTEN.

In another embodiment, the administration of an effective amount the fusion n results in a greater eutic effect in a subject with enteritis compared to the corresponding GLP-2 not linked to XTEN and administered to a comparable subject using a comparable nmoles/kg amount. In the foregoing, the subject is ed from the group consisting of mouse, rat, monkey, and human. In one embodiment of the foregoing, the subject is human and the enteritis is s disease. In another embodiment of the foregoing, the subject is rat subject and the enteritis is induced with indomethacin. In the foregoing embodiments of this paragraph, the greater therapeutic effect is selected from the group consisting of body weight gain, small intestine length, reduction in TNF (1 content of the small intestine , reduced mucosal atrophy, reduced incidence of perforated ulcers, and height of villi. In one embodiment, the administration of a GLP2-XTEN fusion protein to a subject results in an se in small intestine weight of at least about 10%, or at least about 20%, or at least about 30%, or at least about 40% greater ed to that of the corresponding GLP-2 not linked to XTEN. In another embodiment of the administration of a TEN fusion protein to a subject, the administration results in an increase in small intestine length of at least about 5%, or at least about 6%, or at least about 7%, or at least about 8%, or at least about 9%, or at least about 10%, or at least about 20%, or at least about 30%, or at least about 40% greater compared to that of the corresponding GLP-2 not linked to XTEN. In another ment of the administration of a GLP2-XTEN fusion protein to a subject, the administration results in an increase in body weight is at least about 5%, or at least about 6%, or at least about 7%, or at least about 8%, or at least about 9%, or at least about 10%, or at least about 20%, or at least about 30%, or at least about 40% greater compared to that of the corresponding GLP-2 not linked to XTEN. In another embodiment of the administration of a TEN fusion protein to a subject, the administration results a reduction in TNFOL content of at least about 0.5 ng/g, or at least about 0.6 ng/g, or at least about 0.7 ng/g, or at least about 0.8 ng/g, or at least about 0.9 ng/g, or at least about 1.0 ng/g, or at least about 1.1 ng/g, or at least about 1.2 ng/g, or at least about 1.3 ng/g, or at least about 1.4 ng/g of small intestine tissue or greater compared to that of the corresponding GLP-2 not linked to XTEN. In another embodiment of the stration of a GLP2-XTEN fusion protein to a subject, the administration results in an increase in villi height of at least about 5%, or at least about 6%, or at least about 7%, or at least about 8%, or at least about 9%, or at least about 10%, or at least about 11%, or at least about 12% greater compared to that of the ponding GLP-2 not linked to XTEN. In the foregoing embodiments of this paragraph, the fusion protein is administered as 1, or 2, or 3, or 4, or 5 , or 6, or 10, or 12 or more consecutive doses, wherein the dose amount is at least about 5, or least about 10, or least about 25, or least about 100, or least about 200 nmoles/kg.

In one embodiment, the GLP2-XTEN recombinant fusion protein comprises a GLP-2 linked to the XTEN via a cleavage sequence that is cleavable by a mammalian protease including but not limited to factor XIa, factor XIIa, rein, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin), Elastase-2, MMP-12, MMP13, MMP-17 and MMP-20, n cleavage at the cleavage sequence by the mammalian protease releases the GLP-2 sequence from the XTEN ce, and wherein the released GLP-2 sequence exhibits an increase in receptor binding activity of at least about 30% compared to the uncleaved fusion n.

The present invention provides methods of producing the GLPZ-XTEN fusion proteins. In some embodiments, the method of producing a fusion protein sing GLP-Z fused to one or more extended recombinant ptides (XTEN), comprises providing a host cell comprising a recombinant nucleic acid encoding the fusion protein of any ofthe embodiments bed herein; culturing the host cell under conditions permitting the sion of the fusion protein; and recovering the fusion protein.

In one embodiment of the method, the the host cell is a prokaryotic cell. In r embodiment of the method, the host cell is E. coli. In another embodiment of the method, the fusion protein is recovered from the host cell cytoplasm in substantially soluble form. In another ment of the method, the recombinant nucleic molecule has a sequence with at least 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity to a ce selected from the group consisting of the DNA sequences set forth in Table 13, when optimally aligned, or the complement thereof.

The present invention es isolated nucleic acids encoding the GLPZ-XTEN ﬁJsion proteins, vectors, and host cells comprising the vectors and nucleic acids. In one ment, the invention provides an isolated nucleic acid comprising a nucleic acid ce that has at least 70%, or at least about 80%, or at least about 90%,or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to a DNA sequence selected from Table 13, or the complement thereof. In another embodiment, the invention provides a nucleotide sequence encoding the fusion protein of any of fusion protein embodiments described herein, or the complement thereof. In another embodiment, the invention provides an expression vector or isolated host cell comprising the nucleic acid ofthe foregoing embodiments of this aph. In r embodiment, the invention provides a host cell comprising the foregoing expression vector.

Additionally, the present invention provides pharmaceutical compositions comprising the fusion protein of any of the foregoing embodiments described herein and a pharmaceutically acceptable carrier.

In addition, the present invention provides pharmaceutical compositions comprising the fusion protein of any of the foregoing embodiments bed herein for use in treating a gastrointestinal ion in a subject. In one embodiment, administration of a therapeutically effective amount of the ceutical composition to a subject with a gastrointestinal condition results in maintaining blood concentrations of the fusion protein within a therapeutic window for the fusion protein at least three-fold longer compared to the ponding GLP-2 not linked to the XTEN and administered at a comparable amount to the subject. In another embodiment, administration of three or more doses of the pharmaceutical composition to a subject with a gastrointestinal condition using a therapeutically-effective dose n results in a gain in time of at least four-fold between at least two consecutive me peaks and/or Cmin troughs for blood levels of the fusion protein compared to the corresponding GLP-2 not linked to the XTEN and administered using a comparable dose n to a subject. In another embodiment, the intravenous, subcutaneous, or intramuscular administration ofthe pharmaceutical composition comprising at least about 5, or least about 10, or least about 25, or least about 100, or least about 200 nmoles/kg 0f the fusion protein to a subject results in ﬁtsion protein blood levels maintained above 1000 ng/ml for at least 72 hours. In the foregoing embodiments of the paragraph, the gastrointestinal ion is selected from the group consisting of tis, digestion disorders, malabsorption syndrome, short-gut syndrome, short bowel syndrome, cul-de-sac syndrome, inﬂammatory bowel disease, celiac disease, tropical sprue, hypogammaglobulinemic sprue, Crohn's disease, ulcerative colitis, enteritis, chemotherapy-induced enteritis, irritable bowel syndrome, small intestine damage, small intestinal damage due to cancer-chemotherapy, gastrointestinal , diarrheal diseases, intestinal insufﬁciency, acid-induced intestinal injury, arginine deﬁciency, idiopathic hypospermia, obesity, catabolic illness, febrile penia, diabetes, obesity, steatorrhea, autoimmune diseases, food allergies, hypoglycemia, gastrointestinal barrier disorders, sepsis, bacterial peritonitis, bum-induced intestinal damage, decreased gastrointestinal motility, intestinal failure, herapy-associated emia, bowel trauma, bowel ischemia, mesenteric ischemia, malnutrition, necrotizing enterocolitis, necrotizing pancreatitis, neonatal feeding intolerance, NSAID-induced gastrointestinal damage, nutritional insufﬁciency, total parenteral nutrition damage to gastrointestinal tract, neonatal ional insufﬁciency, radiation—induced enteritis, radiation—induced injury to the ines, tis, pouchitis, and gastrointestinal ischemia. In the foregoing embodiments of the paragraph, the subject is selected from mouse, rat, monkey and human.

In another embodiment, the present invention provides a GLPZ-XTEN ﬁtsion protein according to any of the embodiments bed herein for use in the preparation of a ment for the treatment of a gastrointestinal condition described herein.

The present invention es GLPZ-XTEN fusion proteins according to any ofthe embodiments described herein for use in a method of treating a gastrointestinal condition in a subject, comprising administering to the subject a therapeutically ive amount of the fusion protein. In one embodiment, the gastrointestinal condition is selected from the group consisting of gastritis, digestion disorders, malabsorption me, short-gut syndrome, short bowel syndrome, -sac syndrome, inﬂammatory bowel disease, celiac e, tropical sprue, hypogammaglobulinemic sprue, s disease, ulcerative colitis, enteritis, chemotherapy-induced enteritis, irritable bowel syndrome, small intestine damage, small intestinal damage due to cancer-chemotherapy, gastrointestinal injury, diarrheal diseases, intestinal insufﬁciency, acid-induced intestinal injury, arginine deﬁciency, idiopathic hypospermia, obesity, catabolic illness, febrile neutropenia, diabetes, obesity, steatorrhea, autoimmune diseases, food allergies, hypoglycemia, gastrointestinal barrier disorders, sepsis, ial nitis, burn-induced intestinal damage, decreased gastrointestinal ty, intestinal e, chemotherapy- associated bacteremia, bowel trauma, bowel ischemia, mesenteric ischemia, malnutrition, necrotizing enterocolitis, izing pancreatitis, neonatal feeding intolerance, NSAID-induced gastrointestinal 2012/054941 damage, nutritional insufﬁciency, total parenteral nutrition damage to gastrointestinal tract, al nutritional insufﬁciency, radiation-induced enteritis, radiation-induced injury to the intestines, mucositis, pouchitis, and gastrointestinal ischemia. In another embodiment of the fusion protein for use in a method of treating a intestinal condition in a subject, administration of two or more utive doses of the fusion n administered using a therapeutically effective dose n to a subject results in a prolonged period between consecutive Cmax peaks and/or Cmin troughs for blood levels of the ﬁlSlOl’l protein ed to the corresponding GLP-2 that lacks the XTEN and administered using a therapeutically effective dose regimen established for the GLP-Z. In another embodiment of the fusion n for use in a method of treating a gastrointestinal condition in a t, administration of a smaller amount in /kg of the fusion protein to a t in comparison to the corresponding GLP-2 that lacks the XTEN, when administered to a subject under an otherwise equivalent dose regimen, results in the fusion n achieving a comparable therapeutic effect as the corresponding GLP-2 that lacks the XTEN. In the foregoing, the therapeutic effect is selected from the group consisting of blood concentrations of GLP-2, increased mesenteric blood ﬂow, sed inﬂammation, increased weight gain, decreased diarrhea, decreased fecal wet weight, intestinal wound healing, increase in plasma citrulline concentrations, decreased CRP levels, decreased requirement for steroid therapy, enhancing or stimulating mucosal integrity, decreased sodium loss, minimizing, ting, or preventing bacterial translocation in the intestines, enhancing, stimulating or accelerating recovery of the intestines after surgery, preventing relapses of inﬂammatory bowel disease, and maintaining energy homeostasis.

The present invention provides GLPZ-XTEN fusion proteins according to any ofthe ments described herein for use in a pharmaceutical regimen for treatment of a gastrointestinal condition in a subject. In one embodiment, the r pharmaceutical egimen comprises a pharmaceutical composition comprising the GLPZ-XTEN fusion protein. In another ment, the ceutical regimen further comprises the step of determining the amount of pharmaceutical composition needed to achieve a therapeutic effect in the subject, wherein the therapeutic effect is selected from the group ting of increased mesenteric blood ﬂow, decreased inﬂammation, increased weight gain, decreased ea, decreased fecal wet weight, intestinal wound healing, increase in plasma citrulline concentrations, decreased CRP levels, decreased requirement for steroid therapy, enhanced mucosal integrity, decreased sodium loss, preventing bacterial translocation in the intestines, accelerated recovery of the intestines after surgery, prevention of relapses of inﬂammatory bowel disease, and maintaining energy homeostasis. In another embodiment, the pharmaceutical regimen comprises administering the pharmaceutical composition in two or more successive doses to the t at an effective amount, wherein the administration results in at least a 5%, or 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90% greater improvement of at least one, two, or three parameters associated with the gastrointestinal condition compared to the GLP-2 not linked to XTEN and administered using a comparable nmol/kg amount. In one embodiment of the foregoing, the parameter improved is ed from increased blood concentrations of GLP-2, increased mesenteric blood ﬂow, decreased inﬂammation, increased weight gain, decreased diarrhea, sed fecal wet weight, intestinal wound healing, increase in plasma citrulline trations, decreased CRP levels, decreased requirement for steroid y, enhanced mucosal integrity, decreased sodium loss, preventing bacterial translocation in the intestines, accelerated recovery of the intestines after surgery, prevention of relapses of inﬂammatory bowel disease, and maintaining energy homeostasis. In another embodiment, the pharmaceutical regimen comprises administering a eutically effective amount of the pharmaceutical composition once every 7, or 10, or 14, or 21, or 28 or more days. In an embodiment of the foregoing, the effective amount is at least about 5, or least about 10, or least about 25, or least about 100, or least about 200 nmoles/kg.

In the embodiments of the n, the administration is subcutaneous, intramuscular, or intravenous.

The t invention provides methods of treating a gastrointestinal condition in a subject. In some embodiments, the method comprises administering to said subject a composition comprising an effective amount of a pharmaceutical composition comprising a GLP2-XTEN ﬁtsion n described herein. In one embodiment of the method, the effective amount is at least about 5, or least about 10, or least about 25, or least about 100, or least about 200 nmoles/kg. In another embodiment of the method, administration of the pharmaeceutical ition is subcutaneous, intramuscular, or intravenous. In another embodiment of the method, administration of the effective amount results in the fusion protein ting a terminal half-life of greater than about 30 hours in the subject, wherein the subject is ed from the group consisting of mouse, rat, monkey, and human. In the foregoing embodiments, the gastrointestinal condition is selected from the group consisting of gastritis, digestion disorders, malabsorption me, short-gut syndrome, short bowel syndrome, cul-de-sac syndrome, inﬂammatory bowel disease, celiac disease, tropical sprue, hypogammaglobulinemic sprue, Crohn's disease, tive colitis, enteritis, chemotherapy-induced enteritis, ble bowel syndrome, small intestine damage, small intestinal damage due to cancer-chemotherapy, gastrointestinal injury, diarrheal es, intestinal insufﬁciency, acid-induced intestinal injury, arginine deﬁciency, idiopathic hypospermia, obesity, catabolic illness, febrile penia, diabetes, obesity, steatorrhea, autoimmune diseases, food allergies, hypoglycemia, gastrointestinal barrier disorders, sepsis, bacterial peritonitis, burn-induced intestinal , decreased gastrointestinal motility, intestinal failure, chemotherapy- associated bacteremia, bowel trauma, bowel ischemia, mesenteric ischemia, malnutrition, izing enterocolitis, necrotizing atitis, al feeding intolerance, NSAID-induced gastrointestinal damage, nutritional insufﬁciency, total eral nutrition damage to intestinal tract, neonatal nutritional insufﬁciency, radiation-induced enteritis, radiation-induced injury to the intestines, mucositis, pouchitis, and gastrointestinal ischemia. In another ment of the method, the method is used to treat a subject with small intestinal damage due to chemotherapeutic agents such as, but not limited to 5- FU, altretamine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, mbucil, cisplatin, cladribine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, ubicin, docetaxel, doxorubicin, epirubicin, ide, ﬂudarabine, ﬂuorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, liposomal doxorubicin, leucoyorin, lomustine, melphalan, 2012/054941 mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, axel, pemetrexed, pentostatin, procarbazine, raltitrexed, streptozocin, tegafur—uracil, lomide, pa, tioguanine, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, and vinorelbine. In another embodiment of the method, administration ofthe pharmaeceutical composition results in an intestinotrophic effect in said subject. In yet another embodiment of the , administration of the pharmaeceutical composition results in an intestinotrophic effect in said subject, wherein the intestinotrophic effect is at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100% or at least about 120% or at least about 150% or at least about 200% of the intestinotrophic effect compared to the corresponding GLP-2 not linked to XTEN and administered to a subject using a comparable dose. In one embodiment of the foregoing, the intestinotrophic effect is determined after administration of 1 dose, or 3 doses, or 6 doses, or 10 doses, or 12 or more doses of the fusion protein. In r embodiment of the foregoing, the inotrophic effect is selected from the group consisting of intestinal growth, increased hyperplasia of the villus epithelium, increased crypt cell proliferation, increased height of the crypt and villus axis, sed healing after intestinal anastomosis, increased small bowel weight, increased small bowel length, decreased small bowel epithelium apoptosis, and enhancement of intestinal function.

In r embodiment, the present invention provides kits, comprising packaging material and at least a ﬁrst container sing the pharmaceutical composition comprising a GLP2—XTEN fusion protein described herein and a sheet of instructions for the reconstitution and/or administration of the pharmaceutical compositions to a subject.

The following are miting ary embodiments of the invention: Item 1. A recombinant fusion protein comprising a glucagon-like protein-2 (GLP-2) and an extended recombinant ptide (XTEN), wherein the XTEN is characterized in that: (a) the XTEN comprises at least 36 amino acid residues; (b) the sum of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues constitutes more than about 80% of the total amino acid residues of the XTEN; (c) the XTEN is substantially non-repetitive such that (i) the XTEN contains no three contiguous amino acids that are identical unless the amino acids are serine; (ii) at least about 80% of the XTEN sequence consists of non-overlapping sequence motifs, each of the sequence motifs comprising about 9 to about 14 amino acid residues consisting of four to six amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), wherein any two contiguous amino acid residues do not occur more than twice in each of the non-overlapping sequence motifs; or (iii) the XTEN ce has a uence score of less than 10; (d) the XTEN has greater than 90% random coil formation as determined by GOR algorithm; (e) the XTEN has less than 2% alpha helices and 2% beta-sheets as determined by Chou-Fasman algorithm; and (f) the XTEN lacks a predicted T-cell e when analyzed by TEPITOPE algorithm, wherein the TEPITOPE threshold score for said prediction by said algorithm has a threshold of —9, wherein said fusion protein exhibits an apparent molecular weight factor of at least about 4 and exhibits an intestinotrophic effect when administered to a subject using a therapeutically effective .

Item 2. The recombinant fusion n of item 1, wherein the intestinotrophic effect is at least about %, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100% or at least about 120% or at least about 150% or at least about 200% ofthe intestinotrophic effect compared to the corresponding GLP-2 not linked to XTEN when the corresponding GLP-2 is stered to a subject using a comparable dose.

Item 3. The recombinant fusion protein of item 1, wherein the t is selected from the group consisting of mouse, rat, monkey, and human.

Item 4. The recombinant fusion protein of any one of the preceding items, wherein said administration is subcutaneous, intramuscular, or intravenous.

Item 5. The recombinant fusion protein of any one of the preceding items, wherein the intestinotrophic effect is ined after administration of 1 dose, or 3 doses, or 6 doses, or 10 doses, or 12 or more doses of the fusion protein.

Item 6. The recombinant fusion protein of any one of the preceding items, wherein the intestinotrophic effect is selected from the group consisting of intestinal , increased hyperplasia of the villus epithelium, increased crypt cell eration, increased height of the crypt and villus axis, increased healing after intestinal anastomosis, increased small bowel weight, increased small bowel length, decreased small bowel epithelium apoptosis, and enhancement of intestinal on.

Item 7. The recombinant fusion protein of Item 6, wherein the stration results in an increase in small intestine weight of at least about 10%, or at least about 20%, or at least about 30%.

Item 8. The recombinant fusion protein of Item 6, wherein the administration results in an increase in small intestine length of at least about 5%, or at least about 6%, or at least about 7%, or at least about 8%, or at least about 9%, or at least about 10%, or at least about 20%, or at least about 30%.

Item 9. The recombinant fusion protein of any one of the preceding items, wherein the GLP-2 sequence has at least 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to a sequence selected from the group consisting of the sequences in Table 1, when optimally aligned.

Item 10. The recombinant fusion protein of any one of the preceding items, n the GLP-2 comprises human GLP-Z.

Item 11. The recombinant fusion protein of any one of Item 9-Item 11, wherein the GLP-2 is ed from the group consisting of bovine GLP-2, pig GLP-2, sheep GLP-2, chicken GLP-2, and canine GLP- 2012/054941 Item 12. The inant fusion protein of any one of the preceding items, wherein the GLP-2 has an amino acid substitution in place of Alaz, and wherein the substitution is glycine.

Item 13. The recombinant fusion protein of any one of Item 1-Item 9, wherein the GLP-2 has the sequence HGDGSFSDEMNTILDNLAARDFINWLIQTKITD.

Item 14. The recombinant fusion protein any one of the preceding items, wherein the XTEN is linked to the C—terminus of the GLP-2.

Item 15. The recombinant fusion protein of Item 14, further comprising a spacer sequence of l to about 50 amino acid es linking the GLP-2 and XTEN components.

Item 16. The recombinant fusion protein of Item 15, wherein the spacer sequence is a e residue.

Item 17. The recombinant fusion protein of any one of the preceding items, wherein the XTEN is characterized in that: (a) the total XTEN amino acid residues is at least 36 to about 3000 amino acid residues; (b) the sum of e (G), e (A), serine (S), threonine (T), glutamate (E) and proline (P) residues constitutes at least about 90% of the total amino acid residues of the XTEN; Item 18. The recombinant fusion protein of any one of the preceding items, wherein the XTEN is characterized in that the sum of asparagine and glutamine residues is less than 10% of the total amino acid sequence of the XTEN.

Item 19. The recombinant fusion protein of any one of the preceding items, wherein the XTEN is characterized in that the sum of methionine and phan residues is less than 2% of the total amino acid sequence of the XTEN.

Item 20. The inant fusion protein any one of the preceding items, wherein the XTEN has at least 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity when compared to a sequence of comparable length selected from any one of Table 4, Table 8, Table 9, Table 10, Table 11, and Table 12, when optimally aligned.

Item 21. The recombinant fusion protein any one of the preceding items, wherein the XTEN has at least 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity when ed to an AE864 sequence from Table 4, when optimally aligned.

Item 22. The recombinant fusion protein of any one of Item 1-Item 9 or Item 13, wherein the fusion protein sequence has a sequence with at least 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to the sequence set forth in . 2012/054941 Item 23. The recombinant fusion protein of any one of the preceding items, wherein the fusion protein exhibits a terminal half-life that is at least about 30 hours when administered to a subject.

Item 24. The inant fusion protein of any one of the preceding items, wherein the fusion protein binds to a GLP-2 receptor with an EC50 of less than about 30 nM, or about 100 nM, or about 200 nM, or about 300 nM, or about 370 nM, or about 400 nM, or about 500 nM, or about 600 nM, or about 700 nM, or about 800 nM, or about 1000 nM, or about 1200 nM, or about 1400 nM when assayed using an in Vitro GLP2R cell assay wherein the GLP2R cell is a human recombinant GLP-2 on family or calcium-optimized cell.

Item 25. The inant fusion protein of any one of the preceding items, wherein the fusion protein retains at least about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 10%, or about %, or about 30% of the potency of the corresponding GLP-2 not linked to XTEN when assayed using an in Vitro GLP2R cell assay wherein the GLP2R cell is a human recombinant GLP-2 glucagon family receptor calcium-optimized cell.

Item 26. The recombinant fusion protein of any one of the preceding items, characterized in that (a) when an equivalent amount, in nmoles/kg, of the fusion protein and the corresponding GLP-2 that lacks the XTEN are each stered to comparable subjects, the fusion protein es a terminal half-life in the subject that is at least about 3-fold, or at least 4-fold, or at least —fold, or at least 10—fold, or at least 15—fold, or at least 20—fold longer compared to the corresponding GLP—2 that lacks the XTEN; (b) when a 2-fold, or 3-fold, or 4-fold, or 5-fold, or 6-fold smaller amount, in nmoles/kg, of the fusion protein than the corresponding GLP-2 that lacks the XTEN are each stered to comparable subjects with a gastrointestinal condition, the fusion protein achieves a comparable therapeutic effect in the subject as the corresponding GLP-2 that lacks the XTEN; (c) when the fusion protein is stered to a subject in consecutive doses to a subject using a dose interval that is at least about , or at least 3-fold, or at least 4-fold, or at least 5-fold, or at least 10-fold, or at least 15-fold, or at least 20-fold longer as compared to a dose interval for the corresponding GLP-2 that lacks the XTEN and is administered to a comparable subject using an otherwise equivalent nmoles/kg amount, the fusion n achieves a similar blood concentration in the subject as compared to the corresponding GLP-2 that lacks the XTEN; or (d) when the fusion protein is administered to a subject in consecutive doses to a subject using a dose interval that is at least about 3-fold, or at least 4-fold, or at least , or at least 10-fold, or at least 15-fold, or at least 20-fold longer as ed to a dose interval for the corresponding GLP-2 that lacks the XTEN and is administered to a comparable subject using an otherwise equivalent nmoles/kg amount, the ﬁlSlOl’l protein achieves a comparable therapeutic effect in the subject as the corresponding GLP-2 that lacks the XTEN.

Item 27. The recombinant fusion protein of Item 26, wherein the subject is selected from the group consisting of mouse, rat, monkey, and human. 2012/054941 Item 28. The inant fusion protein of Item 27, wherein the subject is rat.

Item 29. The recombinant fusion protein of any one of Item 26-Item 28, wherein the administration results in a greater therapeutic effect compared to the effect seen with the corresponding GLP-2 not linked to XTEN.

Item 30. The recombinant fusion protein of any one of Item 26-Item 29, wherein administration of an ive amount the fusion protein results in a greater therapeutic effect in a subject with enteritis compared to the corresponding GLP-2 not linked to XTEN when the corresponding GLP-2 is administered to a comparable subject using a comparable nmoles/kg amount.

Item 31. The recombinant fusion protein of any one of Item 26-Item 30, wherein the subject is selected from the group consisting of mouse, rat, monkey, and human.

Item 32. The recombinant fusion protein of Item 31, n the subject is human and the enteritis is Crohn’s disease.

Item 33. The recombinant fusion protein of Item 31, wherein the t is rat subject and the enteritis is induced with indomethacin.

Item 34. The recombinant fusion protein of any one of Item 29-Item 33, wherein the greater therapeutic effect is selected from the group consisting of body weight gain, small intestine length, reduction in TNFo. content of the small intestine tissue, reduced mucosal atrophy, reduced incidence of perforated ulcers, and height of Villi.

Item 35. The recombinant fusion protein of Item 34, wherein the administration s in an increase in small intestine weight of at least about 10%, or at least about 20%, or at least about 30%, or at least about 40% greater compared to that of the corresponding GLP-2 not linked to XTEN.

Item 36. The recombinant fusion protein of Item 34, wherein the administration results in an increase in small intestine length of at least about 5%, or at least about 6%, or at least about 7%, or at least about 8%, or at least about 9%, or at least about 10%, or at least about 20%, or at least about 30%, or at least about 40% r ed to that of the corresponding GLP-2 not linked to XTEN.

Item 37. The recombinant fusion protein of Item 34, n the administration results in an increase in body weight is at least about 5%, or at least about 6%, or at least about 7%, or at least about 8%, or at least about 9%, or at least about 10%, or at least about 20%, or at least about 30%, or at least about 40% greater compared to that of the corresponding GLP-2 not linked to XTEN.

Item 38. The inant fusion protein of Item 34, wherein the ion in TNFU. content is at least about 0.5 ng/g, or at least about 0.6 ng/g, or at least about 0.7 ng/g, or at least about 0.8 ng/g, or at least about 0.9 ng/g, or at least about 1.0 ng/g, or at least about 1.1 ng/g, or at least about 1.2 ng/g, or at least about 1.3 ng/g, or at least about 1.4 ng/g of small intestine tissue or greater ed to that of the corresponding GLP-2 not linked to XTEN.

Item 39. The recombinant fusion protein of Item 34, wherein the Villi height is at least about 5%, or at least about 6%, or at least about 7%, or at least about 8%, or at least about 9%, or at least about 10%, or at least about 11%, or at least about 12% greater compared to that of the corresponding GLP-2 not linked to XTEN.

Item 40. The recombinant fusion protein of any one of Item 29-Item 39, wherein the fusion protein is administered as 1, or 2, or 3, or 4, or 5, or 6, or 10, or 12 or more consecutive doses.

Item 41. The recombinant fusion protein of any one of Item 30-Item 40, wherein the effective amount is at least about 5, or least about 10, or least about 25, or least about 100, or least about 200 nmoles/kg.

Item 42. The recombinant fusion n of any one of the preceding items, wherein the GLP-2 is linked to the XTEN via a cleavage sequence that is cleavable by a mammalian protease selected from the group consisting of factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin), Elastase-2, MMP-12, MMP13, MMP-17 and MMP-20, wherein cleavage at the cleavage sequence by the mammalian protease releases the GLP-2 sequence from the XTEN sequence, and wherein the ed GLP-2 sequence exhibits an increase in receptor binding activity of at least about % compared to the uncleaved fusion protein.

Item 43. A method of producing a fusion protein comprising GLP-2 fused to one or more extended inant polypeptides , comprising: (a) providing a host cell comprising a inant nucleic acid encoding the fusion protein of any one of items 1 to Item 41; (b) culturing the host cell under conditions permitting the expression of the fusion protein; and (c) recovering the fusion protein.

Item 44. The method of Item 43, wherein: (a) the host cell is a prokaryotic cell; or (b) the fusion protein is recovered from the host cell asm in ntially soluble form.

Item 45. The method of Item 43, n the recombinant nucleic acid molecule has a ce with at least 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% ce identity to a sequence selected from the group consisting of the DNA sequences set forth in Table 13, when optimally aligned, or the complement thereof.

Item 46. An isolated nucleic acid comprising: (a) a nucleic acid sequence that has at least 70%, or at least about 80%, or at least about 90%,or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity to a DNA sequence selected from Table 13, or the complement thereof; or (b) a nucleotide sequence encoding the fusion protein of any of items 1-Item 41, or the complement thereof.

Item 47. An sion vector or isolated host cell comprising the nucleic acid of any one of Item 43- Item 46.

Item 48. A host cell comprising the expression vector of Item 47.

Item 49. A pharmaceutical composition comprising the ﬁJsion protein of 1-Item 41, and a pharmaceutically acceptable carrier.

Item 50. The recombinant fusion n of item I conﬁgured according to formula V: (a) )-(S)x-(XTEN) (V) wherein independently for each occurrence, (b) GLP-2 is a sequence having at least 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity to a sequence selected from the group consisting of the sequences in Table 1, when optimally aligned; (c) S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a ge sequence from Table 6 or amino acids compatible with restrictions sites; (d) X is either 0 or 1; Item 51. The recombinant fusion protein of Item 50, wherein the GLP-2 comprises human GLP-2.

Item 52. The recombinant fusion n of Item 50, wherein the GLP—2 is selected from the group consisting of bovine GLP—2, pig GLP—2, sheep GLP—2, chicken GLP—2, and canine GLP—2.

Item 53. The recombinant fusion protein of Item 51 or item Item 52, wherein the GLP-2 has an amino acid substitution in place of Alaz, and wherein the tution is glycine.

Item 54. The recombinant fusion protein of Item 50, wherein the GLP-2 has the sequence HGDGSFSDEMNTILDNLAARDFINWLIQTKITD.

Item 55. The recombinant fusion protein of any one of Item 5O-Item 54, comprising a spacer sequence wherein the spacer sequence is a glycine residue.

Item 56. The recombinant fusion n any one of Item m 55, wherein the XTEN has at least 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence ty when compared to a sequence of comparable length selected from any one of Table 4, Table 8, Table 9, Table 10, Table 11, and Table 12, when optimally aligned.

Item 57. The recombinant fusion protein any one of Item 50-Item 55, wherein the XTEN has at least 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity when compared to a AE864 sequence from Table 4, when optimally aligned.

Item 58. The pharmaceutical composition of Item 49, wherein administration of a therapeutically effective amount of the pharmaceutical composition to a subject with a gastrointestinal condition results in maintaining blood trations ofthe fusion protein within a therapeutic window for the fusion WO 40093 protein at least three-fold longer ed to the corresponding GLP-2 not linked to the XTEN and administered at a comparable amount to the subject.

Item 59. The pharmaceutical composition of Item 49, wherein administration of three or more doses of the pharmaceutical composition to a subject with a gastrointestinal condition using a therapeutically- effective dose regimen results in a gain in time of at least four-fold n at least two consecutive Cmax peaks and/or le-n troughs for blood levels of the fusion protein ed to the corresponding GLP-2 not linked to the XTEN and administered using a comparable dose regimen to a subject.

Item 60. The pharmaceutical composition of Item 59 or Item 60, wherein the gastrointestinal condition is ed from the group consisting of gastritis, digestion disorders, malabsorption syndrome, short-gut syndrome, short bowel syndrome, cul-de-sac syndrome, inﬂammatory bowel disease, celiac disease, tropical sprue, hypogammaglobulinemic sprue, Crohn's disease, tive colitis, enteritis, chemotherapy-induced enteritis, irritable bowel syndrome, small intestine damage, small intestinal damage due to cancer-chemotherapy, gastrointestinal injury, diarrheal diseases, intestinal insufﬁciency, acid-induced intestinal , arginine deﬁciency, thic hypospermia, obesity, lic s, febrile neutropenia, diabetes, obesity, steatorrhea, autoimmune diseases, food allergies, hypoglycemia, gastrointestinal barrier disorders, sepsis, bacterial peritonitis, burn-induced inal damage, decreased intestinal motility, intestinal failure, chemotherapy- associated emia, bowel trauma, bowel ischemia, mesenteric ischemia, malnutrition, necrotizing enterocolitis, necrotizing pancreatitis, neonatal feeding intolerance, NSAID—induced gastrointestinal damage, nutritional insufﬁciency, total parenteral nutrition damage to gastrointestinal tract, neonatal nutritional insufﬁciency, radiation-induced enteritis, radiation-induced injury to the intestines, mucositis, pouchitis, and gastrointestinal ischemia.

Item 61. The pharmaceutical composition of Item 49, wherein after intravenous, subcutaneous, or intramuscular administration of the pharmaceutical composition comprising at least about 5, or least about 10, or least about 25, or least about 100, or least about 200 nmoles/kg of the fusion protein to a subject, the fusion protein blood levels are ined above 1000 11ng for at least 72 hours.

Item 62. The pharmaceutical ition of Item 61, wherein the subject is selected from mouse, rat, monkey and human.

Item 63. A recombinant fusion protein according to any one of l-Item 41 for use in the manufacture of a medicament for the treatment of a intestinal condition.

Item 64. The inant fusion protein of Item 63 wherein the gastrointestinal condition is selected from the group consisting of gastritis, digestion disorders, malabsorption syndrome, short-gut syndrome, short bowel syndrome, cul-de-sac syndrome, inﬂammatory bowel disease, celiac disease, al sprue, hypogammaglobulinemic sprue, Crohn's e, ulcerative colitis, enteritis, chemotherapy-induced enteritis, irritable bowel syndrome, small intestine damage, small intestinal damage due to cancer- chemotherapy, gastrointestinal , diarrheal diseases, inal insufﬁciency, acid-induced inal injury, arginine deﬁciency, idiopathic hypospermia, obesity, catabolic illness, febrile neutropenia, diabetes, obesity, steatorrhea, autoimmune diseases, food allergies, ycemia, gastrointestinal barrier WO 40093 disorders, sepsis, bacterial nitis, bum-induced intestinal damage, decreased gastrointestinal motility, inal failure, chemotherapy- associated bacteremia, bowel trauma, bowel ischemia, mesenteric ischemia, malnutrition, necrotizing colitis, necrotizing pancreatitis, neonatal feeding intolerance, NSAID-induced gastrointestinal , nutritional insufﬁciency, total parenteral nutrition damage to gastrointestinal tract, neonatal ional insufﬁciency, radiation-induced enteritis, radiation- induced injury to the intestines, mucositis, pouchitis, ischemia, and stroke.

Item 65. A recombinant fusion protein according to any one of l-Item 41 for use in a method of ng a gastrointestinal condition in a subject, comprising administering to the subject a therapeutically ive amount of the fusion protein.

Item 66. The recombinant fusion protein for use according to item Item 65, wherein the gastrointestinal condition is selected from the group consisting of gastritis, digestion disorders, malabsorption syndrome, short-gut syndrome, short bowel syndrome, cul-de-sac syndrome, inﬂammatory bowel disease, celiac disease, tropical sprue, hypogammaglobulinemic sprue, Crohn's disease, ulcerative colitis, enteritis, chemotherapy-induced enteritis, irritable bowel syndrome, small intestine damage, small intestinal damage due to cancer-chemotherapy, gastrointestinal injury, diarrheal diseases, intestinal insufﬁciency, acid-induced intestinal injury, arginine deﬁciency, idiopathic hypospermia, obesity, catabolic illness, febrile neutropenia, diabetes, obesity, steatorrhea, autoimmune diseases, food allergies, hypoglycemia, gastrointestinal barrier disorders, sepsis, bacterial peritonitis, nduced inal damage, sed gastrointestinal motility, intestinal failure, chemotherapy— associated bacteremia, bowel trauma, bowel ischemia, mesenteric ischemia, malnutrition, necrotizing enterocolitis, necrotizing pancreatitis, neonatal feeding rance, NSAID-induced gastrointestinal damage, nutritional insufﬁciency, total parenteral nutrition damage to gastrointestinal tract, neonatal nutritional ciency, ion-induced enteritis, radiation-induced injury to the intestines, mucositis, pouchitis, ischemia, and stroke.

Item 67. The inant fusion n for use ing to item Item 65 wherein administration of two or more consecutive doses of the fusion protein administered using a therapeutically effective dose regimen to a subject results in a prolonged period between consecutive me peaks and/or Cmin troughs for blood levels of the fusion protein compared to the corresponding GLP-2 that lacks the XTEN and administered using a therapeutically effective dose regimen established for the GLP-Z.

Item 68. The recombinant fusion protein for use according to item Item 65 wherein a smaller amount in nmoles/kg of the fusion protein is stered to a subject in comparison to the ponding GLP-2 that lacks the XTEN administered to a subject under an ise equivalent dose regimen, and the fusion protein achieves a able therapeutic effect as the corresponding GLP-2 that lacks the XTEN.

Item 69. The inant fusion protein for use according to item Item 68, wherein the therapeutic effect is selected from the group consisting of blood concentrations of GLP-2, increased mesenteric blood ﬂow, decreased inﬂammation, sed weight gain, decreased ea, decreased fecal wet weight, intestinal wound healing, increase in plasma citrulline concentrations, decreased CRP levels, decreased requirement for steroid therapy, enhancing or stimulating mucosal integrity, decreased sodium loss, minimizing, mitigating, or preventing ial translocation in the ines, enhancing, stimulating or accelerating recovery of the intestines after surgery, preventing relapses of inﬂammatory bowel disease, and maintaining energy homeostasis.

Item 70. A recombinant fusion protein for use in a pharmaceutical regimen for treatment of a gastrointestinal condition in a subject, said regimen sing a pharmaceutical composition comprising the fusion protein of any one of l-Item 41.

Item 71. The recombinant fusion protein of Item 70, n the pharmaceutical regimen further comprises the step of determining the amount of ceutical composition needed to achieve a therapeutic effect in the subject, wherein the therapeutic effect is selected from the group consisting of increased mesenteric blood ﬂow, decreased inﬂammation, increased weight gain, decreased diarrhea, decreased fecal wet weight, intestinal wound healing, increase in plasma citrulline concentrations, decreased CRP levels, decreased requirement for steroid therapy, enhanced mucosal ity, decreased sodium loss, preventing bacterial translocation in the intestines, accelerated ry of the intestines after surgery, prevention of relapses of inﬂammatory bowel disease, and ining energy homeostasis.

Item 72. The inant fusion protein of Item 70, wherein the gastrointestinal condition is selected from the group consisting of gastritis, digestion disorders, malabsorption syndrome, short-gut me, short bowel syndrome, cul—de—sac syndrome, inﬂammatory bowel disease, celiac disease, tropical sprue, hypogammaglobulinemic sprue, Crohn's disease, ulcerative colitis, enteritis, chemotherapy—induced enteritis, irritable bowel syndrome, small intestine damage, small intestinal damage due to - chemotherapy, gastrointestinal injury, diarrheal diseases, intestinal ciency, acid-induced intestinal injury, ne deficiency, idiopathic hypospermia, obesity, catabolic illness, febrile neutropenia, diabetes, obesity, steatorrhea, autoimmune diseases, food allergies, hypoglycemia, intestinal r disorders, , bacterial peritonitis, bum-induced intestinal damage, decreased gastrointestinal motility, intestinal failure, chemotherapy— associated bacteremia, bowel , bowel ischemia, mesenteric ischemia, malnutrition, izing enterocolitis, necrotizing pancreatitis, al feeding intolerance, NSAID-induced gastrointestinal damage, ional insufficiency, total parenteral nutrition damage to intestinal tract, neonatal nutritional insufficiency, radiation-induced enteritis, radiation- induced injury to the intestines, mucositis, pouchitis, ischemia, and stroke.

Item 73. The recombinant fusion protein of Item 70, wherein the pharmaceutical regimen for treating a subject with a gastrointestinal condition comprises administering the pharmaceutical ition in two or more successive doses to the subject at an effective amount, wherein the administration results in at least a 5%, or 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90% r improvement of at least one, two, or three parameters associated with the gastrointestinal condition compared to the GLP-2 not linked to XTEN and administered using a able nmol/kg amount.

Item 74. The recombinant fusion protein of Item 73, n the parameter improved is selected from increased blood concentrations of GLP-2, increased mesenteric blood ﬂow, decreased inﬂammation, increased weight gain, decreased diarrhea, decreased fecal wet weight, intestinal wound g, increase in plasma citrulline concentrations, decreased CRP levels, decreased requirement for steroid therapy, enhanced mucosal integrity, decreased sodium loss, preventing bacterial translocation in the intestines, accelerated recovery of the intestines after surgery, prevention of relapses of inﬂammatory bowel disease, and maintaining energy tasis.

Item 75. The recombinant fusion protein of Item 70, wherein the regimen comprises stering a therapeutically effective amount of the pharmaceutical composition of Item 49 once every 7, or 10, or 14, or 21, or 28 or more days.

Item 76. The recombinant fusion protein of Item 75, wherein the effective amount is at least about 5, or least about 10, or least about 25, or least about 100, or least about 200 /kg.

Item 77. The recombinant fusion protein of any one of Item 73-Item 76, wherein said administration is subcutaneous, intramuscular, or intravenous.

Item 78. A method of treating a gastrointestinal condition in a subject, comprising administering to said subject a composition comprising an effective amount of the ceutical ition of Item 49.

Item 79. The method of Item 78, wherein the effective amount is at least about 5, or least about 10, or least about 25, or least about 100, or least about 200 nmoles/kg.

Item 80. The method of Item 79, wherein the fusion protein exhibits a terminal half-life of greater than about 30 hours in said t.

Item 81. The method of any one of Item m 80, n the gastrointestinal condition is selected from the group consisting of gastritis, digestion disorders, malabsorption me, short-gut syndrome, short bowel syndrome, cul-de-sac syndrome, inﬂammatory bowel disease, celiac disease, tropical sprue, hypogammaglobulinemic sprue, Crohn's disease, ulcerative colitis, enteritis, chemotherapy-induced enteritis, irritable bowel syndrome, small intestine damage, small intestinal damage due to cancer- chemotherapy, gastrointestinal injury, diarrheal diseases, intestinal insufﬁciency, acid-induced intestinal injury, arginine deficiency, idiopathic hypospermia, obesity, catabolic illness, febrile neutropenia, diabetes, obesity, steatorrhea, mune diseases, food allergies, hypoglycemia, gastrointestinal barrier disorders, , bacterial peritonitis, bum-induced intestinal damage, sed gastrointestinal motility, intestinal failure, chemotherapy— ated bacteremia, bowel trauma, bowel ischemia, mesenteric ischemia, malnutrition, necrotizing enterocolitis, necrotizing pancreatitis, neonatal feeding intolerance, NSAID-induced gastrointestinal damage, nutritional insufficiency, total parenteral ion damage to gastrointestinal tract, al nutritional insufficiency, radiation-induced enteritis, radiation- induced injury to the intestines, mucositis, pouchitis, ischemia, and .

Item 82. The method of Item 81, wherein the gastrointestinal condition is s disease.

Item 83. The method of any one of Item 78-Item 82, wherein the subject is selected from the group consisting of mouse, rat, monkey, and human.

Item 84. The method of any one of Item 78-Item 83, wherein said administration is aneous, intramuscular, or intravenous. 2012/054941 Item 85. The method of any one of Item 78-Item 84, wherein said administration results in an intestinotrophic effect in said subject.

Item 86. The method of Item 85, wherein the intestinotrophic effect is at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100% or at least about 120% or at least about 150% or at least about 200% of the intestinotrophic effect compared to the corresponding GLP-2 not linked to XTEN and administered to a subject using a comparable dose.

Item 87. The method of Item 85 or Item 86, wherein the inotrophic effect is determined after administration of 1 dose, or 3 doses, or 6 doses, or 10 doses, or 12 or more doses of the fusion protein.

Item 88. The method of any one of Item 85-Item 87, wherein the inotrophic effect is selected from the group consisting of intestinal growth, increased hyperplasia ofthe villus epithelium, increased crypt cell proliferation, increased height of the crypt and villus axis, increased healing after intestinal anastomosis, increased small bowel weight, increased small bowel , decreased small bowel epithelium apoptosis, and enhancement of intestinal on.

It is ically contemplated that the recombinant GLP2-XTEN fusion proteins can exhibit one or more or any combination of the properties disclosed herein.

ORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was speciﬁcally and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS The features and advantages of the invention may be ﬁarther explained by reference to the following detailed description and accompanying drawings that sets forth illustrative embodiments. is a tic of the logic ﬂow chart of the algorithm SegScore. In the ﬁgure the following legend applies: i, j - counters used in the control loops that run through the entire sequence; HitCount- this variable is a counter that keeps track ofhow many times a subsequence encounters an identical subsequence in a block; SubSeqX - this variable holds the subsequence that is being checked for redundancy; SubSeqY - this le holds the subsequence that the SubSeqX is checked against; BlockLen - this variable holds the user determined length of the block; SegLen - this variable holds the length of a segment. The program is hardcoded to generate scores for subsequences of lengths 3, 4, 5, 6, 7, 8, 9, and 10; Block - this le holds a string of length BlockLen. The string is composed of letters from an input XTEN sequence and is determined by the position of the i counter; SubSeqList - this is a list that holds all of the generated uence scores. depicts the application of the thm SegScore to a hypothetical XTEN of 11 amino acids in order to determine the tiveness. An XTEN sequence consisting ofN amino acids is d into N-S+l subsequences of length S (823 in this case). A pair-wise comparison of all subsequences is performed and the average number of identical subsequences is calculated to result, in this case, in a subsequence score of 1.89. illustrates the use of donor XTEN sequences to produce truncated XTEN ces. provides the sequence of AG864, with the underlined sequence used to generate an AGS76 sequence. provides the sequence of AG864, with the underlined sequence used to generate an AG288 sequence. provides the ce of AG864, with the ined sequence used to te an AGl44 sequence. es the sequence of AE864, with the underlined sequence used to generate an AES76 sequence. es the sequence of AE864, with the underlined sequence used to generate an AE288 sequence. is a schematic ﬂowchart of representative steps in the assembly, production and the evaluation of an XTEN. is a schematic ﬂowchart of representative steps in the assembly of a GLP2-XTEN polynucleotide construct encoding a fusion protein. Individual oligonucleotides 501 are ed into sequence motifs 502 such as a 12 amino acid motif (“12-mer”), which is ligated to additional sequence motifs from a y to create a pool that encompasses the desired length of the XTEN 504, as well as ligated to a smaller concentration of an oligo containing BbsI, and KpnI restriction sites 503. The resulting pool of on ts is gel-puriﬁed and the band with the desired length of XTEN is cut, resulting in an isolated XTEN gene with a stopper sequence 505. The XTEN gene is cloned into a r vector. In this case, the vector encodes an optional CBD sequence 506 and a GFP gene 508. Digestion is then performed with BbsI/HindIII to remove 507 and 508 and place the stop codon. The resulting product is then cloned into a BsaI/HindIII digested vector containing a gene encoding the GLP-2, resulting in gene 500 encoding a GLP2-XTEN ﬁtsion protein. is a schematic ﬂowchart of representative steps in the ly of a gene encoding fusion protein comprising a GLP-2 and XTEN, its expression and recovery as a fusion n, and its evaluation as a candidate GLP2-XTEN product. shows schematic representations of exemplary GLP2-XTEN fusion proteins (FIGS. 7A- H), all depicted in an N— to C-terminus orientation. shows two different rations of GLP2-XTEN fusion proteins (100), each comprising a single GLP-2 and an XTEN, the ﬁrst of which has an XTEN molecule (102) attached to the C—terminus of a GLP-2 (103), and the second of which has an XTEN molecule attached to the N—terminus of a GLP-2 (103). shows two different conﬁgurations of GLPZ-XTEN fusion proteins (100), each sing a single GLP-2, a spacer sequence and an XTEN, the ﬁrst of which has an XTEN molecule (102) attached to the C-terminus of a spacer sequence (104) and the spacer sequence attached to the C-terminus of a GLP-2 (103) and the second of which has an XTEN molecule attached to the N—terminus of a spacer sequence (104) and the spacer sequence attached to the N—terminus of a GLP-2 (103). shows two different conﬁgurations of GLP2-XTEN fusion ns (101), each comprising two molecules of a single GLP-2 and one molecule of an XTEN, the ﬁrst of which has an XTEN linked to the C-terminus of a ﬁrst GLP-2 and that GLP-2 is 2012/054941 linked to the C—terminus of a second GLP-2, and the second of which is in the opposite orientation in which the XTEN is linked to the inus of a ﬁrst GLP-2 and that GLP-2 is linked to the inus of a second GLP-2. shows two different conﬁgurations of GLP2-XTEN fusion proteins (101), each comprising two molecules of a single GLP-Z, a spacer sequence and one molecule of an XTEN, the ﬁrst of which has an XTEN linked to the C-terminus of a spacer ce and the spacer sequence linked to the C—terminus of a ﬁrst GLP-2 which is linked to the C-terminus of a second GLP-2, and the second of which is in the opposite orientation in which the XTEN is linked to the N—terminus of a spacer sequence and the spacer sequence is linked to the N—terminus of a ﬁrst GLP-2 that that GLP-2 is linked to the N-terminus of a second GLP-2. shows two different conﬁgurations of GLPZ-XTEN fusion proteins (101), each comprising two molecules of a single GLP-2, a spacer sequence and one molecule of an XTEN, the ﬁrst ofwhich has an XTEN linked to the C-terminus of a ﬁrst GLP-2 and the ﬁrst GLP-2 linked to the C-terminus of a spacer sequence which is linked to the C-terminus of a second GLP-2 molecule, and the second of which is in the opposite conﬁguration of XTEN linked to the N-terminus of a ﬁrst GLP-2 which is linked to the N—terminus of a spacer sequence which in turn is linked to the N- terminus of a second molecule of GLP-2. shows a conﬁguration of GLP2-XTEN fusion protein (105), each comprising one molecule of GLP-2 and two molecules of an XTEN linked to the N—terminus and the C-terminus of the GLP-2. shows a conﬁguration (106) of a single GLP-2 linked to two XTEN, with the second XTEN separated from the GLP—2 by a spacer sequence. shows a conﬁguration (106) of a two GLP—2 linked to two XTEN, with the second XTEN linked to the C— terminus ofthe ﬁrst GLP-2 and the N—terminus of the second GLP-2, which is at the C-terminus of the TEN. is a schematic illustration of exemplary polynucleotide constructs (FIGS. 8A—H) of GLP2-XTEN genes that encode the corresponding TEN polypeptides of all depicted in a ’ to 3’ orientation. In these illustrative examples the genes encode GLP2-XTEN fusion proteins with one GLP-2 and XTEN (200); or one GLP-2, one spacer sequence and one XTEN (200); two GLP-2 and one XTEN (201); or two GLP-2, a spacer ce and one XTEN (201); one GLP-2 and two XTEN (205); or two GLP-2 and two XTEN (206). In these depictions, the polynucleotides encode the following components: XTEN (202), GLP-2 (203), and spacer amino acids that can include a cleavage sequence (204), with all sequences linked in frame. is a schematic representation of the design of GLP2-XTEN expression s with different processing strategies. shows an exemplary expression vector ng XTEN fused to the 3’ end ofthe sequence encoding GLP-2. Note that no additional leader sequences are required in this vector. s an expression vector encoding XTEN fused to the 3’ end ofthe sequence encoding GLP-2 with a CBD leader sequence and a TEV protease site. depicts an expression vector where the CBD and TEV processing site have been replaced with an optimized N—terminal leader sequence (NTS). s an expression vector encoding an NTS sequence, an XTEN, a sequence encoding GLP-2, and then a second ce encoding an XTEN. illustrates the process of combinatorial gene assembly of genes ng XTEN. In this case, the genes are assembled from 6 base fragments and each fragment is available in 4 different codon versions (A, B, C and D). This allows for a theoretical ity of 4096 in the assembly of a 12 amino acid motif. shows characteriation data of the fusion protein GLP2-2G_AE864. A is an SDSPAGE gel of GLP2-2G-XTEN_AE864 lot AP690, as described in Example 16. The gels show lanes of lar weight standards and 2 or 10 ug ofreference standard, as indicated. B shows results of a size exclusion chromatography analysis of GLP2-2G-XTEN_AE864 lot AP690, as described in Example 16, ed to molecular weight standards of 667, 167, 44, 17, and 3.5 kDa. shows the ESI-MS analysis of GLP2-2G-XTEN_AE864 lot AP690, as described in Example 16, with a major peak at 83,142 Da, ting full length intact GLP2-2G-XTEN, with an additional minor peak of 83,003 Da detected, representing the des-His GLP2-2G-XTEN at <5% of total protein. shows results of the GLP-2 receptor binding assay, as described in Example 17. shows the results of the pharmacokinetics of GLP2-2G-XTEN_AE864 in C57Bl/6 mice following subcutaneous (SC) administration. The samples were analyzed for fusion protein concentration, performed by both anti-XTEN/anti- XTEN sandwich ELISA and anti-GLP2/anti-XTEN sandwich ELISA, as described in Example 18, with results for both assays plotted. shows the results of the pharmacokinetics of GLP2—2G—XTEN_AE864 in Wistar rats following SC administration of two different dosage levels, performed by both anti-XTEN/anti- XTEN sandwich ELISA and anti-GLP2/anti-XTEN ch ELISA, as described in Example 19, with results for both assays plotted. shows the results of the pharmacokinetics of GLP2-2G-XTEN_AE864 in male cynomolgus monkeys following either subcutaneous (squares) or intravenous (triangles) administration of the fusion protein at a single dosage level (2 mg/kg). The samples were analyzed for fusion protein concentration, performed by anti-GLP2/anti-XTEN ELISA, as described in e 20. shows the linear regression ofthe tric g of GLP2-2G—XTEN half-life from three species used to predict a projected half-life of 240 hours in humans, as described in Example 20. shows the results in rat small intestine weight and length from vehicle and treatment groups, as described in Example 21. shows the results of changes in body weight in a murine dextran sodium sulfate (DSS) model, with groups d with vehicle, GLP2-2G e (no XTEN) or G-XTEN, as bed in Example 21. shows entative histopathology sections of the DSS model mice from vehicle ileum (A) and jejunum (B) and GLP2-2G-XTEN ileum (C) and jejunum (D), as described in Example 21. shows s from Study 1 of a rat model of Crohn’s Disease of indomethacin-induced intestinal inﬂammation, with groups treated with vehicle, GLP2-2G peptide (no XTEN) or GLP2-2G- XTEN and assayed, as described in Example 21. A shows results of the body weight at the termination of the experiment. B shows results of the length of the small intestines from each group. C shows results of the weight of the small intestines from each group. D shows results of the length of ulcerations and the percentage of ulceration in the small intestines from each group. E shows s of the scores of adhesions and transulceration in the small intestines from each group. F shows results of the length and tage of inﬂammation of the small intestines from each group. G shows results of the TNFoc assay of the small intestines from each group. shows s from Study 2 of a rat model of Crohn’s Disease of indomethacin-induced intestinal inﬂammation, with groups treated with vehicle, GLP2-2G peptide (no XTEN) or GLP2-2G- XTEN and assayed, as bed in Example 21. A shows the Trans-Ulceration Score of the small intestines from each group. B shows the Adhesion Score of the small intestines from each group. shows representative histopathology sections from Study 2 of the rat model of Crohn’s Disease of indomethacin-induced intestinal ation from vehicle-no indomethicin (A), vehicle-indomethicin (B) and GLP2-2G-XTEN treatment groups (FIGS. 22C, D), as described in Example 21. shows the results of small intestine length (A), villi height (B) and histopathology scoring (C) of mucosal atrophy, ulceration, inﬁltration measurements from ed, e-treated, GLP2-2G peptide-treated, and GLP2-2G-XTEN—treated rats, as described in Example 21. Asterisks indicate groups with tically signiﬁcant differences from vehicle (diseased) control group. shows results of a size exclusion chromatography analysis of glucagon-XTEN construct samples measured t protein standards n molecular weight (as ted), with the graph output as absorbance versus retention volume, as described in Example 25. The glucagon—XTEN ucts are 1) glucagon—Y288; 2) glucagonY—144; 3) glucagon—Y72; and 4) glucagon—Y36. The results indicate an increase in apparent molecular weight with increasing length ofXTEN moiety. shows the cokinetic proﬁle (plasma concentrations) in cynomolgus monkeys after single doses of different compositions of GFP linked to unstructured polypeptides of varying length, administered either subcutaneously or intravenously, as described in Example 26. The compositions were GFP-L288, GFP-L576, GFP-XTEN_AF576, GFP-Y576 and XTEN_AD836-GFP. Blood samples were analyzed at various times after injection and the tration of GFP in plasma was measured by ELISA using a polyclonal antibody against GFP for capture and a biotinylated preparation of the same polyclonal antibody for detection. Results are presented as the plasma concentration versus time (h) after dosing and show, in particular, a erable increase in half-life for the XTEN_AD836-GFP, the composition with the longest sequence length of XTEN. The construct with the shortest sequence length, the GFP-L288 had the shortest half-life. shows an SDS-PAGE gel of samples from a stability study ofthe fusion protein of E864 fused to the N-terminus of GFP (see Example 27). The GFP-XTEN was incubated in cynomolgus plasma and rat kidney lysate for up to 7 days at 37°C. In addition, GFP-XTEN administered to cynomolgus monkeys was also assessed. Samples were withdrawn at O, l and 7 days and analyzed by SDS PAGE followed by detection using Western is with antibodies against GFP. shows the amino acid ce of GLP2-2G_AE864.

DETAILED DESCRIPTION OF THE INVENTION Before the embodiments ofthe invention are described, it is to be understood that such embodiments are provided by way of example only, and that various alternatives to the embodiments of the invention described herein may be employed in practicing the ion. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention.

Unless otherwise , all technical and scientiﬁc terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or lent to those described herein can be used in the practice or testing ofthe present invention, suitable methods and als are described below. In case of t, the patent speciﬁcation, including deﬁnitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Numerous ions, changes, and substitutions will now occur to those skilled in the art without departing from the invention.

DEFINITIONS In the context of the present application, the ing terms have the meanings ascribed to them unless speciﬁed otherwise: As used in the speciﬁcation and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates ise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

The terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modiﬁed amino acids, and it may be interrupted by ino acids. The terms also encompass an amino acid polymer that has been modiﬁed, for example, by disulﬁde bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as ation with a labeling component.

As used herein, the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including but not limited to both the D or L l isomers, and amino acid s and peptidomimetics. Standard single or three letter codes are used to designate amino acids.

The term “natural L-amino acid” means the L optical isomer forms of glycine (G), proline (P), alanine (A), valine (V), leucine (L), isoleucine (I), nine (M), cysteine (C), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T).

The term aturally occurring,” as applied to sequences and as used herein, means polypeptide or polynucleotide sequences that do not have a counterpart to, are not complementary to, or do not have a high degree of homology with a ype or naturally-occurring sequence found in a mammal. For example, a non-naturally occurring polypeptide or fragment may share no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid sequence identity as compared to a natural sequence when ly aligned.

The terms “hydrophilic” and “hydrophobic” refer to the degree of afﬁnity that a substance has with water. A hydrophilic substance has a strong afﬁnity for water, tending to dissolve in, mix with, or be wetted by water, while a hydrophobic substance substantially lacks afﬁnity for water, tending to repel and not absorb water and tending not to dissolve in or mix with or be wetted by water. Amino acids can be characterized based on their hydrophobicity. A number of scales have been developed. An example is a scale developed by Levitt, M, et al., J Mol Biol (1976) 104:59, which is listed in Hopp, TP, et al., Proc Natl Acad Sci U S A (1981) 78:3 824. Examples of “hydrophilic amino acids” are arginine, lysine, threonine, alanine, asparagine, and glutamine. Of particular interest are the hydrophilic amino acids aspartate, glutamate, and serine, and glycine. Examples of “hydrophobic amino acids” are tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine, and valine.

A “fragment” when applied to a protein, is a truncated form of a native biologically active protein that retains at least a portion of the therapeutic and/or biological activity. A “variant” when applied to a protein, is a n with sequence homology to the native biologically active n that retains at least a portion of the therapeutic and/or biological activity of the biologically active protein. For example, a variant protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity compared with the reference biologically active n. As used herein, the term gically active protein moiety” includes proteins modiﬁed deliberately, as for example, by site directed mutagenesis, synthesis of the encoding gene, insertions, or accidentally through mutations.

The term “sequence variant” means polypeptides that have been d compared to their native or original sequence by one or more amino acid ions, deletions, or substitutions. Insertions may be located at either or both termini of the protein, and/or may be oned within internal regions of the amino acid sequence. A non-limiting e would be insertion of an XTEN sequence within the ce of the biologically-active payload protein. In on variants, one or more amino acid es in a ptide as described herein are removed. Deletion variants, therefore, include all nts of a payload polypeptide ce. In substitution variants, one or more amino acid residues of a polypeptide are removed and replaced with alternative es. In one aspect, the substitutions are conservative in nature and conservative substitutions of this type are well known in the art.

As used herein, “internal XTEN” refers to XTEN ces that have been inserted into the sequence of the GLP-2. Internal XTENs can be constructed by insertion of an XTEN sequence into the sequence of GLP-2 by ion between two adjacent amino acids or wherein XTEN es a partial, internal sequence of the GLP-Z.

As used herein, “terminal XTE ” refers to XTEN sequences that have been fused to or in the N- or C-terminus of the GLP-2 or to a proteolytic cleavage sequence at the N— or C—terminus of the GLP-2.

Terminal XTENs can be fused to the native termini ofthe GLP-Z. Alternatively, terminal XTENs can replace a terminal sequence of the GLP-Z.

The term “XTEN release site” refers to a cleavage sequence in GLPZ-XTEN fusion proteins that can be recognized and d by a mammalian protease, effecting release of an XTEN or a portion of an XTEN from the GLPZ-XTEN fusion protein. As used herein, “mammalian se” means a protease that normally exists in the body ﬂuids, cells or tissues of a mammal. XTEN release sites can be engineered to be cleaved by various mammalian proteases (a.k.a. “XTEN release proteases”) such as FXIa, FXHa, rein, , FVHIa, FXa, FHa (thrombin), se-2, MMP-12, MMP13, , MMP-ZO, or any protease that is present in the subject in proximity to the fusion n. Other equivalent proteases (endogenous or exogenous) that are capable of recognizing a deﬁned cleavage site can be ed. The ge sites can be adjusted and ed to the se utilized.

The term “within”, when referring to a ﬁrst polypeptide being linked to a second polypeptide, encompasses linking that connects the N—terminus of the ﬁrst or second polypeptide to the C—terminus of the second or ﬁrst polypeptide, respectively, as well as insertion of the ﬁrst polypeptide into the sequence of the second polypeptide. For example, when an XTEN is linked “within” a GLP-2 polypeptide, the XTEN may be linked to the N-terminus, the C-terminus, or may be inserted between any two amino acids of the GLP-2 polypeptide.

“Activity” for the purposes herein refers to an action or effect of a component of a fusion protein consistent with that of the corresponding native biologically active protein component of the fusion protein, wherein “biological activity” refers to an in vitro or in vivo biological on or effect, including but not limited to receptor binding, antagonist activity, agonist activity, a cellular or physiologic response, or an effect generally known in the art for the payload GLP-Z.

As used herein, the term "ELISA" refers to an enzyme-linked immunosorbent assay as described herein or as otherwise known in the art.

A “host cell” includes an individual cell or cell culture which can be or has been a recipient for the subject vectors. Host cells include progeny of a single host cell. The progeny may not arily be completely identical (in morphology or in genomic of total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a vector of this invention.

“Isolated,” when used to describe the various polypeptides sed herein, means polypeptide that has been identiﬁed and separated and/or red from a component of its natural environment.

WO 40093 Contaminant components of its natural environment are materials that would lly ere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart. In addition, a ntrated”, “separated” or “diluted” polynucleotide, peptide, polypeptide, n, antibody, or fragments f, is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is generally greater than that of its naturally occurring counterpart. In general, a polypeptide made by recombinant means and expressed in a host cell is considered to be “isolated.” An “isolated” nucleic acid is a nucleic acid molecule that is identiﬁed and separated from at least one contaminant c acid molecule with which it is ordinarily associated in the natural source of the nucleic acid. For example, an ed polypeptide-encoding nucleic acid molecule is other than in the form or g in which it is found in nature. Isolated ptide-encoding nucleic acid molecules therefore are distinguished from the speciﬁc ptide-encoding c acid molecule as it exists in natural cells. However, an isolated polypeptide-encoding nucleic acid molecule includes polypeptide- encoding c acid les ned in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal or extra-chromosomal location different from that of natural cells.

A “chimeric” protein contains at least one fusion polypeptide comprising at least one region in a different on in the sequence than that which occurs in nature. The regions may normally exist in separate proteins and are brought together in the ﬁJsion polypeptide, or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. A chimeric protein may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the e regions are encoded in the desired relationship.

“Conjugated”, “linked,” “fused,” and “fusion” are used interchangeably herein. These terms refer to the joining together of two or more chemical elements, sequences or components, by whatever means including chemical conjugation or recombinant means. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and in reading phase or in-frame.

An ame fusion” refers to the joining oftwo or more open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs. Thus, the resulting recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature).

In the context of polypeptides, a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminus direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide. A “partial sequence” is a linear sequence of part of a polypeptide that is known to comprise additional residues in one or both directions.

“Heterologous” means derived from a genotypically distinct entity from the rest of the entity to which it is being compared. For example, a glycine rich sequence removed from its native coding sequence and operatively linked to a coding ce other than the native sequence is a heterologous glycine rich sequence. The term ologous” as applied to a polynucleotide, a polypeptide, means that the polynucleotide or polypeptide is derived from a genotypically distinct entity from that ofthe rest of the entity to which it is being compared.

The terms “polynucleotides”, ic acids”, “nucleotides” and “oligonucleotides” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may m any on, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) deﬁned from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid , and primers. A polynucleotide may comprise modiﬁed nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modiﬁcations to the nucleotide structure may be imparted before or after assembly of the r. The ce of nucleotides may be interrupted by non—nucleotide components. A cleotide may be ﬁlrther modiﬁed after polymerization, such as by ation with a labeling component.

The term “complement of a polynucleotide” denotes a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence, such that it could hybridize with a reference sequence with complete ﬁdelity.

“Recombinant” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of recombination steps which may include cloning, restriction and/or ligation steps, and other procedures that result in an expression of a recombinant protein in a host cell.

The terms “gene” and “gene fragment” are used interchangeably herein. They refer to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated. A gene or gene fragment may be genomic or cDNA, as long as the polynucleotide ns at least one open reading frame, which may cover the entire coding region or a segment thereof A “fusion gene” is a gene composed of at least two heterologous polynucleotides that are linked together. ogy” or “homologous” or “sequence ty” refers to sequence rity or interchangeability between two or more polynucleotide ces or between two or more polypeptide sequences. When using a program such as BestFit to ine sequence identity, rity or homology between two different amino acid sequences, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, rity or gy scores. Preferably, cleotides that are homologous are those which hybridize under stringent conditions as deﬁned herein and have at least 70%, ably at least 80%, more preferably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, and even more preferably 99% sequence identity compared to those sequences. Polypeptides that are homologous preferably have sequence ties that are at least 70%, preferably at least 80%, even more preferably at least 90%, even more preferably at least 95-99%, and most preferably 100% identical.

"Ligation" refers to the process of forming odiester bonds between two c acid fragments or genes, linking them together. To ligate the DNA fragments or genes together, the ends of the DNA must be compatible with each other. In some cases, the ends will be ly compatible after endonuclease digestion. However, it may be necessary to ﬁrst convert the staggered ends commonly produced after endonuclease digestion to blunt ends to make them compatible for ligation.

The terms “stringent conditions” or “stringent hybridization conditions” includes reference to conditions under which a polynucleotide will hybridize to its target sequence, to a detectably greater degree than other sequences (e. g., at least 2-fold over background). Generally, stringency of hybridization is sed, in part, with reference to the temperature and salt concentration under which the wash step is carried out. Typically, stringent ions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short polynucleotides (e.g., 10 to 50 nucleotides) and at least about 60°C for long polynucleotides (e.g., greater than 50 nucleotides)—for example, “stringent conditions” can include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and three washes for 15 min each in 0.1 XSSC/1% SDS at 60°C to 65°C. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC tration may be varied from about 0.1 to 2XSSC, with SDS being present at about 0.1%. Such wash temperatures are typically selected to be about 5°C to °C lower than the thermal melting point for the speciﬁc sequence at a deﬁned ionic strength and pH.

The Tm is the temperature (under deﬁned ionic th and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid ization are well known and can be found in Sambrook, J. et a]. , “Molecular Cloning: A tory Manual,” 3rd edition, Cold Spring Harbor Laboratory Press, 2001. Typically, blocking reagents are used to block non-speciﬁc hybridization. Such ng reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 [Lg/m1. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA2DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art.

The terms “percent identity, “percentage of sequence identity,” and “% identity,” as applied to polynucleotide sequences, refer to the percentage ofresidue s between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more gful comparison of the two sequences. Percent ty may be measured over the length of an entire deﬁned polynucleotide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, deﬁned polynucleotide sequence, for instance, a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210 or at least 450 uous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown , in the tables, ﬁgures or Sequence Listing, may be used to be a length over which percentage identity may be measured. The percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of matched positions (at which identical residues occur in both polypeptide sequences), dividing the number of matched positions by the total number of positions in the window of ison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. When sequences of different length are to be compared, the shortest sequence deﬁnes the length of the window of comparison. vative substitutions are not considered when calculating sequence identity.

“Percent (%) sequence identity,” with respect to the polypeptide sequences identiﬁed herein, is deﬁned as the tage of amino acid residues in a query ce that are cal with the amino acid residues of a second, reference polypeptide sequence or a portion thereof, after aligning the sequences and introducing gaps, if necessary, to achieve the m percent sequence identity, and not considering any conservative substitutions as part of the sequence identity, thereby ing in optimal alignment. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly ble computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve optimal alignment over the ﬁill length of the sequences being compared. Percent identity may be measured over the length of an entire deﬁned polypeptide sequence, or may be measured over a shorter , for example, over the length of a fragment taken from a larger, deﬁned polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, ﬁgures or Sequence Listing, may be used to be a length over which percentage identity may be measured. itiveness” used in the t of polynucleotide sequences refers to the degree of internal homology in the sequence such as, for example, the frequency of identical nucleotide sequences of a given length. Repetitiveness can, for example, be measured by analyzing the frequency of identical sequences.

