Ligands Modified by Circular Permutation as Agonists and Antagonists

Abstract

The present invention provides fusion polypeptides comprising polypeptide ligands that are modified by circular permutation and fused to at least one polypeptide fusion partner wherein such fusion polypeptides have new, improved or enhanced biological functions or activities. Such improvements include, but are not limited to, increased binding affinity, increased activity, increased agonist activity (super agonist), antagonist activity, increased accessibility, increased flexibility of the active site, increased stability, broader and/or changed substrate specificity, and combinations thereof.

Description

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No. 15/218,193 filed Jul. 25, 2016, which is a continuation of U.S. application Ser. No. 14/182,536, filed Feb. 18, 2014, now U.S. Pat. No. 9,428,563, issued Aug. 30, 2016, which is a continuation of U.S. application Ser. No. 13/911,827, filed Jun. 6, 2013, now U.S. Pat. No. 9,359,415, issued Jun. 7, 2016, which claims the benefit of U.S. Provisional Application No. 61/657,378, filed on Jun. 8, 2012; 61/723,081, filed Nov. 6, 2012; 61/657,264, filed Jun. 8, 2012; 61/778,575, filed Mar. 13, 2013; 61/657,285, filed Jun. 8, 2012 and 61/778,812, filed Mar. 13, 2013. The entire teachings of the above applications are incorporated herein by reference.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 31, 2013, is named 4000.3059WO_SL.txt and is 66,878 bytes in size.

BACKGROUND OF THE INVENTION

[0003] Ligand-receptor interactions are essential to a number of cell signaling pathways. Growth factors, cytokines and other regulatory proteins use these interactions to mediate cell responses. Proteins that inhibit or facilitate these processes have potential as therapeutics.

[0004] Given some of the drawbacks of monoclonal antibody approaches to inhibiting ligand-receptor functions such as expensive manufacturing, large size, limited penetration into tissues and undesirable side effects, researchers have been focusing on the use of non-antibody proteins as therapeutic agents. Furthermore, therapeutic antibody strategies are generally limited to inhibiting, or antagonizing, a signaling pathway and not competent to strategies to enhance, or agonize, a pathway. Thus, new protein engineering approaches are being explored to develop ligands and receptors as agonists and antagonists of clinically important targets as an alternative to antibody strategies.

[0005] Circular permutation involves the linking of the native amino and carboxy ends of a protein, generally with a linker, and creating new amino and carboxy termini by cleaving at a new site within the protein sequence, generally a loop; such that the primary sequence of the resulting protein is reordered, while the secondary structure (and activity) is retained. Thus, creation of the new termini may provide better locations for attachment of a fusion partner relative to the native termini.

[0006] Circular permutation of a protein ligand provides a means by which a protein may be altered to produce new carboxyl and amino termini without diminishing the specificity and binding affinity of the altered protein ligand for its target relative to its native form. Additionally, the new termini can be preferentially moved to a location preferential for incorporating the circularly permuted ligand into a fusion polypeptide, and demonstrate better activity compared with a fusion polypeptide containing the native (non-circularly permuted) ligand.

[0007] The present invention provides fusion polypeptides comprising ligands modified by circular permutation which function as agonists, super agonists or antagonists of a signaling pathway. Such fusion polypeptides are beneficial in the treatment of many disorders, conditions, and diseases that rely on ligand-receptor interaction and signal transduction. For example, such fusion polypeptides that act as antagonists of a target receptor have potential as therapeutics for cancer and autoimmune disorders. Such fusion polypeptides that act as agonists or superagonists of a signaling pathway have the potential, for example, in cancer or regenerative medicine.

SUMMARY OF THE INVENTION

[0008] The present invention provides fusion polypeptides comprising polypeptide ligands that are modified by circular permutation and fused to at least one polypeptide fusion partner wherein such fusion polypeptides have new, improved or enhanced biological functions or activities relative the analogous fusion protein with the native (non-circularly permuted) ligand. Such improvements include, but are not limited to, increased binding affinity, increased activity, increased agonist activity (super agonist), increased antagonist activity, increased accessibility, increased flexibility of the active site, increased stability, broader and/or changed substrate specificity, enhanced tissue targeting, enhanced protein binding, enhanced membrane targeting, improved pharmacokinetic parameters, improved physical properties, and combinations thereof.

[0009] In one embodiment, the circularly permuted ligands comprise all or any portion of their native polypeptide chains, and may optionally include linkers. The circularly permuted ligands of the invention are designed to be optimally oriented such that they may be fused to at least one desired polypeptide fusion partner without compromising the activity, such as the binding affinity of the modified ligand for its target. In one embodiment, the circularly permuted (modified) ligands of the fusion polypeptides are at least as active, and are preferably more active, as compared to their corresponding native proteins. In one embodiment the fusion proteins of the invention have a greater binding affinity for their targets proteins. In one embodiment the binding affinity of the fusion protein for its target protein is at least 5-fold, preferably at least 10-fold, preferably at least 20 fold or more, greater than the affinity of the native ligand for the protein target. In one embodiment the fusion polypeptide of the invention has at least 10 fold greater binding affinity for the receptor.

[0010] In one embodiment, the ligands are selected from the group including, but not limited to, cytokines, lymphokines, chemokines, adipokines, growth factors, hormones, cell adhesion molecules and neurotransmitters. Polypeptide fusion partners may be any polypeptide that provides and enhancement to the native protein. For example, fusion partners may be selected from the group including, but not limited to, all or a portion of: glycoproteins, proteoglycans, cell signaling molecules, accessory proteins, soluble receptors, membrane bound receptors, transmembrane receptors, antibodies, enzymes, targeting polypeptides (e.g., nanobodies), mucins or mucin-like peptides, synthetic polypeptides or any combinations thereof. Enhancements include, but are not limited to, improvements in affinity, agonism, antagonism, addition of synergistic functional activity, tissue targeting, protein targeting, membrane targeting, pharmacokinetic parameters (e.g., half life), or physical properties (e.g., solubility).

[0011] In a preferred embodiment, at least one polypeptide fusion partner comprises all or a portion of a subunit of the target receptor or another molecule involved in its natural signal transduction pathway. It is understood that a polypeptide fusion partner may comprise a polypeptide that is at least 60%, at least 70%, at least 80% or at least 90% homologous to all or a portion of a subunit of a target receptor or another molecule involved in a signal transduction pathway.

[0012] In one embodiment, the invention provides for fusion polypeptides comprising a modified ligand and a polypeptide fusion partner that are further linked to a second fusion partner. Examples of second fusion partners include all or any portion of an antibody (e.g., the Fc region of an antibody) and any of the types of polypeptides suitable as a first fusion partner described above.

[0013] In a preferred embodiment, the fusion polypeptides of the invention function as new and improved agonists (super agonists), or antagonists of a receptor such as a cellular receptor that is involved in signal transduction of a cell signaling pathway. In a preferred embodiment, the fusion polypeptides of the invention can bind a monomeric, dimeric, or a multimeric target receptor and can inhibit or enhance dimerization, trimerization or multimerization of the receptor and/or inhibit or enhance signal transduction and downstream signaling of a cellular pathway.

[0014] In one embodiment, the invention provides a fusion polypeptide comprising, a first polypeptide fusion partner linked to a modified ligand corresponding to a native ligand specific for a target receptor, wherein the modified ligand has been circularly permuted to create a new N-terminus and a new C-terminus as compared to the native ligand, and wherein the new N-terminus or the new C-terminus of the modified ligand is linked to a first polypeptide fusion partner to form a fusion polypeptide that optionally has increased affinity for the target receptor as compared to the native ligand for the receptor, and wherein upon association of the fusion polypeptide with the target receptor the fusion polypeptide super agonizes or antagonizes the activity of the target receptor. In one embodiment, the new C-terminus and the new N-terminus of the modified ligand do not disrupt any binding domain of the modified ligand for the target receptor.

[0015] In one embodiment, the target receptor functions by stepwise formation of a multimeric activation complex to trigger signal transduction of a signaling cellular pathway and wherein upon binding of the fusion polypeptide to the receptor, signal transduction is super agonized or antagonized.

[0016] In one embodiment, the fusion polypeptide binds the receptor and enhances the stepwise formation of the multimeric activation complex thereby super agonizing signal transduction by the target receptor.

[0017] In one embodiment, the fusion polypeptide binds the receptor and sterically hinders the stepwise formation of the multimeric complex thereby antagonizing signal transduction by the target receptor.

[0018] In one embodiment the fusion polypeptide comprises the modified ligand and a first fusion partner wherein the first fusion partner of the modified ligand is derived from all or a portion of the protein with which the native ligand of the target receptor would have associated in the first step of the stepwise formation of the receptor's multimeric activation complex. In one embodiment the fusion polypeptide comprises the modified protein and a fusion partner wherein the fusion partner of the modified protein is derived from all or a portion of the protein with which the native protein of the target receptor would have associated in downstream steps of the stepwise formation of the receptor's multimeric activation complex.

[0019] In one embodiment the first fusion partner of the heterodimer is fused to the modified ligand in a position that is oriented to enhance the stepwise formation of the receptor's multimeric activation complex.

[0020] In one embodiment, the first fusion partner of the heterodimer is fused to the modified ligand in a position that is oriented to sterically hinder the formation of the receptor's multimeric activation complex.

[0021] In one embodiment the fusion polypeptide is a homodimer comprising the modified protein and a fusion partner wherein the fusion partner of the modified ligand is derived from all or a portion of the same ligand where homodimerization is required for formation of the receptor's multimeric activation complex.

[0022] In one embodiment the invention provides a pharmaceutical composition comprising the fusion polypeptide of the invention and a pharmaceutically acceptable carrier.

[0023] In one embodiment the invention provides an isolated or recombinant nucleic acid encoding the fusion polypeptide of the invention; a recombinant vector comprising the nucleic acid encoding a fusion polypeptide of the invention and a host cell comprising a vector of the invention.

[0024] In one embodiment the invention provides a method of super agonizing a target receptor comprising the step of contacting the receptor with the fusion polypeptide of the invention.

