Cytokine conjugates

Abstract

The present invention relates to compositions comprising biologically active proteins, such as cytokines, linked to extended recombinant polypeptide (XTEN), isolated nucleic acids encoding the compositions and vectors and host cells containing the same, and methods of using such compositions in treatment of related disorders and conditions.

Description

CYTOKINE CONJUGATES SEQUENCE LISTING 10001] A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on June 14, 2021 having the file name “776-601_20-1836-WO_ST25 FINAL txt” and is 9R8kb in size. REFERENCE STATEMENT

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 044,335 filed on June 25, 2020 entitled "CYTOKINE CONJUGATES", U.S. Provisional Patent Application No.63 / 197.875 filed on June 7, 2021, entitled "CYTOKINE CONJUGATES"; and U.S. Provisional Patent Application No. 63 / 197,944 filed on June 7. 2021, entitled "CYTOKINE CONJUGATES, all of which are incorporated herein in their entireties. BACKGROUND

[0003] Cytokines can be used to treat a variety of diseases or conditions. such as cancer, inflammatory conditions, autoimmune conditions, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, Alzheimer’s disease, Schizophrenia, viral infections, (e.g., chronic hepatitis C, AIDS), allergic asthma. vetinal veurodegenerative processes, metabolic disorder, insulin resistance. and diabetic cardiomyopathy. However. the therapeutic utility of cytokines can be limited due to the cellular toxicity, short half-life, need for repetitive or frequent dosing, and the potential to elicit undesired immune response in the patients.

[0004] Most cytokine products in the clinical setting are extremely potent. Tnterleukins. such as 11-2 and IL- 12, and IFN-a are cytokines, produced primarily by cells of the immune system to signal and orgamze the immune response. In cancer, cytokines facilitate the ability of the immune system to recognize tumor cells as abnormal and harmful to the host. Cytokines further increase the proliferation of, enhance the survival of, and direct a variety of immune cell types to infiltrate the TME and promote potent anti-tumor immune responses resulting in tumor cell killing and tumor clearance. This limits the practical applications of cytokines in a therapeutic setting, particularly in anti-cancer indications. 10005] Interleukin-12 (IL12) in particular, has been recognized as having potential to be an ideal payload for tumor immunotherapy. It can activate both the innate and the adaptive components of the immune system. 1L12 stimulates the production of IFN-y and activates NK cells, as well as CD8+ and CD4+ T cells. In addition, this cytokine also induces antiangiogenic chemokines, remodeling of the tumor extracellular matrix and stimulation of MHC class I molecules expression, making it an extremely attractive anticancer candidate. However, while researchers have shown encouraging preclinical data, the severe toxicity profile of this cytokine has prevented dose escalation and significantly curbed clinical potential as an anticancer agent. Although multiple clinical trials have been on-going since the first human clinical trial of TL12 in 1996. an FDA-approved 11.12 product remains elusive.

[0006] This presents a significant unmet need for new strategies that can overcome therapeutic index challenges for use of cytokines as anticancer agents. If the potency of cviokines like IL12 could be safely harnessed and the toxicity challenges could be controlled, these agents could serve as powerful therapeutics for potential use against a broad spectrum of cancers. SUMMARY

[0007] The present disclosure includes cytokine-related compositions and related methods that may address one or more drawback. or may provide one or more advantages. In one aspect, disclosed herein is a fusion protein comprising: (a) an extended recombinant polypeptide (X TEN) characterized in that: 1. it comprises at least 12 amino acids; il. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P}; and til it has 4-6 different amino acids selected from G, A, S, T, E and P; and (b) a cytokine linked to the XTEN,

[0008] In some embodiments, the fusion protein further comprises a release segment, wherein the release segment (RS) has at least 88%, at least 94%, or 100% sequence identity to a sequence selected from the sequences set forth in Tables 6-7. fn some embodiments, the fusion protein has a structural arrangement. from N- to C-terminus of X TEN-RS-cytokine or cytokine-RS-X TEN.

[0009] In some embodiments, the cytokine is selected from a group consisting of interleukins, chemokines, interferons, tumor necrosis factors, colony-stimulating factors, or TGF-Beta superfamily members. In some embodiments, the cytokine is an interleukin selected from the group consisting of TL1, IL2, IL3, TL4, ILS. IL6, IL7,108,1L9, IL10,IL11, 1112, [L.13, IL 14, [L15, IL16, and IL17. In some embodiments, the cytokine has at least 90% sequence identity to a sequence selected from Table 3 or Table A. In some embodiments, the cytokine is IL-12 or an [L-12 variant. In some embodiments, the cytokine comprises a first cytokine fragment (Cy1) and a second cytokine fragment (Cy2). In some embodiments, one of the Cy 1 and the Cy2 comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%. 94%, 95%, 96%. 97%. 98%, 99%, or 100% sequence identity to an interleukin-12 subunit beta. In some embodiments, the other one of the Cy1 and the Cy2 comprises an anno acid sequence having at least 70%, 75%, 80%. 85%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an interleukin-12 subunit alpha. In some embodiments, the first cytokine fragment (Cy1) comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence of SEQ ID NO. 5. In some embodiments, the second cytokine fragment (Cy2) comprises an amino acid sequence having at least 70%, 75%. 80%, 85%, 90%, 91%, 92%, 93%, 94%. 95%. 96%. 97%, 98%, 99%, or 100% sequence identity to a sequence of SEQ ID NO. 6. In some embodiments, the cytokine comprises a linker positioned between the first cytokine fragment (Cy 1) and the second cytokine fragment (Cy2). In some embodiments, the cytokine is an TL.-12 variant comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 7.

[0010] In some embodiments, the XTEN sequence consists of multiple non-overlapping sequence motifs, wherein the sequence motifs are selected from the sequence motifs of Tables 2a-2b. In some embodiments, the XTEN has from 40 to 3000 amino acids, or from 100 to 3000 amino acids. [n some embodiments, the XTEN has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%. or at least 99%, or 100% sequence identity to a sequence set forth in Tables 2a-2b. 10011] In some embodiments, a binding activity of the cytokine, when linked to the XTEN in the fusion protein, to a corresponding cytokine receptor can be characterized by a half maximal effective concentration (EC50) at least 1.2 fold greater, at least 1.4 fold greater, at least 1.6 fold greater, at least 1.8 fold greater, at least 2.0 fold greater, at Jeast 3.0 fold greater, at least 4.0 fold greater, at least 5.0 fold greater. at least 6.0 fold greater, at least 7.0 fold greater, at least 8.0 fold greater, at least 9.0 fold greater, or at least 10.0 fold greater than an EC50 characterizing a corresponding binding activity of the cytokine, when not linked to the XTEN, as determined in an ix vitro binding assay. In some embodiments, the cytokine can be interleukin 12 (IL-12) and the corresponding eytokine receptor can be an interleukin 12 receptor (IL-12R). In some embodiments, the in vitro binding assay can utilize a genetically engineered reporter gene cell line configured to respond to binding of the cytokine to the corresponding cytokine receptor with a proportional expression of a reporter protein. In some embodiments, the ix vitro binding assay can be a reporter gene activity assay.

[0012] In another aspect, the present disclosure provides a composition, comprising the fusion protein disclosed herein and at least one pharmaceutically acceptable carrier, In yet another aspect, the present disclosure provides uses of the subject composition in the preparation of a medicament for treating a disease in a subject in need thereof.

[0013] In a related aspect, the present disclosure provides a method of treating or preventing a disease or condition in a subject, the method comprising administering to a subject a therapeutically effective amount of a fusion protein or a composition comprising the fusion protein, all of which are disclosed herein. To some embodiments, the disease or condition can be a cancer, or a cancer-related disease or condition, or an inflammatory or antoimmune disease. In some embodiments, the disease or condition can be a cancer, or a cancer-related disease or condition. The diseases or conditions that can be treated with the subject fusion and composition include without limitation cancer, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, Alzheimer’s disease, Schizophrenia, viral infections, allergic asthma, retinal neurodegenerative processes, metabolic disorder, insulin resistance, and diabetic cardiomyopathy. In some embodiments, the disease or condition can be a cancer or a cancer-related disease or condition. Where desired. the subject fusion and composition can be used in conjunction with a therapeutically effective amount of at least one immune checkpoint inhibitor. Where desired, the mode of administration can be delivered intravenously, subcutaneously, or orally. INCORPORATION BY REFERENCE

[0014] 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

[0015] The features and advantages of the invention may be further explained by reference to the following detailed description and accompanying drawings that sets forth illustrative embodiments. 10016] F1G.1A-FIG. 1G show schematic representations of exemplary BPXTEN fusion proteins (FIGS. 1A- G), all depicted in an N- to C-terminus orientation. FIG. 1A shows two different configurations of BPXTEN fusion proteins (100), each comprising a single biologically active protein (BP) and an XTEN, the first of which has an XTEN molecule (102) attached to the C-terminus of a BP (103), and the second of which has an XTEN molecule attached to the N-terminus of a BP (103). FIG. 1B shows two different configurations of BPXTEN fusion proteins (100), each comprising a single BP, 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 BP (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 BP (103). FIG. 1C shows two different configurations of BPXTEN fusion proteins (101), each comprising two molecules of a single BP and one molecule of an XTEN, the first of which has an XTEN linked to the C-terminus of a first BP and that BP is linked to the C-terminus of a second BP, and the second of which is in the opposite orientation in which the X TEN is linked to the N-terminus of a first BP and that BP is linked to the N-terminus of a second BP. FIG. 1D shows two different configurations of BPXTEN fusion proteins (101), each comprising two molecules of a single BP, a spacer sequence and one molecule of an XTEN, the first of which has an XTEN linked to the C-terminus of a spacer sequence and the spacer sequence linked to the C-terminus of a first BP which is linked to the C-terminus of a second BP, 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 BP that that BP is linked to the N-terminus of a second BP. FIG. 1E shows two different configurations of BPXTEN fusion proteins (101), each comprising two molecules of a single BP, a spacer sequence and one molecule of an XTEN, the first of which has an XTEN linked to the C-terminus of a first BP and the first BP linked to the C-terminus of a spacer sequence which is linked to the C-terminus of a second BP molecule, and the second of which is in the opposite configuration of XTEN linked to the N- terminus of a first BP 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 BP. FIG. 1F shows two different configurations of BPX TEN fusion proteins (105), each comprising two molecules of a single BP, and two molecules of an XTEN, the first of which has a first XTEN linked to the C-terminus of a first BP which is linked to the C-terminus of a second XTEN that is linked to the C-terminus of a second molecule of BP, and the second of which is in the opposite configuration of XTEN linked to the N-terminus of a first BP linked to the N-terminus of a second XTEN linked to the N- terminus of a second BP. FIG. 1G shows a configuration (106) of a single BP linked to two XTEN at the N- and C-termini of the BP.

[0017] FIG. 2A-FIG. 2G is a schematic illustration of exemplary polynucleotide constructs of BPXTEN genes that encode the corresponding BPXTEN polypeptides of FIG. 1A-FIG. 1G; all depicted ina 5” to 3° orientation. In these illustrative examples the genes encode BPXTEN fusion proteins with one BP and XTEN (100); or two BP, one spacer sequence and one XTEN (201); two BP and two XTEN (205); or one BP and two XTEN (206). In these depictions, the polynucleotides encode the following components: XTEN (202), BP (203), and spacer amino acids that can include a cleavage sequence (204), with all sequences linked in frame.

[0018] FIG. 3A-FIG. 3K is a schematic illustration of an exemplary monomeric BPXTEN acted upon by an endogenously available protease and the ability of the monomeric fusion protein or the reaction products to bind to a target receptor on a cell surface, with subsequent cell signaling. FIG. 3A shows a BPXTEN fusion protein (101) in which a BP (103) and an XTEN (102) are linked by spacer sequences that contain a cleavable sequence (104), the latter being susceptible to MMP-13 protease (105). FIG. 3B shows the reaction products of a free BP, spacer sequence and XTEN. FIG. 3C shows the interaction of the reaction product free BP (103) or BPXTEN fusion protein (101) with target receptors (106) to BP on a cell surface (107). In this case, desired binding to the receptor is exhibited when BP has a free C-terminus, as evidenced by the binding of free BP (103) to the receptor while uncleaved fusion protein does not bind tightly to the receptor. FIG. 3D shows that the free BP (103). with high binding affinity, remains bound to the receptor (106), while an intact BPXTEN (101) is released from the receptor. FIG. 3E shows the bound BP has been internalized into an endosome (108) within the cell (107), illustrating receptor-mediated clearance of the bound BP and triggering cell signaling (109), portrayed as stippled cytoplasm.

[0019] FIG. 4 is a schematic flowchart of representative steps in the assembly, production and the evaluation of a XTEN

[0020] FIG. 5 is a schematic flowchart of representative steps in the assembly of a BP-XTEN polynucleotide construct encoding a fusion protein. Individual oligonucleotides 501 are annealed into sequence motifs 502 such as a 12 amino acid motif (“12-mer”), which is subsequently ligated with an oligo containing Bbsl, and Kpnl restriction sites 503. Additional sequence motifs from a library are annealed to the 12-mer until the desired length of the XTEN gene 504 is achieved. The XTEN gene is cloned into a stuffer vector. The vector encodes a Flag sequence 506 followed by a stopper sequence that is flanked by Bsal, Bbsl, and Kpnl sites 507 and a cytokine gene 508, resulting in the gene 500 encoding a BP-XTEN fusion for incorporation into a BPXTEN combination.

[0021] FIG. 6 is a schematic flowchart of representative steps in the assembly of a gene encoding fusion protein comprising a biologically active protein (BP) and X TEN, its expression and recovery as a fusion protein, and its evaluation as a candidate BPX TEN product.

[0022] FIG. 7 illustrates the structural configuration of an exemplified XTENylated cytokine (ic. a “XTENylated IL12” construct), having an amino acid sequence of SEQ ID NO: 2 (see Table B). The exemplified “XTENylated TL.12” construct comprises a cleavage sequence capable of being cleaved by a mammalian protease. Upon the protease cleavage of the exemplified “XTENylated IL12"” construct. a corresponding “de-XTENylated IL12” fragment and an “XTEN fragment” are released. Also illustrated is a reference cytokine construct {i.e. a “Reference IL12” construct), having an amino acid sequence of SEQ [D NO: 4 (see Table B), which contains the same IL 12 moiety,

[0023] FIG. 8 illustrates reduced cytokine activity due to XTENylation. For example, an XTENylated (masked) interleukin-12 (1L12) composition (SEQ ID NO: 2) is at least 2-fold less active in inducing signal transducer and activator of transcription 4 (STAT-4) in 293 HEK IL-12 reporter cells relative to the corresponding protease-activated, de-X TENylated (unmasked) IL-12 composition. The protease treatment to de-XTENylate an XTENylated cytokine composition is illustrated in FIG. 7. The EC50 of the XTENvlated 1L12 (having a value of 167.0) is greater than the EC30 of the corresponding de-XTENylated IL 12 (having a value of 79.4), indicating the masking ability of XTEN on IL12 proteins and, more generally, on cytokines.

[0024] F1G. 9A-FIG. 9B illustrate XTENylation-mediated reduction in cytokine binding. For example, FIG. 9A illustrates binding of an “XTENylated IL12” composition (SEQ ID NO: 2) and a “Reference IL12” composition without XTENylation (SEQ TD NO: 4) to 293 HEK-TL-12 reporter cells (HEK-Blue™ IL-12 cells (Invivogen. San Diego, CA)). The ECS0 of the “XTENylated 1L12” (having a value of ~11.8) is greater than the EC50 of the “Reference IL12" (having a value of ~4.5), indicating the ability (i.e. the masking effect) of an XTEN in interfering with the binding between the IL12 and the corresponding IL12 receptor. FIG. 9B illustrates the lack of binding of the “XTENylated [L12"” and the “Reference [L12"” compositions with [L12 receptor negative 293 HEK cells (control). As a further control, no binding was observed for the corresponding XTEN fragment (see FIG. 7) with either the 1112 reporter cells or the IL12-negative control cells.

[0025] FIG. 10A-10C. TL12-XPAC-4X structure and activity assays. FIG. 10A shows schematic structure of an exemplary [L12-XPAC-4X in which there are 4 XTEN chains on the 1L-12 subunits. FIG. 10B shows schematic of IL12-XPAC-4X shown in FIG. 10A in which a transglutaminase tag (TG) tag is added. The TG tag is shown by the arrow. FIG. 10C HEK Blue activity assay for the PAC and XPACs of the two constructs from FIG. 10A and FIG.108B

[0026] FIG. 11A-11C. All XTENs mask activity. FIG. 11A shows activity with an exemplary construct that contains four XTEN moieties (AP2446). FIG. 11B shows activity with an exemplary construct that contains three XTEN moieties {(AP2447). FIG. 11C shows activity with an exemplary construct that contains one XTEN moiety (AP2450).

[0027] FIG. 12A-12C. Design of three exemplary TL12-XPAC-4X constructs. FIG. 12A design of AC2582 / AC2585. FIG. 12B design of AC3244 / AC3247. FIG. 12C design of AC3245 / AC3246.

[0028] FIG. 13 shows schematic of an exemplary XPAC further comprising a tumor binding domain.

[0029] FIG. 14 shows tumor regression results from an in vivo efficacy study performed in C57 / Blk6 mice bearing MC38 tumors. Once established the tumors were treated with either diluent, rIL-12 at three different concentrations or IL-12-XPAC at two different concentrations. The data shown support the efficacy of 1L-12 XPACs in producing tumor regression.

[0030] FIG. 15A shows the toxicity / body weight data obtained from the tumor-bearing mouse study shown in Figure 14. FIG 15B shows the effects of 11.12 and IL.12 XPAC on the body weight of non-tumor bearing mice. These data demonstrate XPAC safety. DETAILED DESCRIPTION

[0031] While cytokines have potential to be potent therapeutics, even at low concentrations, these agents produce side effects that limit their practical application in a clinical setting. The present disclosure hamesses the therapeutic potential of cytokine-related compositions and related methods while controlling the deleterious effects of those powerful compounds. More specifically, the present disclosure relates to specific BPXTEN molecules known as Xtenylated Protease Activated Cytokines (XPACs) that are conditionally activated in the presence of proteases present in the tumor microenvironment. The present application is directed to methods and compositions for the preparation of XPACs. While the present disclosure presents certain examples with IL12. it should be understood that this disclosure is broadly applicable to any cytokine whose activity should preferably be attenuated until such a time that it is presented at the site of action. XPACs provide an effective method for overcoming tumor-induced immune suppression that can result from the role of IL12 in T- and NK-cell-mediated inflammatory responses.

[0032] As noted above, cytokines are potent immune agonists, however, the relatively narrow therapeutic window of this powerful class of compounds has limited their promise in a therapeutic setting. They have a short half-life, are extremely potent. and produce significant undesirable systemic effects and toxicities. In addition, the therapeutic window was further narrowed by the need to administer large quantities of cytokine in order to achieve the desired levels of cytokine at the intended site of cytokine action in the tumor or tumor microenvironment. As such, cytokines have until now failed to reach their potential in the clinical setting for the treatment of tumors.

[0033] The present invention overcomes the toxicity and short half-life shortcomings that have hampered the clinical use of cytokines in oncology. The XPACs of the present invention contain cytokine polypeptides that have receptor agonist activity. But in the context of the XPAC, the cytokine receptor agonist activity is attenuated and the circulating half-life is extended. The XPACs include protease cleave sites, which are cleaved by proteases that are associated with a desired site of cytokine activity (e.g. a tumor), and are typically enriched or selectively present at the site of desired activity. Thus, the XPACs are preferentially (or selectively) and efficiently cleaved at the desired site of action. This limits the cytokine activity substantially to the desired site of activity, such as the tumor microenvironment. Protease cleavage at the desired site of activity, such as in a tumor microenvironment, releases a form of the cytokine from the XPAC that is much more active as a cytokine receptor agonist than the XPAC which has the XTEN molecule attached. The form of the cytokine that is released upon cleavage of XTEN from the XPAC typically has a short half-life, which is often substantially similar to the half-life of the naturally occurring cytokine. This advantageously limits the cytokine activity to the tumor microenvironment. Even though the half-life of the XPAC is extended, toxicity is dramatically reduced or climinated because the circulating XPAC is attenuated and active cytokine is targeted to the tumor microenvironment. The XPACs described herein, for the first time, enable the administration of an effective therapeutic dose of a cytokine to treat tumors with the activity of the cytokine substantially limited to the tumor microenvironment, and dramatically reduces or eliminates unwanted systemic effects and toxicity of the cytokine,

[0034] Before the embodiments of the 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 invention. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention.

[0035] Unless otherwise defined, 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 equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Definitions

[0036] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0037] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

[0038] The term "cytokine" is well-known to those of skill in the art and refers to any of a class of immunoregulatory proteins that are secreted by cells especially of the immune system and are immunomodulators. Cytokine polypeptides that can be used in the XPACs disclosed herein include, but are not limited to interleukins, such as IL-1, TL.-1L.alpha,, 11-2, IL-3, IL-4, TL.-$, TL-6, IL-7, IL-8, IL-9, TL-10, IL- UU, TL-12, TL-13, IL-14, 1L-15, IL-16, 1-17, 1-8, IL-2] and TL-25, transforming growth factors, such as TGF- alpha. and TGF-beta. (e.g.. TGFbetal, TGFbeta2, TGFbeta3); interferons, such as interferon-.alpha., interferon- beta., interferon-. gamma., interferon-kappa and interferon-omega; tumor necrosis factors, such as tumor necrosis factor alpha and lymphotoxin; chemokines (¢.g., C--X--C motif chemokine 10 (CXCL10), CCL19, CCL20, CCL21), and granulocyte macrophage-colony stimulating factor (GM-CS), as well as functional fragments thereof that retain receptor agonist activity. "Chemokine" is a term of art that refers to any of a family of small cytokines with the ability to induce directed chemotaxis in nearby responsive cells.

[0039] As used herein, the terms “activatable." "activate," "induce," and “inducible” refer to the ability of a protein, i.e. a cytokine, that is part of a XPAC, to bind its receptor and effectuate activity upon cleavage of the XTEN from the XPAC.

[0040] Those of skill in the art understand the term “half-life extension” is used to mean that as compared to a cytokine that is part of the XPAC, the XPAC that increases the serum half-life and improves pK, for example, by altering its size (e.g. lo be above the kidney filtration cutoff), shape, hydrodynamic radios, charge, or parameters of absorption, biodistribution, metabolism. and elimination.

[0041] 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 non-amino 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 conjugation with a labeling component.

[0042] Asused herein the term “amino acid” refers to either natural and / or unnatural or synthetic amino acids, including but not limited to glycine and both the D or L optical isomers. and amino acid analogs and peptidomimetics. Standard single or three letter codes are used to designate amino acids.

[0043] The term “natural L-amino acid” means the L optical isomer forms of glycine (G), proline (P), alanine (A). valine (V), leucine (L), isoleucine (1), methionine (M), cysteine (C). phenylalanine (F). tyrosine (Y). tryptophan (W). histidine (H), lysine (K), arginine (R), ghitamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D), serine (8), and threonine (T).

[0044] The term “non-naturally 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 wild-type or naturally-occurring sequence found in a mammal. For example, a non-naturally occurring polypeptide 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 suitably aligned.

[0045] 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. etal., Proc Natl Acad Sci US A (1981) 78:3824. 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. 10046] A “fragment” 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” is a protein with sequence homology to the native biologically active protein 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 with the reference biologically active protein. As used herein, the term “biologically active protein moiety” includes proteins modified deliberately, as for example, by site directed mutagenesis, insertions, or accidentally through mutations.

[0047] 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 necessarily 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.

[0048] “Isolated,” when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and / or recovered from a coraponent of its natural environment. Contaminant components of its natural environment are materials that would typically interfere 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 “concentrated”. “separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, 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 ™

[0049] An “isolated” polynucleotide or polypeptide-encoding nucleic acid or other polypeptide-encoding nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide-encoding nucleic acid. An isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells. However, an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained 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.

[0050] A “chimeric” protein contains at least one fusion polypeptide comprising regions in a different position in the sequence than that which occurs in nature. The regions may normally exist in separate proteins and are brought together in the fusion 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 peptide regions are encoded in the desired relationship.

[0051] “Conjugated”, “linked.” “fused.” and “fusion” are used interchangeably herein. These terms refer to the joining together of two more chemical elements 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 “in-frame fusion” refers to the joining of two or more open reading frames (ORFs) to forms a continuous longer ORF, in a manuer 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). The terms “link,” “linked,” and “linking” are used in the broadest sense, and are specifically intended to include both covalent and non-covalent attachment of a moiety of the therapeutic agent to another raoicty of the therapeutic agent in a direct or indirect way. The term “linked directly.” as used herein in the context of a therapeutic agent, generally refers to a structure in which a moiety is connected with or attached to another moiety without an intervening tether. The term “linked indirectly,” as used herein in the context of a therapeutic agent, generally refers to a structure in which a moiety of the therapeutic agent is connected with, or attached to, another moiety of the therapeutic agent via an intervening tether.

[0052] In the context of polypeptides, a “linear sequence” or a “sequence” is an order of arsino 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.

[0053] “Heterologous” means derived from a genotypically distinct entity frors 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 sequence other than the native sequence is a heterologous glycine rich sequence. The term “heterologous” as applied to a polynucleotide, a polypeptide, means that the polynucleotide or polypeptide is derived from a genotypically distinct entity from that of the rest of the entity to which itis being compared.

[0054] The terms “polynucleotides”, “nucleic 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 perform any function, 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 probes, 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 polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0055] 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.

[0056] “Recombinant” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of in vitro cloning, restriction and / or ligation steps, and other procedures that result in a construct that can potentially be expressed in a host cell.

[0057] The terms “gene” or “gene fragment” are used interchangeably herein. They refer to a polynucleotide containing at least onc 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 contains 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. 10058] “Homology” or “homologous” refers to sequence similarity or interchangeability between two or more polynucleotide sequences or two or more polypeptide sequences. When using a program such as BestFit to determine sequence identity, similarity 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, similarity or homology scores. Preferably, polynucleotides that are homologous are those which hybridize under stringent conditions as defined herein and have at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, and even more preferably 99% sequence identity to those sequences.

[0059] 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 expressed. in part. with reference to the tersperature and salt concentration under which the wash step is carried out. Typically, stringent conditions 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, | MNaCl, 1% SDS at 37°C., and three washes for 15 min each in 0.1xSSC / 1% SDS at 60 to 65°C. Alternatively, temperatures of about 65°C, 60°C. 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2x8S8C, with SDS being present at about 0.1%. Such wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point © for the specific sequence at a defined ionic strength and pH. The Tr is the teraperature (under defined ionic strength 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 hybridization are well known and can be found in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual , 2 ed., vol. 1-3, Cold Spring Harbor Press, Plainview N.Y. specifically see volume 2 and chapter 9. Typically, blocking reagents are used to block non- specific hybridization. Such blocking reagents include. for instance, sheared and denatured salmon sperm DNA at about 100-200 ug / ml. Organic solvent, such as formamide at a concentration of about 35-50% v / v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art.

[0060] The terms “percent identity” and “% identity,” as applied (0 polynucleotide sequences, refer to the percentage of residue matches 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 meaningful comparison of the two sequences. Percent identity may be measured over the length of an entire defined polynucleotide sequence, for example, as detined by a particular SEQ ID number, 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 43, at least 60, at least 90, at least 120, at least 150, at least 210 or at least 450 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 describe a length over which percentage identity may be measured.

[0061] “Percent (%) amino acid sequence identity,” with respect to the polypeptide sequences identified herein. is defined as the percentage of amino acid residues in a query sequence that are identical with the amino acid residues of a second, reference polypeptide sequence or a portion thereof, atter aligning the sequences and introducing gaps, if necessary. to achieve the maximuam percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. 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 available 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 maximal alignment over the full length of the sequences being compared. Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, 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 describe a length over which percentage identity may be measured.

[0062] The term “non-repetitivencss” as used herein in the context of a polypeptide refers to a lack or limited degree of internal homology in a peptide or polypeptide sequence. The term “substantially non-repetitive” can mean, for example, that there are few or no instances of four contiguous amino acids in the sequence that are identical amino acid types or that the polypeptide has a subsequence score (defined infra) of 10 or less or that there isn’t a pattern in the order, from N- to C-terminus, of the sequence motifs that constitute the polypeptide sequence. The term “repetitiveness™ as used herein in the context of a polypeptide refers to the degree of internal homology in a peptide or polypeptide sequence. In contrast, a “repetitive” sequence may contain multiple identical copies of short amino acid sequences. For instance, a polypeptide sequence of interest may be divided into n-mer sequences and the number of identical sequences can be counted. Highly repetitive sequences contain a large fraction of identical sequences while non-repetitive sequences contain few identical sequences. In the context of a polypeptide, a sequence can contain multiple copies of shorter sequences of defined or variable length, or motifs, in which the motifs themselves have non-repetitive sequences, rendering the full-length polypeptide substantially non-repetitive. The length of polypeptide within which the non- repetitiveness is measured can vary from 3 amino acids to about 200 amino acids, about from 6 to about 50 amino acids. or from about 9 to about 14 amino acids. “Repetitiveness” used in the context 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.

[0063] A “vector” is a nucleic acid molecule, preferably self-replicating in an appropriate host, which transfers an inserted nucleic acid molecule into and / or between host cells. The term includes vectors that function primarily for insertion of DNA or RNA into a cell, replication of vectors that function primarily for the replication of DNA or RNA, and expression 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 functions. 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 vield a desired expression product.

[0064] “Serum degradation resistance,” as applied to a polypeptide, refers to the ability of the polypeptides to withstand degradation in blood or components thereof, which typically involves proteases in the serum or plasma. The serum degradation resistance can be measured by combining 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), typically at about 37°C. The samples for these time points can be run on a Western blot assay and the protein 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 half-life or “serum half-life” of the protein.

[0065] The term “ti; as used herein means the terminal half-life calculated as n(2) / Kei. Ka is the terminal elimination rate constant calculated by linear regression of the terminal linear portion of the log concentration vs. time curve. Half-life 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. The terms “liz”, “terminal half-life”, “elimination half-life” and “circulating half-life” are used interchangeably herein.