A “vector” is a nucleic acid molecule, preferably self-replicating in an appropriate host, which ers an inserted c acid molecule into and/or between host cells. The term includes vectors that ﬁmction primarily for insertion of DNA or RNA into a cell, replication of s that function primarily for the replication of DNA or RNA, and sion vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the above ons. An “expression vector” is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide(s). An “expression system” usually connotes a suitable host cell comprised of an expression vector that can function to yield a desired expression product.

“Serum ation resistance,” as applied to a polypeptide, refers to the ability of the polypeptides to and degradation in blood or components thereof, which typically involves proteases in the serum or plasma. The serum degradation resistance can be measured by ing the protein with human (or mouse, rat, monkey, as appropriate) serum or plasma, typically for a range of days (e.g. 0.25, 0.5, 1, 2, 4, 8, 16 days), lly at about 37°C. The samples for these time points can be run on a Western blot assay and the n is detected with an antibody. The antibody can be to a tag in the protein. If the protein shows a single band on the western, where the protein’s size is identical to that of the injected protein, then no degradation has occurred. In this exemplary method, the time point where 50% of the protein is degraded, as judged by Western blots or equivalent techniques, is the serum degradation ife or “serum half-life” of the protein.

] The terms “tug”, “terminal half-life”, “elimination half-life” and “circulating half-life” are used interchangeably herein and, as used herein mean the al half—life calculated as ln(2)/Kel. K61 is the terminal elimination rate constant calculated by linear regression of the terminal linear portion of the log concentration vs. time curve. ife typically refers to the time required for half the quantity of an administered substance deposited in a living organism to be metabolized or eliminated by normal biological processes.

“Active clearance” means the isms by which a protein is removed from the circulation other than by ﬁltration, and which includes removal from the circulation mediated by cells, receptors, metabolism, or degradation of the protein.

“Apparent molecular weight factor” and “apparent molecular weight” are related terms referring to a measure of the relative increase or se in apparent molecular weight ted by a particular amino acid or polypeptide sequence. The apparent molecular weight is determined using size exclusion chromatography (SEC) or r methods by comparing to globular protein standards and is measured in ent kDa” units. The apparent molecular weight factor is the ratio between the apparent molecular weight and the actual molecular weight; the latter predicted by adding, based on amino acid composition, the calculated molecular weight of each type of amino acid in the composition or by estimation from comparison to molecular weight standards in an SDS electrophoresis gel. Determination of both the apparent molecular weight and apparent molecular weight factor for representative proteins is described in the Examples.

The terms “hydrodynamic radius” or “Stokes radius” is the ive radius (R11 in mm) of a le in a solution measured by assuming that it is a body moving through the solution and resisted by the solution’s Viscosity. In the embodiments of the invention, the hydrodynamic radius measurements of the XTEN fusion ns correlate with the ‘apparent molecular weight factor’, which is a more intuitive measure. The dynamic radius” of a protein affects its rate of diffusion in aqueous solution as well as its y to migrate in gels of macromolecules. The hydrodynamic radius of a protein is determined by its lar weight as well as by its structure, including shape and compactness. Methods for determining the hydrodynamic radius are well known in the art, such as by the use of size exclusion chromatography (SEC), as described in US. Patent Nos. 6,406,632 and 7,294,513. Most proteins have globular structure, which is the most compact three-dimensional ure a protein can have with the smallest ynamic radius. Some proteins adopt a random and open, unstructured, or ‘linear’ conformation and as a result have a much larger hydrodynamic radius compared to typical globular proteins of r molecular weight.

“Physiological conditions” refers to a set of conditions in a living host as well as in vitro conditions, ing temperature, salt concentration, pH, that mimic those conditions of a living subject.

A host of physiologically relevant conditions for use in in vitro assays have been established. Generally, a physiological buffer contains a physiological tration of salt and is adjusted to a neutral pH ranging from about 6.5 to about 7.8, and preferably from about 7.0 to about 7.5. A variety of logical buffers are listed in Sambrook et al. (2001). Physiologically relevant temperature ranges from about 25°C to about 38°C, and preferably from about 350C to about 37°C.

A “reactive group” is a chemical structure that can be coupled to a second reactive group. es for reactive groups are amino groups, carboxyl , sulfhydryl groups, hydroxyl groups, aldehyde groups, azide groups. Some reactive groups can be activated to facilitate coupling with a second reactive group. Non-limiting examples for activation are the reaction of a carboxyl group with carbodiimide, the sion of a carboxyl group into an ted ester, or the conversion of a carboxyl group into an azide function.

“Controlled release agent”, “slow release agent”, “depot formulation” and “sustained release agent” are used interchangeably to refer to an agent capable of ing the duration of release of a polypeptide of the invention relative to the duration of release when the polypeptide is administered in the absence of agent. Different embodiments of the present invention may have ent release rates, resulting in different therapeutic amounts.

The terms “antigen”, “target antigen” and ogen” are used interchangeably herein to refer to the structure or binding determinant that an antibody fragment or an antibody fragment-based therapeutic binds to or has speciﬁcity against.

The term “payload” as used herein refers to a protein or peptide ce that has biological or therapeutic ty; the counterpart to the pharmacophore of small molecules. Examples of ds include, but are not limited to, cytokines, enzymes, hormones, blood coagulation factors, and growth factors. Payloads can further comprise genetically fused or chemically conjugated moieties such as chemotherapeutic agents, antiviral compounds, toxins, or st . These conjugated moieties can be joined to the rest of the polypeptide Via a linker that may be cleavable or non-cleavable.

The term “antagonist”, as used , includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native ptide disclosed herein. Methods for identifying antagonists of a polypeptide may comprise contacting a native ptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally ated With the native polypeptide. In the t of the present invention, antagonists may include proteins, c acids, carbohydrates, antibodies or any other molecules that se the effect of a biologically active protein.

The term “agonist” is used in the broadest sense and includes any molecule that mimics a biological activity of a native polypeptide disclosed herein. Suitable agonist molecules speciﬁcally include agonist antibodies or antibody fragments, fragments or amino acid sequence variants of native ptides, peptides, small organic molecules, etc. Methods for identifying agonists of a native polypeptide may comprise contacting a native polypeptide With a candidate agonist molecule and measuring a detectable change in one or more biological activities ly associated With the native polypeptide.

“Inhibition constant”, or “K”, are used interchangeably and mean the dissociation constant of the enzyme—inhibitor complex, or the reciprocal of the binding y of the inhibitor to the enzyme.

As used herein, “treat” or “treating,” or “palliating” or “ameliorating” are used interchangeably and mean administering a drug or a biologic to achieve a therapeutic beneﬁt, to cure or reduce the severity of an existing condition, or to achieve a prophylactic , prevent or reduce the hood of onset or severity the occurrence of a condition. By therapeutic beneﬁt is meant eradication or amelioration of the underlying condition being treated or one or more of the physiological symptoms associated with the underlying condition such that an improvement is observed in the subject, notwithstanding that the subject may still be afﬂicted With the underlying condition.

A “therapeutic effect” or peutic beneﬁt,” as used herein, refers to a physiologic effect, including but not d to the mitigation, amelioration, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals, resulting from administration of a fusion protein of the invention other than the ability to induce the production of an antibody against an nic epitope sed by the biologically active protein. For prophylactic beneﬁt, the compositions may be administered to a subject at risk of developing a particular condition, or to a subject reporting one or more ofthe physiological symptoms of a condition, even though a diagnosis (e.g., Crohn’s Disease) may not have been made.

The terms “therapeutically ive amount” and “therapeutically effective dose”, as used herein, refer to an amount of a drug or a ically active n, either alone or as a part of a fusion protein composition, that is capable of having any detectable, beneﬁcial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition When administered in one or WO 40093 repeated doses to a subject. Such effect need not be absolute to be beneﬁcial. Determination of a therapeutically effective amount is well within the capability of those d in the art, especially in light of the detailed disclosure provided herein.

The term “therapeutically effective dose regimen”, as used , refers to a schedule for consecutively administered multiple doses (i.e., at least two or more) of a biologically active protein, either alone or as a part of a fusion protein composition, wherein the doses are given in therapeutically effective amounts to result in sustained beneﬁcial effect on any symptom, aspect, measured parameter or characteristics of a e state or condition.

I). GENERAL TECHNIQUES The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell y, cs and inant DNA, which are within the skill of the art. See Sambrook, J. et al., ular Cloning: A Laboratory Manual,” 3rd edition, Cold Spring Harbor Laboratory Press, 2001; “Current protocols in molecular biology”, F. M. Ausubel, et al. eds.,1987; the series ds in Enzymology,” Academic Press, San Diego, CA.; “PCR 2: a practical approach”, M.J. MacPherson, B.D.

Hames and GR. Taylor eds., Oxford University Press, 1995; “Antibodies, a laboratory manual” Harlow, E. and Lane, D. eds., Cold Spring Harbor Laboratory,1988; “Goodman & Gilman’s The Pharmacological Basis of Therapeutics,” 11Th n, McGraw-Hill, 2005; and Freshney, R.I., “Culture of Animal Cells: A Manual of Basic Technique,” 4th edition, John Wiley & Sons, Somerset, NJ, 2000, the contents of which are incorporated in their entirety herein by reference. '1). GLUCAGON-LIKE-2 PROTEIN The present ion relates, in part, to fusion protein compositions comprising GLP-2 and one or more extended recombinant polypeptide , resulting in GLP2-XTEN fusion protein compositions.

“Glucagon—like protein-2” or “GLP-2” means, collectively herein, human glucagon like peptide-2, species gs of human GLP-2, and non-natural sequence variants having at least a n of the biological activity of mature GLP-2 including variants such as, but not limited to, a variant with glycine substituted for alanine at position 2 ofthe mature sequence (“2G”) as well as Val, Glu, Lys, Arg, Leu or Ile substituted for alanine at position 2. GLP-2 or sequence variants have been isolated, synthesized, characterized, or cloned, as described in US. Patent or Application Nos. 5,789,379; 428; 5,990,077; 5,994,500; 6,184,201; 7,186,683; 7,563,770; 20020025933; and 20030162703.

Human GLP-2 is a 33 amino acid e, co-secreted along with GLP-l from intestinal endocrine cells in the epithelium of the small and large intestine. The 180 amino-acid product of the proglucagon gene is post-translationally processed in a tissue-specific manner in pancreatic A cells and inal L cells into the 33 amino acid GLP-2 v et al., FEBS Lett. (1989) 247: 193-196; Hartmann et al., Peptides (2000) 21: 73-80). In pancreatic A cells, the major bioactive hormone is glucagon cleaved by PCSK2/PC2. In the intestinal L cells PCSKl/PCl liberates GLP—l, GLP—2, glicentin and oxyntomodulin. GLP-2 functions as a pleiotropic intestinotrophic hormone with wide-ranging effects that include the promotion of mucosal growth and nutrient absorption, intestinal homeostasis, regulation of gastric motility, gastric acid secretion and inal hexose transport, reduction of intestinal permeability and increase in mesenteric blood ﬂow (Estall JL, Drucker DJ (2006) Glucagon—like e-2. Annual Rev 1391—411), (Guan X, et al. (2006) GLP-2 receptor localizes to enteric neurons and endocrine cells sing vasoactive peptides and mediates increased blood ﬂow.

Gastroenterology 130:150—164; Stephens J, et al. (2006) Glucagon-like peptide-2 acutely increases proximal small inal blood ﬂow in TPN-fed al piglets. Am J Physiol Regul Integr Comp Physiol 290:R283—R289; Nelson DW, et al. (2007) Localization and activation of GLP-2 receptors on vagal afferents in the rat. Endocrinology 148:1954—1962). The effects mediated by GLP-2 are triggered by the binding and activation of the GLP-2 receptor, a member of the glucagon/secretin G ncoupled receptor superfamily that is located on enteric (Bj erknes M, Cheng H (2001) Modulation of speciﬁc inal epithelial progenitors by enteric neurons. Proc Natl Acad Sci USA 98:12497—12502) and vagal (Nelson et al., 2007) nerves, subepithelial myoﬁbroblasts (Orskov C, et al. (2005) GLP-2 stimulates colonic growth Via KGF, released by subepithelial myofibroblasts with GLP-2 receptors.

Regul Pept 124:105—11), and a subset of intestinal epithelial cells (Thulesen J, et al. (2000) Potential targets for glucagon-like peptide 2 (GLP-2) in the rat: distribution and g of i.v. injected (125)1- GLP—2. Peptides 21:151171517). In addition, GLP—2 has an important role in intestinal adaptation, repair and protection during inﬂammatory events, including amelioration of the effects of proinﬂammatory cytokines (Sigalet DL, et al. (2007) Enteric neural pathways mediate the antiinﬂammatory actions of glucagon—like peptide 2. Am J Physiol Gastrointest Liver Physiol 293 :G21 1— G221). GLP-2 also enhances nutrient absorption and gut adaptation in rodents or humans with short bowel syndrome (SBS) (Jeppesen et al., (2001) Gastroenterology 120: 806-815).

In one aspect, the invention contemplates ion of GLP-2 ces in the TEN fusion protein compositions that are identical to human GLP-2, sequences that have homology to GLP-2 ces, ces that are l, such as from humans, non-human primates, mammals (including domestic animals) that retain at least a portion of the biologic activity or biological function of native human GLP-2. In one embodiment, the GLP-2 is a non-natural GLP-2 sequence variant, fragment, or a mimetic of a l sequence that retains at least a portion of the biological activity of the ponding native GLP-2, such as but not limited to the substitution of the alanine at position 2 of the mature GLP-2 peptide ce with glycine (“GLP2G”). In another embodiment, the GLP-2 of the fusion protein has the sequence HGDGSFSDEMNTILDNLAARDFINWLIQTKITD. Sequences with homology to GLP-2 may be found by standard homology searching techniques, such as NCBI BLAST, or in public databases such as Chemical Abstracts Services Databases (e. g., the CAS ry), GenBank, The Universal Protein Resource ot) and subscription provided databases such as GenSeq (e.g., Derwent).

Table 1 provides a non-limiting list of amino acid sequences of GLP-2 that are encompassed by the GLP2-XTEN fusion proteins of the invention. Any of the GLP-2 ces or homologous derivatives to be incorporated into the fusion protein compositions can be constructed by shufﬂing individual mutations into and between the amino acids of the sequences of Table 1 or by replacing the amino acids ofthe sequences of Table 1. The resulting GLP-2 sequences can be evaluated for activity and those that retain at least a portion of the biological activity of the native GLP-2 may be useful for inclusion in the fusion protein compositions of this invention. In some embodiments, GLP-2 that can be incorporated into a GLP2-XTEN include proteins that have at least about 80% ce identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to an amino acid ce selected from Table 1.

Table 1: GLP-Z amino acid seguences Name (source) Amino Acid Sequence GLP-2 (human) HADGSFSDEMNTILD\LAARDFINWLIQTKITD GLP-2 variant 1 SEQ ID NO: 3 US Pat No. 7,186,683 HADGSFSDEMNTILD\LATRDFINWLIQTKITD GLP—2 variant 2 SEQ ID N05 US Pat No. 5,789,379 SDEMNTILD\LAARDFINWLIQTKITD GLP-2 variant 3 HVDGSFSDEMNTILD\LAARDFINWLIQTKITD GLP-2 t 4 HEDGSFSDEMNTILDNLAARDFINWLIQTKITD GLP—2 variant 5 HKDGSFSDEMNTILD\LAARDFINWLIQTKITD GLP-2 variant 6 HRDGSFSDEMNTILD\LAARDFWWLIQTKITD GLP-2 variant 7 HLDGSFSDEMNTILD\LAARDFINWLIQTKITD GLP—2 variant 8 SDEMI\TILDNLAARDFINWLIQTKITD GLP-2 (mouse) HADGSFSDE\/ISTILD\LATRDFINWLIQTKITD GLP-2 (rat) HADGSFSDE\/I\TILDNLATRDFINWLIQTKITD GLP—2 (bovine) HADGSFSDE\/f\TVLDSLATRDFINWLLQTKITD GLP-2 (bovine variant) HGDGSFSDE\/f\TVLDSLATRDFINWLLQTKITD GLP-2 (pig) HADGSFSDE\/I\TVLDNLATRDFINWLLHTKITDSL GLP-2 (pig variant) SDE\/f\TVLDNLATRDFINWLLHTKITDSL GLP-2 (sheep) HADGSFSDE\/f\TVLDSLATRDFINWLLQTKI GLP-2 (sheep variant) HGDGSFSDE\/I\TVLDSLATRDFINWLLQTKI GLP-2 (canine) HADGSFSDE\/f\TVLDTLATRDFINWLLQTKITD GLP-2 (canine variant) HGDGSFSDE\/f\TVLDTLATRDFINWLLQTKITD GLP-2 (chicken) HADGTFTSDII\KILDDMAAKEFLKWLINTKVTQ GLP-2 (chicken variant) HGDGTFTSDII\KILDDMAAKEFLKWLINTKVTQ GLP-2 (turkey) HADGTFTSDII\KILDDMAAKEFLKWLINTKVTQ GLP-2 (turkey variant) HGDGTFTSDII\KILDDMAAKEFLKWLINTKVTQ GLP-2 (Xenopus laevis) HADGSFTNDINKVLDIIAAQEFLDWVINTQETE ] The GLP-2 of the subject compositions are not limited to native, ﬁlll-length GLP-2 ptides, but also include recombinant versions as well as biologically and/or pharmacologically active forms with sequence variants, or fragments thereof. For example, it will be iated that various amino acid deletions, insertions and substitutions can be made in the GLP-2 to create variants that exhibit one or more biological activity or pharmacologic properties of the wild-type GLP-2.

Examples of conservative substitutions for amino acids in polypeptide sequences are shown in Table 2.

In embodiments of the GLPZ-XTEN in which the sequence identity ofthe GLP-2 is less than 100% compared to a speciﬁc sequence disclosed herein, the invention contemplates substitution of any of the other 19 natural L-amino acids for a given amino acid e of a given GLP-Z, which may be at any position within the sequence of the GLP-Z, including adjacent amino acid residues. In some embodiments, the GLP-2 t incorporated into the GLPZ-XTEN has glycine (G), valine (V), glutamate (E), lysine (K), ne (R), leucine (K) or isoleucine (I) substituted for alanine (A) at position 2 of the mature peptide. Such substitution may confer resistance to dipeptidyl peptidase-4 (DPP-4). In one embodiment, glycine is substituted for alanine at position 2 ofthe GLP-2 sequence. If any one substitution s in an undesirable change in biological ty, then one of the alternative amino acids can be employed and the uct protein evaluated by the methods described herein (e.g., the assays of Table 32), or using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in US. Pat. No. 5,364,934 (the content of which is incorporated by reference in its entirety), or using s generally known in the art. In addition, variants can include, for instance, polypeptides wherein one or more amino acid residues are added or deleted at the N— or C-terminus of the ﬁJll-length native amino acid sequence of a GLP-2 that retains some if not all of the ical activity of the native peptide; e.g., the ability to bind GLP-2 receptor and/or the ability to activate GLP-2 receptor.

Table 2: Exemplary conservative amino acid substitutions Originamesmue Ala (A) Arg (R) Asn (N) ‘1’) Cys (C) Ser Gln Q Glu (E) Gly (G) Pro His (H) asn: gin: lys: arg 116 1 Leu (L) Lys (K) arg: gin: asn Met M leu; ohe; ile Phe F Pro (P) Thr (T) Ser T . W Tyr(Y) Val (V) Ile; leu; met; phe; ala; norleucine Sequence variants of GLP-2, whether exhibiting substantially the same or better biological activity than a corresponding wild-type GLP-Z, or, alternatively, exhibiting substantially modiﬁed or reduced biological activity relative to wild-type GLP-2, include, without limitation, polypeptides having an amino acid sequence that differs from the sequence of ype GLP-2 by insertion, deletion, or substitution of one or more amino acids. Such GLP-2 variants are known in the art, including those described in US Patent No. 7,186,683 or US Pat. No. 5,789,379, 500, all ofwhich are incorporated herein by reference.

III). EXTENDED RECOMBINANT POLYPEPTIDES In one aspect, the invention es XTEN polypeptide compositions that are useful as fusion protein partner(s) to link to and/or incorporate within a GLP-2 sequence, resulting in a GLP2-XTEN ﬁlsion n. XTEN are generally polypeptides with non-naturally occurring, substantially non- repetitive ces having a low degree of or no secondary or tertiary structure under physiologic conditions. XTEN typically have from about 36 to about 3000 amino acids of which the majority or the entirety are small hilic amino acids. As used herein, “XTEN” speciﬁcally excludes whole antibodies or antibody fragments (e.g. single-chain antibodies and PC fragments). XTENs have y as a fusion protein partners in that they serve in various roles, conferring certain ble pharmacokinetic, ochemical and pharmaceutical properties when linked to a GLP-2 protein to a create a GLPZ- XTEN fusion protein. Such GLPZ-XTEN fusion protein compositions have enhanced properties ed to the corresponding GLP-2 not linked to XTEN, making them useful in the treatment of certain gastrointestinal conditions, as more fully described below.

The selection criteria for the XTEN to be fused to the biologically active proteins generally relate to attributes of physicochemical properties and conformational ure of the XTEN that is, in turn, used to confer the enhanced properties to the fusion proteins compositions. The unstructured characteristic and physical/chemical properties of the XTEN result, in part, from the overall amino acid composition disproportionately limited to 4-6 hilic amino acids, the linking of the amino acids in a quantiﬁable non-repetitive design, and the length of the XTEN polypeptide. In an ageous feature common to XTEN but uncommon to polypeptides, the properties ofXTEN disclosed herein are not tied to absolute primary amino acid sequences, as evidenced by the diversity of the exemplary sequences of Table 4 that, within varying ranges of length, possess similar ties, many of which are documented in the es. The XTEN of the present invention exhibits one or more of the following advantageous properties: conformational ﬂexibility, reduced or lack of secondary ure, high degree of s solubility, high degree of protease resistance, low immunogenicity, low binding to mammalian receptors, a deﬁned degree of charge, and increased hydrodynamic (or Stokes) radii; properties that make them particularly useful as fusion protein partners. In turn, non-limiting examples of the ed ties of the fusion proteins comprising GLP-2 fused to the XTEN include increases in the overall solubility and/or metabolic stability, reduced susceptibility to proteolysis, reduced immunogenicity, reduced rate of absorption when administered subcutaneously or intramuscularly, reduced nce by the kidney, enhanced interactions with ate, and enhanced pharmacokinetic properties. Enhanced pharmacokinetic properties of the inventive GLPZ-XTEN compositions include longer terminal half-life (e.g., two-fold, three-fold, four-fold or more), increased area under the curve (AUC) (e.g., 25%, 50%, 100% or more), lower volume of distribution, slower absorption after subcutaneous or intramuscular injection (compared to GLP-2 not linked to the XTEN and administered by a similar route) such that the Cmax is lower, which, in turn, results in reductions in adverse effects of the GLP-Z that, collectively, results in an increased period of time that a fusion protein of a GLPZ-XTEN composition administered to a subject provides therapeutic activity. In some embodiments, the TEN compositions se cleavage sequences (described more fully, below) that permits sustained release ogically active GLP-2.A GLPZ-XTEN having such cleavage ce can act as a depot when subcutaneously or intramuscularly administered. It is speciﬁcally contemplated that the t GLPZ-XTEN fusion ns of the disclosure can exhibit one or more or any ation of the improved properties disclosed herein. In some embodiments, GLPZ-XTEN compositions permit less frequent dosing compared to GLP-2 not linked to the XTEN and administered in a able fashion. Such GLPZ- XTEN fusion protein compositions have utility to treat certain related diseases, disorders or conditions, as bed herein.

A y of methods and assays are known in the art for determining the physicochemical properties of proteins such as the compositions comprising the inventive XTEN. Such properties include but are not limited to secondary or tertiary structure, solubility, protein aggregation, melting ties, contamination and water content. Such methods include analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion chromatography (SEC), HPLC-reverse phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, ﬂuorescence, HPLC—ion exchange, IR, NMR, Raman spectroscopy, refractometry, and UVNisible oscopy. Additional methods are disclosed in Arnau, et al., Prot Expr and Purif (2006) 48, 1-13.

The XTEN component(s) of the GLPZ-XTEN are designed to behave like denatured e sequences under physiological conditions, despite the extended length of the polymer. “Denatured” describes the state of a peptide in solution that is characterized by a large mational freedom of the peptide backbone. Most es and proteins adopt a denatured mation in the presence of high concentrations of denaturants or at elevated temperature. Peptides in denatured conformation have, for example, characteristic circular dichroism (CD) spectra and are characterized by a lack of long-range interactions as determined by NMR. “Denatured conformation” and “unstructured conformation” are used synonymously herein. In some embodiments, the invention provides XTEN sequences that, under physiologic conditions, resemble denatured ces that are largely devoid in secondary structure. In other cases, the XTEN sequences are substantially devoid of secondary structure under physiologic conditions. “Largely devoid,” as used in this context, means that less than 50% of the XTEN amino acid residues of the XTEN sequence contribute to secondary structure as measured or determined by the means described herein. “Substantially devoid,” as used in this context, means that at least about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or at least about 99% of the XTEN amino acid residues of the XTEN ce do not contribute to secondary structure, as ed or determined by the methods described herein.

A variety of methods have been established in the art to discern the presence or absence of secondary and tertiary structures in a given ptide. In particular, secondary structure can be measured spectrophotometrically, e.g., by circular dichroism spectroscopy in the “far-UV” spectral region (190-250 nm). Secondary structure elements, such as helix and beta-sheet, each give rise to a characteristic shape and magnitude of CD spectra. ary structure can also be predicted for a polypeptide sequence via certain computer ms or algorithms, such as the well-known Chou- Fasman algorithm (Chou, P. Y., et a]. (1974) Biochemistry, 13: 222-45) and the Garnier—Osguthorpe- Robson algorithm (“Gor algorithm”) (Garnier J, Gibrat JF, Robson B. (1996), GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540-55 3), as described in US Patent Application Publication No. 20030228309A1. For a given sequence, the algorithms can predict whether there exists some or no ary structure at all, expressed as the total and/or percentage of es of the sequence that form, for e, alpha-helices or beta-sheets or the percentage of residues of the sequence predicted to result in random coil formation (which lacks secondary structure). Polypeptide sequences can be analyzed using the Chou-Fasman algorithm using sites on the world wide web at, for example, fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=miscl and the Gor algorithm at npsapbil.ibcp.fr in/npsa_automat.pl?page=npsa_gor4.html (both accessed on September 5, 2012).

In one embodiment, the XTEN sequences used in the subject fusion protein compositions have an alpha—helix percentage ranging from 0% to less than about 5% as determined by the Chou—Fasman algorithm. In another embodiment, the XTEN sequences of the msion protein compositions have a beta- sheet percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman thm.

In some embodiments, the XTEN sequences of the fusion protein compositions have an alpha-helix percentage ranging from 0% to less than about 5% and a beta-sheet percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm. In one ment, the XTEN sequences of the fusion protein compositions have an alpha-helix percentage less than about 2% and a heet tage less than about 2%. The XTEN sequences of the fusion n compositions have a high degree ofrandom coil percentage, as determined by the GOR algorithm. In some embodiments, an XTEN sequence have at least about 80%, more ably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more ably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, and most preferably at least about 99% random coil, as determined by the GOR thm. In one embodiment, the XTEN sequences of the fusion protein compositions have an alpha-helix percentage ranging from 0% to less than about 5% and a beta- sheet percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm and at least about 90% random coil, as determined by the GOR algorithm. In another embodiment, the XTEN sequences of the fusion protein compositions have an alpha-helix percentage less than about 2% and a beta-sheet tage less than about 2% at least about 90% random coil, as determined by the GOR algorithm. 1. Non-repetitive Sequences ] It is contemplated that the XTEN sequences of the GLPZ-XTEN embodiments are substantially non-repetitive. In general, tive amino acid sequences have a cy to aggregate or form higher order ures, as exempliﬁed by natural repetitive ces such as collagens and leucine zippers.

These repetitive amino acids may also tend to form contacts resulting in crystalline or pseudocrystaline structures. In contrast, the low tendency of non-repetitive sequences to aggregate enables the design of long-sequence XTENs with a relatively low frequency of charged amino acids that would otherwise be likely to aggregate if the sequences were repetitive. The non-repetitiveness of a subject XTEN can be observed by assessing one or more of the following es. In one embodiment, a “substantially non- repetitive” XTEN sequence has no three contiguous amino acids in the ce that are of identical amino acid types unless the amino acid is serine, in which case no more than three contiguous amino acids are serine residues. In another embodiment, as described more fully below, a “substantially non- repetitive” XTEN sequence ses motifs of 9 to 14 amino acid residues wherein the motifs consist of 3, 4, 5, or 6 types of amino acids selected from glycine (G), e (A), serine (S), ine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one motif is not repeated more than twice in the sequence motif.

The degree of repetitiveness of a polypeptide or a gene can be measured by computer programs or algorithms or by other means known in the art. According to the current invention, algorithms to be used in ating the degree ofrepetitiveness of a particular polypeptide, such as an XTEN, are disclosed herein, and examples of ces analyzed by algorithms are provided (see Examples, below).

In one embodiment, the repetitiveness of a polypeptide of a predetermined length can be calculated (hereinafter “subsequence score”) according to the formula given by Equation 1: Subsequence score = ET: 3: {Itl‘lt'il'ii g I wherein: m = (amino acid length of polypeptide) — (amino acid length of subsequence) + 1; and Countl- = cumulative number of occurrences of each unique subsequence within sequence;- An algorithm termed ore” was developed to apply the foregoing equation to quantitate repetitiveness of polypeptides, such as an XTEN, providing the subsequence score wherein sequences of a predetermined amino acid length are analyzed for repetitiveness by determining the number of times (a “count”) a unique subsequence of length “s” appears in the set length, divided by the absolute number of subsequences within the predetermined length of the sequence. depicts a logic ﬂowchart of the SegScore algorithm, while portrays a schematic of how a subsequence score is derived for a ﬁctitious XTEN with 11 amino acids and a subsequence length of 3 amino acid es. For example, a predetermined polypeptide length of 200 amino acid es has 192 overlapping 9-amino acid subsequences and 198 3-mer subsequences, but the subsequence score of any given polypeptide will depend on the absolute number of unique uences and how frequently each unique subsequence (meaning a different amino acid sequence) appears in the predetermined length of the sequence.

In the context of the present ion, “subsequence score” means the sum of occurrences of each unique 3-mer frame across 200 consecutive amino acids ofthe cumulative XTEN polypeptide d by the absolute number of unique 3-mer subsequences within the 200 amino acid sequence.

Examples of such subsequence scores derived from 200 consecutive amino acids of repetitive and nonrepetitive polypeptides are presented in e 30. In one embodiment, the invention provides a GLP2-XTEN comprising one XTEN in which the XTEN has a uence score less than 12, more preferably less than 10, more preferably less than 9, more preferably less than 8, more preferably less than 7, more ably less than 6, and most preferably less than 5. In another ment, the invention provides GLP2-XTEN sing two more XTENs in which at least one XTEN has a subsequence score of less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than , or less. In yet r embodiment, the invention provides GLPZ-XTEN comprising at least two XTENs in which each individual XTEN of 36 or more amino acids has a subsequence score of less than , or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less. In the ments of this aph, the XTEN is characterized as substantially petitive.

In one aspect, the non—repetitive characteristic ofXTEN of the present invention together with the particular types of amino acids that predominate in the XTEN, rather than the absolute primary sequence, confers one or more of the enhanced physicochemical and biological properties of the GLP2- XTEN ﬁlSiOIl proteins. These enhanced properties include a higher degree of expression of the fusion protein in the host cell, greater genetic stability ofthe gene encoding XTEN, a greater degree of solubility, less tendency to aggregate, and enhanced pharmacokinetics of the resulting GLP2-XTEN compared to fusion proteins comprising polypeptides having repetitive sequences. These enhanced properties permit more efﬁcient manufacturing, lower cost of goods, and/or facilitate the formulation of XTEN-comprising pharmaceutical preparations containing extremely high protein concentrations, in some cases exceeding 100 mg/ml. In some embodiments, the XTEN polypeptide sequences of the embodiments are designed to have a low degree of internal repetitiveness in order to reduce or substantially eliminate immunogenicity when administered to a mammal. Polypeptide sequences ed of short, repeated motifs largely limited to only three amino acids, such as glycine, serine and glutamate, may result in relatively high dy titers when administered to a mammal despite the absence of predicted T-cell epitopes in these sequences. This may be caused by the repetitive nature of polypeptides, as it has been shown that immunogens with ed epitopes, including protein aggregates, cross-linked immunogens, and repetitive carbohydrates are highly immunogenic and can, for example, result in the cross-linking of B-cell receptors causing B-cell tion. (Johansson, J., et al. (2007) e, 25 :1676-82 ; Yankai, 2., et al. (2006) Biochem Biophys Res Commun, 345 :1365-71 ; Hsu, C. T., et a]. (2000) Cancer Res, 60:3701-5); Bachmann MF, et al. Eur J Immunol. (1995) (12):3445-3451). 2. Exemplaﬂ Seguence Motifs The present invention encompasses XTEN used as fusion partners that comprise multiple units of shorter sequences, or motifs, in which the amino acid sequences of the motifs are substantially non- tive. The petitive property can be met even using a “building block” ch using a y of sequence motifs that are multimerized to create the XTEN sequences. While an XTEN sequence may consist of multiple units of as few as four different types of ce motifs, e the motifs themselves generally consist of non-repetitive amino acid sequences, the overall XTEN sequence is designed to render the sequence ntially non-repetitive.

In one embodiment, an XTEN has a substantially non-repetitive sequence of greater than about 36 to about 3000, or about 100 to about 2000, or about 144 to about 1000 amino acid residues, or even longer wherein at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or about 100% of the XTEN ce consists of non-overlapping sequence motifs, and wherein each of the motifs has about 9 to 36 amino acid residues. As used herein, verlapping” means that the individual motifs do not share amino acid residues but, rather, are linked to other motifs or amino acid residues in a linear fashion. In other embodiments, at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or about 100% ofthe XTEN sequence consists of non—overlapping ce motifs wherein each of the motifs has 9 to 14 amino acid residues.

In still other embodiments, at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or about 100% ofthe XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 12 amino acid residues. In these embodiments, it is preferred that the sequence motifs are composed of substantially (e.g., 90% or more) or exclusively small hydrophilic amino acids, such that the overall sequence has an unstructured, ﬂexible characteristic.

Examples of amino acids that are included in XTEN are, e.g., arginine, lysine, threonine, alanine, asparagine, glutamine, aspartate, glutamate, serine, and glycine. In one embodiment, XTEN sequences have predominately four to six types of amino acids selected from glycine (G), e (A), serine (S), threonine (T), glutamate (E) or proline (P) that are arranged in a substantially non-repetitive sequence that is greater than about 36 to about 3000, or about 100 to about 2000, or about 144 to about 1000 amino acid residues in length. In some embodiments, an XTEN sequence is made of 4, 5 or 6 types of amino acids selected from the group ting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or e (P). In some embodiments, XTEN have sequences of greater than about 36 to about 1000, or about 100 to about 2000, or about 400 to about 3000 amino acid residues wherein at least about 80% of the sequence consists of non-overlapping sequence motifs wherein each of the motifs has 9 to 36 amino acid residues and wherein at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or 100% of each of the motifs consists of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any one amino acid type in the full-length XTEN does not exceed %. In other embodiments, at least about 90% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 9 to 36 amino acid residues wherein the motifs consist of 4 to 6 types of amino acids selected from glycine (G), e (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any one amino acid type in the full-length XTEN does not exceed 40%, or about 30%, or about 25%. In other embodiments, at least about 90% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 12 amino acid residues ting of 4 to 6 types of amino acids selected from glycine (G), e (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any one amino acid type in the ﬁJll-length XTEN does not exceed 40%, or 30%, or about 25%. In yet other embodiments, at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 12 amino acid residues ting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P).

In still other embodiments, XTENs comprise substantially non-repetitive sequences of greater than about 36 to about 3000 amino acid residues wherein at least about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the sequence consists of non—overlapping sequence motifs of 9 to 14 amino acid residues wherein the motifs consist of 4 to 6 types of amino acids selected from e (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one motif is not repeated more than twice in the sequence motif.

In other embodiments, at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of an XTEN sequence consists of non-overlapping sequence motifs of 12 amino acid residues wherein the motifs consist of four to six types of amino acids selected from glycine (G), e (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one sequence motif is not ed more than twice in the sequence motif. In other embodiments, at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of an XTEN sequence ts of non-overlapping sequence motifs of 12 amino acid residues wherein the motifs consist of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and e (P), and wherein the sequence of any two contiguous amino acid residues in any one sequence motif is not repeated more than twice in the sequence motif. In yet other embodiments, XTENs consist of 12 amino acid sequence motifs wherein the amino acids are selected from e (G), e (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one ce motif is not repeated more than twice in the sequence motif, and wherein the content of any one amino acid type in the full-length XTEN does not exceed 30%. The foregoing embodiments are es of substantially non-repetitive XTEN sequences.

Additional examples are detailed below.

In some embodiments, the invention provides GLPZ-XTEN itions comprising one, or two, or three, or four, ﬁve, six or more non-repetitive XTEN sequence(s) of about 36 to about 1000 amino acid residues, or cumulatively about 100 to about 3000 amino acid residues wherein at least about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% of the sequence consists of multiple units of four or more non-overlapping sequence motifs selected from the amino acid ces of Table 3, wherein the overall sequence remains substantially non-repetitive. In some ments, the XTEN comprises non-overlapping sequence motifs in which about 80%, or at least about 85%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% or about 100% of the sequence consists of multiple units of non- overlapping sequences selected from a single motif family ed from Table 3, resulting in a family sequence. Family as applied to motifs means that the XTEN has motifs selected from a motif category of Table 3; i.e., AD, AE, AF, AG, AM, AQ, BC, or BD, and that any other amino acids in the XTEN not from a motif family are selected to achieve a needed property, such as to permit incorporation of a restriction site by the encoding nucleotides, incorporation of a cleavage sequence, or to achieve a better linkage to a GLP—2 component of the GLP2—XTEN. In some embodiments ofXTEN families, an XTEN sequence comprises multiple units of non—overlapping sequence motifs of the AD motif , or of the AE motif , or of the AF motif family, or of the AG motif family, or of the AM motif family, or of the AQ motif family, or of the BC , or of the BD family, with the resulting XTEN exhibiting the range of homology described above. In other embodiments, ofXTEN families, each XTEN of a given family has at least four different motifs of the same family from Table 3; e. g., four motifs ofAD or AE or AF or AG or AM, etc. In other embodiments, the XTEN comprises multiple units of motif sequences from two or more of the motif families of Table 3, selected to achieve d physicochemical characteristics, including such properties as net charge, lack of secondary structure, or lack of repetitiveness that may be conferred by the amino acid composition ofthe motifs, described more fully below. In the embodiments hereinabove described in this paragraph, the motifs or portions of the motifs incorporated into the XTEN can be selected and led using the methods described herein to e an XTEN of about 36, about 42, about 72, about 144, about 288, about 576, about 864, about 1000, about 2000 to about 3000 amino acid residues, or any intermediate length. Non-limiting examples ofXTEN family sequences useful for incorporation into the subject GLP2-XTEN are presented in Table 4. It is intended that a speciﬁed sequence ned relative to Table 4 has that ce set forth in Table 4, while a lized reference to an AE144 sequence, for example, is ed to encompass any AE sequence having 144 amino acid residues; e. g., AE144_1A, AE144_2A, etc., or a generalized reference to an AG144 sequence, for example, is intended to encompass any AG sequence having 144 amino acid residues, e.g., AG144_1, AG144_2, AG144_A, AG144_B, AG144_C, etc. 2012/054941 Table 3: XTEN Seguence Motifs of 12 Amino Acids and Motif Families SSGSES AD GSSESGSSEGGP AE,AM AE, AM, AQ GSEPATSGSETP AAAAAQ AEAAMAAQ AF, AM GSTSESPSGTAP AFAAM AF, AM GSTSSTAESPGP AG,AM AG, AM GSSPSASTGTGP BD GSETATSGSETA a Denotes individual motif sequences that, when used together in various ations, results in a “family sequence” Table 4: XTEN Palmeptides XTEN Amino Acid Sequence Name AE42 GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS AE42 1 TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS AE42—2 PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSG AE42—3 SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSP AG42_1 GAPSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGPSGP AG42:2 GPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASP AG4273 SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA AG42 4 SASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG XTEN Amino Acid Sequence Name AE48 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGS AM48 MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPA AE144 TSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET PGTSTEPSEGSAP SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS AE144_1A EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGP GTSTEPSEGSAPG TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPS 2A TSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP GTSESATPESGPG TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPS 2B EGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP GTSESATPESGPG SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPS AE144_3A EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPG SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPS AE144_3B EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPG TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS AE144_4A EGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGP GTSTEPSEGSAPG TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS AE144_4B EGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGP SEGSAPG TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS AE144_5A EGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEE GSPAGSPTSTEEG TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATS 6B GSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPG GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSST AF144 AESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAP GTSPSGESSTAP SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTG AG144_1 TGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGS GTGPGASP PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASP AG144_2 GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST GSPGTPGSGTASSS GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPS AG144_A ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTG SPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPS AG144_B ASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTG SPGASPGTSSTGSP GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGS AG144_C GTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATG SPGASPGTSSTGSP GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPS AG144_F ASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATG GTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPG AG144_3 TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG SPGASPGTSSTGSP AG144_4 GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPG WO 40093 XTEN Amino Acid Sequence Name PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASS SPGSSTPSGATGSP GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEE AE288_1 GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPA TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSET PGTSESATPESGPGTSTEPSEGSAP GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE AE288_2 GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTE EGTSESATPESGPGTSTEPSEGSAP PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSST PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG AG288_1 TGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGA SPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSAST GTGPGTPGSGTASSSPGSSTPSGATGS GSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPG TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG AG288_2 SPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSS PSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSS TGSPGASPGTSSTGSPGTPGSGTASSSP SSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSXPS ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTG SPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSS TPSGATGSPGSXPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSS AF504 TGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG ASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT SSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS PGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP AESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESPGPGTSTPE SGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAP GSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGSTSES PSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGP AF540 GTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSES PSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASP GSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPE SGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASP GSTSESPSGTAP GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSES GSSEGGPGSSESGSSEGGPGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSSESGSSEG GPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSS ESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSGGEPSE AD576 SGSSGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESG ESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSEGSS GPGESSGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSE SGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGG EPSESGSSGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSEGSSGPGESS GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSES PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS AE576 APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTS TEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESPGPGTSTPE AF576 SGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAP WO 40093 XTEN Amino Acid Sequence Name GSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGSTSES PSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGP SGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSES PSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASP GSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPE SGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASP GSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASP PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSST PSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSS TGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSP GASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPG AG576 SGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSST GSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGS GTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST GSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEEGTS ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG AE624 PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG SAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSP TSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP GSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSGSESGESP GGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGESPGGSS GSESGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGP SSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGG EPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSE GGPGSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPG AD836 SGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGESSGSEGSS GPGESSGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESG SSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGSSESGSSEGGPGSGGEPSESGSSGSEGSSG PGESSGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSES GESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSSESGSSEGGPGSSES GSSEGGPGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSES GSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSS GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE PSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPES GPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG TSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET AE864 PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPA EEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPE SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGS PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSP TSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETP SGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAP PSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPE SGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSPSGESSTAP GTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSTPE AF864 SGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGP GTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPE SGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAP XTEN Amino Acid Sequence Name PSGTAPGSTSESPSGTAPGTSTPESGPXXXGASASGAPSTXXXXSESPSGTAPGSTSE SPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSA SGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTS ESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGS ASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSESPSGTAPGSTSESPSGTAPGT SPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGES STAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSSPSASTGTGPG SSTPSGATGSPGSSTPSGATGSP GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPS ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSST GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGS TGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGT GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGS PGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASP GTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT AG864_2 GSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPG TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGS GTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGS STPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSG ATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS PGSSTPSGATGSPGASPGTSSTGSP GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSE SPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPES GPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEG SSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT SGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGP AM875 GSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGS GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSE TPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTS TEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSE GSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETP GTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSES ATPESGPGTSTEPSEGSAPGTSTEPSEGSAP MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEEGTS ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG AE912 SAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSP SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS APGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAP MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTSTEPSEGSAPGSE PATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSG TAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGT STEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS AM923 EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGS GTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTST EEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSP XTEN Amino Acid Sequence Name AGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGA TGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPG STSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSST AESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPA GSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEG SAPGTSTEPSEGSAP GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSE SPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPES GPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP TEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEG SSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT SGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAPSGGSEPATSGSETP GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSES ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESST APGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSEGSAPGTSESATPESGPGTS AM1318 ESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGTSTEPSEGSAPG SPAGSPTSTEEGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSTPS GATGSPGSSTPSGATGSPGASPGTSSTGSPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPG PGTSPSGESSTAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSST PSGATGSPGASPGTSSTGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPE SGPGSEPATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGS PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATS GSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGTAPGTSPSGESSTAP GSTSSTAESPGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAG SPTSTEEGTSTEPSEGSAP GTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSEPA TSGTEPSGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPG SAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSE PATSGTEPSGTSEPSTSEPGAGSGASEPTSTEPGTSEPSTSEPGAGSEPATSGTEPSGSEPATSG TEPSGTSTEPSEPGSAGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSG SEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEP SEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEP BC 864 SGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGA SEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPG SAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTS EPSTSEPGAGSGASEPTSTEPGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSG GASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSG TSEPSTSEPGAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASE PTSTEPGTSTEPSEPGSA GSETATSGSETAGTSESATSESGAGSTAGSETSTEAGTSESATSESGAGSETATSGSETAGSE TATSGSETAGTSTEASEGSASGTSTEASEGSASGTSESATSESGAGSETATSGSETAGTSTEA SEGSASGSTAGSETSTEAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSES GAGTSTEASEGSASGSETATSGSETAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAG TSESATSESGAGTSTEASEGSASGSETATSGSETAGSTAGSETSTEAGSTAGSETSTEAGSET TAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSESATS BD864 ESGAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGSETATSGSET ATSESGAGSTAGSETSTEAGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGS TAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGSTAGSETSTEAGSETATSGSETAGTSTE ASEGSASGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSG SESATSESGAGSETATSGSETAGTSTEASEGSASGTSTEASEGSASGSTAGSETSTE AGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGS ETATSGSETAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGSETA TSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETA GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAG AE948 EGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSEPATSGSE TPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTS 2012/054941 XTEN Amino Acid Sequence Name TEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSEPATSG SETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG SEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSPAGS PTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSA PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA GSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPE SGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGS EPATSGSETPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATS GSETPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGP GSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAG SPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP GSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTE PSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPES GPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSP AGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPG TSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS PTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSET PGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTST AE1044 EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG SAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGS EPATSGSETPGTSESATPESGPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPS EGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE GTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSES ATPESGPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTST EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTS TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTST GSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPA PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPES SATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTS TEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESA TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTE EGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSE AE1140 SATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPE SGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGS EPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESAT PESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTE PSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPES GPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSP AGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPA GSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSE TPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE GSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG TSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESA TPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE AE1236 EGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSPA GSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGT SESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSESAT TSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSES PGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTST EEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTS XTEN Amino Acid Sequence Name ESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEP PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSTE PSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTST EEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSE PATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSESA TPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSA PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEP AE1332 TPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS ETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT SESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS EGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGP GTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSES ATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTST EEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSE PATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTST GSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTE PSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS TEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG SPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEP SEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSET PGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEP AE1428 ATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPE SGPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGT STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP TSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAP GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSES ATPESGPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTST EEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTS TEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSPA GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAG SPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGS APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTS TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPT STEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEG TSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEP SEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSPAGSPTSTE EGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSE AE1524 SATPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGS ETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGS PAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATS GSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP GSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTE PSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPES GPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTS ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSPA SGSETPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSES ATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGS APGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTS SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSE STEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG AE1620 SEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESA TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE SPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTS TEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGS XTEN Amino Acid Sequence Name PAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETP GSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSES PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPES GPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTS ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAG SPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPES EPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSG SETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPG TSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESG PGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSE AE1716 SATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPE SGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGS EPATSGSETPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGTSTEPS EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEE GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSES ATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS APGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTS ESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSE GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTE PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES GPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSE ETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG TSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESA TPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSA PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPA AE1812 GSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATS GSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE GTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAG SPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGS APGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSP AGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEP GSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAG EGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGS APGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS ESATPESGPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSESATP ESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPG SEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSESA TPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE EGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEP AE1908 ATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTS TEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGT SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESAT PESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE GTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPA TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPES GPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSP AGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEP SEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAG SPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS AE2004A APGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTS ESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSE GSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEG 2012/054941 XTEN Amino Acid Sequence Name TSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESG PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSESATPESGPGTST EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG SAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGT SESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPS EGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP GSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAG SPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTS ESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSE GSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPG SGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGAT GSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGS SPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSAS TGTGPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGS PGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSST PSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTA AG948 SSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPG ASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPS GATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTG SPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTP SSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSAST GTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSP GTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTP SGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP GTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGTPG SGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTG TGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPG ASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGS GTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGAT GSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGS STPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGASPGT SSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGS AG1044 PGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSST PSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSS TGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSP GSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPS ASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSST GSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPG TPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGS GTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSST GASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSTP SGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGAT GSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGS SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSG TASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGS PGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSP SASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTA SPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPG AG1140 TPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGS GTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGAT TPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGS STPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSG ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTG PGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASP SPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSST GSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSST AG1236 GSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASP XTEN Amino Acid Sequence Name GTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTA SSSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPG TPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGS PGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT GPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGA SPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGT ASSSPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGP GSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPS ASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTAS SSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGS STPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSPSAS TGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGS PGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSST PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSS TGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASP GSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSPS ASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGAT PGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGS SPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSG TASSSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSS GTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSP GPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSAST GTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSP AG1332 GATGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPG SGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTA SSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPG ASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGS GTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGT GPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSS TPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS SSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPG GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGTPG SGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGAT GSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG TPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGS GTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGT GPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGA SPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSAS TGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS AG1428 PGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTP GSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSS TGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSP GASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASP GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA SSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPG SSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGS GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASP GSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPG SGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTA SSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPG SSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSPSA STGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATG SPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSS AG1524 PSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTS STGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSP GTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPG SGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTG TGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPG ASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGS XTEN Amino Acid Sequence Name GTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGAT GSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGS SPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGT SSTGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGTPG GSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASP SPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTG TGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPG ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGS GTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGAT GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGS SPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSAS TGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTG AG1 620 PGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASP GTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGAT GSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGS STPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSPSAS TGTGPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSS PGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSST SPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSS TGSPGTPGSGTASSSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSST GASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP SGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTG TGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPG ASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGS GTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTAS SSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGT PGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG ATGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGS AG1716 PGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSP SASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGA TGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSP GSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPG SGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGAT GSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGS SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGT SSTGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPG GSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSPS ASTGTGPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTG TGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPG SSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSA STGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTG SPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGAS PGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSAST GTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSP AG1812 STGTGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPG SGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSST PGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGS SPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGT SSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGS GTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSP SASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSS TGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASP GSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSPS ASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSST GSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPG AG1908 ASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGS GTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGAT GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPG ASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGS XTEN Amino Acid Sequence Name GTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGAT GSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPG ASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA GASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTG SPGTPGSGTASSSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSS PSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTS STGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGP GTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPG SGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSP GSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTP SGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSST GSPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGS SPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSG TASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGS PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSSP SASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTA SSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPG AG2004A SSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSA STGTGPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGT GPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSS TPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTS SSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP GSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGASP GTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTA SSSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASP SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPAT AE72B SGSETPG TSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEP AE72C SEGSAPG TEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGT AE108A SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTS GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPA AE108B TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESAT AE144A PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEE GSPAGSPTSTEEGS SEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGS AE144B PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE EGTSTEPSEGSAPG TSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE AE180A GTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPA TSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSES AE216A PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS ATSGSETPGTSESAT ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG SEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESA AE252A TPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTE EGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSE TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET PGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE AE288A SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPE SGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGT STEPSEGSAPGSEPATSGSETPGTSESA PESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTE AE324A PSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTST EEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE 2012/054941 XTEN Amino Acid Sequence Name PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS TSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEE GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE AE360A TPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS ESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE GSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEE GSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST AE396A EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSG SETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAG SPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE AE432A TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS ESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG SPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSES ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS AE468A TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTS TEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGS ETPGTSESAT EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTE EGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP AE504A TPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS ESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEG TEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS TPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAG SPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPA AE540A GSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES GPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTS TEPSEGSAPGTSTEP TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAG AE576A SPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS APGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA GSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP AE612A SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGP XTEN Amino Acid Sequence Name GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTS ESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG SAPGSEPATSGSETPGTSESAT PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPG TSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESAT TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEG AE648A SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEP SEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSES ATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSP AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG AE684A SETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGS PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATP EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGS EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP ESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEG TSTEPSEGSAPGTSTEPSEGSAPGSEPATS PGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA PGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSES ATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES GPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS SAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS AE720A TEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTS TEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE ETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS ESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTE TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA PGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSES PGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES GPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS TEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS AE756A ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTS TEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS ESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEG SAPGTSTEPSEGSAPGSEPATSGSETPGTSES EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSES ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG AE792A SAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTS TEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTS TEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTS WO 40093 XTEN Amino Acid Sequence Name TEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGS ETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTS TEPS PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG SEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPS EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSES PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSP AE828A AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGS PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGS EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP ESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEG TSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT GPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTP AG72A GSGTASS GSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGS AG72B GTASSSP SPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG AG72C ATGSPGA SASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSST AG108A GSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASP PGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSP AG108B SASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSS PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASP AG144A GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST GSPGTPGSGTASSS PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG AG144B TGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGA SPGTSSTGSPGASP TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT AG180A GPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGAS PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGS SSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGS AG2 1 6A GTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT GPGSSPSASTGTGPGSSTPSG TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT GPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGAS AG252A PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA TGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPG TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT GPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGAS AG288A PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA TGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPG ASPGTSSTGSPGASPGTSSTGSPGTPGS TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSS AG324A TPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTA SSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPG SSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTP GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSS AG360A TGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG ASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSG TASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG XTEN Amino Acid Sequence Name GATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSS TSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSST PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT AG396A GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGS SPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSAS TGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP GASPGT GATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGS PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASP GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT AG432A GSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGS SPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS TGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGP GASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPS TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG SPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSS TPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTA SSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPG AG468A SSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPS GATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS PGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSP SASTGTGPGASPG TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG SPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSS TPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTA SSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPG AG504A SSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPS GATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS PGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSP SASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP TSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG SPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSS TPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSS TGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG AG540A TPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPS GATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSS SGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSST PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT GSPGSSTPSGATGSPGASPG TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGT GPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSS TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGA TGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPG AG576A ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSG TASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTG ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSP GPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSST GSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG TPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP STGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPG TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG SPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSS TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGA AG612A TGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPG ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPS GATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGS PGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSP SASTGTGPGSSPSASTGTGPGASPGTS AG648A GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATG WO 40093 XTEN Amino Acid Sequence Name SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGAS PGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSS TGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPG ATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGT SSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGS PGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSST PSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTAS SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGS STPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATG SPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGAS PGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSS TGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPG TPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGT AG684A SSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGS PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPG SGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST GSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGA SPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGT ASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG SASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGAS PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSS SPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG SSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPS GATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSS AG720A PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSST SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGS SPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSAS TGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP GASPG TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT GPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGAS PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA TGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPG ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT SSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGS AG756A PGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSST PSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTAS SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGS STPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPG TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT GPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGAS PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA TGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPG ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT GSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGS AG792A PGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSST PSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTAS SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGS STPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPS ASTGTGPGASPG TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT AG828A GPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGAS XTEN Ammo ACId Sequence Name PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA SPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPG ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT SSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGS PGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSST SPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTAS SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGS STPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP STGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPS ASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP TASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPG PGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG AG288_D SPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTP GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSS TGSPGTPGSGTASSSPGSSTPSGATGSP In other embodiments, the GLP2-XTEN composition comprises one or more non-repetitive XTEN sequences of about 36 to about 3000 amino acid residues or about 144 to about 2000 amino acid residues or about 288 or about 1000 amino acid residues, wherein at least about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% of the sequence consists of non-overlapping 36 amino acid sequence motifs selected from one or more of the polypeptide sequences of Tables 8-11, either as a family sequence, or where motifs are ed from two or more families of motifs.

In those embodiments wherein the XTEN component of the GLP2—XTEN fusion protein has less than 100% of its amino acids consisting of four to six amino acid selected from glycine (G), alanine (A), serine (S), threonine (T), ate (E) and proline (P), or less than 100% ofthe sequence consisting of the sequence motifs from Table 3 or the sequences of Tables 4, and 8-12 or less than 100% sequence identity compared with an XTEN from Table 4, the other amino acid residues are selected from any other of the 14 l L-amino acids, but are preferentially selected from hydrophilic amino acids such that the XTEN sequence contains at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% hydrophilic amino acids. The XTEN amino acids that are not e (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) are interspersed throughout the XTEN sequence, are located within or between the sequence motifs, or are concentrated in one or more short stretches ofthe XTEN sequence. In such cases where the XTEN component of the TEN comprises amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), it is desirable that the amino acids not be hydrophobic residues and should not substantially confer secondary structure of the XTEN ent. Hydrophobic residues that are less favored in construction ofXTEN include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine. Additionally, one can design the XTEN sequences to contain less than 5% or less than 4% or less than 3% or less than 2% or less than 1% or none of the following amino acids: cysteine (to avoid disulflde formation and oxidation), methionine (to avoid ion), asparagine and glutamine (to avoid desamidation). Thus, in some embodiments, the XTEN component of the GLP2-XTEN fusion protein comprising other amino acids in addition to glycine (G), e (A), serine (S), threonine (T), glutamate (E) and proline (P) would have a sequence with less than 5% of the residues contributing to helices and beta-sheets as measured by the Chou-Fasman algorithm and have at least 90%, or at least about 95% or more random coil formation as measured by the GOR algorithm. 3. Length of Sequence In r aspect, the invention provides XTEN of varying lengths for incorporation into GLP2-XTEN compositions wherein the length of the XTEN sequence(s) are chosen based on the property or function to be achieved in the fusion protein. Depending on the intended property or ﬁanction, the GLP2-XTEN compositions comprise short or intermediate length XTEN and/or longer XTEN sequences that can serve as carriers. While not intended to be limiting, the XTEN or fragments of XTEN include short segments of about 6 to about 99 amino acid residues, intermediate lengths of about 100 to about 399 amino acid residues, and longer lengths of about 400 to about 3000 amino acid residues.

Thus, the subject GLP2-XTEN encompass XTEN or fragments ofXTEN with lengths of about 6, or about 12, or about 36, or about 40, or about 100, or about 144, or about 288, or about 401, or about 500, or about 600, or about 700, or about 800, or about 900, or about 1000, or about 1500, or about 2000, or about 2500, or up to about 3000 amino acid residues in length. In other cases, the XTEN sequences can be about 6 to about 50, or about 100 to 150, about 150 to 250, about 250 to 400, about 400 to about 500, about 500 to 900, about 900 to 1500, about 1500 to 2000, or about 2000 to about 3000 amino acid residues in . The precise length of an XTEN can vary without adversely affecting the biological activity of a GLP2-XTEN ition. In one embodiment, one or more of the XTEN used in the GLP2-XEN disclosed herein has 36 amino acids, 42 amino acids, 144 amino acids, 288 amino acids, 576 amino acids, or 864 amino acids in length and may be selected from one of the XTEN family sequences; i.e., AD, AE, AF, AG, AM, AQ, BC or BD. In another ment, one or more of the XTEN used herein is selected from the group consisting ofXTEN_AE864, XTEN_AE576, XTEN_AE288, XTEN_AE144, E42, XTEN_AG864, XTEN_AG576, XTEN_AG288, G144, and XTEN_AG42 or other XTEN sequences in Table 4. In the embodiments of the GLP2-XTEN, the one or more XTEN or fragments ofXTEN sequences dually t at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to a motif or an XTEN ed from Table 4, or a fragment thereof with comparable length. In some ments, the GLP2-XTEN fusion proteins comprise a first and at least a second XTEN sequence, wherein the cumulative length of the es in the XTEN sequences is greater than about 100 to about 3000 or about 400 to about 1000 amino acid residues and the XTEN can be identical or they can be different in sequence or in length. As used herein, “cumulative length” is intended to encompass the total length, in amino acid residues, when more than one XTEN is incorporated into the GLP2-XTEN fusion protein.

As described more fully below, methods are disclosed in which the GLP2-XTEN is designed by selecting the length of the XTEN to confer a target half-life or other physicochemical property on a fusion n administered to a subject. When XTEN are used as a carrier, the invention takes advantage of the discovery that increasing the length of the non-repetitive, unstructured ptides enhances the unstructured nature of the XTENs and correspondingly enhances the biological and cokinetic properties of fusion proteins comprising the XTEN carrier. In general, XTEN cumulative lengths longer that about 400 residues incorporated into the fusion n compositions result in longer half-life compared to shorter cumulative lengths, e. g., r than about 280 residues.

As described more fully in the Examples, proportional increases in the length of the XTEN, even if created by a repeated order of single family sequence motifs (e.g., the four AE motifs of Table 3), result in a sequence with a higher percentage of random coil ion, as determined by GOR algorithm, or reduced content of alpha-helices or beta-sheets, as determined by Chou-Fasman algorithm, compared to shorter XTEN lengths. In addition, increasing the length of the unstructured polypeptide ﬁJsion partner, as described in the Examples, results in a fusion protein with a disproportionate increase in terminal half- life compared to fusion proteins with unstructured polypeptide partners with shorter ce lengths.

In some embodiments, where the XTEN serve primarily as a carrier, the invention encompasses GLP2-XTEN compositions comprising one or more XTEN wherein the cumulative XTEN sequence length of the fusion protein(s) is greater than about 100, 200, 400, 500, 600, 800, 900, or 1000 to about 3000 amino acid residues, wherein the ﬁision protein exhibits enhanced pharmacokinetic properties when administered to a subject ed to a GLP-2 not linked to the XTEN and administered at a comparable dose. In one embodiment of the foregoing, the one or more XTEN sequences t at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or more identity to a sequence selected from Table 4, and the remainder, if any, of the carrier sequence(s) ns at least 90% hydrophilic amino acids and less than about 2% of the overall ce consists of hobic or aromatic amino acids or cysteine.

The enhanced pharmacokinetic properties of the GLP2-XTEN in comparison to GLP-2 not linked to XTEN are described more fully, below.

In another aspect, the invention provides methods to create XTEN of short or ediate lengths from longer “donor” XTEN sequences, wherein the longer donor sequence is created by truncating at the N—terminus, or the C—terminus, or a fragment is created from the interior of a donor sequence, thereby resulting in a short or intermediate length XTEN. In miting es, as schematically depicted in -C, the AG864 sequence of 864 amino acid residues can be truncated to yield an AG144 with 144 residues, an AG288 with 288 residues, an AG576 with 576 residues, or other intermediate lengths, while the AE864 sequence (as ed in , E) can be truncated to yield an AE288 or AES76 or other intermediate lengths. It is ically contemplated that such an approach can be utilized with any of the XTEN embodiments described herein or with any of the sequences listed in Tables 4 or 8-12 to result in XTEN of a desired length. 4. Net charge ] In other embodiments, the XTEN polypeptides have an ctured characteristic ed by incorporation of amino acid residues with a net charge and containing a low proportion or no hydrophobic amino acids in the XTEN sequence. The overall net charge and net charge density is lled by modifying the content of charged amino acids in the XTEN sequences, either positive or negative, with the net charge typically represented as the percentage of amino acids in the ptide contributing to a charged state beyond those residues that are cancelled by a residue with an opposing charge. In some ments, the net charge y of the XTEN of the compositions may be above +0.1 or below -0.1 charges/residue. By “net charge density” of a protein or e herein is meant the net charge divided by the total number of amino acids in the protein or propeptide. In other embodiments, the net charge of an XTEN can be about 0%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10% about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% or more. In some ments, the XTEN sequence ses charged residues separated by other es such as serine or glycine, which leads to better expression or puriﬁcation behavior. Based on the net charge, some XTENs have an isoelectric point (pI) of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or even 6.5. In one embodiment, the XTEN will have an ctric point n 1.5 and 4.5 and carry a net negative charge under physiologic conditions.

Since most tissues and surfaces in a human or animal have a net negative charge, in some embodiments the XTEN ces are designed to have a net negative charge to minimize non-speciﬁc interactions between the XTEN containing compositions and various surfaces such as blood vessels, y tissues, or various receptors. Not to be bound by a particular theory, an XTEN can adopt open conformations due to electrostatic repulsion between individual amino acids of the XTEN polypeptide that individually carry a net negative charge and that are distributed across the sequence of the XTEN polypeptide. In some embodiments, the XTEN sequence is designed with at least 90% or 95% of the charged residues separated by other residues such as serine, alanine, threonine, proline or glycine, which leads to a more uniform distribution of charge, better expression or puriﬁcation behavior. Such a distribution of net negative charge in the extended sequence lengths ofXTEN can lead to an unstructured conformation that, in turn, can result in an effective increase in hydrodynamic radius. In preferred embodiments, the negative charge of the subject XTEN is conferred by incorporation of glutamic acid residues. Generally, the glutamic residues are spaced uniformly across the XTEN sequence. In some cases, the XTEN can contain about 10-80, or about 15-60, or about 20-50 glutamic residues per 20kDa of XTEN that can result in an XTEN with charged residues that would have very similar pKa, which can increase the charge homogeneity of the product and sharpen its isoelectric point, enhance the physicochemical properties of the resulting GLP2-XTEN fusion protein for, and hence, simplifying puriﬁcation procedures. For example, where an XTEN with a negative charge is desired, the XTEN can be ed solely from an AB family sequence, which has approximately a 17% net charge due to 2012/054941 incorporated glutamic acid, or can include varying proportions of glutamic acid-containing motifs of Table 3 to provide the desired degree of net charge. Non-limiting examples of AE XTEN include, but are not limited to the AE36, AE42, AE48, AE144, AE288, AE576, AE624, AE864, and AE912 polypeptide sequences of Tables 4 or 9, or fragments thereof. In one embodiment, an XTEN sequence of Tables 4 or 9 can be modiﬁed to include additional glutamic acid es to achieve the desired net negative charge. Accordingly, in one ment the invention provides XTEN in which the XTEN sequences contain about 1%, 2%, 4%, 8%, 10%, 15%, 17%, 20%, 25%, or even about 30% ic acid. In some cases, the XTEN can contain about 10-80, or about 15-60, or about 20-50 glutamic residues per 20kDa ofXTEN that can result in an XTEN with charged residues that would have very similar pKa, which can increase the charge homogeneity of the product and sharpen its isoelectric point, enhance the ochemical properties of the resulting GLP2-XTEN fusion protein for, and hence, simplifying puriﬁcation procedures. In one embodiment, the invention contemplates oration of aspartic acid es into XTEN in addition to glutamic acid in order to achieve a net negative charge.

Not to be bound by a particular theory, the XTEN of the GLP2-XTEN compositions with the higher net negative charge are expected to have less non-speciﬁc ctions with s negativelycharged surfaces such as blood vessels, tissues, or various receptors, which would further contribute to reduced active clearance. Conversely, it is believed that the XTEN of the GLP2-XTEN itions with a low (or no) net charge would have a higher degree of interaction with surfaces that can potentiate the biological activity of the associated GLP—2, given the known contribution of phagocytic cells in the inﬂammatory process in the intestines.

In other cases, where no net charge is desired, the XTEN can be selected from, for example, AG family XTEN components, such as the AG motifs of Table 3, or those AM motifs of Table 3 that have approximately no net charge. Non-limiting es ofAG XTEN include, but are not limited to AG42, AG144, AG288, AG576, and AG864 polypeptide sequences of Tables 4 and 11, or fragments thereof. In another embodiment, the XTEN can comprise g proportions ofAE and AG motifs ( in order to have a net charge that is deemed optimal for a given use or to maintain a given physicochemical property.

The XTEN of the compositions of the present invention generally have no or a low t of positively charged amino acids. In some embodiments, the XTEN may have less than about 10% amino acid residues with a positive charge, or less than about 7%, or less than about 5%, or less than about 2%, or less than about 1% amino acid residues with a positive charge. However, the ion contemplates constructs where a d number of amino acids with a positive charge, such as lysine, are incorporated into XTEN to permit conjugation n the n amine of the lysine and a reactive group on a GLP- 2 peptide, a linker bridge, or a reactive group on a drug or small molecule to be conjugated to the XTEN backbone. In one embodiment of the foregoing, the XTEN has between about 1 to about 100 lysine residues, or about 1 to about 70 lysine residues, or about 1 to about 50 lysine residues, or about 1 to about lysine residues, or about 1 to about 20 lysine residues, or about 1 to about 10 lysine residues, or about 1 to about 5 lysine residues, or alternatively only a single lysine residue. Using the foregoing lysine- containing XTEN, fusion proteins are ucted that comprises XTEN, a GLP-2, plus a chemotherapeutic agent useful in the ent of GLPrelated diseases or disorders, wherein the maximum number of molecules of the agent incorporated into the XTEN component is determined by the numbers of lysines or other amino acids with ve side chains (e.g., cysteine) incorporated into the XTEN. Accordingly, the invention also provides XTEN with 1 to about 10 cysteine es, or about 1 to about 5 cysteine residues, or alternatively only a single ne e wherein fusion proteins are constructed that comprises XTEN, a GLP-2, plus a herapeutic agent useful in the treatment of GLPrelated diseases or disorders, wherein the maximum number of molecules of the agent incorporated into the XTEN component is determined by the numbers of cysteines.

As hydrophobic amino acids impart structure to a polypeptide, the invention provides that the content of hydrophobic amino acids in the XTEN will typically be less than 5%, or less than 2%, or less than 1% hydrophobic amino acid content. In one embodiment, the amino acid content of methionine and tryptophan in the XTEN component of a TEN fusion protein is typically less than 5%, or less than 2%, and most preferably less than 1%. In another embodiment, the XTEN will have a sequence that has less than 10% amino acid es with a positive charge, or less than about 7%, or less that about %, or less than about 2% amino acid residues with a positive charge, the sum of nine and tryptophan residues will be less than 2%, and the sum of asparagine and glutamine residues will be less than 5% of the total XTEN sequence.

. Low immunogenicity In another aspect, the invention provides compositions in which the XTEN sequences have a low degree of immunogenicity or are ntially non-immunogenic. Several factors can contribute to the low immunogenicity ofXTEN, e. g., the non-repetitive sequence, the unstructured conformation, the high degree of solubility, the low degree or lack of self-aggregation, the low degree or lack of proteolytic sites within the sequence, and the low degree or lack of epitopes in the XTEN sequence.

Conformational epitopes are formed by regions of the protein surface that are composed of multiple discontinuous amino acid sequences of the protein antigen. The e folding of the protein brings these sequences into a well-deﬁned, stable l configurations, or epitopes, that can be recognized as “foreign” by the host humoral immune system, resulting in the production of antibodies to the protein or the activation of a cell-mediated immune response. In the latter case, the immune response to a protein in an individual is y inﬂuenced by T-cell epitope recognition that is a function of the peptide binding speciﬁcity of that individual’s HLA-DR allotype. Engagement of a MHC Class II peptide complex by a cognate T-cell receptor on the surface of the T-cell, together with the cross-binding of certain other co-receptors such as the CD4 molecule, can induce an ted state within the T-cell.