[0025] In one embodiment the invention provides a method of antagonizing a target receptor comprising the step of contacting the receptor with a fusion polypeptide of the invention.

[0026] In one embodiment, the invention provides a method of making a fusion polypeptide of the invention comprising the steps of: a) selecting a native ligand that binds to a receptor wherein the receptor functions by stepwise formation of a multimeric activation complex to trigger signal transduction of a signaling cellular pathway; b) creating a modified ligand by circular permutation to provide a modified ligand having new N-terminus and a new C-terminus as compared to the native ligand of step (a); and c) linking a first polypeptide fusion partner to the N- or C-terminus of the modified ligand of step (b) to make a fusion polypeptide, wherein the new N- or C-termini of the modified ligand are located to permit the first fusion partner to be linked to the modified ligand in a position oriented to antagonize or super agonize the function of the target receptor upon binding of the fusion polypeptide to the target receptor. In one embodiment, the method further comprises fusing a second fusion partner to the modified ligand of step (b) wherein the second fusion partner provides an additional enhancement to the protein, such as extending the half-life of the fusion polypeptide in vivo. Other enhancements that could be engineered via step (c) include, but are not limited to, addition of synergistic functional activity, organ targeting, tissue targeting, protein targeting, membrane targeting, biological matrix targeting, pharmacokinetic (e.g. percent bioavailability) or physical properties (e.g. solubility).

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1. Structure of the human IL6 hexameric signaling complex (PDB: 1P9M). Side view (top) and top view (bottom) of the hexameric complex consisting of two IL-6 molecules (dark grey), two soluble IL-6R.alpha. molecules (D2-D3 of IL6 receptor subunit a black), and two soluble gp130 molecules (D1-D2-D3, light grey).

[0028] FIG. 2. Illustration of the process of circular permutation utilizing the 4-helix bundle protein, IL-6. Ribbon representation of the IL-6 crystal structure (PDB 1P9M, top left) and of modeled structure of a circularly permuted IL-6 (RDB1503, top right). N and C termini are labeled as are helicies A, B, C, D as per standard IL-6 nomenclature. The circularly permuted protein was engineered by linking the native termini and creating new termini between helicies C and D of native IL-6. The end result of the circular permutation is the relocation of the termini to the opposite face of IL-6. The amino acid sequences for IL-6 (residues 47-212 of SEQ ID NO: 3) and RDB1503 (SEQ ID NO: 1) (middle and bottom, respectively) highlight the reordered sequence. The new N-terminus of RDB1503 immediately precedes helix D. The shaded area within the ribbon representation and the protein sequence of RDB1503 highlight the linker created to connect the native IL-6 N and C termini.

[0029] FIG. 3A is a drawing of a molecular model illustrating the relative orientation of the D1 (domain 1 of gp130) when fused to IL-6, resulting in fusion protein RDB1529. The D1 domain is shaded for highlighting purposes. Portions of gp130 and IL-6R.alpha. in the active hexameric complex are included for reference. The D1 domain of RDB1529 is pointing away from the gp130 binding interface in the hexameric active complex and is therefore predicted to be unable to effectively antagonize the signal.

[0030] FIG. 3B is a drawing of a molecular model illustrating the relative orientation of the D1 (domain 1 of gp130) when fused to RDB1503, resulting in fusion protein RDB1527. The D1 domain is shaded for highlighting purposes. Portions of gp130 and IL-6R.alpha. in the active hexameric complex are included for reference. The D1 domain of RDB1527 both participates in binding to IL-6R.alpha. and occupies the space occupied by the second gp130 molecule in the hexameric complex, thus effectively antagonizing the signal.

[0031] FIG. 4. Dose-response curves for IL-6 (.tangle-solidup.) and RDB1503 () in the HEK-Blue.TM. cell assay. The EC.sub.50 is estimated at 1 pM and 0.6 pM, for IL-6 and RDB1503, respectively.

[0032] FIG. 5. Inhibition of IL6 signaling by RDB1527 in the HEK-Blue.TM. cell assay. Activity of IL6 () as a function of its concentration in the absence of inhibition. Inhibition by RDB1527(), and RDB1529 () were measured in the presence of 12.5 pM of IL6. All measurements were made in duplicate. The estimated value of IC.sub.50 for RDB1527 is 0.22 nM. RDB1529 did not show robust inhibitory activity.

[0033] FIG. 6A is a line graph showing Surface Plasmon Resonance (SPR) measurements of soluble IL-6R.alpha. binding to immobilized IL-6. Sensorgrams and fitted curves are in grey and black, respectively. The kinetic parameters calculated from the data are in the inserted tables.

[0034] FIG. 6 B is a line graph showing Surface Plasmon Resonance (SPR) measurements of soluble IL-6R.alpha. binding to immobilized RDB1529. Sensorgrams and fitted curves are in grey and black, respectively. The kinetic parameters calculated from the data are in the inserted tables.

[0035] FIG. 6C is a line graph showing Surface Plasmon Resonance (SPR) measurements of soluble IL-6R.alpha. binding to immobilized RDB1527. Sensorgrams and fitted curves are in grey and black, respectively. The kinetic parameters calculated from the data are in the inserted tables.

[0036] FIG. 7A is a drawing of the structure of the human IL-1.beta. signaling complex (PDB: 4DEP). IL-1.beta. (highlighted with an arrow in the structure) binds to receptors IL-1RI (black, coming out of the plane) and IL-1RAcP (light grey, going away from the plane). The native N and C termini of IL-1.beta. are not in close proximity to the C-terminus of D1-D2 domain of IL-1RI.

[0037] FIG. 7B is a drawing of the modeled representation of the potential complex formation mediated by RDB1538 (CP_IL-1.beta. IL-1RI (D1-D2); FIG. 7B). The termini of the engineered circularly permuted IL-1.beta. are now proximal to the C-terminus of D1-D2 domain of IL-1RI, thus facilitating the generation of the fusion protein. The shaded area highlights the linker connecting the circularly permuted IL-1.beta. variant to IL-1RI receptor.

[0038] FIG. 8A is a drawing of the structure of the human IL2 signaling complex (PDB: 2ERJ;). IL2 (highlighted with an arrow in the structure) binds to receptors IL2R.alpha. (grey, top left in the complex), IL2R.beta. (light grey, bottom left in the complex) and .gamma..sub.c (black, bottom right in the complex). IL-2R.alpha. stabilizes the conformation of IL-2 to enhance its binding affinity to IL-2R.beta.. The native N and C termini of IL-2 are on the face distal to IL-2/IL-2R.alpha. interface.

[0039] FIG. 8B is a drawing of the modeled signaling complex mediated by RDB1405 (CP_IL-2_IL-2R.alpha.). The termini of the engineered circularly permuted IL-2 are now proximal to the IL-2/IL-2R.alpha. interface, thus facilitating the generation of the fusion protein. The shaded area highlights the linker connecting the circularly permuted IL-2 variant to IL-2R.alpha. receptor.

[0040] FIG. 9. Is a diagram showing representative signaling complexes for cytokines and growth factors illustrating multimeric assembly leading to activation.

[0041] FIG. 10. Is a diagram representing the mechanism of antagonism by Picasso3_D1. Binding determinants from both IL-6 and D1 (domain of gp130) are present in the hybrid fusion protein resulting in high affinity binding to IL-6R.alpha.. Once Picasso3_D1_Fc is bound to IL-6R.alpha., assembly of the signaling complex of IL-6 cannot proceed, resulting in antagonism.

[0042] FIGS. 11A and 11B. Response of HH cells (left) and CTLL-2 cells (right) to wild-type IL-2 (Proleukin) and engineered IL-2 variants.

[0043] FIGS. 12A and 12B Response of HH cells (left) and CTLL-2 cells (right) to wild-type IL-15 and engineered IL-15 variants.

[0044] FIG. 13. Structure of the modeled IL-15 signaling complex for the CP-IL-15-IL-15R.alpha. fusion proteins generated by superposition of the IL-15/IL-15R.alpha. complex (2Z3Q.pdb onto the IL-R.beta. and IL-2R.gamma. chains from the IL-2 ternary signaling complex structure, 2ERJ.pdb). The `Linker` joining the native termini of IL-15 to create the circularly permuted IL-15 variant and `Spacer` to create the CP-IL-15-IL-15R.alpha. fusion are highlighted with arrows. Note that the native termini of IL-15 (originally located at the `Linker` site) are far distally oriented from the IL-15R.alpha. binding interface, thus creating the need for circular permutation of the ligand.

DETAILED DESCRIPTION OF THE INVENTION

[0045] A description of preferred embodiments of the invention follows. For illustrative purposes, polypeptide fusion proteins of the invention featuring a circularly permutated IL-6 ligand fused to a portion of gp130 is used as an exemplary fusion polypeptide of the invention. It is understood that the biological functions, activities and other features of the described embodiments are generally applicable to other fusion polypeptides in accordance with the invention comprising ligands modified by circular permutation fused to polypeptide fusion partners.

[0046] As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide fusion partner" includes a plurality of polypeptide fusion partners. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings, unless a contrary intention is apparent.

Definitions

[0047] The terms "circular permutation" and "circularly permuted" "(CP)" as used herein, refers to the conceptual process of taking a linear protein, or its cognate nucleic acid sequence, and fusing the native N- and C-termini (directly or through a linker, using protein or recombinant DNA methodologies) to form a circular molecule, and then cutting (opening?) the circular molecule at a different location to form a new linear protein, or cognate nucleic acid molecule, with termini different from the termini in the original molecule. Circular permutation thus preserves the sequence, structure, and function of a protein (other than the optional linker), while generating new C- and N-termini at different locations that, in accordance with one aspect of the invention, results in an improved orientation for fusing a desired polypeptide fusion partner as compared to the original ligand. Circular permutation also includes any process that results in a circularly permutated straight-chain molecule, as defined herein. In general, a circularly permuted molecule is de novo expressed as a linear molecule and does not formally go through the circularization and opening steps. The particular circular permutation of a molecule, herein, is designated by brackets containing, in the case of a circularly permuted protein, the amino acid residues between which the peptide bond is eliminated. For example, the designation IL6 (Q182/Q180) designates a circularly permuted IL6 growth factor in which the opening site (position at which the peptide bond is eliminated) occurred between residues Q182 and Q180 of the unpermuted or unmodified native IL6, and thus the newly created N-terminus is a Glutamine which was formerly residue 182, and the newly created C-terminus is a Glutamine which was formerly residue 180.