[0066] “Apparent Molecular Weight Factor” or “Apparent Molecular Weight” arc related terms referring to a measure of the relative increase or decrease in apparent molecular weight exhibited by a particular amino acid sequence. The Apparent Molecular Weight is determined using size exclusion chromatography (SEC) and similar methods compared to globular protein standards and is measured in “apparent kD” 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.

[0067] The “hydrodynamic radius” or “Stokes radius” is the effective radius (Ry, in nm) of a molecule 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 proteins correlate with the “Apparent Molecular Weight Factor’, which is a more intuitive measure. The “hydrodynamic radius” of a protein affects its rate of diffusion in aqueous solution as well as its ability to migrate in gels of macromolecules. The hydrodynamic radius of a protein is determined by its molecular 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 U.S. Patent Nos. 6,406,632 and 7,294.513. Most proteins have globular structure. which is the most compact three-dimensional structure a protein can have with the smallest hydrodynamic 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 similar molecular weight.

[0068] “Physiological conditions” refer to a set of conditions in a living host as well as in vitro conditions, including 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. Geuerally, a physiological buffer contains a physiological concentration 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 physiological buffers is listed in Sambrook et al. (1989). Physiologically relevant temperature ranges from about 25°C to about 38°C, and preferably from about 35°C to about 37°C 10069] A “reactive group is a chemical structure that can be coupled to a second reactive group. Examples for reactive groups are amino groups, carboxyl groups, sulfhydryl groups. hydroxyl groups, aldehyde groups, azide groups. Some reactive groups can be activated to facilitate coupling with a second reactive group. Examples for activation are the reaction of a carboxyl group with carbodiimide, the conversion of a carboxyl group into an activated ester, or the conversion of a carboxyl group into an azide function. 10070] “Controlled release agent”. “slow release agent”. “depot formulation” or “sustained release agent” are used interchangeably to refer to an agent capable of extending the duration of release of a polypeptide of the invention relative (0 the duration of release when the polypeptide is administered in the absence of agent. Different embodiments of the present invention may have different release rates, resulting in different therapeutic amounts.

[0071] The terms “antigen”, “target antigen” or “immunogen” 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.

[0072] The ters “payload” as used herein refers to a protein or peptide sequence that has biological or therapeutic activity; the counterpart to the pharmacophore of small molecules. Examples of payloads include. but are not limited to, cytokines, enzymes, hormones and blood and growth factors. Payloads can further comprise genetically fused or chemically conjugated moieties such as chemotherapeutic agents, antiviral compounds, toxins, or contrast agents. These conjugated moieties can be joined to the rest of the polypeptide via a linker which may be cleavable or non-cleavable.

[0073] The term “antagonist”, as used herein, includes any molecule that partially or fully blocks. inhibits, or neutralizes a biological activity of a native polypeptide disclosed herein. Methods for identifying antagonists of a polypeptide may comprise contacting a native polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the native polypeptide. In the context of the present invention, antagonists may include proteins. nucleic acids. carbohydrates. antibodies or any other molecules that decrease the effect of a biologically active protein.

[0074] 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 [ragments, [ragments or amino acid sequence variants of native polypeptides, 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 normally associated with the native polypeptide.

[0075] “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, wherein “biological activity” refers to an in vitro or in vivo biological function or effect, including but not limited to receptor binding, antagonist activity, agonist activity, or a cellular or physiologic response.

[0076] As used herein, “treatment” or “treating,” or “palliating™ or “ameliorating” is used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and / or a prophylactic bewefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Thus, for example, treatment refers to a method of reducing the effects of a disease or condition or symptom of the disease or condition. Thus, in the disclosed method, treatment can refer to at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or substantially complete reduction in the severity of an established disease or condition or symptom of the disease or condition. For example. a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disease condition such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease or condition, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may vot have been made. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease. condition, or symptoms of the disease or condition.

[0077] A “therapeutic effect”, as used herein, refers to a physiologic effect, including but not limited to the cure, mitigation, amelioration, or prevention of disease or condition in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals, caused by a fusion polypeptide of the invention other than the ability (0 induce the production of an antibody against an antigenic epitope possessed by the biologically active protein. Deternunation of a therapeutically effective amount is well within the capability of those skilled in the art. especially in light of the detailed disclosure provided herein.

[0078] The terms “therapeutically effective amount” and “therapeutically effective dose”, as used herein, refers to an amount of a biologically active protein, 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 repeated doses to a subject. Such effect need not be absolute to be beneficial. The disease or condition can refer to a disorder or a disease.

[0079] The term “therapeutically effective dose regimen”, as used herein, refers to a schedule for consecutively administered doses 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 disease state or condition. 10080] As used herein, the terms “prevent”, "preventing", and “prevention” of a disease or disorder refers to an action, for example, administration of the chimeric polypeptide or nucleic acid sequence encoding the chimeric polypeptide, that occurs before or at about the same time a subject begins to show one or more symptoms of the disease or disorder. which inhibits or delays onset or exacerbation of one or more symptoms of the disease or disorder.

[0081] As used herein, references to "decreasing", "reducing", or "inhibiting" include a change of at least about 10%, of at least about 20%, of at least about 30%, of at least about 40%, of at least about 50%, of at least about 60%, of at least about 70%, of at least about 80%, of at least about 90% or greater as compared to a suitable control level. Such terres can include but do not necessarily include complete elimination of a function or property, such as agonist activity.

[0082] An "attenuated cytokine receptor agonist” is a cytokine receptor agonist that has decreased receptor agonist activity as compared to the cytokine receptors naturally occurring agonist. An attenuated cytokine agonist may have at least about 10 times, at least about 50 times, at least about 100 times, at least about 250 tirses, at least about 500 tires, at least about 1000 times or less agonist activity as compared to the receptor's naturally occurring agonist. When a XPAC that contains a cytokine polypeptide as described herein is described as "attenuated" or having "attenuated activity", it is meant that the XPAC is an attenuated cytokine receptor agonist. General Techniques 10083] The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See Sambrook, J. er al, “Molecular Cloning: A Laboratory Manual,” 3" edition. Cold Spring Harbor Laboratory Press. 2001: “Current protocols in molecular biology”, F. M. Ausubel, er al. eds., 1987, the series “Methods in Enzymology.” Academic Press, San Diego, CA; “PCR 2: a practical approach”, M.J. MacPherson, B.D. Hames and G.R. 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.” 1 1® Edition, McGraw- Hill, 2005; and Freshney, RL, “Culture of Animal Cells: A Manual of Basic Technique.” 4™ edition, John Wiley & Sons, Somerset, NJ, 2000, the contents of which are incorporated in their entirety herein by reference. Cytokines for Use in XPACs

[0084] In general, the therapeutic use of cytokines is strongly linted by their systemic toxicity. TNF, for example, was originally discovered for its capacity of inducing the hemorrhagic necrosis of some tumors, and for its in vitro cytotoxic effect on different tumoral lines, but it subsequently proved to have strong pro- inflammatory activity, which can, in case of overproduction conditions, dangerously affect the human body. As the systemic toxicity is a fundamental problem with the use of pharmacologically active amounts of cytokines in humans, novel derivatives and therapeutic strategies are now under evaluation, aimed at reducing the toxic effects of this class of biological effectors while keeping their therapeutic efficacy.

[0085] A preferred cytokine for use in production of XPACs is Interleukin-12 (IL-12). [L-12 is a disulfide- linked heterodimer of two separately encoded subunits (p3S and p40), which are linked covalently to give rise to the so-called bioactive heterodimeric (p70) molecule. Apart from forming heterodimers (11-12 and TL-23), the p40 subunit is also secreted as a monomer (p40) and a homodimer (p40:). It is known in the art that synthesis of the heterodimer as a single chain with a linker connecting the p33 to the p40 subunit preserves the full biological activity of the heterodimer. IL-12 plays a critical role in the early inflammatory response to infection and in the generation of Thi cells, which favor cell-mediated immunity. Tt has been found that overproduction of IL.-12 can be dangerous to the host because it is involved in the pathogenesis of a number of autoimmune inflammatory diseases (e.g. MS, arthritis, tvpe 1 diabetes).

[0086] The 11-12 receptor (IL-12R) is a heterodimeric complex consisting of IL-12RB 1 and 1L- [2RB2 chains expressed on the surface of activated T-cells and natural killer cells. The IL-12RB1 chain binds to the IL-12p40 subunit, whereas IL-12p35 in association with IL-12RP2 confers an intracellular signaling ability. Signal transduction through I1.-12R induces phosphorylation of Janus kinase (Jak2) and tyrosine kinase (Tyk?2), that phosphorylate and activate signal transducer and activator of transcription (STAT)L, STATS, STAT4. and STATS. The specific cellular effects of IL-12 are due mainly to activation of STAT4. [L-12 induces natural killer and T-cells to produce cytokines, in particular interferon (IFN)y, that mediate many of the proinflamruatory activities of 1L-12, including CD4+ T-cell differentiation toward the Th1 phenotype.

[0087] IL-2 exerts both stimulatory and regulatory functions in the immune system and is, along with other members of the common y-chain cytokine family, central to immune homeostasis. 11.-2 mediates its action by binding to IL-2 receptors (IL-2R), consisting of either trimeric receptors made of TL-2Ra (CD25), IL-2Rp (CD122). and 1L-2R~ (y-c. CD132) chains or dimeric § v IL-2Rs. Both IL-2R variants are able to transmit signal upon TL-2 binding. However, trimeric apy IL-2Rs have a roughly 10-100 times higher affinity for TL-2 than dimeric Py IL-2Rs (3), irplicating that CD25 confers high-affinity binding of IL-2 to its receptor but is not crucial for signal transduction. Trimeric I1.-2Rs are found on activated T cells and CD4+ forkhead box P3 (FoxP3)+ T regulatory cells (Treg), which are sensitive to IL-2 in vitro and in vivo. Conversely, antigen- experienced (memory) CD8+, CD44 high memory-phenotype (MP) CD8+, and natural killer (NK) cells are endowed with high levels of dimeric fv IL-2Rs, and these cells also respond vigorously to fL-2 in vitro and in VIVO.

[0088] Expression of the high-affinity IL-2R is critical for endowing T cells to respond to low concentrations of [L-2 that is transiently available in vivo. IL-2Re expression is absent on naive and memory T cells but is induced after antigen activation. IL-2R is constitutively expressed by NK, NKT, and memory CD8+ T cells but is also induced on naive T cells after antigen activation. -chain is much less stringently regulated and is constitutively expressed by all lymphoid cells. Once the high-affinity IL-2R is induced by antigen, IL-2R signaling upregulates the expression of TL-2Ra in part through StatS-dependent regulation of Il2ra transcription. This process represents a mechanism to maintain expression of the high-affinity [L-2R and sustain TL-2 signaling while there remains a source of TL-2,

[0089] Interleukin-15 (IL-15), another member of the 4-alpha-helix bundle family of cytokines, has also emerged as an immunomodulator for the treatment of cancer. [L-15 is initially captured via IL- I3Ra, which is expressed on antigen-presenting dendritic cells, monocytes and macrophages. IL-15 exhibits broad activity and induces the differentiation and proliferation of T, B and natural killer (NK) cells via signaling through the TL-15 / IL-2-R~f (CD122) and the common v chain (C132). Tt also enhances cytolytic activity of CD8+ T cells and induces long-lasting antigen-experienced CD8+CD44 memory T cells. JL-15 stimulates differentiation and immunoglobulin synthesis by B cells and induces maturation of dendritic cells. Tt does not stimulate mmunosuppressive T regulatory cells (Tregs). Thus, boosting TL-15 activity selectively in the tumor micro- environment could enhance innate and specific immunity and fight tumors.

[0090] Interleukin-7 (IL-7), also of the 1L-2 / IL-15 family, is a well-characterized pleiotropic cytokine, and is expressed by stromal cells, epithelial cells, endothelial cells, fibroblasts, smooth muscle cells and keratinocytes, and following activation, by dendritic cells (Alpdogan et al., 2005). Although it was originally described as a growth and differentiation factor for precursor B lymphocytes, subsequent studies have shown that IL-7 is critically involved in T-lvmphocyte development and differentiation. Interleukin-7 signaling is essential for optimal CD8 T-cell function, homeostasis and establishment of memory (Schluns et al., 2000); it is required for the survival of most T-cell subsets, and its expression has been proposed to be important for regulating T- cell numbers.

[0091] IL-7 has a potential role in enbancing immune reconstitution in cancer patients following cytotoxic chemotherapy. IL-7 therapy enhances immune reconstitution and can augment even limited thymic function by facilitating peripheral expansion of even small numbers of recent thymic emigrants. Therefore, IL-7 therapy could potentially repair the immune system of patients who have been depleted by cytotoxic chemotherapy and may be an attractive candidate for XPAC production.

[0092] Regulatory T cells actively suppress activation of the immune system and prevent pathological self reactivity and consequent autoimmune disease. Developing drugs and methods to selectively activate regulatory T cells for the treatment of autoimmune disease is the subject of intense research and, until the development of the present invention, which can selectively deliver active interleukins at the site of inflammation, has been largely unsuccessful. Regulatory T cells (Treg) are a class of CD4+CD235+ T cells that suppress the activity of other immune cells. Treg are central to immune system homeostasis, and play a major role in maintaining tolerance to self-antigens and in modulating the immune response to foreign antigens Multiple autoimmune and inflammatory diseases, including Type 1 Diabetes (T1D), Systemic Lupus Erythematosus (SLE), and Graft-versus-Host Disease (GVHD) have been shown to have a deficiency of Treg cell numbers or Treg function. 10093] As such, there is great interest in the development of therapies that boost the numbers and / or function of Treg cells. One approach is treatment with low dose Interleukin-2 (IL-2). Treg cells characteristically express high constitative levels of the high affinity IL-2 receptor, IL2Rafy which is composed of the subunits IL2Ra (CD25), TL2RP (CD122), and IL2Ry (CD 132), and Treg cell growth has been shown to be dependent oniL-2. Conversely, immune activation has also been achieved using IL-2, and recombinant IL-2 (Proleukin®) has been approved to treat certain cancers. High-dose TL-2 is used for the treatment of patients with metastatic melanoma and metastatic renal cell carcinoma with a long-ternt impact on overall survival.

[0094] Clinical trials of low-dose 1L-2 treatment of chronic GVHD and HCV-associated autoimmune vasculitis patients demonstrated increased Treg levels and signs of clinical efficacy. The rationale for using so-called low dose IL-2 was to exploit the high IL-2 affinity of the trimeric IL-2 receptor which is constitutively expressed on Tregs while leaving other T cells which do not express the high affinity receptor in the inactivated state. Proleukin® (Prometheus Laboratories, San Diego, Calif), the recombinant form of IL-2 used in these trials, is associated with high toxicity. Aldesleukin, at high doses, is approved for the treatment of metastatic melanoma and metastatic renal cancer, but its side effects are so severe that its use is only recommended in a hospital setting with access (0 intensive care.

[0095] The clinical trials of IL-2 in autoiramune diseases have employed Jower doses of 11-2 in order to target Treg cells, because Treg cells respond to lower concentrations of IL-2 than many other immune cell types due to their expression of IL2R alpha. However, even these lower doses resulted in safety and tolerability issues, and the treatments used have employed daily subcutaneous injections, either chronically or in intermittent 5- day treatment courses. Therefore, there is a need for an autoinunune disease therapy that potentiates Treg cell numbers and function, that targets Treg cells more specifically than [1.-2, that is safer and more tolerable, and that is administered less frequently, This low therapeutic window for IL-2 is played out across other cytokine therapies.

[0096] One approach for improving the therapeutic index of eytokine-based therapy for autoimmune diseases was to usc variants of 1L.-2 that are selective for Treg cells relative to other rumune cells. IL-2 receptors are expressed on a variety of different immune cell types, including T cells, NK cells, eosinophils, and monocytes, and this broad expression pattern likely contributes to its pleiotropic effect on the immune system and high systemic toxicity. In particular, activated T effector cells express [L2Rapy, as do pulmonary epithelial cells. But, activating T effector cells runs directly counter to the goal of down-modulating and controlling an immune response, and activating pulmonary epithelial cells leads to known dose-limiting side effects of 1L-2 including pulmonary edema. In fact, the major side effect of high-dose IL-2 immunotherapy is vascular leak syndrome (VLS), which leads to accumulation of intravascular fluid in organs such as lungs and liver with subsequent pulmonary edema and liver cell damage. There is no treatment of VLS other than withdrawal of IL-2. Low- dose IL-2 regimens have been tested in patients to avoid VLS, however, at the expense of suboptimal therapeutic results.

[0097] Treatment with interleukin cytokines other than IL-2 has been even more limited. IL-15 displays immune cell stimulatory activity similar to that of IL-2 but without the same inhibitory effects, thus making it a promising immunotherapeutic candidate. Clinical trials of recombinant human [L-15 for the treatment of metastatic malignant melanoma or renal cell cancer demonstrated appreciable changes in immune cell distribution, proliferation, and activation and suggested potential antitumor activity. IL-15 therapy is known to be associated with undesired and toxic effects, such as exacerbating certain leukemias, grafi-versus-host disease, hypotension, thrombocytopenia, and liver injury.

[0098] IL-7 promotes lymphocyte development in the thymus and maintains survival of naive and memory T cell homeostasis in the periphery. Moreover, it is important for the organogenesis of lymph nodes (LN) and for the maintenance of activated T cells recruited into the secondary lvmphoid organs (SLOs). In clinical trials of IL-7, patients receiving IL-7 showed increases in both CD4+ and CD8+ T cells, with no significant increase in regulatory T cell numbers as monitored by FoxP3 expression, In clinical trials reported in 2006, 2008 and 2010, patients with different kinds of cancers such as metastatic melanoma or sarcoma were injected subcutaneously with different doses of IL-7. Little toxicity was seen except for transient fevers and mild erythema. Circulating levels of both CD4+ and CD8+ T cells increased significantly and the number of Treg reduced. TCR repertoire diversity increased after JL-7 therapy. However, the anti-tumor activity of IL-7 was not well evaluated. Results suggest that [1-7 therapy could enhance and broaden immune responses. 10099] IL-12 is a pleiotropic cytokine, that creates an interconnection between the innate and adaptive immunity. IL-12 was first described as a factor secreted from PMA-induced EBV-transformed B-cell lines. Based on its actions, TL-12 has been designated as cviotosic lymphocyte maturation factor and natural killer cell stimulatory factor. Due to bridging the innate and adaptive immunity and potently stimulating the production of IFNgamma.. a cytokine coordinating natural mechanisms of anticancer defense, [L-12 seemed ideal candidate for tumor immunotherapy in humans. However, severe side effects associated with systemic administration of TL.-12 in clinical investigations and the very narrow therapeutic index of this cytokine markedly hampered the use of this cytokine in cancer patients. Approaches to [L-12 therapy in which delivery of the cytokine is tumor-targeted, which may diminish some of the previous issues with 1L-12 therapy. are currently in clinical trials for cancers.

[0100] The direct use of IL-2 as an agonist to bind the IL-2R and modulate immune responses therapeutically has been problematic due its well-documented therapeutic risks, e.g., its short serum half-life and high toxicity. These risks have also limited the therapeutic development and use of other cytokines. New forms of cytokines that reduce these risks are needed. Disclosed herein are compositions and methods comprising conditionally active IL-12 and other cytokines designed to address the risks associated with conventional cytokine therapy and provide much needed immunomodulatory therapeutics.

[0101] Cytokines, including interleukins (e.g., IL-2, TL-7, 11-12, IL-15, IL-18, TL-21 IL-23}, interferons (IFN, including [FNalpha, IFNbeta and 1FNgamma), tumor necrosis factors (e.g, TNFalpha, lymphotoxin). transforming growth factors (e.g., TGFbetall, TGFbeta2, TGFbeta3), chemokines (C-X-C motif chemokine 10 (CXCL10), CCL19, CCL20, CCL21), and granulocyte macrophage-colony stimulating factor (GM-CS) are highly potent when administered to patients. Forming XPACs with these molecules could make them more readily amenable for use in a therapeutic setting.

[0102] As used herein, "chemokine" means a family of small cytokines with the ability to induce directed chemotaxis in nearby responsive cells Cytokines can provide powerful therapy, but are accompanied by undesired effects that are difficult to control clinically and which have limited the clinical use of cytokines. This disclosure relates to new forms of cytokines that can be used in patients with reduced or eliminated undesired effects. Tn particular, this disclosure relates to pharmaceutical compositions including chimeric polypeptides (XPACs), nucleic acids encoding XPACs and pharmaceutical formulations of the foregoing that contain cytokines or active fragments or muteins of cytokines that have decreased cytokine receptor activating activity in comparison to the corresponding cytokine. However, under selected conditions or in a selected biological environment the chimeric polypeptides activate their cognate receptors, often with the same or higher potency as the corresponding naturally occurring cytokine. As described herein, this is typically achieved using a cytokine blocking moiety that blocks or inhibits the receptor activating function of the cytokine, active fragment or mutein thereof under general conditions but not under selected conditions, such as those present at the desired site of cytokine activity (e.g. an inflammatory site or a tumor).

[0103] While the present application is exemplified using IL-12 as the exemplary cytokine, those of skill in the art will understand that the teachings provided herein may readily be adapted for and describe and enable the use of XPACs formed from other cytokines, fragments and muteins, such as IL-2, IL-7, IL-12, IL-15, IL- 18, TL-21 TL-23, [FNalpha, [FNbeta, IFNgamma, TNFalpha, lymphotoxin, TGF-beta, TGFbeta2, TGFbeta3, GM-CSF. CXCL10, CCL19, CCL20, CCL2I and functional fragments or muteins of any of the foregoing.

[0104] Various elements ensure the delivery and activity of the cytokine in the XPACs of the invention preferentially at the site of desired cytokine activity and to severely limit systemic exposure to the cytokine via XTENylation which allows serum half-life extension for the cytokine of interest. In this serurs half-life extension strategy, the XPAC may circulates for extended times (preferentially 1-2 or more weeks) but the activated version from which the XTEN sequence has been cleaved has the typical serum half-life of the cytokine.

[0105] By comparison to an XPAC, the serum half-life of the underlying cytokine administered intravenously is only about 10 minutes due to distribution into the total body extracellular space. Subsequently, the cytokine is metabolized by the kidneys with a half-life of 2.5 hours.

[0106] In some embodiments of this invention. the XPAC comprises a release segment which is cleaved at the site of action {e.g., by inflammation-specific or tumor-specific proteases) thereby releasing the cytokines full activity at the desired site and also separating it from the half-life extension of the uncleaved (XPAC) version. In such embodiments, the fully active and free cytokine would have very different pharmacokinetic (pK) properties--a half-life of hours instead of weeks. In addition, exposure to active cytokine is limited to the site of desired cytokine activity {e.g., an inflammatory site or the tumor microenvironment) and systemic exposure to active cytokine, and associated toxicity and side effects, are reduced.

[0107] Creating XPACs from cytokines is an elegant mechanism by which to improve the use of cytokines, as immunostimulatory agents, for example for treating cancer. For example, in this aspect, the pharmacokinetics and / or pharmacodynamics of the cytokine (e.g., IL-2, [L-7, IL-12, IL-15, IL-18, [L-21 IL- 23, IFNalpha, [FNbeta and IFNgamma, TNFalpha, lymphotoxin, TGFbetal, TGFbeta2, TGFbeta3 GM-CSF, CXCL10, CCL19, CCL20, and CCL21 can be tailored to maximally activate effector cells (e.g., effect T cells, NK cells) and / or cytotoxic immune response promoting cells (e.g., induce dendritic cell maturation) at a site of desired activity, such as in a tumor or tumor micrognyironment, but preferably not systemically.

[0108] Thus, provided herein are pharmaceutical compositions comprising XPAces that are comprised of at least one cytokine polypeptide, such as interleukins (e.g, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, [L-23), interferons (IFN, including IFNalpha, IFNbeta and TFNgamma), tumor necrosis factors (e.g., TNFalpha, lymphotoxin). transforming growth factors (e.g., TGFbetal, TGFbeta2, TGFbeta3), chemokines (e.g. CXCL10, CCL19, CCL20, CCL21) and granulocyte macrophage-colony stimulating factor (GM-CS) or a functional fragment or mutein of any of the foregoing.

[0109] Preferably, the cytokine polypeptides (including functional fragments) that are included in the XPACs disclosed herein are not mutated or engineered to alter the properties of the naturally occurring cytokine, including receptor binding affinity and specificity or serum half-life. However, changes in amino acid sequence from naturally occurring (including wild type) cytokine are acceptable to facilitate cloning and to achieve desired expression levels. Extended Recombinant Polypeptides

[0110] The present invention provides compositions comprising extended recombinant polypeptides (“XTEN™ or “XTENs"). In some embodiments, XTEN are generally extended length polypeptides with non-naturally occurring. substantially non-repetitive sequences that are composed mainly of small hydrophilic amino acids, with the sequence having a low degree or no secondary or tertiary structure under physiologic conditions.

[0111] In one aspect of the invention, XTEN polypeptide compositions are disclosed that are useful as fusion partners that can be linked to biologically active proteins (“BP”), resulting in a BPX TEN fusion proteins (e.g, monomeric fusions). XTENs can have utility as fusion protein partners in that they can confer certain chemical and pharmaceutical properties when linked to a biologically active protein to a create a fusion protein. Such desirable properties include but are not limited to enhanced pharmacokinetic parameters and solubility characteristics, amongst other properties described below. Such fusion protein compositions may have utility to treat certain diseases, disorders or conditions, as described herein. As used herein, “XTEN" specifically excludes antibodies or antibody fragments such as single-chain antibodies, Fe fragments of a light chain or a heavy chain.

[0112] In some embodiments, XTEN are long polypeptides having greater than about 100 to about 3000 amino acid residues, preferably greater than 400 to about 3000 residues when used as a single sequence, and cumulatively have greater than about 400 to about 3000 amino acid residues when more thas one XTEN unit is used in a single fusion protein or conjugate. In other cases, where an increase in half-life of the fusion protein is not needed but where an increase in solubility or other physico / chemical property for the biologically active protein fusion partner is desired, an XTEN sequence shorter than 100 amino acid residues, such as about 96, or about 84, or about 72, or about 60, or about 48, or about 36 amino acid residues may be incorporated into a fusion protein composition with the BP to effect the property.

[0113] The selection criteria for the XTEN to be linked to the biologically active proteins to create the inventive fusion proteins generally relate to attributes of physical / chemical properties and conformational structure of the XTEN that can be, in turn, used to confer enhanced pharmaceutical and pharmacokinetic properties to the fusion proteins. The XTEN of the present invention may exhibit one or more of the following advantageous properties: conformational flexibility, enhanced aqueous solubility, high degree of protease resistance, low immunogenicity, low binding to mammalian receptors, and increased hydrodynamic (or Stokes) radii; properties that can make them particularly useful as fusion protein partners. Non-limiting examples of the properties of the fusion proteins comprising BP that may be enhanced by XTEN include increases in the overall solubility and / or metabolic stability, reduced susceptibility to proteolysis, reduced immunogenicity, reduced rate of absorption when administered subcutancously or intramuscularly, and enhanced pharmacokinetic properties such as terminal half-life and area under the curve (AUC), slower absorption after subcutaneous or intramuscular injection (compared to BP not linked to XTEN) such that the Cra 1s lower, which may, in turn, result in reductions in adverse effects of the BP that, collectively, can result in an increased period of time that a fusion protein of a BPXTEN composition administered to a subject remains within a therapeutic window, compared to the corresponding BP component not linked to XTEN,

[0114] A variety of methods and assays are known in the art for determining the physical / chemical properties of proteins such as the fusion protein compositions comprising the inventive XTEN: properties such as secondary or tertiary structure, solubility, protein aggregation, melting properties. contamination and water content. Such methods include analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion, HPLC-reverse phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, HPLC-ion exchange. HPLC-size exclusion, IR, NMR, Raman spectroscopy. refractometry. and UV / Visible spectroscopy. Additional methods are disclosed in Arnau et al. Prot Expr and Purif (2006) 48, 1-13. Application of these methods to the invention would be within the grasp of a person skilled in the art.

[0115] Typically, the XTEN component of the fusion proteins are designed to behave like denatured peptide 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 conformational freedom of the peptide backbone. Most peptides and proteins adopt a denatured conformation 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 cases, the invention provides XTEN sequences that, under physiologic conditions, can resemble denatured sequences largely devoid in secondary structure. In other cases, the XTEN sequences can be 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 sequence do not contribute to secondary structure, as measured or determined by the means described herein.

[0116] A variety of methods have been established iu the art to discern the presence or absence of secondary and tertiary structures in a given polypeptide. 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 alpha-helix and beta-sheet, each give rise to a characteristic shape and magnitude of CD spectra. Secondary structure can also be predicted for a polypeptide sequence via certain computer programs or algorithms, such as the well-known Chou-Fasman algorithm (Chou, P. Y.. er af. (1974) Biochemistry, 13: 222-43) and the Garnier-Osguthorpe-Robson (“GOR”) algorithm (Garnier J, Gibrat JF, Robson B. (1996). GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540-553), as described in US Patent Application Publication No. 20030228309A1. For a given sequence, the algorithms can predict whether there exists some or no secondary structure at all, expressed as the total and / or percentage of residues of the sequence that form, for example, alpha-helices or beta-sheets or the percentage of residues of the sequence predicted to result in random coil formation (which lacks secondary structure).