Activation leads to the release of cytokines further activating other lymphocytes such as B cells to e antibodies or ting T killer cells as a full cellular immune response.

The ability of a peptide to bind a given MHC Class II molecule for presentation on the surface of an APC (antigen presenting cell) is dependent on a number of factors; most notably its primary sequence. In one embodiment, a lower degree of immunogenicity is achieved by designing XTEN sequences that resist antigen processing in antigen presenting cells, and/or choosing sequences that do not bind MHC receptors well. The invention provides TEN fusion proteins with substantially non-repetitive XTEN polypeptides designed to reduce binding with MHC II receptors, as well as avoiding formation of epitopes for T-cell receptor or antibody binding, resulting in a low degree of immunogenicity. Avoidance of immunogenicity can attiibute to, at least in part, a result of the mational ﬂexibility of XTEN sequences; i.e., the lack of secondary structure due to the selection and order of amino acid residues. For example, of particular st are sequences having a low cy to adapt compactly folded conformations in aqueous solution or under physiologic conditions that could result in conformational epitopes. The stration of fusion proteins comprising XTEN, using conventional therapeutic practices and dosing, would generally not result in the formation of neutralizing antibodies to the XTEN sequence, and also reduce the immunogenicity of the GLP-2 fusion partner in the GLP2-XTEN compositions.

In one ment, the XTEN sequences utilized in the subject fusion proteins can be substantially free of epitopes recognized by human T cells. The elimination of such epitopes for the purpose of generating less immunogenic proteins has been disclosed previously; see for example WO 98/52976, WO 02/079232, and WO 00/3317 which are incorporated by reference herein. Assays for human T cell epitopes have been described (Stickler, M., et al. (2003) JImmunol Methods, 281: 95-108). icular interest are peptide sequences that can be oligomerized without generating T cell epitopes or non-human ces. This is achieved by g direct s of these sequences for the presence of T-cell epitopes and for the occurrence of 6 to 15-mer and, in ular, 9-mer sequences that are not human, and then altering the design of the XTEN sequence to eliminate or disrupt the epitope sequence.

In some embodiments, the XTEN sequences are substantially non-immunogenic by the restriction of the numbers of epitopes of the XTEN predicted to bind MHC receptors. With a reduction in the numbers of es capable ofbinding to MHC receptors, there is a concomitant reduction in the potential for T cell activation as well as T cell helper function, reduced B cell activation or upregulation and reduced antibody production. The low degree of predicted T-cell epitopes can be determined by epitope tion algorithms such as, e.g., TEPITOPE iolo, T., et a]. (1999) Nat hnol, 17: 555-61), as shown in Example 31. The TEPITOPE score of a given e frame within a protein is the log of the Kd (dissociation constant, afﬁnity, off-rate) of the binding of that e frame to le of the most common human MHC alleles, as sed in Stumiolo, T. et al. (1999) Nature Biotechnology 171555). The score ranges over at least 20 logs, from about 10 to about -10 (corresponding to binding aints of 10e10 Kd to 10e'10 Kd), and can be reduced by avoiding hydrophobic amino acids that serve as anchor residues during peptide display on MHC, such as M, I, L, V, F. In some embodiments, an XTEN component incorporated into a GLP2-XTEN does not have a predicted T-cell epitope at a TEPITOPE threshold score of about -5, or -6, or -7, or -8, or -9, or at a TEPITOPE score of -10. As used herein, a score of “-9” would be a more stringent TEPITOPE old than a score of -5.

In another embodiment, the inventive XTEN sequences, including those incorporated into the subject GLPZ-XTEN fusion proteins, are rendered ntially munogenic by the restriction of known proteolytic sites from the sequence of the XTEN, reducing the processing ofXTEN into small peptides that can bind to MHC II receptors. In another ment, the XTEN sequence is rendered substantially non-immunogenic by the use a sequence that is substantially devoid of secondary structure, conferring resistance to many proteases due to the high entropy of the structure. Accordingly, the reduced TEPITOPE score and elimination of known proteolytic sites from the XTEN render the XTEN compositions, including the XTEN ofthe GLPZ-XTEN fusion protein itions, substantially unable to be bound by mammalian receptors, including those of the immune system. In one embodiment, an XTEN of a GLPZ-XTEN fusion protein can have >100 nM Kd binding to a mammalian receptor, or greater than 500 nM Kd, or r than 1 uM Kd towards a mammalian cell surface or circulating polypeptide or. onally, the non-repetitive sequence and corresponding lack of epitopes of XTEN limit the ability of B cells to bind to or be activated by XTEN. A repetitive sequence is recognized and can form multivalent contacts with even a few B cells and, as a consequence of the cross-linking of multiple T-cell ndent ors, can stimulate B cell proliferation and antibody production. In st, while a XTEN can make ts with many different B cells over its extended sequence, each individual B cell may only make one or a small number of contacts with an individual XTEN due to the lack of repetitiveness of the sequence. Not being to be bound by any theory, XTENs typically have a much lower tendency to stimulate proliferation of B cells and thus an immune response. In one ment, the GLPZ-XTEN have d immunogenicity as ed to the corresponding GLP-2 that is not fused to an XTEN. In one embodiment, the administration of up to three parenteral doses of a GLPZ- XTEN to a mammal result in detectable anti-GLPZ-XTEN IgG at a serum dilution of 1:100 but not at a dilution of 1:1000. In another embodiment, the administration of up to three parenteral doses of a GLPZ- XTEN to a mammal result in able LP-2 IgG at a serum dilution of 1:1000 but not at a dilution of 1:10,000. In another embodiment, the administration of up to three parenteral doses of a GLPZ-XTEN to a mammal result in detectable anti-XTEN IgG at a serum dilution of 1210,000 but not at a dilution of 0,000. In the foregoing embodiments, the mammal can be a mouse, a rat, a rabbit, or a cynomolgus monkey.

An additional feature ofXTENs with non-repetitive sequences relative to sequences with a high degree ofrepetitiveness is non-repetitive XTENs form weaker contacts with antibodies. Antibodies are multivalent molecules. For instance, IgGs have two identical binding sites and Ing contain 10 identical binding sites. Thus antibodies against repetitive sequences can form multivalent contacts with such repetitive sequences with high avidity, which can affect the potency and/or elimination of such repetitive sequences. In contrast, antibodies against non-repetitive XTENs may yield monovalent interactions, 2012/054941 resulting in less likelihood of immune clearance such that the GLP2-XTEN compositions can remain in ation for an sed period of time. 6. Increased hydrodynamic radius In r aspect, the present ion provides XTEN in which the XTEN polypeptides have a high hydrodynamic radius that confers a corresponding increased apparent molecular weight to the GLP2-XTEN fusion protein incorporating the XTEN. As detailed in Example 25 the linking ofXTEN to therapeutic protein sequences results in GLP2-XTEN compositions that can have increased hydrodynamic radii, increased apparent molecular weight, and increased apparent lar weight factor compared to a eutic protein not linked to an XTEN. For example, in therapeutic applications in which prolonged half-life is d, compositions in which a XTEN with a high hydrodynamic radius is incorporated into a fusion protein comprising a therapeutic protein can effectively enlarge the hydrodynamic radius of the composition beyond the ular pore size of approximately 3-5 nm sponding to an apparent molecular weight of about 70 kDA) eti. 2003.

Pharmacokinetic and biodistribution properties ofpoly(ethylene glycol)-pr0tein conjugates. Adv Drug DeliV Rev 55:1261-1277), resulting in reduced renal nce of circulating proteins with a corresponding increase in terminal half-life and other enhanced pharmacokinetic properties. The hydrodynamic radius of a protein is determined by its lar weight as well as by its structure, including shape or compactness. Not to be bound by a particular theory, the XTEN can adopt open conformations due to electrostatic repulsion between dual s of the peptide or the inherent ﬂexibility imparted by the particular amino acids in the sequence that lack potential to confer secondary structure. The open, extended and unstructured conformation of the XTEN polypeptide can have a greater proportional hydrodynamic radius compared to polypeptides of a comparable sequence length and/or molecular weight that have secondary and/or tertiary structure, such as typical globular proteins.

Methods for determining the hydrodynamic radius are well known in the art, such as by the use of size exclusion chromatography (SEC), as described in US. Patent Nos. 6,406,632 and 7,294,513. As the results of Example 25 demonstrate, the addition of increasing lengths of XTEN s in proportional increases in the parameters of hydrodynamic radius, apparent molecular weight, and apparent molecular weight factor, permitting the ing of GLP2-XTEN to desired characteristic cut-off apparent molecular weights or hydrodynamic radii. Accordingly, in certain embodiments, the GLP2-XTEN fusion protein can be configured with an XTEN such that the fusion protein can have a hydrodynamic radius of at least about 5 nm, or at least about 8 nm, or at least about 10 nm, or 12 nm, or at least about 15 nm. In the foregoing embodiments, the large hydrodynamic radius conferred by the XTEN in a GLP2-XTEN fusion protein can lead to reduced renal clearance of the resulting fusion n, leading to a corresponding increase in terminal half-life, an increase in mean residence time, and/or a decrease in renal clearance rate.

When the molecular weights of the GLP2-XTEN fusion proteins are derived from size exclusion chromatography analyses, the open conformation of the XTEN due to the low degree of secondary structure results in an increase in the apparent molecular weight of the fusion proteins. In some embodiments the GLP2-XTEN comprising a GLP-2 and at least a ﬁrst or multiple XTEN exhibits an apparent molecular weight of at least about 200 kDa, or at least about 400 kDa, or at least about 500 kDa, or at least about 700 kDa, or at least about 1000 kDa, or at least about 1400 kDa. Accordingly, the GLP2-XTEN fusion proteins comprising one or more XTEN exhibit an apparent molecular weight that is about 2-fold greater, or about 3-fold greater or about 4-fold greater, or about 8-fold greater, or about -fold greater, or about 12-fold greater, or about 15 -fold r, or about d r than the actual lar weight of the fusion protein. In one embodiment, the isolated GLP2-XTEN fusion protein of any of the embodiments disclosed herein t an apparent molecular weight factor under physiologic conditions that is greater than about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 10, or about 15, or greater than about 20. In another embodiment, the TEN fusion protein has, under physiologic conditions, an apparent molecular weight factor that is about 3 to about , or is about 5 to about 15, or is about 8 to about 14, or is about 10 to about 12 relative to the actual molecular weight of the fusion n.

IV). GLPZ-XTEN ITIONS The present ion relates in part to fusion protein compositions comprising GLP-2 linked to one or more XTEN, wherein the fusion protein would act to replace or augment existing GLP-2 when administered to a subject. The invention addresses a long-felt need in sing the terminal half-life of ously administered GLP-2 to a subject in need f. One way to increase the circulation half- life of a eutic protein is to ensure that renal clearance of the protein is reduced. Another way to increase the circulation half-life is to reduce the active clearance of the therapeutic protein, whether mediated by receptors, active metabolism of the n, or other endogenous mechanisms. Both may be achieved by conjugating the protein to a r, which, in some cases, is capable of conferring an increased molecular size (or hydrodynamic radius) to the protein and, hence, reduced renal clearance, and, in other cases, interferes with binding of the protein to clearance receptors or other proteins that contribute to metabolism or clearance. Thus, certain objects of the present invention include, but are not limited to, providing improved GLP-2 molecules with a longer circulation or terminal half-life, decreasing the number or frequency of necessary administrations of GLP-2 compositions, retaining at least a portion of the biological activity of the native GLP-2, and enhancing the ability to treat GLP related diseases or gastrointestinal conditions with resulting ement in clinical symptoms and overall well-being more ntly, more effectively, more ically, and with greater safety compared to presently available GLP-2 preparations.

To meet these needs, in a ﬁrst aspect, the invention provides isolated fusion protein itions comprising a biologically active GLP-2 covalently linked to one or more XTEN, resulting in a GLP2-XTEN fusion protein composition. The subject GLPXTEN can mediate one or more biological or therapeutic activities of a wild-type GLP-2. GLP2-XTEN can be produced recombinantly 2012/054941 or by chemical ation of a GLP-2 to and XTEN. In one embodiment, the GLP-2 is native GLP-2.

In another embodiment, the GLP-2 is a sequence t of a natural sequence that retains at least a portion of the biological activity of the native GLP-2. In one ment, the GLP-2 is a sequence having at least 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to a sequence selected from the group consisting of the sequences in Table 1, when optimally aligned. In another embodiment, the GLP-2 is a sequence variant with glycine substituted for alanine at residue number 2 ofthe mature GLP-2 peptide. In one embodiment, the GLP2- XTEN comprises a GLP-2 having the sequence HGDGSFSDEMNTILDNLAARDFINWLIQTKITD. In one ment, the ion provides GLP2-XTEN fusion proteins comprising GLP-2 N- and/or C- terminally modiﬁed forms comprising one or more XTEN.

The GLP-2 of the subject compositions, particularly those disclosed in Table 1, together with their corresponding nucleic acid and amino acid sequences, are well known in the art and descriptions and sequences are available in public databases such as Chemical Abstracts Services Databases (e. g., the CAS Registry), GenBank, The Universal n ce (UniProt) and subscription provided databases such as GenSeq (e.g., Derwent). Polynucleotide sequences may be a wild type polynucleotide sequence encoding a given GLP-2 (e. g., either full length or mature), or in some instances the sequence may be a variant of the wild type polynucleotide sequence (e.g., a polynucleotide which encodes the wild type biologically active protein, wherein the DNA sequence of the polynucleotide has been optimized, for example, for sion in a particular s; or a polynucleotide encoding a variant of the wild type protein, such as a site directed mutant or an allelic variant. It is well within the ability of the skilled artisan to use a wild-type or consensus cDNA sequence or a codon-optimized sequence variant of a GLP- 2 to create GLP2-XTEN constructs contemplated by the invention using methods known in the art and/or in conjunction with the guidance and methods ed herein and described more fully in the Examples.

In some embodiments, the TEN fusion proteins retain at least a portion of the biological activity of native GLP-2. A GLP2-XTEN fusion protein of the invention is capable of binding and activating a GLP-2 receptor. In one embodiment, the GLPZ-XTEN fusion protein ofthe present invention has an EC50 value, when assessed using an in vitro GLP-2 receptor binding assay such as described herein or others known in the art, of less than about 30 nM, or about 100 nM, or about 200 nM, or about 300 nM, or about 400 nM, or about 500 nM, or about 600 nM, or about 700 nM, or about 800 nM, or about 1000 nM, or about 1200 nM, or about 1400 nM. In another embodiment, the TEN fusion protein of the present invention s at least about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 10%, or about 20%, or about 30% of the potency of the corresponding GLP-2 not linked to XTEN when assayed using an in vitro GLP2R cell assay such as described in the es or others known in the art.

In some embodiments, GLP2-XTEN fusion proteins of the disclosure have intestinotrophic, wound healing and anti-inﬂammatory activity. In some ments, the GLP2-XTEN fusion protein compositions exhibit an improvement in one, two, three or more gastrointestinal-related parameters disclosed herein that are at least about 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90%, or 100%, or 120%, or 140%, at least about 150% greater compared to the parameter(s) achieved by the ponding GLP-2 component not linked to the XTEN when administered to a subject. The parameter can be a measured parameter selected from blood trations of GLP-2, sed eric blood ﬂow, decreased inﬂammation, increased weight gain, decreased diarrhea, decreased fecal wet weight, intestinal wound healing, increase in plasma citrulline concentrations, decreased CRP levels, decreased requirement for steroid therapy, enhancing or stimulating mucosal integrity, decreased sodium loss, decreased parenteral nutrition required to maintain body weight, minimizing, ting, or ting bacterial translocation in the intestines, enhancing, stimulating or accelerating recovery of the intestines after surgery, preventing relapses of inﬂammatory bowel disease, or ing or maintaining energy homeostasis, among others. In one embodiment, administration of the GLP2-XTEN ﬁJsion protein to a subject results in a greater ability to se small intestine weight and/or length when administered to a subject with a surgically-resected intestine (e.g., short-bowel syndrome) or Crohn’s Disease, compared to the corresponding GLP-2 not linked to XTEN and administered at a comparable dose in nmol/kg and dose regimen. In another ment, a GLP2-XTEN fusion n exhibits at least about 10%, or %, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or at least about 90% greater ability to reduce ulceration when administered to a subject with Crohn’s Disease (either naturally acquired or experimentally induced) compared to the corresponding GLP—2 ent not linked to the XTEN and administered at a comparable nmol/kg dose and dose regimen. In r embodiment, the ﬁlSiOl’l protein exhibits the ability to reduce inﬂammatory cytokines when stered to a subject with Crohn’s Disease (either naturally ed or experimentally induced) by at least about 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or at least about 90% compared the corresponding GLP-2 component not linked to the XTEN and administered at a able nmol/kg dose and dose regimen. In another embodiment, a GLP2-XTEN fusion protein ts at least about 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or at least about 90% greater ability to reduce mucosal atrophy when stered to a subject with Crohn’s Disease (either naturally acquired or experimentally induced; e. g., administration of indomethacin) ed to the corresponding GLP-2 component not linked to the XTEN and administered at a comparable nmol/kg dose and dose regimen. In another embodiment, a GLP2-XTEN fusion protein exhibits at least about 5%, or at least about 6%, or 7%, or 8%, or 9%, or %, or 11%, or 12%, or 15%, or at least about 20% greater ability to increase height of intestinal villi when administered to a subject with Crohn’s Disease (either naturally ed or experimentally induced; e.g., administration of indomethacin) compared to the corresponding GLP-2 component not linked to the XTEN and administered at a comparable nmol/kg dose and dose regimen. In another embodiment, a GLP2-XTEN fusion protein exhibits at least about 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or at least about 90% greater ability to increase body weight when administered to a subject with Crohn’s Disease (either naturally acquired or mentally induced; e.g., administration of indomethacin) compared to the corresponding GLP-2 component not linked to the XTEN and administered at a comparable nmol/kg dose and dose regimen. In the foregoing embodiments of the paragraph, the t is selected from the group consisting of mouse, rat, monkey and human.

The compositions of the invention include fusion proteins that are useful, when administered to a subject, for mediating or preventing or ameliorating a gastrointestinal condition associated with GLP-2 such as, but not d to ulcers, gastritis, digestion disorders, malabsorption syndrome, short-gut syndrome, short bowel syndrome, cul-de-sac syndrome, inﬂammatory bowel disease, celiac disease, tropical sprue, hypogammaglobulinemic sprue, Crohn's disease, ulcerative colitis, enteritis, chemotherapy-induced enteritis, irritable bowel syndrome, small intestine damage, small inal damage due to -chemotherapy, gastrointestinal , diarrhea] es, inal insufﬁciency, acid-induced intestinal , arginine deﬁciency, idiopathic hypospermia, obesity, catabolic illness, febrile neutropenia, diabetes, obesity, steatorrhea, autoimmune diseases, food allergies, hypoglycemia, gastrointestinal barrier disorders, sepsis, bacterial peritonitis, burn-induced intestinal damage, decreased intestinal motility, intestinal failure, chemotherapy-associated bacteremia, bowel trauma, bowel ischemia, mesenteric ischemia, malnutrition, necrotizing enterocolitis, necrotizing atitis, neonatal feeding rance, NSAID-induced gastrointestinal damage, nutritional insufﬁciency, total eral nutrition damage to gastrointestinal tract, neonatal nutritional insufﬁciency, radiation-induced enteritis, radiation—induced injury to the intestines, mucositis associated with cancer chemotherapy and irritable bowel disease, pouchitis, ia, and stroke.

] Ofparticular interest are GLPZ-XTEN ﬁtsion protein compositions for which an increase in a pharmacokinetic ter, increased solubility, increased stability, or some other enhanced pharmaceutical property compared to native GLP-2 is obtained, providing compositions with enhanced efﬁcacy, safety, or that result in reduced dosing frequency and/or improve patient ment. The GLP2-XTEN fusion proteins of the ments disclosed herein exhibit one or more or any combination of the improved properties and/or the embodiments as detailed herein. Thus, the subject GLPZ-XTEN fusion protein itions are designed and ed with various objectives in mind, including improving the therapeutic efﬁcacy of the ive GLP-2 by, for example, increasing the in vivo exposure or the length that the GLP2-XTEN remains within the therapeutic window when stered to a subject, compared to a GLP-2 not linked to XTEN.

In one embodiment, a GLPZ-XTEN fusion protein comprises a single GLP-2 molecule linked to a single XTEN (e.g., an XTEN as described above). In another embodiment, the GLPZ-XTEN comprises a single GLP-2 linked to two XTEN, wherein the XTEN may be identical or they may be different. In another embodiment, the GLPZ-XTEN fusion protein comprises a single GLP-2 molecule linked to a ﬁrst and a second XTEN, in which the GLP-2 is a sequence that has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity compared to a protein sequence selected from Table l, and the ﬁrst and the second XTEN are each sequences that have 2012/054941 at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence ty ed to one or more sequences selected from Table 4, or fragments thereof. In another embodiment, the GLPZ-XTEN fusion protein comprises a sequence with at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity to a ce from Table 33 and 34. 1. GLP2-XTEN Fusion Protein Conﬁgurations The invention provides GLP2-XTEN fusion n compositions with the GLP-2 and XTEN components linked in speciﬁc N— to inus conﬁgurations.

In one embodiment of the TEN composition, the invention provides a fusion protein of a I: (GLP-2)-(XTEN) I wherein independently for each occurrence, GLP-2 is a GLP-2 protein or variant as defined herein, including sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with sequenced from Table 1, and XTEN is an extended recombinant polypeptide as described herein, including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4.

In another embodiment of the GLP2-XTEN composition, the invention provides a fusion protein of formula II: -(GLP-2) 11 wherein independently for each occurrence, GLP-2 is a GLP-2 protein or variant as defined herein, including sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence ty with sequenced from Table 1, and XTEN is an extended recombinant polypeptide as described herein, ing, but not limited to ces having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4.

In another embodiment of the GLP2-XTEN composition, the invention provides an isolated fusion protein, wherein the fusion protein is of formula III: (XTEN)-(GLP-2)-(XTEN) III wherein independently for each occurrence, GLP-2 is a GLP-2 protein or variant as defined herein, including sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with ced from Table 1, and XTEN is an extended recombinant polypeptide as described herein, including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4..

In another embodiment of the GLPZ-XTEN ition, the invention es an isolated fusion protein, wherein the 11.181011 protein is of formula IV: (GLP-2)-(XTEN)-(GLP-2) IV wherein independently for each occurrence, GLP-2 is a GLP-2 protein or variant as d herein, including ces having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with sequenced from Table 1, and XTEN is an extended recombinant polypeptide as bed herein, including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4.

In another embodiment of the GLPZ-XTEN composition, the ion es an isolated ﬁJsion protein, wherein the fusion protein is of formula V: )-(S)x-(XTEN) V wherein independently for each ence, GLP-2 is a GLP-2 protein or variant as defined herein, ing sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with sequenced from Table 1; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage ce or amino acids compatible with restrictions sites; X is either 0 or 1; and XTEN is an extended recombinant polypeptide as described herein, including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to ces set forth in Table 4.

In another ment of the GLPZ-XTEN composition, the invention provides an isolated ﬁJsion protein, wherein the fusion protein is of formula VI: (XTEN)x-(S)X-(GLP-2)-(S)y-(XTEN)y v1 wherein independently for each occurrence, GLP-2 is a GLP-2 protein or variant as defined herein, including sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with sequenced from Table 1; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; X is either 0 or 1 and y is either 0 or 1 wherein x+y 31; and XTEN is an extended recombinant polypeptide as described herein, including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4.

The embodiments of formulae I-VI ass TEN conﬁgurations wherein one or more XTEN of lengths ranging from about 36 amino acids to 3000 amino acids (e.g., sequences selected from Table 4 or fragments thereof, or sequences exhibiting at least about 90-95% or more sequence identity thereto) are linked to the N- or C-terminus of the GLP-2. The embodiments of a V further provide conﬁgurations wherein the XTEN are linked to GLP-2 via spacer sequences that can optionally comprise amino acids compatible with restrictions sites or can include cleavage sequences (e.g., the sequences of Tables 5 and 6, described more fully below) such that the XTEN encoding sequence can, in the case of a restriction site, be integrated into a GLP2-XTEN construct and, in the case of a cleavage sequence, the XTEN can be ed from the fusion protein by the action of a protease appropriate for the cleavage sequence. In one ment of formula V, the fusion protein comprises a spacer sequence that is a single glycine e. 2. GLP2-XTEN Fusion Protein Conﬁgurations with Spacer and Cleavage Sequences In another aspect, the invention provides TEN conﬁgured with one or more spacer sequences incorporated into or adjacent to the XTEN that are designed to incorporate or enhance a ﬁanctionality or property to the composition, or as an aid in the assembly or manufacture of the fusion protein compositions. Such properties include, but are not limited to, inclusion of cleavage sequence(s), such at TEV or other cleavage sequences of Table 6, to permit release of components, inclusion of amino acids compatible with nucleotide restrictions sites to permit linkage of ncoding nucleotides to GLP—2—encoding tides or that facilitate construction of expression vectors, and linkers designed to reduce steric hindrance in regions of TEN fusion proteins.

In an embodiment, a spacer sequence can be introduced between an XTEN sequence and a GLP-2 component to decrease steric hindrance such that the GLP-2 component may assume its desired tertiary structure and/or interact appropriately with its target receptor. For s and methods of identifying desirable spacers, see, for example, George, et al. (2003) Protein Engineering 152871—879, cally incorporated by reference herein. In one embodiment, the spacer comprises one or more e sequences that are between 1—50 amino acid residues in , or about 1—25 residues, or about 1-10 residues in length. Spacer ces, exclusive of cleavage sites, can se any of the 20 l L amino acids, and will preferably have XTEN-like properties in that 1) they will comprise hydrophilic amino acids that are satirically unhindered such as, but not limited to, glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), proline (P) and aspartate (D); and 2) will be substantially non-repetitive. In addition, spacer sequences are designed to avoid the introduction of T-cell epitopes; determination of which are described above and in the Examples. In some cases, the spacer can be polyglycines or polyalanines, or is predominately a mixture of combinations of glycine, serine and alanine residues. In one embodiment, a spacer sequence, exclusive of ge site amino acids, has about 1 to 10 amino acids that consist of amino acids selected from glycine (G), e (A), serine (S), threonine (T), glutamate (E), and proline (P) and are substantially devoid of secondary structure; e. g., less than about 10%, or less than about 5% as determined by the Chou-Fasman and/or GOR algorithms. In one embodiment, the spacer ce is GPEGPS. In r embodiment, the spacer sequence is a single glycine residue. In another embodiment, the spacer sequence is GPEGPS linked to a cleavage sequence of Table 6.

In a particular embodiment, the GLPZ-XTEN ﬁlSlOl’l protein comprises one or more spacer sequences linked at the junction(s) n the payload GLP-Z sequence and the one more XTEN incorporated into the fusion protein, wherein the spacer sequences comprise amino acids that are compatible with nucleotides encoding restriction sites. In another embodiment, the GLPZ-XTEN fusion protein comprises one or more spacer sequences linked at the junction(s) between the d GLP-2 sequence and a signal sequence incorporated into the fusion protein, wherein the spacer sequences comprise a cleavage sequence (e.g., TEV) to release the TEN after expression. In another embodiment, the TEN fusion protein ses one or more spacer sequences linked at the junction(s) between the payload GLP-2 sequence and the one more XTEN incorporated into the fusion protein wherein the spacer sequences se amino acids that are compatible with nucleotides encoding restriction sites and the amino acids and the one more spacer sequence amino acids are chosen from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P). In another embodiment, the GLPZ-XTEN fusion protein comprises one or more spacer sequences linked at the junction(s) between the payload GLP-2 sequence and the one more XTEN incorporated into the fusion protein wherein the spacer sequences comprise amino acids that are compatible with nucleotides encoding restriction sites and the one more spacer sequences are chosen from the sequences of Table 5.

The exact sequence of each spacer ce is chosen to be ible with cloning sites in expression vectors that are used for a particular GLPZ-XTEN construct. For embodiments in which a single XTEN is attached to the N- or C-terminus, only a single spacer sequence at the on of the two components would be required. As would be apparent to one of ordinary skill in the art, the spacer ces comprising amino acids compatible with restriction sites could be omitted from the construct when an entire GLP2-XTEN gene is synthetically generated, rather than ligated using GLP-2 and XTEN ng genes.

Table 5: Spacer Seguences Compatible with Restriction Sites Spacer Sequence Restriction Enzyme GSPG BsaI ETET BsaI PGSSS BbsI GAP AscI GPA FseI GPSGP SﬁI AAA SacII TG AgeI GT KpnI GAGSPGAETA SﬁI ASS XhoI 2012/054941 In another aspect, the present invention provides GLPZ-XTEN conﬁgurations with cleavage sequences incorporated into the spacer sequences. In some embodiments, a spacer sequence in a GLPZ- XTEN fusion protein composition comprises one or more cleavage sequences, which are identical or different, wherein the cleavage sequence may be acted on by a protease to e the XTEN sequence(s) from the ﬁlSlOl’l protein. In one embodiment, the incorporation of the ge sequence into the GLPZ- XTEN is designed to permit release of a GLP-2 that becomes active or more active upon its release from the XTEN component. The cleavage sequences are located sufficiently close to the GLP-2 ces, generally within 18, or within 12, or within 6, or within 2 amino acids of the GLP-2 sequence, such that any remaining es attached to the GLP-Zs after ge do not appreciably interfere with the activity (e.g., such as binding to a GLP-2 receptor) of the GLP-2, yet provide sufficient access to the protease to be able to effect cleavage of the cleavage ce. In some cases, the GLPZ-XTEN comprising the cleavage sequences will also have one or more spacer sequence amino acids between the GLP-2 and the cleavage sequence or the XTEN and the cleavage ce to facilitate access of the protease to the cleavage sequence; the spacer amino acids sing any natural amino acid, ing glycine, serine and alanine as preferred amino acids. In one embodiment, the cleavage site is a sequence that can be cleaved by a protease endogenous to the mammalian subject such that the GLPZ-XTEN can be cleaved after administration to a subject. In such case, the GLP2—XTEN can serve as a prodrug or a ating depot for the GLP—2. In a particular construct of the foregoing, the GLPZ—XTEN would have one or two XTEN linked to the N— and/or the C-terminus such that the XTEN could be released, g the active form of GLP-2 free. In one embodiment of the foregoing construct, the GLP-2 that is released from the fusion protein by cleavage of the cleavage sequence exhibits at least about a two-fold, or at least about a three-fold, or at least about a four-fold, or at least about a ld, or at least about a six-fold, or at least about a eight-fold, or at least about a ld, or at least about a 20-fold increase in biological activity compared to the intact GLPZ-XTEN fusion n.

] Examples of cleavage sites contemplated by the ion include, but are not limited to, a polypeptide sequence cleavable by a mammalian endogenous protease selected from FXIa, FXIIa, rein, FVIIIa, FVIIIa, FXa, FIIa bin), Elastase-Z, granzyme B, MMP-12, MMP-l3, MMP-l7 or MMP-ZO, or by non-mammalian proteases such as TEV, enterokinase, PreScissionTM protease (rhinovirus 3C protease), and sortase A. Sequences known to be cleaved by the foregoing proteases and others are known in the art. Exemplary cleavage sequences contemplated by the invention and the respective cut sites within the sequences are presented in Table 6, as well as sequence variants thereof.

Thus, cleavage sequences, particularly those of Table 6 that are susceptible to the endogenous proteases present during inﬂammation would provide for release of GLP-2 that, in certain embodiments of the GLPZ-XTEN, provide a higher degree of activity for the GLP-2 component released from the intact form of the GLPZ-XTEN, as well as additional safety margin for high doses of GLPZ-XTEN administered to a subject. For example, it has been demonstrated that many of the metaloproteinases are elevated in WO 40093 Crohn’s Disease and inﬂamed intestines (D Schuppan and T Freitag. Fistulising Crohn’s disease: MMPs gone awry. Gut (2004) 53(5): 622—624). In one embodiment, the invention provides GLPZ-XTEN comprising one or more cleavage sequences operably oned to release the GLP-2 from the fusion protein upon cleavage, wherein the one or more cleavage sequences has at least about 86%, or at least about 92% or greater sequence identity to a sequence selected from Table 6. In another embodiment, the GLPZ-XTEN comprising a cleavage ce would have at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity compared to a sequence selected from Table 34.

In some embodiments, only the two or three amino acids ﬂanking both sides of the cut site (four to six amino acids total) are incorporated into the cleavage sequence that, in turn, is incorporated into the GLPZ-XTEN of the embodiments. In other embodiments, the orated cleavage sequence of Table 6 can have one or more deletions or insertions or one or two or three amino acid substitutions for any one or two or three amino acids in the known sequence, wherein the deletions, insertions or substitutions result in reduced or enhanced susceptibility but not an absence of susceptibility to the protease, ing in an y to tailor the rate of release of the GLP-2 from the XTEN. Exemplary substitutions are shown in Table 6.

Table 6: Protease Cleavage Seguences Protease Acting Upon Exemplary Cleavage Minimal Cut Site* Sequence Sequence KMUTAWAVE/GT/Gv G KD/FL/T/RWA/VE/GT/GV TMTAMVGG NA Kallikrein SPFRlSTGG -/-/FL/RY»LSR/RT/—/— FIXa R¢-/—/—/— FXa IA/E/GFP/RJSTI/VFSHG RtSAG/-/-/- AAA-www- TEV ENLYFQIG lG/s Enterokinase DDDKMVGG DDDKlIVGG (PE:EZZZ:O:§M) LEVLFQJ/GP LEVLFQJ/GP LPKTIGSES L/P/KEAD/TJrG/JEKS/S iindicates cleavage site NA: not applicable * the listing of multiple amino acids , between, or after a slash indicate alternative amino acids that can be substituted at the position; - indicates that any amino acid may be substituted for the corresponding amino acid indicated in the middle column 3. Exemplaﬂ GLP2-XTEN Fusion Protein Sequences Non-limiting examples of sequences of fusion proteins containing a single GLP-2 linked to one or two XTEN, either joined at the N— or ini are presented in Tables 13 and 32. In one embodiment, a GLP2-XTEN composition would comprise a fusion protein having at least about 80% sequence identity compared to a GLP2-XTEN selected from Table 13 or Table 33, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity as compared to a GLP2-XTEN from Table 13 or Table 33.

However, the ion also contemplates substitution of any of the GLP-2 sequences of Table 1 for a GLP-2 component of the GLP2-XTEN of Table 13 or Table 33, and/or substitution of any sequence of Table 4 for an XTEN ent of the GLP2-XTEN of Table 13 or Table 33. In preferred embodiments, the resulting GLP2-XTEN of the foregoing examples retain at least a portion of the biological activity of the corresponding GLP-2 not linked to the XTEN; e. g., the ability to bind and te a GLP-2 receptor and/or result in an intestinotrophic, proliferative, or wound-healing effect. In the foregoing fusion proteins hereinabove described in this paragraph, the GLP2—XTEN fusion protein can ﬁlrther se one or more cleavage sequences; e.g., a sequence from Table 6, the cleavage sequence being located between the GLP-2 and the XTEN. In some embodiments comprising ge sequence(s), the intact GLP2-XTEN composition has less biological activity but a longer half-life in its intact form compared to a corresponding GLP-2 not linked to the XTEN, but is designed such that upon administration to a subject, the GLP-2 component is gradually released from the fusion protein by cleavage at the ge sequence(s) by endogenous proteases, whereupon the GLP-2 component exhibits activity, i.e., the ability to ively bind to the GLP-2 receptor. In non-limiting examples, the GLPZ-XTEN with a cleavage sequence has about 80% sequence identity compared to a sequence from Table 34, or about 85%, or about 90%, or about 95%, or about 97%, or about 98%, or about 99% sequence ty ed to a ce from Table 34. However, the invention also contemplates substitution of any ofthe GLP-2 sequences of Table 1 for a GLP-2 component of the GLP2-XTEN of Table 34, substitution of any sequence of Table 4 for an XTEN component of the GLP2-XTEN of Table 34, and substitution of any cleavage sequence of Table 6 for a ge component ofthe GLP2-XTEN of Table 34. In some cases, the GLP2-XTEN of the foregoing embodiments in this paragraph serve as prodrugs or a circulating depot, resulting in a longer terminal half-life compared to GLP-2 not linked to the XTEN. In such cases, a higher concentration of GLP2-XTEN can be administered to a t to maintain therapeutic blood levels for an extended period of time compared to the corresponding GLP-2 not linked to XTEN because a smaller proportion of the circulating composition is active.

WO 40093 The TEN itions of the embodiments can be evaluated for biological ty using assays or in viva parameters as described herein (e. g., assays ofthe Examples or assays of Table 32), or a pharmacodynamic effect in a preclinical model of GLP-2 deﬁciency or in clinical trials in humans, using s as described in the Examples or other methods lmown in the art for assessing GLP-2 biological activity to determine the suitability of the uration or the GLP-2 sequence variant, and those GLP2-XTEN compositions (including after cleavage of any orated XTEN- releasing ge sites) that retain at least about 40%, or about 50%, or about 55%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95% or more biological activity compared to native GLP-2 sequence are considered suitable for use in the treatment of GLPrelated conditions.

V). PROPERTIES OF THE GLPZ-XTEN COMPOSITIONS OF THE INVENTION (a) Pharmacokinetic Properties of GLPZ-XTEN It is an object of the present invention to provide GLP2-XTEN fusion proteins with ed pharmacokinetics compared to GLP-2 not linked to the XTEN. The pharmacokinetic properties of a GLP-2 that can be enhanced by linking a given XTEN to the GLP-2 include, but are not limited to, terminal half-life, area under the curve (AUC), Cmax, volume of bution, maintaining the biologically active GLP2-XTEN within the therapeutic window above the minimum effective dose or blood unit concentration for a longer period of time compared to the GLP-2 not linked to XTEN, and bioavailability; properties that permits less nt dosing or an enhanced pharmacologic effect, resulting in enhanced utility in the treatment of gastrointestinal conditions.

Native GLP-2 has been ed to have a terminal half-life in humans of approximately seven minutes (Jeppesen PB, et al., Teduglutide (ALX-0600), a dipeptidyl ase IV resistant glucagon-like peptide 2 analogue, improves intestinal function in short bowel syndrome patients. Gut. (2005) 54(9): 1224—123 1; Hartmann B, et al. (2000) Dipeptidyl peptidase IV inhibition enhances the intestinotrophic effect of glucagon—like peptide—2 in rats and mice. Endocrinology 141:401374020), while an analog teduglutide exhibited a terminal half—life of approximately 0.9—2.3 hr in humans (Marier JF, Population pharmacokinetics of teduglutide following repeated subcutaneous administrations in healthy participants and in patients with short bowel syndrome and Crohn's disease. J Clin Pharmacol. (2010) 50(1):36-49). It will be understood by the d artisan that the pharmacokinetic properties ofthe GLP2-XTEN embodiments are to be compared to able forms of GLP-2 not linked to the XTEN, i.e., recombinant, native sequence or a teduglutide-like analog.

As a result of the enhanced properties conferred by XTEN, the GLPZ-XTEN, when used at the dose and dose regimen determined to be appropriate for the composition by the methods bed herein, administration of a GLP2-XTEN fusion protein composition can achieve a circulating concentration resulting in a desired pharmacologic or clinical effect for an extended period of time compared to a comparable dose of the corresponding GLP-2 not linked to the XTEN. As used herein, a “comparable dose” means a dose with an equivalent moles/kg for the active GLP-2 pharmacophore (e.g., GLP-2) that is administered to a subject in a comparable fashion. It will be understood in the art that a "comparable dosage" of GLPZ-XTEN fusion protein would represent a greater weight of agent but would have essentially the same mole-equivalents of GLP-2 in the dose of the fusion protein administered.

In one embodiment, the invention provides TEN that enhance the pharmacokinetics of the fusion protein by linking one or more XTEN to the GLP-Z component of the ﬁJsion n, wherein the fusion protein has an increase in apparent lar weight factor of at least about two-fold, or at least about three-fold, or at least about four-fold, or at least about ﬁve-fold, or at least about six-fold, or at least about fold, or at least about eight-fold, or at least about ten-fold, or at least about twelve-fold, or at least about ﬁfteen-fold, and wherein the terminal half-life of the GLPZ-XTEN when administered to a t is increased at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 6-fold, or at least about 7-fold, or at least about 8-fold, or at least about 10- fold or more compared to the corresponding GLP-2 not linked to the XTEN. In the foregoing embodiment, wherein the fusion protein comprises at least two XTEN molecules incorporated into the GLPZ-XTEN, the XTEN can be identical or they can be of a different sequence composition (and net charge) or length. The XTEN can have at least about 80% sequence identity, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% ce identity compared to a sequence selected from Table 4. Not to be bound by a particular theory, the XTEN of the GLPZ-XTEN itions with the higher net charge are expected, as described above, to have less non—speciﬁc interactions with various negatively—charged es such as blood s, tissues, or various ors, which would further contribute to reduced active clearance. sely, the XTEN of the TEN compositions with a low (or no) net charge are expected to have a higher degree of interaction with surfaces that potentiate the ical activity of the associated GLP-2, given the known association of atory cells in the intestines during an inﬂammatory response. Thus, the invention provides GLP2-XTEN in which the degree of potency, bioavailability, and half-life of the fusion protein can be tailored by the selection and placement of the type and length of the XTEN in the GLPZ-XTEN itions. Accordingly, the invention contemplates compositions in which a GLP-Z from Table l and XTEN from Table 4 are combined and are produced, for example, in a conﬁguration selected from any one of formulae I-VI such that the construct has enhanced pharmacokinetic properties and reduced systemic clearance. The invention further takes advantage of the fact that n ligands with reduced binding to a clearance receptor, either as a result of a decreased on—rate or an increased off-rate, may be effected by the obstruction of either the N- or C-terminus and using that terminus as the linkage to another polypeptide of the composition, whether another molecule of a GLP-Z, an XTEN, or a spacer sequence results in the reduced binding. The choice of the particular conﬁguration of the TEN fusion protein can be tested by methods disclosed herein to conﬁrm those conﬁgurations that reduce the degree ofbinding to a nce receptor such that a reduced rate of active clearance is achieved.

In one embodiment, the invention provides GLPZ-XTEN with enhanced pharmacokinetic properties wherein the GLPZ-XTEN is a sequence that has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to a sequence selected from any one of Tables 13, 32 or 33. In other ments, the GLP2-XTEN with enhanced pharmacokinetic properties comprises a GLP-2 sequence that has at least about 80% sequence ty, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% sequence identity compared to a sequence from Table 1 linked to one or more XTEN that has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% sequence identity compared to a sequence from Table 4.

For the subject compositions, TEN with a longer terminal half-life is generally preferred, so as to improve patient convenience, to increase the interval between doses and to reduce the amount of drug required to achieve a sustained . In the embodiments hereinabove bed in this paragraph the administration of the fusion protein results in an ement in at least one, two, three or more of the parameters disclosed herein as being useful for assessing the t conditions; e.g., maintaining a blood concentration, ining bowel function, preventing onset of a m associated with a gastrointestinal condition such as colitis, short bowel syndrome or Crohn’s Disease, using a lower dose of fusion protein compared to the corresponding GLP-2 component not linked to the fusion protein and administered at a comparable dose or dose regimen to a subject. Alternatively, in the embodiments hereinabove described in this aph the administration of the fusion protein results in an improvement in at least one of the parameters disclosed herein as being useful for assessing the subject conditions using a comparable dose of fusion protein but stered using a dose regimen that has a 2- fold, or 3-fold, or 4-fold, or 5-fold, or 6-fold, or , or 8-fold, or 10-fold, or 20-fold greater interval between dose administrations compared to the corresponding GLP-2 component not linked to the ﬁtsion protein and administered to the subject. In the foregoing embodiments, the total dose in millimoles/kg administered to achieve the improvement in the parameter(s) is at least about three-fold lower, or at least about four-fold, or at least about ld, or at least about ld, or at least about eight-fold, or at least about 10-fold lower compared to the corresponding GLP-2 component not linked to the XTEN.

As described more fully in the Examples pertaining to pharmacokinetic characteristics of fusion proteins comprising XTEN, it was ed that increasing the length ofthe XTEN sequence confers a portionate increase in the terminal half-life of a fusion protein comprising the XTEN.

Accordingly, the invention provides GLP2-XTEN fusion proteins comprising XTEN wherein the XTEN is ed to provide a targeted half-life for the GLP2-XTEN composition administered to a subject. In some embodiments, the invention provides monomeric GLP2-XTEN fusion proteins comprising XTEN wherein the XTEN is selected to confer an se in the terminal half-life for the GLP2-XTEN administered to a subject, compared to the corresponding GLP-2 not linked to the XTEN and stered at a comparable dose, wherein the increase is at least about two-fold longer, or at least about three-fold, or at least about four-fold, or at least about ﬁve-fold, or at least about six-fold, or at least about seven-fold, or at least about eight-fold, or at least about nine-fold, or at least about ten-fold, or at least about 15-fold, or at least a 20-fold, or at least a 40-fold or greater an increase in terminal half-life compared to the GLP-2 not linked to the XTEN. In another embodiment, the administration of a therapeutically effective amount of GLP2-XTEN to a subject in need f results in a terminal half-life that is at least 12 h greater, or at least about 24 h greater, or at least about 48 h greater, or at least about 72 h greater, or at least about 96 h greater, or at least about 144 h r, or at least about 7 days greater, or at least about 14 days greater, or at least about 21 days greater compared to a able dose of the corresponding GLP-2 not linked to the XTEN. In r embodiment, administration of a therapeutically effective dose of a GLP2-XTEN fusion protein to a t in need thereof can result in a gain in time between consecutive doses necessary to maintain a therapeutically effective blood level of the fusion protein of at least 48 h, or at least 72 h, or at least about 96 h, or at least about 120 h, or at least about 7 days, or at least about 14 days, or at least about 21 days between utive doses compared to the corresponding GLP-2 not linked to the XTEN and administered at a comparable dose. It will be understood in the art that the time between consecutive doses to maintain a peutically effective blood level” will vary greatly depending on the logic state of the t, and it will be appreciated that a patient with Crohn’s Disease may require more frequent and longer dosing of a GLP-2 preparation compared to a patient receiving the same preparation for short bowel syndrome. The foregoing notwithstanding, it is believed that the GLP2-XTEN of the present invention permit less frequent dosing, as described above, compared to a GLP—2 not linked to the XTEN. In one embodiment, the GLP2—XTEN administered using a therapeutically—effective amount to a subject results in blood concentrations ofthe GLP2-XTEN fusion protein that remains above at least 500 ng/ml, or at least about 1000 ng/ml, or at least about 2000 ng/ml, or at least about 3000 ng/ml, or at least about 4000 ng/ml, or at least about 5000 ng/ml, or at least about 10000 ng/ml, or at least about 15000 ng/ml, or at least about 20000 ng/ml, or at least about 30000 ng/ml, or at least about 40000 ng/ml for at least about 24 hours, or at least about 48 hours, or at least about 72 hours, or at least about 96 hours, or at least about 120 hours, or at least about 144 hours.

In one embodiment, the present invention provides GLPZ-XTEN fusion proteins that exhibits an increase in AUC of at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about a 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about 500%, or at least about 1000%, or at least about a 2000% compared to the corresponding GLP-2 not linked to the XTEN and administered to a subject at a comparable dose. In another embodiment, the GLPZ-XTEN administered at an riate dose to a subject results in area under the curve concentrations ofthe GLP2-XTEN fusion protein of at least 100000 hr*ng/mL, or at least about 200000 mL, or at least about 400000 hr*ng/mL, or at least about 600000 mL, or at least about 800000 hr*ng/mL, or at least about 1000000 hr*ng/mL, or at least about 2000000 hr*ng/mL after a single dose. The pharmacokinetic parameters of a GLP2-XTEN can be determined by standard methods involving g, the taking of blood samples at times als, and the assaying of the WO 40093 protein using ELISA, HPLC, radioassay, or other methods known in the art or as described herein, followed by standard calculations of the data to derive the half-life and other PK parameters.

The enhanced PK parameters allow for reduced dosing of the GLP2-XTEN itions, compared to GLP-2 not linked to the XTEN, particularly for those subjects receiving doses for routine prophylaxis or c treatment of a gastrointestinal condition. In one embodiment, a r moles- equivalent amount of about two-fold less, or about three-fold less, or about four-fold less, or about ﬁve- fold less, or about six-fold less, or about eight-fold less, or about d less or greater of the fusion protein is administered in comparison to the corresponding GLP-2 not linked to the XTEN under a dose regimen needed to maintain a comparable area under the curve as the corresponding amount of the GLP- 2 not linked to the XTEN. In another embodiment, a smaller amount of moles of about two-fold less, or about three-fold less, or about four-fold less, or about ﬁve-fold less, or about six-fold less, or about eight- fold less, or about 10-fold less or greater of the fusion protein is administered in comparison to the corresponding GLP-2 not linked to the XTEN under a dose regimen needed to maintain a blood concentration above at least about 500 ng/ml, at least about 1000 ng/ml, or at least about 2000 ng/ml, or at least about 3000 ng/ml, or at least about 4000 ng/ml, or at least about 5000 ng/ml, or at least about 10000 ng/ml, or at least about 15000 ng/ml, or at least about 20000 ng/ml, or at least about 30000 ng/ml, or at least about 40000 ng/ml for at least about 24 hours, or at least about 48 h, or at least 72 h, or at least 96 h, or at least 120 h compared to the corresponding amount of the GLP—2 not linked to the XTEN. In another embodiment, the GLP2—XTEN fusion protein requires less frequent administration for treatment of a subject with intestinal condition, wherein the dose is administered about every four days, about every seven days, about every 10 days, about every 14 days, about every 21 days, or about monthly ofthe fusion protein administered to a subject, and the ﬁlsion protein achieves a comparable area under the curve as the ponding GLP-2 not linked to the XTEN. In yet other embodiments, an accumulatively smaller amount of moles of about 5%, or about 10%, or about 20%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% less of the fusion protein is administered to a subject in comparison to the corresponding amount of the GLP-2 not linked to the XTEN under a dose regimen needed to achieve the therapeutic outcome or clinical parameter, yet the fusion protein achieves at least a comparable area under the curve as the corresponding GLP-2 not linked to the XTEN. The accumulative r amount is measure for a period of at least about one week, or about 14 days, or about 21 days, or about one month. (b) Pharmacology and Pharmaceutical Properties of GLP2-XTEN The present invention provides GLP2-XTEN compositions comprising GLP-2 covalently linked to the XTEN that can have enhanced ties compared to GLP-2 not linked to XTEN, as well as methods to enhance the therapeutic and/or ic activity or effect of the respective two GLP-2 components of the compositions. In addition, GLP2-XTEN fusion ns provide signiﬁcant advantages over chemical conjugates, such as pegylated constructs of GLP-2, y the fact that recombinant GLP2-XTEN fusion proteins can be made in host cell expression systems, which can reduce time and cost at both the ch and pment and manufacturing stages of a product, as well as result in a more homogeneous, deﬁned product with less toxicity for both the product and metabolites of the GLPZ-XTEN compared to pegylated conjugates.

As eutic agents, the GLPZ-XTEN possesses a number of advantages over therapeutics not comprising XTEN, including one or more of the following non-limiting ed properties: increased solubility, increased thermal stability, reduced immunogenicity, increased nt molecular weight, reduced renal clearance, reduced proteolysis, reduced metabolism, enhanced therapeutic efﬁciency, a lower ive therapeutic dose, increased bioavailability, increased time between s capable of maintaining a subject without sed symptoms of colitis, enteritis, or Crohn’s Disease, the ability to administer the GLPZ-XTEN composition intravenously, aneously, or intramuscularly, a “tailored” rate of absorption when administered intravenously, aneously, or intramuscularly, enhanced lyophilization stability, enhanced serum/plasma stability, increased terminal half-life, increased solubility in blood stream, decreased binding by neutralizing antibodies, decreased active clearance, reduced side effects, reduced immunogenicity, retention of substrate binding afﬁnity, stability to degradation, stability to freeze-thaw, stability to proteases, stability to ubiquitination, ease of administration, compatibility with other pharmaceutical excipients or carriers, persistence in the subject, increased stability in storage (e.g., increased shelf-life), reduced toxicity in an organism or environment and the like. The GLPZ—XTEN fusion proteins of the embodiments disclosed herein exhibit one or more or any combination of the improved properties and/or the ments as detailed herein. The net effect of the ed properties is that the use of a GLPZ-XTEN ition can result in enhanced therapeutic and/or biologic effect compared to a GLP-2 not linked to the XTEN, result in economic beneﬁts associated with less nt dosing, or result in improved patient compliance when administered to a subject with a GLP-Z-related condition.

] In one embodiment, XTEN as a fusion partner ses the solubility of the GLP-Z d.

Accordingly, where enhancement of the pharmaceutical or physicochemical ties of the GLP-2 is ble, such as the degree of aqueous solubility or stability, the length and/or the motif family composition of the XTEN sequences incorporated into the fusion protein may each be selected to confer a different degree of solubility and/or stability on the respective ﬁlsion proteins such that the overall pharmaceutical properties of the GLPZ-XTEN composition are enhanced. The GLP2-XTEN fusion proteins can be constructed and assayed, using methods described herein, to conﬁrm the physicochemical properties and the XTEN adjusted, as needed, to result in the desired properties. In one embodiment, the GLPZ-XTEN has an aqueous solubility that is at least about 25% greater compared to a GLP-2 not linked to the fusion protein, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 75%, or at least about 100%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500%, or at least about 1000% greater than the corresponding GLP-Z not linked to the ﬁlsion protein.

The invention provides methods to produce and recover expressed GLPZ-XTEN from a host cell with ed solubility and ease of recovery compared to GLP-2 not linked to the XTEN. In one embodiment, the method includes the steps of transforming a host cell with a polynucleotide encoding a GLPZ-XTEN with one or more XTEN ents of cumulative sequence length greater than about 100, or greater than about 200, or greater than about 400, or greater than about 800 amino acid residues, expressing the GLPZ-XTEN fusion protein in the host cell, and recovering the expressed ﬁision protein in soluble form. In the foregoing embodiment, the XTEN of the GLPZ-XTEN fusion proteins can have at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% ce identity compared to one or more XTEN selected from Table 4, and the GLP-2 can have at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or 100% sequence identity compared to a GLP-2 selected from Table l and the TEN components can be in an N— to C-terminus ration ed from any one of formulae I-VI.

The invention provides methods to e the GLP2-XTEN compositions that can maintain the GLP-2 component at therapeutic levels when administered to a subject in need thereof for at least a two-fold, or at least a three-fold, or at least a four-fold, or at least a ﬁve-fold r period of time compared to able dosages of the corresponding GLP—2 not linked to the XTEN. It will be understood in the art that a "comparable dosage" of GLPZ—XTEN fusion protein would ent a greater weight of agent but would have the same approximate moles of GLP-2 in the dose of the ﬁtsion protein and/or would have the same approximate g concentration relative to the dose of GLP-2 not linked to the XTEN. The method to produce the compositions that can maintain the GLP-2 component at therapeutic levels includes the steps of selecting the XTEN appropriate for conjugation to a GLP-2 to provide the desired cokinetic properties in view of a given dose and dose regimen, creating an expression construct that encodes the GLPZ-XTEN using a conﬁguration described , transforming an appropriate host cell with an expression vector comprising the encoding gene, expressing and recovering the GLP2-XTEN, administration of the TEN to a subject followed by assays to verify the cokinetic properties, the activity of the GLPZ-XTEN fusion protein (e.g., the ability to bind receptor), and the safety ofthe administered composition. The subject can be selected from mouse, rat, monkey and human. By the methods, GLPZ-XTEN provided herein can result in increased efficacy of the administered composition by maintaining the circulating concentrations of the GLP-2 at therapeutic levels for an enhanced period oftime.

In another aspect, the GLPZ-XTEN compositions of the invention are capable of resulting in an intestinotrophic effect. As used herein, “intestinotrophic effect” means that a subject, e.g., mouse, rat, monkey or human, exhibits at least one of the ing after administration of a GLP-2 containing composition: intestinal growth, increased hyperplasia of the Villus epithelium, increased crypt cell proliferation, increased the height of the crypt and Villus axis, increased healing after intestinal anastomosis, increased small bowel weight, increased small bowel length, decreased small bowel epithelium apoptosis, or enhancement of intestinal function. The GLPZ-XTEN compositions may act in an endocrine fashion to link inal growth and lism with nt .. GLP-2 and related analogs may be treatments for short bowel syndrome, Crohn's disease, osteoporosis and as adjuvant therapy during cancer chemotherapy, amongst other gastrointestinal conditions described herein. In one embodiment, a GLPZ-XTEN is capable of resulting in at least one, or two, or three or more intestinotrophic effects when administered to a subject using an effective amount.

The characteristics of TEN itions of the invention, including functional characteristics or biologic and pharmacologic ty and ters that result, can be determined by any suitable screening assay known in the art for measuring the d characteristic. The invention provides s to assay the GLPZ-XTEN fusion proteins of differing composition or conﬁguration in order to provide GLPZ-XTEN with the desired degree of biologic and/or therapeutic activity, as well as safety proﬁle. Speciﬁc in vitro, in vivo and ex vivo biological assays are used to assess the activity of each conﬁgured GLPZ-XTEN and/or GLP-2 component to be incorporated into GLPZ-XTEN, including but not limited to the assays of the Examples, assays of Table 32, ination of inflammatory cytokine levels, GLP-2 blood concentrations, ELISA assays, or bowel function tests, as well as clinical endpoints such as bleeding, inﬂammation, colitis, diarrhea, fecal wet weight, weight loss, sodium loss, intestinal ulcers, intestinal obstruction, ﬁstulae, and abscesses, survival, among others known in the art.

The foregoing assays or endpoints can also be used in preclinical assays to assess GLP—2 sequence variants (assayed as single components or as GLPZ-XTEN ﬁlSlOI’l proteins) and can be compared to the native human GLP-2 to determine r they have the same degree of biologic ty as the native GLP-2, or some fraction thereof such that they are suitable for inclusion in GLP2—XTEN. In one ment, the invention provides GLPZ-XTEN fusion ns that exhibit at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100% or at least about 120% or at least about 150% or at least about 200% of the inotrophic effect compared to the corresponding GLP-2 not linked to XTEN and stered to a subject using a comparable dose.

Dose optimization is important for all drugs. A therapeutically effective dose or amount of the GLPZ-XTEN varies according to factors such as the disease state, age, sex, and weight of the individual, and the ability ofthe stered fusion protein to elicit a desired response in the individual. For example, a standardized single dose of GLP-2 for all patients presenting with e pulmonary conditions or abnormal clinical parameters (e.g., neutralizing antibodies) may not always be effective. A consideration of these factors is well within the purview of the ordinarily skilled clinician for the purpose of determining the therapeutically or pharmacologically effective amount of the GLPZ-XTEN and the appropriated dosing schedule, versus that amount that would result in insufﬁcient potency such that clinical improvement is not achieved.

The methods of the invention includes administration of consecutive doses of a therapeutically effective amount of the GLP2-XTEN for a period of time sufficient to achieve and/or maintain the desired parameter or al effect, and such consecutive doses of a therapeutically effective amount establishes the therapeutically effective dose n for the GLP2-XTEN, i.e., the schedule for utively administered doses of the fusion protein composition, wherein the doses are given in amounts to result in a sustained beneﬁcial effect on any clinical sign or symptom, aspect, measured ter or characteristic of a GLPrelated disease state or condition, including, but not limited to, those described herein. A prophylactically effective amount refers to an amount of GLP2-XTEN required for the period of time necessary to prevent a physiologic or clinical result or event; e.g., reduced mesenteric blood ﬂow, bleeding, inﬂammation, colitis, diarrhea, fecal wet , weight loss, sodium loss, intestinal ulcers, intestinal obstruction, fistulae, and abscesses, changed frequency in bowel movements, s, as well growth failure in children, or maintaining blood trations of GLP-2 above a threshold level, e.g., 100 ng/ml of GLP-2 equivalent (or approximately 2200 11ng of GLP 2G_XTEN_AE864) or 30 pmol/L. In the s of treatment, the dosage amount of the TEN that is administered to a subject ranges from about 0.2 to 500 mg/kg/dose (2.5 nmol/kg — 6250 nmol/kg), or from about 2 to 300 dose (25 nmol/kg — 3750 nmol/kg), or from about 6 to about 100 mg/kg/dose (75 nmol/kg/dose — 1250 nmol/kg/dose), or from about 10 to about 60 mg/kg/dose (125 nmol/kg/dose , 750 nmol/kg/dose) for a subject. A le dosage may also depend on other factors that may inﬂuence the response to the drug; e. g., subjects with surgically resected bowel generally requiring higher doses compared to ble bowel syndrome. In some embodiments, the method comprises administering a eutically—effective amount of a ceutical composition comprising a GLP2- XTEN ﬁssion protein composition sing GLP-2 linked to one or more XTEN sequences and at least one pharmaceutically acceptable carrier to a subject in need thereof that results in a greater improvement in at least one of the disclosed parameters or physiologic conditions, or results in a more favorable clinical outcome compared to the effect on the parameter, condition or clinical outcome mediated by administration of a pharmaceutical composition comprising a GLP-2 not linked to XTEN and stered at a comparable dose. In one embodiment of the foregoing, the improvement is ed by administration of the TEN pharmaceutical composition at a therapeutically effective dose. In another embodiment of the foregoing, the improvement is achieved by administration of multiple consecutive doses of the GLP2-XTEN pharmaceutical composition using a therapeutically effective dose regimen (as deﬁned herein) for the length of the dosing period.

] In many cases, the therapeutic levels for GLP-2 in subjects of different ages or degree of disease have been established and are ble in published literature or are stated on the drug label for approved products containing the GLP-2. In other cases, the therapeutic levels can be established for new compositions, including those GLP2-XTEN fusion proteins of the disclosure. The methods for establishing the therapeutic levels and dosing schedules for a given composition are known to those of skill in the art (see, e.g., Goodman & Gilman's The Pharmacological Basis of Therapeutics, llTh Edition, McGraw-Hill (2005)). For e, by using dose-escalation studies in subjects with the target disease or condition to determine efﬁcacy or a desirable pharmacologic effect, appearance of adverse events, and determination of circulating blood levels, the therapeutic blood levels for a given subject or population of subjects can be ined for a given drug or biologic. The dose escalation studies can evaluate the activity of a GLPZ-XTEN through metabolic studies in a subject or group of ts that monitor physiological or biochemical parameters, as known in the art or as described herein for one or more parameters ated with the GLP-Z-related condition, or clinical ters associated with a beneﬁcial outcome for the particular indication, together with observations and/or measured parameters to determine the no effect dose, adverse events, minimum effective dose and the like, together with measurement of pharmacokinetic parameters that establish the determined or derived circulating blood levels. The results can then be correlated with the dose administered and the blood concentrations of the therapeutic that are coincident with the foregoing determined ters or effect levels. By these methods, a range of doses and blood concentrations can be correlated to the minimum effective dose as well as the maximum dose and blood concentration at which a desired effect occurs and the period for which it can be maintained, thereby establishing the eutic blood levels and dosing schedule for the composition. Thus, by the foregoing methods, a Cm blood level is ished, below which the GLP2- XTEN fusion protein would not have the desired pharmacologic effect and a Cmax blood level, above which side effects may occur.

One of skill in the art can, by the means disclosed herein or by other s known in the art, conﬁrm that the stered GLPZ-XTEN remains at therapeutic blood levels yet retains adequate safety (thereby establishing the “therapeutic ”) to maintain biological activity for the desired interval or requires adjustment in dose or length or sequence of XTEN. r, the ination of the appropriate dose and dose frequency to keep the TEN within the therapeutic window establishes the therapeutically effective dose n; the schedule for administration of multiple consecutive doses using a therapeutically effective dose of the fusion n to a subject in need thereof resulting in consecutive Cmax peaks and/or le-n troughs that remain above therapeutically-effective concentrations and result in an improvement in at least one measured parameter relevant for the target condition. In one embodiment, the GLPZ-XTEN administered at an appropriate dose to a subject results in blood concentrations of the GLP2-XTEN fusion protein that remains above the minimum effective concentration to maintain a given activity or effect (as determined by the assays of the Examples or Table 32) for a period at least about two-fold longer compared to the corresponding GLP-2 not linked to XTEN and administered at a comparable dose; alternatively at least about fold longer; alternatively at least about old longer; alternatively at least about ﬁve-fold longer; alternatively at least about six-fold ; alternatively at least about seven-fold longer; alternatively at least about eight-fold longer; atively at least about nine-fold longer, alternatively at least about ten-fold longer, or at least about twenty-fold longer or greater compared to the corresponding GLP-2 not linked to XTEN and administered at a comparable dose. As used herein, an “appropriate dose” means a dose of a drug or biologic that, when administered to a t, would result in a ble therapeutic or pharmacologic effect and/or a blood concentration within the therapeutic window. For example, serum or plasma levels of GLP-2 or XTEN—containing fusion proteins comprising GLP-2 can be ed by nephelometry, ELISA, HPLC, radioimmunoassay or by immunoelectrophoresis (Jeppesen PB. Impaired meal stimulated on-like e 2 response in ileal resected short bowel patients with intestinal failure.

Gut. (1999) 45(4):559-963; assays of Examples 18-21). Phenotypic identiﬁcation of GLP-2 or GLP-2 variants can be accomplished by a number of s including isoelectric focusing (IEF) (Jeppsson et al., Proc. Natl. Acad. Sci. USA, 81 :5690-93, 1994), or by DNA analysis (Kidd et al., Nature, 0-34, 1983; Braun et al., Eur. J. Clin. Chem. Clin. Biochem, 34:761-64, 1996).

In one embodiment, administration of at least two doses, or at least three doses, or at least four or more doses of a GLPZ-XTEN using a therapeutically effective dose regimen results in a gain in time of at least about three-fold longer; alternatively at least about four-fold longer; atively at least about ﬁve-fold longer; alternatively at least about ld longer; atively at least about seven-fold longer; alternatively at least about eight-fold longer; alternatively at least about nine-fold longer or at least about ten-fold longer between at least two consecutive Cm; peaks and/or Cmin troughs for blood levels of the ﬁJsion protein compared to the corresponding ically active protein of the fusion protein not linked to the XTEN and administered at a able dose regimen to a subject. In another embodiment, the GLPZ—XTEN administered at a therapeutically effective dose regimen results in a comparable improvement in one, or two, or three or more measured parameters using less frequent dosing or a lower total dosage in moles of the fusion n of the pharmaceutical composition compared to the corresponding biologically active protein component(s) not linked to the XTEN and administered to a subject using a therapeutically effective dose regimen for the GLP-2. The measured parameters include any of the clinical, biochemical, or physiological parameters disclosed herein, or others known in the art for assessing subjects with GLP-Z-related condition. Non-limiting examples of ters or physiologic effects that can be assayed to assess the activity of the GLPZ-XTEN fusion proteins include assays of the Example, Table 32 or tests or assays to detect reduced mesenteric blood ﬂow, bleeding, inﬂammation, s, diarrhea, fecal wet weight, sodium loss, weight loss, intestinal ulcers, intestinal obstruction, ﬁstulae, and abscesses, changed frequency in bowel movements, uveitis, growth failure in children, or maintaining blood concentrations of GLP-2 above a threshold level, e.g., 100 ng/ml of GLP- 2 equivalent (or approximately 2200 ng/ml of GLP2G_XTEN_AE864), as well as parameters ed from experimental animal models of enteritis such as body weight gain, small intestine , reduction in TNFa content of the small intestine, reduced mucosal atrophy, reduced incidence of perforated ulcers, and height of villi.

In some embodiments, the biological activity of the GLP-2 component is manifested by the intact GLPZ-XTEN fusion n, while in other cases the biological activity of the GLP-Z component is primarily manifested upon cleavage and release of the GLP-2 from the fusion protein by action of a protease that acts on a cleavage sequence incorporated into the GLPZ-XTEN fusion protein using conﬁgurations and sequences described herein. In the foregoing, the GLPZ-XTEN is designed to reduce the binding y of the GLP-2 component for the GLP-2 receptor when linked to the XTEN but have restored or increased afﬁnity when released from XTEN h the cleavage of cleavage sequence(s) incorporated into the GLPZ-XTEN sequence. In one embodiment of the ing, the invention provides an isolated ﬁlsion protein comprising a GLP-2 linked to at least a ﬁrst XTEN by a cleavage sequence, wherein the fusion protein has less than 10% or the biological activity (e. g., or binding) prior to cleavage and wherein the GLP-2 released from the fusion protein by proteolytic cleavage at the cleavage sequence has biological activity that is at least about 40%, at least about 50%, at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95% as active compared to native GLP-2 not linked to the XTEN.

In one aspect, the invention provides GLPZ-XTEN compositions designed to reduce active clearance of the fusion protein, thereby increasing the al half-life of GLPZ-XTEN administered to a subject, while still retaining biological activity. t being bound by any particular theory, it is believed that the GLP2-XTEN of the present invention have comparatively higher and/or sustained activity achieved by reduced active clearance of the molecule by the addition of unstructured XTEN to the GLP-Z. Uptake, elimination, and inactivation of GLP-2 can occur in the atory system as well as in the ascular space.

VI). USES OF THE TEN COMPOSITIONS In r aspect, the invention provides GLP2-XTEN fusion proteins for use in methods of treatment, including treatment for achieving a beneﬁcial effect in a gastrointestinal condition mediated or ameliorated by GLP-Z. As used herein, “gastrointestinal condition” is intended to include, but is not limited to gastritis, digestion disorders, orption syndrome, short-gut syndrome, short bowel syndrome, —sac me, inﬂammatory bowel disease, celiac disease, tropical sprue, mmaglobulinemic sprue, Crohn's disease, ulcerative colitis, enteritis, chemotherapy—induced enteritis, irritable bowel me, small intestine damage, small intestinal damage due to cancer— chemotherapy, gastrointestinal injury, diarrheal diseases, intestinal insufﬁciency, acid-induced intestinal injury, ne deﬁciency, idiopathic hypospermia, obesity, catabolic illness, e neutropenia, obesity, steatorrhea, autoimmune diseases, gastrointestinal barrier disorders, sepsis, bacterial peritonitis, burn-induced intestinal damage, decreased gastrointestinal ty, intestinal failure, chemotherapy- associated bacteremia, bowel trauma, bowel ia, mesenteric ischemia, malnutrition, necrotizing enterocolitis, necrotizing pancreatitis, neonatal feeding intolerance, NSAID-induced intestinal damage, nutritional insufﬁciency, total parenteral nutrition damage to gastrointestinal tract, neonatal nutritional insufﬁciency, radiation-induced enteritis, radiation-induced injury to the intestines, mucositis, pouchitis, and gastrointestinal-induced ischemia, The present invention provides TEN fusion proteins for use in methods for treating a subject, such as a human, with a GLP-Z-related disease, disorder or gastrointestinal condition in order to achieve a beneﬁcial effect, addressing disadvantages and/or limitations of other methods of ent using GLP-Z preparations that have a relatively short terminal half-life, require repeated administrations, or have unfavorable pharmacoeconomics. The fact that GLP-2 native, recombinant or synthetic proteins have a short ife itates frequent dosing in order to achieve clinical beneﬁt, which results in difﬁculties in the management of such patients.