[0048] A "spacer" as used herein and refer to a peptide that joins the proteins comprising a fusion protein. Generally, the spacer has no specific biological activity and its purpose is merely to join the proteins or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of a spacer may be selected based on some properties of the linker or of the resulting molecule such as the flexibility, hydrophilicity, net charge, or proteolytic susceptibility or lack thereof, and lack of immunogenicity.

[0049] The terms "unpermuted", "native", "wild type", or "unmodified" ligand, polypeptide, protein, cytokine, or growth factor, are used herein to provide a reference point for the ligand, cytokine, growth factor or protein prior to its rearrangement into a circularly permuted molecule, as described above. Typically, the unmodified ligand, growth factor or protein has amino and carboxy termini and an amino acid sequence that correspond substantially to the amino and carboxy termini and amino acid sequence of the ligand, growth factor, or protein, or an independent domain of a protein, as it generally occurs in vivo. The unmodified ligand, growth factor, or protein may be a fully mature form or a precursor to the mature form (such as a pro-protein).

[0050] The term "ligand" is used herein generally to denote any polypeptide (whether native, endogenous, or modified in accordance with the invention) that binds to a second protein or receptor and is a component of a biochemical pathways. A ligand directly or indirectly may affect (e.g., induce, inhibit) receptor activity (e.g., signaling, adhesion).

[0051] The term "modified ligand" is used herein to indicate a ligand that has been modified by circular permutation as compared to the corresponding native ligand.

[0052] "Activity" or "biological activity" refer 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.

[0053] An "agonist" is a fusion polypeptide of the invention which is capable of binding to a desired receptor to result in an activated receptor complex. A "superagonist" is a fusion polypeptide of the invention capable of binding the target receptor and that provides enhanced activation of the receptor complex as compared to the native ligand for that target receptor. Activation by the fusion polypeptide superagonist of the invention may be enhanced at least two-fold, and preferably at least 5-fold, preferably at least 10-fold or preferably at least 20-fold or more as compared to activation of the target receptor by the native ligand. A fusion polypeptide of the invention "having agonist activity" refers to the fact that the fusion polypeptides are able to bind to and activate or superagonize at least one receptor.

[0054] An "antagonist" is a fusion polypeptide of the invention which is capable of binding to a desired receptor but incapable of mediating correct conformational or molecular assembly changes of the receptor molecules necessary to result in an activated complex, and whereby native ligand-mediated receptor activation is substantially inhibited. Receptor activation upon binding of a suitable ligand generally involves either a conformational change in the receptor or a difference in association states of the receptor, e.g., oligomerisation of receptor subunits or recruitment of additional proteins or receptors.

[0055] The term "receptor" is understood to indicate a protein present on a cell surface (or a soluble receptor not present on the cell surface but which has or associates with a counterpart cell surface receptor) with which a ligand binds. Cell surface receptors are typically composed of different domains or subunits with different functions, such as an extracellular domain (or domains) containing the region with which the ligand interacts, a transmembrane domain or domains (or in some cases an anchoring lipid) which anchors the receptor in the cell membrane. In some cases, an intracellular effector domain which initiates a cellular signal in response to ligand binding (signal transduction) is also present. Soluble receptors are typically composed of one or more of the extracellular domains resulting from protolytic cleavage from the membrane anchoring region.

[0056] "Target receptors" or "Target ligands" according to the invention are the molecules to which the fusion-polypeptides of the invention are designed to directly bind. In one embodiment "target receptors" according to the invention are capable of ultimately binding or otherwise associating with, signaling molecules (e.g., ligands) in triggering signal transduction of a signaling cellular pathway.

[0057] A receptor that is activated by the "stepwise formation of a multimeric activation complex" is a receptor that in addition to the binding of one or more ligands, requires the interaction of one or more additional protein subunits in a process known as dimerization, trimerization, multimerization, complexation, or oligomerization (also referred to in the art as "clustering") to fully achieve signal transduction of a cell signaling pathway. The receptor may already be in the form of a dimer or multimer prior to ligand binding and upon ligand binding may recruit additional soluble or membrane-anchored proteins in a stepwise fashion to build the fully functioning multimeric activation complex.

[0058] The "hydrodynamic radius" is the apparent radius (R.sub.h in nm) of a molecule in a solution calculated from diffusional properties. The "hydrodynamic radius" of a protein affects its rate of diffusion in aqueous. The hydrodynamic radius of a protein is influenced by its molecular weight as well as by its structure, including shape and compactness, and its hydration state. Methods for determining the hydrodynamic radius are well known in the art, such as by the use of DLS and size exclusion chromatography. 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.

[0059] A "mucin-domain polypeptide" is defined herein as any protein comprising a "mucin domain". A mucin domain is rich in potential glycosylation sites, and has a high content of serine and/or threonine and proline, which can make up greater than 40% of the amino acids. A mucin domain is heavily glycosylated with predominantly O-linked glycans.

[0060] The term "linker" or "linker sequence" as used herein, refers to the peptidic sequence that is used to join the amino and carboxy termini of a protein (or its corresponding nucleic acid sequence encoding the protein) through covalent bonds to both the amino and carboxy terminus. In some embodiments, the circularly permuted protein is produced by linking the ends of the corresponding DNA or RNA sequence, forming various permutants by cutting the circularized nucleic acid sequence, and subsequently translating the nucleic acid sequences to form the circularly permuted protein(s).

[0061] The term "residue" as used herein refers to an amino acid that is incorporated into a peptide. The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.

[0062] The term "opening site", as used herein when referring to circular permutation, refers to the position at which a peptide bond would be eliminated to form new amino and carboxy termini, whether by protein or nucleic acid manipulation. The opening site is designated by the positions of the pair of amino acids, located between the amino and carboxy termini of the unpermuted (native) protein that become the new amino and carboxy termini of the circularly permuted protein. For example, in IL6 (Q182/Q180), the newly created N-terminus (the new starting point of the circularly permuted IL-6) is equivalent (structurally) to Q182 of native IL-6 and the newly created C-terminus (the last residue of the circularly permuted IL-6) is equivalent (structurally) to Q180 of native IL-6. Residue 181 of native IL-6 was eliminated in creating the opening site.

[0063] The term "polypeptides" and "protein" are used interchangeably herein and include proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).

[0064] All amino acid positions described herein use as a frame of reference sequences for the native protein. For example, native IL-1.beta. (SEQ ID NO:19), native IL-6 (SEQ ID NO:3), native IL-2 (SEQ ID NO:20), native gp130 (SEQ ID NO:21), native IL-1RI (SEQ ID NO:22), and native IL-2R.alpha. (SEQ ID NO:23) as presented in the Sequence Listing. For example, an IL-6 molecule "comprising amino acids 47 to 212" would refer to a molecule having amino acids substantially corresponding to those positions in SEQ ID NO:3. Other common references are used herein to indicate deletions or substitutions to a sequence using as reference sequences, the respective native sequences as referenced in the sequence listing or whose GenBank accession number is provided herein. Amino acid substitutions may be indicated by parentheses, for example "(Ser 287)" refers to a molecule having serine at amino acid position 287. Circularly permuted molecules are designated by the native molecule followed by brackets enclosing the amino acid positions that comprise the opening site. Thus, for example, IL6 (182/180) designates a circularly permuted IL6 in which the new amino terminus is at amino acid residue 182, and the new carboxy terminus is at amino acid residue 180 of the unpermuted native IL6. It is recognized that some substitutions, addition, or deletions may be made to any sequences described herein that do not alter the biological activity of the region. Indeed, some such modifications may be required to achieve expression of a particular protein. Thus, for example, a methionine may be added to a sequence to provide an initiator.

[0065] "Variant" refers to a polypeptide that differs from a reference polypeptide, but retains essential properties. A typical variant of a polypeptide differs in its primary amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. In addition, the term "variant" as used herein includes circular permutations of proteins and peptides.

[0066] The term "antibody", as used herein, includes various forms of modified or altered antibodies, such as an intact immunoglobulin, an Fc fragment comprising the constant region of the heavy chains, an Fv fragment containing only the light and heavy chain variable regions, an Fv fragment linked by a disulfide bond an Fab or (Fab)'2 fragment containing the variable regions and parts of the constant regions, a single-chain antibody and the like.

[0067] 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 benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder 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 to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

[0068] A "therapeutic effect", as used herein, refers to a physiologic effect, including but not limited to the cure, mitigation, amelioration, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental well being of humans or animals, caused by a fusion protein of the invention other than the ability to induce the production of an antibody against an antigenic epitope possessed by the active protein. Determination 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.

[0069] The terms "therapeutically effective amount" and "therapeutically effective dose", as used herein, refers to an amount of an 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.

[0070] The term "therapeutically effective dose regimen", as used herein, refers to a schedule for consecutively administered doses of an 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.

[0071] As used herein, the term "dose" refers to the quantity of fusion polypeptide of the invention administered to a subject all at one time (unit dose), or in two or more administrations over a defined time interval. For example, dose can refer to the quantity of fusion polypeptide administered to a subject over the course of one day (24 hours) (daily dose), two days, one week, two weeks, three weeks or one or more months (e.g., by a single administration, or by two or more administrations). The interval between doses can be any desired amount of time.

[0072] The phrase, "half-life," refers to the time taken for the serum concentration of the fusion polypeptide to reduce by 50%, in vivo, for example due to degradation of the ligand and/or clearance or sequestration of the dual-specific ligand by natural mechanisms. The half-life of a fusion polypeptide is increased if presence in a biological matrix (blood, serum, plasma, tissue) persists, in vivo, for a longer period as compared to an appropriate control. Half life may be increased by 10%, 20%, 30%, 40%, 50% or more as compared to an appropriate control.