[0117] In some cases, the XTEN sequences used in the inventive fusion protein compositions can have an alpha-helix percentage ranging from 0% to less than about 5% as determined by a Chou-Fasman algorithm. In other cases, the XTEN sequences of the fusion protein compositions can have a beta-sheet percentage ranging from 0% to less than about 5% as determined by a Chou-Fasman algorithm. Tn some cases, the XTEN sequences of the fusion protein compositions can 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 $% as determined by a Chou-Fasman algorithm. In preferred embodiments, the XTEN sequences of the fusion protein compositions will have an alpha-helix percentage less than about 2% and a beta-sheet percentage less than about 2%. In other cases, the XTEN sequences of the fusion protein compositions can have a high degree of random coil percentage, as determined by a GOR algorithm. ln some embodiments, an XTEN sequence can have at least about 80%, more preferably 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 preferably 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 a GOR algorithm. Non-Repetitive Sequences

[0118] XTEN sequences of the subject compositions can be substantially non-repetitive. In general, repetitive amino acid sequences have a tendency to aggregate or form higher order structures, as exemplified by natural repetitive sequences such as collagens and leucine zippers, or 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 be likely to aggregate if the sequences were otherwise repetitive. Typically, the BPXTEN fusion proteins comprise XTEN sequences of greater than about 100 to about 3000 amino acid residues, preferably greater than 400 to about 3000 residues, wherein the sequences are substantially non-repetitive. In one embodiment, the XTEN sequences can have greater than about 100 to about 3000 amino acid residues, preferably greater than 400 to about 3000 amino acid residues, in which no three contignous amino acids in the sequence are identical amino acid types unless the amino acid is serine, in which case no more than three contiguous amino acids are serine residues. In the foregoing embodiment, the XTEN sequence would be substantially non-repetitive. 10119] 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. Repetitiveness in a polypeptide sequence can, for example, be assessed by determining the number of times shorter sequences of a given length occur within the polypeptide. For example, a polypeptide of 200 amino acid residues has 192 overlapping 9-amino acid sequences (or 9-mer “frames™) and 198 3-mer frames, but the number of unique 9-mer or 3-mer sequences will depend on the amount of repetitiveness within the sequence. A score can be generated (hereinafter “subsequence score”) that is reflective of the degree of repetitiveness of the subsequences in the overall polypeptide sequence. In the context of the present invention, “subsequence score” means the sum. of occurrences of each unique 3-mer frame across a 200 consecutive amino acid sequence of the polypeptide divided by the absolute number of unique 3-mer subsequences within the 200 amino acid sequence. In some embodiments, the present invention provides BPXTEN each comprising XTEN in which the XTEN can have a subsequence 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 preferably less than 6, and most preferably less than 3. To the embodirsents hereinabove described in this paragraph, an XTEN with a subsequence score less than about 10 (e.g. 9, 8. 7, etc.) would be “substantially non-repetitive.”

[0120] The non-repetitive characteristic of XTEN can impart to fusion proteins with BP(s) a greater degree of solubility and less tendency to aggregate compared to polypeptides having repetitive sequences. These properties can facilitate the formulation of XTEN-comprising pharmaceutical preparations containing extremely high drug concentrations, in some cases exceeding 100 mg / ml.

[0121] Furthermore, 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 composed of short, repeated motifs largely limited to three amino acids, such as glycine, serine and glutamate, may result in relatively high antibody 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 repeated 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 activation. (Johansson. J., er af. (2007) Vaccine, 25:1676-82 ; Yankai, Z., ez al. (2006) Biochem Biophys Res Commun, 345 :1365-71 : Hsu, C. T., et al. (2000) Cancer Res, 60:3701-3); Bachmann MF, er al. Ear J Immunol. (1995) 25(12):3445-3451). Exemplary Sequence Motifs

[0122] The present invention encompasses XTEN that can comprise multiple units of shorter sequences, or motifs, in which the amino acid sequences of the motifs are non-repetitive. In designing XTEN sequences, it was discovered that the non-repetitive criterion may be met despite the use of a “building block™ approach using a library of sequence motifs that are multimerized to create the XTEN sequences. Thus, while an XTEN sequence may consist of multiple units of as few as four different types of sequence motifs, because the motifs themselves generally consist of non-repetitive amino acid sequences, the overall XTEN sequence is rendered substantially non-repetitive.

[0123] In one embodiment, XTEN can have a non-repetitive sequence of greater than about 100 to about 3000 amino acid residues, preferably greater than 400 to about 3000 residues, 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 sequence consists of non-overlapping sequence motifs, wherein each of the motifs has about 9 to 36 amino acid residues. 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% of the XTEN sequence consists of non-overlapping sequence 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% of the XTEN sequence component 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 be composed mainly of small hydrophilic amino acids, such that the overall sequence has an unstructured, flexible characteristic. Examples of amino acids that can be included in XTEN, are, ¢.g., arginine, lysine, threonine, alanine, asparagine, glutamine, aspartate, glutamate, serine, and glycine. As a result of testing variables such as codon optimization, assembly polynucleotides encoding sequence motifs, expression of protein, charge distribution and solubility of expressed protein, and secondary and tertiary structure, it was discovered that XTEN compositions with enhanced characteristics mainly include glycine (G). alanine (A), serine (S), threonine (T), glutamate (E} and proline (P) residues wherein the sequences are designed to be substantially non-repetitive. In a preferred embodiment, XTEN sequences have predominately four to six types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P) that are arranged in a substantially non-repetitive sequence that is greater than about 100 to about 3000 amino acid residues, preferably greater than 400 to about 3000 residues in length. In some embodiments, XTEN can have sequences of greater than about 100 to about 3000 amino acid residues, preferably greater than 400 to about 3000 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 wherein 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 30%. 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), alanine (A). serine (8), 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 30%. 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 consisting of 4 to 6 types of amino acids selected from glycine (G), alanine (A). serine (8S). 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 30%. 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 cach of the motifs has 12 amino acid residues consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein in the content of any one amino acid type in the full-length XTEN does not exceed 30%.

[0124] In still other embodiments, XTENs comprise non-repetitive sequences of greater than about 100 to about 3000 amino acid residues, preferably greater than 400 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 glycine (G). alanine (A), serine (8). threonine (T), glutamate (£) 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 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (8). 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 repeated more than twice in the sequence motif. [nother 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 glycine (G), alanine (A), serine (8), threonine (1), glutamate (E) and proline (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 glycine (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 sequence 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%. In the foregoing embodiments hereinabove described in this paragraph, the XTEN sequences would be substantially non-repetitive.

[0125] In some cases, the invention provides compositions comprising a non-repetitive XTEN sequence of greater than about 100 to about 3000 amino acid residues, preferably greater than 400 to about 3000 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 two or more non-overlapping sequence motifs selected from the amino acid sequences of Table 1. In sore cases. the XTEN comprises non-overlapping sequence motifs in which 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 two or more non- overlapping sequences selected from a single motif family of Table 1, resulting in a “family” sequence in which the overall sequence remains substantially non-repetitive. Accordingly, in these embodiments, an XTEN sequence can comprise multiple units of non-overlapping sequence motifs of the AD motif family, or the AE moti family, or the AF motif family, or the AG motif family, or the AM motif family, or the AQ motif family, or the BC family, or the BI) family of sequences of Table 1. In other cases. the XTEN comprises motif sequences from two or more of the motif families of Table 1.

[0126] In some embodiments, where the composition of this disclosure (for example, a fusion protein) comprises an extended recombinant polypeptide (XTEN), the XTEN can be characterized in that: (3). it comprises at least 12 amino acids; (ii). at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99% or 100% of the amino acid residues of the X TEN sequence are selected from glycine (G). alanine (A). serine (S), threonine (T), glutamate (E) and proline (P); and (iii). it has 4-6 different amino acids selected from G, A, S, T, E and P. In some embodiments, the XTEN sequence can consist of multiple non-overlapping sequence motifs, wherein the sequence motifs are (¢.g., each independently) selected from the sequence motifs of Tables 2a-2b. In some embodiments, the XTEN can have fron 40 to 3,000 amino acids, or from 100 to 3,000 amino acids. The XTEN can (e.g. each independently) have at least (about) 40. at least (about) 30, at least (about) 100, at least (about) 1350, at least (about) 200, at least (about) 300, at least (about) 400, at least (about) 500, at least (about) 600, at least (about) 700, at least (about) 800, at least (about) 900, at least (about) 1,000 amino acids, at least (about) 1,500 amino acids, at least (about) 2,000 amino acids, at least (about) 2,500 antino acids, at least (about) 3,000 amino acids, or a range between any of the foregoing. In some embodiments. the XTEN can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%. at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to a sequence set forth in Tables 2a-2b. Table 1: XTEN Sequence Motifs of 12 Amino Acids and Motif Families *Denotes individual motif sequences that, when used together in various permutations, results in a “family sequence” AD 182 GESPGGSSGSES [AD [ig | GSSESGSSEGGR | ATTY 108 ROAD ANQC AD 185% GSGGEPSESGSS AE. AM 186 GSPAGSPTSTEE AE. AM. AQ 187 GSEPATSGSETP AE AM. AQ 188 i GTSESATPESGP ALE, AM, AU 188 SiaboAl ust AE, AM. AQ GTSTEPSEGSAP AT AN PEI ACTOR ADRNTAD AF, AM 190 GSTSESPSGTAP AF, AM 191 GTSTPESGSASP AF, AM i 192 | GTSPSGESSTAP [ AF, AM 193 GETSSTAESPGE CAG,AM bt 194 PF GTPGSGTASSSP AG, AM 1935 GSSTPSGATGSP Motil Family” | SEQIDNG: ALY ARE 3 {27 LQQADIACTOTAD AG, AM 196 GSSPSASTGTGP AG. AM 197 GASPGTSSTGSP AQ 198 GEPAGSPTSTSE AQ 199 , GTGEPSSTPASE AN i779 Flahlros lhAoh [AQ [200 | GSGPSTESAPTE | ATS ~Nt ORT RANADORT TA AQ 201 GSETPSGPSETA AQ 202 GPSETSTSEPGA AQ 203 GSPSEPTEGTSA BC § {81 . GSGASEPTSTEP nt m——g—— a BC 882 GSEPATSGTEPS ne QOS AMOATDATATDNA BC RR3 GTSEPSTSEPGA BC 884 GTSTEPSEPGSA BD 885 GSTAGSETSTEA "BD ; 886 5 GSETATSGSETA em pnp es TS CSD BD 338 GTSTEASEGSAS

[0127] In those embodiments wherein the XTEN component of the BPXTEN fusion protein has less than 100% of its amino acids consisting of four to six amino acid selected [rom glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P). or less than 100% of the sequence consisting of the sequence motifs of Tables 1 or less than 100% sequence identity with an XTEN from Tables 2a-2b, the other amino acid residues can be selected from any other of the 14 natural L-amino acids. The other amino acids may be interspersed throughout the XTEN sequence, may be located within or between the sequence motifs, or may be concentrated in one or more short streiches of the XTEN sequence. In such cases where the XTEN component of the BPXTEN comprises amino acids other than glycine (G), alanine (A). serine (S), threonine (T), glutamate (E) and proline (P), it is preferred that the amino acids not be hvdrophobic residues and should not substantially confer secondary structure of the XTEN component. Thus, in a preferred embodiment of the foregoing, the XTEN component of the BPXTEN fusion protein comprising other amino acids in addition to glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) would have a sequence with less than 5% of the residues contributing to alpha-helices and beta-sheets as measured by Chou-Fasman algorithm and would have at least 90% random coil formation as measured by GOR algorithm. Length of Sequence

[0128] In a particular feature, the invention encompasses BPXTEN compositions comprising XTEN polypeptides with extended length sequences. The present invention makes use of the discovery that increasing the length of non-repetitive, unstructured polypeptides enhances the unstructured nature of the XTENs and the biological and pharmacokinetic properties of fusion proteins comprising the XTEN. As described more fully in the Examples, proportional increases in the length of the XTEN, even if created by a fixed repeat order of single family sequence motifs (e.g, the four AE motifs of Table 1), can result in a sequence with a higher percentage of random coil formation, as determined by GOR algorithm. compared to shorter XTEN lengths In addition. it was discovered that increasing the length of the unstructured polypeptide fusion partuer can, as described in the Examples, result in a fusion protein with a disproportional increase in terminal half-life compared to fusion proteins with unstructured polypeptide partners with shorter sequence lengths.

[0129] Non-limiting examples of XTEN contemplated for inclusion in the BPXTEN of the invention are presented in Tables 2a-2b. Accordingly, the invention provides BPXTEN compositions wherein the XTEN sequence length of the fusion protein(s) is greater than about 100 to about 3000 amino acid residues, and in some cases is greater than 400 to about 3000 amino acid residues, wherein the XTEN confers enhanced pharmacokinetic properties on the BPXTEN in comparison to pavloads not linked to XTEN. In some cases, the XTEN sequences of the BPXTEN compositions of the present invention can be 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 100 to 150, about 150 to 250, about 250 to 400, 401 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 length. In one embodiment, the BPXTEN can comprise an XTEN sequence wherein the sequence exhibits 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 to a XTEN selected from Tables 2a- 2b. In some cases, the XTEN sequence is designed for optimized expression as the N-terminal component of the BPXTEN. In one embodiment of the foregoing, the XTEN sequence has at least 90% sequence identity to the sequence of AE912 or AM923. In another embodiment of the foregoing, the XTEN has the N-terminal residues described in Examples 14-17.

[0130] In other cases, the BPXTEN fusion protein can comprise a first and a second XTEN sequence, wherein the cumulative total of the residues in the XTEN sequences is greater than about 400 to about 3000 amino acid residues. Tn embodiments of the foregoing, the BPXTEN fusion protein can comprise a first and a second XTEN sequence wherein the sequences each exhibit at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%. 88%, 897%, 90%. 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least a first or additionally a second XTEN selected from Tables 2a-2b. Examples where more than one XTEN is used in a BPXTEN composition include, but are not limited to constructs with an XTEN linked to both the N- and C-termini of at least one BP.

[0131] As described more fully below, the invention provides methods in which the BPXTEN is designed by selecting the length of the XTEN to confer a target half-life on a fusion protein administered to a subject. In some cases, the BPXTEN can be designed by selecting the length of the XTEN to confer a target masking effect on the biological polypeptide for administration to a subject. In general, longer XTEN lengths incorporated into the BPX TEN compositions result in longer half-life corapared to shorter XTEN. However, in another embodiment, BPXTEN fusion proteins can be designed to comprise XTEN with a longer sequence length that is selected to confer slower rates of systemic absorption after subcutaneous or intramuscular administration to a subject. In such cases, the Cra is reduced in comparison to a comparable dose of a BP not linked to XTEN, thereby contributing to the ability to keep the BPXTEN within the therapeutic window for the composition. Thus, the XTEN confers the property of a depot to the administered BPXTEN, in addition to the other physical / chemical properties described herein. Table 2A: Exemplary XTEN Polvpeptides | AF144 205 206 207 | AF504 CR 209 Amino Acid Sequence AE144 204 | GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS EGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGT SESATPESGPGSEPATSGSETPGTSTEPSEGSAP [AF 205 | GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESP SGTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGT em ED Ta ry] AE288 206 | GTSESATPESGPGSEPATSGSE TPGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGTSESATPESGPCTSESATPESGPGSEPATSGSETPGSEPATSGSETRPGSPA GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESG PGTSTEPSEGSAP | AFs04 207 | GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSG ATGSPGSXPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGT PGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSCTASSSPGSSTRSGAT GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSXPSASTGTGPGSSP SASTGTGRPGSSTPSCATGSPGSSTPSGATCSPGASPGTSSTGSPGASPGTSSTGS PGASPGTSSTGSPGTPGSGTASS SPGASPGTSSTGSPGASPGTSSTGSPGASPGT SSTGSPGSSPSASTGIGPETPESGTASSSPGASPGTSSTGSPGASPGTSSTGSPG ASPGTSSTGSPGSSTPSGATGSPGSSTPSCATGSPGASPGTSSTGSPGTRPGSGTA SSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGAS repo for Eo AF540 f 208 | GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTA ESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESS TAPGSTSESPSGTAPGS TSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSCESS TAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTS ESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSAS PGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSES PSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTS TPESGSASPG TSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPS GTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTS PSGESSTAPGSTSSTAESPGPGTS TPESCSASPGSTSESPSGTAR ADAT6 209 | GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGSSESGS SEGGPGSSESGSSECGPGSSESCSSECGPGESPGGSSGSESGSEGSSGPGESSES SESGSSEGEPGSSESGSSEGGPGSSESGSSECGPGSGEEPSESCSSGESPGESSG SESGESPGGSSGSESGSGEEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGE EPSESGSSGSEGSSGPGESSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGS SGSGGEPSESGSSGSSESGSSEGEPGESPCGSSGSESCESPGGSSGSESGESPGE SSGSESGESPGGSSGSESGESPGGSSGSESCSSESGSSEGGPGSGGEPSESGSSG SEGSSGPGESSGSSESGSSEGGPGSCGEPSESGSSGSSESGSSEGGPGSGGEPSE SGSSGESPEGSSGSESCES PEGS SGSESGSSESGSSEGGPESGEEPSESGSSESS ESGSSEGGPGSGGEPSESGSSCSGCEPSESCSSGESPGGSSCSESGSEGSSGPGE SSGSSESGSSEGGPGSEGSSGPGESS 211 | AF576 "OY 213 Amino Acid Sequence AES76 210 | GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS EGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTS TEPSEGSAPGSPAGSPTS TEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSE SATPESGPGSEPATSGSETPGISTERSEGSAPGTS TEPSEGSAPGTSESATPESG PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPCTSESATPESGPGSEPATSGSETPGTSESATPESGRGTS TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTST EEGTSESATPESGPGTSTEPSEGSAP | AF576 211 | GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTA ESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGS TSESPSGTAPGTSPSCESSTARPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESS TAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTS ESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSAS PGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSES PSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPG TSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPS GTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTS PSGESSTAPGSTSSTAESPGPGTS TPESGSASPGSTSESPSGTAPGSTSSTAESP a [ABW 212 | GSSESGSSEGGPGSSESGS SEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGS SGSESGESPGGSSGSESGSSESGSSEGGPGSSESCSSEGGPGSSESGSSEGGRPGE SPGGSSGSESGESPEGSSGSESGESPGGSSCSESGSSESGSSEGGPGSSESGSSE GGPGSSESGSSEGGPGSSESGSSECGPGSSESGSSEGGPGSSESGSSEGGRPGSCE EPSESGSSGESPGGSSCSESCESPGGSSGSESGSGGEPSESGSSGSEGSSGPGES SGSSESGSSEGEPGSGEEPSESGSSGSEGSSCPGESSGSSESGSSEGGPGSGGER SESGSSGESPGGSSGSESGSGOEPSESGSSCSGCEPSESGSSGSSESGSSEGGRG SGGEPSESGSSGSGEEPSESGSSGSEGSSGPGESSGESPGESSGSESGSEGSSGR GESSGSEGSSGPGESSGSGOEPSESGSSESSESGSSEGGPESSESGSSEGGRGES PGGSSGSESGSGEEPSESGSSCSECSSCPGESSCESPGGSSGSESGSEGSSGRES SESGSSEGGPGSGGEPSESGSSGSECSSGPGESSGSEGSSGPGESSGSEGSSGRG ESSGSCGEPSESCSSGSCGEPSESGSSGESPEGSSGSESCESPGESSGSESGSGE EPSESGSSGSEGSSGPGESSCESPGGSSGSESGSSESGSSECGPCSSESGSSEGG PGSSESGSSEGGPGSGEEPSESGSSGSSESGSSEGGPGESPGGSSGSESGSGGER SESGSSGSSESCSSEGGPGESPGESSESESGSGGEPSESGSSGESPGGSSGSESG SGGEPSESGSS AES864 213 | GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS EGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS TEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSE SATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSCSETPGTSESA TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATR ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTST EEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE Le |__| GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSP | 216 Amino Acid Sequence TSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGS EPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG SAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP [AF864 214 | GSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGS TSESPSGTAPGTSTPES GSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGS TSESPSGTAPGTSPSGESS TAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESS TAPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTS ESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSAS PGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPE SGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGRG TSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGPXXXGASASGAP STXXXXSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTARGSTS ESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTA PGSTSSTAESPGRGTSESGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSES PSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPG STSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSESPSGTAPGSTSESPS GTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGTS PSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAESE GPGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSCATGSP AG864 215 | GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSE ATGSPGSSPSASTGTGPGASPGTSSTGSPGTRPGSGTASSSPASSTPSGATGSPGT PGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGRPGSSP SASTGTGPGSSTPSGATGCSPESSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS PGASPGTSSTGSPGTPGSGTASS SPGASPGTSSTGSPGASPGTSSTGSPGASPGT SSTGSPGSSPSASTGIGPGTPGSGTASSSPCASPGTSSTGSPGASPGTSSTGSPG ASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTA SSSPGSSTPSGATGSPGSS TPSGATGSPGSSTPSGATGSPESSPSASTGTGRGAS PGTSSTGSPGASPGTSSTGSPCTPESGTASSSPGASPGTSS TGSPGASPGTSSTG SPGASPGTSSTGSPGASPETSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGS GTASSSPGSSTPSGATGSPGTPGSGTASSSPESSTPSGATGSPGSSTPSGATGSE GSSPSASTGIGPGSSPSASTGTGPCASPGTSSTGSPGTPGSGTASSSPGSSTRSG ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGS STPSGATGSPGSSPSASTGTGPGASPETSSTESPGSSPSASTGTGPGTPGSGTAS SSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSE AMST7S 216 | GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPES GSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGS EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTS TEPSEGSAPGTSESATRE SGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST EPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA PGTSESATPESGRPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTRS GATGSPGTPGSGTASSSPGSSTPSCATGSPGTSTEPSEGSAPGTSTEPSEGSARG SEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAP STGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTS ESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTG PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGRPGSTSST AESPGPCTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPG STSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSE GSAPGTSTEPSEGSAPGSSTPSGATGSPESSPSASTGTIGPGASPGTSSTGSPGSE PATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGT Le |__| GPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP 218 219 6 Amino Acid Sequence AE912 217 | MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGS PTSTEEGTSESATPESGPGTSTEPSEGSAPCSPAGSPTSTEEGTS TEPSEGSAPG TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT STEEGTSESATPESGPGTS TEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPES GPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSES ATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTERS EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTERPSEG SAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSE SATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSERPATSG SETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAP AM923 218 | MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTSTEP SEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPG STSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSG SETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS TEPSEGSAPGTSTEPSEGSAPGSPAGSPISTEEGTSTEPSEGSAPGTSTERPSEGS APGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSES ATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSP GTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS GSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTS ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGS TSSTAESPGPGSTSESPSGT APGTSPSGESSTAPGTPGSGTASSSPESSTPSGATGSPGSSPSASTGTGPGSERA TSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGP GTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTA ESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGT STEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGS ETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGRPGASP GTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP AMI129 219 | GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPES 6 GSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGS EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTS TEPSEGSAPGTSESATRE SGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST EPSEGSAPGTISESATPESGPGTSESATPESGPGTS TEPSEGSAPGTSTEPSEGSA PGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPS GATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPG SEPATSGSETPGSPAGSPTSTEEGSPAGSPTS TEEGTSTEPSEGSAPGPERPTGRA PSGGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEECTSESATPESGPGSPAGSPTS TEEGSPAGSPTSTE EGSTSSTAESPGPGSTSESPSCTAPGTSPSGESSTAPGSTSESPSGTAPGSTSES PSGTAPGTSPSGESSTAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG SEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATR ESGPGTSTEPSEGSAPGTSPSCESSTAPGTSPSGESSTAPGTSPSGESSTAPGTS TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATE SPGSSTPSGATCSPESSTPSGATGSPESSTPSGATGSPGASPGTSSTGSPGASAS GAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESATPESGPG 221 Amino Acid Sequence TSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPGTSS TGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSE PATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSA SPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES ATPESGPGSEPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSP GSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTS STGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP BC 864 220 | GTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATS GTEPSGSEPATSGIEPSGSEPATSGTEPSGSGASEPTS TEPGTSTEPSEPGSAGS EPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSER GSAGTSTEPSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSGA SEPTSTEPGTSEPSTSEPGAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSERGS AGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPAT SGTEPSGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTERG TSTEPSEPGSAGSEPATSGTEPSGSCASEPTSTEPGTSTEPSEPGSAGSGASEPT STEPGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGTS TEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTE PSGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGTSTE PSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTS TEPSEPGSAGTSEPSTSEPGA GSGASEPTSTEPGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGSEPATS GTEPSGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGS EPATSGTEPSGTSEPSTSEPCAGSEPATSGTEPSCSGASERTSTEPGTSTEPSER GSAGSEPATSGTEPSGSGASERTSTERPGTSTEPSEPGSA BDS64 221 | GSETATSGSETAGTSESATSESGAGSTAGSETSTEAGTSESATSESGAGSETATS GSETAGSETATSGSETAGTSTEASEGSASGTSTEASEGSASCTSESATSESGAGS ETATSGSETAGTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSESATSE SGAGSETATSGSETAGTSESATSESCAGTSTEASEGSASGSETATSGSETAGSET ATSGSETAGTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSTEASEGSA SGSETATSGSETAGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESA TSESGAGTSESATSESGAGSETATSCSETAGTSESATSESGAGTSESATSESGAG SETATSGSETAGSETATSGSETAGTS TEASEGSASGSTAGSETSTEAGSETATSG SETAGTSESATSESGAGSTAGSETSTEAGSTAGSETSTEAGSTAGSETSTEAGTS TEASEGSASGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGSTAGSETST EAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGTSES ATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGSETATSGSETA GTSTEASEGSASGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGSETATS GSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGS ETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGSETATSGS Ll. | ETAGTSESATSESGAGTSESATSESGAGSETATSGSETA Table 2B. Exemplary XTEN polypeptides 889 On Fremplany Amine Acid Sequence 889 | C-terminal | PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST | previous XTEN EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGS 8001) ETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTERS EGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPA Ce | TSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTERPSEGS | 8002) 801 8003) 892 © 8004) APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEG SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA TPESGPGESEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG PCTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGTSESATPESGPGL tab TSESATPESGPGSEPATSGPTESGSEPATSGSE TPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGEPEA 890 C-terminal PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST | previous XTEN EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGS 8002) ETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPA TSGSETPGTSESATPESCPGISTEPSECSAPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGISESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGISTEPSEGSAPGTSESATPESCPGSPAGSPTSTEEG SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPCTSESATPESG PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGTSESATPESGPGTSESATPESGPGSEPATSGPTESGSEPATSGSETPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT C-terminal PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST 891 XTEN EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGS | (previously ETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGS Bs PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTERPS EGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGTSTEPSEGSAPCGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGISTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEG SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA TPESGPESEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG PCTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGTSESATPESGPGISESATPESGPGSEPATSGSETPGSEPATSGSETPGS PAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESAT 892 N-terminal ASSPAGSPTSTESGTSESATPESGPGTETEPSEGSAPGTSESATPESGPGSEP | (previously iz ATSGSETPCISESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEG $004) SAPGSPAGSPTSTEEGTSESATPESGPGESPATSGSTPEGTSESATPESGPGS PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT PESGPGISESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSEGSAPGGSAP © 8005) 894 8006) 89% 8007) 806 © 8008) 897 © 8009) 898 8010) 893 | N-terminal | ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP | (previously | XTEN ATSGSETPGTSESATPESGPGSEPATSGSETPCTSESATPESGPGTSTERPSEG 8005) SAPGSPAGSPTSTEEGTSESATPESGPGESPATSGSTPEGTSESATPESGPGS PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT PESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSEGSAPGGSAP 894 | N-terminal | ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSER | (previously | XTEN ATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEG 8006) SAPGSPAGSPTSTEEGTSESATPESGPGEEPATSGSTPEGTSESATPESGRGS PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT PESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE GTSTEPSEGSAPGISTEPSEGSAPGGSAP 895 | N-terminal | ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSER | (previously | XTEN ATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEG 8007) SAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGRGS PAGSPTSTEEGSPAGSPISTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT PESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE frome ED a pe a] 896 | C-terminal | PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST | (previously XTEN EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGS 8008) ETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTS TEPSEGSAPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTERS EGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESCPGSEPATSGSETPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEG SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGTSESATPESCPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS PAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESAT frm eR 897 | C-terminal | PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGTST | (previously | XTEN EPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGTSTEPSEGSAPGTSTEPSEG 8009) SAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS EPATSGSETPCTSESATPESGPGSEPATSGSETPCTSESATPESGPGTS TERS EGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEE GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGSEPATSGSETPCTSESATPESGPGTSTEPSEGSAPGSPAGSPTST EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSP AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATR ESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSARG TESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG 898 | N-terminal | SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE | resiousy XTEN GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSTPAESGSETPGSEPA 8010) TSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS APGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS | TEPSEGSAPGTSESATPESCPGSEPATSGSTETPGISTEPSEGSAPGTSTERS C8011) 900 8012) EGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGP GSEPATSGSETPGTISESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE PATSGSETPGISESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPT STEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG TSESATPESGPGSEPATSGESETPGTSESATPESGPGSEPATSGSETPGTSESA TPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSET PCTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSE SATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGS ETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPS EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTE PSEGSAPGTSTEPSEGSAPGSPAGSPISTEEGTSTEPSEGSAPGTSTEPSEGS APGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTESAS 899 C-terminal SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE | (previously | XTEN GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSERPA 8011) TSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS APGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSESETPGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGISESATPESGPGSPAGSPTSTEEGTSESATPESGPG SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTE EGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPCTSTEPSEGSAPCTSESATPESGPGSPAGSPTS TEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGISTEPSEGSAPCGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSES ATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTS ESATPESCGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSE GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGRG SEPATSGSETPGSEPATSGSTETPGSPAGSPTSTEEGTSESATPESGPGTSTE PSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTATESPEGS APGTSESATPESGPGTSTEPSEGSAPGTSAESATPESGPGSEPATSGSETPGT STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTESAS 900 N-terminal GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE | (previously | XTEN PSEGSAPGTSTEPSEGSAPATSESATPESGPGSEPATSGSETPGSEPATSGSE 8012) TPGSPAGSPTSTEEGTSESASPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP ACSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPATSTEPSEGSAPGTSTEPSEGSAPG TSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPAT SCSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGS PAGSPTSTEEGSPAGSPISTEEGTSESATPESGPGTSTEPSEGSAPGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP 8013) oy 8014) 903 8015) 904 8016) GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES GPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATP fromm CSA a 901 N-terminal | GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS TE | (previously | XTEN PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSESATSGSETPGSEPATSGSE 8013) TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGRPGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPG TSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPAT SGSETPGTSESATPESCPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGS PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGR GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES GPGTSESATPESGPGTSESATPESCPGSEPATSGSETPGSEPATSGSETPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATP fot I 902 | N-terminal | SPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPAT | oreviousy XTEN (with | SCSETPGTSESATPESGPGS TRPAESGSETPGTSESATPESGPGTSTEPSEGSA 8014) | Higtag) PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTS TEEGTS TEPSEGSAPGTSESATPESGPGTSESATRE SCPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT STEPSEGSAPGTSTEPSEGSAPGGSAPR 903 | C-terminal | PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST | (previously | XTEN EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG 8015) SAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS EGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEE GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSP AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATP ESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPG TESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA 904 | C-terminal | TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSET | (previously | XTEN PGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST 8016) EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSP TSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTE PSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS ESATPESGPGTSESATPESGPGSEPATSGSETPGSESATSGSETPGSPAGSPT *._ |__| STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA C8017) 906 8018) o)7 8019) 908 8020) 905 | C-terminal | GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG | (previously | XTEN SPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTST 8017 EEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTS ESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP ESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESCPGSEPATSGSETPG TSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA PGTSESATPESGPGSEPATSGSETPGISESATPESGPGSEPATSGSETPGTSE SATPESGPGTSTEPSEGSAPGTSESASPESGPGSPAGSPTSTEEGSPAGSPTS TEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGRGS EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP 906 | C-terminal | GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP | (previously | XTEN GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTE 8018) PSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESCPGTSTEPSEGSAPGTSE SATPESGPGSEPATSGSTETGTSESATPESGPGSEPATSGSETPGTSESATPE from A PATS 907 | C-terminal | EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP | (previously XTEN GSEPATSGSETPGISTEPSEGSAPGTSTEPSEGSAPGTSESATPESGRPGTSES 8019) ATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES GPGTSTEPSEGSAPGTS TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSARPGTS TEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGRG TSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGS PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET PGTSESATPESGPGSEPATSGSETPGTSESASPESGPGTSTEPSEGSAPGSPA GSPISTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTS TEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT 908 | N-terminal | ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSER | (previously ATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGRGTSTERPSEG 8020) SAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGRGS PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT PESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSEGSAPGGSAP Net Charge

[0132] In other cases, the XTEN polypeptides can have an unstructured characteristic imparted by incorporation of amino acid residues with a net charge and / or reducing the proportion of hydrophobic amiso acids in the XTEN sequence. The overall net charge and net charge density may be controlled by modifving the content of charged amino acids in the XTEN sequences. In some cases, the net charge density of the XTEN of the compositions may be above +0.1 or below -0.1 charges / residue. In other cases, the net charge of a XTEN can be about 0%. about 1%, about 2%, about 3%, about 4%. about 3%, 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.