In one embodiment, the method of treatment comprises administering a therapeutically- effective amount of a GLPZ-XTEN composition to a subject with a gastrointestinal condition. In another ment of the method of treatment, the administration ofthe GLPZ-XTEN composition results in the improvement of one, two, three or more biochemical, physiological or clinical parameters associated with the gastrointestinal condition. In the foregoing method, the administered GLPZ-XTEN comprises a GLP-2 with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity to a GLP-2 of Table 1 linked to at least a first XTEN with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity to a XTEN selected from any one of Tables 4, and 8-12. In another embodiment of the foregoing method, the administered GLPZ-XTEN has a sequence with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity to a sequence from Tables 13, 32, or 33. In one embodiment, the method of treatment comprises administering a therapeutically—effective amount of a GLPZ—XTEN composition in one or more doses to a subject with a intestinal condition wherein the stration results in the improvement of one, two, three or more biochemical, physiological or clinical parameters or a therapeutic effect associated with the condition for a period at least two-fold longer, or at least four-fold , or at least ﬁve-fold longer, or at least six-fold longer compared to a GLP-2 not linked to the XTEN and administered using a comparable amount. In another embodiment, the method of ent comprises administering a therapeutically—effective amount of a GLPZ-XTEN composition to a subject ing from GLP-2 deficiency wherein the administration results in ting onset of a clinically relevant parameter or symptom or dropping below a clinically-relevant blood concentration for a duration at least two-fold, or at least fold, or at least four-fold longer compared to a GLP-Z not linked to the XTEN. In another embodiment, the method of treatment comprises administering a therapeutically-effective amount of a GLPZ-XTEN to a subject with a gastrointestinal condition, n the administration s in at least a %, or 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90% greater ement of at least one, two, or three parameters associated with the gastrointestinal condition compared to the GLP- 2 not linked to XTEN and administered using a comparable nmol/kg amount. In the foregoing embodiments of the method of treatment, the stration is subcutaneous, intramuscular, or intravenous. In the foregoing embodiments ofthe method of ent, the subject is selected from the group ting of mouse, rat, monkey, and human. In the foregoing embodiments of the method of treatment, the therapeutic effect or parameter includes, but is not limited to, blood concentrations of GLP-2, increased mesenteric blood ﬂow, sed inﬂammation, increased weight gain, decreased diarrhea, decreased fecal wet weight, intestinal wound healing, increase in plasma citrulline trations, decreased CRP levels, decreased requirement for d therapy, enhancing or stimulating mucosal integrity, decreased sodium loss, minimizing, ting, or preventing bacterial translocation in the intestines, enhancing, stimulating or accelerating recovery of the intestines after surgery; preventing relapses of inﬂammatory bowel disease; or achieving or maintaining energy homeostasis, among others.

In one embodiment, the method of treatment is used to treat a subject with small intestinal damage due to herapeutic agents such as, but not d to 5-FU, altretamine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, crisantaspase, cyclophosphamide, bine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, bine, ﬂuorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, liposomal doxorubicin, leucovorin, ine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, latin, paclitaxel, pemetrexed, tatin, procarbazine, raltitrexed, streptozocin, tegafur-uracil, temozolomide, pa, tioguanine, thioguanine, topotecan, treosulfan, vinblastine, vincristine, ine, and vinorelbine.

Prior to administering treatment by the described methods, a diagnosis of a gastrointestinal condition may be obtained. A gastrointestinal condition can be diagnosed by rd of care means known in the art. Ulcers, for example, may be diagnosed by barium X—ray of the esophagus, stomach, and intestine, by endoscopy, or by blood, breath, and stomach tissue biopsy (e. g., to detect the presence of Helicobacterpylori). Malabsorption syndromes can be diagnosed by blood tests or stool tests that monitor nutrient levels in the blood or levels of fat in stool that are diagnostic of a orption syndrome. Celiac sprue can be sed by antibody tests which may include testing for antiendomysial dy (IgA), antitransglutaminase (IgA), antigliadin (IgA and IgG), and total serum IgA. Endoscopy or small bowel biopsy can be used to detect abnormal intestinal lining where symptoms such as ﬂattening of the villi, which are stic of celiac sprue. Tropical sprue can be diagnosed by detecting malabsorption or infection using small bowel biopsy or response to chemotherapy. Inﬂammatory bowel disease can be detected by scopy or by an x-ray following a barium enema in combination with clinical symptoms, where inﬂammation, bleeding, or ulcers on the colon wall are diagnostic of inﬂammatory bowel es such as ulcerative colitis or Crohn's disease.

In some embodiments of the method of treatment, administration of the TEN to a subject results in an improvement in one or more of the biochemical, physiologic, or clinical parameters that is of greater magnitude than that of the corresponding GLP-2 component not linked to the XTEN, determined using the same assay or based on a measured clinical parameter. In one embodiment of the ing, the administration of a therapeutically effective amount of a GLPZ-XTEN composition to a subject in need thereof results in a greater reduction of parenteral nutrition (PN) dependence in patients with adult short bowel syndrome (SBS) of about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or more in the subject at 2-7 days after administration compared WO 40093 to a comparable amount of the corresponding GLP-2 not linked to the XTEN. In another embodiment, the administration of a GLP2-XTEN to a subject in need thereof using a therapeutically effective dose regimen results in an increase ofbody weight of 10%, or about 20%, or about 30%, or about 40%, or about 50% or more in the subject at 7, 10, 14, 21 or 30 days after initiation of administration compared to a comparable eutically ive dose regimen of the corresponding GLP-2 not linked to the XTEN.

In another embodiment, the administration of a therapeutically effective amount of a GLP2-XTEN composition to a subject in need thereof results in a greater reduction in fecal wet weight in patients with adult short bowel syndrome (SBS) of about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or more in the subject at 2-7 days after administration compared to a comparable amount of the corresponding GLP-2 not linked to the XTEN. In another embodiment, the administration of a therapeutically effective amount of a GLP2-XTEN composition to a subject in need thereofresults in a greater reduction in sodium loss in patients with adult short bowel syndrome (SBS) of about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or more in the t at 2-7 days after administration compared to a comparable amount of the corresponding GLP-2 not linked to the XTEN.

In some embodiments of the method of treatment, (i) a smaller amount of moles of about two- fold less, or about three-fold less, or about four-fold less, or about five-fold less, or about six-fold less, or about eight—fold less, or about 10—fold less of the GLP2—XTEN fusion protein is administered to a subject in need f in comparison to the ponding GLP—2 not linked to the XTEN under an otherwise same dose regimen, and the ﬁJsion protein achieves a comparable area under the curve and/or a comparable therapeutic effect as the corresponding GLP-2 not linked to the XTEN; (ii) the GLP2-XTEN fusion protein is administered less frequently (e. g., every three days, about every seven days, about every days, about every 14 days, about every 21 days, or about monthly) in ison to the corresponding GLP-2 not linked to the XTEN under an otherwise same dose amount, and the fusion protein achieves a able area under the curve and/or a comparable therapeutic effect as the corresponding GLP-2 not linked to the XTEN; or (iii) an accumulative smaller amount of moles of at least about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% less of the fusion protein is administered in comparison to the corresponding GLP-2 not linked to the XTEN under an ise same dose regimen and the GLP2-XTEN fusion protein achieves a comparable area under the curve and/or a comparable therapeutic effect as the ponding GLP-2 not linked to the XTEN. The accumulative smaller amount is measured for a period of at least about one week, or about 14 days, or about 21 days, or about one month. In the ing embodiments ofthe method of treatment, the therapeutic effect can be determined by any of the ed parameters described herein, including but not limited to blood concentrations of GLP-2, assays of Table 32, or assays to detect reduced mesenteric blood flow, bleeding, inﬂammation, colitis, diarrhea, fecal wet weight, weight loss, sodium loss, intestinal ulcers, intestinal obstruction, ﬁstulae, and abscesses, changed frequency in bowel movements, uveitis, growth e in children, or maintaining blood concentrations of GLP-2 above a threshold level, e.g., 100 ng/ml of GLP-2 equivalent (or approximately 2200 ng/ml of GLP2G_XTEN_AE864), among others known in the art for GLPrelated conditions.

] The invention provides GLP2-XTEN fusion proteins for use in a pharmaceutical regimen for treating a t with a intestinal condition. In one embodiment, the regimen comprises a pharmaceutical composition comprising a GLP2-XTEN fusion protein described herein. In another embodiment, the ceutical regimen ﬁirther comprises the step of determining the amount of pharmaceutical composition needed to e a therapeutic effect in the subject. In another embodiment, the pharmaceutical regimen for treating a subject with a gastrointestinal condition comprises administering the pharmaceutical composition in two or more successive doses to the subject at an effective amount, wherein the administration results in at least a 5%, or 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90% greater improvement of at least one, two, or three parameters associated with the gastrointestinal condition ed to the GLP-2 not linked to XTEN and administered using a able nmol/kg amount. In r embodiment of the pharmaceutical nt, the ive amount is at least about 5, or least about 10, or least about 25, or least about 100, or least about 200 nmoles/kg, or any amount intermediate to the ing. In another embodiment, the pharmaceutical regimen for treating a subject with a gastrointestinal condition ses stering a therapeutically effective amount of the pharmaceutical composition once about every 3, 6, 7, 10, 14, 21, 28 or more days. In another embodiment, the pharmaceutical regimen for treating a subject with a gastrointestinal condition comprises administering the GLP2—XTEN pharmaceutical composition wherein said administration is subcutaneous, intramuscular, or intravenous. In another embodiment, the pharmaceutical regimen for treating a subject with a gastrointestinal condition comprises administering a therapeutically effective amount of the pharmaceutical composition, wherein the therapeutically ive amount results in maintaining blood trations ofthe fusion protein within a therapeutic window for the fusion protein at least three-fold longer compared to the corresponding GLP-2 not linked to the XTEN administered at a comparable amount to the subject.

The invention r plates that the GLP2-XTEN used in accordance with the methods provided herein can be administered in conjunction with other treatment methods and compositions (e. g., anti-inﬂammatory agents such as steroids or NSAIDS) useful for treating GLPrelated conditions, or conditions for which GLP-2 is or could be adjunctive therapy.

In another aspect, the invention provides GLP2-XTEN fusion ns for use in a method of preparing a medicament for treatment of a GLPrelated condition In one embodiment, the method of preparing a medicament comprises linking a GLP-2 sequence with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity to a GLP-2 of Table 1 to at least a ﬁrst XTEN with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence ty to a XTEN selected from any one of Tables 4, and 8-12, wherein the GLP2-XTEN retains at least a portion of the biological activity of the native GLP- 2, and further combining the GLP2-XTEN with at least one pharmaceutically acceptable carrier. In WO 40093 another embodiment, the GLP2-XTEN has a sequence With at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity compared to a sequence selected from any one of Tables 13, 32 or 33.

In another , the invention provides a method of designing the GLP2-XTEN compositions to achieve desired pharmacokinetic, cologic or pharmaceutical properties. In general, the steps in the design and production of the ﬁlsion proteins and the inventive compositions, as illustrated in FIGS. 4- 6, include: (1) selecting a GLP-2 (e.g., native proteins, sequences of Table 1, s or derivatives with activity) to treat the particular condition; (2) selecting the XTEN that Will confer the desired PK and physicochemical teristics on the resulting GLP2-XTEN (e.g., the administration ofthe GLP2- XTEN composition to a subject results in the fusion protein being maintained Within the therapeutic Window for a r period compared to GLP-2 not linked to the XTEN); (3) establishing a desired N— to C-terminus conﬁguration ofthe GLP2-XTEN to e the desired efﬁcacy or PK parameters; (4) ishing the design of the expression vector encoding the conﬁgured GLP2-XTEN; (5) transforming a suitable host With the expression vector; and (6) expressing and recovering of the resultant fusion protein. For those TEN for Which an increase in half-life or an increased period of time spent above the minimum effective concentration is desired, the XTEN chosen for incorporation generally has at least about 288, or about 432, or about 576, or about 864, or about 875, or about 912, or about 923 amino acid residues Where a single XTEN is to be incorporated into the GLP2—XTEN. In another embodiment, the GLP2—XTEN comprises a ﬁrst XTEN of the foregoing lengths, and at least a second XTEN ofabout 36, or about 72, or about 144, or about 288, or about 576, or about 864, or about 875, or about 912, or about 923, or about 1000 or more amino acid residues.

In r aspect, the invention provides methods of making GLP2-XTEN compositions to improve ease of manufacture, result in increased stability, increased water solubility, and/or ease of formulation, as compared to the native GLP-2. In one embodiment, the invention includes a method of increasing the water solubility of a GLP-2 comprising the step of linking the GLP-2 to one or more XTEN such that a higher tration in soluble form of the ing GLP2-XTEN can be achieved, under physiologic conditions, compared to the GLP-2 in an un-fused state. In some ments, the method results in a GLP2-XTEN fusion protein wherein the water solubility is at least about 20%, or at least about 30% greater, or at least about 50% greater, or at least about 75% greater, or at least about 90% greater, or at least about 100% r, or at least about 150% greater, or at least about 200% greater, or at least about 400% greater, or at least about 600% greater, or at least about 800% greater, or at least about 1000% greater, or at least about 2000% greater under logic conditions, compared to the un- fused GLP-2. Factors that contribute to the property ofXTEN to confer increased water lity of GLP-2 when incorporated into a fusion protein include the high solubility of the XTEN ﬁlsion partner and the low degree of self-aggregation between molecules of XTEN in solution. In one embodiment of the foregoing, the TEN comprises a GLP-2 linked to an XTEN having at least about 36, or about 48, or about 96, or about 144, or about 288, or about 576, or about 864 amino acid residues in Which the solubility of the ﬁlsion protein under physiologic ions is at least three-fold greater than the ponding GLP-2 not linked to the XTEN, or alternatively, at least four-fold, or ld, or six-fold, or seven-fold, or fold, or nine-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 50-fold, or at least 60-fold or greater than GLP-2 not linked to the XTEN. In one embodiment of the foregoing, the GLP-2 has at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity to a GLP-2 of Table 1 linked to at least an XTEN with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity to a XTEN selected from any one of Tables 4, and 8-12.

In another embodiment, the invention includes a method of increasing the shelf-life of a GLP-2 sing the step of linking the GLP-2 with one or more XTEN selected such that the shelf-life of the resulting GLPZ-XTEN is extended compared to the GLP-2 in an un-fused state. As used herein, shelf- life refers to the period of time over which the functional activity of a GLP-2 or GLPZ-XTEN that is in solution or in some other storage formulation remains stable without undue loss of activity. As used herein, ”functional activity" refers to a pharmacologic effect or biological activity, such as the ability to bind a receptor or ligand, or ate, or trigger an up-regulated activity, or to display one or more known functional activities associated with a GLP-2, as known in the art. A GLP-2 that degrades or aggregates generally has reduced functional activity or reduced bioavailability compared to one that remains in solution. Factors that bute to the ability of the method to extend the shelf life of GLP—2s when incorporated into a fusion protein include increased water solubility, reduced ggregation in on, and increased heat ity of the XTEN fusion partner. In particular, the low tendency of XTEN to aggregate facilitates methods of formulating pharmaceutical preparations containing higher drug concentrations of GLP-2s, and the heat-stability of XTEN contributes to the property of GLPZ- XTEN fusion ns to remain soluble and functionally active for extended periods. In one embodiment, the method results in GLPZ-XTEN fusion proteins with nged” or ”extended" shelf- life that exhibit greater activity relative to a standard that has been subjected to the same storage and handling conditions. The standard may be the un-fused full-length GLP-Z. In one embodiment, the method includes the step of formulating the isolated GLPZ-XTEN with one or more pharmaceutically acceptable ents that enhance the ability of the XTEN to retain its unstructured conformation and for the GLPZ-XTEN to remain soluble in the formulation for a time that is greater than that of the corresponding un-fused GLP-2. In one embodiment, the method comprises linking a GLP-2 to one or more XTEN selected from Table 4 to create a GLPZ-XTEN fusion protein results in a solution that retains greater than about 100% of the functional activity, or r than about 105%, 110%, 120%, 130%, 150% or 200% of the functional activity of a standard when compared at a given time point and when ted to the same storage and handling ions as the standard, thereby increasing its shelf- life.

] Shelf-life may also be ed in terms of functional activity remaining after storage, normalized to functional activity when storage began. GLPZ-XTEN fusion proteins of the invention with prolonged or extended life as exhibited by ged or extended functional activity retain about 50% more onal activity, or about 60%, 70%, 80%, or 90% more ofthe functional activity ofthe equivalent GLP-2 not linked to the XTEN when subjected to the same conditions for the same period of time. For example, a GLPZ-XTEN ﬁtsion n of the invention comprising GLP-2 fused to one or more XTEN ces selected from Table 4 retains about 80% or more of its original ty in solution for periods of up to 2 weeks, or 4 weeks, or 6 weeks, or 12 weeks or longer under various elevated temperature conditions. In some embodiments, the GLPZ-XTEN retains at least about 50%, or about 60%, or at least about 70%, or at least about 80%, and most preferably at least about 90% or more of its original activity in solution when heated at 80°C for 10 min. In other embodiments, the GLPZ- XTEN s at least about 50%, preferably at least about 60%, or at least about 70%, or at least about 80%, or atively at least about 90% or more of its original activity in solution when heated or ined at 37°C for about 7 days. In another embodiment, GLPZ-XTEN fusion protein retains at least about 80% or more of its functional activity after exposure to a temperature of about 30°C to about 70°C over a period of time of about one hour to about 18 hours. In the foregoing embodiments hereinabove described in this paragraph, the retained activity of the GLPZ-XTEN is at least about two-fold, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold greater at a given time point than that of the corresponding GLP-2 not linked to the XTEN.

VII). THE NUCLEIC ACIDS SEQUENCES OF THE INVENTION The present invention provides isolated polynucleic acids encoding GLPZ-XTEN chimeric ﬁtsion proteins and sequences complementary to polynucleic acid molecules encoding GLPZ-XTEN chimeric fusion proteins, including gous variants thereof. In another aspect, the invention encompasses s to produce polynucleic acids encoding GLPZ-XTEN chimeric fusion proteins and sequences complementary to cleic acid molecules ng GLPZ—XTEN chimeric fusion protein, including homologous variants thereof. In general, and as illustrated in FIGS. 4—6, the methods of producing a polynucleotide sequence coding for a GLPZ—XTEN fusion protein and expressing the resulting gene product include assembling nucleotides encoding GLP-2 and XTEN, ligating the components in frame, incorporating the ng gene into an expression vector appropriate for a host cell, transforming the appropriate host cell with the expression vector, and ing the host cell under conditions causing or permitting the fusion protein to be expressed in the ormed host cell, thereby producing the biologically-active GLPZ-XTEN polypeptide, which is recovered as an isolated fusion protein by standard protein puriﬁcation methods known in the art. Standard recombinant techniques in molecular biology are used to make the polynucleotides and expression vectors of the present invention.

In accordance with the invention, nucleic acid sequences that encode GLP2-XTEN (or its complement) are used to generate recombinant DNA molecules that direct the expression of GLP2- XTEN fusion proteins in appropriate host cells. Several cloning gies are suitable for performing the present invention, many of which is used to generate a construct that comprises a gene coding for a ﬁlsion protein of the GLPZ-XTEN ition of the present invention, or its complement. In some ments, the cloning gy is used to create a gene that encodes a monomeric GLPZ-XTEN that comprises at least a ﬁrst GLP-2 and at least a ﬁrst XTEN polypeptide, or their complement. In one embodiment of the foregoing, the gene comprises a sequence ng a GLP-2 or sequence t. In other embodiments, the cloning strategy is used to create a gene that encodes a monomeric GLPZ-XTEN that ses nucleotides encoding at least a ﬁrst molecule of GLP-2 or its ment and a ﬁrst and at least a second XTEN or their complement that is used to transform a host cell for expression ofthe ﬁasion protein of the GLPZ-XTEN composition. In the foregoing embodiments hereinabove described in this paragraph, the genes can further comprise nucleotides encoding spacer ces that also encode cleavage sequence(s).

In designing a desired XTEN sequences, it was discovered that the non-repetitive nature of the XTEN ofthe inventive compositions is achieved despite use of a ”building block” molecular approach in the creation of the XTEN-encoding sequences. This was achieved by the use of a library of cleotides ng peptide sequence motifs, described above, that are then ligated and/or multimerized to create the genes encoding the XTEN sequences (see FIGS. 4, 5, 8, 9 and Examples).

Thus, while the XTEN(s) of the expressed fusion protein may consist of multiple units of as few as four different sequence motifs, because the motifs lves consist of non-repetitive amino acid sequences, the overall XTEN sequence is rendered non—repetitive. Accordingly, in one embodiment, the XTEN— encoding polynucleotides comprise multiple polynucleotides that encode non—repetitive sequences, or motifs, operably linked in frame and in which the resulting expressed XTEN amino acid ces are non-repetitive.

In one approach, a construct is ﬁrst prepared containing the DNA ce corresponding to GLP2-XTEN fusion protein. In those embodiments in which a mammalian native GLP-2 sequence is to be employed in the fusion protein, DNA encoding the GLP-2 of the compositions is obtained from a cDNA library prepared using standard methods from tissue or isolated cells believed to possess GLP-2 mRNA and to express it at a detectable level. Libraries are screened with probes containing, for example, about 20 to 100 bases designed to identify the GLP-Z gene of interest by hybridization using conventional molecular biology techniques. The best ates for probes are those that ent sequences that are highly homologous for GLP-2, and should be of sufﬁcient length and sufﬁciently unambiguous that false ves are minimized, but may be degenerate at one or more positions. If necessary, the coding sequence can be obtained using conventional primer extension procedures as described in Sambrook, et al., supra, to detect precursors and processing intermediates ofmRNA that may not have been reverse-transcribed into cDNA. One can then use polymerase chain reaction (PCR) methodology to amplify the target DNA or RNA coding sequence to obtain sufﬁcient material for the preparation of the GLPZ-XTEN constructs ning the GLP-2 gene. Assays can then be conducted to conﬁrm that the hybridizing full-length genes are the d GLP-2 gene(s). By these conventional s, DNA can be conveniently obtained from a cDNA library prepared from such sources. In those embodiments in which a GLP-2 analog (with one or more amino acid substitutions, such as ces of Table 1) for the preparation of the GLPZ-XTEN constructs, the GLP-2 encoding gene(s) is created by standard synthetic procedures known in the art (e. g., automated nucleic acid synthesis using, for e one of the methods described in Engels et a1. (Agnew. Chem. Int. Ed. Engl., 28:716-734 1989)), using DNA sequences obtained from publicly available databases, s, or literature references. Such procedures are well lmown in the art and well described in the scientiﬁc and patent literature. For example, sequences can be obtained from Chemical Abstracts Services (CAS) Registry Numbers (published by the American Chemical Society) and/or GenBank Accession Numbers (e. g., Locus ID, NP_XXXXX, and XP_XXXXX) Model Protein identiﬁers available through the National Center for Biotechnology Information (NCBI) webpage, available on the world wide web at ncbi.nlm.nih.gov that correspond to entries in the CAS Registry or GenBank database that contain an amino acid ce of the protein of interest or of a fragment or variant of the protein. For such sequence identiﬁers provided herein, the summary pages associated with each ofthese CAS and GenBank and GenSeq ion s as well as the cited l publications (e.g., PubMed ID number (PMID)) are each incorporated by reference in their entireties, particularly with respect to the amino acid sequences described therein. In one embodiment, the GLP-2 encoding gene encodes a protein from any one of Table 1, or a fragment or variant thereof.

A gene or polynucleotide encoding the GLP—2 portion of the t TEN protein, in the case of an expressed fusion protein that comprises a single GLP—2 is then be cloned into a construct, which is a plasmid or other vector under the control of appropriate transcription and translation sequences for high level protein expression in a biological system. In a later step, a second gene or polynucleotide coding for the XTEN is cally fused to the nucleotides encoding the N- and/or C-terminus ofthe GLP-2 gene by cloning it into the construct adjacent and in frame with the gene(s) coding for the GLP-Z.

This second step occurs through a ligation or multimerization step. In the foregoing embodiments above described in this paragraph, it is to be understood that the gene constructs that are created can alternatively be the complement of the tive genes that encode the respective fusion proteins.

The gene encoding for the XTEN can be made in one or more steps, either fully synthetically or by synthesis combined with enzymatic ses, such as restriction enzyme-mediated cloning, PCR and p extension, including methods more fully described in the Examples. The s disclosed herein can be used, for example, to ligate short sequences of polynucleotides encoding XTEN into longer XTEN genes of a desired length and sequence. In one embodiment, the method ligates two or more optimized oligonucleotides encoding XTEN motif or t sequences of about 9 to 14 amino acids, or about 12 to 20 amino acids, or about 18 to 36 amino acids, or about 48 to about 144 amino acids, or about 144 to about 288 or longer, or any combination of the ing ranges of motif or segment lengths.

] Alternatively, the disclosed method is used to multimerize XTEN-encoding sequences into longer sequences of a desired length; e.g., a gene encoding 36 amino acids of XTEN can be dimerized into a gene encoding 72 amino acids, then 144, then 288, etc. Even with multimerization, XTEN ptides can be constructed such that the XTEN-encoding gene has low or virtually no repetitiveness through design of the codons selected for the motifs of the shortest unit being used, which can reduce recombination and se stability of the encoding gene in the transformed host.

Genes encoding XTEN with petitive sequences are assembled from oligonucleotides using standard techniques of gene synthesis. The gene design can be performed using algorithms that optimize codon usage and amino acid composition. In one method of the invention, a library of relatively short XTEN-encoding cleotide constructs is created and then assembled, as described above. The resulting genes are then assembled with genes encoding GLP-2 or regions of GLP-2, as illustrated in FIGS. 5 and 8, and the ing genes used to transform a host cell and produce and recover the GLP2- XTEN for evaluation of its properties, as described herein.

In some embodiments, the GLP2-XTEN sequence is designed for optimized sion by inclusion of an N—terminal sequence (NTS) XTEN, rather than using a leader sequence known in the art.

In one embodiment, the NTS is created by inclusion of encoding nucleotides in the XTEN gene determined to result in zed expression when joined to the gene ng the fusion protein. In one embodiment, the N—terminal XTEN sequence of the expressed GLP2-XTEN is optimized for expression in a otic cell, such as but not limited to CH0, HEK, yeast, and other cell types know in the art.

Polynucleotide libraries In another aspect, the ion provides libraries of polynucleotides that encode XTEN ces that are used to assemble genes that encode XTEN of a desired length and sequence.

In certain embodiments, the XTEN-encoding library constructs comprise polynucleotides that encode polypeptide segments of a ﬁxed length. As an initial step, a library of oligonucleotides that encode motifs of 9-14 amino acid residues can be assembled. In a preferred embodiment, libraries of oligonucleotides that encode motifs of 12 amino acids are assembled.

The XTEN-encoding sequence ts can be dimerized or multimerized into longer encoding sequences. Dimerization or multimerization can be med by ligation, overlap extension, PCR assembly or similar cloning techniques known in the art. This process of can be repeated multiple times until the ing XTEN-encoding sequences have reached the organization of sequence and desired length, providing the XTEN-encoding genes. As will be appreciated, a library of polynucleotides that encodes, e.g., 12 amino acid motifs can be dimerized and/or ligated into a y of polynucleotides that encode 36 amino acids. Libraries encoding motifs of different lengths; e. g., 9-14 amino acid motifs leading to libraries encoding 27 to 42 amino acids are plated by the invention. In turn, the library of polynucleotides that encode 27 to 42 amino acids, and preferably 36 amino acids (as described in the Examples) can be serially dimerized into a library containing successively longer lengths of polynucleotides that encode XTEN sequences of a desired length for incorporation into the gene encoding the GLP2-XTEN fusion protein, as disclosed herein.

WO 40093 A more efﬁcient way to optimize the DNA sequence encoding XTEN is based on combinatorial libraries. The gene encoding XTEN can be designed and synthesized in segment such that le codon versions are obtained for each segment. These segments can be randomly assembled into a library of genes such that each library member encodes the same amino acid sequences but library s comprise a large number of codon ns. Such libraries can be screened for genes that result in high-level expression and/or a low abundance of truncation products. The process of atorial gene assembly is illustrated in . The genes in are assembled from 6 base fragments and each fragment is available in 4 different codon versions. This allows for a tical diversity of 4096.

In some embodiments, libraries are assembled of polynucleotides that encode amino acids that are limited to speciﬁc ce XTEN es; e. g., AD, AE, AF, AG, AM, or AQ ces of Table 3. In other ments, ies comprise sequences that encode two or more of the motif family sequences from Table 3. The names and sequences of representative, miting polynucleotide sequences of ies that encode 36mers are presented in Tables 8-11, and the methods used to create them are described more fully in the respective Examples. In other embodiments, libraries that encode XTEN are constructed from segments of polynucleotide codons linked in a randomized sequence that encode amino acids wherein at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% of the codons are selected from the group consisting of condons for glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) amino acids. The libraries can be used, in turn, for serial dimerization or on to achieve polynucleotide sequence libraries that encode XTEN sequences, for example, of48, 72, 144, 288, 576, 864, 875, 912, 923, 1318 amino acids, or up to a total length of about 3000 amino acids, as well as intermediate lengths, in which the encoded XTEN can have one or more ofthe properties disclosed herein, when expressed as a component of a GLP2-XTEN fusion protein. In some cases, the polynucleotide library sequences may also include additional bases used as ”sequencing islands,” described more fully below. is a schematic ﬂowchart of representative, non-limiting steps in the assembly of an XTEN polynucleotide construct and a GLP2-XTEN polynucleotide construct in the embodiments of the invention. Individual oligonucleotides 501 are annealed into sequence motifs 502 such as a 12 amino acid motif (“12-mer”), which is ligated to additional sequence motifs from a library to create a pool that encompasses the desired length of the XTEN 504, as well as d to a smaller concentration of an oligo containing BbsI, and KpnI restriction sites 503. The resulting pool of ligation products is gel-purified and the band with the desired length ofXTEN is cut, resulting in an isolated XTEN gene with a stopper sequence 505. The XTEN gene is cloned into a stuffer vector. In this case, the vector encodes an optional CBD sequence 506 and a GFP gene 508. Digestion is than performed with BbsI/HindIII to remove 507 and 508 and place the stop codon. The resulting product is then cloned into a BsaI/HindIII digested vector containing a gene encoding the GLP-2, resulting in the gene 500 encoding a GLP2- XTEN fusion protein. A non-exhaustive list of the polynucleotides encoding XTEN and sor sequences is provided in Tables 7-12.

Table 7: DNA seguences of XTEN and sor seguences XTEN DNA Nucleotide Sequence Name AE48 GAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACCCCGGGTAGCGGTACTG CTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTCTCCG GGCACCAGCTCTACCGGTTCT AM48 ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTGCATCCCCGGGCACCAGCT CTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTAGCTCTACC CCGTCTGGTGCTACTGGCTCT AE144 GGTAGCGAACCGGCAACTTCCGGCTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCCTG AGTCTGGCCCAGGTAGCGAACCTGCTACCTCTGGCTCTGAAACCCCAGGTAGCCCGGCAG GCTCTCCGACTTCCACCGAGGAAGGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCAGG TAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAGGTAGCGAACCTGCTACCTCCGGCTCT GAAACTCCAGGTAGCGAACCGGCTACTTCCGGTTCTGAAACTCCAGGTACCTCTACCGAAC AAGGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTA GCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAG CGCACCA AF144 TCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTACTTCTCCTAGCGGTGAATCTT CTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCAGGTTCTACCAGCTCT ACCGCTGAATCTCCTGGCCCAGGTTCTACCAGCGAATCCCCGTCTGGCACCGCACCAGGTT CTACTAGCTCTACCGCAGAATCTCCGGGTCCAGGTACTTCCCCTAGCGGTGAATCTTCTAC TGCTCCAGGTACCTCTACTCCGGAAAGCGGCTCCGCATCTCCAGGTTCTACTAGCTCTACT GCTGAATCTCCTGGTCCAGGTACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAGGTACCT CTCCTAGCGGCGAATCTTCTACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGC ACCA AE288 GGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCT CTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTG CAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGG TACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCC ACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCA ACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTA GCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTAC TGAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGC TACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACT TCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAA CCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAGGCTCTCC GACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCT ACTGAACCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCC CAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGA GGGCAGCGCACCA AE576 GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTG AGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAG GCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGG TACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAA CCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTA CCTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTAC TTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAG CGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTC TCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAC CTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCC GGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCA ACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTT CTACCGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTGAAGGTAGCG CACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAA CCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTC TGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAAC XTEN DNA tide Sequence Name CCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCT GAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTA CCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTC CAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGA GGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACC GAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCA GGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCG GAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAA CTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGG TACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCC ACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTAGCCCGGCAGGC ACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGT ACCTCTACCGAACCGTCTGAGGGCAGCGCACCA AF576 GGTTCTACTAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCCACTAGCTCTACCGCAGAAT CTCCGGGCCCAGGTTCTACTAGCGAATCCCCTTCTGGTACCGCTCCAGGTTCTACTAGCTCT ACCGCTGAATCTCCGGGTCCAGGTTCTACCAGCTCTACTGCAGAATCTCCTGGCCCAGGTA CTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTTCTACCAGCGAATCTCCTTCTGGCACC GGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTTCTACTAGCGAATCTC CTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTACCTCT CCTAGCGGCGAATCTTCTACCGCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCAC CAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTACCTCTCCTAGCGGCGAATC TTCTACCGCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGCG AATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAGG TACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCAGGTTCTACTAGCGAATCTCCTTCTGGTA CCGCTCCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCAGGTTCCACTAGCTCTACC GCTGAATCTCCGGGTCCAGGTTCTACTAGCTCTACTGCAGAATCTCCTGGCCCAGGTACCT CTACTCCGGAAAGCGGCTCTGCATCTCCAGGTACTTCTACCCCTGAAAGCGGTTCTGCATC TCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTACCCCGGAAAGC GGCTCTGCTTCTCCAGGTACTTCTACCCCGGAAAGCGGCTCCGCATCTCCAGGTTCTACTA GCGAATCTCCTTCTGGTACCGCTCCAGGTTCTACCAGCGAATCCCCGTCTGGTACTGCTCC AGGTTCTACCAGCGAATCTCCTTCTGGTACTGCACCAGGTTCTACTAGCTCTACTGCAGAA TCTCCTGGCCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTACTTCTACCC CTGAAAGCGGTTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGG TTCTACCAGCGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCC GCTTCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAAT CTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCAGGTAC TTCTCCGAGCGGTGAATCTTCTACCGCACCAGGTTCTACTAGCTCTACCGCTGAATCTCCG GGCCCAGGTACTTCTCCGAGCGGTGAATCTTCTACTGCTCCAGGTTCCACTAGCTCTACTG CTGAATCTCCTGGCCCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTTCTACT AGCGAATCTCCGTCTGGCACCGCACCAGGTTCTACTAGCTCTACTGCAGAATCTCCTGGCC CAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTACTTCTACCCCTGAAAGCGG TTCTGCATCTCCA AE624 ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACCCCGGGTAGCGGTACTG CTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTCTCCG GGCACCAGCTCTACCGGTTCTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAG GTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGG TAGCGCTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAA CCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTA CTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGA AACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTC TCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAC CTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAG AGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCG TCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTT CTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCG GTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTC TGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTACTTCT ACTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGC CCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCA XTEN DNA Nucleotide Sequence Name ACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAA CCGGCAACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCC CCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGA AGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACT GAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCA GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCT CCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAAA GCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGG TGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCT GAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAAC CGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTA GCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTAC TGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGC AACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCA AM875 GGTACTTCTACTGAACCGTCTGAAGGCAGCGCACCAGGTAGCGAACCGGCTACTTCCGGTT CTGAAACCCCAGGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTC TACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGT TCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGCGAATCCCCGTCTGGTA CTGCTCCAGGTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGGTACCTCTACTCCGGAA AGCGGTTCTGCATCTCCAGGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCAGGTACCT CTGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGA GGAAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACC CCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTA CCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGG AAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACTTCTACCGAACCTTCCGA GGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGA AAGCGCTACTCCTGAATCCGGTCCAGGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCA TCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCT GAATCCGGTCCAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCGAACCTG CTACTTCTGGTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAAGG TACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTT CCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGGTACCTCTACCGAACCG TCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGC GAACCGGCAACCTCCGGTTCTGAAACTCCAGGTAGCCCTGCTGGCTCTCCGACTTCTACTG AGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTC CGAAGGTAGCGCTCCAGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTACTTCTGA AAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAA GGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCTGAAT CTCCTGGCCCAGGTTCTACTAGCGAATCTCCGTCTGGCACCGCACCAGGTACTTCCCCTAG CGGTGAATCTTCTACTGCACCAGGTACCCCTGGCAGCGGTACCGCTTCTTCCTCTCCAGGT AGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTTCTAGCCCGTCTGCATCTACCGGTAC CGGCCCAGGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCAGGTACTTCTGAAAGCGC TACTCCGGAATCCGGCCCAGGTAGCGAACCGGCTACTTCCGGCTCTGAAACCCCAGGTTCC ACCAGCTCTACTGCAGAATCTCCGGGCCCAGGTTCTACTAGCTCTACTGCAGAATCTCCGG GTCCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTAGCGAACCGGCAACCTC TGGCTCTGAAACTCCAGGTAGCGAACCTGCAACCTCCGGCTCTGAAACCCCAGGTACTTCT CCTTCTGAGGGCAGCGCACCAGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTC CAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTC TGGCACTGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACT GAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCA GGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTG GTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTAGCGAACCTGC TACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCAACTCCGGAGTCTGGTCCAGGT AGCCCTGCAGGTTCTCCTACCTCCACTGAGGAAGGTAGCTCTACTCCGTCTGGTGCAACCG GCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCAC CAGCTCTACTGGTTCTCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACC TCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCG CACCA XTEN DNA Nucleotide Sequence Name AE864 GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTG AGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAG GCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGG TACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAA TCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTA CCTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTAC TTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAG CGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTC TCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAC CTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCC GGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCA ACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTT CTACCGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTGAAGGTAGCG CACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAA CCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTC TGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAAC CCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCT AGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTA CCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTC CCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGA CGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACC GAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCA GGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCG GAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAA GCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGG TACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCC ACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTAGCCCGGCAGGC TCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGT ACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAG TCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCG CAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTA CCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAG CGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGC AACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTAC TTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACC GAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTT GCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTT CTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGG CCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCC GGTTCTGAAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTA CTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCC AGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCT GGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA AF864 GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTACCTCTCCTAGCGGCGAATCTT CTACCGCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGCGA ATCCCCGTCTGGTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGGTA CTCCGGAAAGCGGTTCTGCATCTCCAGGTTCTACCAGCGAATCTCCTTCTGGCAC CGCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCGGC GAATCTTCTACCGCACCAGGTTCTACTAGCGAATCTCCGTCTGGCACTGCTCCAGGTACTT CTCCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCCCCTAGCGGCGAATCTTCTACCGCT CCAGGTTCTACTAGCTCTACTGCAGAATCTCCGGGCCCAGGTACCTCTCCTAGCGGTGAAT CTTCTACCGCTCCAGGTACTTCTCCGAGCGGTGAATCTTCTACCGCTCCAGGTTCTACTAGC TCTACTGCAGAATCTCCTGGCCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAG GTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGC ACTGCACCAGGTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTG AAAGCGGTTCCGCTTCTCCAGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTAC CTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACT GCACCAGGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCAGGTTCTACTAGCTCTACCG CTGAATCTCCGGGCCCAGGTACTTCTCCGAGCGGTGAATCTTCTACTGCTCCAGGTACCTC XTEN DNA Nucleotide ce Name TACTCCTGAAAGCGGTTCTGCATCTCCAGGTTCCACTAGCTCTACCGCAGAATCTCCGGGC CCAGGTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCAGGTTCTACTAGCTCTACTGCTGA ATCTCCGGGTCCAGGTTCTACCAGCTCTACTGCTGAATCTCCTGGTCCAGGTACCTCCCCG AGCGGTGAATCTTCTACTGCACCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAG GTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTCC XXXXXXXXXXXXTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAXXXXXXXXTAGCGAAT CTCCTTCTGGTACCGCTCCAGGTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGGTTCT ACCAGCGAATCTCCTTCTGGTACTGCACCAGGTTCTACTAGCGAATCTCCTTCTGGTACCG CTCCAGGTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGGTTCTACCAGCGAATCTCC TTCTGGTACTGCACCAGGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTACTTCTC CTAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCA GGTTCTACCAGCTCTACTGCTGAATCTCCGGGTCCAGGTACTTCCCCGAGCGGTGAATCTT CTACTGCACCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTTCTACCAGCGA ATCTCCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGT ACTTCTCCTAGCGGCGAATCTTCTACCGCACCAGGTTCTACTAGCGAATCCCCGTCTGGTA CCGCACCAGGTACTTCTACCCCGGAAAGCGGCTCTGCTTCTCCAGGTACTTCTACCCCGGA CTCCGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAGGTACT TCTACCCCTGAAAGCGGCTCCGCTTCTCCAGGTTCCACTAGCTCTACCGCTGAATCTCCGG GTCCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCC GTCTGGTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCACCAGGTTCTACC AGCTCTACTGCTGAATCTCCGGGTCCAGGTACTTCCCCGAGCGGTGAATCTTCTACTGCAC CTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTACCTCCCCTAGCGGCGAATC TGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTACCTCCCCTA GCGGTGAATCTTCTACCGCACCAGGTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAGG TTCTACCAGCTCTACTGCTGAATCTCCTGGTCCAGGTACCTCCCCGAGCGGTGAATCTTCTA CTGCACCAGGTTCTAGCCCTTCTGCTTCCACCGGTACCGGCCCAGGTAGCTCTACTCCGTCT GGTGCAACTGGCTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCA XXXX was inserted in two areas Where no sequence information is available.

AG864 GGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTTCTAGCCCGTCTGCTTCTACTGG TACTGGTCCAGGTTCTAGCCCTTCTGCTTCCACTGGTACTGGTCCAGGTACCCCGGGTAGC GGTACCGCTTCTTCTTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAGGTTC TAACCCTTCTGCATCCACCGGTACCGGCCCAGGTGCTTCTCCGGGCACCAGCTCTACTGGT TCTCCAGGTACCCCGGGCAGCGGTACCGCATCTTCTTCTCCAGGTAGCTCTACTCCTTCTGG TGCAACTGGTTCTCCAGGTACTCCTGGCAGCGGTACCGCTTCTTCTTCTCCAGGTGCTTCTC CTGGTACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCGGGCACTAGCTCTACTGGTTCTCCA GGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAAC TCCAGGTGCTTCTCCGGGCACCAGCTCTACCGGTTCTCCAGGTACCCCGGGTAGC GGTACCGCTTCTTCTTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAGGTTC TAACCCTTCTGCATCCACCGGTACCGGCCCAGGTTCTAGCCCTTCTGCTTCCACCGGTACTG GCCCAGGTAGCTCTACCCCTTCTGGTGCTACCGGCTCCCCAGGTAGCTCTACTCCTTCTGGT GGCTCTCCAGGTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTGCATCCC CTGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCTCTACTGGTTCTCCA GGTACTCCTGGCAGCGGTACCGCTTCTTCTTCTCCAGGTGCTTCTCCTGGTACTAGCTCTAC TGGTTCTCCAGGTGCTTCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCCCCGGGCA CTAGCTCTACCGGTTCTCCAGGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTACT CCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTGCATCTCCGGGCACTAGCTCTACTGGTTC TCCAGGTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCT CTACTGGTTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTACT CCTTCTGGTGCTACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCGGTTCTCCAG GTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTAC CGGTTCCCCAGGTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCAGGTAGCTCTACTCCG TCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTG CTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTGCATCCCCGGGTACCAGCTCTACCGG TTCTCCAGGTACTCCTGGCAGCGGTACTGCATCTTCCTCTCCAGGTGCTTCTCCGGGCACCA GCTCTACTGGTTCTCCAGGTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTGCATCC CCTGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCTCTACTGGTTCTCC AGGTACCCCTGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACTCCGTCTGGTGCTA XTEN DNA Nucleotide Sequence Name CCGGTTCTCCAGGTACCCCGGGTAGCGGTACCGCATCTTCTTCTCCAGGTAGCTCTACCCC TGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTA CCCCTTCTGGTGCTACTGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGC TCCCCAGGTTCTAGCCCTTCTGCATCCACCGGTACCGGTCCAGGTTCTAGCCCGTCTGCATC TACTGGTACTGGTCCAGGTGCATCCCCGGGCACTAGCTCTACCGGTTCTCCAGGTACTCCT GGTAGCGGTACTGCTTCTTCTTCTCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGTTCTCC AGGTTCTAGCCCTTCTGCATCCACCGGTACCGGCCCAGGTTCTAGCCCGTCTGCTTCTACCG GTACTGGTCCAGGTGCTTCTCCGGGTACTAGCTCTACTGGTTCTCCAGGTGCATCTCCTGGT ACTAGCTCTACTGGTTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCTCCAGGTTC TAGCCCTTCTGCATCTACCGGTACTGGTCCAGGTGCATCCCCTGGTACCAGCTCTACCGGTT CTCCAGGTTCTAGCCCTTCTGCTTCTACCGGTACCGGTCCAGGTACCCCTGGCAGCGGTAC CGCATCTTCCTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTA CTCCTTCTGGTGCTACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCGGTTCTCCA 1434923 ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTGCATCCCCGGGCACCAGCT CTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTAGCTCTACC CCGTCTGGTGCTACTGGCTCTCCAGGTACTTCTACTGAACCGTCTGAAGGCAGCGCACCAG GTAGCGAACCGGCTACTTCCGGTTCTGAAACCCCAGGTAGCCCAGCAGGTTCTCCAACTTC TACTGAAGAAGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCG GAAAGCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTT CTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGTTCCGC TTCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCTCCAGGTAGCGAACCGGCAACC TCCGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCC CGGCAGGTTCTCCGACTTCCACTGAGGAAGGTACCTCTACTGAACCTTCTGAGGGCAGCGC TCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCC GAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCA GCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCAC CAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCC TGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACCTCTACT GAACCTTCCGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCA GGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTACTTCTACTGAACCTTCCGAAG GTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCCGGCTGG CTCTCCGACCTCCACCGAGGAAGGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGT ACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGG CTCTCCAGGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCG TCTGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACTCCAGGTAGC CCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTACTG AGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTGCAAGCGCAAGCGGCG CGCCAAGCACGGGAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGG CTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGA AGGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCTACTAGCGAATCTCCGTCT GGCACCGCACCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCACCAGGTACCCCTGGCA GCGGTACCGCTTCTTCCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGT TCTAGCCCGTCTGCATCTACCGGTACCGGCCCAGGTAGCGAACCGGCAACCTCCGGCTCTG AAACTCCAGGTACTTCTGAAAGCGCTACTCCGGAATCCGGCCCAGGTAGCGAACCGGCTA CTTCCGGCTCTGAAACCCCAGGTTCCACCAGCTCTACTGCAGAATCTCCGGGCCCAGGTTC CTCTACTGCAGAATCTCCGGGTCCAGGTACTTCTCCTAGCGGCGAATCTTCTACC GCTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCAGGTAGCGAACCTGCAACC TCCGGCTCTGAAACCCCAGGTACTTCTACTGAACCTTCTGAGGGCAGCGCACCAGGTTCTA CCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATC TCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTACTTCTACCGAACCGTCC GAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTA CCGAACCTTCTGAAGGTAGCGCACCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCC AGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCT ACTGGTTCTCCAGGTAGCGAACCTGCTACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAA GCGCAACTCCGGAGTCTGGTCCAGGTAGCCCTGCAGGTTCTCCTACCTCCACTGAGGAAGG TACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTA CTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTACCTCTGAAAGCGC GGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACT TCTACTGAACCGTCCGAAGGTAGCGCACCA WO 40093 2012/054941 XTEN DNA Nucleotide Sequence Name AE912 ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACCCCGGGTAGCGGTACTG CTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTCTCCG GGCACCAGCTCTACCGGTTCTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAG GTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGG TAGCGCTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAA CCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTA CTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGA AACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTC TCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAC CTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAG CGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCG TCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTT CTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCG CACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTC TGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTACTTCT ACTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGC CCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCA ACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAA CCGGCAACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCC CAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGA AGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACT GAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCA GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCT CCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAAA GCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGG TACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCT GAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAAC CGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTA GCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTAC TGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGC AACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTAC CTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAG ACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACC TCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTT AACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCG AAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCT CCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCC GGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAA ACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACC CCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACTTCTG AAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCC AGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAGGCTCTCCGACT TCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTG AACCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAG GTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGG ACCA AM1 3 18 GGTACTTCTACTGAACCGTCTGAAGGCAGCGCACCAGGTAGCGAACCGGCTACTTCCGGTT CTGAAACCCCAGGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTC TACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGT TCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGCGAATCCCCGTCTGGTA CTGCTCCAGGTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGGTACCTCTACTCCGGAA AGCGGTTCTGCATCTCCAGGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCAGGTACCT CTGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGA GGAAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACC CCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTA CCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGG AAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACTTCTACCGAACCTTCCGA GGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGA AAGCGCTACTCCTGAATCCGGTCCAGGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCA 2012/054941 XTEN DNA Nucleotide Sequence Name GGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCT GAATCCGGTCCAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCGAACCTG CTACTTCTGGTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAAGG TACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTT CCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGGTACCTCTACCGAACCG TCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGC GAACCGGCAACCTCCGGTTCTGAAACTCCAGGTAGCCCTGCTGGCTCTCCGACTTCTACTG AGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTC CGAAGGTAGCGCTCCAGGTCCAGAACCAACGGGGCCGGCCCCAAGCGGAGGTAGCGAAC CGGCAACCTCCGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCC AGGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAAGGTACTTCTGAAAGCGCTACTCCT GAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCT GGCTCTCCAACTTCTACTGAAGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAG GTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTC TACTGAAGAAGGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCTACTAGCGAA TCTCCGTCTGGCACCGCACCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCACCAGGTT CTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTAC AGGTACTTCTCCTAGCGGCGAATCTTCTACCGCACCAGGTACTTCTACCGAACCT TCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTT CTGAAAGCGCTACTCCTGAATCCGGTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAA CCCCAGGTACCTCTGAAAGCGCTACTCCGGAATCTGGTCCAGGTACTTCTGAAAGCGCTAC TCCGGAATCCGGTCCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCT GAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCA CCAGGTACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAAT CTTCTACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGCACCAGGTACTTCTACC GAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAA GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTTCTAGCCCTTCTGCTTCCACCG GTACCGGCCCAGGTAGCTCTACTCCGTCTGGTGCAACTGGCTCTCCAGGTAGCTCTACTCC GTCTGGTGCAACCGGCTCCCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGT AGCTCTACCCCGTCTGGTGCAACCGGCTCCCCAGGTGCATCCCCGGGTACTAGCTCTACCG GTTCTCCAGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTACTTCTCCGAGCGGTG AATCTTCTACCGCACCAGGTTCTACTAGCTCTACCGCTGAATCTCCGGGCCCAGGTACTTCT CCGAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCC CAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGA AGGTAGCGCACCAGGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTAGCTCTACT CCTTCTGGTGCTACCGGCTCTCCAGGTGCTTCTCCGGGTACTAGCTCTACCGGTTCTCCAGG TACTTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTACTTCTCCTAGCGGTGAATCTTCTA CTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCAGGTACTTCTGAAAGCGC AACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACT TCTACCGAACCGTCCGAAGGTAGCGCACCAGGTTCTACCAGCGAATCCCCTTCTGGTACTG CTCCAGGTTCTACCAGCGAATCCCCTTCTGGCACCGCACCAGGTACTTCTACCCCTGAAAG CGGCTCCGCTTCTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCT GCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCA CCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACC CCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTAGCTCT ACCCCGTCTGGTGCTACCGGTTCCCCAGGTGCTTCTCCTGGTACTAGCTCTACCGGTTCTCC AGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTTCTACTAGCGAATCCCCGTCT GGTACTGCTCCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGGTTCTACCAGCTC TACCGCAGAATCTCCGGGTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGT GCATCCCCGGGTACCAGCTCTACCGGTTCTCCAGGTACTCCGGGTAGCGGTACCGCTTCTT CCTCTCCAGGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTC TCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCA BC864 TCCACCGAACCATCCGAACCAGGTAGCGCAGGTACTTCCACCGAACCATCCGAA AGCGCAGGTAGCGAACCGGCAACCTCTGGTACTGAACCATCAGGTAGCGGCGCA TCCGAGCCTACCTCTACTGAACCAGGTAGCGAACCGGCTACCTCCGGTACTGAGCCATCAG GTAGCGAACCGGCAACTTCCGGTACTGAACCATCAGGTAGCGAACCGGCAACTTCCGGCA CTGAACCATCAGGTAGCGGTGCATCTGAGCCGACCTCTACTGAACCAGGTACTTCTACTGA ACCATCTGAGCCGGGCAGCGCAGGTAGCGAACCAGCTACTTCTGGCACTGAACCATCAGG TACTTCTACTGAACCATCCGAACCAGGTAGCGCAGGTAGCGAACCTGCTACCTCTGGTACT XTEN DNA Nucleotide Sequence Name GAGCCATCAGGTAGCGAACCGGCTACCTCTGGTACTGAACCATCAGGTACTTCTACCGAAC CATCCGAGCCTGGTAGCGCAGGTACTTCTACCGAACCATCCGAGCCAGGCAGCGCAGGTA GCGAACCGGCAACCTCTGGCACTGAGCCATCAGGTAGCGAACCAGCAACTTCTGGTACTG AACCATCAGGTACTAGCGAGCCATCTACTTCCGAACCAGGTGCAGGTAGCGGCGCATCCG AACCTACTTCCACTGAACCAGGTACTAGCGAGCCATCCACCTCTGAACCAGGTGCAGGTA GCGAACCGGCAACTTCCGGCACTGAACCATCAGGTAGCGAACCGGCTACCTCTGGTACTG AACCATCAGGTACTTCTACCGAACCATCCGAGCCTGGTAGCGCAGGTACTTCTACCGAACC ATCCGAGCCAGGCAGCGCAGGTAGCGGTGCATCCGAGCCGACCTCTACTGAACCAGGTAG CGAACCAGCAACTTCTGGCACTGAGCCATCAGGTAGCGAACCAGCTACCTCTGGTACTGA ACCATCAGGTAGCGAACCGGCTACTTCCGGCACTGAACCATCAGGTAGCGAACCAGCAAC CTCCGGTACTGAACCATCAGGTACTTCCACTGAACCATCCGAACCGGGTAGCGCAGGTAG CGAACCGGCAACTTCCGGCACTGAACCATCAGGTAGCGGTGCATCTGAGCCGACCTCTACT GAACCAGGTACTTCTACTGAACCATCTGAGCCGGGCAGCGCAGGTAGCGAACCTGCAACC TCCGGCACTGAGCCATCAGGTAGCGGCGCATCTGAACCAACCTCTACTGAACCAGGTACTT AACCATCTGAGCCAGGCAGCGCAGGTAGCGGCGCATCTGAACCAACCTCTACTG AACCAGGTAGCGAACCAGCAACTTCTGGTACTGAACCATCAGGTAGCGGCGCATCTGAGC CTACTTCCACTGAACCAGGTAGCGAACCGGCAACTTCCGGCACTGAACCATCAGGTAGCG GTGCATCTGAGCCGACCTCTACTGAACCAGGTACTTCTACTGAACCATCTGAGCCGGGCAG CGCAGGTAGCGAACCGGCAACTTCCGGCACTGAACCATCAGGTAGCGGTGCATCTGAGCC TACTGAACCAGGTACTTCTACTGAACCATCTGAGCCGGGCAGCGCAGGTAGCGA ACCAGCTACTTCTGGCACTGAACCATCAGGTACTTCTACTGAACCATCCGAACCAGGTAGC GCAGGTAGCGAACCTGCTACCTCTGGTACTGAGCCATCAGGTACTTCTACTGAACCATCCG AGCCGGGTAGCGCAGGTACTTCCACTGAACCATCTGAACCTGGTAGCGCAGGTACTTCCAC TGAACCATCCGAACCAGGTAGCGCAGGTACTTCTACTGAACCATCCGAGCCGGGTAGCGC AGGTACTTCCACTGAACCATCTGAACCTGGTAGCGCAGGTACTTCCACTGAACCATCCGAA CCAGGTAGCGCAGGTACTAGCGAACCATCCACCTCCGAACCAGGCGCAGGTAGCGGTGCA TCTGAACCGACTTCTACTGAACCAGGTACTTCCACTGAACCATCTGAGCCAGGTAGCGCAG GTACTTCCACCGAACCATCCGAACCAGGTAGCGCAGGTACTTCCACCGAACCATCCGAAC CTGGCAGCGCAGGTAGCGAACCGGCAACCTCTGGTACTGAACCATCAGGTAGCGGTGCAT CCGAGCCGACCTCTACTGAACCAGGTAGCGAACCAGCAACTTCTGGCACTGAGCCATCAG GTAGCGAACCAGCTACCTCTGGTACTGAACCATCAGGTAGCGAACCGGCAACCTCTGGCA CTGAGCCATCAGGTAGCGAACCAGCAACTTCTGGTACTGAACCATCAGGTACTAGCGAGC CATCTACTTCCGAACCAGGTGCAGGTAGCGAACCTGCAACCTCCGGCACTGAGCCATCAG GTAGCGGCGCATCTGAACCAACCTCTACTGAACCAGGTACTTCCACCGAACCATCTGAGCC AGGCAGCGCAGGTAGCGAACCTGCAACCTCCGGCACTGAGCCATCAGGTAGCGGCGCATC AACCTCTACTGAACCAGGTACTTCCACCGAACCATCTGAGCCAGGCAGCGCA IHR64 GGTAGCGAAACTGCTACTTCCGGCTCTGAGACTGCAGGTACTAGTGAATCCGCAACTAGC GAATCTGGCGCAGGTAGCACTGCAGGCTCTGAGACTTCCACTGAAGCAGGTACTAGCGAG TCCGCAACCAGCGAATCCGGCGCAGGTAGCGAAACTGCTACCTCTGGCTCCGAGACTGCA GGTAGCGAAACTGCAACCTCTGGCTCTGAAACTGCAGGTACTTCCACTGAAGCAAGTGAA GGCTCCGCATCAGGTACTTCCACCGAAGCAAGCGAAGGCTCCGCATCAGGTACTAGTGAG TCCGCAACTAGCGAATCCGGTGCAGGTAGCGAAACCGCTACCTCTGGTTCCGAAACTGCA GGTACTTCTACCGAGGCTAGCGAAGGTTCTGCATCAGGTAGCACTGCTGGTTCCGAGACTT CTACTGAAGCAGGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAAT CCAGCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGACTGCAG GTACTAGCGAGTCCGCTACTAGCGAATCTGGCGCAGGTACTTCCACTGAAGCTAGTGAAG GTTCTGCATCAGGTAGCGAAACTGCTACTTCTGGTTCCGAAACTGCAGGTAGCGAAACCGC TACCTCTGGTTCCGAAACTGCAGGTACTTCTACCGAGGCTAGCGAAGGTTCTGCATCAGGT AGCACTGCTGGTTCCGAGACTTCTACTGAAGCAGGTACTAGCGAGTCCGCTACTAGCGAAT CTGGCGCAGGTACTTCCACTGAAGCTAGTGAAGGTTCTGCATCAGGTAGCGAAACTGCTAC TTCTGGTTCCGAAACTGCAGGTAGCACTGCTGGCTCCGAGACTTCTACCGAAGCAGGTAGC ACTGCAGGTTCCGAAACTTCCACTGAAGCAGGTAGCGAAACTGCTACCTCTGGCTCTGAGA CTGCAGGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCCGCTA CCAGCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGACTGCAGGTACTA GCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCCGCTACCAGCGAATCCG GTAGCGAAACTGCAACCTCTGGTTCCGAGACTGCAGGTAGCGAAACCGCTACCT CTGGTTCCGAAACTGCAGGTACTTCTACCGAGGCTAGCGAAGGTTCTGCATCAGGTAGCAC TTCCGAGACTTCTACTGAAGCAGGTAGCGAAACTGCTACTTCCGGCTCTGAGACT GCAGGTACTAGTGAATCCGCAACTAGCGAATCTGGCGCAGGTAGCACTGCAGGCTCTGAG XTEN DNA Nucleotide Sequence Name ACTTCCACTGAAGCAGGTAGCACTGCTGGTTCCGAAACCTCTACCGAAGCAGGTAGCACT GCAGGTTCTGAAACCTCCACTGAAGCAGGTACTTCCACTGAGGCTAGTGAAGGCTCTGCAT GCACTGCTGGTTCCGAAACCTCTACCGAAGCAGGTAGCACTGCAGGTTCTGAAA CCTCCACTGAAGCAGGTACTTCCACTGAGGCTAGTGAAGGCTCTGCATCAGGTAGCACTGC AGGTTCTGAGACTTCCACCGAAGCAGGTAGCGAAACTGCTACTTCTGGTTCCGAAACTGCA GGTACTTCCACTGAAGCTAGTGAAGGTTCCGCATCAGGTACTAGTGAGTCCGCAACCAGC GAATCCGGCGCAGGTAGCGAAACCGCAACCTCCGGTTCTGAAACTGCAGGTACTAGCGAA TCCGCAACCAGCGAATCTGGCGCAGGTACTAGTGAGTCCGCAACCAGCGAATCCGGCGCA GGTAGCGAAACCGCAACCTCCGGTTCTGAAACTGCAGGTACTAGCGAATCCGCAACCAGC GAATCTGGCGCAGGTAGCGAAACTGCTACTTCCGGCTCTGAGACTGCAGGTACTTCCACCG AAGCAAGCGAAGGTTCCGCATCAGGTACTTCCACCGAGGCTAGTGAAGGCTCTGCATCAG GTAGCACTGCTGGCTCCGAGACTTCTACCGAAGCAGGTAGCACTGCAGGTTCCGAAACTTC CACTGAAGCAGGTAGCGAAACTGCTACCTCTGGCTCTGAGACTGCAGGTACTAGCGAATC TGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCCGCTACCAGCGAATCCGGCGCAGG TAGCGAAACTGCAACCTCTGGTTCCGAGACTGCAGGTAGCGAAACTGCTACTTCCGGCTCC GAGACTGCAGGTAGCGAAACTGCTACTTCTGGCTCCGAAACTGCAGGTACTTCTACTGAGG AAGGTTCCGCATCAGGTACTAGCGAGTCCGCAACCAGCGAATCCGGCGCAGGTA GCGAAACTGCTACCTCTGGCTCCGAGACTGCAGGTAGCGAAACTGCAACCTCTGGCTCTGA AACTGCAGGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCCGC TACCAGCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGACTGCA One may clone the library of XTEN—encoding genes into one or more expression vectors known in the art. To facilitate the identiﬁcation of well—expressing library members, one can uct the library as fusion to a reporter protein. Non—limiting examples of suitable er genes are green ﬂuorescent protein, luciferace, alkaline phosphatase, and alactosidase. By screening, one can identify short XTEN sequences that can be expressed in high concentration in the host organism of choice. Subsequently, one can generate a library of random XTEN dimers and repeat the screen for high level of sion. Subsequently, one can screen the resulting constructs for a number of properties such as level of expression, protease stability, or binding to antiserum.

One aspect ofthe invention is to provide polynucleotide sequences encoding the components of the fusion protein wherein the on of the sequence has undergone codon optimization. Of particular interest is codon zation with the goal of improving expression of the polypeptide compositions and to improve the genetic stability of the encoding gene in the production hosts. For example, codon optimization is of particular importance for XTEN ces that are rich in e or that have very repetitive amino acid sequences. Codon optimization is performed using computer programs (Gustafsson, C., et al. (2004) Trends Biotechnol, 22: 346-53), some of which minimize ribosomal g (Coda Genomics Inc.). In one embodiment, one can perform codon optimization by constructing codon libraries where all members of the library encode the same amino acid sequence but where codon usage is varied. Such libraries can be screened for highly expressing and genetically stable members that are particularly suitable for the large-scale production of XTEN-containing products. When designing XTEN sequences one can consider a number of properties. One can minimize the repetitiveness in the encoding DNA ces. In addition, one can avoid or minimize the use of codons that are rarely used by the production host (e. g. the AGG and AGA ne codons and one leucine codon in E. coli). In the case of E. coli, two glycine , GGA and GGG, are rarely used in highly expressed proteins. Thus codon optimization of the gene encoding XTEN sequences can be very desirable. DNA sequences that have a high level of glycine tend to have a high GC content that can lead to instability or low expression levels. Thus, when possible, it is preferred to choose codons such that the tent ofXTEN- encoding sequence is suitable for the production organism that will be used to manufacture the XTEN.

Optionally, the full-length XTEN-encoding gene comprises one or more sequencing islands. In this t, sequencing islands are short-stretch sequences that are distinct from the XTEN library construct sequences and that include a restriction site not present or expected to be present in the full- length XTEN-encoding gene. In one embodiment, a sequencing island is the sequence ’-AGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGT-3’. In another embodiment, a sequencing island is the sequence ’-AGGTCCAGAACCAACGGGGCCGGCCCCAAGCGGAGGT-3 ’.

] In one ment, polynucleotide libraries are constructed using the disclosed methods wherein all members ofthe library encode the same amino acid sequence but the codon usage for the respective amino acids in the sequence is varied. Such libraries can be screened for highly expressing and genetically stable members that are particularly suitable for the large-scale production ofXTEN- containing products.

] Optionally, one can sequence clones in the library to eliminate isolates that n undesirable ces. The initial library of short XTEN sequences allows some variation in amino acid sequence.

For instance one can randomize some codons such that a number of hydrophilic amino acids can occur in a particular on. During the process of iterative multimerization one can screen the resulting library s for other characteristics like solubility or protease resistance in addition to a screen for high- level expression.

Once the gene that encodes the XTEN of desired length and properties is selected, it is genetically fused at the desired location to the nucleotides encoding the GLP-2 ) by cloning it into the construct adjacent and in frame with the gene coding for GLP-Z, or atively in frame with tides encoding a spacer/cleavage sequence linked to a terminal XTEN. The invention provides various permutations of the foregoing, depending on the GLPZ-XTEN to be encoded. For example, a gene encoding a GLPZ-XTEN fusion protein comprising a GLP-2 and two XTEN, such as embodied by formula III, as depicted above, the gene would have polynucleotides encoding GLP-2, and cleotides encoding two XTEN, which can be identical or different in ition and sequence length. In one non-limiting embodiment of the ing, the GLP-2 polynucleotides would encode native GLP-2 and the polynucleotides encoding the C-terminus XTEN would encode AE864 and the polynucleotides encoding an N—terminal XTEN AE912. The step of cloning the GLP-2 genes into the XTEN construct can occur through a ligation or erization step, as shown in in a schematic ﬂowchart of representative steps in the assembly of a GLPZ-XTEN polynucleotide construct. Individual oligonucleotides 501 are annealed into sequence motifs 502 such as a 12 amino acid motif (“12-mer”), which is ligated to additional sequence motifs from a library that can multimerize to create a pool that encompasses the desired length of the XTEN 504, as well as ligated to a smaller tration of an oligo containing BbsI, and KpnI ction sites 503. The motif libraries can be limited to speciﬁc sequence XTEN families; e.g., AD, AE, AF, AG, AM, or AQ sequences of Table 3. As illustrated in the XTEN polynucleotides encode a length, in this case, of 36 amino acid residues, but longer lengths can be achieved by this process. For example, multimerization can be performed by ligation, overlap extension, PCR ly or similar cloning techniques known in the art. The resulting pool of ligation products is gel-puriﬁed and the band with the d length of XTEN is cut, resulting in an isolated XTEN gene with a stopper sequence 505. The XTEN gene can be cloned into a stuffer vector. In this case, the vector encodes an optional CBD ce 506 and a GFP gene 508. Digestion is than performed with BbsI/HindIII to remove 507 and 508 and place the stop codon. The resulting product is then cloned into a BsaI/HindIII digested vector ning a gene encoding the GLP-2, resulting in the gene 500 encoding a GLPZ-XTEN fusion protein. As would be apparent to one of ordinary skill in the art, the methods can be applied to create constructs in alternative conﬁgurations and with varying XTEN lengths.

The constructs encoding GLPZ-XTEN fusion proteins can be ed in different conﬁgurations of the components XTEN, GLP—2, and spacer sequences, such as shown in In one embodiment, the construct comprises polynucleotide sequences complementary to, or those that encode a monomeric polypeptide of components in the following order (5’ to 3’) GLP-2 and XTEN. In r embodiment, the construct comprises polynucleotide sequences complementary to, or those that encode a monomeric polypeptide of ents in the following order (5’ to 3’) XTEN and GLP—2. In another embodiment, the construct comprises polynucleotide sequences complementary to, or those that encode a monomeric polypeptide of components in the following order (5’ to 3’) XTEN, GLP-2, and a second XTEN. In another embodiment, the uct comprises polynucleotide ces complementary to, or those that encode a monomeric polypeptide of ents in the following order (5’ to 3’) GLP-Z, spacer sequence, and XTEN. In another embodiment, the construct comprises polynucleotide sequences complementary to, or those that encode a ric polypeptide of components in the following order (5’ to 3’) XTEN, spacer sequence, and GLP-2. The spacer polynucleotides can optionally comprise sequences encoding cleavage sequences. As will be apparent to those of skill in the art, other permutations or multimers of the foregoing are possible.

The invention also encompasses polynucleotides comprising XTEN-encoding polynucleotide ts that have a high tage of sequence identity compared to (a) a polynucleotide sequence from Table 7, or (b) sequences that are complementary to the polynucleotides of (a). A polynucleotide with a high percentage of sequence identity is one that has at least about an 80% nucleic acid sequence identity, alternatively at least about 81%, alternatively at least about 82%, alternatively at least about 83%, alternatively at least about 84%, alternatively at least about 85%, alternatively at least about 86%, atively at least about 87%, atively at least about 88%, alternatively at least about 89%, alternatively at least about 90%, alternatively at least about 91% at least about 92%, , alternatively alternatively at least about 93%, alternatively at least about 94%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, and alternatively at least about 99% nucleic acid sequence identity ed to (a) or (b) of the foregoing, or that can hybridize with the target polynucleotide or its complement under stringent conditions.

Homology, sequence rity or sequence identity of nucleotide or amino acid sequences may also be determined conventionally by using known software or computer programs such as the BestFit or Gap pairwise comparison programs (GCG Wisconsin Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. 53711). BestFit uses the local homology thm of Smith and Waterman (Advances in Applied Mathematics. 1981. 2: 482-489), to ﬁnd the best segment of identity or similarity between two sequences. Gap performs global alignments: all of one sequence with all of another similar sequence using the method of Needleman and Wunsch, (Journal of Molecular Biology. 1970. 48:443- 453). When using a sequence alignment program such as BestFit, to ine the degree of ce homology, similarity or identity, the default setting may be used, or an appropriate scoring matrix may be selected to optimize identity, similarity or homology scores.

Nucleic acid sequences that are “complementary” are those that are capable of airing according to the standard Watson-Crick complementarity rules. As used herein, the term ementary sequences” means nucleic acid sequences that are substantially mentary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the polynucleotides that encode the GLPZ—XTEN sequences under stringent conditions, such as those described herein.

The resulting polynucleotides ng the GLPZ-XTEN chimeric fusion ns can then be individually cloned into an expression vector. The nucleic acid sequence is ed into the vector by a y of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a ription termination sequence (. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan. Such techniques are well known in the art and well described in the scientiﬁc and patent literature.