[0073] Sequences similar or homologous (e.g., at least about 70% sequence identity) to the sequences disclosed herein are also part of the invention. In some embodiments, the sequence identity at the amino acid level can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. At the nucleic acid level, the sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively, substantial identity exists when the nucleic acid segments will hybridize under selective hybridization conditions (e.g., very high stringency hybridization conditions), to the complement of the strand. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.

[0074] Calculations of "homology" or "sequence identity" or "similarity" between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. In the case of circularly related proteins, the sequence of one of the partners needs to be appropriately split and aligned in two sections to achieve optimal alignment of the functionally equivalent residues necessary to calculate the percent identity.

[0075] Amino acid and nucleotide sequence alignments and homology, similarity or identity, as defined herein are preferably prepared and determined using the algorithm BLAST 2 Sequences, using default parameters (Tatusova, T. A. et al., FEMS Microbiol Lett, 174:187-188 (1999)). Alternatively, the BLAST algorithm (version 2.0) is employed for sequence alignment, with parameters set to default values. BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx; these programs ascribe significance to their findings usingthe statistical methods of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87(6):2264-8.

[0076] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc. which are incorporated herein by reference) and chemical methods.

Circular Permutation of a Reference Ligand

[0077] Circular permutation is functionally equivalent to taking a straight-chain molecule, fusing the ends to form a circular molecule, and then cutting the circular molecule at a different location to form a new straight chain molecule with different termini. Circular permutation thus has the effect of essentially preserving the sequence and identity of the amino acids of a protein while generating new termini at different locations.

[0078] Engineered fusion proteins aim to combine the beneficial properties of two polypeptides into a single protein, however, the construction of the fusion protein comes with various challenges and risks. Often, the functional activity of the fusion protein is compromised relative that of the unmodified protein potentially due to a negative effect of the fusion partner on the integrity of the tertiary structure of the protein or on the proteins ability to bind to cognate partners (for example, due to steric hindrance) to elicit its biological function. Furthermore, inclusion of spacers between the fusion partners can increase the potential for susceptibility to proteolysis or, in the case of therapeutic proteins, also increase the potential for immunogenicity; the longer the spacer, the greater the risk. Thus, in generating fusion proteins, preserving the structural integrity of the fusion peptide, maintaining unobstructed access for binding to the necessary cognate partners, and minimizing the length of spacer sequences are important design goals. Towards these aims, utilizing circular permutation of a ligand as described herein provides preferential locations for fusion to a second protein.

[0079] Preferential locations for the new termini are geometrically, structurally, and functionally favored (relative to the native termini) for the fusion of a desired polypeptide fusion partner, and reduce the length of the required spacer. In one embodiment, the location of the new termini is more proximal to the native position of a potential fusion partner to which the ligand may normally associate with during the stepwise formation of a cellular receptor activation complex. The orientation of the modified ligand and the fusion partner in the fusion polypeptide may be optimal to either enhance agonistic activity of the ligand to the receptor activation complex, or provide steric hindrance of the stepwise formation of the activation complex thereby providing antagonism of the activation complex.

[0080] The process of circular permutation for IL6 is schematically illustrated in FIG. 2. The constituent amino acid residues of the native IL6 protein are numbered sequentially 47 through 212 from the amino to the carboxyl terminus.

[0081] To circularly permute IL6, recombinant constructs are engineered such that the native amino and carboxy termini of IL6 are joined by a linker sequence, and new amino and carboxy termini are engineered at amino acid residues 182 and 180, respectively (FIG. 2). Thus, circular permutation produces a new linear protein (IL-6 (182/180), aka Picasso3) which, proceeding from the amino to the carboxy terminus, comprises the segment of the original protein corresponding to residues 182 through 212 (now 1 through 31) followed by the linker, followed by a segment of the original protein corresponding to residues 49 through 180 (now 39 through 107) (FIG. 2).

[0082] It is important to create a permutation of a native ligand that will retain the biological activity of the native form of the ligand while providing an optimal termini for fusing a desired polypeptide fusion partner. If the new termini interrupt a critical region of the native protein, activity may be lost. Similarly, if linking the original termini destroys activity, then no permutation will retain biological activity. Thus, there are two requirements for the creation of an active circularly permuted protein: 1) The termini in the native protein must be favorably located so that creation of a linkage does not destroy biological activity; and 2) There must exist an "opening site" where new termini can be formed without disrupting a region critical for protein folding and desired biological activity.

[0083] In one embodiment, the new N-terminus and C-terminus of the modified ligand do not disrupt any binding domain of the modified ligand for the target receptor.

[0084] In one embodiment, the modified ligands are as fully active as the original ligands. In one embodiment the modified ligands have enhanced activity as compared to the original ligands. In one embodiment the enhanced activity is increased binding affinity for the target receptor.

[0085] Thus, in general, good candidates for circular permutation are proteins in which the termini of the original protein are in close proximity and favorably oriented. In one embodiment, the termini of the original protein are equal to or less than 20 .ANG. apart. Where the termini are naturally situated close together, it is expected that direct fusion of the termini to each other is possible and the introduction of a linker will have relatively little effect. However, because the linker may be of any length, close proximity of the native termini is not an absolute requirement.

[0086] In a preferred embodiment, it is desirable to use a linker sequence in the permuted protein that preserves the spacing between the amino and carboxy termini that is comparable to the spacing between the amino and carboxy termini found in the unpermuted or native molecule. In a preferred embodiment, the linker sequence will itself be between at least about one amino acid to at least about 10 amino acids. In a preferred embodiment, a small number of amino acids from either terminus may be removed (trimmed back) to bring the termini closer together. For example, in the crystal complex of IL-6 with IL-6R and gp130, the termini of the cytokine IL6 are 16 .ANG. apart (Brevnova, et al. (2003) Science 300:2102). Removal of the first two N-terminal residues, which are not required structurally or functionally, reduces the distance between the termini to 10.2 .ANG.. A linkage that essentially preserves this spacing is made with the peptide sequence SGGSGGG (SEQ ID NO: 14). Similarly, a preferred linker for circularly permuted IL-113 and IL-2 are GGSGGSG and GG, respectively (SEQ ID NO: 15 and SEQ ID NO: 16, respectively).

[0087] The selection of an opening site may be determined by a number of factors. Where the three dimensional conformation of the protein is known or predicted, preferred opening sites will be located in connecting loops or regions that do not show a highly regular three-dimensional structure. Thus, it is preferred that opening sites be selected in regions of the protein that do not contain defined secondary structures such as alpha helices, .beta. strands, and the like. Methods of identifying regions of defined secondary structure based on amino acid sequence are widely available on the World Wide Web. Furthermore, various programs are available for predicting the three-dimensional structure of proteins, recently reviewed in Nayeem et al., Protein Science, 808-24 (2006).

[0088] When retention or enhancement of the bioactivity of the native molecule is desired in the circularly permuted molecule, it is preferable that the opening site not be involved directly or indirectly in interactions with its protein partners. In one embodiment, the choice of the new opening site does not disrupt a binding domain present in the native ligand that is involved directly or indirectly in the binding affinity of the native ligand for its target receptor. Alternatively, where the substitution of certain amino acids or the modification of the side chains of certain amino acids does not change the activity of a protein, it is expected that those amino acids are not critical to the protein's activity. Thus, amino acids that can be mutated (in vitro) or are actually modified in vivo, with little impact on the protein's activity, are potentially good candidates for opening sites. Preferred opening sites in IL-6 are between residues 131 and 135 and between residues 180 and 182. A preferred opening site in IL-1.beta. is between residues 179 and 180, and also between residues 223 and 224. A preferred opening site in IL-2 is between residues 94 and 95.

[0089] Where the protein is a member of a family of related proteins within a species, one may infer that the highly conserved sequences are critical for biological activity, while the variable regions are not. Likewise, one may infer that highly conserved sequences of a protein which is functionally conserved across mammalian species, particularly if there is cross-species pharmacological activity, are critical for biological activity. Preferred opening sites are then selected in regions of the protein that do not show highly conserved sequence identity between various members of the protein family, either within or between species. Alternatively, preferred opening sites that are identified in a protein provide good candidate locations for opening sites in homologous proteins. Methods of determining sequence identity are well known to those of skill in the art and are described above.

[0090] One of skill in the art will recognize that other modifications may be made. Thus for example, amino acid substitutions may be made that increase the specificity or binding affinity of the ligand modified by circular permutation. Thus where there are regions of the ligand that are not themselves involved in the activity of the ligand, those regions may be eliminated or replaced with shorter segments that merely serve to maintain the correct special relationships between the ligand and the proteins that it is intended to associate with.

[0091] For a number of native ligands (e.g. growth factors, cytokines, and other proteins), the carboxy and amino termini are situated such that when fusion polypeptides are formed by joining a second polypeptide or molecule to either terminus of the native ligand, the desired downstream activity of the second polypeptide is significantly decreased or absent. Aberrant protein folding or steric hindrance is often ascribed to account for the decreased or absent activity of the second polypeptide. In other cases, fusion of a second polypeptide to either terminus of the native protein is tolerated (i.e. the functional activity of the native protein is not significantly impacted), however the orientation of the fusion polypeptide does not impart the desired activity to the fusion protein, such as in the case where the fusion polypeptide is meant to interfere (i.e. antagonize) with the formation of a signaling complex through steric interference where the location of the fusion polypeptide occupies the space that a downstream signaling molecule would occupy in the assembly of the active signaling complex.

[0092] In contrast, circular permutation of a ligand as described here provides a means by which the ligand may be altered to produce new carboxy and amino termini that permit fusion of the second molecule or polypeptide without diminishing the specificity and binding affinity of the altered ligand relative to its native form, and that also permits that the fused second molecule or polypeptide to impart, for example, superagonism or antagonism of a signaling activation complex. In one embodiment the fusion polypeptide of the invention converts a native ligand that is an agonist of a target signaling activation complex to an antagonist of the signaling activation complex. This is illustrated in the context of the cytokine, IL-6, in FIGS. 1-5.