[0133] Since most tissues and surfaces in a human or animal have a net negative charge, the XTEN sequences can be designed to have a net negative charge to minimize non-specific interactions between the XTEN containing compositions and various surfaces such as blood vessels. healthy tissues, or various receptors. Not to be bound by a particular theory, the XTEN can adopt open conformations due to electrostatic repulsion between individual amino acids of the XTEN polypeptide that individually carry a high net negative charge and that are distributed across the sequence of the XTEN polypeptide. Such a distribution of net negative charge in the extended sequence lengths of XTEN can lead to an unstructured conformation that, in turn, can result in an effective increase in hydrodynamic radius. Accordingly. in one embodiment the invention provides XTEN in which the XTEN sequences contain about 8, 10, 15, 20, 25, or even about 30% glutamic acid. The XTEN of the compositions of the present invention generally have no or a low content of positively charged amino acids. In some cases 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% amino acid residues with a positive charge. However, the invention contemplates constructs where a limited number of amino acids with a positive charge, such as lysine, may be incorporated into XTEN to permit conjugation between the epsilon amine of the lysine and a reactive group on a peptide, a linker bridge, or a reactive group on a drug or small molecule to be conjugated to the XTEN backbone. In the foregoing, a fusion proteins can be constructed that comprises XTEN, a biologically active protein, plus a chemotherapeutic agent useful in the treatment of 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 reactive side chains (e.g., cysteine) incorporated into the XTEN.

[0134] In some cases, an XTEN sequence may comprise charged residues separated by other residues such as serine or glycine. which may lead to better expression or purification behavior. Based on the net charge, XTENs of the subject compositions may have an isoelectric point (pl) 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. [n preferred embodiments, the XTEN will have an isoelectric point between 1.5 and 45. In these embodiments, the XTEN incorporated into the BPXTEN fusion protein compositions of the present invention would carry a net negative charge under physiologic conditions that may contribute to the unstructured conformation and reduced binding of the XTEN component to mammalian proteins and tissues.

[0135] As hydrophobic amino acids can 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 BPXTEN 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 residues with a positive charge, or less than about 7%, or less that about 3%, or less than about 2% amino acid residues with a positive charge, the sum of methionine and tryptophan residues will be less than 2%, and the sum of asparagine and glutamine residues will be less than 10% of the total XTEN sequence. Low Immunogenicity

[0136] Tn another aspect, the invention provides compositions in which the XTEN sequences have a low degree of insmunogenicity or are substantially non-imumunogenic. Several factors can contribute to the low immunogenicity of XTEN. ¢.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 conformational epitopes in the XTEN sequence.

[0137] Conformational epitopes are formed by regions of the protein surface that are composed of multiple discontinuous amino acid sequences of the protein antigen. The precise folding of the protein brings these sequences into a well-defined, stable spatial 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 triggering a cell-mediated immune response. In the latter case, the immune response to a protein in an individual is heavily influenced by T-cell epitope recognition that is a function of the peptide binding specificity of that individuals HLA-DR allotype. Engagement of an 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 activated state within the T-cell. Activation leads to the release of cytokines further activating other lymphocytes such as B cells to produce antibodies or activating T killer cells as a full cellular immune TeSponse.

[0138] The ability of a peptide to bind a given MHC Class 11 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 may be 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 BPXTEN 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 is, in part, a direct result of the conformational flexibility of XTEN sequences; e.g., the lack of secondary structure due to the selection and order of amino acid residues. For example, of particular interest are sequences having a low tendency to adapt compactly folded conformations in aqueous solution or under physiologic conditions that could result in conformational epitopes. The administration 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 may also reduce the immunogenicity of the BP fusion partner in the BPXTEN compasitions. 10139] In one embodiment, 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 bas 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., er al. (2003) J Immunol Methods, 281: 95-108). Of particular interest are peptide sequences that can be oligomerized without generating T cell epitopes or non-human sequences. This can be achieved by testing direct repeats of these sequences for the presence of T-cell epitopes and for the occurrence of 6 to |3-mer and, in particular, 9-raer sequences that are not human. and then altering the design of the XTEN sequence to eliminate or disrupt the epitope sequence. In some cases, 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 epitopes capable of binding 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 deternuned by epitope prediction algorithms such as, e.g., TEPITOPE (Sturniolo, T., eral. (1999) Nat Biotechnol, 17: 555- 61). The TEPITOPE score of a given peptide frame within a protein is the log of the Kq (dissociation constant, affinity, off-rate) of the binding of that peptide frame to multiple of the most common human MHC alleles, as disclosed in Sturniolo, T. er al. (1999) Nature Biotechnology 17.555). The score ranges over at least 20 logs, from about 10 to about -10 (corresponding to binding constraints of 10e'® Ky to 10e7” Kg), and can be reduced by avoiding hydrophobic amino acids that can serve as anchor residues daring peptide display on MHC, such as M, I, L, V, F. In some embodiments, an XTEN component incorporated into a BPXTEN does not have a predicted T-cell epitope at a TEPITOPE score of about -3 or greater, or -6 or greater, or ~7 or greater, or -8 or greater, or at a TEPITOPE score of -9 or greater. As used herein. a score of “9 or greater” would encompass TEPITOPE scores of 10 to -9, inclusive, but would not encompass a score of -10, as -10 is less than -9.

[0140] In another embodiment, the inventive XTEN sequences, including those incorporated into the subject BPXTEN fusion proteins, can be rendered substantially non-immunogenic by the restriction of known proteolytic sites from the sequence of the XTEN., reducing the processing of XTEN into small peptides that can bind to MHC 1l receptors. In another embodiment, the XTEN sequence can be 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 may render the XTEN compositions, including the XTEN ofthe BPX TEN fusion protein compositions, substantially unable to be bound by mammalian receptors, including those of the immune system. In one embodiment, an XTEN of a BPXTEN fusion protein can have >100 nM Ka binding to a mammalian receptor, or greater than 500 nM Ka, or greater than 1 uM Ka towards a mammalian cell surface or circulating polypeptide receptor.

[0141] Additionally, the non-repetitive sequence and corresponding tack of epitopes of XTEN can 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 independent receptors, can stimulate B cell proliferation and antibody production. In contrast, while a XTEN can make contacts 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. As a result, XTENS typically may have a much lower tendency to stimulate proliferation of B cells and thus an immune response. In one embodiment, the BPXTEN may have reduced tmmunogenicity as compared to the corresponding BP that is not fused. In one embodiment, the administration of up to three parenteral doses of a BPX TEN to a mammal may result in detectable anti-BPXTEN 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 BPXTEN to a mammal may result in detectable anti-BP IgG at a serum dilution of 1:100 but not at a dilution of 1:1000. fo another embodiment, the administration of up to three parenteral doses of a BPXTEN to a mammal may result in detectable anti-XTEN 1gG at a serum dilution of 1:100 but not at a dilution of 1:1000. In the foregoing embodiments, the mammal can be a mouse, a rat, a rabbit, or a cynomolgus monkey.

[0142] An additional feature of XTENSs with non-repetitive sequences relative to sequences with a high degree of repetitiveness can be that non-repetitive XTENSs form weaker contacts with antibodies. Antibodies are multivalent molecules. For instance, IgGs have two identical binding sites and IgMs 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, resulting in less likelihood of immune clearance such that the BPXTEN compositions can remain in circulation for an increased period of time. Increased Hydrodynamic Radius

[0143] In another aspect, the present invention provides XTEN in which the XTEN polypeptides can have a high hydrodynamic radius that confers a corresponding increased Apparent Molecular Weight to the BPXTEN fusion protein incorporating the XTEN. The linking of XTEN to BP sequences can result in BPXTEN compositions that can have increased hydrodynamic radii, increased Apparent Molecular Weight, and increased Apparent Molecular Weight Factor compared to a BP not linked to an XTEN. For example, in therapeutic applications in which prolonged half-life is desired, compositions in which a XTEN with a high hydrodynamic radius is incorporated into a fusion protein comprising one or more BP can effectively enlarge the hydrodynamic radius of the composition beyond the glomerular pore size of approximately 3-5 nm (corresponding to an apparent molecular weight of about 70 kDA) (Caliceti. 2003. Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates. Adv Drug Deliv Rev 55:1261-1277), resulting in reduced renal clearance of circulating proteins. The hydrodynamic radius of a protein is determined by its molecular weight as well as by its structure. including shape and compactness. Not to be bound by a particular theory, the XTEN can adopt open conformations due to electrostatic repulsion between individual charges 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 U.S, Patent Nos. 6,406,632 and 7.294.513. The addition of increasing lengths of XTEN results in proportional increases in the parameters of hydrodynaruic radius, Apparent Molecular Weight, and Apparent Molecular Weight Factor, permitting the tailoring of BPXTEN to desired characteristic cut-off Apparent Molecular Weights or hydrodynamic radii. Accordingly, in certain embodiments, the BPXTEN 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 BPXTEN fusion protein can lead to reduced renal clearance of the resulting fusion protein, leading to a corresponding increase in terminal half-life, an increase in mean residence time, and / or a decrease in renal clearance rate. In another embodiment, an XTEN of a chosen length and sequence can be selectively incorporated into a BPXTEN to create a fusion protein that will have, under physiologic conditions, an Apparent Molecular Weight of at least about 100 kDa, at least about 150 kDa, or at least about 300 kDa, or at least about 400 kDa, or at least about 500 kDA, or at least about 600 kDa, or at least about 700 kDA, or at least about 800 kDa. or at least about 900 kDa, or at least about 1000 kDa, or at least about 1200 kDa, or at least about 1500 kDa, or at least about 1800 kDa. or at least about 2000 kDa, or at least about 2300 kDa or more. In another embodiment, an XTEN of a chosen length and sequence can be selectively linked to a BP to result in a BPXTEN fusion protein that has, under physiologic conditions, an Apparent Molecular Weight Factor of at least three, alternatively of at least four, altematively of at least five, alternatively of at least six, alternatively of at least eight, alternatively of at least 10, alternatively of at least 15, or an Apparent Molecular Weight Factor of at least 20 or greater. In another embodiment, the BPXTEN fusion protein has. under physiologic conditions, an Apparent Molecular Weight Factor that is about 4 to about 20, or is about 6 to about 15, or is about 8 to about 12, or is about 9 to about 10 relative to the actual molecular weight of the fusion protein. BIOLOGICALLY ACTIVE PROTEINS OF THE BPXTEN FUSION PROTEIN COMPOSITIONS

[0144] The present invention relates in part to fusion protein compositions comprising biologically active proteins and XTEN and the uses thereof for the treatment of diseases, disorders or conditions of a subject.

[0145] In one aspect, the invention provides at least a first biologically active protein (hereinafter “BP”) covalently linked to a fusion protein comprising one or more extended recombinant polypeptides (“XTEN™), resulting in an XTEN fusion protein composition (hereinafter “BPXTEN”). As described more fully below, the fusion proteins can optionally include spacer sequences that can further comprise cleavage sequences to release the BP from the fusion protein when acted on bv a protease.

[0146] The term “BPXTEN™, as used herein, is meant to encompass fusion polypeptides that comprise one or two payload regions each comprising a biologically active protein that mediates one or more biological or therapeutic activities and at Jeast one other region comprising at least one XTEN polypeptide.

[0147] The BP of the subject compositions. particularly those disclosed in Tables 6, 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 Protein Resource (UniProt) and subscription provided databases such as GenSeq (e.g., Derwent). Polynucleotide sequences may be a wild type polynucleotide sequence encoding a given BP (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 expression in a particular species; 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 variant of a BP to create BPXTEN constructs contemplated by the invention using methods known in the art and / or in conjunction with the guidance and methods provided herein, and described more fully in the Examples.

[0148] The BP for inclusion in the BPXTEN of the invention can include any protein of biologic. therapeutic, prophylactic, or diagnostic interest or function, or that is useful for mediating a biological activity or preventing or ameliorating a disease, disorder or conditions when administered to a subject. Of particular interest are BP for which an increase in a pharmacokinetic parameter, increased solubility, increased stability, or some other enhanced pharmaceutical property is sought, or those BP for which increasing the terminal half-life would improve efficacy, safety, or result in reduce dosing frequency and / or improve patient compliance. Thus, the BPXTEN fusion protein compositions are prepared with various objectives in mind, including improving the therapeutic efficacy of the bioactive compound by, for example, increasing the in vive exposure or the length that the BPXTEN remains within the therapeutic window when administered to a subject, compared to a BP not linked to XTEN.

[0149] A BP of the invention can be a native, full-length protein or can be a fragment or a sequence variant of a biologically active protein that retains at least a portion of the biological activity of the native protein.

[0150] In one embodiment, the BP incorporated into the subject compositions can be a recombinant polypeptide with a sequence corresponding to a protein found in nature. In another embodiment, the BP can be sequence variants, fragments, homologs, and mimetics of a natural sequence that retain at least a portion of the biological activity of the native BP. In non-limiting examples, a BP can be a sequence that exhibits 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 to a protein sequence selected from Tables 6. In one embodiment, a BFXTEN fusion protein can comprise a single BP molecule linked to an XTEN (as described more fully below). In another embodiment, the BPXTEN can comprise a first BP and a second molecule of the same BP, resulting in a fusion protein comprising the two BP linked to one or more XTEN (for example, two molecules of TL-1ra, or two molecules of [L.-10). Biologically active proteins including those as therapeutics are typically labile molecules exhibiting short shelf-fives. particularly when formulated in aqueous solutions. In addition, many biologically active peptides and proteins have limited solubility, or become aggregated during recombinant productions, requiring complex solubilization and refolding procedures. Various chemical polymers can be attached to such proteins to modify their properties. Of particular interest are hydrophilic polymers that have flexible conformations and are well hydrated in aqueous solutions. A frequently used polymer is polyethylene glycol (PEG). These polymers tend to have large hydrodynamic radii relative to their molecular weight (Kubetzko, S., er al. (2005) Mol Pharmacol, 68: 1439-54), and can result in enhanced pharmacokinetic properties. Depending on the points of attachment, the polymers tend to have limited interactions with the protein that they have been attached to such that the polymer-modified protein retains its relevant functions. However, the chemical conjugation of polymers to proteins requires complex multi-step processes. Typically, the protein component needs to be produced and purified prior to the chemical conjugation step. In addition, the conjugation step can result in the formation of heterogeneous product mixtures that need to be separated, leading to significant product loss. Alternatively, such mixtures can be used as the final pharmaceutical product. but are difficult to standardize. Some examples are currently marketed PEGylated Interferon-alpha products that are used as mixtures (Wang. B. L, ef al. (1998) J Submicrosc Cytol Pathol, 30: 503-9; Dhalluin, C., er al. (2005) Bioconjug Chem, 16: 504-17). Such mixtures are difficult to reproducibly manufacture and characterize as they contain isomers with reduced or no therapeutic activity.

[0151] In general, BP will exhibit a binding specificity to a given target or another desired biological characteristic when used 7» vivo or when utilized in an in virro assay. For example, the BP can be an agonist, areceptor, a ligand, an antagonist, an enzyme, or a hormone. Of particular interest are BP used or known to be useful for a disease or disorder wherein the native BP have a relatively short terminal half-life and for which an enhancement of a pharmacokinetic parameter (which optionally could be released from the fusion protein by cleavage of a spacer sequence) would permit less frequent dosing or an enhanced pharmacologic effect. Also of interest are BP that have a narrow therapeutic window between the minimum effective dose or blood concentration (Cm) and the maximum tolerated dose or blood concentration (Cras). In such cases, the linking of the BP to a fusion protein comprising a select XTEN sequence(s) can result in an improvement in these properties, making them more useful as therapeutic or preventive agents compared to BP not linked to XTEN. 10152] The BP can be a cytokine. Cytokines encompassed by the inventive compositions can have utility in the treatment in various therapeutic or disease categories, including but not limited to cancer, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, Alzheimer’s disease, Schizophrenia, viral infections {e.g., chronic hepatitis C, AIDS), allergic asthma, retinal neurodegenerative processes, metabolic disorder, insulin resistance, and diabetic cardiomyopathy. Cytokines can be especially useful in treating inflammatory conditions and autoimmune conditions.

[0153] The BP can be one or more cytokines. The cytokines refer to proteins (¢.g., chemokines. interferons, lymphokines, interleukins, and tumor necrosis factors) released by cells which can affect cell bebavior. Cytokines can be produced by a broad range of cells, including but not limited to immune cells such as macrophages, B lymphocytes, T lymphocytes, microglia cells, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. A given cytokine can be produced by more than one type of cell. Cytokines can be involved in producing systemic or local immunomodulatory effects.

[0154] Certain cytokines can function as pro-inflammatory cytokines. Pro-inflammatory cytokines refer to cytokines involved in inducing or amplifying an inflammatory reaction. Pro-inflammatory cytokines can work with various cells of the immune system, such as neutrophils and leukocytes, to generate an immune response. Certain cytokines can function as anti-inflammatory cytokines. Anti-inflammatory cytokines refer to cytokines imvolved in the reduction of an inflammatory reaction. Anti-inflammatory cytokines, in some cases, can regulate a pro-inflammatory cytokine response. Some cytokines can function as both pro- and anti- inflammatory cytokines.

[0155] Examples of cytokines that are regulatable by systems and compositions of the present disclosure include. but are not limited to lymphokines, monokines, and traditional polypeptide hormones except for human growth hormone. Included among the cytokines are parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha ; mullerian-inhibiting substance; mouse gonadotropin- associated peptide; inhibin: activin; vascular endothelial growth factor: integrin; thrombopoietin (TPO): nerve growth factors such as NGF-alpha; platelet-growth factor: transforming growth factors (TGFs) such as TGF- alpha, TGF-beta, TGF-betal, TGF-beta, and TGF-beta3; insulin-like growth factor-I and -[I; erythropoietin (EPO); F1t-3L: stem cell factor (SCF); osteoinductive factors; interferons (IFNs) such as IFN-a, IFN-B, [FN-y; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM- CSF); granulocyte-CSF (G-CSF). macrophage stimulating factor (MSP): interleukins (ILs) such as IL-1, IL- 1a, [L-1b, IL-1RA. IL-18, IL-2, IL-3, IL-4, IL-5, 1L-6, IL-7, IL-8, IL-9, IL-10, IL-11. IL-12, 1L-12b, IL-13, IL-14, IL-15, IL-16, IL-17, 1L-20; a tumor necrosis factor such as CD154, LT-beta, TNF-alpha, TNF-beta, 4- BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE; and other polypeptide factors including LIF. oncostatin M (OSM) and kit ligand (KL). Cytokine receptors refer to the receptor proteins which bind cytokines. Cytokine receptors may be both membrane-bound and soluble.

[0156] The target polynucleotide can encode for a cytokine. Non-limiting examples of cytokines include 4- 1BBL, activin BA, activin BB, activin BC, activin BE, artemin (ARTN), BAFF / BLyS / TNFSF138, BMP10, BMP15, BMP2, BMP3, BMP4, BMPS, BMP6, BMP7, BMP8a, BMP8b, bone morphogenetic protein 1 (BMP1), CCLI / TCA3, CCLI11, CCL12 / MCP-5,CCLI3 / MCP-4, CCL14, CCL15, CCL16, CCLI7 / TARC, CCLI8, CCL19, CCL2 / MCP-1, CCL20, CCL2}, CCL22MDC, CCL23, CCL24. CCL23, CCL26, CCL27, CCL28. CCL3, CCL3L3, CCL4, CCLALI / LAG-1, CCLS, CCL6, CCL7. CCL8, CCL9, CD153 / CD30L / TNFSF8, CD40L / CD154 / TNFSFS, CD40LG, CD70. CD70 / CD27L / TNFSF7, CLCFL. ¢- MPL / CD110 / TPOR, CNTF, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCLIS, CXCL16, CXCL17, CXCL2 / MIP-2, CXCL3, CXCL4, CXCL3, CXCL6, CXCL7 / Ppbp, CXCLY, EDA-AL, FAMI9AL, FAM19A2, FAMI9A3, FAM19A4, FAMI9AS, Fas Ligand / FASLG / CDISL / CDI78, GDF10, GDFi1, GDF15, GDF2, GDF3, GDF4, GDF3, GDF6, GDF7, GDF8. GDF9, glial cell line-derived neurotrophic factor (GDNF), growth differentiation factor 1 (GDFI), IFNAL. [FNA10. [FNAI13, [FNA14, IFNA2, IFNA4, IFNAS / IFNaG, IFNA7, IFNAS, IFNBI, IFNE, IFNG, IFNZ, IFNo / IFNW1, IL11, IL18, IL18BP, ILIA, ILIB, ILIF10, ILIF3 / ILIRA, IL1FS, ILIF6, TLIF7, TLIF8, ILIF9, IL1RL2, TL31, IL33, IL6, IL8 / CXCLSR, inhibin-A. inhibin-B, Leptin, LIF, LTA / INFB / INFSF1, LTB / TNFC, neurturin (NRTN), OSM, OX-40L / TNFSF4 / CD252, persephin (PSPN}), RANKL / OPGL / TNFSF11(CD254), TLIA / TNFSF1S, TNFA, TNF-alpha / TNFA, TNFSFI10 / TRAIL / APO-2L{CD253), TNFSF12, TNFSF13, TNFSFI14 / LIGHT / CD258, XCL1, and XCL2. In some embodiments, the target gene encodes for an immune checkpoint inhibitor. Non- limiting examples of such immune checkpoint inhibitors include PI>-1. CTLA-4, LAG3, TIM-3. A2AR, B7- H3. B7-H4, BTLA, IDO, KIR, and VISTA. In some embodiments. the target gene encodes for a T cell receptor (TCR) alpha, beta, gamma, and / or delta chain.

[0157] In some cases, the cytokine can be a chemokine. The chemokine can be selected from a group including, but not limited to, ARMCX2, BCA-1 / CXCL13, CCL11, CCL12 / MCP-5, CCL.13 / MCP-4, CCL15 / MIP- 5 / MIP-1 delta, CCL16 / HCC-4 / NCC4, CCLI7 / TARC. CCL18 / PARC / MIP-4, CCLI19 / MIP-3b, CCL2 / MCP-1, CCL20 / MIP-3 alpha / MIP3A, CCL21 / 6Ckine. CCL22 / MDC, CCL23 / MIP 3, CCL24 / Eotaxin- 2 / MPIF-2, CCL2S / TECK, CCL26 / Eotaxin-3, CCL27 / CTACK, CCL28, CCL3 / Mipla, CCL4 / MIPIB, CCL4L1 / LAG-1, CCL3 / RANTES, CCL6 / C10, CCL8 / MCP-2, CCLY, CML5, CXCL1, CXCLI10 / Crg-2, CXCL12 / SDF-1 beta, CXCL14 / BRAK, CXCL15 / Lungkine, CXCL16 / SR-PSOX, CXCL17, CXCL2 / MIP- 2, CXCL3 / GRO gamma, CXCL4 / PF4, CXCL3, CXCL6 / GCP-2, CXCLY / MIG, FAMI9A1, FAMI9A2, FAMI9A3, FAM19A4 / TAFA4, FAMI19AS. Fractalkine / CX3CL1, I-309 / CCL1 / TCA-3, IL-8 / CXCL8, MCP- 3 / CCL7, NAP-2 / PPBP / CXCL7, XCL2, and Armo IL10.

[0158] Table 3 provides a non-limiting list of such sequences of BPs that are encompassed by the BPXTEN fusion proteins of the invention. Metabolic proteins of the inventive BPXTEN compositions can be a protein that exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%. 84%, 83%, 86%, 87%. 88%. 89%. 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%, 99%, or 100% sequence identity to a protein sequence selected from Table 3. Table 3: Cytokines for Conjugation Anakinm 12 subunit beta (11-12 p40) Interlenkin- | 2 6 12 subnpnit Synonym {L-lra, buman | MEICRGLRSHLITLLLFLFHSETICRPSGRKS SKMQAFRIWDVNQKTFYLRNNQLVAGY LQGPNV full length NLEEKIDVVPIEPHALFLGIHGGKMCLSCVKS GDETRLQLEAVNI TDLSENRKQDKRFAFIRSDS GPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SID ID NO: 132) 1L-1ra, Dog METCRCPLSYLISKFLLFLPHSETACRLGKRPCRMQAFRIWDVNQK FY LRNNQLVAGY LOQGSNTK LEEKLDVVPVEPHAVFLGIHGGKLCLACVKSGDETRLQLEAVNITDLSKNKDQDKRFTFILSDSG PTTSFESAACPGWFLCTALEADRPVS LTNRPEEAMMYTKEYFQKE (SEQ ID No: 153) ! TL-1ra, Rabbit MRPSRSTRRHLISLLLFLFHSETACRPS GKRPCRMQAFRIWDVNOKTFY LRNNQLVAGY LOGPNA KLEERIDVVPLEPQLLFLGIQRGKLCLSCVKS GDKMKLHLEAVNI TDLGKNKEQDKRFTFIRSNS GPTTTFESASCPGWFLCTALEADQPVSLTNTPDDS IVVTKFYFQED (SEQ ID NO: 154) 1L-1ta, Rat MEICRGPYSHLISLLLILLFRSESAGHI PAGKRPCKMQAFRINDTNQK TF Y LRNNQLIAGY LQGP NTKLEEKIDMVPIDFRNVFLGIHGGKLCLSCVKSGDDTKLQLEEVNITDLNKNKEEDKRFTFIRS ETGPTTSFESLACPGWFLCTTLEADHEVS LINTPKERPCTVIKFYFQED (SEQ ID NO: 155! TL.-1ra, Mouse MEICWGPYSHLISLLLILLFHSEAACRPSGKRPCKMOAFRIWDINQKTFY LRNNQLIAGY LOGPN IKLEEKIDMVPIDLHSVFLGIHGGKLCLSCAKSGDDIKLOLEEVNITDLSKNKEEDKRF TF IRSE KGPTTSFESAACPGWFLCTTLEADRPVSLENTPEEPLIVIKFYFORDQ (SEQ ID NO: 156) Anakinm MRPSGRKSSKMQAFRIWDVNQKL¥Y LRNNQLVAGY LOGPNVNLERK IDVVPIEPHALFLGLHGGK MCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQ PVSLTNMPDEGVMYVTKEYFQEDE (SEG ID NO: 157) TL-10 MHSSALLCCLVLLTGVRASPGQGTQSENSCTHF PGNLPNMLRDLRDAF SRVKTFFQMKDQLDNLL LKESLLEDFKGYLGCQALSEMIQFY LEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFL PCENKSKAVEQVKNAFNKLORKGLYKAMSEFDIFINY ITEAYMTMKIRN (SEQ ID NO: 138) Table A. Amino acid sequences of exemplary interleukin-12 (11-12) or fragments thereof Name SEQ ID NO. Amino Acid Sequence Interleukin- 5 MWELEKDVYVVEVDWIPDAPGETVNLTCDTPEEDDI TWISDQREGVIGSGKTLT 12 subunit ITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWS TEI LKNFKNKTFLKCEA beta (11-12 | PNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVICGMASLSAEKVTLDQRDY p40) EKYSVSCQEDVICPTAEETLPIELALEARQONKYENY STSFFIRDI IKPDPPKN LQMKPLKNSQVEVSWEY PDSWSTPHSY FSLKFFVRIQRKKEKMKETEEGCNQKG AFLVEXTSTEVQCKGGNVCVOAQDRY YNSSCSKWACVPCRVRS Ham EE 12 subunit TLRTCLPLELHKNESCLATRETS STTRGSCLPPOKTS LMMILCLGS TYEDLKMY alpha (IL-12 QTEFQAINAALONHNHQOI I LDKGMLVAIDELMOS LNHNGETLROKPPVGEADP 35) YRVKMKLCILLHAFSTRVVTINRVMGYLSSA TL-12 variant 7 MWELEKDVYVVEVDWIPDAPGETVNLTCDTPEEDDI TWTSDQREGVIGSGKTLT ITVKEFLDAGQY TCHKGGRT LSHSHLLLHKKENGIWS TET LKNFKNKTF LKCEA PNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVICGMASLSAEKVTLDQRDY EKYSVSCQEDVICPTAEETLPIELALEARQONKYENY STSFFIRDI IKPDPPKN LQMKPLKNSQVEVSWEY PDSWS TPHSY FSLKFFVRIQRKKEKMKETEEGCNQKG AFLVEKTSTEVQCKGENVCVQAQDRY YNSSCSKWACVRCRVRS GEEGSGEGESG GGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHY SCTAEDIDHEDITR DQTSTLXICLPLELHKNESCLATRETS STTRGS CLPPOKTSLMMILCLGS IYED LKMYQTEFQAINAALQONHNHQQI I LDKGMLVAIDELMQS LNHNGETLRQKPPVG EADPYRVKMKLCTLLHAFSTRVVEINRVMGYLS SA

[0159] Table A provides a non-limiting list of interleukin- 12 sequences (or fragments thereof). The inventive BPXTEN compositions of this disclosure can contain an amino acid sequence that exhibits 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 to a protein sequence selected from Table A.