Various vectors are ly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage that may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.

Thus, the vector may be an autonomously replicating vector, i.e., a vector, which exists as an hromosomal , the replication of which is independent of chromosomal replication, e. g., a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

The invention provides for the use of plasmid vectors containing replication and l sequences that are compatible with and ized by the host cell, and are operably linked to the GLP2- 2012/054941 XTEN gene for controlled expression of the GLP2-XTEN fusion proteins. The vector ordinarily carries a replication site, as well as sequences that encode ns that are capable of providing phenotypic selection in transformed cells. Such vector ces are well known for a y of bacteria, yeast, and viruses. Useful expression s that can be used include, for example, segments of chromosomal, non-chromosomal and tic DNA sequences. "Expression vector” refers to a DNA construct containing a DNA sequence that is operably linked to a suitable control sequence capable of ing the expression of the DNA encoding the fusion protein in a suitable host. The requirements are that the s are replicable and viable in the host cell of choice. Low- or high-copy number vectors may be used as desired. le vectors include, but are not limited to, derivatives of SV40 and pcDNA and known bacterial plasmids such as col El, pCRl, pBR322, pMal-C2, pET, pGEX as described by Smith, et al., Gene 57:31-40 (1988), pMB9 and derivatives thereof, plasmids such as RP4, phage DNAs such as the numerous derivatives of phage I such as NM98 9, as well as other phage DNA such as M13 and ﬁlamentous single stranded phage DNA; yeast plasmids such as the 2 micron plasmid or derivatives of the 2m plasmid, as well as centomeric and integrative yeast shuttle vectors; vectors useful in eukaryotic cells such as s useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modiﬁed to employ phage DNA or the sion control sequences; and the like. Yeast expression systems that can also be used in the present invention include, but are not limited to, the non—ﬁlsion pYES2 vector (lnvitrogen), the fusion pYESHisA, B, C (Invitrogen), pRS vectors and the like.

The control sequences of the vector include a promoter to effect transcription, an optional operator sequence to l such transcription, a ce encoding suitable mRNA me binding sites, and sequences that control termination of transcription and translation. The promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of the DNA encoding the GLP2- XTEN in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1 (1981), 854-864), the MT-l (metallothionein gene) promoter (Palmiter et al., Science 222 , 809-814), the CMV promoter (Boshart et al., Cell 41 2521-5 30, 1985) or the adenovirus 2 major late promoter (Kaufman and Sharp, M01. Cell. Biol, 2:1304-1319, 1982). The vector may also carry sequences such as UCOE (ubiquitous tin opening elements).

Examples of suitable promoters for use in ﬁlamentous fungus host cells are, for instance, the ADH3 promoter or the tpiA promoter. Examples of other useful promoters are those derived from the gene encoding A. orjyzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral 0t- amylase, A. niger acid stable a-amylase, A. niger or A. awamoriglucoamylase (gluA), Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae tliose phosphate isomerase or A. nidulans acetamidase. red are the TAKA-amylase and gluA promoters. Yeast sion systems that can also be used in the t invention include, but are not limited to, the non-fusion pYES2 vector (Invitrogen), the ﬁlsion pYESHisA, B, C (Invitrogen), pRS s and the like.

Promoters le for use in expression vectors with prokaryotic hosts include the ﬁ-lactamase and lactose promoter systems [Chang et al., Nature, 275 :615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a phan (trp) promoter system [G0eddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci.

USA, 25 (1983)], all is operably linked to the DNA encoding GLP2-XTEN polypeptides.

Promoters for use in bacterial systems can also contain a Shine-Dalgarno (S.D.) ce, ly linked to the DNA encoding GLP2-XTEN polypeptides.

The ion contemplates use of other expression systems including, for example, a baculovirus expression system with both non-fusion transfer vectors, such as, but not limited to pVL941 Summers, et al., Virology 84:390-402 (1978)), pVL1393 (Invitrogen), pVL1392 (Summers, et al., Virology 84:390- 402 (1978) and Invitrogen) and acHl (Invitrogen), and fusion transfer vectors such as, but not limited to, pAc7 00 (Summers, et al., Virology 84:390-402 (1978)), pAc701 and pAc70- 2 (same as pAc700, with different g frames), pAc36O Invitrogen) and acHisA, B, C (Invitrogen) can be used.

The DNA sequences encoding the GLP2-XTEN may also, if necessary, be ly connected to a suitable terminator, such as the hGH terminator (Palmiter et al., Science 222, 1983, pp. 809-814) or the TH] terminators (Alber and Kawasaki, J. Mol. App]. Gen. 1, 1982, pp. 419-434) or ADH3 (McKnight et al., The EMBO J. 4, 1985, pp. 2093-2099). Expression vectors may also contain a set of RNA splice sites located downstream from the promoter and upstream from the insertion site for the GLP2-XTEN sequence itself, including splice sites obtained from adenovirus. Also contained in the sion vectors is a polyadenylation signal located downstream of the insertion site. Particularly preferred polyadenylation signals include the early or late polyadenylation signal from SV4O (Kaufman and Sharp, ibid.), the polyadenylation signal from the adenovirus 5 Elb region, the hGH terminator (DeNoto et al. Nucl. Acids Res. 9:3719—3730, 1981). The expression vectors may also include a noncoding viral leader sequence, such as the irus 2 tripartite leader, located between the promoter and the RNA splice sites; and enhancer sequences, such as the SV40 enhancer.

In one embodiment, the polynucleotide encoding a GLP2-XTEN ﬁlSiOl’l protein composition is fused C-terminally to an inal signal sequence appropriate for the expression host system. Signal sequences are typically proteolytically removed from the protein during the translocation and ion process, generating a deﬁned N-terminus. A wide variety of signal ces have been bed for most expression systems, including bacterial, yeast, insect, and mammalian systems. A non-limiting list of preferred examples for each expression system follows . Preferred signal sequences are OmpA, PhoA, and DsbA for E. coli expression. Signal peptides preferred for yeast expression are ppL-alpha, DEX4, ase signal peptide, acid phosphatase signal peptide, CPY, or 1NU1. For insect cell expression the preferred signal sequences are sexta adipokinetic hormone precursor, CP1, CP2, CP3, CP4, TPA, PAP, or gp67. For mammalian expression the preferred signal sequences are IL2L, SV40, IgG kappa and IgG lambda.

In another embodiment, a leader sequence, potentially comprising a well-expressed, independent n , can be fused to the N-terminus of the GLPZ-XTEN sequence, separated by a protease ge site. While any leader peptide sequence which does not inhibit cleavage at the designed proteolytic site can be used, sequences in preferred embodiments will comprise stable, wellexpressed sequences such that expression and folding of the overall composition is not signiﬁcantly adversely affected, and preferably expression, solubility, and/or folding efﬁciency are signiﬁcantly improved. A wide variety of suitable leader sequences have been described in the literature. A nonlimiting list of suitable sequences includes e binding n, cellulose binding domain, glutathione S-transferase, 6xHis tag, FLAG tag, utinin tag, and green ﬂuorescent protein. The leader sequence can also be further improved by codon optimization, especially in the second codon position following the ATG start codon, by methods well described in the literature and hereinabove.

The procedures used to ligate the DNA sequences coding for the GLP2-XTEN, the promoter and optionally the ator and/or secretory signal sequence, respectively, and to insert them into suitable vectors containing the ation ary for replication, are well known to persons skilled in the art (of, for instance, ok, J. et al., “Molecular Cloning: A Laboratory Manual,” 3rd edition, Cold Spring Harbor tory Press, 2001).

In other embodiments, the invention provides constructs and methods of making constructs comprising an polynucleotide sequence optimized for expression that encodes at least about 20 to about 60 amino acids with XTEN characteristics that can be included at the N—terminus of an XTEN carrier encoding sequence (in other words, the polynucleotides encoding the 20-60 encoded optimized amino acids are linked in frame to cleotides encoding an XTEN component that is N—terminal to GLP-2) to promote the initiation slation to allow for expression of XTEN fusions at the N—terminus of proteins without the presence of a helper domain. In an advantage ofthe foregoing, the sequence does not require subsequent cleavage, thereby reducing the number of steps to manufacture XTEN-containing itions. As described in more detail in the Examples, the optimized N—terminal sequence has attributes of an unstructured protein, but may include nucleotide bases encoding amino acids selected for their ability to promote tion of ation and enhanced expression. In one ment of the foregoing, the optimized polynucleotide encodes an XTEN sequence with at least about 90% sequence identity compared to AE912. In another embodiment of the foregoing, the optimized cleotide encodes an XTEN sequence with at least about 90% sequence identity compared to AM923. In another embodiment of the foregoing, the optimized polynucleotide encodes an XTEN sequence with at least about 90% sequence identity compared to AE48. In r embodiment of the foregoing, the optimized polynucleotide encodes an XTEN sequence with at least about 90% sequence identity compared to AM48. In one embodiment, the zed polynucleotide NTS comprises a sequence that exhibits at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least 2012/054941 about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity compared to a sequence or its complement selected from AB 48: 5’- ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACCCCGGGTAGCGGTACTGC TTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTCTCCGGG CACCAGCTCTACCGGTTCTCCA-3’ AM 48: 5 ’- ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTGCATCCCCGGGCACCAGCTC TACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTAGCTCTACCCC GTCTGGTGCTACTGGCTCTCCA-3 ’.

In this manner, a chimeric DNA molecule coding for a monomeric GLP2-XTEN ﬁJsion protein is generated. Optionally, this chimeric DNA molecule may be transferred or cloned into another construct that is a more riate expression vector. At this point, a host cell capable of sing the chimeric DNA molecule can be transformed With the chimeric DNA molecule. The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, Whereas calcium phosphate treatment, lipofection, or electroporation may be used for other cellular hosts. Other methods used to transform mammalian cells include the use of polybrene, protoplast , liposomes, oporation, and microinjection. See, generally, Sambrook, et al., supra.

The transformation may occur With or t the utilization of a r, such as an expression vector. Then, the ormed host cell is cultured under conditions suitable for the expression of the chimeric DNA molecule encoding of GLP2-XTEN.

The present invention also provides a host cell for expressing the monomeric fusion protein compositions disclosed . Examples ofmammalian cell lines for use in the present invention are the COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), BHK-21 (ATCC CCL 10)) and BHK-293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977), BHK-570 cells (ATCC CRL 10314), CHO-Kl (ATCC CCL 61), CHO-S (Invitrogen 11619-012), and 293-F (Invitrogen R790-7). A 3 BHK cell line is also available from the ATCC under accession number CRL 1632. In addition, a number of other cell lines may be used Within the present invention, ing Rat Hep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat ma; ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1), CHO (ATCC CCL 61) and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980).

Examples of suitable yeasts host cells include cells of Saccharomyces spp. or Schizosaccharomyces spp., in particular strains of Saccharomyces cerevisiae or romyces kluyveri.

Other yeasts include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 ; EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis 8C, CBS683, CBS4574; court et al., J. Bacteriol, 737 ), K fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K waltii (ATCC 56,500), K drosophilarum (ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)), K. tolerans and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol, 28:265-278 [1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces ntalis (EP 394,538 published 31 Oct. 1990).

Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis , and Rhodotorula. Further examples of suitable yeast cells are strains of Kluyveromyces, such as Hansenula H. polymorpha or Pichia P. pastoris (cf. Gleeson et al., , e.g. , , e.g.

J. Gen. Microbiol. 132, 1986, pp. 3459-3465; U.S. Pat. No. 4,882,279). A list of speciﬁc species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982). Methods for transforming yeast cells With heterologous DNA and producing heterologous polypeptides there from are bed, e.g. in U.S. Pat. No. 4,599,311, U.S. Pat. No. 4,931,373, U.S. Pat.

No. 4,870,008, 5,037,743, and U.S. Pat. No. 4,845,075, all of Which are hereby incorporated by reference.

] Examples of other fungal cells are cells of ﬁlamentous fungi, e. g. Aspergillus spp., Neurospora spp., Fusarium spp. or derma spp., in particular strains of A. , A. nidulans or A. niger. The use ofAspergillus spp. for the expression of proteins is described in, e.g., EP 272 277, EP 238 023, EP 184 438 The transformation of F. rum may, for instance, be carried out as described by Malardier et al., 1989 Gene 78: 147-156. The transformation of Trichoderma spp. may be performed for instance as described in EP 244 234.

Other suitable cells that can be used in the present invention include, but are not limited to, prokaryotic host cells strains such as Escherichia coli, (e. g., strain DH5-0t), Bacillus subtilis, Salmonella typhimurium, or strains of the genera of monas, Streptomyces and Staphylococcus. Non-limiting examples of le prokaryotes include those from the : Actinoplanes; Archaeoglobus; Bdellovibrio; Borrelia; Chloroﬂexus; Enterococcus; Escherichia; Lactobacillus; Listeria; Oceanobacillus; Paracoccus; Pseudomonas; lococcus; Streptococcus; Streptomyces; Thermoplasma; and Vibrio.

Transformed cells are selected by a phenotype determined by a able marker, commonly drug resistance or the ability to grow in the e of a particular nutrient, e.g. leucine. A preferred vector for use in yeast is the POT1 vector disclosed in U.S. Pat. No. 4,931,373. The DNA sequences encoding the GLPZ-XTEN may be preceded by a signal sequence and ally a leader sequence, e. g. as described above. Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g., Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982), 422- 426; Wigler et a1., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb, Virology 52 , 456; and Neumann et al., EMBOJ. 1 (1982), 841-845.

Cloned DNA sequences are introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell 14:725-732, 1978; Corsaro and n, c Cell Genetics 7:603 -616, 1981; Graham and Van der Eb, Virology 52d1456-467, 1973), transfection with many commercially available reagents such as FuGENEG Roche Diagnostics, Mannheim, Germany) or lipofectamine (lnvitrogen) or by oporation (Neumann et al., EMBO J. 1:841-845, 1982). To identify and select cells that s the exogenous DNA, a gene that confers a selectable phenotype (a selectable ) is generally introduced into cells along with the gene or cDNA of interest. Preferred selectable markers include genes that confer resistance to drugs such as neomycin, hygromycin, puromycin, zeocin, and methotrexate. The selectable marker may be an ampliﬁable able marker. A preferred ampliﬁable selectable marker is a dihydrofolate reductase (DHFR) sequence. Further examples of selectable s are well known to one of skill in the art and e reporters such as enhanced green ﬂuorescent protein (EGFP), beta-galactosidase (B-gal) or mphenicol acetyltransferase (CAT). Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., incorporated herein by reference). A person skilled in the art will easily be able to choose suitable selectable markers. Any known selectable marker may be employed so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. able markers may be introduced into the cell on a separate plasmid at the same time as the gene of st, or they may be introduced on the same plasmid. On the same plasmid, the selectable marker and the gene of st may be under the control of different promoters or the same promoter, the latter arrangement produces a dicistronic message. Constructs of this type are known in the art (for example, Levinson and en, US. Pat. No. 4,713,339). It may also be advantageous to add additional DNA, known as “carrier DNA,” to the mixture that is introduced into the cells.

After the cells have taken up the DNA, they are grown in an appropriate growth , typically 1-2 days, to begin expressing the gene of interest. As used herein the term “appropriate growth medium” means a medium ning nutrients and other ents required for the growth of cells and the expression ofthe GLP2-XTEN of interest. Media generally include a carbon source, a nitrogen source, essential amino acids, essential sugars, vitamins, salts, phospholipids, protein and growth factors.

For production of carboxylated proteins, the medium will contain vitamin K, preferably at a concentration of about 0.1 [Lg/ml to about 5 [Lg/ml. Drug selection is then applied to select for the growth of cells that are expressing the selectable marker in a stable fashion. For cells that have been transfected with an ampliﬁable selectable marker the drug tration may be increased to select for an increased copy number of the cloned sequences, thereby increasing expression levels. Clones of stably transfected cells are then screened for expression of the GLP-2 polypeptide variant of interest.

The transformed or transfected host cell is then ed in a suitable nutrient medium under conditions permitting expression ofthe GLP2-XTEN fusion protein after which the resulting peptide may WO 40093 be recovered from the culture. The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the an Type Culture Collection). The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

Gene expression may be ed in a sample directly, for example, by conventional Northern blotting to tate the transcription ofmRNA [Thomas, Proc. Natl. Acad. Sci. USA, 1-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can ize speciﬁc duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of dy bound to the duplex can be detected.

Gene expression, atively, may be measured by immunological of cent methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body ﬂuids or the ion of selectable markers, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample ﬂuids may be either monoclonal or polyclonal, and may be prepared in any . Conveniently, the antibodies may be prepared against a native sequence GLP-2 polypeptide or against a synthetic peptide based on the DNA ces provided herein or against exogenous sequence fused to GLP-2 and encoding a speciﬁc antibody epitope.

Examples of selectable s are well known to one of skill in the art and include reporters such as enhanced green ﬂuorescent protein (EGFP), beta-galactosidase (B-gal) or chloramphenicol acetyltransferase (CAT).

Expressed GLP2-XTEN polypeptide product(s) may be puriﬁed via methods known in the art or by s disclosed herein. Procedures such as gel ﬁltration, afﬁnity puriﬁcation (e. g., using an anti-GLP-2 antibody column), salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxyapatite adsorption chromatography, hydrophobic interaction chromatography and gel electrophoresis may be used; each tailored to recover and purify the fusion protein produced by the respective host cells. Additional puriﬁcation may be ed by conventional al puriﬁcation means, such as high performance liquid chromatography. Some expressed GLP2-XTEN may require refolding during isolation and puriﬁcation. Methods of puriﬁcation are described in Robert K. Scopes, n Puriﬁcation: Principles and Practice, Charles R. Castor (ed.), Springer-Verlag 1994, and Sambrook, et £11., supra. Multi-step puriﬁcation separations are also described in Baron, er al., Crit. Rev. hnol. 10:179-90 (1990) and Below, er al., J. Chromatogr. A. 679:67-83 (1994). For therapeutic purposes it is preferred that the GLP2-XTEN fusion proteins of the invention are substantially pure.

Thus, in a preferred embodiment of the invention the GLPZ-XTEN of the invention is puriﬁed to at least about 90 to 95% homogeneity, ably to at least about 98% neity. Purity may be assessed by, e. g., gel ophoresis, HPLC, and amino-terminal amino acid sequencing.

VIII). PHARMACEUTICAL COMPOSITIONS The t invention provides pharmaceutical compositions comprising GLPZ-XTEN. In one embodiment, the pharmaceutical composition comprises a GLP2-XTEN fusion protein disclosed herein and at least one pharmaceutically acceptable carrier. GLPZ-XTEN polypeptides of the present ion can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the polypeptide is ed in admixture with a pharmaceutically acceptable carrier vehicle, such as aqueous solutions, buffers, solvents and/or pharmaceutically acceptable suspensions, emulsions, stabilizers or excipients. Examples of non-aqueous solvents include propylethylene glycol, polyethylene glycol and vegetable oils. Formulations of the pharmaceutical compositions are prepared for storage by mixing the active GLPZ-XTEN ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients (e.g., sodium chloride, a calcium salt, sucrose, or polysorbate) or stabilizers (e.g., sucrose, trehalose, raffmose, arginine, a m salt, glycine or histidine), as described in Remington's ceutical Sciences 16th edition, Osol, A. Ed. (1980), in the form of lyophilized formulations or aqueous solutions.

] In one embodiment, the pharmaceutical composition may be supplied as a lyophilized powder to be reconstituted prior to administration. In another embodiment, the pharmaceutical composition may be supplied in a liquid form, which can be administered directly to a patient. In another embodiment, the composition is supplied as a liquid in a pre-filled syringe for administration of the composition. In another embodiment, the composition is supplied as a liquid in a pre-ﬁlled vial that can be incorporated into a pump.

The pharmaceutical compositions can be administered by any suitable means or route, including subcutaneously, subcutaneously by infusion pump, intramuscularly, intravenously, or via the pulmonary route. It will be iated that the preferred route will vary with the disease and age of the recipient, and the severity of the condition being treated.

In one embodiment, the GLPZ-XTEN pharmaceutical composition in liquid form or after reconstitution (when ed as a lized ) comprises GLP-2 linked to XTEN, which composition is capable of increasing GLPrelated activity to at least 10% of the normal GLP-2 plasma level in the blood for at least about 72 hours, or at least about 96 hours, or at least about 120 hours, or at least about 7 days, or at least about 10 days, or at least about 14 days, or at least about 21 days after administration of the GLP-Z ceutical composition to a subject in need. In another embodiment, the GLPZ-XTEN ceutical composition in liquid form or after reconstitution (when ed as a lyophilized powder) and administration to a subject is capable of increasing GLPZ-XTEN concentrations to at least 500 ng/ml, or at least 1000 ng/ml, or at least about 2000 ng/ml, or at least about 3000 ng/ml, or at least about 4000 ng/ml, or at least about 5000 ng/ml, or at least about 10000 ng/ml, or at least about 2012/054941 15000 ng/ml, or at least about 20000 ng/ml, or at least about 30000 , or at least about 40000 ng/ml for at least about 24 hours, or at least about 48 hours, or at least about 72 hours, or at least about 96 hours, or at least about 120 hours, or at least about 144 hours after stration of the GLP-2 pharmaceutical composition to a subject in need. It is speciﬁcally contemplated that the pharmaceutical compositions of the foregoing embodiments in this paragraph can be formulated to include one or more excipients, buffers or other ingredients known in the art to be compatible with administration by the intravenous route or the subcutaneous route or the intramuscular route. Thus, in the embodiments hereinaboye described in this paragraph, the pharmaceutical composition is administered subcutaneously, intramuscularly, or intravenously.

The compositions of the invention may be formulated using a variety of excipients. Suitable excipients include microcrystalline cellulose (e.g. Avicel PH102, Avicel PHlOl), polymethacrylate, poly(ethyl acrylate, methyl methacrylate, trimethylammonioethyl methacrylate chloride) (such as Eudragit RS-30D), hydroxypropyl methylcellulose (Methocel K100M, Premium CR Methocel K100M, Methocel E5, ®), magnesium stearate, talc, triethyl citrate, aqueous ellulose dispersion (Surelease®), and protamine sulfate. The slow release agent may also se a carrier, which can comprise, for example, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. Pharmaceutically acceptable salts can also be used in these slow release agents, for example, l salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as the salts of organic acids such as acetates, proprionates, malonates, or benzoates. The composition may also n liquids, such as water, saline, glycerol, and ethanol, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents. Liposomes may also be used as a carrier.

In another embodiment, the itions of the present ion are encapsulated in liposomes, which have demonstrated utility in delivering beneficial active agents in a controlled manner over prolonged s of time. Liposomes are closed bilayer nes containing an entrapped aqueous volume. Liposomes may also be unilamellar vesicles possessing a single membrane bilayer or multilamellar vesicles with multiple membrane bilayers, each separated from the next by an aqueous layer. The ure of the resulting membrane bilayer is such that the hydrophobic (non-polar) tails of the lipid are oriented toward the center of the bilayer while the hydrophilic ) heads orient s the aqueous phase. In one embodiment, the liposome may be coated with a ﬂexible water soluble polymer that avoids uptake by the organs of the mononuclear phagocyte system, primarily the liver and spleen. Suitable hydrophilic polymers for surrounding the liposomes include, without limitation, PEG, polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, p0lyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxethylacrylate, hydroxymethylcellulose hydroxyethylcellulose, polyethyleneglycol, partamide and hydrophilic e sequences as described in U.S. Pat. Nos. 6,316,024; 6,126,966; 6,056,973; 6,043,094, the contents of which are orated by reference in their entirety. mes may be comprised of any lipid or lipid ation known in the art. For example, the vesicle-forming lipids may be naturally-occurring or synthetic lipids, including phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phasphatidylglycerol, phosphatidylinositol, and sphingomyelin as disclosed in US. Pat. Nos. 6,056,973 and 5,874,104. The vesicle-forming lipids may also be glycolipids, cerebrosides, or cationic lipids, such as 1,2-dioleyloxy—3-(trimethylamino) propane (DOTAP); N-[1-(2,3,-ditetradecyloxy)propyl]-N,N- yl-N-hydroxyethylammonium bromide (DMRIE); N-[1 [(2,3,-dioleyloxy)propyl]-N,N-dimethyl- N-hydroxy ethylammonium bromide (DORIE); 2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA); 3 [N-(N',N'—dimethylaminoethane) carbamoly] cholesterol ol); or yldioctadecylammonium (DDAB) also as disclosed in US. Pat. No. 973.

Cholesterol may also be present in the proper range to impart stability to the e as disclosed in U.S.

Pat. Nos. 5,916,588 and 5,874,104.

Additional liposomal technologies are described in U.S. Pat. Nos. 6,759,057; 6,406,713; 6,352,716; 6,316,024; 6,294,191; 6,126,966; 6,056,973; 6,043,094; 5,965,156; 5,916,588; 5,874,104; 680; and 4,684,479, the contents of which are incorporated herein by reference. These be liposomes and lipid-coated microbubbles, and methods for their manufacture. Thus, one skilled in the art, considering both the disclosure of this invention and the disclosures of these other s could produce a liposome for the extended e of the polypeptides of the t invention.

For liquid formulations, a desired property is that the formulation be supplied in a form that can pass through a 25, 28, 30, 31, 32 gauge needle for intravenous, intramuscular, intraarticular, or subcutaneous administration. In another embodiment, a desired property is that the formulation be supplied in a form that can be nebulized into an aerosal of suitable particle size for inhalation therapy.

Osmotic pumps may be used as slow release agents in the form of tablets, pills, capsules or implantable devices. Osmotic pumps are well known in the art and readily available to one of ordinary skill in the art from companies experienced in ing osmotic pumps for extended release drug delivery. Examples are ALZA‘s DUROSTM; ALZA's OROSTM; Osmotica Pharmaceutical's OsmodexTM system; Shire tories' EnSoTrolTM system; and AlzetTM. Patents that describe c pump logy are US. Pat. Nos. 6,890,918; 6,838,093; 6,814,979; 6,713,086; 090; 6,514,532; 6,361,796; 6,352,721; 6,294,201; 6,284,276; 6,110,498; 5,573,776; 4,200,0984; and 4,088,864, the contents of which are orated herein by reference. One skilled in the art, considering both the disclosure of this invention and the disclosures of these other patents could produce an osmotic pump for the extended release of the polypeptides of the present invention.

Syringe pumps may also be used as slow release agents. Such devices are described in US.

Pat. Nos. 4,976,696; 4,933,185; 5,017,378; 6,309,370; 6,254,573; 4,435,173; 4,398,908; 6,572,585; ,298,022; 5,176,502; 534; 5,318,540; and 4,988,337, the contents of which are incorporated herein by reference. One skilled in the art, considering both the disclosure of this invention and the disclosures of these other patents could produce a syringe pump for the extended release ofthe compositions of the t invention.

IX). PHARMACEUTICAL KITS In another aspect, the invention provides a kit to facilitate the use of the GLPZ-XTEN polypeptides. The kit comprises the pharmaceutical composition provided herein, a label identifying the pharmaceutical composition, and an instruction for storage, reconstitution and/or administration of the ceutical compositions to a subject. In some embodiment, the kit comprises, preferably: (a) an amount of a GLPZ-XTEN fusion protein composition sufﬁcient to treat a gastrointestinal condition upon administration to a subject in need thereof; (b) an amount of a pharmaceutically able carrier; and (0) together in a formulation ready for injection or for reconstitution with sterile water, buffer, or dextrose; together with a label identifying the GLPZ-XTEN drug and storage and handling ions, and a sheet of the approved indications for the drug, instructions for the reconstitution and/or administration of the GLPZ-XTEN drug for the use for the prevention and/or treatment of an approved indication, appropriate dosage and safety information, and information identifying the lot and expiration of the drug. In another embodiment of the foregoing, the kit can comprise a second container that can carry a suitable diluent for the GLPZ-XTEN composition, the use of which will e the user with the appropriate concentration of GLPZ-XTEN to be delivered to the t.

EXAMPLES Example 1: Construction of D36 motif segments The following example describes the construction of a collection of optimized genes encoding motif sequences of 36 amino acids. As a ﬁrst step, a stuffer vector pCWO359 was constructed based on a pET vector and that includes a T7 er. pCWO359 encodes a cellulose binding domain (CBD) and a TEV protease recognition site followed by a stuffer sequence that is ﬂanked by BsaI, BbsI, and KpnI sites. The BsaI and BbsI sites were ed such that they generate compatible overhangs after digestion. The stuffer sequence is ed by a truncated version of the GFP gene and a His tag. The stuffer sequence contains stop codons and thus E. coli cells carrying the r plasmid pCWO359 form non-ﬂuorescent colonies. The r vector pCW0359 was digested with BsaI and KpnI to remove the stuffer segment and the resulting vector fragment was isolated by agarose gel purification. The sequences were designated XTEN_AD36, reﬂecting the AD family of motifs. Its segments have the amino acid sequence [X]3 where X is a 12mer e with the sequences: GESPGGSSGSES, GSEGSSGPGESS, GSSESGSSEGGP, or GSGGEPSESGSS. The insert was obtained by ing the following pairs of orylated synthetic oligonucleotide pairs: AD1 for: AGGTGAATCTCCDGGTGGYTCYAGCGGTTCYGARTC AD1 rev: ACCTGAYTCRGAACCGCTRGARCCACCHGGAGATTC AD2for: AGGTAGCGAAGGTTCTTCYGGTCCDGGYGARTCYTC AD2rev: ACCTGARGAYTCRCCHGGACCRGAAGAACCTTCGCT : AGGTTCYTCYGAAAGCGGTTCTTCYGARGGYGGTCC AD3rev: ACCTGGACCRCCYTCRGAAGAACCGCTTTCRGARGA : AGGTTCYGGTGGYGAACCDTCYGARTCTGGTAGCTC We also annealed the phosphorylated oligonucleotide 3KpnlstopperFor: AGGTTCGTCTTCACTCGAGGGTAC and the non-phosphorylated oligonucleotide pr_3KpnIstopperRev: CCTCGAGTGAAGACGA. The annealed oligonucleotide pairs were ligated, which resulted in a mixture of ts with varying length that represents the varying number of 12mer repeats ligated to one BbsI/Kpnl segment. The products corresponding to the length of 36 amino acids were isolated from the mixture by preparative agarose gel electrophoresis and ligated into the Bsal/KpnI digested stuffer vector pCW035 9. Most of the clones in the resulting y designated LCW0401 showed green ﬂuorescence after ion, which shows that the sequence ofXTEN_AD36 had been ligated in frame with the GFP gene and that most sequences ofXTEN_AD36 had good expression levels.

We screened 96 isolates from library LCW0401 for high level of cence by stamping them onto agar plate containing IPTG. The same isolates were evaluated by PCR and 48 isolates were ﬁed that contained segments with 36 amino acids as well as strong ﬂuorescence. These isolates were sequenced and 39 clones were identiﬁed that contained correct XTEN_AD36 segments. The ﬁle names of the nucleotide and amino acid ucts for these segments are listed in Table 8.

Table 8: DNA and Amino Acid Seguences for 36-mer motifs File name Amino acid sequence Nucleotide sequence LCW0401_001_ GSGGEPSESGSSGESPGG GGTTCTGGTGGCGAACCGTCCGAGTCTGGTAGC GFP-N_A01.abl SSGSESGESPGGSSGSES TCAGGTGAATCTCCGGGTGGCTCTAGCGGTTCC GAGTCAGGTGAATCTCCTGGTGGTTCCAGCGGT TCCGAGTCA LCW0401_002_ GSEGSSGPGESSGESPGG GGTAGCGAAGGTTCTTCTGGTCCTGGCGAGTCT B01.abl SSGSESGSSESGSSEGGP TCAGGTGAATCTCCTGGTGGTTCCAGCGGTTCT GAATCAGGTTCCTCCGAAAGCGGTTCTTCCGAG GGCGGTCCA LCW0401_003_ GSSESGSSEGGPGSSESG GGTTCCTCTGAAAGCGGTTCTTCCGAAGGTGGT GFP-NiCO l .abl SSEGGPGESPGGSSGSES CCAGGTTCCTCTGAAAGCGGTTCTTCTGAGGGT GGTGAATCTCCGGGTGGCTCCAGCGGT TCCGAGTCA LCW0401_004_ SESGSSGSSESG GGTTCCGGTGGCGAACCGTCTGAATCTGGTAGC GFP-N_D01.abl SSEGGPGSGGEPSESGSS TCAGGTTCTTCTGAAAGCGGTTCTTCCGAGGGT GGTCCAGGTTCTGGTGGTGAACCTTCCGAGTCT GGTAGCTCA LCW0401_007_ SSEGGPGSEGSS GGTTCTTCCGAAAGCGGTTCTTCTGAGGGTGGT GFP-N_F01.abl GPGESSGSEGSSGPGESS CCAGGTAGCGAAGGTTCTTCCGGTCCAGGTGAG GGTAGCGAAGGTTCTTCTGGTCCTGGT GAATCTTCA LCW0401_008_ GSSESGSSEGGPGESPGG GGTTCCTCTGAAAGCGGTTCTTCCGAGGGTGGT GFP-N_G01.abl SSGSESGSEGSSGPGESS CCAGGTGAATCTCCAGGTGGTTCCAGCGGTTCT GAGTCAGGTAGCGAAGGTTCTTCTGGTCCAGGT GAATCCTCA LCW0401_012_ GSGGEPSESGSSGSGGEP GGTTCTGGTGGTGAACCGTCTGAGTCTGGTAGC GFP—N_H01.abl SESGSSGSEGSSGPGESS TCAGGTTCCGGTGGCGAACCATCCGAATCTGGT GGTAGCGAAGGTTCTTCCGGTCCAGGT GAGTCTTCA LCW0401_015_ GSSESGSSEGGPGSEGSS GGTTCTTCCGAAAGCGGTTCTTCCGAAGGCGGT WO 40093 File name Amino acid sequence Nucleotide sequence GFP-N_A02.ab1 GPGESSGESPGGSSGSES CCAGGTAGCGAAGGTTCTTCTGGTCCAGGCGAA TCTTCAGGTGAATCTCCTGGTGGCTCCAGCGGT TCTGAGTCA LCW0401_016_ GSSESGSSEGGPGSSESG GGTTCCTCCGAAAGCGGTTCTTCTGAGGGCGGT GFP—N_B02.ab1 SSEGGPGSSESGSSEGGP CCAGGTTCCTCCGAAAGCGGTTCTTCCGAGGGC GGTCCAGGTTCTTCTGAAAGCGGTTCTTCCGAG GGCGGTCCA LCW0401_020_ GSGGEPSESGSSGSEGSS GGTTCCGGTGGCGAACCGTCCGAATCTGGTAGC GFP-N_E02.ab1 GPGESSGSSESGSSEGGP TCAGGTAGCGAAGGTTCTTCTGGTCCAGGCGAA TCTTCAGGTTCCTCTGAAAGCGGTTCTTCTGAG GGCGGTCCA LCW040170227 GSGGEPSESGSSGSSESG GGTTCTGGTGGTGAACCGTCCGAATCTGGTAGC GFP-N_F02.ab1 SSEGGPGSGGEPSESGSS TCAGGTTCTTCCGAAAGCGGTTCTTCTGAAGGT GGTCCAGGTTCCGGTGGCGAACCTTCTGAATCT GGTAGCTCA LCW0401_024_ GSGGEPSESGSSGSSESG GGTTCTGGTGGCGAACCGTCCGAATCTGGTAGC GFP-N_G02.ab1 SSEGGPGESPGGSSGSES TCCTCCGAAAGCGGTTCTTCTGAAGGT GGTCCAGGTGAATCTCCAGGTGGTTCTAGCGGT TCTGAATCA LCW0401_026_ GSGGEPSESGSSGESPGG GGTTCTGGTGGCGAACCGTCTGAGTCTGGTAGC GFP-N_H02.ab1 SSGSESGSEGSSGPGESS TCAGGTGAATCTCCTGGTGGCTCCAGCGGTTCT GAATCAGGTAGCGAAGGTTCTTCTGGTCCTGGT LCW0401_027_ GSGGEPSESGSSGESPGG GGTTCCGGTGGCGAACCTTCCGAATCTGGTAGC GFP-N_A03.ab1 SSGSESGSGGEPSESGSS TCAGGTGAATCTCCGGGTGGTTCTAGCGGTTCT GAGTCAGGTTCTGGTGGTGAACCTTCCGAGTCT LCW0401_028_ GSSESGSSEGGPGSSESG GGTTCCTCTGAAAGCGGTTCTTCTGAGGGCGGT GFP-N_BO3.ab1 SSEGGPGSSESGSSEGGP CCAGGTTCTTCCGAAAGCGGTTCTTCCGAGGGC GGTCCAGGTTCTTCCGAAAGCGGTTCTTCTGAA GGCGGTCCA LCW0401_030_ GESPGGSSGSESGSEGSS GGTGAATCTCCGGGTGGCTCCAGCGGTTCTGAG CO3.ab1 GPGESSGSEGSSGPGESS TCAGGTAGCGAAGGTTCTTCCGGTCCGGGTGAG TCCTCAGGTAGCGAAGGTTCTTCCGGTCCTGGT GAGTCTTCA LCW0401_031_ GSGGEPSESGSSGSGGEP GGTTCTGGTGGCGAACCTTCCGAATCTGGTAGC GFP-N_D03.ab1 SESGSSGSSESGSSEGGP TCAGGTTCCGGTGGTGAACCTTCTGAATCTGGT AGCTCAGGTTCTTCTGAAAGCGGTTCTTCCGAG GGCGGTCCA LCW040170337 GSGGEPSESGSSGSGGEP GGTTCCGGTGGTGAACCTTCTGAATCTGGTAGC GFP-N_E03.ab1 GSGGEPSESGSS TCAGGTTCCGGTGGCGAACCATCCGAGTCTGGT AGCTCAGGTTCCGGTGGTGAACCATCCGAGTCT GGTAGCTCA LCW0401_037_ GSGGEPSESGSSGSSESG GGTTCCGGTGGCGAACCTTCTGAATCTGGTAGC GFP-N_F03.ab1 SSEGGPGSEGSSGPGESS TCCTCCGAAAGCGGTTCTTCTGAGGGC GGTAGCGAAGGTTCTTCTGGTCCGGGC GAGTCTTCA LCW0401_038_ GSGGEPSESGSSGSEGSS GGTTCCGGTGGTGAACCGTCCGAGTCTGGTAGC GFP-N_G03.ab1 GPGESSGSGGEPSESGSS TCAGGTAGCGAAGGTTCTTCTGGTCCGGGTGAG TCTTCAGGTTCTGGTGGCGAACCGTCCGAATCT GGTAGCTCA LCW0401_039_ SESGSSGESPGG GGTGGCGAACCGTCCGAATCTGGTAGC GFP-N_H03.ab1 SSGSESGSGGEPSESGSS TCAGGTGAATCTCCTGGTGGTTCCAGCGGTTCC GAGTCAGGTTCTGGTGGCGAACCTTCCGAATCT GGTAGCTCA LCW0401_040_ GSSESGSSEGGPGSGGEP TCCGAAAGCGGTTCTTCCGAGGGCGGT GFP-N_A04.ab1 SESGSSGSSESGSSEGGP CCAGGTTCCGGTGGTGAACCATCTGAATCTGGT AGCTCAGGTTCTTCTGAAAGCGGTTCTTCTGAA GGTGGTCCA File name Amino acid sequence Nucleotide sequence LCW0401_042_ GSEGSSGPGESSGESPGG GGTAGCGAAGGTTCTTCCGGTCCTGGTGAGTCT GFP-N_C04.ab1 SSGSESGSEGSSGPGESS TCAGGTGAATCTCCAGGTGGCTCTAGCGGTTCC GAGTCAGGTAGCGAAGGTTCTTCTGGTCCTGGC GAGTCCTCA 1_046_ GSSESGSSEGGPGSSESG GGTTCCTCTGAAAGCGGTTCTTCCGAAGGCGGT GFP-N_D04.ab1 GSSESGSSEGGP CCAGGTTCTTCCGAAAGCGGTTCTTCTGAGGGC GGTCCAGGTTCCTCCGAAAGCGGTTCTTCTGAG GGTGGTCCA LCW0401_047_ GSGGEPSESGSSGESPGG GGTTCTGGTGGCGAACCTTCCGAGTCTGGTAGC GFP-N_EO4.ab1 SSGSESGESPGGSSGSES TCAGGTGAATCTCCGGGTGGTTCTAGCGGTTCC GAGTCAGGTGAATCTCCGGGTGGTTCCAGCGGT TCTGAGTCA LCW0401_051_ GSGGEPSESGSSGSEGSS GGTGGCGAACCATCTGAGTCTGGTAGC GFP-N_F04.ab1 GPGESSGESPGGSSGSES TCAGGTAGCGAAGGTTCTTCCGGTCCAGGCGAG TCTTCAGGTGAATCTCCTGGTGGCTCCAGCGGT TCTGAGTCA LCW0401_053_ GESPGGSSGSESGESPGG GGTGAATCTCCTGGTGGTTCCAGCGGTTCCGAG GFP-N_H04.ab1 SSGSESGESPGGSSGSES TCAGGTGAATCTCCAGGTGGCTCTAGCGGTTCC GAGTCAGGTGAATCTCCTGGTGGTTCTAGCGGT TCTGAATCA LCW0401_054_ GSEGSSGPGESSGSEGSS GGTAGCGAAGGTTCTTCCGGTCCAGGTGAATCT GFP-N_A05.ab1 GPGESSGSGGEPSESGSS TCAGGTAGCGAAGGTTCTTCTGGTCCTGGTGAA TCCTCAGGTTCCGGTGGCGAACCATCTGAATCT GGTAGCTCA LCW0401_059_ GSGGEPSESGSSGSEGSS GGTTCTGGTGGCGAACCATCCGAATCTGGTAGC GFP-N_D05.ab1 GPGESSGESPGGSSGSES TCAGGTAGCGAAGGTTCTTCTGGTCCTGGCGAA TCTTCAGGTGAATCTCCAGGTGGCTCTAGCGGT TCCGAATCA LCW0401_060_ GSGGEPSESGSSGSSESG GGTTCCGGTGGTGAACCGTCCGAATCTGGTAGC GFP-N_E05.ab1 SSEGGPGSGGEPSESGSS TCAGGTTCCTCTGAAAGCGGTTCTTCCGAGGGT GGTCCAGGTTCCGGTGGTGAACCTTCTGAGTCT GGTAGCTCA LCW0401_061_ GSSESGSSEGGPGSGGEP GGTTCCTCTGAAAGCGGTTCTTCTGAGGGCGGT GFP-N_F05.ab1 SESGSSGSEGSSGPGESS TCTGGTGGCGAACCATCTGAATCTGGT AGCTCAGGTAGCGAAGGTTCTTCCGGTCCGGGT 1_063_ GSGGEPSESGSSGSEGSS GGTTCTGGTGGTGAACCGTCCGAATCTGGTAGC GFP-N_H05.ab1 GPGESSGSEGSSGPGESS TCAGGTAGCGAAGGTTCTTCTGGTCCTGGCGAG TCTTCAGGTAGCGAAGGTTCTTCTGGTCCTGGT GAATCTTCA 1_066_ GSGGEPSESGSSGSSESG GGTTCTGGTGGCGAACCATCCGAGTCTGGTAGC GFP-N_B06.ab1 SSEGGPGSGGEPSESGSS TCAGGTTCTTCCGAAAGCGGTTCTTCCGAAGGC GGTCCAGGTTCTGGTGGTGAACCGTCCGAATCT GGTAGCTCA LCW0401_067_ GSGGEPSESGSSGESPGG GGTTCCGGTGGCGAACCTTCCGAATCTGGTAGC GFP-N_C06.ab1 SSGSESGESPGGSSGSES TCAGGTGAATCTCCGGGTGGTTCTAGCGGTTCC GAATCAGGTGAATCTCCAGGTGGTTCTAGCGGT TCCGAATCA LCW0401_069_ GSGGEPSESGSSGSGGEP GGTGGTGAACCATCTGAGTCTGGTAGC GFP-N_D06.ab1 SESGSSGESPGGSSGSES TCAGGTTCCGGTGGCGAACCGTCCGAGTCTGGT AGCTCAGGTGAATCTCCGGGTGGTTCCAGCGGT TCCGAATCA LCW0401_070_ GSEGSSGPGESSGSSESG GGTAGCGAAGGTTCTTCTGGTCCGGGCGAATCC GFP-N_EO6.ab1 SSEGGPGSEGSSGPGESS TCCTCCGAAAGCGGTTCTTCCGAAGGT GGTAGCGAAGGTTCTTCCGGTCCTGGT GAATCTTCA LCW0401_078_ GSSESGSSEGGPGESPGG GGTTCCTCTGAAAGCGGTTCTTCTGAAGGCGGT GFP-N_F06.ab1 SSGSESGESPGGSSGSES GAATCTCCGGGTGGCTCCAGCGGTTCT GAATCAGGTGAATCTCCTGGTGGCTCCAGCGGT TCCGAGTCA —Amino acid sequence Nucleotide ce LCW0401_079_ GSEGSSGPGESSGSEGSS GGTAGCGAAGGTTCTTCTGGTCCAGGCGAGTCT GFP-N_G06.abl GPGESSGSGGEPSESGSS TCAGGTAGCGAAGGTTCTTCCGGTCCTGGCGAG TCTTCAGGTTCCGGTGGCGAACCGTCCGAATCT GGTAGCTCA Example 2: uction of XTEN_AE36 segments A codon library encoding XTEN sequences of 36 amino acid length was constructed. The XTEN sequence was designated XTENiAE36. Its ts have the amino acid sequence [X]3 where X is a 12mer peptide with the sequence: GSPAGSPTSTEE, GSEPATSGSE TP, GTSESA TPESGP, or GTSTEPSEGSAP. The insert was obtained by ing the following pairs of phosphorylated synthetic oligonucleotide pairs: AEl for: AGGTAGCCCDGCWGGYTCTCCDACYTCYACYGARGA AB 1 rev: ACCTTCYTCRGTRGARGTHGGAGARCCWGCHGGGCT AEZfor: AGGTAGCGAACCKGCWACYTCYGGYTCTGARACYCC : ACCTGGRGTYTCAGARCCRGARGTWGCMGGTTCGCT AE3 for: AGGTACYTCTGAAAGCGCWACYCCKGARTCYGGYCC AE3rev: ACCTGGRCCRGAYTCMGGRGTWGCGCTTTCAGARGT AE4for: AGGTACYTCTACYGAACCKTCYGARGGYAGCGCWCC AE4reV: ACCTGGWGCGCTRCCYTCRGAMGGTTCRGTAGARGT We also annealed the phosphorylated oligonucleotide 3KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC and the non-phosphorylated oligonucleotide pr_3KpnIstopperRev: GTGAAGACGA. The annealed oligonucleotide pairs were ligated, which resulted in a mixture of products with g length that represents the varying number of 12mer repeats d to one BbsI/Kpnl segment. The products corresponding to the length of 36 amino acids were isolated from the mixture by ative agarose gel electrophoresis and ligated into the Bsal/Kpnl digested stuffer vector pCWO35 9. Most of the clones in the resulting library designated LCW0402 showed green ﬂuorescence after induction which shows that the sequence ofXTEN_AE36 had been ligated in frame with the GFP gene and most sequences ofXTEN_AE36 show good expression.

We screened 96 isolates from library LCW0402 for high level of ﬂuorescence by stamping them onto agar plate containing IPTG. The same isolates were evaluated by PCR and 48 isolates were identiﬁed that contained ts with 36 amino acids as well as strong cence. These isolates were sequenced and 37 clones were identiﬁed that contained correct XTEN_AE36 ts. The ﬁle names of the nucleotide and amino acid constructs for these segments are listed in Table 9.

Table 9: DNA and Amino Acid Seguences for 36—mer motifs File name Amino acid sequence Nucleotide sequence LCW0402_002_ GSPAGSPTSTEEGTSE GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAA GFP-N_A07 . ab 1 SATPESGPGTSTEPSE GGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCA GSAP GGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCA LCW0402_003_ GTSTEPSEGSAPGTST GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCA GFP-N_BO7 . ab 1 EPSEGSAPGTSTEPSE TCTACTGAACCTTCCGAGGGCAGCGCTCCA GSAP GGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCA File name Amino acid sequence Nucleotide sequence Lcw0402_004_ SEGSAPGTSE GGTACCTCTACCGAACCGTCTGAAGGTAGCGCACCA GFP-\_C07.ab1 SATPESGPGTSESATP GGTACCTCTGAAAGCGCAACTCCTGAGTCCGGTCCA ESGP GGTACTTCTGAAAGCGCAACCCCGGAGTCTGGCCCA LCW0402_005_ GTSTEPSEGSAPGTSE GGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCA GFP—\_D07.ab1 SATPESGPGTSESATP GGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCCA ESGP GGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCA LCW0402_006_ SGSETPGTSE GGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCA EO7.ab1 SATPESGPGSPAGSPT GGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCCA STEE GGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAA LCW0402_008_ GTSESATPESGPGSEP TCTGAAAGCGCAACCCCTGAATCCGGTCCA GFP-\_F07.ab1 TPGTSTEPSE GGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCA GSAP GGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCA LCW0402_009_ GSPAGSPTSTEEGSPA GGTAGCCCGGCTGGCTCTCCAACCTCCACTGAGGAA GFP—\_G07.ab1 GSPTSTEEGSEPATSG GGTAGCCCGGCTGGCTCTCCAACCTCCACTGAAGAA SETP GGTAGCGAACCGGCTACCTCCGGCTCTGAAACTCCA LCW0402_011_ GSPAGSPTSTEEGTSE GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAA GFP-\_A08.ab1 GPGTSTEPSE GGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCA GSAP GGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCA Lcw0402_012_ PTSTEEGSPA GGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAA B08eab1 GSPTSTEEGTSTEPSE GGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAA GSAP GGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCA LCW0402_013_ GTSESATPESGPGTST GGTACTTCTGAAAGCGCTACTCCGGAGTCCGGTCCA GFP-\_C08.ab1 EPSEGSAPGTSTEPSE GGTACCTCTACCGAACCGTCCGAAGGCAGCGCTCCA GSAP GGTACTTCTACTGAACCTTCTGAGGGTAGCGCTCCA LCW0402_014_ GTSTEPSEGSAPGSPA GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCA GFP-\_D08.ab1 GSPTSTEEGTSTEPSE GGTAGCCCGGCAGGTTCTCCTACTTCCACTGAGGAA GSAP GGTACTTCTACCGAACCTTCTGAGGGTAGCGCACCA LCW040270157 GSEPATSGSETPGSPA GGTAGCGAACCGGCTACTTCCGGCTCTGAGACTCCA GFP-\_E08.ab1 GSPTSTEEGTSESATP GGTAGCCCTGCTGGCTCTCCGACCTCTACCGAAGAA ESGP GGTACCTCTGAAAGCGCTACCCCTGAGTCTGGCCCA 2_016_ GTSTEPSEGSAPGTSE GGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCA GFP-\_F08.ab1 SATPESGPGTSESATP GGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCA ESGP GGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCA 2_020_ GTSTEPSEGSAPGSEP GGTACTTCTACTGAACCGTCTGAAGGCAGCGCACCA GFP-\7G08.ab1 ATSGSETPGSPAGSPT GGTAGCGAACCGGCTACTTCCGGTTCTGAAACCCCA STEE GGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAA Lcw0402_023_ GSPAGSPTSTEEGTSE GGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAA GFP-\_A09.ab1 SATPESGPGSEPATSG GGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCA SETP GGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCA LCW0402_024_ GTSESATPESGPGSPA GGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCA GFP-\_B09.ab1 GSPTSTEEGSPAGSPT GGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAA STEE GGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAA 2_025_ GTSTEPSEGSAPGTSE GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCA GFP-\7CO9.ab1 SATPESGPGTSTEPSE GGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCA GSAP GGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA LCW0402_026_ GSPAGSPTSTEEGTST GGTAGCCCGGCAGGCTCTCCGACTTCCACCGAGGAA GFP-\_D09.ab1 EPSEGSAPGSEPATSG GGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCA SETP GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCA LCW0402_027_ GSPAGSPTSTEEGTST GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAA GFP-\_E09.ab1 EPSEGSAPGTSTEPSE GGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCA GSAP GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCA LCW0402_032_ GSEPATSGSETPGTSE GAACCTGCTACCTCCGGTTCTGAAACCCCA GFP—\_H09.ab1 SATPESGPGSPAGSPT GGTACCTCTGAAAGCGCAACTCCGGAGTCTGGTCCA STEE GGTAGCCCTGCAGGTTCTCCTACCTCCACTGAGGAA Lcw0402_034_ GTSESATPESGPGTST GGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCA GFP-\_A10.ab1 EPSEGSAPGTSTEPSE GGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCA GSAP GGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA 2_036_ GSPAGSPTSTEEGTST GGTAGCCCGGCTGGTTCTCCGACTTCCACCGAGGAA GFP-\_C10.ab1 EPSEGSAPGTSTEPSE GGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCA File name Amino acid sequence Nucleotide ce GSAP GGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCA LCW0402_039_ GTSTEPSEGSAPGTST GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCA GFP-\_E10.abl EPSEGSAPGTSTEPSE GGTACTTCTACTGAACCTTCTGAAGGCAGCGCTCCA GSAP GGTACTTCTACTGAACCTTCCGAAGGTAGCGCACCA LCW0402_040_ GSEPATSGSETPGTSE GGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCA GFP-\_F10.ab1 SATPESGPGTSTEPSE GGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCA GSAP TCTACTGAACCGTCCGAGGGCAGCGCACCA LCW0402_041_ GTSTEPSEGSAPGSPA GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCA GFP—\_G10.abl GSPTSTEEGTSTEPSE GGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAA GSAP GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCA LCW0402_050_ GSEPATSGSETPGTSE GGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCA GFP-\_Al l.abl SATPESGPGSEPATSG GGTACTTCTGAAAGCGCTACTCCGGAATCCGGCCCA SETP GGTAGCGAACCGGCTACTTCCGGCTCTGAAACCCCA LCW0402_051_ GSEPATSGSETPGTSE GGTAGCGAACCGGCAACTTCCGGCTCTGAAACCCCA GFP-\_B11.ab1 SATPESGPGSEPATSG GGTACTTCTGAAAGCGCTACTCCTGAGTCTGGCCCA SETP GGTAGCGAACCTGCTACCTCTGGCTCTGAAACCCCA LCW0402_059_ GSEPATSGSETPGSEP GGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCA El l.ab1 ATSGSETPGTSTEPSE GAACCTGCAACCTCCGGCTCTGAAACCCCA GSAP GGTACTTCTACTGAACCTTCTGAGGGCAGCGCACCA LCW0402_060_ GTSESATPESGPGSEP GGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCA GFP-\_Fl l.ab1 ATSGSETPGSEPATSG GGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCA SETP GGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCA LCW0402_061_ GTSTEPSEGSAPGTST GGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCA GFP-\_G1 labl EPSEGSAPGTSESATP GGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCA ESGP GGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCA 2_065_ GSEPATSGSETPGTSE GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCA GFP-\_A12.abl SATPESGPGTSESATP GGTACCTCTGAAAGCGCTACTCCGGAATCTGGTCCA ESGP GGTACTTCTGAAAGCGCTACTCCGGAATCCGGTCCA LCW0402_066_ GSEPATSGSETPGSEP GGTAGCGAACCTGCTACCTCCGGCTCTGAAACTCCA GFP-\_B12.abl TPGTSTEPSE GGTAGCGAACCGGCTACTTCCGGTTCTGAAACTCCA GSAP TCTACCGAACCTTCCGAAGGCAGCGCACCA LCW0402_067_ GSEPATSGSETPGTST GGTAGCGAACCTGCTACTTCTGGTTCTGAAACTCCA C12‘abl EPSEGSAPGSEPATSG GGTACTTCTACCGAACCGTCCGAGGGTAGCGCTCCA SETP GGTAGCGAACCTGCTACTTCTGGTTCTGAAACTCCA LCW0402_069_ GTSTEPSEGSAPGTST GGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCA GFP-\_D12.abl EPSEGSAPGSEPATSG GGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCA SETP GGTAGCGAACCGGCAACCTCCGGTTCTGAAACTCCA LCW0402_073_ GTSTEPSEGSAPGSEP GGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCA GFP-\_F12.abl ATSGSETPGSPAGSPT GGTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCA STEE GGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAA LCW0402_074_ GSEPATSGSETPGSPA GGTAGCGAACCGGCTACTTCCGGCTCTGAGACTCCA GFP-\_G12.abl GSPTSTEEGTSESATP GGTAGCCCAGCTGGTTCTCCAACCTCTACTGAGGAA ESGP GGTACTTCTGAAAGCGCTACCCCTGAATCTGGTCCA LCW0402_075_ GTSESATPESGPGSEP GGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCA GFP-\_H12.abl ATSGSETPGTSESATP GGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCA ESGP GGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCA Example 3: Construction of XTEN_AF36 ts A codon library encoding sequences of 36 amino acid length was constructed. The sequences were designated XTEN_AF3 6. Its segments have the amino acid sequence [X]3 where X is a 12mer peptide with the sequence: GSTSESPSGTAP, GTSTPESGSASP, GTSPSGESSTAP, or GSTSSTAESPGP. The insert was ed by ing the following pairs of phosphorylated tic oligonucleotide pairs: AFl for: AGGTTCTACYAGCGAATCYCCKTCTGGYACYGCWCC AF 1 rev: WGCRGTRCCAGAMGGRGATTCGCTRGTAGA AFZfor: AGGTACYTCTACYCCKGAAAGCGGYTCYGCWTCTCC AFZrev: ACCTGGAGAWGCRGARCCGCTTTCMGGRGTAGARGT AF3 for: AGGTACYTCYCCKAGCGGYGAATCTTCTACYGCWCC AF3rev: ACCTGGWGCRGTAGAAGATTCRCCGCTMGGRGARGT AF4for: AGGTTCYACYAGCTCTACYGCWGAATCTCCKGGYCC : ACCTGGRCCMGGAGATTCWGCRGTAGAGCTRGTRGA We also annealed the orylated oligonucleotide 3KpnlstopperFor: AGGTTCGTCTTCACTCGAGGGTAC and the non-phosphorylated oligonucleotide pr_3KpnIstopperRev: CCTCGAGTGAAGACGA. The annealed oligonucleotide pairs were ligated, which resulted in a mixture of products with varying length that represents the varying number of 12mer repeats ligated to one BbsI/Kpnl segment The products corresponding to the length of 36 amino acids were isolated from the mixture by preparative agarose gel electrophoresis and ligated into the BsaI/Kpnl ed stuffer vector pCWO35 9. Most of the clones in the resulting y designated LCW0403 showed green ﬂuorescence after induction which shows that the sequence ofXTEN_AF36 had been ligated in frame with the GFP gene and most sequences ofXTEN_AF36 show good expression.

We screened 96 isolates from library 3 for high level of ﬂuorescence by stamping them onto agar plate containing lPTG. The same es were evaluated by PCR and 48 isolates were identiﬁed that contained segments with 36 amino acids as well as strong ﬂuorescence. These isolates were sequenced and 44 clones were identiﬁed that contained correct XTEN_AF36 segments. The ﬁle names of the nucleotide and amino acid constructs for these segments are listed in Table 10.

Table 10: DNA and Amino Acid Seguences for 36-mer motifs File name Amino acid SCI uence Nucleotide SQ a_uence 370047 GTSTPESGSASPGTSP GGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCA GFP-\_A0 l i ab 1 SGESSTAPGTSPSGES GGTACTTCTCCTAGCGGTGAATCTTCTACTGCTCCAG STAP GTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCA LCW0403_005_ GTSPSGESSTAPGSTS GGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCA B0 l . ab 1 STABSPGPGTSPSGES GGTTCTACTAGCTCTACCGCTGAATCTCCGGGCCCAG STAP CTCCGAGCGGTGAATCTTCTACTGCTCCA LCW0403_006_ AESPGPGTSP GGTTCCACCAGCTCTACTGCTGAATCTCCTGGTCCAG GFP-\iCO l .abl SGESSTAPGTSTPESG GTACCTCTCCTAGCGGTGAATCTTCTACTGCTCCAGG SASP TACTTCTACTCCTGAAAGCGGCTCTGCTTCTCCA LCW0403_007_ GSTSSTAESPGPGSTS ACCAGCTCTACTGCAGAATCTCCTGGCCCAG GFP-\_D01.abl STAESPGPGTSPSGES GTTCCACCAGCTCTACCGCAGAATCTCCGGGTCCAG STAP GTACTTCCCCTAGCGGTGAATCTTCTACCGCACCA LCW0403_008_ GSTSSTAESPGPGTSP GGTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCAG GFP-\_E01.abl SGESSTAPGTSTPESG GTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGG SASP TACCTCTACTCCGGAAAGCGGTTCTGCATCTCCA 3_010_ GSTSSTAESPGPGTST GGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAG GFP-\_F01.abl PESGSASPGSTSESPS GTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAG GTAP GTTCTACTAGCGAATCTCCTTCTGGCACTGCACCA 3_011_ GSTSSTAESPGPGTST GGTTCTACTAGCTCTACTGCAGAATCTCCTGGCCCAG GFP-\_G01.abl PESGSASPGTSTPESG GTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAG SASP GTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCA LCW0403_012_ GSTSESPSGTAPGTSP GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAG GFP-\_H01.abl SGESSTAPGSTSESPS GTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGG File name Amino acid sequence Nucleotide sequence GTAP TTCTACTAGCGAATCTCCTTCTGGCACTGCACCA LCW0403_013_ GSTSSTAESPGPGSTS GGTTCCACCAGCTCTACTGCAGAATCTCCGGGCCCA GFP-\_A02.ab1 STAESPGPGTSPSGES GGTTCTACTAGCTCTACTGCAGAATCTCCGGGTCCAG STAP GTACTTCTCCTAGCGGCGAATCTTCTACCGCTCCA LCW0403_014_ GSTSSTAESPGPGTST GGTTCCACTAGCTCTACTGCAGAATCTCCTGGCCCAG GFP-\_B02.ab1 PESGSASPGSTSESPS GTACCTCTACCCCTGAAAGCGGCTCTGCATCTCCAG GTAP GTTCTACCAGCGAATCCCCGTCTGGCACCGCACCA LCW0403_015_ AESPGPGSTS ACTAGCTCTACTGCTGAATCTCCGGGTCCAG GFP—\_C02.ab1 STAESPGPGTSPSGES GTTCTACCAGCTCTACTGCTGAATCTCCTGGTCCAGG STAP TACCTCCCCGAGCGGTGAATCTTCTACTGCACCA LCW0403_017_ GSTSSTAESPGPGSTS GGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAG D02.ab1 ESPSGTAPGSTSSTAE GTTCTACCAGCGAATCCCCGTCTGGCACCGCACCAG SPGP GTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCA LCW0403_018_ GSTSSTAESPGPGSTS GGTTCTACCAGCTCTACCGCAGAATCTCCTGGCCCA GFP-\_E02.ab1 STAESPGPGSTSSTAE GGTTCCACTAGCTCTACCGCTGAATCTCCTGGTCCAG SPGP GTTCTACTAGCTCTACCGCTGAATCTCCTGGTCCA 3_019_ GSTSESPSGTAPGSTS GGTTCTACTAGCGAATCCCCTTCTGGTACTGCTCCAG GFP—\_F02.ab1 STAESPGPGSTSSTAE GTTCCACTAGCTCTACCGCTGAATCTCCTGGCCCAGG SPGP TAGCTCTACTGCAGAATCTCCTGGTCCA LCW0403_023_ GSTSESPSGTAPGSTS GGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAG GFP-\_H02.ab1 ESPSGTAPGSTSESPS GTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGG GTAP TTCTACCAGCGAATCTCCTTCTGGTACTGCACCA 3_024_ GSTSSTAESPGPGSTS GGTTCCACCAGCTCTACTGCTGAATCTCCTGGCCCAG GFP-\_A03.ab1 STAESPGPGSTSSTAE GTTCTACCAGCTCTACTGCTGAATCTCCGGGCCCAGG SPGP TTCCACCAGCTCTACCGCTGAATCTCCGGGTCCA LCW0403_025_ GSTSSTAESPGPGSTS GGTTCCACTAGCTCTACCGCAGAATCTCCTGGTCCAG GFP-\_B03.ab1 STAESPGPGTSPSGES CTAGCTCTACTGCTGAATCTCCGGGTCCAGG STAP TACCTCCCCTAGCGGCGAATCTTCTACCGCTCCA LCW0403_028_ GSSPSASTGTGPGSST GGTTCTAGCCCTTCTGCTTCCACCGGTACCGGCCCAG D03.ab1 PSGATGSPGSSTPSGA CTACTCCGTCTGGTGCAACTGGCTCTCCAGG TGSP TAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCA LCW0403_029_ GTSPSGESSTAPGTST GGTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAG GFP-\_E03.ab1 PESGSASPGSTSSTAE GTACCTCTACTCCGGAAAGCGGCTCCGCATCTCCAG SPGP GTTCTACTAGCTCTACTGCTGAATCTCCTGGTCCA LCW0403_030_ GSTSSTAESPGPGSTS GGTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCAG GFP-\_F03.ab1 STAESPGPGTSTPESG GTTCTACCAGCTCTACTGCAGAATCTCCTGGCCCAGG SASP TACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCA 3_031_ GTSPSGESSTAPGSTS GGTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAG GFP-\_G03.ab1 STAESPGPGTSTPESG GTTCTACCAGCTCTACTGCTGAATCTCCTGGCCCAGG SASP TACTTCTACCCCGGAAAGCGGCTCCGCTTCTCCA 3_033_ GSTSESPSGTAPGSTS GGTTCTACTAGCGAATCCCCTTCTGGTACTGCACCAG GFP-\_H03.ab1 GPGSTSSTAE GTTCTACCAGCTCTACTGCTGAATCTCCGGGCCCAGG SPGP TTCCACCAGCTCTACCGCAGAATCTCCTGGTCCA LCW0403_035_ GSTSSTAESPGPGSTS ACCAGCTCTACCGCTGAATCTCCGGGCCCA GFP-\_A04.ab1 ESPSGTAPGSTSSTAE GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACCA SPGP GGTTCTACTAGCTCTACCGCAGAATCTCCGGGCCCA LCW0403_036_ GSTSSTAESPGPGTSP GGTTCTACCAGCTCTACTGCTGAATCTCCGGGTCCAG GFP-\7BO4.ab1 SGESSTAPGTSTPESG GTACTTCCCCGAGCGGTGAATCTTCTACTGCACCAG SASP GTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCA LCW0403_039_ GSTSESPSGTAPGSTS GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAG GFP-\_C04.ab1 APGTSPSGES GTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAG STAP GTACTTCTCCTAGCGGCGAATCTTCTACCGCACCA LCW0403_041_ GSTSESPSGTAPGSTS GGTTCTACCAGCGAATCCCCTTCTGGTACTGCTCCAG GFP-\_D04.ab1 ESPSGTAPGTSTPESG GTTCTACCAGCGAATCCCCTTCTGGCACCGCACCAG SASP GTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCA LCW0403_044_ GTSTPESGSASPGSTS GGTACCTCTACTCCTGAAAGCGGTTCTGCATCTCCAG GFP-\_EO4.ab1 STAESPGPGSTSSTAE GTTCCACTAGCTCTACCGCAGAATCTCCGGGCCCAG SPGP GTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCA LCW0403_046_ GSTSESPSGTAPGSTS GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACCA File name Amino acid sequence Nucleotide sequence GFP-\_F04.ab1 ESPSGTAPGTSPSGES GGTTCTACTAGCGAATCCCCTTCTGGTACCGCACCAG STAP GTACTTCTCCGAGCGGCGAATCTTCTACTGCTCCA LCW0403_047_ GSTSSTAESPGPGSTS GGTTCTACTAGCTCTACCGCTGAATCTCCTGGCCCAG GFP-\_G04.ab1 STAESPGPGSTSESPS CTAGCTCTACCGCAGAATCTCCGGGCCCAG GTAP GTTCTACTAGCGAATCCCCTTCTGGTACCGCTCCA LCW0403_049_ GSTSSTAESPGPGSTS GGTTCCACCAGCTCTACTGCAGAATCTCCTGGCCCA GFP-\_H04.ab1 STAESPGPGTSTPESG GGTTCTACTAGCTCTACCGCAGAATCTCCTGGTCCAG SASP GTACCTCTACTCCTGAAAGCGGTTCCGCATCTCCA LCW0403_051_ GSTSSTAESPGPGSTS GGTTCTACTAGCTCTACTGCTGAATCTCCGGGCCCAG GFP-\_A05.ab1 STAESPGPGSTSESPS GTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCAGG GTAP TTCTACTAGCGAATCTCCTTCTGGTACCGCTCCA LCW0403_053_ ESSTAPGSTS GGTACCTCCCCGAGCGGTGAATCTTCTACTGCACCA GFP-\_B05‘ab1 ESPSGTAPGSTSSTAE GGTTCTACTAGCGAATCCCCTTCTGGTACTGCTCCAG SPGP GTTCCACCAGCTCTACTGCAGAATCTCCGGGTCCA LCW0403_054_ PSGTAPGTSP GGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAG GFP-\_C05.ab1 SGESSTAPGSTSSTAE GTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGG SPGP TTCTACCAGCTCTACCGCAGAATCTCCGGGTCCA 3_057_ GSTSSTAESPGPGSTS GGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAG GFP-\_D05.ab1 ESPSGTAPGTSPSGES GTTCTACTAGCGAATCTCCGTCTGGCACCGCACCAG STAP GTACTTCCCCTAGCGGTGAATCTTCTACTGCACCA LCW0403_058_ GSTSESPSGTAPGSTS ACTAGCGAATCTCCTTCTGGCACTGCACCAG GFP-\_E05.ab1 ESPSGTAPGTSTPESG GTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAG SASP GTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCA LCW0403_060_ GTSTPESGSASPGSTS GGTACCTCTACTCCGGAAAGCGGTTCCGCATCTCCA GFP-\_F05.ab1 ESPSGTAPGSTSSTAE GGTTCTACCAGCGAATCCCCGTCTGGCACCGCACCA SPGP GGTTCTACTAGCTCTACTGCTGAATCTCCGGGCCCA LCW0403_063_ GSTSSTAESPGPGTSP GGTTCTACTAGCTCTACTGCAGAATCTCCGGGCCCA GFP-\7G05.ab1 APGTSPSGES GGTACCTCTCCTAGCGGTGAATCTTCTACCGCTCCAG STAP CTCCGAGCGGTGAATCTTCTACCGCTCCA LCW0403_064_ ESSTAPGTSP GGTACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAG GFP-\_H05.ab1 SGESSTAPGTSPSGES CTCCTAGCGGCGAATCTTCTACCGCTCCAGG STAP CCCTAGCGGTGAATCTTCTACCGCACCA LCW0403_065_ GSTSSTAESPGPGTST ACTAGCTCTACTGCTGAATCTCCTGGCCCAG GFP-\_A06.ab1 PESGSASPGSTSESPS GTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGG GTAP TTCTACTAGCGAATCTCCGTCTGGCACCGCACCA LCW0403_066_ GSTSESPSGTAPGTSP GGTTCTACTAGCGAATCTCCGTCTGGCACTGCTCCAG GFP-\_BO6.ab1 SGESSTAPGTSPSGES GTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGG STAP TACTTCCCCTAGCGGCGAATCTTCTACCGCTCCA LCW0403_067_ GSTSESPSGTAPGTST GGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAG GFP-\_CO6.ab1 PESGSASPGSTSSTAE GTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCAGG SPGP TTCCACTAGCTCTACCGCTGAATCTCCGGGTCCA LCW0403_068_ GSTSSTAESPGPGSTS ACTAGCTCTACTGCTGAATCTCCTGGCCCAG GFP-\_DO6.ab1 STAESPGPGSTSESPS GTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGG GTAP TTCTACCAGCGAATCTCCGTCTGGCACCGCACCA LCW0403_069_ GSTSESPSGTAPGTST GGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCA E06.ab1 PESGSASPGTSTPESG GGTACTTCTACCCCGGAAAGCGGCTCTGCTTCTCCAG SASP GTACTTCTACCCCGGAAAGCGGCTCCGCATCTCCA LCW0403_070_ GSTSESPSGTAPGTST GGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAG GFP-\_F06.ab1 PESGSASPGTSTPESG GTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGG SASP TACCTCTACTCCGGAAAGCGGTTCTGCATCTCCA Example 4: Construction of XTEN_AG36 segments A codon library encoding sequences of 36 amino acid length was constructed. The sequences were designated G36. Its segments have the amino acid sequence [X]3 Where X is a 12mer peptide With the sequence: GTPGSGTASSSP, GSSTPSGATGSP, GSSPSASTGTGP, or GASPGTSSTGSP. The insert was obtained by annealing the following pairs of phosphorylated synthetic oligonucleotide pairs: AG] for: AGGTACYCCKGGYAGCGGTACYGCWTCTTCYTCTCC AGlreV: ACCTGGAGARGAAGAWGCRGTACCGCTRCCMGGRGT AGZfor: AGGTAGCTCTACYCCKTCTGGTGCWACYGGYTCYCC : ACCTGGRGARCCRGTWGCACCAGAMGGRGTAGAGCT AG3for: AGGTTCTAGCCCKTCTGCWTCYACYGGTACYGGYCC AG3reV: ACCTGGRCCRGTACCRGTRGAWGCAGAMGGGCTAGA : AGGTGCWTCYCCKGGYACYAGCTCTACYGGTTCTCC AG4reV: AGAACCRGTAGAGCTRGTRCCMGGRGAWGC We also annealed the orylated ucleotide 3KpnlstopperFor: AGGTTCGTCTTCACTCGAGGGTAC and the non-phosphorylated oligonucleotide pr_3Kpnlst0pperReV: CCTCGAGTGAAGACGA. The ed oligonucleotide pairs were ligated, which resulted in a mixture of products with varying length that represents the varying number of 12mer repeats ligated to one Bbsl/Kpnl segment. The products corresponding to the length of 36 amino acids were isolated from the mixture by preparative agarose gel electrophoresis and ligated into the Bsal/Kpnl digested stuffer vector pCWO35 9. Most of the clones in the resulting library designated LCW0404 showed green ﬂuorescence after induction which shows that the sequence ofXTENiAG36 had been ligated in frame with the GFP gene and most sequences ofXTEN_AG36 show good expression.