[0093] One feature of the invention is that fusion polypeptides comprised of a circularly permuted ligand fused to a fusion partner, enhance the binding affinity of the fusion polypeptide to the native ligand's native receptor relative to the binding affinity of the native (unfused, unmodified, unpermuted) ligand for its native receptor. For example, Example 3 compares the binding affinity to the IL6 receptor of, 1) a fusion polypeptide comprised of circularly permuted IL6 fused to domain one of the transmembrane signaling molecule gp130, with 2) native IL-6. The binding affinity of the fusion polypeptide is seen to be more than 200 fold greater than the binding affinity of native IL6 to the IL6 receptor (FIGS. 6A and 6C).

Fusion Polypeptides

[0094] The present invention provides for novel fusion polypeptides comprising circularly permuted (modified) ligands and at least one polypeptide fusion partner, wherein the fusion polypeptide optionally possesses specificity and binding affinity greater than the specificity and binding affinity of the native (unpermuted) ligand for its native target receptor. Additionally, the fusion polypeptide may for example, be further engineered to generate an antagonist of a pathway where the native ligand functioned as an agonist through binding a target receptor as described herein.

[0095] Many receptors bind native ligands and cluster, i.e., form dimers, trimers or multimers, upon binding their native ligands (dimeric or multimeric receptor). For example, the IL-1 family cytokines, fibroblast growth factors, and 4-helix cytokines form multimeric signaling complexes of incorporating various numbers of ligands and receptors (FIG. 9). Ligand-induced clustering (e.g., dimerization, multimerization) often leads to higher affinity complexes and initiates signal transduction. Accordingly, the fusion polypeptides of the invention can, for example, antagonize signaling by, for example, inhibiting binding of the native ligand, or inhibiting receptor clustering (e.g., dimerization, trimerization, multimerization) with or without also inhibiting native ligand binding (FIG. 10). Alternatively, the fusion polypeptides of the invention can enhance signaling by, for example, facilitating the progression of the clustering, through generation of ligands with greater affinity for target receptors or pre-association of components leading to a signaling complex. In a preferred embodiment, the fusion polypeptides of the invention can bind a monomeric ligand or receptor, or a dimeric, or multimeric complex and can inhibit or enhance one or more steps in the assembly of signaling complexes and thereby inhibit or enhance signal transduction of a cellular pathway.

[0096] For example, in the stepwise build up of higher order complexes leading to a final active complex as set forth in Scheme 1, which is representative of the pathway leading to signaling by IL-2 where IL-2 is "A", IL-2R.alpha. is "B", IL-2R.beta. is "C" and .gamma.c is "D":

A+B.fwdarw.AB(step 1);

AB+C.fwdarw.ABC(step 2);

ABC+D.fwdarw.ABCD(step 3); Scheme 1:

[0097] where ABCD is the signaling complex and signaling is initiated by bringing C and D proximal to one another. (The signaling complex is illustrated in FIG. 9 and the structure of the extracellular components of the complex are in FIG. 8).

[0098] A pre-assembled, single chain `AB` would be expected to be a superagonist as it would possess a higher affinity to C than either A or B and thus facilitate assembly of ABCD at lower concentrations. In the case where the native termini of "A" are not positioned to enable the fusion protein, a fusion protein of the ligand "A" that has been modified by circular permutation in accordance with the invention to be optimally oriented to be fused with "B" enables the generation of the single chain `AB` protein. FIG. 8 illustrates this for the case of an engineered IL-2 superagonist (RDB1405; SEQ ID NO: 12 (protein) and SEQ ID NO: 13 (DNA)). On activated T cells, IL-2 signals through the `high affinity` quaternary complex consisting of IL-2, IL-2R.alpha. (also termed CD25), IL-2R.beta. and .gamma.c (FIG. 8A). Although IL-2 can much more weakly bind to IL-2R.beta. in the absence of IL-2R.alpha., the binding of IL-2R.alpha. to IL-2 stabilizes the conformation of IL-2 for presentation to IL-2R.beta. with much greater affinity. .gamma.c is then recruited to the composite surface formed by the IL-2/IL-2R.beta. complex. Expression of a fusion protein of native IL-2 with IL-2R.alpha. is challenging because the IL-2 termini are at the polar opposite face to which IL-2R.alpha. interacts, requiring a spacer to span greater than 50 angstroms and likely disrupting the ability for the fusion protein to bind (FIG. 8A, the IL-2 termini are at the bottom pointing away from IL-2R.alpha. at the top). In fact, an IL-2/IL-2R.alpha. fusion protein with a long spacer has recently been described, and it is incapable of promoting a signal in the absence of a protease-mediated cleavage of the linker with subsequent release of IL-2 (Puskas et al., Immunology, 133(2), 206-220 (2011)). The termini of circularly permuted IL-2(95/94) are engineered to be on the face proximal to the binding interface with IL-2R.alpha., significantly reducing the length of the spacer required to generate a fusion protein that can assemble as an activated complex and may function as a super agonist (FIG. 8B). (The distance between the engineered C-terminus of the circularly permuted IL-2 and the N-terminus of IL-2R.alpha. is about 11 angstroms; the design fusion construct, RDB1405 contains a 6 amino acid spacer between IL-2 and IL-2R.alpha.).

[0099] Alternatively, the stepwise build up of multimeric activation complexes for signal transduction offers the opportunity to create potent antagonists. In this case, the ligand is modified by circular permutation to provide an N- or C-terminus which facilitates linking a fusion partner in an orientation that sterically hinders the stepwise formation of a multimeric activation complex of a target receptor, and in some cases the fusion partner can furthermore augment the binding affinity to the target receptor, if for example, the fusion partner is a protein or domain that in itself contains binding determinants to the target receptor. This latter case is illustrated in the context of the cytokine, IL-6, in FIGS. 1-5 and 10. In this example, the fusion polypeptide comprises circularly permuted interleukin 6 (IL-6) fused to a polypeptide comprising the D1 domain of the transmembrane receptor gp130, which is a natural component of the hexameric IL-6 signaling complex. The circularly permuted ligand (RDB1503) (FIG. 2), in absence of a fusion partner, retains the identical agonistic activity as wild-type IL-6 (FIG. 4), and thus retains the necessary interactions with IL-6R.alpha. and gp130. The hexameric signaling complex of IL-6 is composed of 2 molecules each of IL-6, IL-6R.alpha., and gp130 (FIG. 1). Signal is initiated through the cytoplasmic domain of the two gp130 molecules when two heterotrimers (each with one molecule each of IL-6, IL-6R.alpha., and gp130) come together (FIG. 1). The driving force for the final step in complex formation is the symmetrical interactions between the D1 domains of gp130 from one heterotrimer with IL-6 and IL-6R.alpha. of the other heterotrimer. A fusion of the D1 domain of gp130 to native IL-6 orients the D1 domain away from the gp130 interface (FIG. 3A) and results in a protein (RDB1529) that does not antagonize IL-6-mediated signaling (FIG. 5). In contrast, a fusion of the D1 domain to the circularly permuted IL-6 (RDB1527) orients the D1 such that it can interact with IL-6R.alpha. in an analogous fashion to the hexameric complex, and more importantly sterically prevent the binding of the D1 domain from a second heterotrimer (FIG. 3B), and thus is a potent antagonist of IL-6-mediated signaling (FIG. 5). As the CP_IL-6_D1 fusion protein now carries the combined IL-6R.alpha. binding determinants of the unmodified IL-6 and the native gp130-D1 domain in a single polypeptide, the binding affinity of the fusion polypeptide is measured to be 40 pM, more than 200 fold greater than the binding affinity of native IL6 to the IL-6R.alpha. (FIGS. 6A and 6C).

[0100] In one embodiment, the invention provides for fusion polypeptides comprising the modified ligand and a first fusion partner wherein the first fusion partner of the modified ligand is derived from all or a portion of a protein with additional binding determinants to the target receptor, for example as in the case of a protein or domain which is a component of the natural multimeric signaling complex, and the first fusion partner sterically prevents the assembly of the full signaling complex, thereby acting as an antagonist.

[0101] The ligands modified by circular permutation comprising the fusion polypeptides of the invention include soluble proteins whose binding to cell surface receptors initiate a signaling cascade or serve as natural negative regulators of a signaling cascade (e.g., antagonists), including, but not limited to, cytokines, chemokines, adipokines, growth factors, hormones, soluble receptors, cytokine binding proteins (e.g., IL-18 bp).

[0102] Preferred ligands and proteins modified by circular permutation comprising the fusion polypeptides of the invention include helix bundle proteins and cytokines (including, but not limited to, growth hormone, IL-2, IL-4, IL-5, IL-6, IL-10, IL-22, IL-23p19, IL-11, IL-13, IL-15, IL-12p35, IL-21, IL-30 (IL27p28), IL-34, IL-35, IL-35p35, IFN-.beta., IFN.gamma., LIF, CNTF, Oncostatin M, CLCF-1, GCSF, GM-CSF, EPO, ferritin, leptin, placental lactogen, prolactin, apolipoprotein e), b-trefoil proteins (including, but not limited to, IL-1.alpha., IL-1.beta., IL-1Ra, IL18, IL-33, IL-36Ra, IL-36a, IL-36b, IL-36g, IL-37, IL-38, IL1Hy2, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8a, FGF-8b, FGF-8e, FGF-8f, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23), .alpha./.beta. (TIM) barrel proteins (including, but not limited to, triosephosphate isomerase), beta sandwich proteins (including, but not limited to, galectin-1, galectin-3, TNF-beta, seven .beta.-propeller proteins, class 1 MHC .alpha.1.alpha.2 domain, integrin I domain, GYF domain, C1 domain, C2 domain (for example, from cPLA2, PKC, synaptotagmin), PDZ domains, C3d, C5a.