[0160] In some embodiments, where the composition of this disclosure (such as a fusion protein) comprises acytokine, the cytokine can be selected from a group consisting of interleukins, chemokines, interferons, tumor necrosis factors, colony-stimulating factors, or transforming growth factor beta (TGF-beta) superfamily members. In some embodiments, the cytokine can be an interleukin selected from the group consisting of IL1, IL2, IL3, IL4, ILS, IL6, IL7, IL8, IL9, IL10, IL11. IL12, ILI3, [L14, IL15, IL16. and IL17. In some embodiments, the cviokine can have at least (about) 80%, at least (about) 81%, at least (about) 82%, at least {about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%. at least (about) 91%. at least (about) 92%, at least (about) 93%. at least (about) 94%. at least (about) 93%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or 100% sequence identity to a sequence selected from Table 3 or Table A. In some embodiments, the cytokine can have at least (about) 80%, at least (about) 81%, at least (about) 82%, atleast (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 93%, at least (about) 96%. at least (about) 97%, at least {about) 98%, at least (about) 99%, or 100% sequence identity to a sequence selected from Table 3. In some embodiments, the cytokine can have at least (about) 80%, at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%. at least (about) 83%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%. at least (about) 95%, at least (about) 96%, at least (about) 97%, at least {about) 98%, at least (about) 99%, or 100% sequence identity to a sequence selected from Table A. In some embodiments, the cytokine can be IL-12 or an TL-12 variant. In some embodiments. the cytokine can comprise a first cytokine fragment (Cy1) and a second cytokine fragment (Cy2). In some embodiments, one of the Cyl and the Cy2 can comprise an amino acid sequence having at least 70% sequence identity to an interleukin-12 subunit beta. In some embodiments, the other one of the Cy 1 and the Cy2 can comprise an amino acid sequence having at least (about) 70%, at least (about) 75%, at least (about) 80%, at least (about) 85%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%. at least (about) 98%, at least (about) 99%. or 100% sequence identity to an interleukin- 12 subunit alpha. In some embodiments, the first cytokine fragment (Cy1) can comprise an amino acid sequence having at least (about) 70%, at least (about) 75%, at least (about) 80%, at least (about) 85%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 93%, at least (about) 96%, at Jeast (about) 97%, at least (about) 98%. at least (about) 99%. or 100% sequence identity to a sequence of SEQ ID NO. 5. In some embodiments. the second cytokine fragment {Cy2) can comprise an amino acid sequence having at least (about) 70%, at least (about) 75%, at least (about) 80%, at Jeast (about) 85%, at least (about) 90%. at least (about) 91%, at least (about) 92%. at least (about) 93%, at least (about) 94%, at least (about) 93%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or 100% sequence identity to a sequence of SEQ ID NO. 6. In some embodiments, the cytokine can comprise a linker positioned between the first cvtokine fragment (Cy) and the second cytokine fragment (Cy2). In some embodiments, the cytokine can be an IL-12 variant comprising an amino acid sequence having at least (about) 70%, at least (about) 75%, at Jeast (about) 80%. at least (about) 85%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or 100% sequence identity to SEQ ID NO. 7. The linker can be a GS linker (such as (GGGGS)«(SEQ ID NO: 273), (GGGGS)(SEQ ID NO: 273), (GGGGS):(SEQ ID NO: 273), (GGGGS)«(SEQ ID NO: 273), (GGGGS)s(SEQ 1D NO: 273), ete.)

[0161] “1L-lra” means the human IL-1 receptor antagonist protein and species and sequence variants thereof, including the sequence variant anakinra (Kineret®), having at least a portion of the biological activity of mature IL-Ira. Human IL-Ira is a mature glycoprotein of 152 amino acid residues. The inhibitory action of IL-1ra results from its binding to the type [ IL-1 receptor. The protein has a native molecular weight of 25 kDa, and the molecule shows limited sequence homology to [L-lo (19%) and IL-1B (26%). Anakinra is a nonglycosylated, recombinant human IL-1ra and differs from endogenous human IL-1ra by the addition of an N-terminal methionine. A commercialized version of anakinra is marketed as Kineret®. It binds with the same avidity to IL-1 receptor as native IL-1ra and IL.-1b, but does not result in receptor activation (signal transduction), an effect attributed to the presence of only one receptor binding motif on [L-1ra versus two such motifs on IL-1 ¢ and IL-18. Anakinra has 153 amino acids and 17.3 kD in size, and has a reported half-life of approximately 4-6 hours.

[0162] Increased TL-1 production has been reported in patients with various viral, bacterial, fungal, and parasitic infections; intravascular coagulation; high-dose 1-2 therapy; solid tumors; leukemias; Alzheimer’s disease; HIV- 1 infection; autoimmune disorders; trauma (surgery), hemodialysis; ischemic diseases (myocardial infarction); noninfectious hepatitis, asthma; UV radiation: closed head injury: pancreatitis; peritonitis; grafl-versus-host disease; transplant rejection; and in healthy subjects afier strenuous exercise. There is an association of increased TL-1b production in patients with Alzheimer’s disease and a possible role for IL 1 in the release of the amyloid precursor protein. IL-1 has also been associated with diseases such as type 2 diabetes, obesity. hyperglycemia, hyperinsulinemia, type 1 diabetes, insulin resistance, retinal neurodegenerative processes, disease states and conditions characterized by insulin resistance, acute myocardial infarction (AMI), acute coronary svndrome (ACS), atherosclerosis, chronic inflammatory disorders, rheumatoid arthritis, degenerative intervertebral disc disease. sarcoidosis, Crohn's disease, ulcerative colitis, gestational diabetes, excessive appetite, insufficient satiety, metabolic disorders, glucagonomas, secretory disorders of the airway, osteoporosis, central nervous system disease, restenosis, neurodegenerative disease, renal failure, congestive heart failure. nephrotic syndrome, cirthosis, pulmonary edema, hypertension, disorders wherein the reduction of food intake is desired, irritable bowel syndrome, myocardial infarction, stroke, postsurgical catabolic changes, hibernating myocardium, diabetic cardiomyopathy, insufficient urinary sodium excretion, excessive urinary potassium concentration, conditions or disorders associated with toxic hypervolemia, polycystic ovary syndrome, respiratory distress, chronic skin ulcers. nephropathy, left ventricular systolic dysfunction. gastrointestinal diarrhea, postoperative duruping syndrome, irritable bowel syndrome, critical illness polyneuropathy (CIPN), systemic inflammatory response syndrome (SIRS), dyslipidemia, reperfusion injury following ischemia, and coronary heart disease risk factor (CHDRF) syndrome. [L-lra-containing fusion proteins of the invention may find particular use in the treatment of any of the foregoing diseases and disorders. [L-lra has been cloned, as described in U.S. Pat. Nos. 5,075.222 and 6,858.409.

[0163] In some cases, the BP can be IL-10. IL-10 can be an effective anti-inflammatory cytokine that represses the production of the proinflammatory cytokines and chemokines. IL-10 is the one of the major TH2-type cytokine that increases humoral immune responses and lowers cell-mediated immune reactions. JL-10 can be useful for the treatment of autoimmune diseases and inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis. systemic lupus erythematosus, Alzheimer’s disease. Schizophrenia, allergic asthma, retinal neurodegenerative processes, and diabetes.

[0164] In some cases, IL-10 can be modified to improve stability and decrease prolytic degradation. The modification can be one or more amide bond substitution. In some cases, one or more amide bonds within backbone of IL-10 can be substituted to achieve the abovementioned effects. The one or more amide linkages —CO—NH—) in IL-10 can be replaced with a linkage which is an isostere of an amide linkage, such as — CHINH ~~, ~~ CH28~-, ~~ CH2CH2-, ~~ CH~~CH-(cis and trans), ~~ COCH2-, ~~ CH(OH)CH2 or ~~ CH2SO ~~. Furthermore, the amide linkages in IL-10 can also be replaced by a reduced isostere pseudopeptide bond. See Couder et al. (1993) Int. J. Peptide Protein Res. 41:181-184, which is hereby incorporated by reference in its entirety.

[0165] The one or more acidic amino acids, including aspartic acid. glutamic acid. homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, and heteroaryl sulfonamides of 2.4-diaminopriopionic acid, omithine or lysine and tetrazole-substituted alkyl amino acids; and side chain amide residues such as asparagine, glutamine, and alkyl or aromatic substituted derivatives of asparagine or glutamine; as well as hydroxyl-containing amino acids, including serine, threonine, homoserine, 2,3-diaminopropionic acid, and alky! or aromatic substituted derivatives of serine or threonine can be substituted.

[0166] The one or more hydrophobic amino acids in IL-10 such as alanine, leucine, isoleucine, valine, norleucine, ($)-2-aminobutyric acid, (S)-cyclohexylalanine or other simple alpha-amino acids can be substituted with amino acids including, but not limited to, an aliphatic side chain from C1-C10 carbons including branched. cyclic and straight chain alkyl, alkenyl or alkynyl substitutions

[0167] In some cases, the one or more hydrophobic amino acids in IL-10 such as can be substituted substitution of aromatic-substituted hydrophobic amino acids. including phenylalanine, tryptophan, tyrosine, sulfotyrosine, biphenylalanine, 1-naphthylalanine, 2-naphthylalanine, 2-benzothienylalanine, 3- benzothienylalanine, histidine, including amino, alkylamino, dialkylamino, aza. halogenated (fluoro, chloro, bromo, or iodo) or alkoxy (from C1-C4)-substituted forms of the above-listed aromatic amino acids, illustrative examples of which are: 2-, 3- or 4-aminopbenylalanine, 2-, 3- or d-chlorophenylalanine, 2-. 3- or 4- methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, S-amino-. S-chloro-, 5-methyl- or 35- methoxy tryptophan, 2°-, 3°, or 4’-amino-, 2°-, 3’, or 4’-chloro-, 2, 3, or 4-biphenylalanine, 2°-, 3"-, or 4'- methvl-, 2-, 3- or 4-biphenylalanine, and 2- or 3-pyvridvlalanine;

[0168] The one or more hydrophobic amino acids in 1L-10 such as phenylalanine, tryptophan, tyrosine. sulfotyrosine, biphenylalanine, l-naphthylalanine, 2-vapbthylalanine, 2-benzothienylalanine, 3- benzothienylalanine, histidine, including amino, alkylamino, dialkylamino. aza, halogenated (fluoro, chloro, bromo, or iodo) or alkox can be substituted by aromatic amino acids including: 2-, 3- or 4-aminophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3- or 4-methy phenylalanine, 2-, 3- or 4-methoxyphenylalanine, S-amino-, 5-chloro-, S-methyl- or 5-methoxytryptophan, 2°-, 3’-, or 4’-amino-, 2’, 3’, or 4’-chloro-, 2, 3, or 4- biphenylalanine, 2’-, 3°, or 4’-methyl-, 2-, 3- or 4-biphenylalanine. and 2- or 3-pyridylalanine

[0169] The amino acids comprising basic side chains, including arginine, lysine, histidine, omithine, 2,3- diaminopropionic acid, homoarginine, including alkyl, alkenyl, or aryl-substituted derivatives of the previous amino acids, can be substituted. Examples are N-epsilon-isopropyl-lysine, 3-(4-tetrahydropyridyl)-glycine, 3- (4-tetrahydropyridyl)-alanine, N,N-gamma, gamma’-dicthyl-homoarginine, alpha-methyl-arginine, alpha- methyl-2,3-diaminopropionic acid, alpha-methyl-histidine, and alpha-methyl-omithine where the alkyl group occupies the pro-R position of the alpha-carbon. The modified IL-10 can comprise amides formed from any combination of alkyl, aromatic, heteroaromatic, ornithine, or 2,3-diaminopropionic acid, carboxylic acids or any of the many well-known activated derivatives such as acid chlorides, active esters, active azolides and related derivatives, lysine, and ornithine.

[0170] In some cases, IL-10 comprises can comprise one or more naturally occurring L-amino acids, synthetic L-amino acids, and / or D-enantiomers of an amino acid. The IL-10 polypeptide can comprise one or more of the following amino acids: m-aminodecanoic acid, w-aminotetradecanoic acid, cyclohexylalanine, o,y- diaminobutyric acid, «.p-diaminopropionic acid, é-amino valeric acid, t-butylalanive, t-butylglycine, N- methylisoleucine, phenylglycine, cyclohexylalanine, norleucine, naphthylalanine, ornithine. citrulline, 4- chlorophenylalanine, 2-fluorophenylalanine, pyridylalanine 3-benzothieny! alanine, hydroxyproline, B-alanine, o-aminobenzoic acid, m-aminobenzoic acid, p-aminobenzoic acid, m-aminomethylbenzoic acid, 2.3- diaminopropionic acid. o-aminoisobutyric acid, N-methylglycine(sarcosine), 3-fluorophenylalanine, 4- fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, B-2-thienylalanine, methionine sulfoxide, homoarginine, N-acetyl Ivsine, 2,4-diamino butyric acid, rho-aminophenylalanine, N- methylvaline, homocysteine, homoserine, e-amino hexanoic acid, o-aminohexanoic acid, w-aminoheptanoic acid, @-aminooctanoic acid, and 2,3-diaminobutvric acid.

[0171] IL-10 can comprise a cysteine residue or a cysteine which can act as linker to another peptide via a disulfide linkage or to provide for cyclization of the IL-10 polypeptide. Methods of introducing a cysteine or cysteine analog are known in the art; see, e.g., U.S. Pat. No. 8.067.532. An IL-10 polypeptide can be cyclized. Other means of cyclization include introduction of an oxime linker or a lanthionine linker; see, ¢.g., U.S. Pat. No. 8,044,175. Any combination of amino acids (or non-amino acid moieties) that can form a cyclizing bond can be used and / or introduced. A cyclizing bond can be generated with any combination of amino acids (or with an amino acid and —(CH2)n-CO— or —(CH2)n-C6H4—CO0—) with functional groups which allow for the introduction of a bridge. Some examples are disulfides, disulfide mimetics such as the —(CH2)n-carba bridge, thioacetal, thioether bridges (cystathionine or lanthionine) and bridges containing esters and ethers.

[0172] The [L-10 can be substituted with an N-atkyl, aryl, or backbone crosslinking to construct lactams and other cyclic structures, C-terminal hydroxymethyl derivatives, o-modified derivatives, N-terminally modified derivatives including substituted amides such as atkylamides and hydrazides. To some cases, an TL-10 polypeptide is a retroinverso analog, 10173] IL-10 can be IL-10 can be native protein. peptide fragment IL-10, or modified peptide, having at least a portion of the biological activity of native 1L-10. IL-10 can be modified to improve intracellular uptake. One such modification can be attachment of a protein transduction domain, The protein transduction domain can be attached to the C-terminus of the IL-10. Alternatively, the protein transduction domain can be attached to the N-terminus of the IL-10. The protein transduction domain can be attached to 1L-10 via covalent bond. The protein transduction domain can be chosen from any of the sequences listed in Table 9. Table 9. Exemplary protein transduction domains 13) 14) 1%) Amino Acid Sequence J OREERROBRR SEG ID 01 Bo rmermmemmene SWILNSAGYLLGRINLEALARLRKRIL 58 ID NO: 13). KALAWEAKLAKALAKALAKHLAKALAKALKCEA | SEQ 10 No: 11} RQIKIWFQNRRMKWKK {SEQ ID NC: 12} YGRKKRRQRRR (SEQ ID NO: 13) RKKRRQRRR (SEQ ID NO: 14} YGRKKRRQRRR (SEQ ID NO: 1%) RKKRRQRR (SEQ 1D NO: 16) YARAAARQARA (SEQ ID NO: 17) GGRRARRRRRR (SER ID NO: 1%) BPXTEN STRUCTURAL CONFIGURATIONS AND PROPERTIES

[0174] The BP of the subject compositions are not limited to native, full-length polypeptides, but also include recombinant versions as well as biologically and / or pharmacologically active variants or fragments thereof. For example, it will be appreciated that various amino acid substitutions can be made in the GP to create variants without departing from the spirit of the invention with respect to the biological activity or pharmacologic properties of the BP. Examples of conservative substitutions for amino acids in polypeptide sequences are shown in Table 4. However, in embodiments of the BPXTEN in which the sequence identity of the BP is less than 100% compared to a specific sequence disclosed herein, the invention conternplates substitution of any of the other 19 natural L-amino acids for a given amino acid residue of the given BP, which may be at any position within the sequence of the BP, including adjacent amino acid residues. If any one substitution results in an undesirable change in biological activity, then one of the alternative amino acids can be employed and the construct evaluated by the methods described herein, or using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Pat. No. 5,364,934, the contents of which is incorporated by reference in its entirety, or using methods generally known to those of skill in the art. In addition, variants can also include, for instance, polypeptides wherein one or more amino acid residues are added or deleted at the N- or C-terminus of the full-length native amino acid sequence of a BP that retains at least a portion of the biological activity of the native peptide. Table 4: Exemplary conservative amino acid substitutions Original Residue Exeniplary Substitutions val; leu; ile Ala (A) Eales] Wells [Lite [EL Ac FEN Bebe nts orn Arg (R) lys; gin; asn Asn (N gin; his; Tys; arg asn Asp (D) la Cys (C ser 3 TY VSN GG) pro His (H asn: gin: Tys: arg xlle 0 leu; val; met; ala; phe: norleucine Yai TS arcs arenreems This avenlle onsavtv ra¥ oso wolaes Leu (L norleucine: ile: val; met; ala: phe ny (KD ATE GINIASE Met (M leu; phe; ile Dive (IY Raupes wres¥e Foxe wafer Phe (F) leu: val: ile; ala Pro (P, oly thr Thr (T) ser Tro (W) tyr ee ODL UDIPREIIONISOE Val (V) ile; leu; met; phe; ala; norleucine BPXTEN Fasion Protein Configurations

[0175] The invention provides BPXTEN fusion protein compositions comprising BP linked to one or more XTEN polypeptides useful for preventing, treating, mediating, or ameliorating a disease, disorder or condition related to glucose homeostasis, insulin resistance. or obesity. In some cases, the BPXTEN is a monomeric fusion protein with a BP linked to one or more XTEN polypeptides. 1n other cases, the BPXTEN composition can include two BP molecules linked to one or more XTEN polypeptides. The invention contemplates BPXTEN comprising. but not limited to BP selected from Table 3 or Table A (or fragments or sequence variants thereof), and XTEN selected from Tables 2a-2b or sequence variants thereof. In some cases, at least a portion of the biological activity of the respective BP is retained by the intact BPXTEN, In other cases, the BP component either becomes biologically active or has an increase in activity upon its release from the XTEN by cleavage of an optional cleavage sequence incorporated within spacer sequences into the BPXTEN, described more fully below.

[0176] In some embodiments, the BPXTEN fusion protein composition comprises (a) an XTEN (such as one disclosed herein) and (b) a cytokine linked to the XTEN.

[0177] In one embodiment of the BPX TEN composition. the invention provides a fusion protein of formula 1:

[0178] (BP)-(S)=(XTEN)

[0179] wherein independently for each occurrence, BP is a is a biologically active protein as described hereinabove: S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence (as described more fully below); x is either 0 or 1; and XTEN is an extended recombinant polypeptide as described hereinabove. The embodiment has particular utility where the BP requires a free N-terminus for desired biological activity, or where linking of the C-terminus of the BP to the fusion protein reduces biological activity and it is desired to reduce the biological activity and / or side effects of the administered BPXTEN

[0180] In another embodiment of the BPXTEN composition, the invention provides a fusion protein of formula 1I (components as described above):

[0181] (XTEN)-(S)-(BP) I

[0182] wherein independently for each occurrence, BP is a is a biologically active protein as described hereinabove: S is a spacer sequence having between 1 to about 30 amino acid residues that can optionally include a cleavage sequence (as described more fully below); x is either 0 or 1; and XTEN is an extended recombinant polypeptide as described hereinabove. The embodiment has particular utility where the BP requires a free C-terminus for desired biological activity, or where linking of the N-terminus of the BP to the fusion protein reduces biological activity and it is desired to reduce the biological activity and / or side effects of the administered BPXTEN {0183] Thus, the BPXTEN having a single BP and a single XTEN can have at least the following permutations of configurations, each listed in an N- to C-terminus orientation: BP-XTEN; XTEN-BP; BP-S-XTEN: or XTEN-S-BP.

[0184] Iv another embodiraent, the invention provides an isolated fusion protein, wherein the fusion protein is of formula 111: (BP)-(S)-(XTEN}-(S)y~(BP)~(S),-(XTEN), 1

[0185] wherein independently for each occurrence. BP is a is a biologically active protein as described hereinabove; S is a spacer sequence having between | to about 50 amino acid residues that can optionally include a cleavage sequence {as described more fully below); x is either 0 or 1; y is either 0 or 1; z is either 0 or 1; and XTEN is an extended recombinant polypeptide as described hereinabove.

[0186] In another embodiment, the invention provides an isolated fusion protein, wherein the fusion protein is of formula IV (components as described above):

[0187] (XTEN)-(S)-(BP)-(S)-(XTEN)-(BP) vv

[0188] In another embodiment, the invention provides an isolated fusion protein, wherein the fusion protein is of formula V (components as described above):

[0189] (BP) (S)(BP)-(S)~(XTEN) Vv

[0190] In another embodiment, the invention provides an isolated fusion protein, wherein the fusion protein is of formula VI (components as described above):

[0191] (XTEN)-(S)-(BP)(S)~(BP) VI

[0192] Tn another embodiment, the invention provides an isolated fusion protein, wherein the fusion protein is of formula VII (components as described above): 10193] (XTEN)-(S)-(BP)-(8)s-(BP)-(XTEN) VII

[0194] In some cases, the BP can comprise a first fragment and a second cytokine fragment, and the XTEN is positioned between the first fragment and the second fragment. When desired, the BP can be cytokine. In some cases, the cytokine can be 11-10.

[0195] In the foregoing embodiments of fusion proteins of formulas I-V1l, administration of a therapeutically effective dose of a fusion protein of an embodiment 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 more spent within a therapeutic window for the fusion protein compared 10 the corresponding BP not linked to the XTEN of and administered at a comparable dose to a subject.

[0196] Any spacer sequence group is optional in the fusion proteins encompassed by the invention. The spacer may be provided to enhance expression of the fusion protein from a host cell or to decrease steric hindrance such that the BP component may assume its desired tertiary structure and / or interact appropriately with its target molecule. For spacers and methods of identifving desirable spacers, see, for example, George, et al. (2003) Protein Engineering 15:871-879. specifically incorporated by reference herein. In one embodiment, the spacer comprises one or more peptide sequences that are between 1-50 amino acid residues in length, or about 1-25 residues, or about 1-10 residues in length. Spacer sequences, exclusive of cleavage sites, can comprise any of the 20 natural L amino acids, and will preferably comprise hydrophilic amino acids that are sterically unhindered that can include, but not be limited to, glycine (G), alanine (A), serine (S). threonine (T), glutamate (E) and proline (P). In some cases, the spacer can be polyglycines or polyalanines, or is predominately a mixture of combinations of glycine and alanine residues. The spacer polypeptide exclusive of a cleavage sequence is largely to substantially devoid of secondary structure. In one embodiment, one or both spacer sequences in a BPXTEN fusion protein composition may cach further contain a cleavage sequence, which may be identical or may be different, wherein the cleavage sequence may be acted on by a protease to release the BP from the fusion protein.

[0197] In some cases, the incorporation of the cleavage sequence into the BPXTEN is designed to permit release of a BP that becomes active or mare active upon its release from the XTEN. The cleavage sequences are located sufficiently close to the BP sequences, generally within 18, or within 12, or within 6, or within 2 amino acids of the BP sequence terminus, such that any remaining residues attached to the BP after cleavage do not appreciably interfere with the activity (e.g. such as binding to a receptor) of the BP, yet provide sufficient access to the protease to be able to effect cleavage of the cleavage sequence. In some embodiments, the cleavage site is a sequence that can be cleaved by a protease endogenous to the mammalian subject such that the BPXTEN can be cleaved after administration to a subject. In such cases, the BPXTEN can serve as a prodrug or a circulating depot for the BP. Examples of cleavage sites contemplated by the invention include, but are not limited to, a polypeptide sequence cleavable by a mammalian endogenous protease selected from FXIa, FX1Ia, kallikrein, FVIla, F1Xa, FXa, Flla (thrombin), Elastase-2, granzyme B, MMP-12, MMP-13, MMP-17 or MMP-20, or by non-mammalian proteases such as TEV, enterokinase, PreScission™ protease {rhinovirus 3C protease), and sortase A. Sequences known to be cleaved by the foregoing proteases are known in the art. Exemplary cleavage sequences and cut sites within the sequences are presented in Table 5, as well as sequence variants. For example, thrombin (activated clotting factor II) acts on the sequence LTPRSLLV (SEQ ID NO: 230) [Rawlings N.D., et al. (2008) Nucleic Acids Res., 36: D320], which would be cut after the arginine at position 4 in the sequence. Similarly, incorporation of other sequences into BPXTEN that are acted upon by endogenous proteases would provide for sustained release of BP that may, in certain cases, provide a higher degree of activity for the BP from the “prodrug” form of the BPXTEN.