We screened 96 es from library LCW0404 for high level of ﬂuorescence by stamping them onto agar plate containing lPTG. The same es were evaluated by PCR and 48 isolates were identiﬁed that contained segments with 36 amino acids as well as strong ﬂuorescence. These isolates were sequenced and 44 clones were identiﬁed that contained correct XTEN_AG36 segments. The ﬁle names of the nucleotide and amino acid constructs for these segments are listed in Table 11.

Table 11: DNA and Amino Acid Seguences for 36-mer motifs File name Amino acid ce Nucleotide sequence LCW0404_001_ GASPGTSSTGSPGTPG GGTGCATCCCCGGGCACTAGCTCTACCGGTTCTCCAGGTA TGSP ACTCCTTCTGGTGCTACTGGTTCTCCA LCW0404_003_ GSSTPSGATGSPGSSP GGTAGCTCTACCCCTTCTGGTGCTACCGGCTCTCCAGGTT B07.abl SASTGTGPGSSTPSGA CTAGCCCGTCTGCTTCTACCGGTACCGGTCCAGGTAGCTC TGSP TACCCCTTCTGGTGCTACTGGTTCTCCA LCW0404_006_ GASPGTSSTGSPGSSP GGTGCATCTCCGGGTACTAGCTCTACCGGTTCTCCAGGTT GFP-\_CO7.abl SASTGTGPGSSTPSGA CTAGCCCTTCTGCTTCCACTGGTACCGGCCCAGGTAGCTC TGSP TACCCCGTCTGGTGCTACTGGTTCCCCA LCW0404_007_ TASSSPGSST GGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTA GFP-\_D07.abl PSGATGSPGASPGTSS GCTCTACCCCTTCTGGTGCAACTGGTTCCCCAGGTGCATC CCCTGGTACTAGCTCTACCGGTTCTCCA LCW0404_009_ GTPGSGTASSSPGASP GGTACCCCTGGCAGCGGTACTGCTTCTTCTTCTCCAGGTG E07.abl GTSSTGSPGSRPSAST CTTCCCCTGGTACCAGCTCTACCGGTTCTCCAGGTTCTAG ACCTTCTGCATCCACCGGTACTGGTCCA LCW0404_01 l_ GASPGTSSTGSPGSST GGTGCATCTCCTGGTACCAGCTCTACCGGTTCTCCAGGTA GFP-\_F07.abl PSGATGSPGASPGTSS GCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCC TGSP CCGGGTACCAGCTCTACCGGTTCTCCA WO 40093 File name Amino acid sequence Nucleotide sequence Lcw0404_012_ GTPGSGTASSSPGSST GGTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAGGTA GFP-\_G07.ab1 TGSP TACCCCGTCTGGTGCAACCGGCTCCCCA 4_014_ GASPGTSSTGSPGASP TCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTG GFP—\_H07.ab1 GTSSTGSPGASPGTSS CATCCCCTGGCACTAGCTCTACTGGTTCTCCAGGTGCTTC TGSP TCCTGGTACCAGCTCTACTGGTTCTCCA LCW0404_015_ GSSTPSGATGSPGSSP GGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTT GFP-\_A08.ab1 SASTGTGPGASPGTSS CTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTC TGSP CCCGGGCACCAGCTCTACTGGTTCTCCA LCW0404_016_ GSSTPSGATGSPGSST GGTAGCTCTACTCCTTCTGGTGCTACCGGTTCCCCAGGTA GFP-\_B08.ab1 PSGATGSPGTPGSGT GCTCTACTCCTTCTGGTGCTACTGGTTCCCCAGGTACTCC ASSSP GGGCAGCGGTACTGCTTCTTCCTCTCCA LCW0404_017_ GSSTPSGATGSPGSST GGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGTA GFP—\_C08.ab1 PSGATGSPGASPGTSS GCTCTACTCCTTCTGGTGCTACTGGCTCCCCAGGTGCATC TGSP CCCTGGCACCAGCTCTACCGGTTCTCCA LCW0404_018_ GFP-\_D08.ab1 SASTGTGPGSSTPSGA CTAGCCCTTCTGCATCTACCGGTACCGGTCCAGGTAGCTC TGSP TACTCCTTCTGGTGCTACTGGCTCTCCA Lcw0404_023_ GASPGTSSTGSPGSSP GGTGCTTCCCCGGGCACTAGCTCTACCGGTTCTCCAGGTT GFP-\_F08.ab1 SASTGTGPGTPGSGT CTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTACTCC ASSSP GGGCAGCGGTACTGCTTCTTCCTCTCCA LCW0404_025_ GATGSPGSST GGTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAGGTA GFP-\_G08.ab1 PSGATGSPGASPGTSS GCTCTACCCCTTCTGGTGCAACCGGCTCCCCAGGTGCTTC TGSP TCCGGGTACCAGCTCTACTGGTTCTCCA LCW0404_029_ GTPGSGTASSSPGSST GGTACCCCTGGCAGCGGTACCGCTTCTTCCTCTCCAGGTA A09.ab1 PSGATGSPGSSPSAST CCCCGTCTGGTGCTACTGGCTCTCCAGGTTCTAG GTGP CCCGTCTGCATCTACCGGTACCGGCCCA LCW040470307 GSSTPSGATGSPGTPG GGTAGCTCTACTCCTTCTGGTGCAACCGGCTCCCCAGGTA GFP-\_B09.ab1 SGTASSSPGTPGSGTA GCAGCGGTACCGCATCTTCCTCTCCAGGTACTCC GGGTAGCGGTACTGCTTCTTCTTCTCCA 4_031_ GTPGSGTASSSPGSST GGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGGTA GFP-\_C09.ab1 PSGATGSPGASPGTSS GCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTC TCCGGGCACCAGCTCTACCGGTTCTCCA 4_034_ GSSTPSGATGSPGSST GGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTA GFP-\7D09.ab1 PSGATGSPGASPGTSS GCTCTACCCCGTCTGGTGCAACCGGCTCCCCAGGTGCATC TGSP CCCGGGTACTAGCTCTACCGGTTCTCCA LCW0404_035_ GFP-\_E09.ab1 SGTASSSPGSSTPSGA CCCCGGGCAGCGGTACCGCATCTTCTTCTCCAGGTAGCTC TGSP TACTCCTTCTGGTGCAACTGGTTCTCCA 4_036_ GSSPSASTGTGPGSST GGTTCTAGCCCGTCTGCTTCCACCGGTACTGGCCCAGGTA GFP-\_F09.ab1 PSGATGSPGTPGSGT GCTCTACCCCGTCTGGTGCAACTGGTTCCCCAGGTACCCC ASSSP TGGTAGCGGTACCGCTTCTTCTTCTCCA LCW0404_037_ GASPGTSSTGSPGSSP GGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAGGTT GFP-\7G09.ab1 SASTGTGPGSSTPSGA CTAGCCCTTCTGCATCCACCGGTACCGGTCCAGGTAGCTC TGSP TACCCCTTCTGGTGCAACCGGCTCTCCA LCW0404_040_ GASPGTSSTGSPGSST GGTGCATCCCCGGGCACCAGCTCTACCGGTTCTCCAGGTA GFP-\_H09.ab1 PSGATGSPGSSTPSGA GCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTAGCTC TGSP TACCCCGTCTGGTGCTACTGGCTCTCCA LCW0404_041_ GFP-\_A10.ab1 PSGATGSPGTPGSGT GCTCTACTCCGTCTGGTGCTACCGGTTCTCCAGGTACCCC ASSSP GGGTAGCGGTACCGCATCTTCTTCTCCA LCW0404_043_ GSSPSASTGTGPGSST GGTTCTAGCCCTTCTGCTTCCACCGGTACTGGCCCAGGTA C10.ab1 PSGATGSPGSSTPSGA GCTCTACCCCTTCTGGTGCTACCGGCTCCCCAGGTAGCTC TGSP TACTCCTTCTGGTGCAACTGGCTCTCCA LCW0404_045_ GASPGTSSTGSPGSSP GGTGCTTCTCCTGGCACCAGCTCTACTGGTTCTCCAGGTT GFP-\_D10.ab1 SASTGTGPGSSPSAST CTAGCCCTTCTGCTTCTACCGGTACTGGTCCAGGTTCTAG GTGP CCCTTCTGCATCCACTGGTACTGGTCCA 4_047_ GFP-\_F10.ab1 GTSSTGSPGASPGTSS CTTCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTGCTTCT 2012/054941 File name Nucleotide sequence CCGGGCACTAGCTCTACTGGTTCTCCA GFP-\_G10.ab1 GTSSTGSPGSSTPSGA CTTCTCCTGGTACTAGCTCTACCGGTTCTCCAGGTAGCTC TGSP TACCCCGTCTGGTGCTACTGGCTCTCCA LCW0404_049_ GSSTPSGATGSPGTPG GGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTA TGSP TACCCCTTCTGGTGCTACTGGCTCTCCA LCW0404_050_ GASPGTSSTGSPGSSP GGTGCATCTCCTGGTACCAGCTCTACTGGTTCTCCAGGTT GFP—\_A1 1.ab1 SASTGTGPGSSTPSGA CTAGCCCTTCTGCTTCTACCGGTACCGGTCCAGGTAGCTC TGSP TACTCCTTCTGGTGCTACCGGTTCTCCA LCW0404_051_ GSSTPSGATGSPGSST GGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTA GFP-\_B1 1.ab1 PSGATGSPGSSTPSGA GCTCTACTCCTTCTGGTGCTACTGGTTCCCCAGGTAGCTC TGSP TACCCCGTCTGGTGCAACTGGCTCTCCA 4_052_ GASPGTSSTGSPGTPG GGTGCATCCCCGGGTACCAGCTCTACCGGTTCTCCAGGTA TGSP TCCGGGCACCAGCTCTACTGGTTCTCCA LCW0404_053_ GSSTPSGATGSPGSSP GGTAGCTCTACTCCTTCTGGTGCAACTGGTTCTCCAGGTT GFP—\_D1 1.ab1 SASTGTGPGASPGTSS CTAGCCCGTCTGCATCCACTGGTACCGGTCCAGGTGCTTC TGSP CCCTGGCACCAGCTCTACCGGTTCTCCA GFP-\_E1 1.ab1 PSGATGSPGSSPSAST GCTCTACTCCGTCTGGTGCAACCGGCTCTCCAGGTTCTAG GTGP CCCTTCTGCATCTACCGGTACTGGTCCA LCW0404_060_ GTPGSGTASSSPGSST GGTACTCCTGGCAGCGGTACCGCATCTTCCTCTCCAGGTA TGSP TCCGGGTACCAGCTCTACCGGTTCTCCA LCW0404_062_ GSSTPSGATGSPGTPG GGTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCAGGTA GFP-\_G1 1.ab1 SGTASSSPGSSTPSGA CTCCTGGTAGCGGTACCGCTTCTTCTTCTCCAGGTAGCTC TGSP TACTCCGTCTGGTGCTACCGGCTCCCCA LCW0404_066_ STGTGPGSSP GGTTCTAGCCCTTCTGCATCCACCGGTACCGGCCCAGGTT GFP-\_H1 1.ab1 GPGASPGTSS CTAGCCCGTCTGCTTCTACCGGTACTGGTCCAGGTGCTTC TGSP TCCGGGTACTAGCTCTACTGGTTCTCCA LCW0404_067_ TASSSPGSST GGTACCCCGGGTAGCGGTACCGCTTCTTCTTCTCCAGGTA A12.ab1 PSGATGSPGSNPSAST GCTCTACTCCGTCTGGTGCTACCGGCTCTCCAGGTTCTAA CCCTTCTGCATCCACCGGTACCGGCCCA LCW0404_068_ STGTGPGSST GGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTA GFP-\_B12.ab1 PSGATGSPGASPGTSS GCTCTACTCCTTCTGGTGCTACCGGCTCTCCAGGTGCTTCT CCGGGTACTAGCTCTACCGGTTCTCCA LCW0404_069_ GATGSPGASP GGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTG GFP-\_C12.ab1 GTSSTGSPGTPGSGTA CATCCCCGGGTACCAGCTCTACCGGTTCTCCAGGTACTCC SSSP CGGTACCGCTTCTTCCTCTCCA LCW0404_070_ GSSTPSGATGSPGSST GGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGTA GFP-\_D12.ab1 SPGSSTPSGA GCTCTACCCCTTCTGGTGCAACCGGCTCCCCAGGTAGCTC TGSP TACCCCTTCTGGTGCAACTGGCTCTCCA LCW0404_073_ GASPGTSSTGSPGTPG GGTGCTTCTCCTGGCACTAGCTCTACCGGTTCTCCAGGTA GFP-\_E12.ab1 SGTASSSPGSSTPSGA CCCCTGGTAGCGGTACCGCATCTTCCTCTCCAGGTAGCTC TGSP TACTCCTTCTGGTGCTACTGGTTCCCCA LCW0404_075_ GSSTPSGATGSPGSSP GGTAGCTCTACCCCGTCTGGTGCTACTGGCTCCCCAGGTT GFP-\7F12.ab1 SASTGTGPGSSPSAST CTAGCCCTTCTGCATCCACCGGTACCGGTCCAGGTTCTAG GTGP CCCGTCTGCATCTACTGGTACTGGTCCA LCW0404_080_ GASPGTSSTGSPGSSP GGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTT GFP-\_G12.ab1 SASTGTGPGSSPSAST CTAGCCCGTCTGCTTCTACTGGTACTGGTCCAGGTTCTAG GTGP CCCTTCTGCTTCCACTGGTACTGGTCCA LCW0404_081_ GASPGTSSTGSPGSSP GGTGCTTCCCCGGGTACCAGCTCTACCGGTTCTCCAGGTT GFP-\_H12.ab1 SASTGTGPGTPGSGT CTAGCCCTTCTGCTTCTACCGGTACCGGTCCAGGTACCCC ASSSP TGGCAGCGGTACCGCATCTTCCTCTCCA Example 5: uction of XTEN_AE864 E864 was constructed from serial dimerization of XTEN_AE36 to AE72, 144, 288, 576 and 864. A collection ofXTEN_AE72 segments was ucted from 37 different segments of XTEN_AE36. Cultures of E. coli harboring all 37 ent 36-amino acid segments were mixed and plasmids were isolated. This plasmid pool was digested with BsaI/Ncol to generate the small fragment as the insert. The same plasmid pool was ed with BbsI/Ncol to generate the large fragment as the vector. The insert and vector fragments were ligated resulting in a doubling ofthe length and the ligation mixture was transformed into BL2lGold(DE3) cells to obtain colonies _AE72.

] This library of XTEN_AE72 segments was designated LCWO406. All clones from LCWO406 were combined and zed again using the same process as described above yielding library O ofXTEN_AE144. All clones from LCWO410 were combined and dimerized again using the same process as described above yielding library LCWO414 _AE288. Two es LCWO414.001 and LCWO414.002 were randomly picked from the library and sequenced to verify the identities. All clones from LCWO414 were combined and dimerized again using the same process as described above yielding library LCWO418 ofXTEN_AES76. We screened 96 isolates from library LCWO418 for high level of GFP ﬂuorescence. 8 isolates with right sizes of inserts by PCR and strong ﬂuorescence were sequenced and 2 isolates (LCWO418.018 and LCWO418.052) were chosen for future use based on sequencing and expression data.

The speciﬁc clone pCWO432 ofXTENiAE864 was constructed by combining LCWO418.018 ofXTEN_AE576 and LCWO414.002 ofXTEN_AE288 using the same dimerization process as described above.

Example 6: Construction of XTEN_AM144 A tion ofXTEN_AM144 segments was constructed starting from 37 different segments ofXTEN_AE36, 44 segments of XTEN_AF36, and 44 ts of XTEN_AG36.

Cultures of E. coli harboring all 125 different 36-amino acid segments were mixed and plasmids were isolated. This plasmid pool was digested with BsaI/Ncol to generate the small nt as the insert. The same plasmid pool was digested with BbsI/Ncol to generate the large fragment as the . The insert and vector fragments were ligated resulting in a doubling ofthe length and the ligation mixture was transformed into BL2lGold(DE3) cells to obtain colonies ofXTEN_AM72.

This library of XTEN_AM72 segments was designated LCWO461. All clones from LCWO461 were combined and dimerized again using the same process as described above ng library LCWO462. 1512 Isolates from library LCWO462 were screened for protein expression. Individual colonies were transferred into 96 well plates and cultured ght as starter cultures. These starter cultures were d into fresh autoinduction medium and cultured for 20-3 Oh. Expression was measured using a ﬂuorescence plate reader with excitation at 395 nm and emission at 510 nm. 192 isolates showed high level sion and were submitted to DNA sequencing. Most clones in library LCWO462 showed good expression and similar physicochemical properties suggesting that most combinations of M36 segments yield useful XTEN sequences. 30 isolates from LCWO462 were chosen as a preferred collection ofXTEN_AM144 segments for the construction of multifunctional proteins that contain multiple XTEN segments. The ﬁle names ofthe nucleotide and amino acid constructs for these segments are listed in Table 12.

T—q—g_able12.DNAandamino acid se uences for AM144 se ments DNA Sequence Protein Sequence LCW462_rl GGTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAGGTA GTPGSGTASSSPGSSTPS GCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTAGCTC GATGSPGSSTPSGATGS TACCCCGTCTGGTGCAACCGGCTCCCCAGGTAGCCCGGCT PGSPAGSPTSTEEGTSES GGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCG ATPESGPGTSTEPSEGS CTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAACCGTC APGSSPSASTGTGPGSS CGAAGGTAGCGCTCCAGGTTCTAGCCCTTCTGCATCCACC TGPGASPGTSS GGTACCGGCCCAGGTTCTAGCCCGTCTGCTTCTACCGGTA STEPSEGSAPG CTGGTCCAGGTGCTTCTCCGGGTACTAGCTCTACTGGTTC TSTEPSEGSAPGSEPATS TCCAGGTACCTCTACCGAACCGTCCGAGGGTAGCGCACC GSETP CTCTACTGAACCGTCTGAGGGTAGCGCTCCAGG TAGCGAACCGGCAACCTCCGGTTCTGAAACTCCA _r5 GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACCAGGTT GSTSESPSGTAPGSTSES GCGAATCCCCTTCTGGTACCGCACCAGGTACTTC PSGTAPGTSPSGESSTAP TCCGAGCGGCGAATCTTCTACTGCTCCAGGTACCTCTACT SEGSAPGTSTEP GAACCTTCCGAAGGCAGCGCTCCAGGTACCTCTACCGAA SEGSAPGTSESATPESG CCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCA PGASPGTSSTGSPGSSTP ACCCCTGAATCCGGTCCAGGTGCATCTCCTGGTACCAGCT SGATGSPGASPGTSSTG CTACCGGTTCTCCAGGTAGCTCTACTCCTTCTGGTGCTAC SPGSTSESPSGTAPGSTS TGGCTCTCCAGGTGCTTCCCCGGGTACCAGCTCTACCGGT ESPSGTAPGTSTPESGS TCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCAC ASP CAGGTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAGG TACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCA LCW462_r9 TCTACCGAACCTTCCGAGGGCAGCGCACCAGGT GTSTEPSEGSAPGTSES ACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTT ATPESGPGTSESATPES CTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACCTCTAC GPGTSTEPSEGSAPGTS TGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAG ESATPESGPGTSTEPSEG CGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCG SAPGTSTEPSEGSAPGS TCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCTTCCG EPATSGSETPGSPAGSP AAGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTC TSTEEGASPGTSSTGSP TGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCTCCACC GSSPSASTGTGPGSSPS GAGGAAGGTGCTTCTCCTGGCACCAGCTCTACTGGTTCTC ASTGTGP CAGGTTCTAGCCCTTCTGCTTCTACCGGTACTGGTCCAGG TTCTAGCCCTTCTGCATCCACTGGTACTGGTCCA LCW462_r10 GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAGGT GSEPATSGSETPGTSES ACCTCTGAAAGCGCTACTCCGGAATCTGGTCCAGGTACTT ATPESGPGTSESATPES CTGAAAGCGCTACTCCGGAATCCGGTCCAGGTTCTACCA GPGSTSESPSGTAPGSTS GCGAATCTCCTTCTGGCACCGCTCCAGGTTCTACTAGCGA ESPSGTAPGTSPSGESST ATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCGGC APGASPGTSSTGSPGSS GAATCTTCTACCGCACCAGGTGCATCTCCGGGTACTAGCT PSASTGTGPGSSTPSGA CTACCGGTTCTCCAGGTTCTAGCCCTTCTGCTTCCACTGGT TGSPGSSTPSGATGSPG CCAGGTAGCTCTACCCCGTCTGGTGCTACTGGTT SSTPSGATGSPGASPGT GTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCC SSTGSP AGGTAGCTCTACTCCTTCTGGTGCTACTGGCTCCCCAGGT GCATCCCCTGGCACCAGCTCTACCGGTTCTCCA LCW462_r15 GGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAGGTT GASPGTSSTGSPGSSPS CTAGCCCTTCTGCATCCACCGGTACCGGTCCAGGTAGCTC ASTGTGPGSSTPSGATG TACCCCTTCTGGTGCAACCGGCTCTCCAGGTACTTCTGAA SPGTSESATPESGPGSEP ACCCCGGAATCTGGCCCAGGTAGCGAACCGGCT ATSGSETPGSEPATSGS ACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCT ESATPESGPGTS CCGGTTCTGAAACTCCAGGTACTTCTGAAAGCGCTACTCC TEPSEGSAPGTSTEPSEG GGAGTCCGGTCCAGGTACCTCTACCGAACCGTCCGAAGG SAPGTSTEPSEGSAPGT CAGCGCTCCAGGTACTTCTACTGAACCTTCTGAGGGTAGC STEPSEGSAPGSEPATS GCTCCAGGTACCTCTACCGAACCGTCCGAGGGTAGCGCA GSETP Clone DNA ce Protein ce CCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCA GAACCGGCAACCTCCGGTTCTGAAACTCCA LCW462_I1 6 GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGT GTSTEPSEGSAPGSPAG GCAGGTTCTCCTACTTCCACTGAGGAAGGTACTT SPTSTEEGTSTEPSEGSA CTACCGAACCTTCTGAGGGTAGCGCACCAGGTACCTCTG PGTSESATPESGPGSEP AAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTG ATSGSETPGTSESATPES CTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGC GPGSPAGSPTSTEEGTS AACCCCGGAATCTGGTCCAGGTAGCCCGGCTGGCTCTCCT ESATPESGPGTSTEPSEG ACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTG SAPGSEPATSGSETPGT AGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTA GSAPGSEPATS GCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGAAAC GSETP TACTTCTACCGAACCGTCCGAGGGTAGCGCTCCA GGTAGCGAACCTGCTACTTCTGGTTCTGAAACTCCA LCW462_I20 GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGT GTSTEPSEGSAPGTSTEP ACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCT SEGSAPGTSTEPSEGSA CTACCGAACCTTCTGAAGGTAGCGCACCAGGTACTTCTAC PGTSTEPSEGSAPGTSTE CGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGA PSEGSAPGTSTEPSEGS ACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCT APGTSTEPSEGSAPGTS TCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCTTCCG ESATPESGPGTSESATPE AGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTG SGPGTSTEPSEGSAPGS AGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATC EPATSGSETPGSPAGSP CGGTCCAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCT TSTEE CCAGGTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCAG GTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAA LCW462_I23 GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAGGT GTSTEPSEGSAPGTSTEP ACTGAACCTTCTGAAGGCAGCGCTCCAGGTACTT SEGSAPGTSTEPSEGSA CTACTGAACCTTCCGAAGGTAGCGCACCAGGTTCTACCA PGSTSESPSGTAPGSTSE GCGAATCCCCTTCTGGTACTGCTCCAGGTTCTACCAGCGA SPSGTAPGTSTPESGSAS ATCCCCTTCTGGCACCGCACCAGGTACTTCTACCCCTGAA PGSEPATSGSETPGTSES AGCGGCTCCGCTTCTCCAGGTAGCGAACCTGCAACCTCTG ATPESGPGTSTEPSEGS GCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGA EPSEGSAPGTS ATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAG ESATPESGPGTSESATPE CGCACCAGGTACTTCTACTGAACCGTCTGAAGGTAGCGC SGP ACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCC AGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCA LCW462_I24 GGTAGCTCTACCCCTTCTGGTGCTACCGGCTCTCCAGGTT GSSTPSGATGSPGSSPS CTAGCCCGTCTGCTTCTACCGGTACCGGTCCAGGTAGCTC PGSSTPSGATG TACCCCTTCTGGTGCTACTGGTTCTCCAGGTAGCCCTGCT SPGSPAGSPTSTEEGSPA GGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTT GSPTSTEEGTSTEPSEGS CTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTC APGASPGTSSTGSPGSS CGAAGGTAGCGCTCCAGGTGCTTCCCCGGGCACTAGCTCT PSASTGTGPGTPGSGTA ACCGGTTCTCCAGGTTCTAGCCCTTCTGCATCTACTGGTA SSSPGSTSSTAESPGPGT CTGGCCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTC SPSGESSTAPGTSTPESG TCCAGGTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCA SASP GGTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGGTA CCTCTACTCCGGAAAGCGGTTCTGCATCTCCA LCW462_I27 GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTA GTSTEPSEGSAPGTSES CTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTC ATPESGPGTSTEPSEGS TACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACT APGTSTEPSEGSAPGTS GAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGC ESATPESGPGTSESATPE CCGGAATCCGGCCCAGGTACCTCTGAAAGCGCA SGPGTPGSGTASSSPGA ACCCCGGAGTCCGGCCCAGGTACTCCTGGCAGCGGTACC SPGTSSTGSPGASPGTSS GCTTCTTCTTCTCCAGGTGCTTCTCCTGGTACTAGCTCTAC TGSPGSPAGSPTSTEEG TGGTTCTCCAGGTGCTTCTCCGGGCACTAGCTCTACTGGT SPAGSPTSTEEGTSTEPS TCTCCAGGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGG EGSAP AAGGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAG GTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCA LCW462_I28 GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGT GSPAGSPTSTEEGTSTEP ACTGAACCTTCCGAAGGCAGCGCACCAGGTACCT SEGSAPGTSTEPSEGSA CTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACCTCTAC PGTSTEPSEGSAPGTSES 2012/054941 Clone DNA Sequence n Sequence CGAACCGTCTGAAGGTAGCGCACCAGGTACCTCTGAAAG ATPESGPGTSESATPES CGCAACTCCTGAGTCCGGTCCAGGTACTTCTGAAAGCGC GPGTPGSGTASSSPGSS AACCCCGGAGTCTGGCCCAGGTACCCCGGGTAGCGGTAC TPSGATGSPGASPGTSS TGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAA STEPSEGSAPG CCGGCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACCGG TSESATPESGPGTSTEPS TTCTCCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCT EGSAP CCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCA GGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA LCW462_I3 8 GGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCAGGT GSEPATSGSETPGTSES ACTTCTGAAAGCGCTACTCCGGAATCCGGCCCAGGTAGC ATPESGPGSEPATSGSE GAACCGGCTACTTCCGGCTCTGAAACCCCAGGTAGCTCTA TPGSSTPSGATGSPGTP CTGGTGCAACCGGCTCCCCAGGTACTCCTGGTAG GSGTASSSPGSSTPSGA CGGTACCGCTTCTTCTTCTCCAGGTAGCTCTACTCCGTCTG TGSPGASPGTSSTGSPG GTGCTACCGGCTCCCCAGGTGCATCTCCTGGTACCAGCTC SSTPSGATGSPGASPGT TACCGGTTCTCCAGGTAGCTCTACTCCTTCTGGTGCTACT SSTGSPGSEPATSGSETP GGCTCTCCAGGTGCTTCCCCGGGTACCAGCTCTACCGGTT GTSTEPSEGSAPGSEPA CTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGAAACTCC TSGSETP TTCTACCGAACCGTCCGAGGGTAGCGCTCCAGG TAGCGAACCTGCTACTTCTGGTTCTGAAACTCCA LCW462_I39 GGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGT SEGSAPGTSTEP ACCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACT SEGSAPGTSESATPESG TCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCCCT PGSPAGSPTSTEEGSPA GCTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTG GSPTSTEEGTSTEPSEGS GTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACC APGSPAGSPTSTEEGTS TTCCGAAGGTAGCGCTCCAGGTAGCCCGGCTGGTTCTCCG TEPSEGSAPGTSTEPSEG ACCGAGGAAGGTACCTCTACTGAACCTTCTGAGG SAPGASPGTSSTGSPGS CTCCAGGTACCTCTACTGAACCTTCCGAAGGCA SPSASTGTGPGSSPSAST GCGCTCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTC GTGP TCCAGGTTCTAGCCCGTCTGCTTCTACTGGTACTGGTCCA GGTTCTAGCCCTTCTGCTTCCACTGGTACTGGTCCA LCW462_I4 1 GGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTG GSSTPSGATGSPGASPG CTTCTCCTGGTACTAGCTCTACCGGTTCTCCAGGTAGCTC TSSTGSPGSSTPSGATGS TACCCCGTCTGGTGCTACTGGCTCTCCAGGTAGCCCTGCT PGSPAGSPTSTEEGTSES CCAACCTCCACCGAAGAAGGTACCTCTGAAAGC ATPESGPGSEPATSGSE GCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACC TPGASPGTSSTGSPGSST TCCGGTTCTGAAACCCCAGGTGCATCTCCTGGTACTAGCT PSGATGSPGSSPSASTG CTACTGGTTCTCCAGGTAGCTCTACTCCGTCTGGTGCAAC TGPGSTSESPSGTAPGS CGGCTCTCCAGGTTCTAGCCCTTCTGCATCTACCGGTACT TSESPSGTAPGTSTPESG GGTCCAGGTTCTACCAGCGAATCCCCTTCTGGTACTGCTC SASP CAGGTTCTACCAGCGAATCCCCTTCTGGCACCGCACCAGG TACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCA LCW462_I42 GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTT GSTSESPSGTAPGSTSES CTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTC PSGTAPGTSPSGESSTAP TCCTAGCGGCGAATCTTCTACCGCACCAGGTACCTCTGAA GTSESATPESGPGTSTEP AGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAAC SEGSAPGTSTEPSEGSA CGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTC PGTSTEPSEGSAPGTSES CGAAGGTAGCGCACCAGGTACCTCTACTGAACCTTCTGA ATPESGPGTSTEPSEGS GGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGA APGSSTPSGATGSPGAS GTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGC PGTSSTGSPGSSTPSGAT GCACCAGGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCC GSP CAGGTGCTTCTCCTGGTACTAGCTCTACCGGTTCTCCAGG TAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCA LCW462_I43 GGTTCTACTAGCTCTACTGCAGAATCTCCGGGCCCAGGTA GSTSSTAESPGPGTSPSG CTAGCGGTGAATCTTCTACCGCTCCAGGTACTTC ESSTAPGTSPSGESSTAP CGGTGAATCTTCTACCGCTCCAGGTTCTACTAGC GSTSSTAESPGPGSTSST TCTACCGCTGAATCTCCGGGTCCAGGTTCTACCAGCTCTA AESPGPGTSTPESGSASP CTGCAGAATCTCCTGGCCCAGGTACTTCTACTCCGGAAAG GTSPSGESSTAPGSTSST CGGTTCCGCTTCTCCAGGTACTTCTCCTAGCGGTGAATCT AESPGPGTSTPESGSASP TCTACCGCTCCAGGTTCTACCAGCTCTACTGCTGAATCTC GSTSSTAESPGPGSTSES CTGGCCCAGGTACTTCTACCCCGGAAAGCGGCTCCGCTTC PSGTAPGTSPSGESSTAP Clone DNA Sequence Protein ce TCCAGGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCA ACTAGCGAATCTCCGTCTGGCACCGCACCAGGTA CTTCCCCTAGCGGTGAATCTTCTACTGCACCA _I45 GGTACCTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTT GTSTPESGSASPGSTSES CTACCAGCGAATCCCCGTCTGGCACCGCACCAGGTTCTAC PSGTAPGSTSSTAESPGP TAGCTCTACTGCTGAATCTCCGGGCCCAGGTACCTCTACT SEGSAPGTSTEP GAACCTTCCGAAGGCAGCGCTCCAGGTACCTCTACCGAA SEGSAPGTSESATPESG CCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCA PGTSESATPESGPGTSTE ACCCCTGAATCCGGTCCAGGTACCTCTGAAAGCGCTACTC PGTSTEPSEGS CGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGG APGTSESATPESGPGTS GTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTA TEPSEGSAPGTSTEPSEG GCGCACCAGGTACTTCTGAAAGCGCTACTCCGGAGTCCG SAP GTCCAGGTACCTCTACCGAACCGTCCGAAGGCAGCGCTC CAGGTACTTCTACTGAACCTTCTGAGGGTAGCGCTCCC LCW4627I47 GGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCAGGT GTSTEPSEGSAPGTSTEP ACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGC SEGSAPGSEPATSGSET GAACCGGCAACCTCCGGTTCTGAAACTCCAGGTACTTCTA PGTSTEPSEGSAPGTSES CTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAA ATPESGPGTSESATPES GCGCAACCCCGGAATCCGGCCCAGGTACCTCTGAAAGCG GPGASPGTSSTGSPGSS CAACCCCGGAGTCCGGCCCAGGTGCATCTCCGGGTACTA PSASTGTGPGSSTPSGA GCTCTACCGGTTCTCCAGGTTCTAGCCCTTCTGCTTCCACT TGSPGSSTPSGATGSPG GGTACCGGCCCAGGTAGCTCTACCCCGTCTGGTGCTACTG SSTPSGATGSPGASPGT GTTCCCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGTTC SSTGSP CCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGCTCCCCA GGTGCATCCCCTGGCACCAGCTCTACCGGTTCTCCA LCW462_I54 GGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCAGGT GSEPATSGSETPGSEPA AGCGAACCTGCAACCTCCGGCTCTGAAACCCCAGGTACT TSGSETPGTSTEPSEGSA TCTACTGAACCTTCTGAGGGCAGCGCACCAGGTAGCGAA PGSEPATSGSETPGTSES ACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAA ATPESGPGTSTEPSEGS GCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACC APGSSTPSGATGSPGSS GTCCGAGGGCAGCGCACCAGGTAGCTCTACTCCGTCTGG TPSGATGSPGASPGTSS TGCTACCGGCTCTCCAGGTAGCTCTACCCCTTCTGGTGCA TGSPGSSTPSGATGSPG ACCGGCTCCCCAGGTGCTTCTCCGGGTACCAGCTCTACTG ASPGTSSTGSPGSSTPSG GTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGTTC ATGSP TGCTTCTCCTGGTACTAGCTCTACCGGTTCTCCA GGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCA LCW462_r55 GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAGGT SEGSAPGTSTEP ACTTCTACTGAACCTTCTGAAGGCAGCGCTCCAGGTACTT GTSTEPSEGSA CTACTGAACCTTCCGAAGGTAGCGCACCAGGTACTTCTGA PGTSESATPESGPGTSTE AAGCGCTACTCCGGAGTCCGGTCCAGGTACCTCTACCGA PSEGSAPGTSTEPSEGS ACCGTCCGAAGGCAGCGCTCCAGGTACTTCTACTGAACCT APGSTSESPSGTAPGTSP TCTGAGGGTAGCGCTCCAGGTTCTACTAGCGAATCTCCGT SGESSTAPGTSPSGESST CTGGCACTGCTCCAGGTACTTCTCCTAGCGGTGAATCTTC APGSPAGSPTSTEEGTS TACCGCTCCAGGTACTTCCCCTAGCGGCGAATCTTCTACC ESATPESGPGTSTEPSEG GCTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGG SAP AAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGG TACCTCTACTGAACCGTCCGAAGGTAGCGCTCCA LCW462_I57 GGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTA GTSTEPSEGSAPGSEPA GCGAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCC TSGSETPGSPAGSPTSTE GGCTGGCTCTCCGACCTCCACCGAGGAAGGTAGCCCGGC EGSPAGSPTSTEEGTSES AGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAG ATPESGPGTSTEPSEGS CGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACC APGTSTEPSEGSAPGTS GGGCAGCGCACCAGGTACCTCTACTGAACCTTCC TEPSEGSAPGTSESATPE GAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAG SGPGSSTPSGATGSPGS GGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAA SPSASTGTGPGASPGTS TCCGGTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCT STGSP CCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCC AGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCA LCW462_I6 1 GAACCGGCTACTTCCGGCTCTGAGACTCCAGGT GSEPATSGSETPGSPAG AGCCCTGCTGGCTCTCCGACCTCTACCGAAGAAGGTACCT SPTSTEEGTSESATPESG Clone DNA Sequence Protein Sequence CTGAAAGCGCTACCCCTGAGTCTGGCCCAGGTACCTCTAC PGTSTEPSEGSAPGTSTE TGAACCTTCCGAAGGCAGCGCTCCAGGTACCTCTACCGA PSEGSAPGTSESATPES ACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGC GPGTSTPESGSASPGSTS AACCCCTGAATCCGGTCCAGGTACCTCTACTCCGGAAAG ESPSGTAPGSTSSTAESP CGGTTCCGCATCTCCAGGTTCTACCAGCGAATCCCCGTCT GPGTSESATPESGPGTS GGCACCGCACCAGGTTCTACTAGCTCTACTGCTGAATCTC SAPGTSTEPSEG CGGGCCCAGGTACTTCTGAAAGCGCTACTCCGGAGTCCG SAP GTCCAGGTACCTCTACCGAACCGTCCGAAGGCAGCGCTC CTTCTACTGAACCTTCTGAGGGTAGCGCTCCA LCW462_I64 TCTACCGAACCGTCCGAGGGCAGCGCTCCAGGT GTSTEPSEGSAPGTSTEP ACTTCTACTGAACCTTCTGAAGGCAGCGCTCCAGGTACTT SEGSAPGTSTEPSEGSA CTACTGAACCTTCCGAAGGTAGCGCACCAGGTACCTCTAC PGTSTEPSEGSAPGTSES CGAACCGTCTGAAGGTAGCGCACCAGGTACCTCTGAAAG ATPESGPGTSESATPES CGCAACTCCTGAGTCCGGTCCAGGTACTTCTGAAAGCGC GPGTPGSGTASSSPGSS AACCCCGGAGTCTGGCCCAGGTACTCCTGGCAGCGGTAC TPSGATGSPGASPGTSS CGCATCTTCCTCTCCAGGTAGCTCTACTCCGTCTGGTGCA TGSPGSTSSTAESPGPG ACTGGTTCCCCAGGTGCTTCTCCGGGTACCAGCTCTACCG TSPSGESSTAPGTSTPES CAGGTTCCACCAGCTCTACTGCTGAATCTCCTGG GSASP TCCAGGTACCTCTCCTAGCGGTGAATCTTCTACTGCTCCA GGTACTTCTACTCCTGAAAGCGGCTCTGCTTCTCCA LCW462_I67 GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGT GSPAGSPTSTEEGTSES ACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACC ATPESGPGTSTEPSEGS TCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCT APGTSESATPESGPGSE GAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCG PATSGSETPGTSTEPSEG GCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAAC SAPGSPAGSPTSTEEGT CGTCCGAAGGTAGCGCACCAGGTAGCCCGGCTGGTTCTC STEPSEGSAPGTSTEPSE CGACTTCCACCGAGGAAGGTACCTCTACTGAACCTTCTGA GSAPGTSTEPSEGSAPG GGGTAGCGCTCCAGGTACCTCTACTGAACCTTCCGAAGG TSTEPSEGSAPGTSTEPS CAGCGCTCCAGGTACTTCTACCGAACCGTCCGAGGGCAG EGSAP CGCTCCAGGTACTTCTACTGAACCTTCTGAAGGCAGCGCT CCAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCACCA LCW4627I69 GGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCAGGTT GTSPSGESSTAPGSTSST CTACTAGCTCTACCGCTGAATCTCCGGGCCCAGGTACTTC AESPGPGTSPSGESSTAP TCCGAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTGAA GTSESATPESGPGTSTEP AGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAAC GTSTEPSEGSA CGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTC PGSSPSASTGTGPGSSTP CGAAGGTAGCGCACCAGGTTCTAGCCCTTCTGCATCTACT SGATGSPGASPGTSSTG GGCCCAGGTAGCTCTACTCCTTCTGGTGCTACCG PESGSASPGTSP GCTCTCCAGGTGCTTCTCCGGGTACTAGCTCTACCGGTTC SGESSTAPGTSPSGESST TCCAGGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCA AP TCTCCTAGCGGTGAATCTTCTACTGCTCCAGGTA CCTCTCCTAGCGGCGAATCTTCTACTGCTCCA LCW462_I70 GGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGT GTSESATPESGPGTSTEP ACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTT SEGSAPGTSTEPSEGSA CTACTGAACCGTCCGAAGGTAGCGCACCAGGTAGCCCTG PGSPAGSPTSTEEGSPA CTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGG GSPTSTEEGTSTEPSEGS TTCTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCT APGSSPSASTGTGPGSS TCCGAAGGTAGCGCTCCAGGTTCTAGCCCTTCTGCTTCCA TPSGATGSPGSSTPSGA CCGGTACTGGCCCAGGTAGCTCTACCCCTTCTGGTGCTAC TGSPGSEPATSGSETPG CGGCTCCCCAGGTAGCTCTACTCCTTCTGGTGCAACTGGC TSESATPESGPGSEPATS TCTCCAGGTAGCGAACCGGCAACTTCCGGCTCTGAAACC GSETP CCAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGCCCAG GTAGCGAACCTGCTACCTCTGGCTCTGAAACCCCA _r72 GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGT GTSTEPSEGSAPGTSTEP ACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCT SEGSAPGTSTEPSEGSA CTACCGAACCTTCTGAAGGTAGCGCACCAGGTAGCTCTA PGSSTPSGATGSPGASP CTGGTGCTACCGGTTCCCCAGGTGCTTCTCCTGG GTSSTGSPGSSTPSGAT TACTAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCT GSPGTSESATPESGPGS ACTGGCTCTCCAGGTACTTCTGAAAGCGCAACCC EPATSGSETPGTSTEPSE CTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTC GSAPGSTSESPSGTAPG Clone DNA Sequence n Sequence TGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAG STSESPSGTAPGTSTPES CGCACCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCA GSASP CCAGGTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAG GTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCA LCW462_I73 GGTACCTCTACTCCTGAAAGCGGTTCTGCATCTCCAGGTT GTSTPESGSASPGSTSST CCACTAGCTCTACCGCAGAATCTCCGGGCCCAGGTTCTAC AESPGPGSTSSTAESPGP TAGCTCTACTGCTGAATCTCCTGGCCCAGGTTCTAGCCCT GSSPSASTGTGPGSSTPS TCTGCATCTACTGGTACTGGCCCAGGTAGCTCTACTCCTT GASPGTSSTGS CTGGTGCTACCGGCTCTCCAGGTGCTTCTCCGGGTACTAG TSGSETPGTSES CTCTACCGGTTCTCCAGGTAGCGAACCGGCAACCTCCGGC ATPESGPGSPAGSPTST TCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAAT EEGSTSESPSGTAPGSTS CCGGCCCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGA ESPSGTAPGTSTPESGS GGAAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCA ASP GGTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAGGTA CCTCTACCCCTGAAAGCGGTTCCGCTTCTCCC LCW462_I78 GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTA GSPAGSPTSTEEGTSES CTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTC ATPESGPGTSTEPSEGS TACTGAACCGTCCGAAGGTAGCGCTCCAGGTTCTACCAG APGSTSESPSGTAPGSTS CGAATCTCCTTCTGGCACCGCTCCAGGTTCTACTAGCGAA ESPSGTAPGTSPSGESST TCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCGGCG APGTSTEPSEGSAPGSP AATCTTCTACCGCACCAGGTACCTCTACCGAACCTTCCGA AGSPTSTEEGTSTEPSE AGGTAGCGCTCCAGGTAGCCCGGCAGGTTCTCCTACTTCC GSAPGSEPATSGSETPG ACTGAGGAAGGTACTTCTACCGAACCTTCTGAGGGTAGC TSESATPESGPGTSTEPS GCACCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACC EGSAP CCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAG GTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA LCW462_I79 GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGT GTSTEPSEGSAPGSPAG AGCCCGGCAGGTTCTCCTACTTCCACTGAGGAAGGTACTT EGTSTEPSEGSA CTACCGAACCTTCTGAGGGTAGCGCACCAGGTACCTCCCC PGTSPSGESSTAPGTSPS TAGCGGCGAATCTTCTACTGCTCCAGGTACCTCTCCTAGC GESSTAPGTSPSGESST GGCGAATCTTCTACCGCTCCAGGTACCTCCCCTAGCGGTG APGSTSESPSGTAPGSTS AATCTTCTACCGCACCAGGTTCTACCAGCGAATCCCCTTC ESPSGTAPGTSTPESGS TGGTACTGCTCCAGGTTCTACCAGCGAATCCCCTTCTGGC ASPGSEPATSGSETPGT ACCGCACCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTT SESATPESGPGTSTEPSE CTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCC GSAP AGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGT ACTTCTACTGAACCGTCCGAGGGCAGCGCACCA LCW462_I87 GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAGGT GSEPATSGSETPGTSES ACCTCTGAAAGCGCTACTCCGGAATCTGGTCCAGGTACTT ATPESGPGTSESATPES CTGAAAGCGCTACTCCGGAATCCGGTCCAGGTACTTCTCC GPGTSPSGESSTAPGSTS GAGCGGTGAATCTTCTACCGCACCAGGTTCTACTAGCTCT STAESPGPGTSPSGESST ACCGCTGAATCTCCGGGCCCAGGTACTTCTCCGAGCGGTG APGSTSESPSGTAPGTSP AATCTTCTACTGCTCCAGGTTCTACTAGCGAATCCCCGTC SGESSTAPGSTSSTAESP TGGTACTGCTCCAGGTACTTCCCCTAGCGGTGAATCTTCT GPGSSTPSGATGSPGSS ACTGCTCCAGGTTCTACCAGCTCTACCGCAGAATCTCCGG GSPGSSTPSGA GTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCC NWLS AGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCCCCAGGT AGCTCTACCCCTTCTGGTGCAAACTGGCTCTCC LCW462_I8 8 GGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTA GSPAGSPTSTEEGSPAG CTGGTTCTCCGACTTCTACTGAGGAAGGTACTTC SPTSTEEGTSTEPSEGSA TACCGAACCTTCCGAAGGTAGCGCTCCAGGTACCTCTACT PGTSTEPSEGSAPGTSTE GAACCTTCCGAAGGCAGCGCTCCAGGTACCTCTACCGAA PSEGSAPGTSESATPES CCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCA GTSSTGSPGSS ACCCCTGAATCCGGTCCAGGTGCATCTCCTGGTACCAGCT TPSGATGSPGASPGTSS CTACCGGTTCTCCAGGTAGCTCTACTCCTTCTGGTGCTAC TGSPGSSTPSGATGSPG TGGCTCTCCAGGTGCTTCCCCGGGTACCAGCTCTACCGGT TPGSGTASSSPGSSTPSG TCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTC ATGSP CAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGG TACCCCTTCTGGTGCTACTGGCTCTCCA LCW462_I89 GGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTA GSSTPSGATGSPGTPGS DNA Sequence Protein Sequence CTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTC GTASSSPGSSTPSGATG TTCTGGTGCTACTGGCTCTCCAGGTAGCCCGGCT SPGSPAGSPTSTEEGTSE GGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCG SATPESGPGTSTEPSEGS CTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAACCGTC APGTSESATPESGPGSE TAGCGCTCCAGGTACCTCTGAAAGCGCAACTCC ETPGTSESATPE TGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCT SGPGTSTEPSEGSAPGT GAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCT ESGPGTSESATP GGTCCAGGTACTTCTACTGAACCGTCTGAAGGTAGCGCA ESGP CCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCCA GGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCA Example 7: Construction of XTEN_AM288 The entire library LCWO462 was dimerized as described in Example 6 resulting in a library of XTEN_AM288 clones designated LCWO463. 1512 isolates from library LCWO463 were screened using the protocol described in Example 6. 176 highly expressing clones were sequenced and 40 preferred XTEN_AM288 segments were chosen for the construction of unctional proteins that contain le XTEN segments with 288 amino acid residues.

Example 8: Construction of M432 We ted a library ofXTEN_AM432 segments by recombining segments from y LCWO462 ofXTEN_AM144 ts and ts from library LCWO463 ofXTEN_AM288 segments. This new library of XTEN_AM432 segment was designated LCWO464. Plasmid was isolated from cultures of E. coli harboring LCWO462 and LCWO463, respectively. 1512 isolates from library LCWO464 were screened using the protocol described in Example 6. 176 highly expressing clones were sequenced and 39 preferred XTEN_AM432 t were chosen for the construction of longer XTENs and for the construction of multifunctional proteins that contain multiple XTEN segments with 432 amino acid residues.

In parallel we constructed library LMSOlOO ofXTENiAM432 segments using preferred segments of XTENiAM144 and XTENiAM288. Screening of this library d 4 isolates that were selected for further construction Example 9: Construction of M875 The stuffer vector pCW0359 was digested with Bsal and Kpnl to remove the r segment and the resulting vector fragment was isolated by agarose gel ation.

We annealed the phosphorylated oligonucleotide BsaI-AscI-KpnlforP: AGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTTCGTCTTCACTCGAGGGTAC and the non-phosphorylated oligonucleotide Bsal-Ascl-Kpnlrev: CCTCGAGTGAAGACGAACCTCCCGTGCTTGGCGCGCCGCTTGCGCTTGC for introducing the sequencing island A (SI-A) which encodes amino acids GASASGAPSTG and has the restriction enzyme AscI recognition nucleotide sequence GGCGCGCC inside. The annealed oligonucleotide pairs were ligated with BsaI and KpnI digested stuffer vector pCW0359 prepared above to yield pCWO466 containing Sl-A. We then generated a library of XTEN_AM443 segments by recombining 43 preferred XTEN_AM432 segments from e 8 and Sl-A segments from pCWO466 at C-terminus using the WO 40093 same dimerization process described in Example 5. This new library of XTEN_AM443 segments was designated LCW0479.

We generated a library ofXTEN_AM875 segments by recombining ts from library LCW0479 ofXTEN_AM443 segments and 43 preferred XTEN_AM432 segments from Example 8 using the same dimerization process described in Example 5. This new library _AM875 segment was ated LCW0481.

Example 10: Construction of XTEN_AM1318 We annealed the phosphorylated oligonucleotide BsaI-Fsel-KpnlforP: AGGTCCAGAACCAACGGGGCCGGCCCCAAGCGGAGGTTCGTCTTCACTCGAGGGTAC and the non-phosphorylated oligonucleotide Bsal-FseI-Kpnlrev: CCTCGAGTGAAGACGAACCTCCGCTTGGGGCCGGCCCCGTTGGTTCTGG for introducing the sequencing island B (SI-B) which encodes amino acids GPEPTGPAPSG and has the ction enzyme Fsel recognition nucleotide ce GGCCGGCC inside. The annealed oligonucleotide pairs were ligated with BsaI and Kpnl digested stuffer vector pCWO359 as used in Example 9 to yield pCWO467 containing Sl-B. We then generated a library ofXTEN_AM443 segments by recombining 43 preferred XTEN_AM432 ts from Example 8 and Sl-B segments from 7 at C-terminus using the same dimerization process described in Example 5. This new library of XTEN_AM443 segments was designated LCW0480.

We generated a library ofXTEN_AM13 l 8 segments by recombining ts from library LCW0480 ofXTEN_AM443 segments and ts from library 1 ofXTEN_AM875 segments using the same dimerization process as in Example 5. This new library of XTEN_AM13 l 8 t was designated LCW0487.

Example 11: Construction of XTEN_AD864 ] Using the several consecutive rounds of dimerization, we assembled a collection of D864 sequences starting from segments ofXTEN_AD36 listed in Example 1. These sequences were assembled as bed in Example 5. Several isolates from XTEN_AD864 were evaluated and found to show good expression and excellent lity under physiological conditions. One intermediate construct of XTEN_AD576 was ced. This clone was evaluated in a PK experiment in cynomolgus monkeys and a half-life of about 20h was measured.

Example 12: Construction of XTEN_AF864 Using the several consecutive rounds of dimerization, we assembled a collection of XTEN_AF864 sequences starting from segments ofXTEN_AF36 listed in Example 3. These sequences were assembled as described in Example 5. Several isolates from F864 were evaluated and found to show good expression and excellent solubility under physiological conditions. One intermediate construct of XTEN_AF54O was sequenced. This clone was evaluated in a PK experiment in cynomolgus monkeys and a half-life of about 20h was measured. A full length clone of XTEN_AF864 had excellent solubility and showed half-life exceeding 60h in cynomolgus monkeys. A second set of XTEN_AF sequences was assembled including a sequencing island as described in Example 9.

Example 13: Construction of XTEN_AG864 Using the several utive rounds of dimerization, we assembled a collection of XTEN_AG864 sequences starting from segments ofXTEN_AG36 listed in Example 4. These ces were assembled as described in Example 5. Several es from XTEN_AG864 were evaluated and found to show good expression and ent solubility under physiological conditions. A full-length clone ofXTEN_AG864 had excellent lity and showed half-life exceeding 60h in cynomolgus monkeys.

Example 14: Methods of producing and evaluating GLP2-XTEN containing GLP-2 and ] A l schema for producing and evaluating GLP2-XTEN compositions is presented in and forms the basis for the general description of this Example. The GLP-2 peptides and sequence variants may be prepared recombinantly. Exemplary recombinant methods used to prepare GLP-2 peptides include the following, among others, as will be apparent to one skilled in the art. Typically, a GLP-2 peptide or sequence variant as deﬁned and/or described herein is prepared by constructing the nucleic acid encoding the desired peptide, cloning the nucleic acid into an expression vector in frame with nucleic acid encoding one or more XTEN, orming a host cell (e.g., bacteria such as Escherichia coli such as Saccharomyces cerevisiae or mammalian cell such as Chinese r , yeast , ovary cell or baby hamster kidney cell), and expressing the c acid to produce the desired GLP2- XTEN. Methods for producing and expressing recombinant polypeptides in vitro and in prokaryotic and eukaryotic host cells are known to those of ordinary skill in the art. See, for example, U.S. Pat. No. 122, and Sambrook et al., Molecular g—A Laboratory Manual (Third Edition), Cold Spring Harbor Laboratory Press (2001).

Using the disclosed methods and those known to one of ordinary skill in the art, together with guidance provided in the illustrative examples, a skilled artesian can create and evaluate GLPZ-XTEN ﬁasion proteins comprising XTENs, GLP-2 and variants of GLP-2 disclosed herein or otherwise known in the art. The Example is, ore, to be construed as merely illustrative, and not limitative of the s in any way whatsoever; numerous variations will be apparent to the ordinarily skilled artisan. In this Example, 3 GLP2-XTEN comprising a GLP-2 linked to an XTEN of the AE family of motifs is created.

] The general scheme for producing polynucleotides encoding XTEN is presented in FIGS. 4 and . is a schematic rt of representative steps in the assembly of a XTEN polynucleotide construct in one of the embodiments of the invention. Individual oligonucleotides 501 are annealed into sequence motifs 502 such as a 12 amino acid motif er”), which is ligated to additional sequence motifs from a library that can multimerize to create a pool that encompasses the desired length of the XTEN 504, as well as ligated to a smaller concentration of an oligo containing BbsI, and KpnI restriction sites 503. The motif libraries can be limited to speciﬁc sequence XTEN families; e.g., AD, AE, AF, AG, AM, or AQ sequences of Table 3. As illustrated in the XTEN length in this case is 864 amino acid residues, but shorter or longer lengths can be achieved by this process. For e, multimerization can be performed by ligation, overlap extension, PCR assembly or similar cloning techniques lmown in the art. The resulting pool of ligation products is gel-purified and the band with the desired length ofXTEN is cut, resulting in an isolated XTEN gene with a stopper sequence 505. The XTEN gene can be cloned into a stuffer vector. In this case, the vector encodes an optional CBD sequence 506 and a GFP gene 508. Digestion is than performed with BbsI/HindIII to remove 507 and 508 and place the stop codon. The resulting t is then cloned into a BsaI/HindIII digested vector containing a gene encoding the GLP-Z, ing in the gene 500 encoding a GLPZ-XTEN fusion protein.

As would be apparent to one of ordinary skill in the art, the methods can be applied to create constructs in alternative conﬁgurations and with varying XTEN lengths.

DNA sequences encoding GLP-2 can be conveniently obtained by standard procedures known in the art from a cDNA library prepared from an appropriate cellular source, from a genomic library, or may be created synthetically (e.g., automated nucleic acid synthesis), particularly where sequence variants (e. g., 2G) are to be incorporated, using DNA sequences obtained from publicly available databases, patents, or ture references. In the present example, the GLP2G sequence is utilized. A gene or polynucleotide encoding the GLP—2 portion of the protein or its complement can be then be cloned into a construct, such as those described herein, which can be a plasmid or other vector under control of appropriate transcription and translation ces for high level protein expression in a biological system. A second gene or polynucleotide coding for the XTEN portion or its complement can be genetically fused to the nucleotides encoding the terminus of the GLP-2 gene by cloning it into the construct adjacent and in frame with the gene coding for the GLP-Z, through a ligation or multimerization step. In this manner, a chimeric DNA molecule coding for (or complementary to) the GLP2-XTEN fusion protein is ted within the uct. Optionally, a gene encoding for a second XTEN is inserted and ligated in-frame ally to the nucleotides encoding the GLP-Z-encoding region.

Optionally, this chimeric DNA molecule is transferred or cloned into r construct that is a more riate expression vector; e. g., a vector appropriate for a prokaryotic host cell such as E. coli, a eukaryotic host cell such as yeast, or a mammalian host cell such as CHO, BHK and the like. At this point, a host cell capable of sing the chimeric DNA molecule is ormed with the chimeric DNA molecule. The s containing the DNA segments of interest can be erred into an appropriate host cell by well-known methods, depending on the type of cellular host, as described supra.

Host cells containing the GLPZ-XTEN expression vector are cultured in conventional nutrient media modified as appropriate for activating the promoter. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. After expression of the fusion n, culture broth is harvested and ted from the cell mass and the ing crude t retained for purification of the fusion protein.

Gene sion is ed in a sample directly, for example, by tional Southern blotting, Northern blotting to quantitate the transcription ofmRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided . Alternatively, gene expression is measured by immunological of ﬂuorescent methods, such as immunohistochemical staining of cells to quantitate directly the expression of gene product. Antibodies useﬁll for immunohistochemical staining and/or assay of sample ﬂuids may be either monoclonal or polyclonal, and may be prepared in any mammal.

Conveniently, the antibodies may be prepared t the GLP-2 sequence polypeptide using a synthetic peptide based on the sequences provided herein or against exogenous sequence fused to GLP-2 and encoding a speciﬁc antibody epitope. Examples of selectable markers are well known to one of skill in the art and include reporters such as enhanced green ﬂuorescent protein (EGFP), beta-galactosidase (B- gal) or chloramphenicol acetyltransferase (CAT).

The GLP2-XTEN polypeptide product is puriﬁed via methods known in the art. Procedures such as gel ﬁltration, y puriﬁcation, salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxyapatite adsorption chromatography, hydrophobic interaction tography or gel electrophoresis are all techniques that may be used in the puriﬁcation. Speciﬁc s of puriﬁcation are bed in Robert K. Scopes, Protein Puriﬁcation: Principles and ce, s R. Castor, ed., Springer—Verlag 1994, and Sambrook, et al., supra. Multi—step puriﬁcation tions are also described in Baron, er al., Crit. Rev. Biotechnol. —90 (1990) and Below, er al., J. Chromatogr. A. 679:67-83 (1994).

As illustrated in the isolated GLP2-XTEN fusion proteins would then be characterized for their chemical and activity properties. Isolated fusion protein is characterized, e.g., for sequence, purity, apparent molecular weight, solubility and stability using standard methods known in the art. The fusion protein meeting expected standards would then be evaluated for activity, which can be measured in vitro or in vivo by measuring one of the GLP-Z-associated parameters described herein, using one or more assays disclosed herein, or using the assays of the es or the assays of Table 32.

In addition, the GLPZ-XTEN fusion protein is administered to one or more animal species to determine standard cokinetic parameters and pharmacodynamic properties, as described in Examples 18-21.

] By the iterative s of producing, expressing, and recovering GLP2-XTEN constructs, followed by their characterization using methods disclosed herein or others known in the art, the GLP2- XTEN compositions comprising GLP-2 and an XTEN can be produced and evaluated by one of ry skill in the art to conﬁrm the expected ties such as enhanced solubility, enhanced stability, improved pharmacokinetics and reduced immunogenicity, leading to an overall enhanced therapeutic activity compared to the corresponding unfused GLP-2. For those fusion ns not possessing the desired properties, a different sequence can be constructed, expressed, isolated and evaluated by these s in order to obtain a composition with such properties.

Example 15: Construction of GLPZ-XTEN genes and vectors Oligonucleotides were designed and constructed such that the entire GLP-Z gene could be assembled through the tiling together of these oligonucleotides via designed complementary over hang regions under conditions of a 48°C annealing temperature. The complementary regions were held constant, but the other regions of the oligonucleotides were varied such that a codon library was d with ~50% of the codons in the gene varied instead of the single native gene sequence. A PCR was performed to create a combined gene library which, as is typical, contained a variety of combinations of the oligonucleotides and presented as a smear on an agarose gel. A polishing PCR was performed to amplify those assemblies that had the correct termini using a set of ampliﬁcation s complimentary to the 5’ and 3’ ends of the gene. The product of this PCR was then gel d, taking only bands at the N] 00 bp length of the expected GLP-2 ﬁnal gene product. This gel-puriﬁed product was digested with Bsal and Ndel and ligated into a rly digested construct containing DNA encoding a CBD leader sequence and the AE864 XTEN, to produce a GLP2-XTEN_AE864 gene, and transformed in BL21 gold competent cells. Colonies from this transformation were picked into 500 pl cultures of SB in 96 deep well plates and grown to tion overnight. These cultures were stored at 4°C after 20 ul of these cultures was used to inoculate 500 pl of auto-induction media and these cultures were grown at 26°C for >24 hours. Following the grth the GFP ﬂuorescence of 100 pl of these auto-induction media cultures was measured using a fluorescence plate reader. The GFP fluorescence is proportional to the number of les of GLP2—XTEN_AE464 made and is therefore a read—out of total expression. The highest sing clones were identiﬁed, and a new 1 ml overnight was started in SB from the original saturated overnight culture of that clone. Mini-preps were performed with these new cultures and the derived plasmids were sequenced to determine the exact tide composition. An E. coli isolate was designated strain AC453 and was identiﬁed as a strain that ed the desired GLP-2_2G- XTEN_AE864 fusion protein. The DNA and amino acid sequences of the pre-cleavage expressed product (with a CBD leader and TEV cleavage sequence) and the amino acid sequence of the ﬁnal product GLP-Z-ZG—XTEN_AE864 (after TEV cleavage) are ed in Table 13.

Table 13: GLPZ-XTEN DNA and amino acid seguences Clone DNA Sequence Amino Acid Sequence Name CBD-TEV- ATGGCAAATACACCGGTATCAGGCAATTTGAAGGTTGAAT MANTPVSGNLKVEF 2G— TCTACAACAGCAATCCTTCAGATACTACTAACTCAATCAA YNSNPSDTTNSNPQ AE864 GTTCAAGGTTACTAATACCGGAAGCAGTGCAATT FKVTNTGSSAIDLSK (pCW812/ GATTTGTCCAAACTCACATTGAGATATTATTATACAGTAGA LTLRYYYTVDGQKD AC453) CGGACAGAAAGATCAGACCTTCTGGGCTGACCATGCTGCA QTFWADHAAHGSN ATAATCGGCAGTAACGGCAGCTACAACGGAATTACTTCAA GSYNGITSNVKGTF ATGTAAAAGGAACATTTGTAAAAATGAGTTCCTCAACAAA VKMSSSTNNADTYL TAACGCAGACACCTACCTTGAAATCAGCTTTACAGGCGGA ACTCTTGAACCGGGTGCACATGTTCAGATACAAGGTAGAT EISFTGGTLEPGAHV TTGCAAAGAATGACTGGAGTAACTATACACAGTCAAATGA QIQGRFAKNDWSNY CTACTCATTCAAGTCTGCTTCACAGTTTGTTGAATGGGATC TQSNDYSFKSASQF AGGTAACAGCATACTTGAACGGTGTTCTTGTATGGGGTAA TAYLNGV CGGTGGCAGTGTAGTAGGTTCAGGTTCAGGATCC LVWGKEPGGSVVGS GAAAATCTGTATTTTCAACATGGTGACGGCTCTTTTAGCGA GSGSENLYFQHGDG TGAAATGAATACTATACTGGACAACCTTGCGGCACGCGAC Clone DNA Sequence Amino Acid ce Name TTCATTAACTGGCTGATCCAGACAAAAATCACCGATGGAG SFSDEMNTILDNLA GTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTAC ARDFINWLIQTKITD TTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTA GGSPAGSPTSTEEGT CTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGG SESATPESGPGTSTE CTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTT PSEGSAPGSPAGSPT CCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGA STEEGTSTEPSEGSA GGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAA PGTSTEPSEGSAPGT TCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAA CCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCC SESATPESGPGSEPA AGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGT TSGSETPGSEPATSG GAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCT SETPGSPAGSPTSTE CTACCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCTAC EGTSESATPESGPGT GTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGG STEPSEGSAPGTSTE TTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGT PSEGSAPGSPAGSPT CCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTCTGA STEEGTSTEPSEGSA GGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAG PGTSTEPSEGSAPGT TCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCG SESATPESGPGTSTE CACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCC PSEGSAPGTSESATP AGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGT ACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTACTT EPATSGSET CTACTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGA PGTSTEPSEGSAPGT AAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGAAAGC STEPSEGSAPGTSES GCAACCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTC ATPESGPGTSESATP CAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCC ESGPGSPAGSPTSTE TGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCT ATPESGPGS GAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTG EPATSGSETPGTSES GCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCC ATPESGPGTSTEPSE AGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGT GSAPGTSTEPSEGSA ACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCT PGTSTEPSEGSAPGT CTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTAC CGAACCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAA STEPSEGSAPGTSTE CCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTC PSEGSAPGTSTEPSE CCACCGAGGAAGGTACTTCTACCGAACCGTCCGA GSAPGSPAGSPTSTE GGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAG EGTSTEPSEGSAPGT TCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGA SESATPESGPGSEPA CTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCC TSGSETPGTSESATP CGAACCTGCAACCTCTGGCTCTGAAACCCCAGGT ESGPGSEPATSGSET ACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTT PGTSESATPESGPGT CTACTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGA STEPSEGSAPGTSES AAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGC ATPESGPGSPAGSPT TCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTC CAACTTCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGAC STEEGSPAGSPTSTE CTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAG EGSPAGSPTSTEEGT TCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCG SESATPESGPGTSTE CACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCC PSEGSAPGTSESATP AGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGT ESGPGSEPATSGSET ACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCG PGTSESATPESGPGS AACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGA EPATSGSETPGTSES AAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAA PGTSTEPSE CCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTC GSAPGSPAGSPTSTE CAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCC EGTSESATPESGPGS TGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCT GAAACCCCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCG EPATSGSETPGTSES GCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGA ATPESGPGSPAGSPT AGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGT STEEGSPAGSPTSTE ACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTT EGTSTEPSEGSAPGT CTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGA SESATPESGPGTSES AAGCGCTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGC PGTSESATP CCGGAATCTGGCCCAGGTAGCGAACCGGCTACTT ESGPGSEPATSGSET 13:33: DNA Sequence Amino Acid ce CTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGG PGSEPATSGSETPGS TTCTGAAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCC PAGSPTSTEEGTSTE ACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCG PSEGSAPGTSTEPSE CACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCC GSAPGSEPATSGSET AGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGT ACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTT EggsggégiggGPGT CTACTGAACCGTCCGAGGGCAGCGCACCAGGT GLP-2—2G— CATGGTGACGGCTCTTTTAGCGATGAAATGAATACTATAC HGDGSFSDEMNTIL AE864 TGGACAACCTTGCGGCACGCGACTTCATTAACTGGCTGAT DNLAARDFINWLIQ CCAGACAAAAATCACCGATGGAGGTAGCCCGGCTGGCTCT TKITDGGSPAGSPTS CCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTC TEEGTSESATPESGP CTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGG GTSTEPSEGSAPGSP TAGCGCTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACT AGSPTSTEEGTSTEP GAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCAC SEGSAPGTSTEPSEG CAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGG TACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGC SAPGTSESATPESGP GAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAAC GSEPATSGSETPGSE CGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGG ETPGSPAGS CTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCA PTSTEEGTSESATPE ACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTG SGPGTSTEPSEGSAP AGGGCAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGG SEGSAPGSP TAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACC AGSPTSTEEGTSTEP GAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCAC SEGSAPGTSTEPSEG CAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGG SAPGTSESATPESGP TACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACT GTSTEPSEGSAPGTS TCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTG AAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGC ESATPESGPGSEPAT TACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGT SGSETPGTSTEPSEG CCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTGA SAPGTSTEPSEGSAP CGCACCAGGTACTTCTGAAAGCGCAACCCCGGAA GTSESATPESGPGTS TCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCG ESATPESGPGSPAGS GCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGA PTSTEEGTSESATPE AGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGT SGPGSEPATSGSETP AGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACCT GTSESATPESGPGTS GCGCTACTCCGGAGTCTGGCCCAGGTACCTCTAC SAPGTSTEP TGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAA SEGSAPGTSTEPSEG CCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGT CCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGA SAPGTSTEPSEGSAP GGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGT GTSTEPSEGSAPGTS AGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCG TEPSEGSAPGSPAGS CACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGA GTSTEPSEG AGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGT SAPGTSESATPESGP ACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCG GSEPATSGSETPGTS AACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGA ESATPESGPGSEPAT AAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCA SGSETPGTSESATPE ACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTA SGPGTSTEPSEGSAP CTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGA GTSESATPESGPGSP CGCACCAGGTACTTCTGAAAGCGCTACTCCTGAG TCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCG AGSPTSTEEGSPAGS AGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGA PTSTEEGSPAGSPTS AGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGT TEEGTSESATPESGP ACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCT SEGSAPGTS CTACCGAACCGTCTGAGGGCAGCGCACCAGGTACCTCTGA SGPGSEPAT AAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCT SGSETPGTSESATPE ACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAA SGPGSEPATSGSETP CCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGG GTSESATPESGPGTS CTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAA TEPSEGSAPGSPAGS CCAGGTACTTCTACTGAACCGTCCGAGGGCAGCG PTSTEEGTSESATPE CACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGA WO 40093 Clone DNA ce Amino Acid Sequence Name AGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGT SGPGSEPATSGSETP AGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACTT GTSESATPESGPGSP CTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGC AGSPTSTEEGSPAGS TGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGC PTSTEEGTSTEPSEG TCTCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTC SAPGTSESATPESGP CGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCT GTSESATPESGPGTS GGCCCAGGTACTTCTGAAAGCGCTACTCCTGAAT ESATPESGPGSEPAT CAGGTACTTCTGAAAGCGCTACCCCGGAATCTGG CCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCA SGSETPGSEPATSGS GGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTA ETPGSPAGSPTSTEE GCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTC GTSTEPSEGSAPGTS TACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACT TEPSEGSAPGSEPAT TCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAA SGSETPGTSESATPE CCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTAC SGPGTSTEPSEGSAP TCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAG GGCAGCGCACCAGGT Example 16: Expression and puriﬁcation of fusion proteins comprising GLP2G fused to XTEN_AE864.