[0103] In the most preferred embodiments, the ligand modified by circular permutation comprising the fusion polypeptides of the invention is selected from IL-6, IL-2, IL-15, IL-1.alpha., IL-1.beta., IL-1Ra, IL-18, FGF-19, FGF-21, FGF-23.

[0104] The ligands modified by circular permutation comprising the fusion polypeptides of the invention can have binding specificity for a receptor, or for a receptor that binds a native ligand in the following list: ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2, FGF-acidic, FGF-basic, fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-.beta.1, insulin, IFN-.gamma., IGF-I, IGF-II, IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin .alpha., Inhibin .beta., IP-10, keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian inhibitory substance, monocyte colony inhibitory factor, monocyte attractant protein, M-CSF, MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a.), MIG, MIP-1.alpha., MIP-1.beta., MIP-3.alpha., MIP-3.beta., MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1), NAP-2, Neurturin, Nerve growth factor, .beta.-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1.alpha., SDF1.beta., SCF, SCGF, stem cell factor (SCF), TARC, TGF-.alpha., TGF-.beta., TGF-.beta.2, TGF-.beta.3, tumor necrosis factor (TNF), TNF-.alpha., TNF-.beta., TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-.beta., GRO-.gamma., HCC1, 1-309, HER1, HER2, HER3, and HER4.

[0105] Additional receptors that the modified ligand can have binding specificity for include the receptors in the following list, or a receptor that binds a native ligand included in the following list: EpoR, TACE recognition site, TNF BP-I, TNF BP-II, IL-1R1, IL-6R, IL-10R, IL-18R, IL-1, IL-19, IL-20, IL-21, IL-23, IL-24, IL-25, IL-27, IFN-.gamma., IFN-.alpha./.beta., CD4, CD89, CD19, HLA-DR, CD38, CD138, CD33, CD56, CEA, and VEGF receptor.

[0106] Further receptors that the modified ligands of the fusion polypeptides of the invention can have binding specificity for include gastrin releasing peptide receptor, neurotensin receptor, adrenomedullin receptor, H2 histamine receptor, HCG receptor, MET receptor, sphingosine 1-phosphate receptor, CD126, CD213a1, and KDR, among others.

[0107] The modified ligand of the polypeptide fusion protein of the invention can have binding specificity for a receptor that dimerizes upon binding to a native ligand (a dimeric receptor), or a receptor that forms multimers, such as trimers, upon binding to a native ligand (a multimeric receptor). Many cytokine receptors and growth factor receptors, such as members of the TNF receptor superfamily (e.g., TNFR1, TNFR2) and members of the receptor tyrosine kinase family (e.g., EGFR, PDGFR, M-CSF receptor (c-Fms)) form dimers or multimers upon binding their native ligands. The TNF receptor superfamily is an art recognized group of proteins that includes TNFR1 (p55, CD120a, p60, TNF receptor superfamily member 1A, TNFRSF1A), TNFR2 (p'75, p80, CD120b, TNF receptor superfamily member 1B, TNFRSF1B), CD (TNFRSF3, LT.beta.R, TNFR2-RP, TNFR-RP, TNFCR, TNF-R-III), OX40 (TNFRSF4, ACT35, TXGP1L), CD40 (TNFRSF5, p50, Bp50), Fas (CD95, TNFRSF6, APO-1, APTI), DcR3 (TNFRSF6B), CD27 (TNFRSF7, Tp55, S152), CD30 (TNFRSF8, Ki-1, D1S166E), CD137 (TNFRSF9, 4-1BB, ILA), TRAILR-1 (TNFRSF10A, DR4, Apo2), TRAIL-R2 (TNFRSF10B, DR5, KILLER, TRICK2A, TRICKB), TRAILR3 (TNFRSF10C, DcR1, LIT, TRID), TRAILR4 (TNFRSF10D, DcR2, TRUNDD), RANK (TNFRSF11A), OPG (TNFRSF11B, OCIF, TR1), DR3 (TNFRSF12, TRAMP, WSL-1, LARD, WSL-LR, DDR3, TR3, APO-3), DR3L (TNFRSF12L), TAC1 (TNFRSF13B), BAFFR (TNFRSF13C), HVEM (TNFRSF14, ATAR, TR2, LIGHTR, HVEA), NGFR (TNFRSF16), BCMA (TNFRSF17, BCM), AITR (TNFRSF18, GITR), TNFRSF19, FLJ14993 (TNFRSF19L, RELT), DR6 (TNFRSF21), SOBa (TNFRSF22, Tnfrh2, 2810028K06Rik), and mSOB (THFRSF23, Tnfrh1). The receptor tyrosine kinase family is an art recognized group of proteins that includes EGFR (ERBB1, HER1), PDGFR, c-Fms, FGFR1, FGFR2, FGFR3, FGFR4, Insulin receptor, and Insulin-like growth factor receptors (IGF1R, IGF2R). See, Grassot et al., Nucleic Acids Research, 31(1):353-358 (2003).

[0108] In one embodiment, the first polypeptide fusion partner comprises all or any portion of the extracellular domains of the natural receptors or accessory proteins for growth hormone, IL-2, IL-4, IL-5, IL-6, IL-10, IL-11, IL-12, IL-13, IL-15, IL-21, IL-22, IL-23, IL-30 (IL27p28), IL-34, IL-35, IFN-.beta., IFN.gamma., LIF, CNTF, Oncostatin M, CLCF-1, GCSF, GM-CSF, EPO, placental lactogen, prolactin, apolipoprotein, IL-1.alpha., IL-1.beta., IL-1Ra, IL18, IL-33, IL-36Ra, IL-36a, IL-36b, IL-36g, IL-37, IL1Hy2, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8a, FGF-8b, FGF-8e, FGF-8f, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23, TNF-beta.

[0109] In the most preferred embodiments, the fusion partner is the extracellular domain or a domain thereof selected from gp130 (most preferentially the D1 domain), IL-2R.alpha., IL-15R.alpha., IL-1RI, IL-1RII, IL-18R.alpha., IL-18R.beta., IL1RAcP, FGFR1b, FGFR1c, FGFR2b, FGFR2c, FGFR3b, FGFR3c, FGFR4, .alpha.-Klotho, and .beta.-Klotho.

[0110] In one embodiment, the protein modified by circular permutation comprising the fusion polypeptides of the invention and the fusion partner may originate from the same original protein such that the fusion generates a single chain "homodimer".

[0111] In one embodiment, the fusion partner to the circularly permuted polypeptide may also require circular permutation to enable the fusion. Thus both partners of the fusion protein of the invention may be circularly permuted, if necessary.

[0112] In one embodiment the polypeptide fusion partner provides other novel or improved/enhanced functions or behavior to the fusion polypeptide. In addition to, or alternatively, a second fusion partner may be added to the fusion polypeptide of the invention to provide other novel and improved/enhanced functions or behavior to the fusion polypeptide of the invention. For example, the fusion partners may provide extended half life to the fusion polypeptide of the invention. Addition of fusion partners to extend in vivo half-life is particularly useful when the fusion polypeptide of the invention is of a size that is rapidly cleared from the body, which can limit clinical use.

[0113] A polypeptide of the invention can be modified such that it has a larger hydrodynamic size by for example, coupling to polymers or carbohydrates (such as polyethyleneglycol (PEG), colominic acid, or hydroxyethyl starch), incorporation of N-glycosylation sites, or through recombinant PEG mimetics produced through fusion of a long, flexible polypeptide sequence, such as those described in U.S. 2010/0239554 A1, Hydrodynamic size of a polypeptide fusion protein of the invention may be determined using methods which are well known in the art. For example, gel filtration chromatography may be used to determine the hydrodynamic size. Suitable gel filtration matrices for determining the hydrodynamic sizes of ligands, such as cross-linked agarose matrices, are well known and readily available.

[0114] In one preferred embodiment, a fusion polypeptide of the invention is designed to incorporate a mucin-domain polypeptide as is described in U.S. Ser. No. 61/657,264 entitled "Fusion Polypeptides Comprising an Active Protein Linked to a Mucin-Domain Polypeptide" filed on even date herewith, bearing attorney docket number 4000.3058 US, and incorporated by reference herein in its entirety.

[0115] In one embodiment, a fusion polypeptide of the invention can be fused to proteins, protein domains, or peptides that that enhance serum half-life through FcRn-mediated recycling, including immunoglobulins, the Fc domain of immunoglobulins (most notably IgG1 and IgG2), serum albumin, serum albumin domains (most notably DIII), peptides with binding affinity to FcRn, or proteins or peptides with binding affinity to immunoglobulins or serum albumin (such as nanobodies).

[0116] Methods for pharmacokinetic analysis and determination of ligand half-life will be familiar to those skilled in the art. Details may be found in Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al., Pharmacokinetc analysis: A Practical Approach (1996). Reference is also made to "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2.sup.nd Rev. ex edition (1982), which describes pharmacokinetic parameters such as t alpha and t beta half lives and area under the curve (AUC).

[0117] In one embodiment, a fusion polypeptide of the invention can be fused to proteins, protein domains, or peptides that that target (i.e. have affinity for) specific organs, tissues, cells, or physiological matrices (such as collagen), carbohydrates, or lipids as a means for localizing, distributing, or retaining the fusion polypeptide of the invention in a particular region of the body.

[0118] Additional sequences also can be included as part of the fusion polypeptide such as affinity tag sequences that can be provided to facilitate the purification or isolation of the fusion polypeptide such as those known in the art. Stability sequences can also be added to the fusion polypeptide to protect the molecule from degradation (e.g., by a protease). Suitable stability sequences include, but are not limited to, glycine molecules incorporated after the initiation methionine (e.g., MG (SEQ ID NO: 17), or MGG (SEQ ID NO: 18) to protect the fusion molecule from ubiquitination; two prolines incorporated at the C-terminus (conferring protection against carboxypeptidase action), and the like.

[0119] In order to test the biological activity, binding specificity and binding affinity of a fusion polypeptide of the invention, an appropriate biological assay may be used. Assays for biological activities of various kinds are well known to those of skill in the art. The particular assay depends on the particular activity of the molecule.