[0198] In some cases. only the two or three amino acids flanking both sides of the cut site (four to six amino acids total) would be incorporated into the cleavage sequence. In other cases, the known cleavage sequence 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, resulting in an ability to tailor the rate of release of the BP from the XTEN. Exemplary substitutions are shown in Table 5. Table 5: Protease Cleavage Sequences : SEQ ID Exemplary SEQ ID Protease Act % Co . Urn Son NO Suey No Minimal Cut Site* quence FXla 274 KLTRVVVGG 869 KD / FL / T / RYVA / VE / GT / GV EXIla 275 TMTRYIVGG NA NA Kallikrein 276 SEFRYSTGG 870 - / - / FL / RYVSR / RT / - / ~ I FVlia T2717 1 1ovrRytvee | NAT NA 1 aA she Ade VARY Ly ser Glial FIXa 278 PLGRYIVGG 871 - / = / G / RY= / =]~]~ FXa 279 IEGRITVGE 872 1a / E / GFP / RVSTI / VFS / - / G Flla (thrombin) 280 LTPRYSLLV 873 ~ / - / e1a / Rlsac / - / - / - S—————————. S——— —————— Elastase-2 281 LGPVIseve 874 fm) VIAN f= =) = Granzyme-B 282 VAGDVSLEE 875 Vf [DN fmf) ~ MMP-12 283 GPAGYLGGA 876 G / PA / - / GVL / - / G / - mmr mm ee NA i age a FEET APLGVLRLR t 878 I - / p8 / ~ / =v1.0 / - / LT / ~ 1 287 | ewntvyroda fF 870 TEV AvlaraL mas a ARE AAT ARRAY oh AREF Yl FRA A= - AE Ee TE ah ATA Ee VI F Enterokinase f 288 + DDDRYIVGE + 988 f DDDKY IVGE { Enterokinase ! 288 |! DpDpprYIves | 28% Protease 3C 867 867 (PreScission™) | LEVLEQIGE LEVLEQIGP Sortase A 868 LPKTVGSES 880 L / P / KEAD / TYG / ~ / EKS / S pm l——t Tete wt, Sore ETE I Vindicates cleavage site NA: not applicable * the listing of multiple amino acids before, 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

[0199] In another aspect, the disclosure provides fusion protein comprising multiple release segment (RS) wherein each RS sequence is selected from the group of sequences set forth in Table 6 and the RS are linked to each other by 1 to 6 amino acids selected from glycine, serine, alanine, and threonine. In one erabodiment, the fusion protein comprises a first RS and a second RS different from the first RS wherein each RS sequence is selected from the group of sequences set forth in Table 6 and the RS are linked to each other by 1 to 6 amino acids selected from glycine, serine, alanine, and threonine. In another embodiment, the fusion protein comprises a first RS, a second RS different from the first RS, and a third RS different from the first and the second RS wherein cach sequence is selected from the group of sequences set forth in Table 6 and the first and the second and the third RS are linked to each other by 1 to 6 amino acids selected from glycine, serine, alanine, and threonine. Tt is specifically intended that the multiple RS of the fusion protein can be concatenated to form a sequence that can be cleaved by multiple proteases at different rates or efficiency of cleavage. In another embodiment, the disclosure provides fusion protein comprising an RS1 and an RS2 selected from the group of sequences set forth in Tables 6 and 7 and an XTEN1 and XTEN2 selected from current disclosure wherein the RS1 is fused between the XTENI and the binding moieties and the RS2 is fused between the XTEN2 and the binding moieties. Tt is contemplated that such compositions would be more readily cleaved by diseased target tissues that express multiple proteases, compared with healthy tissues or when in the normal circulation, with the result that the resulting fragments bearing the binding moieties would more readily penetrate the target tissue: ¢.g.. a tumor, and have an enhanced ability to bind and tink the target cell and the effector cell (or just the target cell in the case of fusion protein designed with a single binding moiety. In some embodiments, where the composition of this disclosure (such as a fusion protein) comprises a release segment, the release segment (RS) can have at least 82%, at least 88%, at least 94%, or 100% sequence identity to a sequence selected from the sequences set forth in Tables 6-7. Tn some embodiments, the composition of this disclosure (such as a fusion protein) can have a structural arrangement, from N- to C-terminus of XTEN-RS- cviokine or cytokine-RS-XTEN. Table 6. Release Segment Sequences. BSRS-6 AC1604 BSRS-Al-1 AC1605 BSRS-A2-1 AC 1606 BSRS-A3-1 | ACle07 RSR-1512 AC1610 RSR-1517 AC1611 VP-2 AC1612 RSR-I018 AC1613 RSR-1053 | AC1614 RSR-1167 ACl617 RSR-1201 ACI6I18 RSR-1218 ACL619 RSR-1226 | ACIL1620 RSR-1261 AC1623 RSR-1293 AC1624 RSR-1309 AC1625 RSR-1326 AC1626 RSR-134% | AC1627 RSR-1478 AC1630 RSR-1479 AC1631 RSR-1496 AC1632 RSR-1508 | ACl633% RSR-1524 AC1636 RSR-1622 AC1637 RSR-1629 AC1638 RSR-1664 AC1639 RSR-1667 | AC1640 Name Construct ID | Amino Acid Sequence BSRS-1 BSRS-6 AC1604 LAGRSDNHEPLELVAG (SEQ ID NG: 22) BSRS-Al-1 AC1605 ASCRSTNAGPSGLAGP (S5Q ID NG: 23) BSRS-A2-1 AC1606 ASGRSTNAGPQGLAGQ (SEQ ID NC: 24! RSR-1752 AC1609 ASSRTTNTGPSTLTGP (SEQ 1D NO: 27) RSR-1512 ACI610 AAGRSDNGTPLELVAP (5EQ ID NO: 28) RSR-1517 ACI61L EAGRSANHEPLGLVAT (SEG ID NO: 298) VP-2 AC1612 ASGRGTNAGPAGLTGP (SEC IT NC: 30) RSR-1018 AC1613 LFGRNDNHEPLELGGE (SEQ IT NO: 31} RSR-1059 AC1615 LDGRSDNFHPPELVAG (SEQ IL NO: 33} RSR-1167 ACI617 LKGRSDNNAPLALVAG (SEQ ID NG: 35) RSR-1201 ACI6I8 VYSRGTNAGPHGLYTGR (S5Q ID NG: 36) RSR-1218 ACL619 ANSRGTNKGFAGLIGP (SEQ ID NG: #7) RSR-1256 ACl622 ESSRGINIGQGGLTGP (5uQ ID NO: 40) RSR-1261 AC1623 SSSRGINQDPAGLTIP (5EQ ID NO: 41) RSR-1293 AC1624 ASSRGONHSPMGLTGR (SEG ID NO: 42) RSR-1309 AC1625 AYSRGPNAGPAGLEGR (SEC IT NO: 43) RSR-1326 AC1626 ASERGNNAGPANLTGY (SEC IT NO: 44) RSR-1354 AC1628 MSSRRTNANPAQLTGP (SEQ ID NO: 4€) RSR-1478 AC1630 LAGRSENTAPLELTAG (SEQ ID NG: 4%) RSR-1479 AC1631 LEGRPDONHEPLALVAS (SEQ ID NC: 4%) RSR-1496 AC1632 LSGRSDNEEPLALPAG (SEQ ID NG: 50) RSR-1516 ACI633 LSGRSDNEAPLELEAG (55g 1D NO: 53) RSR-1524 AC1636 LGGRADNHEPPELGAG (3EQ 1D NO: 54) RSR-1622 AC1637 PPSRGINAEPAGLIGE (55¢ ID NO: 55) RSR-1629 AC1638 ASTRGENAGPAGLEAP (SEC IT NO: 56) RSR-1664 AC1639 ESSRGTNGAFEGLTGP (SEC IT NO: 57) RSR-1709 ACl641 ASSRGENPPPGGLTGP (SEQ IL NO: 58) RSR-1727 ACL643 AGSRTTNAGPGGLEGP (SEC ID NG: 61: RSR-1754 ACl644 RSR-1819 AC 1645 RSR-1832 | ACl|6d6 RSR-1951 AC1650 RSR-2295 AC1761 RSR-2298 AC1762 ESGRAANTTPAGLTEP (SEC ID NC: 70% RSR-2038 | AC1679 TTGRATEAANLTPAGLTGP (SES ID NO: 71% RSR-2302 AC1682 TTGRATEAANATPAGLTGP (SED ID NO: 74) RSR-3047 AC1697 RSR-3082 AC 1698 RSR-3043 AC 1699 RSR-3041 | ACIL700 RSR-3058 AC1703 RSR-2485 AC1763 RSR-2486 AC1764 RSR-2488 | AC1688 RSR-2707 AC1717 RSR-2708 ACIL7I8 RSR-270% ACITI9 RSR-2710 ACIT20 RSR-2711 + AC1721 RSR-2714 AC1724 RSR-2715 AC1725% RSR-2716 AC1726 RSR-2717 | AC1727 RSR-2720 AC1730 RSR-2721 ACI1731 RSR-2722 ACI1T732 RSR-2723 ACI1T733 RSR-2724 | ACI1734 "RSR.27T27 LL AC1737 RSR-2727 AC1737 RSR-2728 AC1738 RSR-2729 AC1739 RSR-2730 | AC1740 RSR-2733 AC1743 RSR-2734 AC1744 RSR-2735 AC 17458 RSR-2737 ACI1747 RSR-2738 | AC|1748 RSR-2742 AC1752 RSR-2743 AC1753 RSR-2744 AC1754 RSR-274% | AC17%5 RSR-2748 AC1758 RSR-2749 ACL1759 RSR-2750 AC 1760 RSR-2781 AC L765 RSR-2754 | ACIR01 RSR-2757 AC1804 RSR-2758 AC1805% RSR-2759 AC1806 RSR-2760 | ACI1807 RSR-2763 ACIR10 RSR-2764 ACISI1 RSR-2765 ACI812 RSR-2766 ACI813 RSR-2767 | ACISI4 RSR-2770 ACI1817 RSR-2771 ACISIR RSR-2772 AC1819 RSR-2773 | AC1820 [Name [ConstructID | Amino AcidSequence ~~ = | RSR-2736 | _ACI746 RSR2757 _ _ACIT47 RSR-2740 ACI750 TTGRAGGAANLABTOLTEP (SLy iD Wo: 420) RSR-2742 ACL752 RSR2743 _ _ACI753 RSR-2744 _ _ACI7SI RSR-2747 AC1757 TYGRTTTPGNEPPASLEAR (SEQ 1D 80: a7) | RSR-2748 | _ACI7SK RSR2749 _ _ACI759 RSR-2750 _ _ACI760 RSR-2751 ACT765 RSR2755 | ACI802 | TTGRAGEAANLIBAALTES (Sig 1b Wo: 133) RSR2757 | _ACI804 RSR-2758 _ __ACL80 RSR-2759 _ __ACI806 | RSR2760_ |" ACI8(7 _ | TTGRAGEAANLTPEPLIGA (SEC ID NO: 3%) RSR2762 | ACIS09 | TTGRAGEAANLTPEGLTEA Su 15 Noi 40) RSR-276) | _ACISI0 RSR-2764 _ _ACISIL RSR-2765 _ _ACISI2 RSR-2766 _ _ACISI3 RSR2768 | ACISLS | TTGRAGEAANLIPEALTGG (Sig 1b Wo: 146) RSR2770 | _ACISL7 RSR-2771 _ __ACISIS RSR-2772 _ _ACI819 Table 7. Release Segment Sequences RSN-0002 RSN-0003 RSN-0004 RSN-O005 RSN-0006 RSN-0007 RSN-0009 RSN-0010 RSNA0011 RSN-0012 RSN0013 RSN-0014 RSN-0015 RSN-0016 RSN-0017 RSN-0018 RSN-0019 RSN-0020 RSN-D021 RSN-0022 RSN-0023 RSN-0D024 RSN-D025 RSN-D026 RSN-0027 RSN-0028 RSN-0029 RSN-030 RSN-0031 RSN-032 RSN-0033 RSN-0034 ons i RSN-0022 eeu mALIGME RSC.0022 IYI LIARS RSN-0023 SN SSWIATSSWETP RSC0023 a RSN-0024 a ammnioe RSC-0024 i RSN-0025 EN TIMSTMATSIEI RSC0025 TE RL AELRAONITY RSN-0028 NN SoTMTSSWEIR RSC-0028 Tama a RSN-0036 RSN-0037 RSN-O038 RSN-0039 RSN-0040 RSN-0041 RSN-0042 RSN-0043 RSN-0044 RSN-0045 RSN-0046 RSN-0047 RSN-D048 RSN-D049 RSN-D050 RSN-D0O51 RSN-D052 RSN-D053 RSN-D054 RSN-0055 RSN-056 RSN-057 RSN-0058 RSN-0059 RSN-0060 RSN-0061 or PE RSN-0049 yap vmuewE RSC:0049 yw ugane RSN-0050 EN oWATSSWET® RSC0050 Tama i RSN-0051 ls RSC-0051 wav RSN-0052 ni raed RSC0052 i RSN-0055 EN ATSRWETR RSC-0055 Tama pa GGAAPG oo a — RSN-0063 RSN-0064 RSNAO065 RSN-0066 RSN-0067 RSN-0068 RSN-0069 RSN-0070 RSN-0071 RSN-0072 RSN-0O073 RSN-0074 RSN-D0OT5 RSN-D0T6 RSN-D077 RSN-DOT8 RSN-D079 RSN-D0S0O RSN-D0OS1 RSN-0082 RSN-0083 RSN-0084 RSN-0085 RSN-086 RSN-00R7 RSN-088 RSN-0076 en wsewe RSC0076 ry unwrRRy RSN-0077 maT RSC0077 i all Pre RR Tcl re RSN-0079 oy mwATSIER RSC0079 SE STIICITGLIGIENNTR RSN-0082 A PoomATSIERR RSC-0082 EN auRsvEIIe RSN-0090 RSN-0091 RSN-0092 RSN-0093 RSN-0094 RSN-0095 RSN-0096 RSN-1517 VP-1 RSN-1752 RSN-1512 RSN-1517 VP-2 RSN-1018 RSN-1053 RSN-1059 RSN-10635 RSN-1167 RSN-1201 RSN-1218 RSN-1226 RSN-1254 RSN-1256 Amine Acid Sequence Amino Acid Sequence GSAPGGTGGAGNLVRGGPAATSGSETP GTAEAASASGGGTGGAGNLVRGGPARG GSAPGEAGGAPNLVRGGSIATSGSETP GTAEAASASGGEAGGAPNLVRGGSIPG RSN-00%0 | Gp 55g 10 vo: 424) RSCO0%0 | spismg 10 wor e72; ji GSAPGPGGGAVNLVRGGTAATSGSETP GTAEAASASGGPGGGAVNLVRGGTARG GSAPGSEGGALNLVRGGAIATSGSETR GTARAASASGGSEGGALNLVRGGAIRG GSAPGTPGGASNLVRGGGAATSGSETP GTAEAASASGGTPGGASNLVRGGGAPG GSAPGASGGATNLVRGGEIATSGSETP GTAEAASASGGASGGATNLVRGGEIPG . | GSAPGGTGGAQNLVRGGPRATSGSETR on GTARAASASGGGIGGAQNLVRGGPARG GSAPGEAGGAENLVRGGSIATSGSETR GTARAASASGGEAGGAENLVRGGSIRG RSN-00% | gr (ss 10 vo: 430) RSCO09 | 5p (sug 1p No: €78) | GSAPEAGRSANHEPLGLVATATSGSET 1517 | GTARAASASGEAGRSANHEPLGLVATR OY GSAPASGRSTNAGPSGLAGPATSGSET cnc al | GTARAASASGASGRSINAGPSGLAGPR BSRS-AL2 | par (sig 10 wo: 432) BSRS-ALS | gop sg 10 vo: €80) oe GSAPASGRSTNAGPQGLAGQATSGSET coc 2 | GTAEAASASGASGRSTNAGPQGLAGQP SRS-A2- A2- BSRS-A22 | par (sug 1p vo: 433) BSRS-A23 | gep sug in vo: es) oc ax | GSAPASGRSTNAGPPGLTGPATSGSET coc sx | GTAEAASASGASGRSTNAGPPGLTGPP VB GSAPASSRGTNAGPAGLIGPATS GSET vel GTAEAASASGASSRGINAGPAGLIGRP PGT (SEQ ID NO: 435} GSP {SEQ ID No: €83) ny 1m GSAPASSRTTNTGPSTLTGPATSGSET oo GTAEAASASGASSRTTNTGPSTLTGEP RSN-1752 | pgp (sxq 1p no: 436) RSC1752 | gop (smo 10 nox esd) < GSAPAAGRSDNGTPLELVAPATSGSET oo GTAEAASASGAAGRSDNGTPLELVAPP RSN-I512 | pgp (sxq 1p wo: 437) RSC-1512 | gop (sup 10 no: 93) o ., | GSAPEAGRSANHEPLGLVATATSGSET i 1xim | CTAEAASASGEAGRSANHEPLGLVATP RSN-I317 | pa isxg 1p no: 436) RSC-IS17 | gap issg to vor 696) _— GSAPASGRGTNAGPAGLTGPATSGSET _— GTAEAASASGASGRGTNAGPAGLTGEP - PGT (SEQ ID NO: 438) = GSP SEQ ID No: €87) « GSAPLFGRNDNHEPLELGGGATSGSET oo GTAEAASASGLFGRNDNHEPLELGGGP RSN-1018 | pgp (sxq 1p no: 440) RSC-1018 | gop (sup 10 ox esa) o GSAPTAGRSDNLEPLGLVFGATSGSET » GTAEAASASGTAGRSDNLEPLGLVFGP RSN-10S3 | pay isre 1p no: ssi; RSC-I083 | gap isco vo vor a99) . GSAPLDGRSDNFHPPELVAGATSGSET GTAEAASASGLDGRSDNFHPPELVAGP LOA CLAD RSN-109 | pep sxe 1p no: 442; RSC-1089 | gap sco ovo: avn) . | GSAPLEGRSDNEEPENLVAGATSGSET . | GTAEARSASGLEGRSDNEEPENLVAGP RSN-1065 | par ise 15 no: 443; RSC-1065 | gop iseo 10 wo: 650) RSN-1167 | CSAPLEGRSDNNAPLALVAGATSGSET | poi). | OTAEAASASGLKGRSDNNAPLALVAGP PUT | PeTisEQ ID NO: 444) TM | espisEe TD No: 651) GSAPVYSRGTNAGPHGLTGRATSGSET GTAEAASASGVY SRGTNAGPHGLTGRE N-12X 2 RSN-1201 | pgp srg 10 no: 443) RSC-1201 | gop iseg 15 no: 883) RSN-1218 | CSAPANSRGINKGFAGLIGPATSGSET | co, | GTAEAASASGANSRGINKGFAGLIGPR Dr PGT (SEQ ID NO: 446) oe GSP {SEQ ID NO: 894) GSAPASSRLTNEAPAGLTIPATSGSET GTAEAASASGAS SRLTNEAPAGLTIPP N-12; 12 RSN-1226 | pag seo 15 nos 447; RSC-I26 | gop iseo 15 wo: 69%) GSAPDQSRGTNAGPEGLTDPATSGSET GTAEAASASGDQSRGTNAGPEGLTDPP N-12. J GSAPESSRGTNIGQGGLTGPATSGSET p GTAEAASASGES SRGTNIGQGGLTGPP N-12 12 RSN-1293 RSN-1309 RSN-1326 RSN-1345 RSN-1354 RSN-1426 RSN-1478 RSN-1479 RSN-1496 RSN-1508 RSN-1513 RSN-1516 RSN-1524 RSN-1622 RSN-1629 RSN-1664 RSN-1667 RSN-1709 RSN-1712 RSN-1727 RSN-1754 RSN-I819 RSN-1832 RSN-1855 RSN-1911 RSN-1929 RSN-162 | er any oer | RsCelez2 | gp RR ART REERGLTGED MO a EE RIN-I66 | BC an dag ont | RSCal6G | GR on PRGLGRD RSN-1667 | Soier To nor gent RSET | Ge payee RSN-1727 SSN EAR TINA SOL RIPALEISRT RSC-1727 GIAEAASAS! GASSRTINMIDIGLEIRE PGT (SEC ID NO: 470; GSP SEQ ID No: 7.8) RSN-229% RSN-2038 RSN-2072 RSN-2089 RSN-2302 RSN-3047 RSN-3052 RSN-3043 RSN-3041 RSN-3044 RSN-3057 RSN-3058 RSN-2485 RSN-2486 RSN-2488 RSN-2399 RSN-2706 RSN-2707 RSN-2708 RSN-2709 RSN-2710 RSN-2711 RSN-2712 RSN-2713 RSN-2714 rv im RSN-2485 i RSC-2485 ay waw RSN-2486 pip id RSC-2456 I pvasuTar® RN2488 | DE or dag oot | RSCdS8 | ggpnir eI TS ER SOLEGR RSN-2T08_| Corre to vor aan | RSC | er ran RSN-2717 RSN-2718 RSN-2719 RSN-2720 RSN-2721 RSN-2722 RSN-2723 RSN-2724 RSN-2725 RSN-2726 RSN-2727 RSN-2728 RSN-2729 RSN-2730 RSN-2731 RSN-2732 RSN-2733 RSN-2734 RSN-2735 RSN-2736 RSN-2737 RSN-2738 RSN-2739 RSN-2740 RSN-2741 Amine Acid Sequence Amino Acid Sequence RSN-271s | CSAPATGRPGEPANTIPEGLEGPATSG | ci, o | GTAEAASASGATGRPGEPANTTPEGLE ak SETPGT (SEQ 1D NO: 504) et GPPGSP (SEQ ID NO: 752) GSAPSTGRSGEPANATPGGLTGPATSG GIAEAASASGSTGRSGEPANATPGGLT N-27 27 RSN2716 | spear (seg 10 wo: 505) RSC-2716 | Gppasp (seg 1p wo: 753) GSAPPTGRGGEGANTIPTGLEGPATSG GTAEAASASGPIGRGGEGANTTPTGLE N-27 27 GSAPPTGRSGEGANATPSGLIGPATSG GTARAASASGPTGRSGEGANATPSGLT RSN-2718 | cpmpar azo 10 No: 5% RSC-2718 SEQ ID NO: 755) SEQ ID NO: 507) GPPGSP (SEQ ID NO: 755) GSAPTTGRASEGANSTPAPLTEPATSG GIAEAASASGTTGRASEGANSTPAPLT N-27 27 RSN-2719 | sprpar (seg 10 no: 508) RSC-2719 | gppasp (seg 1p wo: 756) RIE FERPA ARARR ER ARES RSC2720 | CLARAASASGIYGRAARAANTTPAGLT (SEQ 1D NO: 509) APPGSP (SEQ ID NO: 757) GSAPTTGRATEGANATPAELTEPATSG on GTARAASASGTTGRATEGANATPAELT RSN-2721 ee Tr Tit RSC2721 ip SETPGT (SEQ 1D NO: 510) EPPGSP (SEQ ID NO: 753) RSN-2722 | CORPTVGRASERANTIPASLIGEATSG | po), | CTARAASASGIVGRASEERNTTPASLY SETPGT (SEQ 1D NO: 511) GPPGSP (SEQ ID NO: 759) GSAPTTGRAPEAANATPAPLTGPATSG om GTARAASASGTTGRAPEAANATPARLT RSN-2723 | gperpar (920 1p NG: 5-2 R8C-2723 TSEC. IT Wis D801 (JEG ID NC: 512) GPPGSP (SEQ ID NO: 760) RSN-2724 | CSAPTWGRATEPANATPAPLUSPAYSG | poi.) | GTAEAASASGUWGRATEPANATPAFLT SETPGT (SEQ 1D NO: 513) SPPGSP (SEQ ID NO: 761) GSAPTVGRASESANATPAELTSPATSG GTAEAASASGTVGRASESANATPAELT N-2723 8-27 RSN-2725 | sprpar (sig 1p no: 514) RSC-2725 | 5ppasp (seg 1p wo: 752) GSAPTVGRAPEGANSTPAGLTGPATSG GTAEAASASGTVGRAPEGANSTPAGLT N-272 $0.07 RSN-2726 | sprpor (se 10 No: 5:9) RSC-2726 | gepose (sre nwo: 763) RSN.2727 | GSAPTWGRATEAPNLEPATLYTPATSG | oc... | GIAEAASASGIWGRATEAPNLEPATLT SNelSl | SETPGT (SEQ ID NG: 518) “4'50 | TPPGSP (SEQ ID NO: 784) oom GSAPTTGRATEAPNLTPAPLTEPATSG oo GTAEAASASGTTGRATEAPNLTPAPLT RSN-2728 | smrpor (sme 1o No: 57) RSC-2728 | popgsp srg In no: 765) < GSAPTQGRATEAPNLSPARLTSPATSG on GTAEAASASGTQGRATEAPNLSPAALT N-2728 $C-2728 po RSN-2729 | smrper(sze io No: 5:4) RSC-2729 | spposp srg Ib no: 76%) cn.ymap | GSAPTQGRAREAPNLTPATLTAPATSG oma | GIAEAASASGTQGRAAEAPNLTPATLT RSN-2730 | sgeper (sie io No: 5.6) RSC2730 | appar sre 10 wo: 767 cn.ymn; | GSAPTSGRAPEATNLAPAPLTGPATSG omy | GCTAEAASASGTSGRAPEATNLAPAPLT RSN-2T3L | speper (sae 10 No: 520) RSC | gppgap srg 10 no: 768) cn ons | GSAPTQGRAAEAANLTPAGLTEPATSG oo | GTAEAASASGTQGRAAEAANLTPAGLT RSN-2732 | smrper(sze 1p No: 520) RSC-2732 | ppposp srg Ib no: 763) cn.ymaz | GSAPTTGRAGSAPNLPPTGLTIPATSG mn | GIAEAASASGTTGRAGSAPNLPPTGLT RSN-2733 | speper (sie io no: 52) RSC2733 | yppgap srg 10 wo: 770) cn ons | GSAPTTGRAGGAENLPPEGLTAPATSG ov: | GTAEAASASGTTGRAGGAENLPPEGLT RSN-273 | speper (sie io No: 523) RSC2734 | appar srg 10 no: 771 RSN-2735 | CSAPTISRAGIATNLTPEGLTAPATSG | "| GIAEAASASGTTSRAGTATNLIPEGLT PRET | spTPGT SEQ TH NG: 524) “07 | APPGSP (SEQ ID No: 772) +c | GSAPTTGRAGTATNLPPSGLTTPATSG GTAEAASASGTTGRAGTATNLPPSGLT RSN2736 | spapey (sz ro no: 525) RSC-2736 | yppagp seg tn wo: 773; GSAPTTARAGEAENLSPSGLIAPATSG GTAEAASASGTTARAGEAENLS PSGLT 3 - RSN2737 | spapariseg 10 no: 526) RSC-2737 | appesp seg 10 no: 7743 ~e | GSAPTTGRAGGAGNLAPGGLTEPATSG GTAEAASASGTTGRAGGAGNLAPGGLT | GSAPTTGRAGTATNLPPEGLTGPATSG GTAEAASASGTTGRAGTATNLPPEGLT RSN2739 | spapey (sz ro no: 528) RSC-2739 | gppasp seg tn wo: 776; GSAPTTGRAGGAANLAPTGLIEPATSG GTAEAASASGTTGRAGGAANLAPTGLT GSAPTTGRAGTAENLAPSGLITPATSG GTAEAASASGTTGRAGTAENLAPSGLT RSN-2743 RSN-2744 RSN-2745 RSN-2746 RSN-2747 RSN-2748 RSN-2749 RSN-2750 RSN-2751 RSN-2754 RSN-2755 RSN-2756 RSN-2757 RSN-2758 RSN-2759 RSN-2760 RSN-2761 RSN-2762 RSN-2763 RSN-2764 RSN-2765 RSN-2766 RSN-2767 RSN-2768 RSN-2769 RSN-2770 RST | re Nor ta | rscarss | Ge ee RSN-2772 RSN-2773 RSN-3047 RSN-2783 RSN-3107 RSN-3103 RSN-3102 RSN-3119 RSN-3043 RSN-2780 RSN-3109 RSN-3110 RSN-3111 RSN-3112 RSN-3113 RSN-3114 RSN-3115 RSN-3125 RSN-3126 RSN-3127 RSN-3131 RSN-3132 RSN-3133 RSN-3138 SETPGT (3EQ ID NO: 558) EGPGSP {SEQ ID NO: 80) SETPGT (3EQ ID NO: 560) EGPGSP {SEQ ID NO: 808) RSN-3112 amy RSC1 0 ae SoLIRraeeY Ba | res | sama | ar oe RIN | ar arts Toons | RSC | or rosey RSN-3115 mappa aii RSC 15 SE umNmLOSROGSI® RSN-3127 or Cr owERATIONRY RSC-3127 Mo surssemue®

[0200] The RS of the disclosure are useful for inclusion in recombinant polypeptides as therapeutics for treatment of cancers, autoimmune diseases, inflammatory diseases and other conditions where localized activation of the recombinant polypeptide is desirable. The subject compositions address an unmet need and are superior in one or more aspects including enhanced terminal half-life, targeted delivery, and improved therapeutic ratio with reduced toxicity to healthy tissues compared to conventional antibody therapeutics or bispecific antibody therapeutics that are active upon injection.

[0201] Iv one erubodiment. a BP incorporated into a BPX TEN fusion protein can have a sequence that exhibits at least about 80% sequence identity to a sequence from Table 3 or Table A, alternatively at least about 81%, or about 82%, or about 83%, or about 84%, or about 83%, or about 86%, or about 87%, or about 88%6, or about 89%, 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 about 100% sequence identity as compared with a sequence froma Table 3 or Table A. The BP of the foregoing embodiment can be evaluated for activity using assays or nacasured or determined parameters as described herein, and those sequences 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 activity compared to the corresponding native BP sequence would be considered suitable for inclusion in the subject BPXTEN. The BP found to retain a suitable level of activity can be linked to one or more XTEN polypeptides described hereinabove. In one embodiment, a BP found to retain a suitable level of activity can be linked to one or more XTEN polypeptides having at least about 80% sequence identity to a sequence from Tables 2a-2b, 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 with a sequence of Tables 2a-2b, resulting in a chimeric fusion protein.

[0202] The disclosure contemplates substitution of other BP selected from Table 3 or Table A linked to one or two XTEN, which may be the same or different, selected from Tables 2a-2b. In the foregoing fusion proteins hereinabove described in this paragraph, the BPXTEN fusion protein can further comprise a cleavage sequence from Table 5; the cleavage sequence being located between the BP and the XTEN or between adjacent BP. Tn some cases, the BPXTEN comprising the cleavage sequences will also have one or more spacer sequence amino acids between the BP and the cleavage sequence or the XTEN and the cleavage sequence to facilitate access of the protease; the spacer amino acids comprising any natural amino acid, including glycine and alanine as preferred amino acids. Targeting Moieties

[0203] In certain embodiments, it is contemplated that the XPACs of the present invention also may further comprise a tumor targeting moiety that allows the XPAC to bind to an antigen expressed on the tumor. This can be achieved by including one further domain in the chimeric polypeptide (XPAC) to influence its movements within the body. For example, the chimeric nucleic acids can encode a domain that directs the polypeptide to a location in the body, e.g. tursor cells or a site of inflammation. Exemplary and suitable targeting moieties domains comprise those that have a cognate ligand that is overexpressed in inflamed tissues, e.g., the IL-1 receptor, or the IL-6 receptor. In other embodiments, the suitable targeting moieties comprise those who have a cognate ligand that is overexpressed in tumor tissue, ¢.g., Epcam, CEA or mesothelin. To some embodiments, the targeting domain is linked to the cytokine via a linker which is cleaved at the site of action (e.g., by inflammation or cancer specific proteases) releasing the cytokine full activity at the desired site. In some embodiments, the targeting and / or retention domain is linked to the interleukin via a linker which is not cleaved at the site of action (c.g. by inflammation or cancer specific proteases), causing the cytokine to remain at the desired site.

[0204] Particularly preferred targeting moieties target antigens expressed on the surface of a diseased cell or tissue, for example a tumor or a cancer cell. Antigens useful for tumor targeting and retention include but are not limited to EpCAM, EGFR, HER-2, HER-3, c-Met, FOLRI, and CEA. Pharmaceutical compositions disclosed herein, also include proteins comprising two targeting and / or retention domains that bind to two different target antigens known to be expressed on a diseased cell or tissue. Exemplary pairs of antigen binding domains include but are not limited to EGFR / CEA, EpCAM / CEA, and HER-2 / HER-3.

[0205] Suitable targeting moieties include antigen-binding domains, such as antibodies and fragments thereof including, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single chain variable fragment (scFv), single-domain antibody such as a heavy y chain variable domain (VH), a light chain variable domain {VL) and a variable domain of camelid-type nanobody (VHH), a dAb and the like. Other suitable antigen-binding domain include non-immunoglobulin proteins that mimic antibody binding and / or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers. DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocallin and CTLA4 scaffolds. Further examples of antigen-binding polypeptides include a ligand for a desired receptor, a ligand-binding portion of a receptor, a lectin, and peptides that binds to or associates with one or more target antigens

[0206] In some embodiments, the targeting moieties specifically bind to a cell surface molecule. In some embodiments, the targeting and / or retention domains specifically bind to a tumor antigen. In some embodiments, the targeting polypeptides specifically and independently bind to a tumor antigen selected from at least one of Fibroblast activation protein alpha (FAPa), Trophoblast glycoprotein (ST4), Tumor-associated calcium signal transducer 2 (Trop2), Fibronectin EDB (EDB-FN, see US Publication 20200397915), fibronectin EIB domain, CGS-2. EpCAM. EGFR, HER-2. HER-3, ¢cMet, CEA, and FOLRL In some embodiments, the targeting polvpeptides specifically and independently bind to two different antigens, wherein at least one of the antigens is a tumor antigen selected from EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FOLRI

[0207] The targeted antigen can be a tumor antigen expressed on a tumor cell. Tumor antigens are well known in the art and include, for example, EpCAM, EGFR, HER-2, HER-3. c-Met, FOLR1, PSMA, CD38, BCMA, and CEA. 5T4, AFP, B7-H3, Cadherin-6, CAIX, CD117, CDI123, CDI138, CD166, CD19, CD20, CD203, CD22, CD30, CD33, D332, CD37, CD44, CD32, CD36, CD70, CD71, CD74, CD79, DLL3, EphA2, FAP, FGFR2, FGFR3. GPC3, gpA33, FLT-3, gpNMB, HPV-16 E6, HPV-16 E7, ITGA2, ITGA3, SLC39A6, MAGE, mesothelin, Mucl. Muc16, NaPi2b, Nectin-4, P-cadherin, NY-ESO-1, PRLR, PSCA, PTK7, ROR1, SLC44A4, SLTRKS, SLTRK6, STEAPI, TIMI, Trop2, WT1.