The host strain for expression, AmE025, was derived from E. coli W3110, a strain with a K-12 background, having a deletion of theﬂmA gene and with the lambda DE3 prophage integrated onto the chromosome. The host cell contained the plasmid pCWlOlO (AC616), ng an amino acid sequence that is identical to that d by pCW812 (AC453). The ﬁnal construct comprised the gene encoding the cellulosome anchoring protein cohesion region cellulose binding domain (CBD) from Clostridium thermocellum (accession #ABN54273), a tobacco etch virus (TEV) protease recognition site (ENLYFQ), the GLPZ-ZG sequence, and an AE864 amino acid XTEN sequence under control of a T7 er. The protein was expressed in a 5L glass jacketed fermentation vessel with a B. Braun Biostat B controller.

Brieﬂy, a starter culture of host strain AmE025 was used to ate 2L of fermentation batch media.

After 6 hours of culture at 37°C, a 50% glucose feed was initiated. After 20 hours of culture, the temperature was reduced to 26°C and 1M IPTG was added to induce expression. After a total fermentation run time of 45 hours, the culture was harvested by centrifugation yielding cell pellets ~1 kg in wet weight. The pellets were stored frozen at -80°C until puriﬁcation was initiated.

Lysis, heat ation and clariﬁcation The ing cell paste was resuspended at ambient temperature in 20 mM Cl pH 7.5, 50 mM NaCl, at a ratio of N4 ml per 1 g of cell paste. The cells were lysed by 2 passes through an APV 2000 homogenizer at an operating pressure of 800-900 bar. After lysis, the homogenate was heated to ~85°C in a heat exchanger and held for 20 minutes to coagulate host cell protein, then rapidly cooled to ~10°C. The cooled homogenate was clariﬁed by centriﬁugation at 4,000 rpm for 60 min using a Sorvall H6000A rotor in a Sorvall RC-3C fuge. The supernatant was decanted, passed through a 6OSPO3A Zeta Plus EXT depth ﬁlter (3M), followed by passage through a 0.2 um LifeASSURE PDA sterile capsule and stored at 4°C overnight.

] Initial Anion Exchange capture with arl SuperQ-65OM resin WO 40093 GLP2-2G-XTEN was ed out of the clariﬁed lysate using 3 columns steps at ambient temperature. GLP2-2G-XTEN was captured using Toyopearl SuperQ-650M (Tosoh) anion exchange resin, which selects for the negatively charged XTEN polypeptide tail and removes the bulk of host cell protein. An appropriately scaled SuperQ-650M column was equilibrated with 5 column volumes of 20 mM Tris-HCl pH 7.5, 50 mM NaCl and the lysate was loaded onto the column at a linear ﬂow rate of 120 cm/hr. The column was then washed with 3 column s of 20 mM Tris-HCl pH 7.5, 50 mM NaCl and 3 column volumes of 20 mM Tris-HCl pH 7.5, 150 mM NaCl, until the UV absorbance returned to baseline. GLP2-2G-XTEN protein was eluted with a 7 column volume linear gradient from 150 mM NaCl to 300 mM NaCl in 20 mM NaCl Tris-HCl, pH 7.5. Fractions were collected throughout and analyzed by SDS-PAGE for pooling and storage at 2-8°C. Product purity was determined to be ~80% after the Super Q e step.

Intermediate Anion Exchange capture with GE MacroCap Q resin The resulting SuperQ pool was diluted ~4-fold with 20 mM Tris-HCl pH 7.5 to reduce the conductivity to < 10 mS/cm. An appropriately scaled MacroCap Q anion ge column (GE Life Sciences) selects for the full-length intact XTEN polypeptide tail and removes the bulk of endotoxin and any al host cell protein and DNA. The column was equilibrated with 5 column volumes of 20 mM Tris-HCl pH 7.5, 50 mM NaCl. The diluted SuperQ pool was loaded at a linear ﬂow rate of 120 cm/hr.

The column was then washed with 3 column volumes of 20 mM Tris—HCl pH 7.5, 50 mM NaCl, and then 3 column volumes of20mM Tris—HCl pH 7.5, 150 mM NaCl, until the UV absorbance returned to ne. GLP2-2G-XTEN protein was eluted with a 12 column volume linear gradient from 150 mM NaCl to 300 mM NaCl in 20 mM Tris-HCl pH 7.5. Fractions were collected hout and analyzed by SDS-PAGE for pooling and storage at 2-8°C. Product purity was determined to be >95% after the ap Q intermediate step.

Hydrophobic Interaction Chromatography [HIC] using Toyopearl Phenyl-650M resin An appropriate amount of solid NaCl salt was dissolved in the MacroCap Q pool to adjust load to 4 M NaCl. and then was sterile d through a 0.2 um ﬁlter. An appropriately scaled Toyopearl Phenyl-650M (Tosoh) column selects for the hydrophobic residues of the GLP2 payload and removes al XTEN fragments and endotoxin. The column was equilibrated with 5 column volumes of 20 mM Tris-HCl pH 7.5, 4 M NaCl. The MacroCap Q pool was loaded at a linear ﬂow rate of 60 cm/hr. The column was then washed with 3 column volumes of 20 mM Tris-HCl pH 7.5, 4 M NaCl. G- XTEN n was eluted with a step-down gradient to 1.2 M NaCl in 20 mM Tris-HCl pH 7.5. The elution peak was fractionated and analyzed by SDS-PAGE to conﬁrm successful capture and elution of GLP2-2G-XTEN. Product purity was determined to be >95% after the ﬁnal polishing step. The resulting pool was concentrated to ~11 mg/ml and buffer exchanged into 20 mM Tris-HCl pH 7.5, 135 mM NaCl formulation buffer using a 30 KDa MWCO Pellicon XL 50 Ultraﬁltration Cassette (Millipore). The puriﬁed lot of GLP2-2G-XTEN was designated AP69O and stored at -80°C until r use.

SDS-PAGE Analysis SDS-PAGE analysis was conducted with 2 pg, 5 pg and 10 pg of AP690 loaded onto a NuPAGE 4-12% Bis Tris Gel (Invitrogen) and then run for 35 minutes at a constant 200V. The results (A) showed that the AP690 protein was free from host cell ties and that it migrated near the 160 kDa marker, the expected result for a payload-XTEN fusion protein of this lar weight and composition.

Endotoxin Content Endotoxin levels of lot AP690 was assessed using an fe PTS test cartridge (Carles River) and determined to be 3.5 EU/mg of protein, making the AP690 lot appropriate for injection into test animals for pharmacokinetic or pharmacodynamic studies.

Analy_tical size exclusion HPLC ] Gel ﬁltration analysis was performed using a Phenomenex BioSep-SEC-s4000 (7.8mm x 600mm) . 20 pg or AP690 G-XTEN fusion protein were analyzed at a ﬂowrate of 0.5 ml/min with 50 mM Phosphate pH 6.5, 300 mM NaCl mobile phase. Elution was monitored using OD215nm. Column calibration was performed using a size exclusion check standard from Phenomenex, with the following markers: lobulin (670 kDa), IgG (156 kDa), BSA (66 kDa) and ovalbumin (17 kDa). The result (B) indicated an apparent molecular weight of 1002 kDa for the fusion protein of 83.1 kDa actual weight, for an apparent molecular weight factor of 12.5.

Intact mass determination by ESI—MS 200 pg of AP690 GLP2—2G—XTEN protein was desalted by solid phase extraction using an Extract-Clean C18 column (Discovery Sciences). The ed protein solution in 0.1% formic acid, 50% acetonitrile was inﬁlsed at 4 pl/min into a QSTAR XL mass spectrometer (AB Sciex). Multicharge TOF spectrum was acquired in 800-1400 amu range. A zero-charge um was obtained by Bayesian reconstruction in 10-100 kDa range (). The experimental mass ofthe full length intact GLP2-2G- XTEN was determined to be 83,142 Da, with an additional minor peak of 83,003 Da detected, representing the des-His G-XTEN at <5% of total protein.

Example 17: Characterization of GLP2-XTEN in vitro receptor binding by calcium ﬂux potency assay A receptor binding assay was performed using a GPCRProﬁler assay (Millipore) to assess GLP2-2G-XTEN preparations (including AP690). The assay employed a transfected GLP2R cell line (Millipore, Cat# HTS164C) ting of a Chem-11 human cell stably transfected with the GLP2 G- protein d receptor and a G alpha protein that ates calcium ﬂux upon agonism of the GLP2 receptor. Assays were performed by addition of serial dilutions of GLP2-2G-XTEN, synthetic GLP2-2G peptide (without XTEN) and synthetic native GLP2 peptide, and the m ﬂux was monitored in real- time by a FLIPR TETRA instrument (Molecular Devices) using the no wash calcium assay kit (Molecular devices). The s, presented in , were used to derive EC50 values of 370 nM for GLP2-2G-XTEN and 7 nM for GLP2-2G peptide. The s indicate that the GLP2-2G-XTEN was able to bind and activate the GLP-2 receptor, with about 2% ofthe potency compared to GLP2-2G.

WO 40093 Example 18: Pharmacokinetic evaluation of GLPZ-XTEN in mice The fusion protein GLP2-2G-XTEN_AE864 was evaluated for its cokinetic properties in C57Bl/6 mice following subcutaneous (SC) administration. Female C57Bl/6 mice were injected SC with 2 mg/kg (25 nmol/kg) of the GLP2-2G—XTEN (lot AP498A) at 0.25mg/mL (8 mL/kg). Three mice were sacriﬁced at each of the following time points: Predose, 0.08, 4, 8, 24, 48, 72, 96 and 120 hours post-dose. Blood samples were collected from the mice and placed into prechilled heparinized tubes at each interval and were separated by centrifugation to recover the plasma. The samples were analyzed for ﬁasion protein concentration, performed by both anti-XTEN/anti- XTEN sandwich ELISA (AS1405) and anti-GLP2/anti-XTEN sandwich ELISA (AS1717), and the results were analyzed using WinNonLin to obtain the PK parameters. Terminal half-life was ﬁt from 24 to 120 hours. The s are presented in Table 14 and , with both assays showing essentially lent results, with a terminal half-life of 31.6-33.9 h determined.

Table 14: GLP2-2G—XTEN-864 Pharmacokinetics (m1) GLPZ-XTEN 11,200 720,000 33.9 3.4 ELISA e 19: cokinetic evaluation of GLPZ-XTEN in rats The fusion protein G-XTEN_AE864 was evaluated for its pharmacokinetic properties in Wistar rats following SC administration of two different dosage levels. Prior to the ment, catheters were surgically implanted into the r vein of female Wistar rats. The catheterized animals were ized into two groups containing three rats each. The fusion protein GLP2-2G-XTEN (lot AP510) was administered to each rat via SC injection as follows: 1) Low Dose 2 mg/kg (25 nmol/kg); or 2) High Dose 16 mg/kg (200 nmol/kg). Blood samples (~O.2mL) were collected through the jugular vein catheter from each rat into prechilled nized tubes at pre-dose, 0.08, 4, 8, 24, 48, 72, 96, 120 and 168 hours after test nd stration (10 time points). Blood was processed into plasma by centrifugation, split into two ts for analysis by ELISA. The samples were analyzed for fusion protein concentration, performed by both anti-XTEN/anti-XTEN sandwich ELISA (AS1602) and anti- GLP2/anti-XTEN sandwich ELISA (AS1705) and the results were analyzed using WinNonLin to obtain the PK parameters. Terminal half life was ﬁt from 48 to 168 hours. The results are presented in Table 15 and , with both assays g essentially equivalent results and with a terminal half—life of 37.5— 49.7 h determined, greatly exceeding the reported terminal half—life for GLP—2 and for GLP2—2G. In addition, the pharmacokinetic proﬁle of GLP2—2G—XTEN after single subcutaneous administration to rats at 25 nmol/kg and 200 nmol/kg was dose proportional with the CH1“ and AUC increasing in an approximately linear manner.

Table 15: GLP2-2G—XTEN-864 Pharmacokinetics T 1/2 Cmax AUCInf Vz Cl (hr) (n ml) (hr*n mL) (mL) (mL/hr) TEN ELISA High Dose 42.0 37900 0 65.0 1.07 (l6mg/kg) Low Dose 42.6 6270 530000 43.4 0.71 (2 mg/kg) ANTI-GLPZ-XTEN ELISA High Dose 49.7 40300 3660000 70.2 0.972 (Mme/kg) Low Dose 37.5 6900 530000 43.4 0.797 (2 mg/kg) e 20: Pharmacokinetic evaluation of TEN in cynomolgus monkeys The fusion protein GLP2-2G-XTEN_AE864 was evaluated for its pharmacokinetic properties in male cynomolgus s following either subcutaneous or intravenous administration of the fusion protein at a single dosage level. Three male cynomolgus monkeys were injected IV and 3 male cynomolgus monkeys were injected SC with 2 mg/kg (25 nmol/kg) GLP2-2G-XTEN at time 0. Blood samples were collected from each monkey into prechilled heparinized tubes at se and at approximately 0.083 h (5 min), 1, 2, 4, 8, 24, 48, 72, 96, 120, 168, 216, 264, and 336 hours after administration ofthe fusion n for the ﬁrst phase of the study. Animals were allowed to “wash-out” for a 6 week period (4 weeks post-last collection time point of Phase 1), the groups were crossed over (SC to IV and IV to SC), and dosed again with the same dose of GLP2-2G-XTEN fusion protein. Blood samples were collected at pre-dose and at approximately 0.083 h (5 min), 1, 2, 4, 8, 24, 48, 72, 96, 120, 168, 216, 264, 336, 384, 432, and 504 hours ose in the second phase of the study. All blood samples were processed into plasma by centrifugation and split into two aliquots for analysis by ELISA. The samples were analyzed for fusion protein concentration, performed by anti-GLP2/anti-XTEN ELISA (AS1705) and the results were analyzed using WinNonLin to obtain the PK parameters. The results are presented in Table 16 and , with a terminal half-life for the GLP2-2G-XTEN_AE864 fusion protein of 110 h for IV and 120 h for SC administration determined. The bioavailability was 96% demonstrating that GLP2-2G-XTEN is rapidly and near completely absorbed after subcutaneous administration.

Table 16: G—XTEN-864 cokinetics GROUP Tl/z Cm AUCInf Vd (:1 (hr) (ng/ml) (hr*ng/mL) (mL/kg) (mL/hr) The cumulative results of the PK analyses were used to perform allometric scaling of GLP2- 2G_AE864 terminal half-life, clearance and volume of distribution using data from three species (mouse, rat and monkey). Pharmacokinetic values for a 70 kg human were predicted by extrapolating the log linear relationship between body weight and each pharmacokinetic parameter, as shown in . The data for terminal half life, volume of distribution and clearance are presented in Table 17. The ted terminal half-life in humans of 240 h, greatly exceeds the reported 3.2 h terminal half-life of teduglutide in humans r, J-F, et al. Pharmacokinetics, Safety, and Tolerability of Teduglutide, a Glucagon- Like Peptide-2 (GLP-2) Analog, Following le Ascending aneous Administrations in Healthy Subjects. J Clin Pharmacol (2008) 48: 1289-1299). The terminal half-life in humans can also be estimated using the predicted values for clearance (Cl) and volume of distribution (Vd) as 0.693 X Vd/Cl. ng this formula yields a predicted terminal half-life of 230 h in humans, which agrees well with the extrapolation from the animal Tl/z data, and which greatly exceeds the reported terminal half-life for native GLP-2 and for GLP2-2G.

Table 17: Allometric scalin 0f GLP2-2G—XTEN-864 harmacokinetics *predicted value Example 21: Pharmacodynamic evaluation of GLPZ-XTEN in animal models The in vivo pharmacologic activity of the GLP2-2G-XTEN_AE864 fusion protein was assessed using preclinical models of intestinotrophic growth in normal rats and efﬁcacy in mouse DSS-colitis and rat Crohn’s Disease.

In vivo evaluation of GLP2-2G-XTEN-AE864 in normal rats ] To determine the intestinotrophic ties of GLPZ-XTEN, small intestine grth in rats was measured as a primary pharmacodynamic nt. GLP2—2G-XTEN—AE864 fusion protein GLP2-2G peptide, or vehicle was administered via subcutaneous injection into male Sprague-Dawley rats ng 200-220 grams (10-12 rats per group). GLP2-2G e was dosed using the previously published n of 12.5 nmol/kg (0.05 mg/kg) twice daily for 12 days. GLP2-2G—XTEN was dosed at nmol/kg once daily for 12 days. After sacriﬁce, a midline incision was made, the small intestines were d, stretched to their maximum length and the length recorded. The fecal material was ﬂushed from the lumen and the small intestinal wet weight ed. The small intestine length and weight data were ed with an ANOVA model with a Tukey/Kramer post-hoc test for pairwise comparisons, with cance at p = 0.05.

: Treatment with GLP2-2G e for 12 days (12.5 nmol/kg/dose using the standard twice daily dosing regimen) resulted in a signiﬁcant increase in small intestine weight of 24% (FIG.

???A). There were no signiﬁcant effects on small intestine length. stration of equal moles GLP2- 2G-XTEN over the 12 day study (25 nmol/kg/dose, once daily) resulted in a similar signiﬁcant increase in small intestine weight of 31%. In contrast to the results seen with GLP2-2G peptide, the small intestine of GLP2-2G-XTEN treated rats showed a signiﬁcant increase in length of 9% (10 cm), and was visibly thicker than the tissues from vehicle-treated control animals. ().

Conclusions: The results of the study show that GLP2-2G-XTEN induced small intestine growth that was as good or better than G peptide, using equal nmol/kg dosing.

In vivo evaluation of G-XTEN-AE864 in murine acute DSS-induced colitis model To determine the efﬁcacy of GLP2-XTEN, the GLP2-2G—XTEN-AE864 fusion protein was evaluated in a mouse model of intestinal inﬂammatory colitis. Intestinal colitis was induced in female C57B1/6 mice (9—10 weeks of age) by feeding mice with 4.5% n sodium sulfate (DSS) dissolved in drinking water for 10 days, until ~20% body weight loss is observed. A naive, non-treated control group (group 1) was given normal drinking water for the on of the experiment. The DSS treated groups (groups 2-7) were treated SC with vehicle (group2), GLP2-2G peptide (no XTEN) (group 3) or GLP2- 2G-XTEN (lot AP5100 (groups 4-7). The treatment doses and regimens are outlined in Table 18, below; the GLP-2G peptide was administered BID days 1-10 while the fusion protein was stered QD in the morning with vehicle control administered in the evening days 1-10. Measured parameters included body weights (recorded daily) and the following terminal endpoints, determine at day 10 of the experiment: colon weight and length, small intestine weight and length, and stomach weight. Tissues were ﬁxed in formalin and then transferred to ethanol for ng and histopathology. The anatomical data was analyzed with an ANOVA model with a Tukey/Kramer post-hoc test for pairwise comparisons, with signiﬁcance at p = 0.05.

Table 18: Treatment groups GROUP N Treatment Dose Route Regimen Normal water + NA BID (10-12h) Vehicle DSS + Vehicle BID (10—12h) DSS + 0.05 mg/ gk 3 10 sc BID (10-12h) GLP2-2G peptide (12.5 nmol/kg) DSS + 6 mg/kg Fusion protein AM GLP2-2G-XTEN (75 nmol/kg) Vehicle PM DSS + 2 mg/kg Fusion protein AM l O GLP2-2G-XTEN (25 nmol/kg) Veh1cle PM.

DSS + 0.2 mg/kg Fusion protein AM GLP2-2G-XTEN (2.5 nmol/kg) Vehicle PM DSS + 0.02 mg/kg Fusion protein AM GLP2-2G-XTEN (0.25 nmol/kg) Vehicle PM Results: Treatment effects on body weight colon length and weight, small intestine weight and length and stomach weight were assessed on the day of sacriﬁce. Although DSS-treated mice showed the expected signiﬁcant decrease in body weight as compared to the l mice (see ), neither the mice treated with GLP2-2G peptide nor any of the groups of mice treated with any dose of GLP2-2G- XTEN mice showed a reduced loss ofbody weight loss over the course of the experiment. With respect to ent effects on colon, small intestine and stomach, the parameter with a statistically signiﬁcant change was an increase in small ine weight in the GLP2-2G-XTEN high dose group (6 mg/kg), compared to the l groups 1 and 2 and the GLP2-2G-XTEN medium dose group (2 mg/kg), compared to group 1 (data not shown). The GLP2-2G peptide did not induce cant growth in the d tissues in the current study. Histopathology examination was performed on group 2 (DSS/vehicle treated) and group4 (DSS/GLP2-2G-XTEN 6 mg/kg qd treated). Results of the examination indicated that small intestine samples from the vehicle treated mice show mild-moderate and marked s of mucosal atrophy (see A, B). The mucosa were sparsely lined by stunted villi (diminished height) and sed l thickness. In contrast, small intestine samples from mice treated with G-XTEN at 6 mg/kg qd showed normal mucosal architecture with elongated villi densely populated with columnar epithelial and goblet cells (see C, D). The results support the conclusion that, under the ions of the experiment, treatment with the GLP2-2G—XTEN fusion protein protected the intestines from the inﬂammatory effects of DSS, with maintenance of normal villi and mucosal architecture.

Efﬁcacy of GLP2-2G-XTEN vs. GLP2-2G peptide in rat Crohn’s Disease indomethacin induced inﬂammation model To determine the efﬁcacy of GLP2-XTEN using single dose or qd dosing, the GLP2-2G— XTEN—AE864 fusion protein was evaluated in a rat model of s Disease of indomethacin—induced intestinal inﬂammation in three separate s.

M: Intestinal ation was induced in eighty male Wistar rats (Harlan Sprague Dawley) using indomethacin administered on Days 0 and l of the experiment. The rats were divided into seven treatment groups for treatment according to Table 19.

Table 19: Treatment groups “Route Regimen + Indomethacin 1 10 ml/kg sc BID No 2 10 ml/kg sc BID Yes 0.05 mg/kg LP2-2 SC BID Yes <12~5nmovkg> --0.5 mg/kg 4 LP2—2 SC BID Yes <125nmol/kg) 2 mg/kg - GLPZ-ZG-XTEN SC QD Y“ l/kg) -6 mg/kg QD G-XTEN SC Yes <75nmol/kg) 7 Prednisolone 10 mg/kg PO QD Yes All treatments were administered per the le starting on Day -3 of the experiment. Body weights were determined daily. Groups 3 and 5 were dosed equimolar/day. On Day 2 (24 hours post-2nd indomethacin dose), the animals were prepped for sacriﬁce and is. Thirty minutes prior to sacriﬁce, the rats were injected intravenously with 1 ml 1% Evans Blue dye, in order to visualize ulcers and extent of inﬂammation by athology analysis. The rats were anesthetized (SOP 1810), blood samples were removed to determine the concentration of GLP2G-XTEN using the anti-XTEN/anti-GLP2 ELISA method. The rats were euthanized then necropsied and scored by gross ation of the intestines for the presence of adhesions; i.e., none = 0, mild = 1, moderate = 2, or severe = 3. The small intestines were removed and the length of each was recorded. In each small intestine, a longitudinal incision was made and the interior was examined. The degree and length of the ted area was recorded as a score; i.e., none = 0, few = 1, multiple = 2, or continuous = 3. For TNFOL determination, intestinal samples were thawed and homogenized in a total of 20 ml with DPBS. The supernatants were equilibrated to room temperature and assayed for TNFOL by ELISA (R&D Systems, Cat. RTAOO, lot 281687, exp. 07SEP11). The s for Group 1 were assayed ted. The samples for Groups 2-7 were diluted 1:4. For histopathology, the small intestines were gently washed with saline to remove the fecal al and were blotted to remove excess ﬂuid. Each small intestine was weighed then processed for histopathology examination to quantitate the degree of inﬂammation; i.e., .0% = 0, 1-33% = 1, 34- 66% = 2, 67-100% = 3.

RLults: The values and scores for the body weight and various small intestine parameters are presented graphically in . The changes in parameters and scores for Group 2 control animals versus Group 1 healthy controls indicates that the model is representative ofthe disease process. Results of body weights (A) indicate that the GLP2-2G did not have a cant increase in body weight ed to disease control (Group 2), while the GLP2G-XTEN groups demonstrated a signiﬁcant increase. Results from the small intestine length (B) showed a signiﬁcant increase for both the GLP2G peptide and GLP2G-XTEN fusion protein treatments, with the latter resulting in length equivalent to the non-diseased control (Group 1). Results from the small intestine weight (C) WO 40093 showed a signiﬁcant increase for the 0.5 mg/kg GLP2G peptide and both GLP2G—XTEN fusion protein groups, compared to diseased control Group 2. Based on gross ogy scoring of the small intestine, both the GLP2G peptide and 2G-XTEN fusion protein treatments resulted in signiﬁcant decreases in ulceration (D), with the 6 mg/kg ﬁlSlOl’l protein resulting in a score that was not signiﬁcantly different from the non-diseased control (Group 1). Based on scoring of adhesions and transulceration (E), both the GLP2G peptide and GLP2G-XTEN fusion protein treatments showed signiﬁcant decreases ed to diseased l (Group 2), with the 2 and 6 mg/kg ﬁJsion n resulting in scores that were not signiﬁcantly ent from the non-diseased control (Group 1). Based on scoring of small ine ation (F), neither the GLP2G peptide nor the GLP2G-XTEN fusion protein ents showed a signiﬁcant effect on inﬂammation. Based on TNFoc assays (G), both the GLP2G peptide and GLP2G-XTEN fusion protein treatments showed signiﬁcantly decreased cytokine levels compared to the diseased control Group 2.

Conclusions: The results of the study show that GLP2-2G-XTEN provided efﬁcacy that was as good or better than GLP2-2G peptide, using equal nmol/kg dosing, in improving indomethacin-induced small intestine damage.

M: Intestinal inﬂammation was induced in eighty male Wistar rats (Harlan Sprague Dawley) using indomethacin administered on Days 0 and 1 of the experiment. The rats were divided into eight treatment groups for treatment according to Table 20.

Table 20: Treatment groups 1 10 ml/kg sc BID N 2 10 ml/kg sc BID Yes 0.05 mg/kg 3 LP2—2 sc BID Yes <12-5nmol/kg) 2 mg/kg Once daily 4 -GLP2-2G-XTE\ SC Y65 <25nmol/kg) (QB) (23:35.: All treatments were administered per the le starting on Day -3 of the experiment. Body weights were determined daily. On Day 2 (24 hours post-2nd indomethacin dose), the animals were prepped for sacriﬁce and is. Thirty minutes prior to ce, the rats were injected intravenously with 1 ml 1% Evans Blue dye, in order to visualize ulcers and extent of inﬂammation by histopathology analysis. The rats were anesthetized and blood samples were removed to determine the concentration of 2012/054941 GLP2G-XTEN using the anti-XTEN/anti-GLP2 ELISA method. The rats were euthanized then sied and scored by gross examination of the intestines for the presence of ons; i.e., none = 0, mild = 1, moderate = 2, or severe = 3. The small intestines were d and the length of each was recorded. In each small intestine, a longitudinal incision was made and the interior was examined. The degree and length of the ulcerated area was recorded as a score; i.e., none = O, few = 1, multiple = 2, or continuous = 3. The fecal material was washed away with saline and d to remove excess ﬂuid and each small intestine was weighed then processed for histopathology examination to quantitate the degree ofinﬂammation; i.e., .O% = O, l-33% = 1, 34-66% = 2, 67-100% = 3.

RLults: The scores for the various ters are presented graphically in . In the vehicle negative control group, the gross pathologic changes due to indomethacin treatment were most severe in the ileum and m, with a total disease score of 8.5-9 by assessment of this group. Of the s GLP2G peptide and GLP2G-XTEN treatment groups, the GLP2G peptide red bid, the GLP2G-XTEN delivered qd, and the single doses of GLP2G-XTEN at 6 or 2 mg/kg resulted in signiﬁcantly improved scores compared to the indomethacin-treated vehicle control group. In the trans- ulceration scores, the same treatment groups as per the total disease score d tical signiﬁcance (A, with star indicating statistically signiﬁcant difference compared to vehicle . In the adhesions score analysis, the indomethacin-treated vehicle l group approached the maximum score of 3 (B). Once—daily treatment with the GLP—2—2G—XTEN provided nearly te protection from adhesions, and the single high—dose 6 mg/kg GLP—2—2G—XTEN group reached statistically signiﬁcant difference compared to vehicle control (star in ﬁgure indicating statistically signiﬁcant difference), as did the daily bid dosed GLP2G peptide group. In the small intestine length analysis (with the non-indomethacin treated group ized to 100%), the once-daily treatment with the GLP- 2-2G-XTEN group and the daily bid dosed GLP2G peptide group reached statistically signiﬁcant difference compared to indomethacin-treated vehicle control group. The histopathology assessment ﬁnding were essentially similar to the gross pathology ﬁndings. The histopathologic changes in the e control group due to indomethacin treatment were most severe in the ileum and jejunum. The vehicle control group showed severe mucosal atrophy, ulceration and inﬁltration (A). The protective effects of the daily bid GLP2G peptide and once-daily GLP2G-XTEN treatments were most nced in the ileum, but were also seen in the jejunum. Group 3 had one rat with essentially normal tissue (B) while two rats each showed ulceration and inﬁltration but no atrophy and two rats had histopathologic changes similar to the vehicle control disease group 2. Group 4 (D) showed protective effects with two rats with essentially normal tissue, one rat showing no atrophy or ulceration but with slight inﬁltration, one rat with no atrophy but slight ulceration and inﬁltration, and one rat had histopathologic changes similar to the vehicle control e group 2. Group 7 showed tive effects with one rat with essentially normal tissue, two rats with no ulceration or inﬁltration but showing muscular atrophy, and two rats had histopathologic changes similar to the vehicle control e group 2. Group 8 (C) showed protective effects with one rat with no ulceration or inﬁltration, one rat with reduced tion and inﬁltration, and three rats had histopathologic changes similar to the vehicle control e group 2. The ELISA results te that the GLP2G-XTEN fusion n was detectable at Day 2 in all animals of Group 4 and Group 8, and three rats in Group 7.

The results t the conclusion that, under the conditions of the experiment, treatment with the GLP2-2G-XTEN fusion protein provided signiﬁcant protection to the intestines from the atory effects of indomethacin, with daily dosing at 2 mg/kg showing the greatest efﬁcacy and single doses of 6 mg/kg or 2 mg/kg showing signiﬁcant efﬁcacy in some parameters.

Stidxﬁ: A third indomethacin-induced inﬂammation study was performed to verify previous results and test additional dose regimens. Intestinal inﬂammation was induced in male Wistar rats (Harlan Sprague Dawley) using indomethacin administered on Days 0 and 1 ofthe experiment ing to Table 21.

Table 21: Treatment groups ——10 Mg QD ND --0.05 mg/kg 2 LP2-2 SC BID 125““)ng <12-5nmol/kg) --2 mg/kg Once daily 3 GLP2-2 -XTEN SC 1251““ gl k <25nmol/kg) (QD) --2 mg/kg Day —3 —1 1 4 GLP2-2G-XTEN SC ’ ’ 75m” gl k <25nmol/kg) <sz 6 mg/kg Once day —3 GLP2-2G-XTEN SC 75 nmol/kg (75 nmol/kg) only ] All treatments were administered per the schedule starting on Day —3 of the experiment. Body weights were determined daily. On Day 2 (24 hours post—2nd indomethacin dose), the animals were d for sacriﬁce and analysis. The small intestines were removed and the length of each was recorded. Quantitative histopathology was performed on a subset of samples. Rat small intestine s consisted of a 3 cm section of proximal jejunum and a 3 cm section of mid-j ejunum collected 15 cm and cm from the pylorus, respectively. Samples were ﬁxed in 10% neutral buffered formalin. Samples were trimmed into multiple ns without bias toward lesion ce or absence. These sections were placed in cassettes, embedded in parafﬁn, microtomed at approximately 4 microns thickness, and stained with hematoxylin and eosin (H&E). The slides were evaluated microscopically by a board certiﬁed veterinary pathologist and scored for villous height as well as inﬁltration/inﬂammation, mucosal atrophy, villi/crypt ance, abscesses/ulceration. A 1 to 4 ty grading scale was used, where 1 = l, 2 = mild, 3 = moderate, 4 = marked/severe, reﬂecting the combination ofthe cellular reactions seen histopathologically. Small intestine length was analyzed with an ANOVA model with a Tukey/Kramer post-hoc test for se comparisons, with signiﬁcance at p = 0.05. Non-parametric histology score variables were compared with the vehicle control using a Mann Whitney U test with a Bonferroni correction for the p-value to create an overall alpha of 0.05. s: As seen in the initial studies, there was an increase in small intestine length in the GLP2-2G-XTEN—treated ed rats as compared to vehicle-treated diseased rats (A). This increase correlated with a signiﬁcant increase in villi height (B). Both high (total dose of 125 nmol/kg) and low (total dose of 75 nmol/kg) dose GLP2-2G-XTEN-treated groups showed a signiﬁcant increase in villi height; the increase in villi height seen in e treated rats was not signiﬁcant. There was also a signiﬁcant decrease in l atrophy as both high and low dose GLP2-2G-XTEN-treated rats showed a cantly lower mucosal atrophy score than vehicle-treated ed rats (C).

Although there was a trend showing a reduction in mucosal tion and mixed cell inﬁltrate following G-XTEN and GLP2-2G peptide treatment, these results were not signiﬁcant for any of the three ent groups.

Conclusions: Histopathological results t the conclusion that GLP2-2G-XTEN provided efﬁcacy that was as good or better than GLP2-2G peptide in improving indomethacin-induced small intestine damage. Furthermore, G-XTEN dosed once at 75 nmol/kg or three times at 25 nmol/kg is as effective as GLP2-2G peptide dosed ten times at 12.5 g.

Example 22: Human Clinical Trial Designs for Evaluating GLPZ-XTEN comprising GLP- As demonstrated in Examples 18-20, fusion ofXTEN to the C-terminus of GLP2glycine results in improved half—life compared to that known for the native form of the GLP—2 or the GLP—2—2G peptide, which, it is believed, would enable a reduced dosing frequency yet still result in clinical efﬁcacy when using such GLP2-XTEN—containing fusion protein compositions. Clinical trials in humans comparing a GLP2-XTEN fusion protein to GLP-2 (or GLP2G peptide) formulations are performed to establish the efﬁcacy and advantages, compared to current or experimental modalities, of the GLP2- XTEN binding fusion protein compositions. Such studies comprise three phases. First, a Phase I safety and cokinetics study in adult patients is conducted to determine the maximum tolerated dose and pharmacokinetics and codynamics in humans (e.g., normal healthy volunteer subjects), as well as to deﬁne ial toxicities and e events to be tracked in future studies. A Phase I study is conducted in which single rising doses of a GLPZ-XTEN composition, such as are disclosed herein, are administered by the desired route (e.g., by subcutaneous, intramuscular, or intravenous routes) and biochemical, PK, and clinical parameters are measured at deﬁned intervals, as well as adverse events. A Phase Ib study will multiple doses would follow, also measuring the biochemical, PK, and clinical parameters at deﬁned intervals. This would permit the determination of the minimum effective dose and the maximum tolerated dose and establishes the threshold and maximum concentrations in dosage and circulating drug that constitute the therapeutic window for the active component. From this information, the dose and dose schedule that permits less frequent administration of the GLP2-XTEN compositions (compared to GLP-2 not linked to XTEN), yet retains the pharmacologic response, is obtained.

Thereafter, Phase II and III clinical trials are conducted in patients with the GLP-2 associated condition, ing the effectiveness and safety of the TEN compositions under the dose conditions. al trials could be conducted in patients suffering from any disease in which native GLP-2 or the standard of care for the given condition may be expected to provide al beneﬁt. For e, such indications include gastritis, digestion disorders, malabsorption syndrome, gut syndrome, short bowel syndrome, cul-de-sac syndrome, inﬂammatory bowel disease, celiac e, tropical sprue, hypogammaglobulinemic sprue, Crohn's disease, ulcerative colitis, enteritis, chemotherapy-induced enteritis, ble bowel syndrome, small intestine damage, mucosal damage of the small intestine, small intestinal damage due to cancer-chemotherapy, intestinal injury, diarrhea] diseases, intestinal insufﬁciency, acid-induced intestinal injury, arginine deﬁciency, thic hypospermia, obesity, catabolic illness, febrile neutropenia, es, obesity, steatorrhea, autoimmune diseases, food allergies, hypoglycemia, gastrointestinal r disorders, sepsis, bacterial peritonitis, burn-induced intestinal damage, decreased gastrointestinal motility, intestinal failure, chemotherapy-associated bacteremia, bowel trauma, bowel ischemia, mesenteric ischemia, malnutrition, necrotizing enterocolitis, necrotizing pancreatitis, neonatal feeding intolerance, NSAlD-induced gastrointestinal damage, nutritional insufﬁciency, total parenteral ion damage to gastrointestinal tract, neonatal nutritional insufﬁciency, radiation-induced enteritis, radiation-induced injury to the intestines, mucositis, pouchitis, ischemia, and stroke. Trials monitor patients before, during and after treatment for s in physiologic and clinical parameters associated with the respective indications; e. g., weight gain, inﬂammation, cytokine levels, pain, bowel function, appetite, febrile episodes, wound healing, glucose levels; enhancing or rating hunger satiety; parameters that are tracked relative to the placebo or positive control groups. Efﬁcacy outcomes are determined using standard statistical methods. Toxicity and adverse event markers are also followed in the study to verify that the compound is safe when used in the manner described.

Example 23: GLPZ-XTEN with cleavage sequences C-terminal XTEN able by FXIa An GLPZ-XTEN fusion protein ting of an XTEN protein ﬁlsed to the inus of GLP-2 can be created with a XTEN release site ge sequence placed in between the GLP-2 and XTEN components, as depicted in Exemplary ces are provided in Table 34. In this case, the release site cleavage sequence can be incorporated into the GLP2-XTEN that contains an amino acid sequence that is recognized and cleaved by the FXIa se (EC 3.4.21.27, t P03951).

Speciﬁcally the amino acid sequence KLTRAET is cut after the arginine of the sequence by FXIa protease. FXI is the pro-coagulant protease located immediately before FVIII in the intrinsic or contact activated coagulation pathway. Active FXIa is produced from FXI by proteolytic cleavage of the zymogen by FXHa. Production of FXIa is tightly controlled and only occurs when coagulation is necessary for proper hemostasis. Therefore, by incorporation of the KLTRAET cleavage sequence, the XTEN domain is removed from GLP-2 concurrent with activation of the intrinsic coagulation y in proximity to the GLPZ-XTEN. inal XTEN releasable by Elastase-2 An GLP2-XTEN fusion protein consisting of an XTEN protein fused to the C-terminus of GLP-2 can be created with a XTEN release site cleavage sequence placed in between the GLP-2 and XTEN components, as depicted in Exemplary sequences are provided in Table 34. In this case, the release site contains an amino acid sequence that is recognized and cleaved by the elastase-2 protease (EC 3.4.21.37, Uniprot P08246). Speciﬁcally the sequence LGPVSGVP [Rawlings N.D., et al. (2008) Nucleic Acids Res, 36: D320], is out after position 4 in the sequence. Elastase is constitutively expressed by neutrophils and is present at all times in the circulation, but particularly during acute inﬂammation.

Therefore as the long lived GLP2-XTEN circulates, a fraction of it is cleaved, particularly locally during atory responses (e.g., inﬂammation ofthe bowel), creating a pool of r-lived GLP-2 at the site of inﬂammation, e. g., in s e, where the GLP-2 is most needed. inal XTEN releasable by MMP-12 An TEN fusion protein consisting of an XTEN protein fused to the C-terminus of GLP-2 can be created with a XTEN release site cleavage ce placed in n the GLP-2 and XTEN components, as depicted in Exemplary sequences are provided in Table 34. In this case, the release site ns an amino acid sequence that is recognized and cleaved by the MMP-12 protease (EC 3.4.24.65, Uniprot P39900). Speciﬁcally the ce GPAGLGGA [Rawlings N.D., et al. (2008) Nucleic Acids Res, 36 : D320], is out after on 4 of the sequence. MMP—l2 is constitutively expressed in whole blood. Therefore as the GLP2—XTEN circulates, a fraction of it is cleaved, creating a pool of shorter-lived GLP-2 to be used. In a desirable feature of the inventive composition, this creates a circulating ug depot that constantly releases a prophylactic amount of GLP-2, with higher amounts ed during an inﬂammatory response, e.g., in Crohn’s e, where the GLP-2 is most needed.

C-terminal XTEN able by MMP-13 An TEN fusion protein consisting of an XTEN protein fused to the C-terminus of GLP-2 can be created with a XTEN release site cleavage sequence placed in between the GLP-2 and XTEN components, as depicted in Exemplary sequences are provided in Table 34. In this case, the release site contains an amino acid sequence that is recognized and cleaved by the MMP-l 3 protease (EC 3.4.24.-, Uniprot P45452). Speciﬁcally the sequence GPAGLRGA [Rawlings N.D., et a1. (2008) Nucleic Acids Res, 36: D320], is out after position 4. MMP-13 is constitutively expressed in whole blood. Therefore as the long lived GLP2-XTEN circulates, a fraction of it is cleaved, creating a pool of shorter-lived GLP-2 to be used. In a desirable feature of the inventive composition, this creates a circulating pro-drug depot that constantly releases a prophylactic amount of GLP-2, with higher s released during an inﬂammatory se, e.g., in Crohn’s Disease, where the GLP-2 is most needed.

C-terminal XTEN releasable by MMP-17 A GLP2-XTEN fusion protein consisting of an XTEN protein fused to the C-terminus of GLP- 2 can be created with a XTEN release site cleavage sequence placed in between the GLP-2 and XTEN components, as depicted in Exemplary sequences are provided in Table 34. In this case, the e site contains an amino acid sequence that is recognized and cleaved by the MMP-20 protease (EC.3.4.24.-, Uniprot Q9ULZ9). Speciﬁcally the sequence LR [Rawlings N.D., et a1. (2008) Nucleic Acids Res, 36: D320], is cut after position 4 in the sequence. MMP-17 is constitutively expressed in whole blood. Therefore as the GLPZ-XTEN circulates, a fraction of it is cleaved, creating a pool of shorter-lived GLP-2 to be used. In a desirable feature of the inventive composition, this creates a circulating pro-drug depot that ntly releases a lactic amount of GLP-2, with higher amounts released during an inﬂammatory response, e.g., in Crohn’s Disease, where the GLP-2 is most needed.

] C-terminal XTEN releasable by MMP-20 A GLPZ-XTEN fusion protein consisting of an XTEN protein fused to the C-terminus of GLP- 2 can be created with a XTEN release site cleavage sequence placed in between the GLP-2 and XTEN components, as depicted in Exemplary sequences are provided in Table 34. In this case, the e site contains an amino acid sequence that is recognized and cleaved by the MMP-ZO protease 4.24.-, Uniprot 060882). Speciﬁcally the sequence AQ [Rawlings N.D., et al. (2008) Nucleic Acids Res, 36: D320], is out after position 4 (depicted by the arrow). MMP-20 is constitutively expressed in whole blood. Therefore as the TEN circulates, a fraction of it is cleaved, creating a pool of r-lived GLP-2 to be used. In a ble e of the inventive composition, this creates a circulating pro-drug depot that constantly releases a prophylactic amount of GLP-2, with higher amounts ed during an inﬂammatory response, e.g., in Crohn’s Disease, where the GLP—2 is most needed.

Optimization of the release rate of C-terminal XTEN Variants of the foregoing constructs of the Examples can be created in which the release rate of C-terminal XTEN is altered. As the rate ofXTEN release by an XTEN release protease is dependent on the sequence of the XTEN release site, by varying the amino acid sequence in the XTEN release site one can control the rate ofXTEN release. The sequence speciﬁcity ofmany proteases is well known in the art, and is documented in several data bases. In this case, the amino acid speciﬁcity of proteases is mapped using combinatorial libraries of substrates [Harris, JL, et al. (2000) Proc Natl Acad Sci US A, 97: 7754] or by following the cleavage of ate mixtures as illustrated in [Schellenberger, V, et a1. (1993) mistry, 32: 4344]. An alternative is the ﬁcation of optimal protease cleavage ces by phage y [Matthews, D., et a1. (1993) Science, 260: 1113]. Constructs are made with variant sequences and assayed for XTEN release using standard assays for detection of the XTEN.

] Example 24: Biodistribution of large XTEN molecules To verify that constructs with long XTEN fusions can penetrate into tissue, the biodistribution of three ﬂuorescently tagged constructs were tested in mice, aHer2-XTEN—864-Alexa 680, aHer2-XTEN- 576-Alexa 680, and aHer2-XTENAlexa 680, using fluorescence imaging. The aHer2 payload is a scFv fragment speciﬁc for binding the Her2 antigen, which is not found on normal tissues (and hence should not affect biodistribution in normal animals). This study also included ﬂuorescently tagged Herceptin-Alexa 680 as a control antibody. The mice were given a single intravenous injection of each agent. After 72 hours, all groups were euthanized and liver, lung, heart, spleen and kidneys were ex vivo imaged using ﬂuorescence imaging. The data are shown Table 22.

Conclusions: All constructs showed signiﬁcant penetration into all tissues assayed. The lower overall ﬂuorescence signals of the XTEN_S76 and XTEN_288 groups are attributed to the increased clearance of the shorter XTEN constructs over the 72 hour bution period. Similar proportions for lung ﬂuorescence relative to total signal were ed for all groups, including the antibody control, supportng that XTEN fusion protein constructs are bioavailable in tissue under these conditions.

Table 22: Fluorescence Signals by Organ Test Material scFv-XTEN— 6.7 28 130 16 120 864-Alexa 680 scFv—XTEN— Wiemso"m scFv-XTEN- 288—A1exa680 mAb'Alexa680 3.3 32 150 25 370 110 Example 25: Analytical size exclusion tography of XTEN fusion proteins with e payloads Size ion chromatography analyses were performed on fusion proteins containing various therapeutic proteins and unstructured recombinant proteins of increasing length. An exemplary assay used a -G4000 SWXL (7.8mm x 30cm) column in which 40 ug of puriﬁed glucagon fusion protein at a concentration of 1 mg/ml was separated at a ﬂow rate of 0.6 ml/min in 20 mM phosphate pH 6.8, 114 mM NaCl. Chromatogram proﬁles were monitored using OD214nm and OD280nm. Column calibration for all assays were performed using a size exclusion calibration standard from ; the markers e thyroglobulin (670 kDa), bovine gamma-globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobuin (17 kDa) and vitamin B12 (1.35 kDa). entative chromatographic proﬁles of Glucagon—Y288, Glucagon—Y144, Glucagon—Y72, Glucagon—Y36 are shown as an overlay in . The data show that the nt molecular weight of each compound is proportional to the length of the attached XTEN sequence. However, the data also show that the apparent molecular weight of each construct is signiﬁcantly larger than that expected for a globular protein (as shown by ison to the standard proteins run in the same assay). Based on the SEC analyses for all constructs evaluated, the apparent molecular weights, the apparent molecular weight factor (expressed as the ratio of apparent molecular weight to the calculated lar weight) and the hydrodynamic radius (RH in nm) are shown in Table 23. The results indicate that oration of different XTENs of 576 amino acids or r confers an apparent molecular weight for the fusion protein of imately 339 kDa to 760, and that XTEN of 864 amino acids or greater confers an apparent molecular weight greater than at least approximately 800 kDA. The results of proportional increases in apparent molecular weight to actual 2012/054941 molecular weight were tent for fusion proteins created with XTEN from several ent motif families; i.e., AD, AE, AF, AG, and AM, with increases of at least four-fold and ratios as high as about 17-fold. Additionally, the incorporation of XTEN fusion partners with 576 amino acids or more into fusion proteins with the various ds (and 288 residues in the case of glucagon fused to Y288) resulted with a hydrodynamic radius of 7 nm or greater; well beyond the glomerular pore size of approximately 3-5 nm. Accordingly, it is expected that fusion proteins comprising growth and XTEN have reduced renal clearance, contributing to increased terminal ife and improving the eutic or biologic effect relative to a corresponding un-fused biologic payload protein.

Table 23: SEC analysis of s polypeptides uct XEEEHM Therapeutic APWM $593161: RH Name Protein r (kl)a) 18:23:: (1111]) AC14 Y288 Glucagon 28.7 370 12.9 7.0 AC33 Y36 Glucagon 6.8 29.4 4.3 2.6 AC89 AF120 Glucagon 14.1 76.4 5.4 4.3 AC88 AF108 Glucagon 13.1 61.2 4.7 3.9 AC73 AF144 Glucagon 16.3 95.2 5.8 4.7 AC53 AG576 GFP 74.9 339 4.5 7.0 AC39 AD576 GFP 76.4 546 7.1 7.7 AC41 AE576 GFP 80.4 760 9.5 8.3 AC52 AF576 GFP 78.3 526 6.7 7.6 AC398 AE288 FVII 76.3 650 8.5 8.2 AC404 AE864 FVII 129 1900 14.7 10.1 AC85 AE864 Exendin-4 83.6 93 8 11.2 8.9 AC114 AM875 Exendin-4 82.4 1344 16.3 9.4 AC143 AM875 hGH 100.6 846 8.4 8.7 AC227 AM875 IL-lra 95.4 1103 11.6 9.2 AC228 AM1318 IL—lra 134.8 2286 17.0 10.5 Example 26: Pharmacokinetics of extended polypeptides fused to GFP in cynomolgus monkeys The pharmacokinetics of GFP-L288, GFP-L576, GFP-XTEN_AF576, GFP-XTEN_Y576 and XTEN_AD836-GFP were tested in lgus monkeys to determine the effect of composition and length of the unstructured polypeptides on PK parameters. Blood samples were analyzed at various times after injection and the concentration of GFP in plasma was measured by ELISA using a polyclonal antibody against GFP for capture and a biotinylated preparation of the same polyclonal antibody for detection. Results are summarized in . They show a surprising increase of half-life with increasing length of the XTEN sequence. For example, a half-life of 10 h was determined for GFP- XTEN_L288 (with 288 amino acid residues in the XTEN). Doubling the length of the unstructured polypeptide fusion partner to 576 amino acids increased the half-life to 20-22 h for multiple fusion protein constructs; i.e., GFP-XTEN_L576, GFP-XTEN_AF576, GFP-XTEN_Y576. A further increase of the unstructured polypeptide fusion partner length to 836 residues resulted in a half-life of 72-75 h for XTEN_AD836-GFP. Thus, increasing the polymer length by 288 residues from 288 to 576 residues sed in vivo half-life by about 10 h. However, increasing the ptide length by 260 residues from 576 residues to 836 es increased half-life by more than 50 h. These results show that there is a surprising threshold of unstructured polypeptide length that results in a greater than proportional gain in in viva ife. Thus, fusion ns comprising extended, unstructured polypeptides are expected to have the property of enhanced pharmacokinetics compared to polypeptides of shorter lengths.

] Example 27: Serum stability of XTEN A fusion protein containing XTEN_AE864 fused to the N-terminus of GFP was incubated in monkey plasma and rat kidney lysate for up to 7 days at 37°C. Samples were withdrawn at time 0, Day 1 and Day 7 and analyzed by SDS PAGE followed by detection using Western is and detection with antibodies against GFP as shown in . The ce of XTEN_AE864 showed negligible signs of degradation over 7 days in plasma. However, XTEN_AE864 was rapidly degraded in rat kidney lysate over 3 days. The in vivo stability of the fusion protein was tested in plasma samples wherein the GFPiAE864 was immunoprecipitated and analyzed by SDS PAGE as described above. Samples that were withdrawn up to 7 days after injection showed very few signs of degradation. The results trate the resistance of GLP2-XTEN to degradation due to serum proteases; a factor in the enhancement of pharmacokinetic properties of the GLP2-XTEN fusion proteins.

Example 28: sing solubility and stability of a peptide payload by linking to XTEN In order to evaluate the ability ofXTEN to enhance the physicochemical properties of solubility and stability, fusion proteins of glucagon plus shorter-length XTEN were prepared and evaluated. The test articles were ed in Tris-buffered saline at neutral pH and characterization of the Gog-XTEN solution was by reverse-phase HPLC and size exclusion chromatography to afﬁrm that the n was neous and non-aggregated in solution. The data are presented in Table 24. For comparative purposes, the solubility limit of unmodiﬁed glucagon in the same buffer was measured at 60 MM (0.2 mg/mL), and the result demonstrate that for all lengths ofXTEN added, a substantial se in solubility was attained. Importantly, in most cases the glucagon-XTEN fusion proteins were prepared to achieve target concentrations and were not evaluated to determine the maximum solubility limits for the given construct. r, in the case of on linked to the AF-l44 XTEN, the limit of solubility was determined, with the result that a 60-fold increase in solubility was achieved, compared to glucagon not linked to XTEN. In on, the glucagon-AF144 GLP2-XTEN was evaluated for stability, and was found to be stable in liquid formulation for at least 6 months under refrigerated ions and for approximately one month at 37°C (data not shown).

WO 40093 2012/054941 The data support the conclusion that the linking of short-length XTEN polypeptides to a biologically active protein such as glucagon can markedly enhance the solubility properties of the protein by the resulting fusion protein, as well as confer stability at the higher protein concentrations.

Table 24: Solubility of Glucagon-XTEN constructs ] Example 29: Analysis of sequences for secondary structure by prediction algorithms ] Amino acid sequences can be assessed for secondary structure via certain computer programs or algorithms, such as the nown Chou-Fasman algorithm (Chou, P. Y., et a]. (1974) Biochemistry, 13: 222-45) and the Gamier-Osguthorpe-Robson, or “GOR” method (Gamier J, Gibrat JF, Robson B. (1996). GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540-553). For a given sequence, the algorithms can t whether there exists some or no secondary structure at all, expressed as total and/or percentage of residues of the sequence that form, for example, alpha—helices or beta—sheets or the percentage of es of the sequence predicted to result in random coil formation.

Several representative sequences from XTEN “families” have been assessed using two algorithm tools for the Chou-Fasman and GOR methods to assess the degree of secondary structure in these ces. The Chou-Fasman tool was provided by William R. Pearson and the University of Virginia, at the “Biosupport” intemet site, URL located on the World Wide Web at .fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=misc1 as it existed on June 19, 2009. The GOR tool was provided by Pole atique Lyonnais at the Network Protein ce Analysis internet site, URL located on the World Wide Web at .npsa—pbilibcp.fr/cgi-bin/secpred_g0r4.pl as it existed on June 19, 2008.

As a ﬁrst step in the analyses, a single XTEN sequence was ed by the two algorithms.

The AE864 composition is a XTEN with 864 amino acid residues created from multiple copies of four 12 amino acid sequence motifs consisting of the amino acids G, S, T, E, P, and A. The sequence motifs are characterized by the fact that there is limited repetitiveness within the motifs and within the overall sequence in that the sequence of any two utive amino acids is not repeated more than twice in any one 12 amino acid motif, and that no three contiguous amino acids of full-length the XTEN are cal.

Successively longer portions of the AF 864 sequence from the inus were analyzed by the Chou- Fasman and GOR algorithms (the latter requires a minimum length of 17 amino acids). The sequences were analyzed by entering the FASTA format sequences into the prediction tools and g the analysis. The results from the analyses are presented in Table 25.

The results indicate that, by the Chou-Fasman calculations, short XTEN of the AE and AG families, up to at least 288 amino acid residues, have no alpha-helices or beta sheets, but amounts of predicted percentage of random coil by the GOR algorithm vary from 78-99%. With increasing XTEN lengths of 504 residues to r than 1300, the XTEN ed by the asman algorithm had ted percentages of alpha-helices or beta sheets of 0 to about 2%, while the calculated percentages of random coil increased to from 94-99%. Those XTEN with alpha-helices or beta sheets were those sequences with one or more instances of three contiguous serine residues, which resulted in predicted beta-sheet formation. However, even these sequences still had approximately 99% random coil formation.

The data provided herein ts that 1) XTEN created from le sequence motifs of G, S, T, E, P, and A that have limited repetitiveness as to contiguous amino acids are predicted to have very low s of alpha-helices and beta-sheets; 2) that increasing the length of the XTEN does not appreciably increase the ility of alpha—helix or beta—sheet formation; and 3) that ssively increasing the length of the XTEN sequence by addition of non—repetitive 12—mers consisting of the amino acids G, S, T, E, P, and A results in increased percentage of random coil formation. Results ﬁarther indicate that XTEN sequences deﬁned herein (including e.g., XTEN created from sequence motifs of G, S, T, E, P, and A) have limited repetitiveness (including those with no more than two identical contiguous amino acids in any one motif) are expected to have very limited secondary ure.

Any order or combination of sequence motifs from Table 3 can be used to create an XTEN polypeptide that will result in an XTEN sequence that is substantially devoid of secondary structure, though three contiguous s are not preferred. The unfavorable property ofthree contiguous series however, can be ameliorated by increasing the length ofthe XTEN. Such sequences are expected to have the characteristics described in the GLP2-XTEN embodiments of the invention disclosed herein.