Preparation of Circularly Permuted Proteins

[0120] Circularly permuted proteins may be made by a number of means known to those of skill in the art. These include chemical synthesis, modification of existing proteins, and expression of circularly permuted proteins using recombinant DNA methodology.

Where the protein is relatively short (i.e., less than about 50 amino acids) the circularly permuted protein may be synthesized using standard chemical peptide synthesis techniques. If the linker is a peptide, it may be incorporated during the synthesis. If the linker is not a peptide, it may be coupled to the peptide after synthesis. Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is the preferred method for the chemical synthesis of the circularly permuted ligands and fusion proteins of this invention. Techniques for solid phase, synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984) which are incorporated herein by reference.

[0121] Alternatively, the circularly permuted protein may be made by chemically modifying a native protein. Generally, this requires reacting the native protein in the presence of the linker to form covalent bonds between the linker and the carboxyl and amino termini of the protein, thus forming a circular protein. New termini are then formed by opening the peptide bond joining amino acids at another location. This may be accomplished chemically or enzymatically using, for example, a peptidase.

[0122] In a preferred embodiment, the circularly permuted protein, or fusion polypeptides comprising the circularly permuted protein fused to at least one fusion partner, will be synthesized using recombinant DNA methodology. Generally, this involves creating a DNA sequence that encodes the circularly permuted ligand (or entire fusion polypeptide containing the circularly permuted ligand and fusion partner), placing the DNA in an expression vector under the control of a particular promoter, expressing the protein in a host, isolating the expressed protein and, if required, renaturing the protein.

[0123] DNA encoding the circularly permuted ligand may be produced by gene synthesis, or by using DNA amplification methods, for example polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR). DNA encoding a signal sequence such that the properly processed circularly permuted fusion protein is secreted from the cell can optionally be added.

[0124] One of skill will appreciate that the circularly permuted ligand and the other molecule comprising the fusion polypeptides of the invention may be joined together in any order. Thus, the second molecule is preferably joined to either the amino (N-terminal fusion) or carboxy (C-terminal fusion) terminus of the circularly permuted ligand.

[0125] The circularly permuted ligands and their fusion proteins may be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell lines. The recombinant protein gene will be operably linked to appropriate expression control sequences for each host. For E. coli this includes a promoter such as the T7, trp, or lambda promoters, a ribosome binding site and preferably a transcription termination signal. For eukaryotic cells, the control sequences will include a promoter and preferably an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences.

[0126] The plasmids of the invention can be transferred (transfected) into the chosen host cell by well-known methods such as calcium chloride transformation for E. coli and calcium phosphate treatment, electroporation, lipofectamine treatment, or PEI treatment for mammalian cells. Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo and hyg genes.

[0127] Once expressed, the recombinant fusion proteins can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity chromatography, column chromatography with ionic or hydrophobic resins, gel electrophoresis and the like (see, generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982), Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. N.Y. (1990)). Substantially pure compositions of at least about 90 to 95% purity are preferred, and 98 to 99% or higher purity are most preferred for pharmaceutical uses. Once purified, the polypeptides may be tested in preclinical models, tested clinically, or used therapeutically.

[0128] One of skill would recognize that modifications can be made to the circularized protein sequence without diminishing its biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of the circularly permuted ligand into a fusion protein. Such modifications are well known to those of skill in the art and include, addition of residues for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids placed on either terminus to protect the protein from exopeptidases. For example, circularly permuted IL6 may optionally have an additional methionine (Met) codon at the amino terminus to provide an initiation site for translation.

[0129] One of skill will recognize that other modifications may be made. Thus, for example, amino acid substitutions may be made that increase specificity or binding affinity of the circularly permuted protein, etc. Alternatively, non-essential regions of the molecule may be shortened or eliminated entirely. Thus, where there are regions of the molecule that are not themselves involved in the activity of the molecule, they may be eliminated or replaced with shorter segments that merely serve to maintain the correct spatial relationships between the active components of the molecule.

[0130] The two proteins may be fused together directly or joined by means of a peptide spacer. The peptide spacer may range from about 1 to 40 residues in length. In a preferred embodiment, the peptide spacer is 20 .ANG. or less in length.

[0131] Generally, the spacer has no biological activity itself and functions only to link and provide some distance between the two active proteins comprising the fusion protein. However, one of skill will recognize that the residues of the spacer may be chosen to optimize a property of the fusion protein. For example, a spacer containing polar or charged residues in the spacer may enhance solubility in aqueous solutions. Similarly, the spacer residues may be chosen for their effect on the folding of the fusion protein.

[0132] It is understood that the invention includes the above-described nucleic acids encoding the fusion polypeptides of the inventions such as recombinant nucleic acids produced by recombinant DNA methodology, as well as expression vectors comprising the nucleic acids of the invention and host cells comprising the vectors of the invention.

Therapeutic Uses

[0133] The fusion polypeptides of the invention compositions described herein are particularly well suited as therapeutic agents targeting cells of interest in vivo (i.e., target cells) since they exhibit, among other properties, higher binding affinities for native receptors than native ligands, and super agonist and antagonistic activities. Thus, the compositions and pharmaceutical compositions containing the present fusion polypeptides can be administered to a patient in need for therapeutic treatments. In therapeutic applications, fusion polypeptides of the invention comprising circularly permuted ligands, and various compositions containing these molecules are administered to a patient suffering from a disease or disorder in a therapeutically effective amount.

[0134] The invention provides compositions comprising the fusion polypeptides of the invention and a pharmaceutically acceptable carrier, diluent or excipient, and therapeutic and diagnostic methods that employ the ligands or compositions of the invention.

[0135] Therapeutic and prophylactic uses of ligands of the invention involve the administration of ligands according to the invention to a recipient mammal, such as a human. The fusion polypeptides of the invention preferably bind to targets with high affinity and/or avidity. Substantially pure ligands of at least 90 to 95% homogeneity are preferred for administration to a mammal, and 98 to 99% or more homogeneity is most preferred for pharmaceutical uses, especially when the mammal is a human. Once purified, partially or to homogeneity as desired, the fusion polypeptides of the invention may be used diagnostically or therapeutically (including extracorporeally) or in developing and performing assay procedures, immunofluorescent stainings and the like.

[0136] For example, the fusion polypeptides of the present invention will typically find use in preventing, suppressing or treating disease states. For example, fusion polypeptides can be administered to treat, suppress or prevent a disease or disorder caused by receptor activity, or characterized by expression or overexpression of receptor, such as chronic inflammation or chronic inflammatory diseases, cardiovascular diseases, metabolic diseases (e.g., obesity, Type II diabetes, metabolic syndrome), respiratory diseases (e.g., asthma, COPD), ophthalmic diseases (e.g., AMD, glaucoma), hematopoietic disorders, immunosuppression, organ transplant rejection, graft versus host disease, bone and cartilage diseases (osteoporosis, osteoarthritis), allergic hypersensitivity, cancer, bacterial or viral infection, autoimmune disorders (which include, but are not limited to, Type I diabetes, asthma, multiple sclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, spondylarthropathy (e.g., ankylosing spondylitis), autoinflammatory disorders, systemic lupus erythematosus, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis), myasthenia gravis and Behcet's syndrome), psoriasis, endometriosis, and abdominal adhesions (e.g., post abdominal surgery).

[0137] One preferred application is, through the use of the circularly permuted IL-2 fused to IL-2R.alpha. generating an IL-2 super agonist), the treatment of cancer, or of autoimmune conditions such as graft-versus-host disease, organ transplant rejection.

[0138] Another preferred application is, through the use of a circularly permuted IL-6 fused to the D1 domain of gp130 (generating a very potent IL-6 antagonist), the treatment of chronic inflammatory diseases, autoimmune diseases (including, but not limited to, rheumatoid arthritis, psoriasis, psoriatic arthritis, juvenile rheumatoid arthritis, Crohn's disease, inflammatory bowel syndrome), cancer (including multiple myeloma), and Castleman's disease. As described in Examples 2 and 3, the circularly permuted ligand-gp130 fusion protein of the present invention (RDB1527) shows greater specific binding affinity to the native IL6 receptor (FIGS. 6A vs. 6C) and target cell inhibition (FIG. 5), as compared to native ligands. The increased binding affinity and growth inhibition of circularly permuted IL6-gp130 fusion polypeptides may allow these fusion proteins to be administered at lower dosages as compared to other inhibitors of IL6 signaling, while achieving the same therapeutic efficacy. Alternatively, administration at the same dosages results in prolonged therapeutic efficacy as the fusion proteins must be cleared from the circulation to a lower concentration before they cease to show significant efficacy. In addition, the increased therapeutic efficacy is not accompanied by an increase in undesired side effects due to non-specific binding and cytotoxicity.

[0139] Another preferred application is, through the use of a circularly permuted IL-1 fused to a domain of either IL-1RI, IL-1RII, or IL-1RAcP (generating a very potent IL-1 antagonist), the treatment of autoinflammatory diseases, Type I diabetes, chronic inflammatory diseases, autoimmune diseases (including, but not limited to, rheumatoid arthritis, psoriasis, psoriatic arthritis, juvenile rheumatoid arthritis, Crohn's disease, inflammatory bowel syndrome), cancer, gout, and osteoarthritis.

[0140] Another preferred application is, through the use of the circularly permuted IL-15 fused to IL-15.alpha. (generating an IL-15 super agonist), the treatment of cancer, of autoimmune conditions such as graft-versus-host disease, organ transplant rejection, or of infection.

[0141] The circularly permuted ligand portion of the fusion polypeptide is chosen according to the intended use. Proteins that may serve as targets for the circularly permuted ligands include but are not limited to signaling molecules such as growth factors or biologically active fragments or mutants thereof. For example, the growth factor can be a cytokine (e.g., an interleukin or chemokine). While one of ordinary skill in the art can readily determine whether a molecule is a signaling molecule (i.e., whether it is produced and secreted by a first cell type and exerts an effect on itself or (autocrine) or on a second cell type (paracrine), usually by specifically binding a receptor), various particular signaling molecules may be properly placed in two or more categories. For example, IL-1 may be properly referred to as a cytokine or interleukin and erythropoietin may be properly referred to as a growth factor or a hormone; etc.