[0208] The targeted antigen can be an immune checkpoint protein. Examples of immune checkpoint proteins include but are not limited to CD27, CD137, 2B4. TIGIT, CD155, ICOS, HVEM, CD4OL, LIGHT. TIM-1, 0X40, DNAM-|, PD-L1, PDL, PD-L2, CTLA-4, CDS, CD40, CEACAMI, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA. [DO1, IDO2. TDO. KIR, LAG-3, TIM-3. or VISTA.

[0209] The targeted antigen can be a cell surface molecule such as a protein, lipid or polysaccharide. In some embodiments, such an antigen on a tumor cel, virally infected cell, bacterially infected cell, damaged red blood cell, arterial plaque cell, inflamed or fibrotic tissue cell. Such an antigen can comprise an immune response modulator such as for example, including but not limited to granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 12 (IL-12), interleukin 15 (IL-15), B7-1 (CD80), B7-2 (CD86), GITRL, CD23, or GITR. Pharmacokinetic Properties of BPXTEN 10210] The invention provides BPXTEN fusion proteins with enhanced pharmacokinetics compared to the BP not linked to XTEN that, when used at the dose determined for the composition by the methods described herein, can achieve a circulating concentration resulting in a pharmacologic effect, yet stay within the safety range for biologically active component of the composition for an extended period of time compared to a comparable dose of the BP not linked to XTEN. In such cases, the BPXTEN remains within the therapeutic window for the fusion protein composition for the extended period of time. As used herein, a “comparable dose” means a dose with an equivalent moles / kg for the active BP pharmacophore that is administered to a subject in a comparable fashion. Tt will be understood in the art that a "comparable dosage" of BPXTEN fusion protein would represent a greater weight of agent but would have essentially the same mole-equivalents of BP in the dose of the fusion protein and / or would have the same approximate molar concentration relative to the BP

[0211] The pharmacokinetic properties of a BP that can be enhanced by linking a given XTEN to the BP include terminal half-life, area under the curve (AUC), Cruax volume of distribution, and bioavailability

[0212] As described more fully in the Exarsples pertaining to pharmacokinetic characteristics of fusion proteins comprising XTEN, it was surprisingly discovered that increasing the length of the XTEN sequence could confer a disproportionate increase in the terminal half-life of a fusion protein comprising the XTEN. Accordingly, the invention provides BPXTEN fusion proteins comprising XTEN wherein the XTEN can be selected to provide a targeted half-life for the BPXTEN composition administered to a subject. In some embodiments, the invention provides monomeric fusion proteins comprising XTEN wherein the XTEN is selected to confer an increase in the terminal half-life for the administered BPXTEN, compared to the corresponding BP not linked to the fusion protein, of at least about two-fold longer, or at Jeast 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 greater an increase in terminal half-life compared to the BP not linked to the fusion protein. Similarly. the BPXTEN fusion proteins can have an increase in AUC of at least about 50%, or at least about 60%. or at least about 70%, or at feast about 80%. or at least about 90%, or at Jeast about 100%, or at least about 150%, or at least about 200%, or at least about 300% increase in AUC compared to the corresponding BP not linked to the fusion protein. The pharmacokinetic parameters of a BPXTEN can be determined by standard methods involving dosing, the taking of blood samples at times intervals, and the assaying of the 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.

[0213] The invention further provides BPXTEN comprising a first and a second BP molecule, optionally separated by a spacer sequence that may further comprise a cleavage sequence, or separated by a second XTEN sequence. In one embodiment, the BP has less activity when linked to the fusion protein compared 0 a corresponding BP not linked to the fusion protein. In such case, the BPXTEN can be designed such that upon administration to a subject, the BP component is gradually released by cleavage of the cleavage sequences), whereupon it regains activity or the ability to bind to its target receptor or ligand. Accordingly, the BPXTEN of the foregoing serves as a prodrug or a circulating depot, resulting in a longer terminal half-life compared to BP not linked to the fusion protein.

[0214] As described herein, in exemplary embodiments, the BPXTEN is an XPAC in which the BP is a cytokine. In preferred embodiments, the activity of the cytokine polypeptide in the context of the XPAC is attenuated, and protease cleavage at the desired site of activity, such as in a tumor microenvironment, releases a form of the cytokine from the XPAC that is much more active as a cytokine receptor agonist than the XPAC For example. the cytokine-receptor activating (agonist) activity of the fusion polypeptide can be at least about 10 times, at least about 50 times, at least about 100 times, at least about 250 times, at least about 500 times. or at least about 1000 times less than the cytokine receptor activating activity of the cytokine polypeptide as a separate molecular entity. The cytokine polypeptide that is part of the XPAC exists as a separate molecular entity when it contains an amino acid that is substantially identical to the cytokine polypeptide and does not substantially include additional amino acids and is vot associated (by covalent or non-covalent bonds) with other molecules. If necessary, a cytokine polypeptide as a separate molecular entity may include some additional amino acid sequences, such as a tag or short sequence to aid in expression and / or purification.

[0215] In other examples, the cytokine-receptor activating (agonist) activity of the fusion polypeptide is at least about 0 times, at least about 50 times, at least about 100 times, at least about 230 times, at least about 500 times, or about 1000 times less than the cytokine receptor activating activity of the polypeptide that contains the cytokine polypeptide that is produced by cleavage of the protease cleavable linker in the XPAC. In other words. the cytokine receptor activating (agonist) activity of the polypeptide that contains the cytokine polypeptide that is produced by cleavage of the protease cleavable linker in the XPAC is at least about 10 times, at least about 50 times, at least about 100 times, at least about 250 times, at least about 500 times, or at least about 1000 times greater than the cytokine receptor activating activity of the XPAC. Pharmacology and Pharmaceutical Properties of BPXTEN

[0216] The present invention provides BPXTEN compositions comprising BP covalently linked to XTEN that can have enhanced properties compared to BP not linked to XTEN, as well as methods to enhance the therapeutic and / or biologic activity or effect of the respective two BP components of the compositions. In addition, the invention provides BPXTEN compositions with enhanced properties compared to those art- known fusion proteins containing immunoglobulin polypeptide partners. polypeptides of shorter length and / or polypeptide partners with repetitive sequences. In addition, BPXTEN fusion proteins provide significant advantages over chemical conjugates, such as pegylated constructs, notably the fact that recombinant BPXTEN fusion proteins can be made in bacterial cell expression systems, which can reduce time and cost at both the research and development 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 BPXTEN compared to pegylated conjugates.

[0217] As therapeutic agents, the BPXTEN may possess a number of advantages over therapeutics not comprising XTEN including, for example, increased solubility, increased thermal stability, reduced immunogenicity, increased apparent molecular weight, reduced renal clearance, reduced proteolysis, reduced metabolism, enhanced therapeutic efficiency, a lower effective therapeutic dose, increased bioavailability, increased time between dosages to maintain blood levels within the therapeutic window for the BP, a “tailored” rate of absorption, enhanced Ivophilization stability, enhanced serum / plasma stability, increased terminal half- life, increased solubility in blood stream, decreased binding by neutralizing antibodies, decreased receptor- mediated clearance, reduced side effects, retention of receptor / ligand binding affinity or receptor / ligand activation, 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 net effect of the enhanced properties is that the BPXTEN may result in enhanced therapeutic and / or biologic effect when administered to a subject with a metabolic disease or disorder.

[0218] In other cases where, where enhancement of the pharmaceutical or physicochemical properties of the BP is desirable, (such as the degree of aqueous solubility or stability), the length and / or the motif family composition of the first and the second XTEN sequences of the first and the second fusion protein may each be selected to confer a different degree of solubility and / or stability on the respective fusion proteins such that the overall pharmaceutical properties of the BPXTEN composition are enhanced. The BPXTEN fusion proteins can be constructed and assaved, 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 XTEN sequence of the BPXTEN is selected such that the fusion protein has an aqueous solubility that is within at least about 25% greater compared to a BP 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 BP not linked to the fusion protein. In the erabodiments hereinabove described in this paragraph. the XTEN of the 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% sequence identity to an XTEN selected from Tables 2a-2b.

[0219] In one embodiment. the invention provides BPXTEN compositions that can maintain the BP component within a therapeutic window for a greater period of time compared to comparable dosages of the corresponding BP not linked to XTEN. It will be understood in the art that a "comparable dosage" of BPXTEN fusion protein would represent a greater weight of agent but would have the same approximate mole- equivalents of BP in the dose of the fusion protein and / or would have the same approximate molar concentration relative to the BP. 10220] The invention also provides methods to select the XTEN appropriate for conjugation to provide the desired pharmacokinetic properties that, when matched with the selection of dose, enable increased efficacy of the administered composition by maintaining the circulating concentrations of the BP within the therapeutic window for an enhanced period of time. As used herein. “therapeutic window” means that amount of drug or biologic as a blood or plasma concentration range, that provides efficacy or a desired pharmacologic effect over time for the disease or condition without unacceptable toxicity: the range of the circulating blood concentrations between the minimal amount to achieve any positive therapeutic effect and the maximum amount which results in a response that is the response immediately before toxicity to the subject (at a higher dose or concentration). Additionally. therapeutic window generally encompasses an aspect of time; the maximum and minimum concentration that results in a desired pharmacologic effect over time that does not result in unacceptable toxicity or adverse events. A dosed composition that stays within the therapeutic window for the subject could also be said to be within the “safety range.”

[0221] Dose optimization is important for all drugs, especially for those with a narrow therapeutic window. For example, many peptides involved in glucose homeostasis have a narrow therapeutic window. For a BP with a narrow therapeutic window, such as glucagon or a glucagon analog, a standardized single dose for all patients presenting with a variety of symptoms may not always be effective. Since different glucose regulating peptides are often used together in the treatment of diabetic subjects, the potency of each and the interactive effects achieved by combining and dosing ther together must also be taken into account. 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 BPXTEN, versus that amount that would result in unacceptable toxicity and place it outside of the safety range.

[0222] In many cases, the therapeutic window for the BP components of the subject compositions have been established and are available in published literature or are stated on the drug label for approved products containing the BP. In other cases, the therapeutic window can be established. The methods for establishing the therapeutic window for a given compasition are known to those of skill in the art (see. ¢.g.. Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11% Edition, McGraw-Hill (2005)). For example, by using dose-escalation studies in subjects with the target disease or disorder to determine efficacy or a desirable pharmacologic effect, appearance of adverse events, and determination of circulating blood levels, the therapeutic window for a given subject or population of subjects can be determined for a given drug or biologic, or combinations of biologics or drugs. The dose escalation studies can evaluate the activity of a BPXTEN through metabolic studies in a subject or group of subjects that monitor physiological or biochemical parameters, as known in the art or as described herein for one or more parameters associated with the metabolic disease or disorder, or clinical parameters associated with a beneficial outcome for the particular indication, together with observations and / or measured parameters to determine the no effect dose, adverse events, maximum tolerated 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 parameters 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 above which toxicity occurs, thereby establishing the therapeutic window for the dosed therapeutic. Blood concentrations of the fusion protein (or as measured by the BP component) above the maximum would be considered outside the therapeutic window or safety range. Thus, by the foregoing methods, a Cun blood level would be established, below which the BPXTEN fusion protein would not have the desired pharmacologic effect, and a Cmax blood level would be established that would represent the highest circulating concentration before reaching a concentration that would elicit unacceptable side effects, toxicity or adverse events, placing it outside the safety range for the BPXTEN. With such concentrations established, the frequency of dosing and the dosage can be further refined by measurement of the Car and Cuin to provide the appropriate dose and dose frequency to keep the fusion protein(s) within the therapeutic window. One of skill in the art can, by the means disclosed herein or by other methods known in the art, confirm that the administered BPXTEN remains in the therapeutic window for the desired interval or requires adjustment in dose or length or sequence of XTEN. Further, the determination of the appropriate dose and dose frequency to keep the BPXTEN within the therapeutic window establishes the therapeutically effective dose regimen; the schedule for administration of multiple consecutive doses using a therapeutically effective dose of the fusion protein to a subject in need thereof resulting in consecutive Ciux peaks and / or Can troughs that remain within the therapeutic window and results in an improvement in at least one measured parameter relevant for the target disease, disorder or condition. In some cases, the BPXTEN administered at an appropriate dose to a subject may result in blood concentrations of the BPXTEN fusion protein that remains within the therapeutic window for a period at least about two-fold longer compared to the corresponding BP not linked to XTEN and administered at a comparable dose: alternatively at least about three-fold longer; alternatively at least about four-fold longer; alternatively at least about five-fold longer; alternatively at least about six-fold longer; alternatively 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 or greater compared to the corresponding BP 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 subject. would result in a desirable therapeutic or pharmacologic effect and a blood concentration within the therapeutic window.

[0223] In one embodiment, the BPXTEN administered at 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; alternatively at least about five-fold longer; alternatively at least about six-fold longer; alternatively at least about seven-fold longer; alternatively at least about cight-fold longer; alternatively at least about nine-fold longer or at least about ten-fold longer between at least two consecutive Cmax peaks and / or Coma troughs for blood levels of the fusion protein compared to the corresponding biologically active protein of the fusion protein not linked to the fusion protein and administered at a comparable dose regimen to a subject. In another embodiment, the BPXTEN administered at a therapeutically effective dose regimen results in a comparable improvement in one, or two, or three or more measured parameter using less frequent dosing or a lower total dosage in moles of the fusion protein of the pharmaceutical composition compared to the corresponding biologically active protein component(s) not linked to the fusion protein and administered to a subject using a therapeutically effective dose regimen for the BP. The measured parameters may include any of the clinical, biochemical, or physiological parameters disclosed herein, or others known in the art for assessing subjects with glucose- or imsulin-related disorders, metabolic diseases or disorders, coagulation or bleeding disorders, or growth hormone-related disorders.

[0224] The activity of the BPXTEN compositions of the invention, including functional characteristics or biologic and pharmacologic activity and parameters that result, may be determined by any suitable screening assay known in the art for measuring the desired characteristic. The activity and structure of the BPXTEN polypeptides comprising BP components may be measured by assays described herein, or by methods known in the art to ascertain the degree of solubility, structure and retention of biologic activity. Assays can be conducted that allow determination of binding characteristics of the BPXTEN for BP receptors or a ligand, including binding constant (Kg), ECs values, as well as their half-life of dissociation of the ligand-receptor complex (Ty). Binding affinity can be measured. for example, by a competition-type binding assay that detects changes in the ability to specifically bind to a receptor or ligand. Additionally, techniques such as flow cytometry or surface plasmon resonance can be used to detect binding events. The assays may comprise soluble receptor molecules, or may determine the binding to cell-expressed receptors. Such assays may include cell-based assays, including assays for proliferation, cell death, apoptosis and cell migration. Other possible assays may determine receptor binding of expressed polypeptides, wherein the assay may comprise soluble receptor molecules, or may determine the binding to cell-expressed receptors. The binding affinity of a BPXTEN for the target receptors or ligands of the corresponding BP can be assayed using binding or competitive binding assays, such as Biacore assays with chip-bound receptors or binding proteins or ELISA assays, as described in US Patent 5,534,617, assays described in the Examples herein, radio-receptor assays, or other assays known in the art. In addition, BP sequence variants (assayed as single components or as BPXTEN fusion proteins) can be compared to the native BP using a competitive ELISA binding assay to determine whether they have the same binding specificity and affinity as the native BP, or some fraction thereof such that they are suitable for inclusion in BPXTEN.

[0225] The invention provides isolated BPXTEN in which the binding affinity for BP target receptors or ligands by the BPXTEN can be at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 30%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%. or at least about 95%, or at least about 99%, or at least about 100% or more of the affinity of a native BP not bound to XTEN for the target receptor or ligand. In some cases, the binding affinity Kg between the subject BPXTEN and a native receptor or ligand of the BPXTEN is at least about 10M, alternatively at least about 10% M, altematively at least about 10° M, or at least about 10” M of the affinity between the BPXTEN and a native receptor or ligand. 10226] In some embodiments, where a composition of this disclosure (such as a fusion protein) comprises a cylokine, a binding activity of the cytokine (when linked to an XTEN in the fusion protein) to a corresponding cytokine receptor can be characterized by a half maximal effective concentration (EC50) at least (about) 1.1 fold greater, at least (about) 1.2 fold greater. at least (about) 1.3 fold greater, at least (about) 1.4 fold greater, at least (about) 1.5 fold greater, at least (about) 1.6 fold greater, at least (about) 1.7 fold greater, at least (about) 1.8 fold greater, at least (about) 1.9 fold greater, or at least (about) 2.0 fold greater than an EC50 characterizing a corresponding binding activity of the cytokine (when not linked to the XTEN). In some embodiments, a binding activity of the cytokine (when linked to an XTEN in the fusion protein) to a corresponding cytokine receptor can be characterized by a half maximal effective concentration (EC30) of (about) 1.1 fold greater, (about) 1.2 fold greater, (about) 1.3 fold greater, (about) 1.4 fold greater. (about) 1.5 fold greater. (about) 1.6 fold greater, (about} 1.7 fold greater, (about) 1.8 fold greater, (about) 1.9 fold greater, or (about) 2.0 fold greater, or a range between any two of the foregoing, than an EC50 characterizing a corresponding binding activity of the cytokine (when not linked to the XTEN). In some embodiments, the EC30 vatue(s) can be determined in an in virro binding assay. In some embodiments, the cytokine can be interleukin 12 (IL-12), and the corresponding cytokine receptor can be an interleukin 12 receptor (IL-12R). In some embodiments, the in vitro binding assay can utilize a genetically engineered reporter gene cell line configured to respond to binding of the cytokine to the corresponding cytokine receptor with a proportional expression of a reporter protein. The term “ECs” generally refers to the concentration needed to achieve half of the maximum biological response of the active substance, and can be generally determined by ELISA or cell-based assays, including the methods of the Examples described herein. In some embodiments, the in vitro binding assay can be a reporter gene activity assay (such as one disclosed in Example 8). For example, an exemplary reporter gene activity assay can be based on genetically engineered cell(s), generated by stably introducing relevant gene(s) for the receptor(s)-of-interest and the signaling pathway (s)-of-interest, such that binding to the engineered receptor triggers a signaling cascade leading to the activation of the engineered gene pathway with a subsequent production of signature polypeptide(s) (such as an enzyme).

[0227] Tn other cases, the invention provides isolated BPXTEN in which the fusion protein is designed to bind with high affinity to a target receptor, thereby resulting in antagonistic activity for the native ligand. A non-limiting example of such a BPXTEN is IL-1raX TEN, which is configured to bind to an IL-1 receptor such that the bound composition substantially interferes with the binding of TL-1 « and / or TL-1p to IL-1 receptor. In certain cases, the interference by an antagonist BPXTEN (such as, but not limited to IL-1raX TEN) with the binding the native ligand to the target receptor can be at least about 1%, or about 10%, or about 20%, or about 30%, or about 40%, or about 30%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 99%, or about 100%. In other embodiments, the invention provides isolated BPXTEN fusion proteins (such as, but not limited to IL.-1raX TEN) wherein the binding of the isolated fusion protein to a cellular receptor elicits less than 20%, or less than 10%, or less than 5% activation of the signaling pathways of the cell with bound BPXTEN antagonist in comparison to those evoked by the native ligand. In other cases, the antagonistic BPXTEN compositions bind to the target receptor with a dissociation constant of about 10 nM or less, about SnM or less, about 1 nM or less, about 500 pM or less, about 250 pM or less, about 100 pM or less, about 50 PM or less, or about 25 pM or less. Non-limiting examples of specific constructs of antagonistic BPXTEN can include IL-1ra-AMS873, [L-1ra-AE864, or IL-1ra-AM 1296.

[0228] In some cases, the BPXTEN fusion proteins of the invention retain at least about 10%, or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%. 98%, or 99% percent of the biological activity of the corresponding BP not linked to the fusion protein with regard to an in vitro biologic activity or pharmacologic effect known or associated with the use of the native BP in the treatment and prevention of metabolic conditions and disorders. In some cases of the foregoing embodiment. the activity of the BP component may be manifest by the intact BPX TEN fusion protein, while in other cases the activity of the BP component would be primarily manifested upon cleavage and release of the BP from the fusion protein by action of a protease that acts on a cleavage sequence incorporated into the BPXTEN fusion protein. In the foregoing. as illustrated in FIG. 3A- FIG. 3E, the BPXTEN can be designed to reduce the binding affinity of the BP component for the receptor or ligand when linked to the XTEN but have increased affinity when released from XTEN through the cleavage of cleavage sequence(s) incorporated into the BFXTEN sequence, as described more fully above.

[0229] In other cases, the BPXTEN are designed to reduce the binding affinity of the BP component when linked to the XTEN to, for example, increase the terminal half-life of BPXTEN administered to a subject by reducing receptor-mediated clearance or to reduce toxicity or side effects due to the administered composition. Where the toxicological no-effect dose or blood concentration of a BP not linked to an XTEN is low (meaning that the native peptide has a high potential to result in side effects), the invention provides BPXTEN fusion proteins in which the fusion protein is configured to reduce the biologic potency or activity of the BP component.

[0230] In some cases. it has been found that a BPXTEN can be configured to have a substantially reduced binding affinity (expressed as Kd) and a corresponding reduced bioactivity, compared to the activity of a BPXTEN wherein the configuration does not result in reduced binding affinity of the corresponding BP component, and that such configuration is advantageous in terms of having a composition that displays both a long terminal half-life and retains a sufficient degree of bioactivity. Linking a single XTEN to the C-terminus of a BP (e.g., [L-10) can result in the retention of significant binding affinity to its target receptor, linking an XTEN to the N-terminus decreases its binding affinity and corresponding biological activity, compared to constructs where the XTEN is bound to the C-terminus. In another example, it has been found. as described in the Examples, that while linking of BP to the C-terminus of an XTEN molecule does not substantially interfere with the binding to the BP receptors, the addition of a second XTEN to the C-terminus of the same molecule (placing the second XTEN to the C-terminus of hGH) reduced the affinity of the molecule to the BP receptor and also resulted in an increase in terminal half-life of the XTEN-BP-XTEN configuration compared to XTEN-BP configuration. The ability to reduce binding affinity of the BP to its target receptor may be dependent on the requirement to have a free N- or C-terminus for the particular BP. Accordingly, the invention provides a method for increasing the terminal half-life of a BPX TEN by producing a single-chain fusion protein construct with a specific N- to C-terminus configuration of the components comprising at least a first biologically active protein and one or more XTEN, wherein the fusion protein in a first N- to C-terminus configuration of the biologically active protein and XTEN components has reduced receptor-mediated clearance (RMC) and a corresponding increase in terminal half-life compared to a BPXTEN in a second N- to C-terminus configuration. In one embodiment of the foregoing, the BPX TEN ts configured, N- to C-terminus as BP-XTEN. In another embodiment of the foregoing, the BPXTEN is configured XTEN-BP. In another embodiment of the foregoing. the BPXTEN is configured XTEN-BP-XTEN. In the latter embodiment, the two XTEN molecules can be identical or they can be of a different sequence composition or length. Non- limiting exaruples of the foregoing embodiment with twa XTEN linked to a single BP. Non-lumiting examples of the foregoing embodiment with one BP linked to one XTEN include AM875-IL-1ra, AE864-IL-1ra, AMS75-IL10, or AES64-IL10. The invention contemplates other such constructs in which a BP from Table 3 or Table A and XTEN from Tables 2a-2b are substituted for the respective components of the foregoing examples, and configured such that the construct has reduced receptor mediated clearance compared to an alternate configuration of the respective components.

[0231] Tn some cases, the method provides configured BPXTEN in which the reduced receptor mediated clearance can result in an increase in the ternunal half-life of at least two-fold, or at least three-fold, or at least four-fold, or at least five-fold compared to the half-life of a BPXTEN in a second configuration where RMC is not reduced. The invention takes advantage of BP ligands wherein reduced binding affinity to a 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 BP, an XTEN, or a spacer sequence. The choice of the particular configuration of the BPXTEN fusion protein can reduce the degree of binding affinity to the receptor such that a reduced rate of receptor-mediated clearance can be achieved. Generally, activation of the receptor is coupled to RMC such that binding of a polypeptide to its receptor without activation does not lead to RMC, while activation of the receptor leads to RMC. However, in some cases, particularly where the ligand has an increased off rate, the ligand may nevertheless be able to bind sufficiently to initiate cell signaling without triggering receptor mediated clearance, with the net result that the BPXTEN remains bioavailable. In such cases, the configured BPXTEN has an increased half-life compared to those configurations that lead to a higher degree of RMC.

[0232] In cases where a reduction in binding affinity is desired in order to reduce receptor-mediated clearance but retention of at least a portion of the biological activity is desired, it will be clear that sufficient binding affinity to obtain the desired receptor activation must nevertheless be maintained. Thus. in one embodiment, the invention provides a BPXTEN configured such that the binding affinity of the BPXTEN for a target receptor is in the range of about 0.01%-40%, or about 0.1%-30%, or about 19-20% of the binding affinity compared to a corresponding BPXTEN in a configuration wherein the binding affinity is not reduced. The binding affinity of the configured BXTEN is thus preferably reduced by at least about 80%. or at least about 85%, or at least about 90%, or at least about 953%, or at least about 99%, or at least about 99.9%, or at least about 99.99% as compared to the binding affinity of a corresponding BPXTEN in a configuration wherein the binding affinity of the BP component to the target receptor is not reduced or compared to the BP not linked to the fusion protein, determined under comparable conditions. Expressed differently, the BP component of the configured BPXTEN may have a binding affinity that is as small as about 0.01%. or at least about 0.1%, or at least about 1%, or at least about 2%, or at least about 3%, or at least about 4%, or at least about 5%, or at least about 10%, or at least about 20% of that of the corresponding BP component of a BPXTEN in a configuration wherein the binding affinity of the BP component is not reduced. In the foregoing embodiments hereinabove described in this paragraph. the binding affinity of the configured BPXTEN for the target receptor would be “substantially reduced” compared to a corresponding native BP or a BPXTEN with a configuration in which the binding affinity of the corresponding BP component is not reduced. Accordingly, the present invention provides compositions and methods to produce compositions with reduced RMC by configuring the BPXTEN 50 as to be able to bind and activate a sufficient number of receptors to obtain a desired in vivo biological response yet avoid activation of more receptors than is required for obtaining such response. In one embodiment, the BPXTEN is configured such that the subject BP is at the N-terminus of the BPXTEN, wherein the RMC of the administered BPXTEN is reduced compared to a BPXTEN configured with the subject BP linked to the C-terminus of an XTEN and at least a portion of the biological activity of the native BP is retained. In another embodiment, the BPXTEN is configured such that the subject BP is at the C-terminus of the BPXTEN, wherein the RMC of the administered BPXTEN is reduced compared to a BPXTEN configured with the subject BP is at the N-terminus of the BPXTEN and at least a portion of the biological activity of the native BP is retained. In another embodiment, the BPXTEN is configured, N- to C-terminus, as XTEN-BP- XTEN, wherein the RMC of the adnunistered BPX TEN is reduced compared to a BPXTEN configured with one XTEN and at least a portion of the biological activity of the native BP is retained. It will be apparent to one of skill in the art that other configurations to achieve this property are contemplated by the invention; e.g., addition of a second molecule of the BP or a spacer sequence. In the foregoing embodiments hereinabove described in this paragraph, the half-life of the BPXTEN can be increased at least about 50%, or at least about 75%, or at least about100%, or at least about 150%, or at least about 200%, or at least about 300% compared to a BPXTEN configured wherein the binding affinity and RMC of the BP component is not reduced. In the foregoing embodiments hereinabove described in this paragraph, the increased half-life can permit higher dosages and reduced frequency of dosing compared to BP not linked to XTEN or compared to BPXTEN configurations wherein the BP component retains a binding affinity to the receptor comparable to the native BP

[0233] Specific in vive and ex vivo biological assays may also be used to assess the biological activity of each configured BPXTEN and / or BP component to be incorporated into BPXTEN. For example, the increase of insulin secretion and / or transcription from the pancreatic beta cells can be measured by methods known in the art. Glucose uptake by tissues can also be assessed by methods such as the glucose clamp assay and the like. Other in vivo and ex vivo parameters suitable to assess the activity of administered BPXTEN fusion proteins in treatment of metabolic diseases and disorders include fasting glucose level, peak postprandial glucose level, glucose homeostasis, response to oral glucose tolerance test, response to insulin challenge, HA, caloric intake, satiety, rate of gastric emptying. pancreatic secretion, insulin secretion, peripheral tissue insulin sesitivity, beta cell mass, beta cell destruction, blood lipid levels or profiles. body mass index, or body weight. Based on the results of these assays or other assays known in the art, the BPXTEN configuration or composition can be confirmed or, if needed, adjusted and re-assayed to confirm the target binding affinity or biologic activity.

[0234] Specific assays and methods for measuring the physical and structural properties of expressed proteins are known in the art, including methods for determining properties such as protein aggregation, solubility, secondary and tertiary structure, melting properties, contamination and water content, ete. Such methods include analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion, HPLC-reverse phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-size exclusion, [R, NMR, Raman spectroscopy, refractometry, and UVVisible spectroscopy. Additional methods are disclosed in Amau et al, Prot Expr and Purif (2006) 48, 1- 13. Application of these methods to the invention would be within the grasp of a person skilled in the art. USES OF THE COMPOSITIONS OF THE PRESENT INVENTION 10235] In another aspect, the invention provides a method of for achieving a beneficial effect in a disease, disorder or condition mediated by BP. The present invention addresses disadvantages and / or limitations of BP that have a relatively short terminal half-life and / or a narrow therapeutic window between the minimum effective dose and the maximum tolerated dose.