Table 25: CHOU-FASMAN and GOR prediction calculations of polypeptide ces SEQ N0. Chan-Fasman GOR Sequence NAME Residues Calculation Calculation A1336: GSPAGSPTSTEEGTSESATPESGPGTST 36 e totals1H: 0 E: 0 94.44% LCW0402 EPSEGSAP percent: H: 0.0 E; 0.0 A1336: GTSTEPSEGSAPGTSTEPSEGSAPGTST 36 Residue totals: H: 0 E: 0 94.44% L&\§0402 EPSEGSAP percent: H: 0.0 E: 0.0 AG36: GASPGTSSTGSPGTPGSGTASSSPGSST 36 Residue totals: H: 0 E: 0 77.78% 1453?]0404 PSGATGSP percent: H; 0.0 E; 0.0 AG36: GSSTPSGATGSPGSSPSASTGTGPGSST 36 Residue totals: H; 0 E; 0 83.33 % SEQ N0. asman GOR Se uence‘1 NAME Residues Calculation calculation LCW0404 SP perceit1H: 0.0 E: 0.0 7003 AE42_1 TEPSEGSAPGSPAGSPTSTEEGTSESAT 42 Residue : H: 0 E: 0 90.48% PESGPGSEPATSGS perce it: H; 0.0 E; 0.0 AE42_1 TEPSEGSAPGSPAGSPTSTEEGTSESAT 42 Residue : H; 0 E; 0 90.48% PESGPGSEPATSGS perce 1t: H: 0.0 E; 0.0 AG42_1 GAPSPSASTGTGPGTPGSGTASSSPGS 42 Residue t0ta1s:H: 0 E; 0 88.10% STPSGATGSPGPSGP perce 1t: H: 0.0 E; 0.0 AG42_2 GPGTPGSGTASSSPGSSTPSGATGSPG 42 Residue totals: H; 0 E: 0 88.10% SSPSASTGTGPGASP perce 1t: H: 0.0 E; 0.0 AE144 SGSETPGTSESATPESGPGSEP 144 e totals: H: 0 E: 0 98.61% ATSGSETPGSPAGSPTSTEEGTSTEPSE percent H; 0.0 E; 0.0 EPATSGSETPGSEPATSGSETP GSEPATSGSETPGTSTEPSEGSAPGTSE SATPESGPGSEPATSGSETPGTSTEPSE GSAP AG144_1 PGSSPSASTGTGPGSSPSASTGTGPGTP 144 Residue t0ta1s:H: 0 E: 0 91.67% GSGTASSSPGSSTPSGATGSPGSSPSAS percent; H; 0.0 E; 0.0 TGTGPGASPGTSSTGSPGTPGSGTASS SPGSSTPSGATGSPGTPGSGTASSSPG ASPGTSSTGSPGASPGTSSTGSPGTPGS GTASSS AE288 GTSESATPESGPGSEPATSGSETPGTSE 288 Residue totals; H; 0 E; 0 99.31% SATPESGPGSEPATSGSETPGTSESATP percent; H; 0.0 E; 0.0 ESGPGTSTEPSEGSAPGSPAGSPTSTEE GTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGP GTSESATPESGPGTSESATPESGPGSEP ATSGSETPGSEPATSGSETPGSPAGSPT STEEGTSTEPSEGSAPGTSTEPSEGSAP GSEPATSGSETPGTSESATPESGPGTST EPSEGSAP AG288_2 GSSPSASTGTGPGSSPSASTGTGPGTP 288 Residue t0ta1s:H: 0 E; 0 92.71 GSGTASSSPGSSTPSGATGSPGSSPSAS percent; H; 0.0 E; 0.0 TGTGPGASPGTSSTGSPGTPGSGTASS SPGSSTPSGATGSPGTPGSGTASSSPG ASPGTSSTGSPGASPGTSSTGSPGTPGS GTASSSPGSSTPSGATGSPGASPGTSST GSPGTPGSGTASSSPGSSTPSGATGSP GSSPSASTGTGPGSSPSASTGTGPGSST PSGATGSPGSSTPSGATGSPGASPGTS STGSPGASPGTSSTGSPGASPGTSSTGS PGTPGSGTASSSP AF504 GASPGTSSTGSPGSSPSASTGTGPGSSP 504 Residue totals: H; 0 E; 0 94.44% SASTGTGPGTPGSGTASSSPGSSTPSG percent: H: 0.0 E: 0.0 ATGSPGSNPSASTGTGPGASPGTSSTG SGTASSSPGSSTPSGATGSPGT PGSGTASSSPGASPGTSSTGSPGASPG TSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGASPGTSSTGSPGTPGSGTASSSP GSSTPSGATGSPGSNPSASTGTGPGSS PSASTGTGPGSSTPSGATGSPGSSTPSG ATGSPGASPGTSSTGSPGASPGTSSTG SPGASPGTSSTGSPGTPGSGTASSSPG ASPGTSSTGSPGASPGTSSTGSPGASP SEQ N0. Chou—Fasman GOR Sequence NAME Residues Calculation Calculation SPGSSPSASTGTGPGTPGSGT ASPGTSSTGSPGASPGTSSTGS PGASPGTSSTGSPGSSTPSGATGSPGSS TPSGATGSPGASPGTSSTGSPGTPGSG TASSSPGSSTPSGATGSPGSSTPSGATG SPGSSTPSGATGSPGSSPSASTGTGPG ASPGTSSTGSP AD 576 GSSESGSSEGGPGSGGEPSESGSSGSSE 576 Residue totals:H: 7 E; 0 99.65% GPGSSESGSSEGGPGSSESGSS t: H; 1.2 E; 0.0 EGGPGSSESGSSEGGPGSSESGSSEGG PGESPGGSSGSESGSEGSSGPGESSGSS ESGSSEGGPGSSESGSSEGGPGSSESGS SEGGPGSGGEPSESGSSGESPGGSSGS ESGESPGGSSGSESGSGGEPSESGSSGS SESGSSEGGPGSGGEPSESGSSGSGGE SGSEGSSGPGESSGESPGGSSG SESGSGGEPSESGSSGSGGEPSESGSSG SGGEPSESGSSGSSESGSSEGGPGESPG GSSGSESGESPGGSSGSESGESPGGSS SPGGSSGSESGESPGGSSGSES GSSESGSSEGGPGSGGEPSESGSSGSE GSSGPGESSGSSESGSSEGGPGSGGEP SESGSSGSSESGSSEGGPGSGGEPSESG SSGESPGGSSGSESGESPGGSSGSESGS SESGSSEGGPGSGGEPSESGSSGSSESG SSEGGPGSGGEPSESGSSGSGGEPSES GSSGESPGGSSGSESGSEGSSGPGESS GSSESGSSEGGPGSEGSSGPGESS AE576 GSPAGSPTSTEEGTSESATPESGPGTST 576 Residue totals:H: 2 E: 0 99.65% EPSEGSAPGSPAGSPTSTEEGTSTEPSE percent: H: 0.4 E: 0.0 GSAPGTSTEPSEGSAPGTSESATPESGP GSEPATSGSETPGSEPATSGSETPGSPA GSPTSTEEGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSEGSAPGTSE SATPESGPGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGTSE SATPESGPGSPAGSPTSTEEGTSESATP ESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSE GSAPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGTSTEPSEGSA PGTSESATPESGPGSPAGSPTSTEEGSP AGSPTSTEEGSPAGSPTSTEEGTSESAT PESGPGTSTEPSEGSAP AG576 PGTPGSGTASSSPGSSTPSGATGSPGSS 576 Residue totals; H; 0 E; 3 99.31% PSASTGTGPGSSPSASTGTGPGSSTPSG percent: H: 0.4 E; 0.5 ATGSPGSSTPSGATGSPGASPGTSSTG GTSSTGSPGASPGTSSTGSPGT PGSGTASSSPGASPGTSSTGSPGASPG TSSTGSPGASPGTSSTGSPGSSPSASTG TGPGTPGSGTASSSPGASPGTSSTGSP GASPGTSSTGSPGASPGTSSTGSPGSST PSGATGSPGSSTPSGATGSPGASPGTS SEQ N0. Chou—Fasman GOR Sequence NAME Residues Calculation Calculation STGSPGTPGSGTASSSPGSSTPSGATGS PGSSTPSGATGSPGSSTPSGATGSPGSS PSASTGTGPGASPGTSSTGSPGASPGT SSTGSPGTPGSGTASSSPGASPGTSSTG SPGASPGTSSTGSPGASPGTSSTGSPG ASPGTSSTGSPGTPGSGTASSSPGSSTP SGATGSPGTPGSGTASSSPGSSTPSGA TGSPGTPGSGTASSSPGSSTPSGATGSP GSSTPSGATGSPGSSPSASTGTGPGSSP SASTGTGPGASPGTSSTGSPGTPGSGT ASSSPGSSTPSGATGSPGSSPSASTGTG ASTGTGPGASPGTSSTGS AF540 GSTSSTAESPGPGSTSSTAESPGPGSTS 540 Residue totals; H; 2 E; 0 99.65 ESPSGTAPGSTSSTAESPGPGSTSSTAE t: H: 0.4 E; 0.0 SPGPGTSTPESGSASPGSTSESPSGTAP GTSPSGESSTAPGSTSESPSGTAPGSTS ESPSGTAPGTSPSGESSTAPGSTSESPS GTAPGSTSESPSGTAPGTSPSGESSTAP GSTSESPSGTAPGSTSESPSGTAPGSTS APGTSTPESGSASPGSTSESPS GTAPGTSTPESGSASPGSTSSTAESPGP GSTSSTAESPGPGTSTPESGSASPGTST PESGSASPGSTSESPSGTAPGTSTPESG SASPGTSTPESGSASPGSTSESPSGTAP GSTSESPSGTAPGSTSESPSGTAPGSTS STAESPGPGTSTPESGSASPGTSTPESG SASPGSTSESPSGTAPGSTSESPSGTAP SGSASPGSTSESPSGTAPGSTS ESPSGTAPGTSTPESGSASPGTSPSGES STAPGSTSSTAESPGPGTSPSGESSTAP AESPGPGTSTPESGSASPGSTS AD836 GSSESGSSEGGPGSSESGSSEGGPGESP 836 e totals:H: 0 E: 0 98.44% GGSSGSESGSGGEPSESGSSGESPGGS percent: H: 0.0 E: 0.0 SGSESGESPGGSSGSESGSSESGSSEGG PGSSESGSSEGGPGSSESGSSEGGPGES PGGSSGSESGESPGGSSGSESGESPGG SSGSESGSSESGSSEGGPGSSESGSSEG GPGSSESGSSEGGPGSSESGSSEGGPG SSESGSSEGGPGSSESGSSEGGPGSGG EPSESGSSGESPGGSSGSESGESPGGSS GSESGSGGEPSESGSSGSEGSSGPGESS GSSESGSSEGGPGSGGEPSESGSSGSE GSSGPGESSGSSESGSSEGGPGSGGEP SESGSSGESPGGSSGSESGSGGEPSESG SSGSGGEPSESGSSGSSESGSSEGGPGS GGEPSESGSSGSGGEPSESGSSGSEGSS GPGESSGESPGGSSGSESGSEGSSGPG ESSGSEGSSGPGESSGSGGEPSESGSSG SSESGSSEGGPGSSESGSSEGGPGESPG GSSGSESGSGGEPSESGSSGSEGSSGP GESSGESPGGSSGSESGSEGSSGPGSSE SGSSEGGPGSGGEPSESGSSGSEGSSG PGESSGSEGSSGPGESSGSEGSSGPGES SGSGGEPSESGSSGSGGEPSESGSSGES PGGSSGSESGESPGGSSGSESGSGGEP SESGSSGSEGSSGPGESSGESPGGSSGS ESGSSESGSSEGGPGSSESGSSEGGPGS SEQ N0. Chou—Fasman GOR Sequence NAME Residues Calculation Calculation SESGSSEGGPGSGGEPSESGSSGSSESG SSEGGPGESPGGSSGSESGSGGEPSES GSSGSSESGSSEGGPGESPGGSSGSES GSGGEPSESGSSGESPGGSSGSESGSG GEPSESGSS AE864 GSPAGSPTSTEEGTSESATPESGPGTST 864 Residue totals; H; 2 E; 3 99.77% EPSEGSAPGSPAGSPTSTEEGTSTEPSE percent: H: 0.2 E; 0.4 GSAPGTSTEPSEGSAPGTSESATPESGP GSEPATSGSETPGSEPATSGSETPGSPA GSPTSTEEGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSEGSAPGTSE SATPESGPGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGTSE SATPESGPGSPAGSPTSTEEGTSESATP ESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSE STEPSEGSAPGSPAGSPTSTEE SEGSAPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGTSTEPSEGSA PGTSESATPESGPGSPAGSPTSTEEGSP TEEGSPAGSPTSTEEGTSESAT PESGPGTSTEPSEGSAPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGTSTEPS SPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGTSESATPESGPGS PAGSPTSTEEGSPAGSPTSTEEGTSTEP SEGSAPGTSESATPESGPGTSESATPES GPGTSESATPESGPGSEPATSGSETPGS EPATSGSETPGSPAGSPTSTEEGTSTEP SEGSAPGTSTEPSEGSAPGSEPATSGSE TPGTSESATPESGPGTSTEPSEGSAP AF864 PSGTAPGTSPSGESSTAPGSTS 875 Residue totals; H; 2 E; 0 95.20% ESPSGTAPGSTSESPSGTAPGTSTPESG percent: H: 0.2 E: 0.0 SASPGTSTPESGSASPGSTSESPSGTAP GSTSESPSGTAPGTSPSGESSTAPGSTS ESPSGTAPGTSPSGESSTAPGTSPSGES STAPGSTSSTAESPGPGTSPSGESSTAP ESSTAPGSTSSTAESPGPGTST PESGSASPGTSTPESGSASPGSTSESPS GTAPGSTSESPSGTAPGTSTPESGSASP GSTSSTAESPGPGTSTPESGSASPGSTS ESPSGTAPGTSPSGESSTAPGSTSSTAE SPGPGTSPSGESSTAPGTSTPESGSASP AESPGPGSTSSTAESPGPGSTS STAESPGPGSTSSTAESPGPGTSPSGES STAPGSTSESPSGTAPGSTSESPSGTAP GTSTPESGPXXXGASASGAPSTXXXX SESPSGTAPGSTSESPSGTAPGSTSESP SGTAPGSTSESPSGTAPGSTSESPSGTA PGSTSESPSGTAPGTSTPESGSASPGTS PSGESSTAPGTSPSGESSTAPGSTSSTA ESPGPGTSPSGESSTAPGTSTPESGSAS PGSTSESPSGTAPGSTSESPSGTAPGTS SEQ N0. Chou—Fasman GOR Sequence NAME Residues Calculation Calculation TAPGSTSESPSGTAPGTSTPES GSASPGTSTPESGSASPGSTSESPSGTA PGTSTPESGSASPGSTSSTAESPGPGST SESPSGTAPGSTSESPSGTAPGTSPSGE SSTAPGSTSSTAESPGPGTSPSGESSTA PGTSTPESGSASPGTSPSGESSTAPGTS PSGESSTAPGTSPSGESSTAPGSTSSTA STSSTAESPGPGTSPSGESSTA PGSSPSASTGTGPGSSTPSGATGSPGSS TPSGATGSP AG864 GASPGTSSTGSPGSSPSASTGTGPGSSP 864 e totals: H: 0 E: 0 94.91% GPGTPGSGTASSSPGSSTPSG percent: H: 0.0 E: 0.0 SSPSASTGTGPGASPGTSSTG SPGTPGSGTASSSPGSSTPSGATGSPGT PGSGTASSSPGASPGTSSTGSPGASPG TSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGASPGTSSTGSPGTPGSGTASSSP GSSTPSGATGSPGSSPSASTGTGPGSSP SASTGTGPGSSTPSGATGSPGSSTPSG ATGSPGASPGTSSTGSPGASPGTSSTG SPGASPGTSSTGSPGTPGSGTASSSPG ASPGTSSTGSPGASPGTSSTGSPGASP GTSSTGSPGSSPSASTGTGPGTPGSGT ASSSPGASPGTSSTGSPGASPGTSSTGS PGASPGTSSTGSPGSSTPSGATGSPGSS TPSGATGSPGASPGTSSTGSPGTPGSG TASSSPGSSTPSGATGSPGSSTPSGATG SPGSSTPSGATGSPGSSPSASTGTGPG ASPGTSSTGSPGASPGTSSTGSPGTPGS GTASSSPGASPGTSSTGSPGASPGTSST GSPGASPGTSSTGSPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGTP GSGTASSSPGSSTPSGATGSPGTPGSG GSSTPSGATGSPGSSTPSGATG SPGSSPSASTGTGPGSSPSASTGTGPG ASPGTSSTGSPGTPGSGTASSSPGSSTP SGATGSPGSSPSASTGTGPGSSPSAST GTGPGASPGTSSTGSPGASPGTSSTGS PGSSTPSGATGSPGSSPSASTGTGPGA SPGTSSTGSPGSSPSASTGTGPGTPGSG TASSSPGSSTPSGATGSPGSSTPSGATG SPGASPGTSSTGSP AM875 GTSTEPSEGSAPGSEPATSGSETPGSPA 875 Residue totals: H: 7 E; 3 98.63% GSPTSTEEGSTSSTAESPGPGTSTPESG percent: H: 0.8 E; 0.3 SASPGSTSESPSGTAPGSTSESPSGTAP GTSTPESGSASPGTSTPESGSASPGSEP ATSGSETPGTSESATPESGPGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGSPA GSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGTSE SATPESGPGTSTEPSEGSAPGSEPATSG SETPGSPAGSPTSTEEGSSTPSGATGSP GTPGSGTASSSPGSSTPSGATGSPGTS TEPSEGSAPGTSTEPSEGSAPGSEPATS GSETPGSPAGSPTSTEEGSPAGSPTSTE EGTSTEPSEGSAPGASASGAPSTGGTS SEQ N0. Chou—Fasman GOR Sequence NAME Residues Calculation Calculation ESATPESGPGSPAGSPTSTEEGSPAGSP TSTEEGSTSSTAESPGPGSTSESPSGTA PGTSPSGESSTAPGTPGSGTASSSPGSS TPSGATGSPGSSPSASTGTGPGSEPAT SGSETPGTSESATPESGPGSEPATSGSE TPGSTSSTAESPGPGSTSSTAESPGPGT SPSGESSTAPGSEPATSGSETPGSEPAT SGSETPGTSTEPSEGSAPGSTSSTAESP GPGTSTPESGSASPGSTSESPSGTAPGT STEPSEGSAPGTSTEPSEGSAPGTSTEP SEGSAPGSSTPSGATGSPGSSPSASTGT GPGASPGTSSTGSPGSEPATSGSETPG TSESATPESGPGSPAGSPTSTEEGSSTP SGATGSPGSSPSASTGTGPGASPGTSS TGSPGTSESATPESGPGTSTEPSEGSAP SEGSAP AM1318 GTSTEPSEGSAPGSEPATSGSETPGSPA 1318 Residue t0ta1s1H: 7 E; 0 99.17% GSPTSTEEGSTSSTAESPGPGTSTPESG percent: H: 0.7 E; 0.0 TSESPSGTAPGSTSESPSGTAP SGSASPGTSTPESGSASPGSEP ATSGSETPGTSESATPESGPGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGSPA GSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGTSE SATPESGPGTSTEPSEGSAPGSEPATSG SETPGSPAGSPTSTEEGSSTPSGATGSP GTPGSGTASSSPGSSTPSGATGSPGTS TEPSEGSAPGTSTEPSEGSAPGSEPATS GSETPGSPAGSPTSTEEGSPAGSPTSTE PSEGSAPGPEPTGPAPSGGSEP ATSGSETPGTSESATPESGPGSPAGSPT STEEGTSESATPESGPGSPAGSPTSTEE GSPAGSPTSTEEGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGSTSSTAE SPGPGSTSESPSGTAPGTSPSGESSTAP GSTSESPSGTAPGSTSESPSGTAPGTSP SGESSTAPGTSTEPSEGSAPGTSESATP ESGPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSESATPESGPGTST EPSEGSAPGTSESATPESGPGTSTEPSE GSAPGTSPSGESSTAPGTSPSGESSTAP GTSPSGESSTAPGTSTEPSEGSAPGSPA EEGTSTEPSEGSAPGSSPSAST GTGPGSSTPSGATGSPGSSTPSGATGS PGSSTPSGATGSPGSSTPSGATGSPGA SPGTSSTGSPGASASGAPSTGGTSPSG ESSTAPGSTSSTAESPGPGTSPSGESST APGTSESATPESGPGTSTEPSEGSAPG TSTEPSEGSAPGSSPSASTGTGPGSSTP SGATGSPGASPGTSSTGSPGTSTPESG SASPGTSPSGESSTAPGTSPSGESSTAP GTSESATPESGPGSEPATSGSETPGTST EPSEGSAPGSTSESPSGTAPGSTSESPS GTAPGTSTPESGSASPGSPAGSPTSTEE GTSESATPESGPGTSTEPSEGSAPGSPA GSPTSTEEGTSESATPESGPGSEPATSG WO 40093 2012/054941 SEQ N0. Chou—Fasman GOR Sequence NAME Residues Calculation ation STPSGATGSPGASPGTSSTGSP GSSTPSGATGSPGSTSESPSGTAPGTSP SGESSTAPGSTSSTAESPGPGSSTPSGA TGSPGASPGTSSTGSPGTPGSGTASSSP GSPAGSPTSTEEGSPAGSPTSTEEGTST EPSEGSAP AM923 MAEPAGSPTSTEEGASPGTSSTGSPGS 924 Residue totals; H; 4 E; 3 98.70% STPSGATGSPGSSTPSGATGSPGTSTEP percent: H: 0.4 E; 0.3 SEGSAPGSEPATSGSETPGSPAGSPTST EEGSTSSTAESPGPGTSTPESGSASPGS TSESPSGTAPGSTSESPSGTAPGTSTPE SGSASPGTSTPESGSASPGSEPATSGSE TPGTSESATPESGPGSPAGSPTSTEEGT GSAPGTSESATPESGPGTSTEP SEGSAPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGTSTEPSEGSAPGT SESATPESGPGTSESATPESGPGTSTEP SEGSAPGTSTEPSEGSAPGTSESATPES GPGTSTEPSEGSAPGSEPATSGSETPGS PAGSPTSTEEGSSTPSGATGSPGTPGS PGSSTPSGATGSPGTSTEPSEG SAPGTSTEPSEGSAPGSEPATSGSETPG SPAGSPTSTEEGSPAGSPTSTEEGTSTE PSEGSAPGASASGAPSTGGTSESATPE SGPGSPAGSPTSTEEGSPAGSPTSTEEG STSSTAESPGPGSTSESPSGTAPGTSPS GESSTAPGTPGSGTASSSPGSSTPSGA TGSPGSSPSASTGTGPGSEPATSGSETP GTSESATPESGPGSEPATSGSETPGSTS STAESPGPGSTSSTAESPGPGTSPSGES STAPGSEPATSGSETPGSEPATSGSETP GTSTEPSEGSAPGSTSSTAESPGPGTST PESGSASPGSTSESPSGTAPGTSTEPSE GSAPGTSTEPSEGSAPGTSTEPSEGSAP GSSTPSGATGSPGSSPSASTGTGPGAS PGTSSTGSPGSEPATSGSETPGTSESAT PESGPGSPAGSPTSTEEGSSTPSGATGS PGSSPSASTGTGPGASPGTSSTGSPGTS ESATPESGPGTSTEPSEGSAPGTSTEPS EGSAP AE912 MAEPAGSPTSTEEGTPGSGTASSSPGS 913 e totals:H: 8 E: 3 99.45% STPSGATGSPGASPGTSSTGSPGSPAG percent: H: 0.9 E; 0.3 SPTSTEEGTSESATPESGPGTSTEPSEG SAPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSEGSAPGTSESATPESGPGSEPA TSGSETPGSEPATSGSETPGSPAGSPTS TEEGTSESATPESGPGTSTEPSEGSAPG TSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPE SGPGTSTEPSEGSAPGTSESATPESGPG SEPATSGSETPGTSTEPSEGSAPGTSTE PSEGSAPGTSESATPESGPGTSESATPE SGPGSPAGSPTSTEEGTSESATPESGPG SEPATSGSETPGTSESATPESGPGTSTE PSEGSAPGTSTEPSEGSAPGTSTEPSEG SAPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSESATPESGPGSEPATSGS SEQ N0. Chou—Fasman GOR Sequence NAME Residues Calculation Calculation ETPGTSESATPESGPGSEPATSGSETPG PESGPGTSTEPSEGSAPGTSES ATPESGPGSPAGSPTSTEEGSPAGSPTS TEEGSPAGSPTSTEEGTSESATPESGPG EGSAPGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGTSTEPSEGSAPG SPAGSPTSTEEGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGSPAGSPTS TEEGSPAGSPTSTEEGTSTEPSEGSAPG TSESATPESGPGTSESATPESGPGTSES ATPESGPGSEPATSGSETPGSEPATSGS ETPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSEGSAPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAP BC 864 GTSTEPSEPGSAGTSTEPSEPGSAGSEP Residue totals: H: 0 E: 0 99.77% PSGSGASEPTSTEPGSEPATS percent: H: 0 E; 0 GTEPSGSEPATSGTEPSGSEPATSGTEP SGSGASEPTSTEPGTSTEPSEPGSAGSE PATSGTEPSGTSTEPSEPGSAGSEPATS GTEPSGSEPATSGTEPSGTSTEPSEPGS AGTSTEPSEPGSAGSEPATSGTEPSGS EPATSGTEPSGTSEPSTSEPGAGSGAS EPTSTEPGTSEPSTSEPGAGSEPATSGT EPSGSEPATSGTEPSGTSTEPSEPGSAG TSTEPSEPGSAGSGASEPTSTEPGSEPA TSGTEPSGSEPATSGTEPSGSEPATSGT EPSGSEPATSGTEPSGTSTEPSEPGSAG GTEPSGSGASEPTSTEPGTSTE PSEPGSAGSEPATSGTEPSGSGASEPTS TEPGTSTEPSEPGSAGSGASEPTSTEPG SEPATSGTEPSGSGASEPTSTEPGSEPA TSGTEPSGSGASEPTSTEPGTSTEPSEP GSAGSEPATSGTEPSGSGASEPTSTEP GTSTEPSEPGSAGSEPATSGTEPSGTST EPSEPGSAGSEPATSGTEPSGTSTEPSE PGSAGTSTEPSEPGSAGTSTEPSEPGSA GTSTEPSEPGSAGTSTEPSEPGSAGTST EPSEPGSAGTSEPSTSEPGAGSGASEPT STEPGTSTEPSEPGSAGTSTEPSEPGSA GTSTEPSEPGSAGSEPATSGTEPSGSG TEPGSEPATSGTEPSGSEPATS GTEPSGSEPATSGTEPSGSEPATSGTEP SGTSEPSTSEPGAGSEPATSGTEPSGSG ASEPTSTEPGTSTEPSEPGSAGSEPATS GTEPSGSGASEPTSTEPGTSTEPSEPGS * H: alpha-helix E: beta-sheet Example 30: Analysis of polypeptide sequences for repetitiveness ] In this Example, different polypeptides, including several XTEN sequences, were assessed for repetitiveness in the amino acid sequence. Polypeptide amino acid sequences can be ed for repetitiveness by quantifying the number of times a shorter subsequence appears Within the overall polypeptide. For e, a polypeptide of 200 amino acid residues length has a total of 165 overlapping 36-amino acid “blocks” (or “36-mers”) and 198 3-mer “subsequences”, but the number of unique 3-mer uences will depend on the amount of repetitiveness within the sequence. For the analyses, different polypeptide sequences were ed for repetitiveness by determining the uence score obtained by application of the following equation: Y 3 H 5:; if} if??? {7 E Subsequence score = “3:”:I I 3‘31 wherein: m 2 (amino acid length of polypeptide) — (amino acid length of subsequence) + 1; and Countl- = cumulative number of occurrences of each unique subsequence within sequence,- In the analyses of the t Example, the subsequence score for the ptides of Table 26 were determined using the foregoing equation in a computer program using the algorithm depicted in wherein the subsequence length was set at 3 amino acids. The resulting subsequence score is a reﬂection of the degree ofrepetitiveness within the polypeptide.

The results, shown in Table 26, indicate that the ctured polypeptides consisting of 2 or 3 amino acid types have high subsequence scores, while those of ting of the 12 amino acid motifs of the six amino acids G, S, T, E, P, and A with a low degree of internal repetitiveness, have subsequence scores of less than 10, and in some cases, less than 5. For e, the L288 sequence has two amino acid types and has short, highly tive sequences, resulting in a subsequence score of 50.0. The polypeptide J288 has three amino acid types but also has short, repetitive ces, resulting in a subsequence score of 33.3. Y576 also has three amino acid types, but is not made of internal repeats, reﬂected in the subsequence score of 15.7 over the first 200 amino acids. W576 consists of four types of amino acids, but has a higher degree of internal repetitiveness, e. g., “GGSG”, resulting in a uence score of 23.4. The AD576 consists of four types of 12 amino acid motifs, each consisting of four types of amino acids. Because of the low degree of internal repetitiveness of the individual motifs, the l subsequence score over the ﬁrst 200 amino acids is 13.6. In contrast, XTEN’s consisting of four motifs contains six types of amino acids, each with a low degree of internal repetitiveness have lower subsequence scores; i.e., AE864 (6.1), AF864 (7.5), and AM875 (4.5), while XTEN consisting of four motifs containing five types of amino acids were intermediate; i.e., AE864, with a score of 7.2.

Conclusions: The results indicate that the combination of 12 amino acid subsequence motifs, each consisting of four to six amino acid types that are non-repetitive, into a longer XTEN ptide results in an overall sequence that is substantially non-repetitive, as indicated by overall average subsequence scores less than 10 and, in many cases, less than 5. This is despite the fact that each subsequence motif may be used multiple times across the sequence. In contrast, polymers created from smaller numbers of amino acid types resulted in higher average subsequence scores, with polypeptides consisting of two amino acid type having higher scores that those consisting of three amino acid types.

Table 26: Average subseguence score calculations of polypeptide seguences Seq SEQ ID Score Name Amino Acid Sequence J288 783 GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEG 33.3 GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEG GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEG GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEG GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEG GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEG K288 784 GEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGE 46.9 GEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGE GGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGG GEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGE GGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGG EGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEG EGGGEG L288 785 SSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSS 50.0 SESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSE SSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSE SSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSES SSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSS Y288 786 GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEGGS 26.8 EGSEGEGSGEGSEGEGGSEGSEGEGSGEGSEGEGSEGGSEGEGGSEGSEG EGSGEGSEGEGGEGGSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSGE GSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGE GSGEGSEGEGGGEGSEGEGSGEGGEGEGSEGGSEGEGGSEGGEGEGSEG SGEGEGSEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGE Q576 787 GGKPGEGGKPEGGGGKPGGKPEGEGEGKPGGKPEGGGKPGGGEGGKPE 18.5 GGKPEGEGKPGGGEGKPGGKPEGGGGKPEGEGKPGGGGGKPGGKPEGE GKPGGGEGGKPEGKPGEGGEGKPGGKPEGGGEGKPGGGKPGEGGKPGE GKPGGGEGGKPEGGKPEGEGKPGGGEGKPGGKPGEGGKPEGGGEGKPG GKPGEGGEGKPGGGKPEGEGKPGGGKPGGGEGGKPEGEGKPGGKPEGG GEGKPGGKPEGGGKPEGGGEGKPGGGKPGEGGKPGEGEGKPGGKPEGE GKPGGEGGGKPEGKPGGGEGGKPEGGKPGEGGKPEGGKPGEGGEGKPG GGKPGEGGKPEGGGKPEGEGKPGGGGKPGEGGKPEGGKPEGGGEGKPG GGKPEGEGKPGGGEGKPGGKPEGGGGKPGEGGKPEGGKPGGEGGGKPE GEGKPGGKPGEGGGGKPGGKPEGEGKPGEGGEGKPGGKPEGGGEGKPG GKPEGGGEGKPGGGKPGEGGKPEGGGKPGEGGKPGEGGKPEGEGKPGG GEGKPGGKPGEGGKPEGGGEGKPGGKPGGEGGGKPEGGKPGEGGKPEG U576 788 GKPGSGGGKPGEGGKPGSGEGKPGGKPGSGGSGKPGGKPGEG 18.1 SGGKPGGGGKPGGKPGGEGSGKPGGKPEGGGKPEGGSGGKPG GKPEGGSGGKPGGKPGSGEGGKPGGGKPGGEGKPGSGKPGGEGSGKPG GKPEGGSGGKPGGKPEGGSGGKPGGSGKPGGKPGEGGKPEGGSGGKPG GSGKPGGKPEGGGSGKPGGKPGEGGKPGSGEGGKPGGGKPGGEGKPGS GKPGGEGSGKPGGKPGSGGEGKPGGKPEGGSGGKPGGGKPGGEGKPGS GGKPGEGGKPGSGGGKPGGKPGGEGEGKPGGKPGEGGKPGGEGSGKPG GGGKPGGKPGGEGGKPEGSGKPGGGSGKPGGKPEGGGGKPEGSGKPGG GGKPEGSGKPGGGKPEGGSGGKPGGSGKPGGKPGEGGGKPEGSGKPGG GSGKPGGKPEGGGKPEGGSGGKPGGKPEGGSGGKPGGKPGGEGSGKPG GKPGSGEGGKPGGKPGEGSGGKPGGKPEGGSGGKPGGSGKPGGKPEGG GSGKPGGKPGEGGKPGGEGSGKPGGSGKPG W576 789 GGSGKPGKPGGSGSGKPGSGKPGGGSGKPGSGKPGGGSGKPGSGKPGG 23.4 SGKPGGGGKPGSGSGKPGGGKPGGSGGKPGGGSGKPGKPGSG GSGKPGSGKPGGGSGGKPGKPGSGGSGGKPGKPGSGGGSGKPGKPGSG GSGGKPGKPGSGGSGGKPGKPGSGGSGKPGSGKPGGGSGKPGSGKPGSG GSGKPGKPGSGGSGKPGSGKPGSGSGKPGSGKPGGGSGKPGSGKPGSGG SGKPGKPGSGGGKPGSGSGKPGGGKPGSGSGKPGGGKPGGSGGKPGGS GGKPGKPGSGGGSGKPGKPGSGGGSGKPGKPGGSGSGKPGSGKPGGGS Seq SEQ ID Score Amino Acid Sequence Name N0: KPGSGGSGKPGKPGSGGSGGKPGKPGSGGGKPGSGSGKPGGG KPGSGSGKPGGGKPGSGSGKPGGGKPGSGSGKPGGSGKPGSGKPGGGSG GKPGKPGSGGSGKPGSGKPGSGGSGKPGKPGGSGSGKPGSGKPGGGSGK PGSGKPGGGSGKPGSGKPGGGSGKPGSGKPGGGGKPGSGSGKPGGSGG KPGKPGSGGSGGKPGKPGSGGSGKPGSGKPGGGSGGKPGKPGSGG Y576 790 GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEGGE 15.7 GSGEGEGSGEGSEGEGGGEGSEGEGSGEGGEGEGSEGGSEGEGGSEGGE GEGSEGSGEGEGSEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGEGEGSE GSGEGEGSEGSGEGEGSEGGSEGEGGSEGSEGEGSGEGSEGEGGSEGSEG EGGGEGSEGEGSGEGSEGEGGSEGSEGEGGSEGSEGEGGEGSGEGEGSE GSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSGEGSEG EGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEGGSEGSEGEGGEG SGEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGE EGEGGEGSGEGEGSGEGSEGEGGGEGSEGEGSEGSGEGEGSEGS GEGEGSEGGSEGEGGSEGSEGEGSEGGSEGEGSEGGSEGEGSEGSGEGEG SEGSGEGEGSGEGSEGEGGSEGGEGEGSEGGSEGEGSEGGSEGEGGEGSG EGEGGGEGSEGEGSEGSGEGEGSGEGSE AE288 288 TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES 6.0 ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG TSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPAT SGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSET PGTSESATPESGPGTSTEPSEGSAP AG288_ 288 PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTP 6.9 1 GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSS PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGAS PGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSAST GTGPGTPGSGTASSSPGSSTPSGATGS ADS76 791 GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGSSE 13.6 SGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSEGSSGP GESSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSS GESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGSGG EPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSGGEPSE SGSSGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSES GESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSE SGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSE SGSSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSES GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGG EPSESGSSGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSEGSSGP GESS AE576 792 AGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS 6.1 TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATS GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTST EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEG ESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGS PTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSA AF540 793 GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSS 8.8 PGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGT APGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTS PSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESG SASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPG Seq SEQ ID Score Amino Acid Sequence Name NO: TSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPE SGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPG PGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTST PESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESS TAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGS TSESPSGTAP AF504 794 GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSST 7.0 PSGATGSPGSNPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA TGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSP GSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSNP SASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSS TGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSP GASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASP GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGA TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP GSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP AE864 795 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST 6.1 EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSG PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG SPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGS SETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTE EGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPT STEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG TSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPAT SGSETPGSPAGSPTSTEEGTSTEPSEGSAP AF864 796 GSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTP 7.5 ESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESST APGSTSESPSGTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTS PSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESG SASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPG TSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSG ESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPG PGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTST PESGPXXXGASASGAPSTXXXXSESPSGTAPGSTSESPSGTAPGSTSESPS GTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASP GTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTP ESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGT APGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGST SSTAESPGPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAE SPGPGTSPSGESSTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPG TSPSGESSTAPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSSPSA GSSTPSGATGSPGSSTPSGATGSP AG864 864 GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSST 7.2 PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA PGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSP GSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSP GPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSS TGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSP GASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASP GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGA TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP GSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPG WO 40093 Seq SEQ ID Score Amino Acid Sequence Name NO: SGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSS TGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSP GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSP SASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST GTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSP GSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSST PSGATGSPGSSTPSGATGSPGASPGTSSTGSP AG868 797 GGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSP 7.5 GSSTPSGATGSPGSNPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSST PSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTA SSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP GSNPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASP GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSS TGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSP GASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSST PSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGA TGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSP GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASP GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGA TGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGP GSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSP SASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGA TGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSP GSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP AM875 798 GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTP 4.5 ESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSA SPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTS ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPA GSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSE GSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEE GTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAG EGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASS SPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGS EPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATS GSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASP GSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTP SGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPE AGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPG TSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP AE912 913 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSP 4.5 AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETP PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEG SAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPG TSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEG SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA TPESGPGTSTEPSEGSAP Seq SEQ ID Score Amino Acid Sequence Name NO: AM923 924 MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTS 4.5 TEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESG SASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPG SEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESA TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA PSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTST EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTS TEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPG TSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEP SEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE EGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSST PSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSG SETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPG SEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSES PSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATG SPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSP AGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESAT PESGPGTSTEPSEGSAPGTSTEPSEGSAP AM1296 799 GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTP 4.5 ESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSA SPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTS ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE STEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPA GSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSE STEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEE GTSTEPSEGSAPGPEPTGPAPSGGSEPATSGSETPGTSESATPESGPGSPAG SPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTS PSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPS GESSTAPGTSPSGESSTAPGTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGS STPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASASGAPSTGGTSPSGE SSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESATPESGPGTSTEPSEGSAP GTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTST SPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSEPATSGS ETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGS PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESA GSEPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATG SPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGAS PGTSSTGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE GSAP Example 31: Calculation of TEPITOPE scores TEPITOPE scores of 9mer peptide ce can be calculated by adding pocket potentials as described by Stumiolo [Sturniolo, T., et a1. (1999) Nat Biotechnol, 17: 555]. In the present Example, separate Tepitope scores were calculated for individual HLA alleles. Table 27 shows as an example the pocket potentials for HLA*0101B, which occurs in high frequency in the ian population. To calculate the TEPITOPE score of a peptide with sequence Pl-P2-P3-P4-P5-P6-P7-P8-P9, the corresponding individual pocket potentials in Table 27 were added. The HLA*0101B score of a 9mer peptide with the sequence FDKLPRTSG is the sum of 0, -1.3, 0, 0.9, 0, -1.8, 0.09, 0, 0.

To evaluate the TEPITOPE scores for long peptides one can repeat the process for all 9mer subsequences of the sequences. This process can be repeated for the proteins encoded by other HLA alleles. Tables 28-31 give pocket potentials for the protein products of HLA s that occur with high frequency in the Caucasian tion.

TEPITOPE scores calculated by this method range from approximately -10 to +10. However, 9mer peptides that lack a hydrophobic amino acid WY) in P1 position have calculated TEPITOPE scores in the range of -1009 to -989. This value is biologically meaningless and reﬂects the fact that a hydrophobic amino acid serves as an anchor residue for HLA binding and peptides lacking a hydrophobic e in P1 are considered non binders to HLA. Because most XTEN sequences lack hydrophobic residues, all combinations of 9mer subsequences will have TEPITOPEs in the range in the range of -1009 to -989. This method conﬁrms that XTEN polypeptides may have few or no predicted T- cell epitopes.

Table 27: Pocket otential for HLA*0101B .

Amino Acid U0 0 - 0 0 —I-— —I-— 0 —I-— —I-— "UZKT‘WHE —I-—-1 I-— —I-— '1—I-— -1 08-.— —I-— —I-— 01730 . . -l.8 0.2 . -l.8 0.09 -0 6 -0 2 Table 28: Pocket otential for HLA*0301B allele.

—Amino acid "UIx.) "UI» "U Ln ’1: ox "U \1 "Uo .- I I—I U) I I—A U.) N U.) .0 b—K I I—A N I ._I ._I .0 W l ._I G O U} ._I HoooOmowqowmgﬂomwww ._I O U. ._I ._I l ._I O 535.082.0952:mougcm'o ._I ._I O I-I ._I 63.0‘U‘oo OOI—l O [\J s5._. | .0 ON — | )—A m- oc'sNr«Di—.HLJNN .0535:”1199.) oo )_A .53 «a oDl—K.\])l—ko I I-I ._I S5I—I I .0 LI] o I .0 U) I I-I .0 00 .0 L» ’12\9 LIL. \l\l I I-I WO 40093 Amino acid pg*4 pd 00 *UZ I0.0.0ogoxoo *<€< Amino acid P1 P3 A -999 0 0 FHUO -999 0 0 -999 -1.3 -1.3 —-n— —_*UZEFWHEQTJ —-n—730 <am -999 -O.3 0.2 . . . . —_m<2 Table 31: Pocket potential for HLA*1501B allele.

Amino acid "d4; "Cl6 ’TJU'JUO L. U) ll l—Kl—K we; $3.0 OOl—t s5 \l .'_..'_. \oo .0 00 N636:-..oo#04; 3'3 4; Wl—l ._i ._i ._i LII I .0 \l b—K t—n O 33.0 \10 L 0 1 l 0.5 - : WOPUZE t—I t—I ._.

.O'oNo'oNe'osDsop—t‘wNN‘moo OOHGenerous I.0 N L. [\J I.0 W L. ._.

I.0 00 L. O\ GO .0 [\J .53 LI] .

Hm $5.53 UJUJ .0 N <e< .0 U} 9.53.53“No.7 O 9.0 #N .0 00 .N Ur 3:; ®4> Table 32: Exem lar Biolo icalActivit Exem la Assa s and Indications Biologically Active Protein Bi010_ calActivit Exemnla Activit Assa s Indication: Glucagon-Like- Stimulates proliferation intestinal lial cell Gastrointestinal conditions Peptide 2 (GLPZ; and inhibits apoptosis proliferation can be including, but not limited Glyt (ill—2) of intestinal lial measured using methods to: gastrointestinal cells; reduces lial lrnown in the art, including epithelial inj ury; recovery permeability; decreases the cell proliferation from bowel resection; gastric acid secretion assays described in Dig. enteritis; colitis; gastritis; and gastrointestinal Dis. Sci. 47(5): l lSSMl 140 elreinotlierapyindnced motility; promotes “2092). tnttcositis; short bowel wound g. Protection of intestinal syndrome; intestinal epithelium can he atrophy; inflammatory evaluated using methods bowel disease; Crohn's known in the art: including disease; Ulcerative the in Vitro intestinal colitis; acid ; peptic injury model described in ulcers; diabetes-associated .l} Sing. Res ltl7t'l): 44—9 bowel growth; intestinal Biologically Active Protein Bi010_'calActivit Exemla Activit Assa s Indication: {2092). iscbernia syndromes; GLP~2 can be assayed by maintenance of gut radieinnnnneassay ity after major burn bed in Regu. trauma; regulatien ef l’l’iysioll 278(4): Ellie"?— i nal Rll‘s63 . permeability anti nutrient Cnntractility of intestinal abserptien. tissue by GLP—Z can be Hyperglycemia; Diabetes; measured as described in Diabetes lnsipidus; US Pat Ne. 7,498,l 4i; Diabetes mellitus; Type l Measurement 0f CAMP diabetes; Ty e 2 diabetes; levels in isolated rat small n resistance; insulin intestinal deficiency; muensal cells expressing llyperl ini d emia; {ELF—2 receptors or in llyperltetnnemia; Non— COS eells insulin dependent ected Witlt tile GLP- Diabetes Mellitus ’2 receptor, or AP-l NDDM); n; luciferase rennrter gene dependent Diabetes activity in Bill: Mellitus ODDM); ﬁbroblast cells Cnntlitions asseeiated with nously expressing Diabetes including, but the (ELF-2 receptor as not limited to Dbesity.‘ described in US Pat App. l-leart N0. Elli llll’fl lb-l; Disease, Hyperglycemia, ECSG determinations by infections” patby, Flipper assay measuring And/0r Ulcers; Metabolic calcium ﬂux by Disorders; immune ﬂuorescence triggered by Disnrders; Obesity; binding of (ELF-2 tn an ar Diserders; ered cell line with a ssion ef Bedy stable GEM—R and G (1 Weight; Suppressinn nf q/‘l l expressien. Appetite; Synrlrmne Xv Table 33: Exemplary GLPZ-XTEN comprising GLP-2 and terminal XTEN GLP2— Amino Acid Sequence XTEN Names“ GLP-Z- HADGSFSDEMNTILDNLAARDFINWLIQTKITDGGSEPATSGSETPGTSESATPESGPGSEPATS AE144 GSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPG TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP GLP-2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGGSEPATSGSETPGTSESATPESGPGSEPATS variant 2- GSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPG AE144 TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP GLP-Z- HADGSFSDEMNTILDNLAARDFINWLIQTKITDGGTSESATPESGPGSEPATSGSETPGTSESAT AE288 PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG SEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP ESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT STEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GLP-2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGGTSESATPESGPGSEPATSGSETPGTSESAT variant 2— PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG AE288 SEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP GLPZ- Amino Acid ce XTEN Name" ESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT STEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GLP—Z— HADGSFSDEMNTILDNLAARDFINWLIQTKITDGGTSTPESGSASPGTSPSGESSTAPGTSPSGE AF144 SSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGS TSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAP GLP-2 SDEMNTILDNLAARDFINWLIQTKITDGGTSTPESGSASPGTSPSGESSTAPGTSPSGE variant 2- SSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGS AF144 TSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAP GLP-Z- HADGSFSDEMNTILDNLAARDFINWLIQTKITDGGSSESGSSEGGPGSGGEPSESGSSGSSESGS AD576 SEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESG SEGSSGPGESSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGS SGSESGESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSG SEGSSGPGESSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSGGEPSESGSSGSSESGS SEGGPGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESG SSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSSESGS SEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSG SSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGSEGSSGPGESSGSSESGS SEGGPGSEGSSGPGESS GLP-2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGGSSESGSSEGGPGSGGEPSESGSSGSSESGS variant 2- SEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESG ADS76 SEGSSGPGESSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGS ESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSG SEGSSGPGESSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSGGEPSESGSSGSSESGS ESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESG SSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSSESGS SEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSG SSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGSEGSSGPGESSGSSESGS SEGGPGSEGSSGPGESS GLP-Z- HADGSFSDEMNTILDNLAARDFINWLIQTKITDGGSPAGSPTSTEEGTSESATPESGPGTSTEPS AE576 EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG SEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG SEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPE SGPGTSTEPSEGSAP GLP-2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGGSPAGSPTSTEEGTSESATPESGPGTSTEPS variant 2- EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG AE576 SEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG SEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPE SGPGTSTEPSEGSAP GLP-Z- HADGSFSDEMNTILDNLAARDFINWLIQTKITDGGSTSSTAESPGPGSTSSTAESPGPGSTSESP AF576 SGTAPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGS TSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESS TAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTST PESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTA PGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSST AESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPG STSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGES STAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTS GLPZ- Amino Acid Sequence XTEN Name" TPESGSASP GLP-2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGGSTSSTAESPGPGSTSSTAESPGPGSTSESP variant 2— SGTAPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGS AF576 TSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESS TAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTST PESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTA PGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSST AESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPG STSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGES STAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTS GLP-2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGMAEPAGSPTSTEEGTPGSGTASSSPGSSTP variant 2- SGATGSPGASPGTSSTGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE AE624 EGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG SPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP GLP-Z- HADGSFSDEMNTILDNLAARDFINWLIQTKITDGGSSESGSSEGGPGSSESGSSEGGPGESPGG AD836 GSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGP GSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSSESG SSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSS GESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEP SESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSS GSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGG SSGSESGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGP GESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGSSESGSSEG GPGSGGEPSESGSSGSEGSSGPGESSGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSG GEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSG SESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGES PGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSG SESGSGGEPSESGSS GLP-2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGGSSESGSSEGGPGSSESGSSEGGPGESPGG variant 2- SSGSESGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGP AD83 6 GSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSSESG SSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSS GESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEP SESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSS GSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGG SSGSESGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGP GESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGSSESGSSEG EPSESGSSGSEGSSGPGESSGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSG GEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSG SESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGES PGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSG GEPSESGSS GLP-Z- HADGSFSDEMNTILDNLAARDFINWLIQTKITDGGSPAGSPTSTEEGTSESATPESGPGTSTEPS AE864 EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG SEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG SEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPE GLPZ- Amino Acid Sequence XTEN Name" SGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSE GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS APGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG GLP-2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGGSPAGSPTSTEEGTSESATPESGPGTSTEPS variant 2- EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG AE864 SEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG SEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPE SGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSE SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS APGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG GLP—2 HADGSFSDEMNTILDNLATRDFINWLIQTKITDGGSPAGSPTSTEEGTSESATPESGPGTSTEPS variant 1- EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG AE864 GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG SEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT GSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPE SGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSE SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS APGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG GLP-2 HVDGSFSDEMNTILDNLAARDFINWLIQTKITDGGSPAGSPTSTEEGTSESATPESGPGTSTEPS variant 3- EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG AE864 SEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG SEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPE SGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSE SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS APGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG GLP-Z- HADGSFSDEMNTILDNLAARDFINWLIQTKITDGGSTSESPSGTAPGTSPSGESSTAPGSTSESP AF864 SGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGT SPSGESSTAPGSTSESPSGTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESS PSGESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTS ESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTA PGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSST AESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGPXXX GASASGAPSTXXXXSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESP SGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGT SPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSG TAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTS 2012/054941 GLPZ- Amino Acid Sequence XTEN Name" ESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSAS PGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAESPGPGTSPSG ESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSP GLP-Z HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGGSTSESPSGTAPGTSPSGESSTAPGSTSESP variant 2- SGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGT AF864 SPSGESSTAPGSTSESPSGTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESS TAPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTS ESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTA PGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSST AESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGPXXX APSTXXXXSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESP SGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGT SPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSG TAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTS ESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSAS GESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAESPGPGTSPSG ESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSP GLP-Z- HADGSFSDEMNTILDNLAARDFINWLIQTKITDGGASPGTSSTGSPGSSPSASTGTGPGSSPSAS AG864 TGTGPGTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTGPGASPGTSSTGSPGTPGSGTASSSP GSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPS GATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTGPGSSPSASTGTG PGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGS GTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSS PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPG TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTG PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPG TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGS PGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPG TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS PGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGS GTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP GLP—2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGGASPGTSSTGSPGSSPSASTGTGPGSSPSAS variant 2- TGTGPGTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTGPGASPGTSSTGSPGTPGSGTASSSP AG864 GSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPS GATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTGPGSSPSASTGTG PGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGS GTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSS PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPG TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTG PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPG TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGS GTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPG PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS PGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGS GTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP GLP-2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGGTSTEPSEGSAPGSEPATSGSETPGSPAGSP variant 2- TSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGT AM875 STPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS ESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGS APGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTST EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS APGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSE SPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSET PGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPAT SGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPG TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTS STGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPG GLPZ- Amino Acid Sequence XTEN Name" STGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP GLP-Z HADGSFSDEMNTVLDSLATRDFINWLLQTKITDGGSPAGSPTSTEEGTSESATPESGPGTSTEP bovine— SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP AE864 GSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP GSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGS PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG TSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATP ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTS SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS APGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG GLP-2 pig- HADGSFSDEMNTVLDNLATRDFINWLLHTKITDSLGGASPGTSSTGSPGSSPSASTGTGPGSSP AG864 SASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTGPGASPGTSSTGSPGTPGSGTAS SSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTGPGSSPSASTG TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTP GSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTAS SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGAS PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTG TGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGAS GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT GSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTP GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP GLP-Z rat- HADGSFSDEMNTILDNLATRDFINWLIQTKITDGGSPAGSPTSTEEGTSESATPESGPGTSTEPS AE576 EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG SEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG SEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPE SGPGTSTEPSEGSAP GLP—2 HKDGSFSDEMNTILDNLAARDFINWLIQTKITDGGSTSESPSGTAPGTSPSGESSTAPGSTSESP variant 5- SGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGT AF864 SPSGESSTAPGSTSESPSGTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESS TAPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTS ESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTA PGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSST AESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGPXXX GASASGAPSTXXXXSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESP SGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGT SPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSG TAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTS ESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSAS PGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAESPGPGTSPSG ESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSP GLP-2 HRDGSFSDEMNTILDNLAARDFINWLIQTKITDGGSPAGSPTSTEEGTSESATPESGPGTSTEPS variant 6- EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG AE864 SEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG GLPZ- Amino Acid Sequence XTEN Name" SEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPE SGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSE SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS APGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG GLP-2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSP variant 2- TSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG AE1236 TSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSP TSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG SPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATP ESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGS EPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGT STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPE SGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSP AGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEG SAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSE ETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS ETPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTS TEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSPAGSPTS TEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSE GLP-2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGGSPAGSPTSTEEGTSTEPSEGSAPGTSESAT variant 2- PESGPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEG AE1332 EGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSE GSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGT ESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEG SAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTS TEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPES GPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTST EPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTST EEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTST EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTST EEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPA GSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSE TPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSPA GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPES ATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTST GLP-2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGGSETPGTSTEPSEGSAPGTSTEPSEGSAPGT variant 2- SESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE AE612A TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT GLP-2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGTSGSETPGSEPATSGSETPGSPAGSPTSTEE variant 2- GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS AE720A EGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPG TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATS GSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG WO 40093 GLPZ- Amino Acid Sequence XTEN Name" EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGS PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGS SETPGSPAGSPTSTEEGTSTE GLP-2 HGDGSFSDEMNTILDNLAARDFlNWLIQTKITDGSTGSPGTPGSGTASSSPGSSTPSGATGSPG variant 2- ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSG AG6 12A ATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPG ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTS STGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPG TPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS STGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT ASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG TPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS GLP-2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGTSSTGSPGSSPSASTGTGPGSSPSASTGTGP variant 2- GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPS AG792A GATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPS GATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSP GASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGT SSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGT SSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSG TASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGT SSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPG * Sequence name s N— to C-terminus conﬁguration of the GLP-2 and XTEN (by family name and length) Table 34: Exemplary GLPZ-XTEN comprising GLP-2, cleavage sequences and XTEN seguences GLP2- Amino Acid Sequence XTEN Name" GLPZ- HADGSFSDEMNTILDNLAARDFINWLIQTKITDGLTPRSLLVGGGGSSESGSSEGGPGSSESGS Thrombin- SEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPG AD83 6 SSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGS SEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPG SGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGSSESGS SEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESG SGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSG PGESSGESPGGSSGSESGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSSESGSSEGGPG SSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSG PGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSEGSSGPGESSGSEGSSGPGESSGSGGE SGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGES SGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGSSES GSSEGGPGESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGS SGESPGGSSGSESGSGGEPSESGSS GLPZ— HADGSFSDEMNTILDNLAARDFlNWLIQTKITDGGGKLTRVVGGGGSPAGSPTSTEEGTSESA FXIa- TPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP AE864 GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPS GLPZ- Amino Acid Sequence XTEN Name" EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPG TSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATP ESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGT STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTS TEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS SGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG GLPZ- HADGSFSDEMNTILDNLAARDFINWLIQTKITDGGGLGPVSGVPGGSTSESPSGTAPGTSPSGE Elastase- SSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGS AP864 GTAPGTSPSGESSTAPGSTSESPSGTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAES PGPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTS ESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTA PGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSST AESPGPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPG TSTPESGPXXXGASASGAPSTXXXXSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPS GTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGST SSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESST APGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSS TAESPGPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAP GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTA ESPGPGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSP GLPZ- HADGSFSDEMNTILDNLAARDFINWLIQTKITDGAPLGLRLRGGGGASPGTSSTGSPGSSPSAS MMP TGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTGPGASPGTSSTGSP AG864 GTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSG TASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTGP GSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGT SSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGP TASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPS GATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSP GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGT SSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSP GSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGP GASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSA STGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP GLP-2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGLTPRSLLVGGGGSSESGSSEGGPGSSESGS variant 2- SEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPG Thrombin— SEGGPGSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGS ADS3 6 SEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPG SGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGSSESGS SEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESG SGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSG PGESSGESPGGSSGSESGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSSESGSSEGGPG SSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSG PGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSEGSSGPGESSGSEGSSGPGESSGSGGE PSESGSSGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGES SGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGSSES GSSEGGPGESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGS SGESPGGSSGSESGSGGEPSESGSS GLP-Z HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGGGKLTRVVGGGGSPAGSPTSTEEGTSESA variant 2- TPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP FXIa- GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPS AE864 EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPG TSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATP GLPZ- Amino Acid Sequence XTEN Name" ESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGT STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS TEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS SGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTS TEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS SGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG GLP-2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGGGLGPVSGVPGGSTSESPSGTAPGTSPSGE variant 2- STSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGS Elastase- TSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAES AP864 PGPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTS ESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTA PGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSST AESPGPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPG TSTPESGPXXXGASASGAPSTXXXXSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPS GTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGST SSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESST APGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSS TAESPGPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAP SGSASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTA ESPGPGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSP GLP-2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITDGAPLGLRLRGGGGASPGTSSTGSPGSSPSAS t 2— TGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTGPGASPGTSSTGSP MMP GTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSG AG864 TASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTGP GSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGT SSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGP GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPS GATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSP GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGT SSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSP GSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA STGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGP GASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSA STGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP AE912- MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEEGTSES Thrombin- ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG GLP2 PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEP SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESA TPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS PTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGS PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP GTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS EGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGLTPRSLLVGGG HADGSFSDEMNTILDNLAARDFINWLIQTKITD AE912- MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEEGTSES FXIa-GLP- ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG 2 variant 2 TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEP SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESA TPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GLPZ- Amino Acid Sequence XTEN Name" GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS PTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGS PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP GTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS EGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGGGKLTRVVGGG HGDGSFSDEMNTILDNLAARDFINWLIQTKITD AE912- MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEEGTSES Elastase- ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG GLP-2 PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEP variant 2 SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESA TPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS PTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGS PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPAT GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP GTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS EGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGGGLGPVSGVPG HGDGSFSDEMNTILDNLAARDFINWLIQTKITD GLP—2 SDEMNTILDNLAARDFlNWLIQTKITDGAPLGLRLRGGGGSPAGSPTSTEEGTSESAT variant 2- PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG MMP- l 7- SEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSE AE864 GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPE SGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS TEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS SGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTS TEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS ESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG * Sequence name reﬂects N— to C-terminus conﬁguration of the GLP-Z, XTEN (by family name and length) and cleavage ce d by protease name active on the sequence.

Claims

1. A composition for use in achieving an intestinotrophic effect in a subject comprising a inant fusion protein comprising (i) a glucagon-like protein-2 (GLP-2) sequence selected from the group consisting of the sequences of SEQ ID NOS: 1 and 3-23, and (ii) an extended recombinant polypeptide (XTEN), n the XTEN is a sequence exhibiting at least 90% sequence identity to a sequence ed from the group consisting of the sequences in Table 4, and wherein the XTEN is further characterized in that: (a) the XTEN comprises at least 36 amino acid residues; (b) the sum of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues constitutes more than 80% of the total amino acid residues of the XTEN; (c) the XTEN is substantially non-repetitive such that (i) the XTEN contains no three contiguous amino acids that are identical unless the amino acids are serine; (ii) at least 80% of the XTEN ce consists of non-overlapping sequence motifs, each of the sequence motifs comprising 9 to 14 amino acid residues consisting of four to six amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), wherein any two contiguous amino acid residues do not occur more than twice in each of the non-overlapping sequence motifs; or (iii) the XTEN sequence has a uence score of less than 10; (d) the XTEN has greater than 90% random coil formation as determined by GOR thm; (e) the XTEN has less than 2% alpha helices and 2% heets as determined by Chou-Fasman algorithm; and (f) the XTEN lacks a predicted T-cell e when ed by TEPITOPE thm, wherein the TEPITOPE threshold score for said prediction by said algorithm has a threshold of –9, wherein said fusion protein exhibits an apparent molecular weight factor of at least 4 and is capable of achieving an intestinotrophic effect in a subject using a dosage of 2.5 nmol/kg to 6250 nmol/kg, or 25 nmol/kg to 3750 nmol/kg, or 75 nmol/kg/dose to 1250 nmol/kg/dose, or 125 g/dose to 750 nmol/kg/dose.

2. The composition of claim 1, wherein the intestinotrophic effect is at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100% or at least 120% or at least 150% or at least 200% of the intestinotrophic effect compared to the corresponding GLP-2 not linked to XTEN upon administration of said corresponding GLP-2 to a subject using comparable dose.

3. The ition of claim 2, wherein the subject is selected from the group consisting of mouse, rat, monkey, pig, bovine, sheep, and human.

4. The composition of claim 2, wherein the fusion protein is formulated for delivery by aneous, intramuscular, or intravenous doses.

5. The composition of claim 2, wherein the intestinotrophic effect is determined after administration of 1 dose, or 3 doses, or 6 doses, or 10 doses, or 12 or more doses of the composition.

6. The ition of claim 5, wherein the intestinotrophic effect is selected from the group consisting of intestinal growth, increased hyperplasia of the villus epithelium, increased crypt cell proliferation, increased height of the crypt and villus axis, increased healing after intestinal anastomosis, increased small bowel weight, increased small bowel length, decreased small bowel epithelium apoptosis, and enhancement of intestinal on.

7. The composition of claim 6, wherein the intestinotrophic effect is an increase in small intestine weight of at least 10%, or at least 20%, or at least 30%.

8. The composition of claim 6, wherein the intestinotrophic effect is an increase in small intestine length of at least 5%, or at least 6%, or at least 7%, or at least 8%, or at least 9%, or at least 10%, or at least 20%, or at least 30%.

9. The composition of any one of claims 1-8, n the GLP-2 is ed from the group consisting of bovine GLP-2, pig GLP-2, sheep GLP-2, n GLP-2, and canine GLP-2.

10. The composition of any one of claims 1-8, wherein the GLP-2 has an amino acid substitution in place of Ala2, and wherein the substitution is e.

11. The composition of any one of claims 1-8, wherein the GLP-2 has the sequence HGDGSFSDEMNTILDNLAARDFINWLIQTKITD.

12. The composition of any one of claims 1-8, wherein the XTEN is linked to the C- terminus of the GLP-2.

13. The ition of claim 12, further comprising a spacer sequence of 1 to 50 amino acid residues linking the GLP-2 and XTEN.

14. The composition of claim 13, wherein the spacer sequence ses a glycine residue.

15. The composition of any one of claims 1-8, wherein the XTEN has at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity when ed to a sequence of comparable length selected from any one of Table 4, Table 8, Table 9, Table 10, Table 11, and Table 12, when optimally aligned.

16. The composition of claim 15, wherein the XTEN has at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity when compared to an AE864 sequence from Table 4, when optimally aligned.

17. The composition of any one of claims 1-8, wherein the fusion protein ce has a sequence with at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence ty to SEQ ID NO: 741, 743, 745, 747, 749, 751-752, 754, 756-758, 760, 762-774, and 798.

18. The composition of any one of claims 1-8, wherein the fusion protein exhibits a terminal half-life that is at least 30 hours when administered to the subject.

19. The composition of any one of claims 1-8, wherein the fusion protein binds to a GLP-2 or with an EC50 of less than 30 nM, or 100 nM, or 200 nM, or 300 nM, or 370 nM, or 400 nM, or 500 nM, or 600 nM, or 700 nM, or 800 nM, or 1000 nM, or 1200 nM, or 1400 nM when assayed using an in vitro GLP2R cell assay wherein the GLP2R cell is a human recombinant GLP-2 glucagon family receptor calcium-optimized cell.

20. The composition of any one of claims 1-8, wherein the fusion protein retains at least 1%, or 2%, or 3%, or 4%, or 5%, or 10%, or 20%, or at least 30% of the potency of the corresponding GLP-2 not linked to XTEN when d using an in vitro GLP2R cell assay wherein the GLP2R cell is a human inant GLP-2 glucagon family receptor calciumoptimized cell.

21. The composition of any one of claims 1-8 or 17, characterized in that (a) an equivalent amount, in nmoles/kg, of the fusion protein compared to the corresponding GLP-2 that lacks the XTEN has a terminal half-life that is at least , or at least 4-fold, or at least 5-fold, or at least 10-fold, or at least 15-fold, or at least 20-fold longer compared to the corresponding GLP-2 that lacks the XTEN; or (b) an equivalent amount, in nmoles/kg, of the fusion protein compared to the corresponding GLP-2 that lacks the XTEN achieves a greater intestinotrophic effect in a subject compared to the corresponding GLP-2 that lacks the XTEN.

22. The composition of claim 21, wherein the greater intestinotrophic effect is selected from the group consisting of body weight gain, small intestine length, reduction in TNFα content of the small intestine tissue, reduced l atrophy, reduced nce of perforated ulcers, and height of villi.

23. The composition of claim 22, wherein the greater intestinotrophic effect is an increase in small intestine weight of at least 10%, or at least 20%, or at least 30%, or at least 40% r compared to that of the corresponding GLP-2 not linked to XTEN.

24. The composition of claim 22, n the greater intestinotrophic effect is an increase in small intestine length of at least 5%, or at least 6%, or at least 7%, or at least 8%, or at least 9%, or at least 10%, or at least 20%, or at least 30%, or at least 40% greater compared to that of the corresponding GLP-2 not linked to XTEN.

25. The composition of claim 22, wherein the greater intestinotrophic is an increase in body weight is at least 5%, or at least 6%, or at least 7%, or at least 8%, or at least 9%, or at least 10%, or at least 20%, or at least 30%, or at least 40% r compared to that of the corresponding GLP-2 not linked to XTEN.

26. The composition of claim 22, wherein the greater intestinotrophic effect is a reduction in TNFα content is at least 0.5 ng/g, or at least 0.6 ng/g, or at least 0.7 ng/g, or at least 0.8 ng/g, or at least 0.9 ng/g, or at least 1.0 ng/g, or at least 1.1 ng/g, or at least 1.2 ng/g, or at least 1.3 ng/g, or at least 1.4 ng/g of small intestine tissue or greater ed to that of the corresponding GLP-2 not linked to XTEN.

27. The ition of claim 22, wherein the greater intestinotrophic effect is villi height is at least 5%, or at least 6%, or at least 7%, or at least 8%, or at least 9%, or at least 10%, or at least 11%, or at least 12% greater compared to that of the corresponding GLP-2 not linked to XTEN.

28. A method of producing a fusion protein comprising GLP-2 fused to one or more extended recombinant polypeptides (XTEN), comprising: (a) providing a prokaryotic host cell comprising a recombinant nucleic acid encoding the fusion protein of any one of claims 1-8 or 17; (b) culturing the host cell under conditions permitting the expression of the fusion protein; and (c) recovering the fusion protein.

29. The method of claim 28, wherein the fusion protein is recovered from the host cell cytoplasm in substantially soluble form.

30. The method of claim 28, wherein the recombinant c acid le has a sequence with at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to a sequence ed from the group consisting of the DNA sequences set forth in Table 13, when optimally aligned, or the complement thereof.

31. An isolated nucleic acid comprising: (a) a nucleic acid sequence that has at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% ce identity to a DNA sequence selected from Table 13, or the complement thereof; or (b) a nucleotide sequence encoding the fusion protein of any one of claims 1 to 8 or 17, or the complement thereof.

32. An sion vector or isolated host cell comprising the nucleic acid of claim 31.

33. A prokaryotic host cell comprising the expression vector of claim 32.

34. A pharmaceutical composition sing the fusion protein of any one of claims 1-8 or 17, and a pharmaceutically acceptable carrier.

35. The composition of any one of claims 1-8 or 17 ured according to formula (GLP-2)-(S)x-(XTEN) (V) wherein S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence from Table 6 or amino acids compatible with restrictions sites; x is either 0 or 1.

36. The ition of claim 35, wherein the GLP-2 is selected from the group consisting of bovine GLP-2, pig GLP-2, sheep GLP-2, chicken GLP-2, and canine GLP-2.

37. The composition of claim 35, wherein the GLP-2 has an amino acid substitution in place of Ala2, and wherein the substitution is glycine.

38. The composition of claim 35, wherein the GLP-2 has the sequence HGDGSFSDEMNTILDNLAARDFINWLIQTKITD.

39. The composition of claim 35, comprising a spacer sequence wherein the spacer ce comprises a glycine residue.

40. The composition of claim 35, wherein the XTEN has at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity when ed to a AE864 ce from Table 4, when optimally d.

41. The composition of claim 17 for use in the manufacture of a medicament for the treatment of a gastrointestinal condition.

42. The composition of claim 41 wherein the gastrointestinal condition is selected from the group ting of gastritis, ion disorders, malabsorption syndrome, short-gut syndrome, short bowel syndrome, cul-de-sac syndrome, inflammatory bowel disease, celiac disease, tropical sprue, hypogammaglobulinemic sprue, Crohn's disease, ulcerative colitis, enteritis, chemotherapy-induced enteritis, irritable bowel syndrome, small intestine damage, small intestinal damage due to cancer-chemotherapy, gastrointestinal injury, diarrheal diseases, intestinal insufficiency, acid-induced intestinal , ne deficiency, idiopathic hypospermia, obesity, catabolic illness, e neutropenia, diabetes, obesity, steatorrhea, autoimmune diseases, food allergies, ycemia, gastrointestinal r disorders, sepsis, bacterial nitis, burn-induced intestinal damage, decreased gastrointestinal motility, intestinal failure, chemotherapy- associated bacteremia, bowel trauma, bowel ischemia, mesenteric ischemia, malnutrition, necrotizing enterocolitis, necrotizing pancreatitis, neonatal feeding intolerance, NSAID-induced gastrointestinal damage, nutritional insufficiency, total parenteral nutrition damage to gastrointestinal tract, al nutritional insufficiency, radiation-induced enteritis, radiation-induced injury to the intestines, mucositis, tis, and gastrointestinal ischemia.

43. A kit, comprising packaging material and at least a first container comprising the composition of claim 17, an amount of a pharmaceutically acceptable carrier, and a sheet of ctions for the reconstitution and/or administration of the composition to a subject.

44. Use of a recombinant fusion protein in the manufacture of a medicament for achieving an intestinotrophic effect in a subject, wherein the recombinant fusion protein comprises (i) a glucagon-like n-2 (GLP-2) sequence selected from the group consisting of the sequences of SEQ ID NOS: 1 and 3-23, and (ii) an extended recombinant polypeptide (XTEN), wherein the XTEN is a sequence exhibiting at least 90% sequence identity to a sequence selected from the group ting of the sequences in Table 4, and wherein the XTEN is further characterized in that: (a) the XTEN comprises at least 36 amino acid residues; (b) the sum of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues constitutes more than 80% of the total amino acid residues of the XTEN; (c) the XTEN is substantially non-repetitive such that (i) the XTEN contains no three contiguous amino acids that are cal unless the amino acids are serine; (ii) at least 80% of the XTEN sequence ts of non-overlapping ce motifs, each of the sequence motifs comprising 9 to 14 amino acid residues consisting of four to six amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), wherein any two contiguous amino acid residues do not occur more than twice in each of the non-overlapping sequence motifs; or (iii) the XTEN ce has a subsequence score of less than 10; (d) the XTEN has greater than 90% random coil formation as determined by GOR algorithm; (e) the XTEN has less than 2% alpha helices and 2% beta-sheets as determined by Chou-Fasman algorithm; and (f) the XTEN lacks a predicted T-cell epitope when analyzed by TEPITOPE algorithm, wherein the PE old score for said prediction by said algorithm has a threshold of –9, wherein said fusion protein exhibits an apparent molecular weight factor of at least 4 and is prepared for administration to a subject at a dosage of 2.5 nmol/kg to 6250 nmol/kg, or 25 nmol/kg to 3750 nmol/kg, or 75 nmol/kg/dose to 1250 g/dose, or 125 nmol/kg/dose to 750 g/dose.

45. The use of claim 1, wherein the intestinotrophic effect is at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100% or at least 120% or at least 150% or at least 200% of the intestinotrophic effect compared to a corresponding GLP-2 not linked to XTEN upon administration of said corresponding GLP-2 to a subject using comparable dose.

46. The use of claim 45, wherein the subject is selected from the group consisting of mouse, rat, monkey, pig, bovine, sheep, and human.

47. The use of claim 45, wherein the fusion protein is formulated for delivery by subcutaneous, intramuscular, or intravenous doses.

48. The use of claim 45, wherein the fusion protein is formulated for administration of 1 dose, or 3 doses, or 6 doses, or 10 doses, or 12 or more doses.

49. The use of claim 48, wherein the intestinotrophic effect is selected from the group consisting of intestinal growth, increased hyperplasia of the villus epithelium, increased crypt cell proliferation, sed height of the crypt and villus axis, sed healing after intestinal anastomosis, increased small bowel weight, increased small bowel length, decreased small bowel epithelium apoptosis, and enhancement of inal function.

50. The use of claim 49, wherein the intestinotrophic effect is an se in small intestine weight of at least 10%, or at least 20%, or at least 30%.

51. The use of claim 49, wherein the intestinotrophic effect is an increase in small intestine length of at least 5%, or at least 6%, or at least 7%, or at least 8%, or at least 9%, or at least 10%, or at least 20%, or at least 30%.

52. The use of any one of claims 44-51, wherein the GLP-2 is selected from the group ting of bovine GLP-2, pig GLP-2, sheep GLP-2, chicken GLP-2, and canine GLP-2.

53. The use of any one of claims 44-51, wherein the GLP-2 has an amino acid substitution in place of Ala2, and wherein the substitution is glycine.

54. The use of any one of claims 44-51, wherein the GLP-2 has the sequence HGDGSFSDEMNTILDNLAARDFINWLIQTKITD.

55. The use of any one of claims 44-51, wherein the XTEN is linked to the C- terminus of the GLP-2.

56. The use of claim 55, wherein the fusion protein further comprises a spacer sequence of 1 to 50 amino acid residues linking the GLP-2 and XTEN.

57. The use of claim 56, wherein the spacer sequence ses a e e.

58. The use of any one of claims 44-51, wherein the XTEN has at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity when compared to a sequence of comparable length selected from any one of Table 4, Table 8, Table 9, Table 10, Table 11, and Table 12, when optimally aligned.

59. The use of claim 58, wherein the XTEN has at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity when compared to an AE864 sequence from Table 4, when optimally aligned.

60. The use of any one of claims 44-51, wherein the fusion protein sequence has a sequence with at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 741, 743, 745, 747, 749, 751-752, 754, 756-758, 760, 762- 774, and 798.

61. The use of any one of claims 44-51, wherein the fusion n ts a terminal half-life that is at least 30 hours when administered to a non-human subject.

62. The use of any one of claims 44-51, wherein the fusion protein binds to a GLP-2 receptor with an EC50 of less than 30 nM, or 100 nM, or 200 nM, or 300 nM, or 370 nM, or 400 nM, or 500 nM, or 600 nM, or 700 nM, or 800 nM, or 1000 nM, or 1200 nM, or 1400 nM when assayed using an in vitro GLP2R cell assay wherein the GLP2R cell is a human recombinant GLP-2 glucagon family receptor calcium-optimized cell.

63. The use of any one of claims 44-51, wherein the fusion protein retains at least 1%, or 2%, or 3%, or 4%, or 5%, or 10%, or 20%, or at least 30% of the potency of a corresponding GLP-2 not linked to XTEN when d using an in vitro GLP2R cell assay wherein the GLP2R cell is a human recombinant GLP-2 glucagon family receptor calciumoptimized cell.

64. The use of any one of claims 44-51 or 60, characterized in that (a) an equivalent amount, in nmoles/kg, of the fusion protein compared to a corresponding GLP-2 that lacks the XTEN has a terminal half-life that is at least 3-fold, or at least 4-fold, or at least 5-fold, or at least 10-fold, or at least 15-fold, or at least 20-fold longer compared to the corresponding GLP-2 that lacks the XTEN; or (b) an equivalent , in nmoles/kg, of the fusion protein compared to the corresponding GLP-2 that lacks the XTEN es a greater instestinotrophic effect in a subject compared to the corresponding GLP-2 that lacks the XTEN.

65. The use of claim 64, wherein the greater intestinotrophic effect is selected from the group consisting of body weight gain, increased small intestine length, reduction in TNFα content of the small intestine tissue, reduced mucosal atrophy, d incidence of perforated ulcers, and increased height of villi.

66. The use of claim 65, wherein the r intestinotrophic effect is an increase in small intestine weight of at least 10%, or at least 20%, or at least 30%, or at least 40% greater ed to that of the corresponding GLP-2 not linked to XTEN.

67. The use of claim 65, wherein the greater intestinotrophic effect is an increase in small ine length of at least 5%, or at least 6%, or at least 7%, or at least 8%, or at least 9%, or at least 10%, or at least 20%, or at least 30%, or at least 40% greater compared to that of the corresponding GLP-2 not linked to XTEN.

68. The use of claim 65, wherein the greater intestinotrophic is an increase in body weight is at least 5%, or at least 6%, or at least 7%, or at least 8%, or at least 9%, or at least 10%, or at least 20%, or at least 30%, or at least 40% greater compared to that of the corresponding GLP-2 not linked to XTEN.

69. The use of claim 65, wherein the greater intestinotrophic effect is a reduction in TNFα content is at least 0.5 ng/g, or at least 0.6 ng/g, or at least 0.7 ng/g, or at least 0.8 ng/g, or at least 0.9 ng/g, or at least 1.0 ng/g, or at least 1.1 ng/g, or at least 1.2 ng/g, or at least 1.3 ng/g, or at least 1.4 ng/g of small intestine tissue or greater compared to that of the corresponding GLP-2 not linked to XTEN.

70. The use of claim 65, wherein the greater inotrophic effect is increased villi height of at least 5%, or at least 6%, or at least 7%, or at least 8%, or at least 9%, or at least 10%, or at least 11%, or at least 12% compared to that of the corresponding GLP-2 not linked to XTEN.

71. The use of any one of claims 44-51 or 60, wherein producing the fusion protein comprising GLP-2 fused to one or more extended recombinant polypeptides (XTEN), comprises: (a) providing a yotic host cell comprising a recombinant nucleic acid encoding the fusion protein; (b) culturing the host cell under ions permitting the expression of the fusion protein; and (c) recovering the fusion n.

72. The use of claim 71, wherein the fusion protein is recovered from the host cell cytoplasm in substantially soluble form.

73. The use of claim 71, wherein the recombinant nucleic acid molecule has a sequence with at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% ce identity to a sequence selected from the group consisting of the DNA sequences set forth in Table 13, when optimally aligned, or the complement thereof.

74. The use of any one of claims 44-51 or 60, wherein the medicament comprising the fusion protein further comprises a pharmaceutically acceptable carrier.

75. The use of any one of claims 44-51 or 60, wherein the fusion protein is configured according to formula V: (GLP-2)-(S)x-(XTEN) (V) wherein S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence from Table 6 or amino acids compatible with restrictions sites; x is either 0 or 1.

76. The use of claim 75, wherein the GLP-2 is selected from the group ting of bovine GLP-2, pig GLP-2, sheep GLP-2, chicken GLP-2, and canine GLP-2.

77. The use of claim 75, wherein the GLP-2 has an amino acid substitution in place of Ala2, and wherein the substitution is glycine.

78. The use of claim 75, wherein the GLP-2 has the sequence HGDGSFSDEMNTILDNLAARDFINWLIQTKITD.

79. The use of claim 75, wherein the fusion protein comprises a spacer sequence and wherein the spacer ce ses a glycine residue.

80. The use of claim 75, wherein the XTEN has at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity when compared to a AE864 sequence from Table 4, when optimally aligned.

81. The use of claim 60, n the intestinotrophic effect ses treatment of a gastrointestinal condition.

82. The use of claim 81 wherein the gastrointestinal condition is selected from the group consisting of gastritis, digestion disorders, malabsorption syndrome, short-gut syndrome, short bowel syndrome, cul-de-sac syndrome, inflammatory bowel disease, celiac disease, tropical sprue, hypogammaglobulinemic sprue, Crohn's e, ulcerative colitis, enteritis, chemotherapy-induced enteritis, irritable bowel syndrome, small intestine damage, small intestinal damage due to cancer-chemotherapy, gastrointestinal injury, eal diseases, intestinal insufficiency, acid-induced intestinal injury, ne deficiency, idiopathic ermia, obesity, catabolic illness, febrile neutropenia, diabetes, obesity, steatorrhea, autoimmune diseases, food allergies, hypoglycemia, intestinal barrier disorders, sepsis, bacterial peritonitis, burn-induced intestinal damage, decreased gastrointestinal motility, intestinal failure, chemotherapy- associated bacteremia, bowel trauma, bowel ischemia, mesenteric ischemia, malnutrition, necrotizing colitis, necrotizing pancreatitis, neonatal feeding intolerance, NSAID-induced gastrointestinal , nutritional insufficiency, total parenteral nutrition damage to gastrointestinal tract, neonatal nutritional insufficiency, radiation-induced enteritis, radiation-induced injury to the intestines, mucositis, pouchitis, and gastrointestinal ischemia.

83. The use of claim 60, wherein the medicament is provided in a kit, sing ing material and at least a first container comprising the fusion protein, an amount of a pharmaceutically acceptable r, and a sheet of instructions for the reconstitution and/or administration of the composition to a t.

84. The use of claim 44, n the fusion protein is ed for administration to a subject at a dosage of 2.5 nmol/kg to 125 nmol/kg/dose.