[0236] In one embodiment. the invention provides a method for achieving a beneficial effect in a subject comprising the step of administering to the subject a therapeutically- or prophylactically-effective amount of aBPXTEN. The effective amount can produce a beneficial effect in helping to treat a disease or disorder. In some cases, the method for achieving a beneficial effect can inclade administering a therapeutically effective amount of a BPXTEN [fusion protein composition to treat a subject with.

[0237] In one embodiment, the method comprises administering a therapeutically-effective amount of a pharmaceutical composition comprising a BPXTEN fusion protein composition comprising a BP linked to an XTEN sequence(s) and at least one pharmaceutically acceptable carrier to a subject in need thereof that results in greater improvement in at least one parameter, physiologic condition, or clinical outcome mediated by the BP component(s) compared to the effect mediated by adnunistration of a pharmaceutical composition comprising a BP not linked to XTEN and administered at a comparable dose. In one embodiment, the pharmaceutical composition is administered at a therapeutically effective dose. In another embodiment, the pharmaceutical composition is administered using multiple consecutive doses using a therapeutically effective dose regimen (as defined herein) for the length of the dosing period.

[0238] As a result of the enbanced PK parameters of BPXTEN, as described herein, the BP may be administered using longer intervals between doses compared to the corresponding BP not linked to XTEN to prevent, treat, alleviate, reverse or ameliorate symptoms or clinical abnormalities of the metabolic disease, disorder or condition or prolong the survival of the subject being treated.

[0239] The methods of the invention may include administration of consecutive doses of a therapeutically effective amount of the BPXTEN for a period of time sufficient to achieve and / or maiutain the desired parameter or clinical effect. and such consecutive doses of a therapeutically effective amount establishes the therapeutically effective dose regimen for the BPXTEN; e.g. the schedule for consecutively administered doses of the fusion protein composition, wherein the doses are given in therapeutically effective amounts to result in a sustained beneficial effect on any clinical sign or symptom, aspect, measured parameter or characteristic of a metabolic disease state or condition, including, but not limited to, those described herein.

[0240] A therapeutically effective amount of the BPXTEN may vary according to factors such as the disease state, age, sex. and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the BPXTEN are outweighed by the therapeutically beneficial effects. A prophylactically effective amount refers to an amount of BPXTEN required for the period of time necessary to achieve the desired prophylactic result.

[0241] For the inventive methods, longer acting BPXTEN compositions are 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 effect. In one embodiment. a method of treatment comprises administration of a therapeutically effective dose of a BPXTEN to a subject in need thereof that results in a gain in time spent within a therapeutic window established for the fusion protein of the composition compared to the corresponding BP component(s) not linked to the fusion protein and administered at a comparable dose to a subject. In some cases, the gain in time spent within the therapeutic window is 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 10-fold, or at least about 20-fold, or at least about 40-fold compared to the corresponding BP component not linked to the fusion protein and administered at a comparable dose to a subject. The methods further provide that administration of multiple consecutive doses of a BPXTEN administered using a therapeutically effective dose regimen to a subject in need thereof can result in a gain in time between consecutive Cmax peaks and / or Cumin troughs for blood levels of the fusion protein compared to the corresponding BP(s) not linked to the fusion protein and administered using a dose regimen established for that BP. In the foregoing embodiment, the gain in time spent between consecutive Cua peaks and / or Cain troughs can be 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 10- fold, or at least about 20-fold, or at least about 40-fold compared to the corresponding BP component(s) not linked to the fusion protein and administered using a dose regimen established for that BP. In the embodiments hereinabove described in this paragraph the administration of the fusion protein can result in an improvement in at least one of the parameters (disclosed herein as being useful for assessing the subject diseases, conditions or disorders) using a Jower unit dose in moles of fusion protein compared to the corresponding BP component(s) not linked to the fusion protein and administered at a comparable unit dose or dose regimen to a subject.

[0242] In one embodiment, the BPXTEN can have activity that results in an improvement in one of the clinical, biochemical or physiologic parameters that is greater than the activity of the BP component not linked to XTEN, determined using the same assay or based on a measured clinical parameter. In another embodiment, the BPXTEN can have activity in two or more clinical or metabolic-related parameters (e.g., glucose homeostasis and weight control in a diabetic subject, or reduced prothrombin and bleeding times in a hemophiliac subject, or increased muscle mass and bone density in a growth-hormone deficient subject), each mediated by one of the different BP that collectively result in an enhanced effect compared the BP component not linked to XTEN., determined using the same assays or based on measured clinical parameters. In another embodiment, administration of the BPXTEN can result in activity in one or more of the clinical or biochemical or physiologic pararseters that is of longer duration than the activity of one of the single BP components not linked to XTEN, determined using that same assay or based on a measured clinical parameter.

[0243] In some embodiments, the present disclosure provides a method of treating or preventing a disease or condition in a subject, the method comprising administering to a subject a therapeutically effective amount of a fusion protein or a composition comprising the fusion protein, all of which are disclosed herein. In some embodiments, the disease or condition can be a cancer, or a cancer-related disease or condition. or an inflammatory or autoimmune disease. In sore embodiments, the disease or condition can be a cancer, or a cancer-related disease or condition. In some embodiments, the disease or condition can be a cancer or a cancer- related disease or condition. Where desired, the subject fusion and composition can be used in conjunction with a therapeutically effective amount of at least one immune checkpoint inhibitor.

[0244] The invention further contemplates that BPXTEN used in accordance with the methods provided herein may be administered in conjunction with other treatment methods and pharmaceutical compositions useful for treating cancer, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, Alzheimer’s disease, Schizophrenia, viral infections (e.g., chronic hepatitis C, AIDS). allergic asthma, retinal newrodegenerative processes. metabolic disorder, insulin resistance, and diabetic cardiomyopathy. inflammatory conditions and autoimmune conditions.

[0245] In some cases, the administration of a BPXTEN may permit use of lower dosages of the co- administered pharmaceutical composition to achieve a comparable clinical effect or measured parameter for the disease, disorder or condition in the subject.

[0246] The foregoing notwithstanding. in certain embodiments, the BPXTEN used in accordance with the methods of the present invention may prevent or delay the need for additional treatment methods or use of drugs or other pharmaceutical compositions in subjects with glucose-related diseases, metabolic diseases or disorders, coagulation disorders, or growth-hormone deficiency or growth disorders. In other embodiments, the BPXTEN may reduce the amount, frequency or duration of additional treatment methods or drugs or other pharmaceutical compositions required to treat the underlying disease. disorder or condition.

[0247] In another aspect, the invention provides a method of designing the BPXTEN compositions with desired pharmacologic or pharmaceutical properties. The BPXTEN fusion proteins are designed and prepared with various objectives in mind (compared to the BP components not linked to the fusion protein), including improving the therapeutic efficacy for the treatment of metabolic diseases or disorders, enhancing the pharmacokinetic characteristics of the fusion proteins compared to the BP, lowering the dose or frequency of dosing required to achieve a pharmacologic effect, enhancing the pharmaceutical properties. and to eshance the ability of the BP components to remain within the therapeutic window for an extended period of time.

[0248] In general, the steps in the design and production of the fusion proteins and the inventive compositions may, as illustrated in FIGS, 4-6, include: (1) the selection of BPs (e.g., native proteins, peptide hormones, peptide analogs or derivatives with activity. peptide fragments, etc.) to treat the particular disease, disorder or condition; (2) selecting the XTEN that will confer the desired PK and physicochemical characteristics on the resulting BPXTEN (e.g, the administration of the composition to a subject results in the fusion protein being maintained within the therapeutic window for a greater period compared to BP not linked to XTEN}; (3) establishing a desired N- to C-terminus configuration of the BPXTEN to achieve the desired efficacy or PK pararseters; (4) establishing the design of the expression vector encoding the configured BPXTEN: (5) transforming a suitable host with the expression vector; and (6) expression and recovery of the resultant fusion protein. For those BPXTEN for which an increase in half-life (greater than 16 h) or an increased period of time spent within a therapeutic window is desired, the XTEN chosen for incorporation will generally have at least about 500, or about 376, or about 864, or about 875, or about 913, or about 924 amino acid residues where a single XTEN is to be incorporated into the BPXTEN. In another embodiment, the BPXTEN can comprise a first XTEN of the foregoing lengths, and a second XTEN of about 144, or about 288, or about 576, or about 864. or about 875, or about 913, or about 924 amino acid residues.

[0249] In other cases, where in increase in half-life is not required, but an increase in a pharmaceutical property (e.g., solubility) is desired, a BPXTEN can be designed to include XTEN of shorter lengths. In some embodiments of the foregoing, the BPXTEN can comprise a BP linked to an XTEN having at least about 24, or about 36, or about 48, or about 60, or about 72, or about 84, or about 96 amino acid residues, in which the solubility of the fusion protein under physiologic conditions is at least three-fold greater than the corresponding BP not linked to XTEN., or alternatively, at least four-fold, or five-fold, or six-fold, or seven-fold, or eight-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 glucagon not linked to XTEN. In still other cases. where a half-life of 2-6 hours for a glucagon- containing BPXTEN fusion protein is desired (e.g., in the treatment of nocturnal hypoglycemia), a fusion protein can be designed with XTEN of intermediate lengths such as about 100 amino acids, or about 144 amino acids, or about 156 amino acids, or about 168 amino acids, or about 180 amino acids, or about 196 amino acids in the XTEN component of the glucagon-containing BPX TEN,

[0250] In another aspect, the invention provides methods of making BPXTEN compositions to improve case of manufacture, result in increased stability, increased water solubility, and / or ease of formulation, as compared to the native BPs. In one embodiment, the invention includes a method of increasing the water solubility of a BP comprising the step of linking the BP to one or more XTEN such that a higher concentration in soluble form of the resulting BPXTEN can be achieved, under physiologic conditions, compared to the BP in an un- fused state. Factors that contribute to the property of XTEN to confer increased water solubility of BPs when incorporated into a fusion protein include the high solubility of the XTEN fusion partner and the low degree of self-aggregation between molecules of XTEN in solution. In some embodiments. the method results in a BPXTEN fusion protein wherein the water solubility is at least about 30%, or at least about 60% greater, or at least about 70% greater, or at least about 80% greater, or at least about 90% greater, or at least about 100% greater, 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, or at least about 4000% greater, or at least about 6000% greater under physiologic conditions, compared to the un-fused BP.

[0251] Tn another embodiment, the invention includes a method of enhancing the shelf-life of a BP comprising the step of linking the BP with one or more XTEN selected such that the shelf-life of the resulting BPXTEN is extended compared to the BP in an un-fused state. As used herein, shelf-life refers to the period of time over which the functional activity of a BP or BPXTEN 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 an enzymatic activity, or to display one or more known functional activities associated with a BP, as known in the art. A BP that degrades or aggregates generally has reduced functional activity or reduced bioavailability compared to one that remains in solution. Factors that contribute to the ability of the method to extend the shelf life of BPs when incorporated into a fusion protein include the increased water solubility, reduced self-aggregation in solution, and increased heat stability of the XTEN fusion parter. Iu particular, the low tendency of XTEN to aggregate facilitates methods of formulating pharmaceutical preparations containing higher drug concentrations of BPs, and the heat-stability of XTEN contributes to the property of BPXTEN fusion proteins to remain soluble and functionally active for extended periods. In one embodiment, the method results in BPXTEN fusion proteins with “prolonged” 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 BP. Tu one embodiment, the method includes the step of formulating the isolated BPXTEN with one or more pharmaceutically acceptable excipients that enhance the ability of the XTEN to retain its unstructured conformation and for the BPXTEN to remain soluble in the formulation for a time that is greater than that of the corresponding un-fused BP. Tn one embodiment, the method encompasses linking a BP to an XTEN to create a BPXTEN fusion protein results in a solution that retains greater than about 100% of the functional activity, or greater 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 subjected to the same storage and handling conditions as the standard, thereby enhancing its shelf-life.

[0252] Shelf-life may also be assessed in terms of functional activity remaining after storage, normalized to functional activity when storage began. BPXTEN fusion proteins of the invention with prolonged or extended shelf-life as exhibited bv prolonged or extended functional activity may retain about 30% more functional activity, or about 60%, 70%. 80%, or 90% more of the functional activity of the equivalent BP not linked to XTEN when subjected to the same conditions for the same period of time. For example, a BPXTEN fusion protein of the invention comprising exendin-4 or glucagon fused to a XTEN sequence may retain about 80% or more of its original activity in solution for periods of up to 5 weeks or more under various temperature conditions. Tn some embodiments. the BPXTEN 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 BPXTEN retains at least about 50%, preferably at least about 60%, or at least about 70%, or at least about 80%, or altematively at least about 90% or more of its original activity in solution when heated or maintained at 37°C for about 7 days. Tn another embodiment, BPXTEN fusion protein retains at least about 80% or more of its functional activity after exposure to a teraperature of about 30°C to about 70°C over a period of time of about one hour to about I8 hours. In the foregoing embodiments hereinabove described in this paragraph. the retained activity of the BPXTEN would be 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 BP not linked to the fusion protein. THE DNA SEQUENCES OF THE INVENTION 10253] The present invention provides isolated polynucleic acids encoding BPXTEN chimeric polypeptides and sequences complementary to polynucleic acid molecules encoding BPXTEN chimeric polypeptides, including homologous variants. In another aspect, the invention encompasses methods to produce polynucleic acids encoding BPX TEN chimeric polypeptides and sequences complementary to polysucleic acid molecules encoding BPXTEN chimeric polypeptides, including homologous variants. In general, and as illustrated in FIGS. 4-6, the methods of producing a polynucleotide sequence coding for a BPXTEN fusion protein and expressing the resulting gene product include assembling nucleotides encoding BP and XTEN, linking the components in frame, incorporating the encoding gene into an appropriate expression vector, transforming an appropriate host cell with the expression vector, and causing the fusion protein to be expressed in the transformed host cell. thereby producing the biologically -active BFXTEN polypeptide. Standard recombinant techniques in molecular biology can be used to make the polynucleotides and expression vectors of the present invention.

[0254] In accordance with the invention, nucleic acid sequences that encode BPXTEN may be used to generate recombinant DNA molecules that direct the expression of BPXTEN fusion proteins in appropriate host cells. Several cloning strategies are envisioned to be suitable for performing the present invention, many of which can be used to generate a construct that comprises a gene coding for a fusion protein of the BPXTEN composition of the present invention, or its complement. In one embodiment, the cloning strategy would be used to create a gene that encodes a monomeric BPXTEN that comprises at least a first BP and at least a first XTEN polypeptide, or its complement. In another embodiment, the cloning strategy would be used to create a gene that encodes a monomeric BPXTEN that comprises a first and a second molecule of the one BP and at least a first XTEN (or its complement) that would be used to transform a host cell for expression of the fusion protein used to formulate a BPXTEN composition. Tn the foregoing embodiments hereinabove described in this paragraph, the gene can further comprise nucleotides encoding spacer sequences that may also encode cleavage sequence(s).

[0255] In designing a desired XTEN sequences, it was discovered that the non-repetitive nature of the XTEN of the inventive compositions can be 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 polynucleotides encoding sequence motifs that are then multimerized to create the genes encoding the XTEN sequences (see FIGS. 4 and 5). Thus. while the expressed XTEN may consist of multiple units of as few as four different sequence motifs, because the motifs themselves 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 sequences are non-repetitive. 10256] In one approach, a construct is first prepared containing the DNA sequence corresponding to BPXTEN fusion protein. DNA encoding the BP of the compositions may be obtained from a cDNA library prepared using standard methods from tissue or isolated cells believed to possess BP mRNA and to express it at a detectable level. If necessary, the coding sequence can be obtained using conventional primer extension procedures as described in Sambrook, ez al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transeribed into cDNA. Accordingly, DNA can be conveniently obtained from a cDNA library prepared from such sources. The BP encoding gene(s} may also be obtained from a genomic library or created by standard synthetic procedures known in the art (e.g., automated nucleic acid synthesis) using DNA sequences obtained from publicly available databases. patents, or literature references. Such procedures are well known 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 nebi.nlm nil. gov that correspond to entries in the CAS Registry or GenBank database that contain an amino acid sequence of the BAP or of a fragment or variant of the BAP. For such sequence identifiers provided herein, the summary pages associated with each of these CAS and GenBank and GenSeq Accession Numbers as well as the cited journal publications (e.g., PubMed ID nuwber (PMID) are each incorporated by reference in their entireties, particularly with respect to the anino acid sequences described therein. In one embodiment, the BP encoding gene encodes a protein from any one of Table 3 or Table A, or a fragment or variant thereof.

[0257] A gene or polynucleotide encoding the BP portion of the subject BPXTEN protein, in the case of an expressed fusion protein that will comprise a single BP can then be cloned into a construct. which can be a plasmid or other vector under control of appropriate transcription and translation sequences for high level protein expression in a biological system. Tn a later step, a second gene or polynucleotide coding for the XTEN is genetically fused to the nucleotides encoding the N- and / or C-terminus of the BP gene by cloning it iuto the construct adjacent and in frame with the gene(s) coding for the BP. This second step can occur through a ligation or multimerization step. In the foregoing embodiments hereinabove described in this paragraph. it is to be understood that the gene constructs that are created can alternatively be the complement of the respective genes that encode the respective fusion proteins.

[0258] The gene encoding for the XTEN can be made in one or more steps, either fully synthetically or by synthesis corsbined with enzymatic processes, such as restriction enzyme-mediated cloning, PCR and overlap extension. XTEN polypeptides can be constructed such that the XTEN-encoding gene has low repetitiveness while the encoded amino acid sequence has a degree of repetitiveness. Genes encoding XTEN with non- repetitive sequences can be 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 polynucleotide constructs is created and then assembled, as illustrated in FIGS. 4 and 5. This can be a pure codon library such that each library member has the same amino acid sequence but many different coding sequences are possible. Such libraries can be assembled from partially randomized oligonucleotides and used to generate large libraries of XTEN segments comprising the sequence motifs. The randomization scheme can be optimized to control amino acid choices for each position as well as codon usage. Polynucleotide Libraries

[0259] In another aspect, the invention provides libraries of polynucleotides that encode XTEN sequences that can be used to assemble genes that encode XTEN of a desired length and sequence.

[0260] 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. [n a preferred embodiment, libraries of oligonucleotides that encode motifs of 12 amino acids are assembled.

[0261] The XTEN-encoding sequence segments can be dimerized or multimerized into longer encoding sequences. Dimerization or multimerization can be performed by ligation, overlap extension, PCR assembly or similar cloning techniques known in the art. This process of can be repeated multiple times until the resulting 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 12 amino acids can be dimerized into a library of polynucleotides that encode 36 amino acids. In turn, the library of polynucleotides that encode 36 amino acids can be serially dimerized into a library containing successively longer lengths of polynucleotides that encode XTEN sequences. In some embodiments, Libraries can be assembled of polvnucleotides that encode amino acids that are limited to specific sequence XTEN families; e.g. AD, AE, AF, AG, AM, or AQ sequences of Table 1. In other embodiments, libraries can comprise sequences that encode two or more of the motif family sequences from Table 1. The libraries can be used. in turn, for serial dimerization or ligation to achieve polynucleotide sequence libraries that encode XTEN sequences, for example, of 72. 144. 288. 576, 864, 912, 923, 1296 anno acids, or up to a total length of about 3000 amino acids. as well as intermediate lengths. 1n some cases, the polynucleotide library sequences may also include additional bases used as "sequencing islands.” described more fully below.

[0262] FIG. 5 is a schematic flowchart of representative, non-limiting steps in the assembly of a XTEN polynucleotide construct and a BPXTEN polynucleotide construct in the embodiments of the invention. Individual oligonucleotides 501 can be annealed into sequence motifs 502 such as a 12 amino acid motif ("12- mer"). which is subsequently ligated with an oligo containing Bbsl, and KpnI restriction sites 503. Additional sequence motifs from a library are annealed to the 12-mer until the desired length of the XTEN gene 504 is achieved. The XTEN gene is cloned into a stuffer vector. The vector can optionally encode a Flag sequence 506 followed by a stuffer sequence that is flanked by Bsal, Bbsl. and Kpnl sites 507 and, in this case, a single BP gene (encoding exendin-4 in this example) S08, resulting in the gene encoding a BPXTEN comprising a single BP 500. A non-exhaustive list of the XTEN names and SEQ ID NOS. for polynucleotides encoding XTEN and precursor sequences is provided in Table 8 Table 8: DNA sequences of XTEN and precursor sequences AF144 | 248 249 XTEN | SEQID \ AE144 247 | GGTAGCGRACCGGCAACTTCCGGCTCTGARACCCCAGGTACTTCTGAAAGCGCTACTC CTGAGTCTGECCCAGATAGCEGAACCTGCTACCTCTGECTCTGAAACCCCAGETAGCCC GGCAGGCTCTCCGACTTCCACCGAGGAAGGTACCTCTACTGAACCTTCTGAGGGTAGC GCTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAGGTAGCGAACCTGCTA CCTCCGGCTCTGAAACTCCAGGTAGCGAACCGGCTACTTCCGGTTCTGAAACTCCAGG TACCTCTACCGAACCTTCCGAAGGCAGCECACCAGGTACT TCTCGAAAGCGCAACCCCT GAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTA CCGAACCGTCCGAMGGTAGCGCACCA [TAF144 248 | GGTACTTCTACTCCGGARAGCGET TCCGCATCTCCAGGTACT ICTCCTAGCGGTGART | CTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCAGGTTCTAC CAGCTCTACCGCTGAATCTCCTEGECCCAGGTTCTACCAGCGAATCCCCGTCTGGCACC GCACCAGGTTCTACTAGCTCTACCGCAGAATCTCCGGGTCCAGETACTTCCCCTAGCG GTGAATCTTCTACTGCTCCAGGTACCTCTACTCCGGARAGCGGCTCCGCATCTCCAGG TTCTACTAGCTCTACTGCTGAATCTCCTGGTCCAGGTACCTCCCCTAGCGGCGAATCT TCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTACCTCCC CTAGCGGTGAATCTTCTACCGCACCA GCTCTGAGACTCCAGATACCTCTGAMAGCGCAACCCCGGAATCTGGTCCAGETAGCGA ACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGARAGCGCTACTCCTGAATCT GGCCCAGGTACTTCTACTGAACCETCCGAGGGCAGCECACCAGGTAGCCCTECTGGCT CTCCAACCTCCACCGAAGARAGGTACCTCTGAAAGCGCAACCCCTGAATCCGECCCAGG 250 XTEN | SEQID GAGTCCGGCCCAGGTAGCCCEGCTEGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGG CTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGC ACCAGGTACTTCTGARAGCGCTACCCCTEGAGTCCGGCCCAGGTACTTCTGAAAGCGCT ACTCCTGAATCCGETCCAGGTACTTCTGAAAGCECTACCCCGGAATCTGGCCCAGGTA GCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTC TGAAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACT GAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTC CAGGTAGCGAACCTGCAACCTCTGECTCTGARACCCCAGGTACCTCTGAAAGCGCTAC TCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA AEST6 550 | GGTAGCCCGECTGECTCTCCTACCTCTACTGAGGAAGG TACT TCTGAAAGCGCTACTC CTGAGTCTGETCCAGETACCTCTACTGAACCGTCCGAAGETAGCGCTCCAGETAGCCC AGCAGGCTCTCCGACTTCCACTGAGEAAGGTACTTCTACTGAACCTTCCGAAGGCAGC GCACCAGGTACCTCTACTGAACCTTCTGAGEGGCAGCAGCTCCAGGTACTTCTGAAAGCG CTACCCCGGAATCTGGCCCAGGTAGCGARCCGECTACTTCTGETTCTGARACCCCAGG TAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACT TCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTA CCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGC ACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCCAGCGAAGGTACTTCTACCGAACCG TCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTA CTTCTGAAAGCCCTACCCCGGAGTCCGETCCAGCTACTTCTACTGAACCGTCCCGAAGG ...

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A fusion protein comprising: (a) an extended recombinant polypeptide (XTEN) characterized in that: i it comprises at least 12 amino acids; il. at least 90%, 91%, 92%, 93%, 949%, 953%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN sequence are selected from glveine (G). alanine (A), serine (8), threonine (1), glutamate (E) and proline (P); and iil. it has 4-6 different amino acids selected from G, A, S. T, E and P; and ®) a cytokine linked to at least one XTEN. 2 The fusion protein of claim 1, wherein said fusion protein comprises 1, 2, 3. 4 or more XTENs. 3 The fusion protein of claim 1 wherein said fusion protein further comprises a tumor targeting domatn. 4 The fusion protein of claim 1, further comprising a release segment, wherein the release segment (RS) has at least 88%, at least 94%, or 100% sequence identity to a sequence selected from the sequences set forth in Tables 6-7. 3 The fusion protein of claim 3. wherein said tumor targeting domain is linked one of the XTENs that are linked to the cytokine.

6. The fusion protein of claim S, wherein the C-terminus of the tumor targeting domain is linked to a further XTEN. 7 The fusion protein of claim 6, further comprising a release site between the C-terminus of the tumor targeting domain and the N-terminus of said further XTEN. 8 The fusion protein of claim 4, wherein the fusion protein has a structural arrangement, from N- to C- terminus of XTEN-RS-cytokine or cytoking-RS-XTEN. 9 The fusion protein of any one of claims 1-8, wherein the cytokine is selected from a group consisting of interleukins, chemokines, interferons, tumor necrosis factors, colony-stimulating factors, or TGF-Beta superfamily members.

10. The fusion protein of claim 9, wherein the cytokine is an interleukin selected from the group consisting of IL1, IL2, 1L3, 1L4, ILS, IL6, IL7, IL8, 1L9, [L10, IL11, 1112, [L.13, IL 14. IL15, IL16, and 1L17. u The fusion protein of claim 9, wherein the cytokine has at least 90% sequence identity to a sequence selected from Table 3 or Table A.

12. The fusion protein of claim 9, wherein the cytokine is IL-12 or an IL-12 variant. 13 The fusion protein of claim 12, wherein the cytokine comprises a first cytokine fragment (Cy {) and a second cytokine fragment (Cy2).

14. The fusion protein of claim 13, wherein one of the Cy1 and the Cy2 comprises an amino acid sequence having at least 70%, 75%, $0%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an interleukin-12 subunit beta.

15. The fusion protein of claim 14, wherein the other one of the Cy1 and the Cy2 comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or 100% sequence identity to an interleukin-12 subunit alpha.

16. The fusion protein of claim 13, wherein the first cytokine fragment (Cy 1) comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%, or 100% sequence identity to a sequence of SEQ ID NO.

5.

17. The fusion protein of claim 13, wherein the second cytokine fragment (Cy2) comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence of SEQ ID NO.

6.

18. The fusion protein of any one of claims 13-17, wherein the cytokine comprises a tinker positioned between the first cytokine fragment (Cy 1) and the second cytokine fragment (Cy2).

19. The fusion protein of claim 18 wherein said fusion protein comprises a Cyl fragment that comprises a XTEN at the N terminus and an XTEN at the C-terminus.

20. The fusion protein of claim 18 wherein said fusion protein comprises a Cy2 fragment that comprises a XTEN at the N terminus and an XTEN at the C-termings. It The fusion protein of claim 18, wherein the cytokine is an IL-12 variant comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO.

7.

2. The fusion protein of any one of claims 1-21. wherein the XTEN sequence consists of multiple non- overlapping sequence motifs, wherein the sequence motifs are selected from the sequence motifs of Tables 2a-2b.

2. The fusion protein of any one of claims 1-22, wherein the XTEN has from 40 to 3000 amino acids, or from 100 to 3000 amino acids.

24. The fusion protein of any one of claims 1-23, wherein the XTEN has at least 90%, at least 91%, at least 92%. at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to a sequence set forth in Tables 2a-2b. 25 The fusion protein of any one of claims 1-24, wherein a binding activity of the cytokine, when linked to the XTEN in the fusion protein. to a corresponding cytokine receptor is characterized by a half maximal effective concentration (ECso) at least 1.2 fold greater, at least 1.4 fold greater, at least 1.6 fold greater, at least 1.8 fold greater, at least 2.0 fold greater, at least 3.0 fold greater, at least 4.0 fold greater, at least 5.0 fold greater, at least 6.0 fold greater, at least 7.0 fold greater, at least 8.0 fold greater, at least 9.0 fold greater, or at least 10.0 fold greater than an ECs, characterizing a corresponding binding activity of the cytokine when not linked to the XTEN as determined in an iz vitro binding assay.

26. The fusion protein of claim 25, wherein said cytokine is interleakin 12 (IL-12) and said corresponding cytokine receptor is an interleukin 12 receptor (IL-12R).

27. The fusion protein of claim 25 or claim 26, wherein the in virro binding assay utilizes a genetically engineered reporter gene cell line configured to respond to binding of the cytokine to the corresponding cytokine receptor with a proportional expression of a reporter protein.

28. A pharmaceutical composition, comprising the fusion. protein of any one of the claims §-27 and at least one pharmaceutically acceptable carrier.

29. Use of a composition of claim. 28 in the preparation of a medicament for treating a disease or condition in a subject in need thereof, 30. The use of claim 29, wherein the disease or condition is selected from cancer, theumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, Alzheimer’s disease, Schizophrenia, viral infections, allergic asthma, retinal neurodegenerative processes, metabolic disorder, insulin resistance, and diabetic cardiomyopathy. 3L A method of treating or preventing a disease or condition in a subject, the method comprising administering to a subject a therapeutically effective amount of the fusion protein of any one of claims 1-27 or the composition of claim 28 32. The method of claim 31. wherein the disease or condition is a cancer or a cancer-related disease or condition. 33, The method of claim 31 or claim 32, further comprising administering to the subject a therapeutically effective amount of at least one immune checkpoint inhibitor. 34, The method of any one of claims 31-33, wherein the fusion protein is delivered intravenously, subcutaneously. or orally.

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