[0142] An cytokine includes but is not limited to, IL-1.alpha., IL-1.beta., IL-1Ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-12p35, IL-13, IL-15, IL-17 family members, IL18, IL-21, IL-22, IL-23, IL-23p19, IL-30 (IL27p28), IL-33, IL-34, IL-35, IL-35p35, IL-36Ra, IL-36a, IL-36b, IL-36g, IL-37, IL-38, LIF, CNTF, Oncostatin M, CLCF-1, GCSF, GM-CSF, ferritin, placental lactogen, apolipoprotein e, interferon-alpha (IFN.alpha.), interferon-beta (IFN.beta.), or interferon-gamma (IFN.gamma.). A chemokine can be a member of the a subfamily and/or can bind a CXCR1, CXCR2, CXCR3, CXCR4, or CXCR5 receptor; it can be a member of the .beta. subfamily and/or can bind a CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11 molecule. A chemokine can also be lymphotactin or another chemokine that binds a XCR1 receptor; a chemokine can also be fractalkine or can bind a CX3CR1 receptor. For example, the chemokine can be CCL7, CCL23, CCL27, CCL28, CXCL12, CXCL14, or CXCL15.

[0143] Growth factors include but are not limited to members of the tumor necrosis factor (TNF) family, members of the nerve growth factor (NGF) family, members of the transforming growth factor (TGF) family, members of the GDF family, members of the BMP family, members of the fibroblast growth factor (FGF) family (including FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8a, FGF-8b, FGF-8e, FGF-8f, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23, members of the insulin-like growth factor (IGF) family, members of the epidermal growth factor (EGF) family, or members of the platelet-derived growth factor (PDGF) family. For example, the growth factor can be TNF, EGF, TGF.alpha., TGF.beta., FGF, NGF, erythropoietin, IGF-1, or IGF-2.

[0144] A hormone can be a hormone produced by the adrenal gland, parathyroid gland, pituitary gland, or thyroid gland; it can also be produced by the hypothalamus, the ovary, the testicle, the pancreas, the pineal body, or the thymus. For example, the hormone can be a thyroid-stimulating hormone, a follicle-stimulating hormone, a leuteinizing hormone, prolactin, growth hormone, adrenocorticotropic hormone, antidiuretic hormone, oxytocin, thyrotropin-releasing hormone, gonadotropin-releasing hormone, growth hormone-releasing hormone, corticotropin-releasing hormone, somatostatin, dopamine, melatonin, thyroxine, calcitonin, parathyroid hormone, a glucocorticoid, a mineralocorticoid, an androgen, adrenaline, an estrogen, progesterone, human chorionic gonadotropin, insulin, glucagons, somatostatin, erythropoietin, calcitriol, atrial-natriuretic peptide, gastrin, secretin, cholecystokinin, somatostatin, neuropeptide Y, ghrelin, PYY3-36, insulin-like growth factor-1, angiotensinogen, thrombopoietin, or leptin.

[0145] Neurotransmitters include acetylcholine, dopamine, norepinephrine, serotonin, histamine, or epinephrine. The neurotransmitter can also be a neuroactive peptide (e.g., bradykinin, cholecystokinin, gastrin, secretin, oxytocin, a sleep peptide, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, neurotensin, motilin, thyrotrop in, neuropeptide Y, leuteinizing hormone, calcitonin, or vasoactive intestinal peptide). Suitable co-stimulatory molecules include B7-1 and B7-2.

Pharmaceutical Compositions

[0146] The present invention provides pharmaceutical compositions comprising fusion proteins of the invention. In one embodiment, the pharmaceutical composition comprises the fusion protein and at least one pharmaceutically acceptable carrier. Fusion proteins of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the polypeptide is combined with a pharmaceutically acceptable carrier vehicle, such as aqueous solutions or buffers, pharmaceutically acceptable suspensions and emulsions. Examples of non-aqueous solvents include propyl ethylene glycol, polyethylene glycol and vegetable oils. Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers, as described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), in the form of lyophilized formulations or aqueous solutions.

[0147] More particularly, the present pharmaceutical compositions may be administered for therapy by any suitable route including subcutaneous, subcutaneous or intrathecally by infusion pump, intramuscular, intravenous, intradermal, intravitreal, nasal, and pulmonary. It will also be appreciated that the preferred route will vary with the therapeutic agent, condition and age of the recipient, and the disease being treated.

[0148] In one embodiment, the pharmaceutical composition is administered subcutaneously. In this embodiment, the composition may be supplied as a lyophilized powder to be reconstituted prior to administration. The composition may also be supplied in a liquid form, which can be administered directly to a patient. In one embodiment, the composition is supplied as a liquid in a pre-filled syringe such that a patient can easily self-administer the composition.

[0149] In another embodiment, the compositions of the present invention are encapsulated in liposomes, which have demonstrated utility in delivering beneficial active agents in a controlled manner over prolonged periods of time. Liposomes are closed bilayer membranes containing an entrapped aqueous volume. Liposomes may also be unilamellar vesicles possessing a single membrane bilayer or multilamellar vesicles with multiple membrane bilayers, each separated from the next by an aqueous layer. The structure of the resulting membrane bilayer is such that the hydrophobic (non-polar) tails of the lipid are oriented toward the center of the bilayer while the hydrophilic (polar) heads orient towards the aqueous phase. In one embodiment, the liposome may be coated with a flexible water soluble polymer that avoids uptake by the organs of the mononuclear phagocyte system, primarily the liver and spleen. Suitable hydrophilic polymers for surrounding the liposomes include, without limitation, PEG, polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxethylacrylate, hydroxymethylcellulose hydroxyethylcellulose, polyethyleneglycol, polyaspartamide and hydrophilic peptide sequences as described in U.S. Pat. Nos. 6,316,024; 6,126,966; 6,056,973 and 6,043,094, the contents of which are incorporated by reference in their entirety.

[0150] Liposomes may be comprised of any lipid or lipid combination known in the art. For example, the vesicle-forming lipids may be naturally-occurring or synthetic lipids, including phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phasphatidylglycerol, phosphatidylinositol, and sphingomyelin as disclosed in U.S. Pat. Nos. 6,056,973 and 5,874,104. The vesicle-forming lipids may also be glycolipids, cerebrosides, or cationic lipids, such as 1,2-dioleyloxy-3-(trimethylamino) propane (DOTAP); N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE); N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethylammonium bromide (DOME); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); 3 [N--(N',N'-dimethylaminoethane) carbamoly] cholesterol (DC-Chol); or dimethyldioctadecylammonium (DDAB) also as disclosed in U.S. Pat. No. 6,056,973. Cholesterol may also be present in the proper range to impart stability to the vesicle as disclosed in U.S. Pat. Nos. 5,916,588 and 5,874,104.

[0151] For liquid formulations, a desired property is that the formulation be supplied in a form that can pass through a 25, 28, 30, 31, 32 gauge needle for intravenous, intramuscular, intraarticular, or subcutaneous administration.

[0152] In other embodiments, the composition may be delivered via intranasal to enable transfer of the active agents through the olfactory passages into the CNS and reducing the systemic administration. Devices commonly used for this route of administration are included in U.S. Pat. No. 6,715,485. Compositions delivered via this route may enable increased CNS dosing or reduced total body burden reducing systemic toxicity risks associated with certain drugs. Preparation of a pharmaceutical composition for delivery in a subdermally implantable device can be performed using methods known in the art, such as those described in, e.g., U.S. Pat. Nos. 3,992,518; 5,660,848; and 5,756,115.

[0153] A typical pharmaceutical composition for parenteral administration would be about 0.1 to 3 mg/kg per patient per day. Methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980).

[0154] Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the proteins of this invention to effectively treat the patient.

EXAMPLES

[0155] The following examples are offered by way of illustration and are not to be construed as limiting the invention as claimed in any way.

Example 1. Design, Preparation, Expression, and Purification of Picasso Fusion Constructs

1. Design of Circularly Permutated IL-6 and Fusion to Domain D1 of Gp130 Receptor

[0156] The crystal structure of the hexameric IL-6 signaling complex (1P9M.pdb; FIG. 1) was utilized to design a circularly permutated variant of human IL6, named picasso3 (FIG. 2; RDB1503; SEQ ID NO: 1 (protein) and SEQ ID NO: 2 (nucleic acid)), such that the engineered C-terminus can be fused to the N-terminus of the D1 domain of gp130 in the complex through a short spacer. Briefly, the engineered N and C termini in picasso3 correspond to residues 182 and 180 in wild-type IL-6 (SEQ ID NO: 3), and the native IL-6 termini were joined through a 7 amino acid linker. The D1 domain of gp130 was chosen as the fusion partner such that not only would it interfere sterically with hexamer formation, but also enhance the binding affinity to IL-6R through native interactions present in the complex (FIG. 3B). The engineered C-terminus of picasso3 was fused to the D1 domain through a two amino acid spacer to form RDB1527 (FIG. 3B; SEQ ID NO: 4 (protein) and SEQ ID NO: 5 (nucleic acid)). As a control an analogous fusion protein consisting of native IL6 fused to D1 (RDB1529) was designed (FIG. 3A; SEQ ID NO: 6 (protein) and SEQ ID NO: 7 (nucleic acid)).

2. Gene Synthesis

[0157] Synthesis of the genes for expression of the designed constructs was carried out using standard methods.

3. Subcloning of the Synthesized Gene into a Mammalian Expression Vector

[0158] A) Preparation of the Expression Vector pcDNA.TM. (Invitrogen).

[0159] 5 .mu.g of pcDNA was digested with BamHI and HindIII for two hours at 37.degree. C. The digest was treated with calf alkaline phosphatase to remove the 5' phosphate, thus preventing religation of vector on itself. Buffer was exchange to remove salts from calf alkaline phosphatase reaction. Qiagen's PCR cleanup kit was used following the manufacturer's suggested protocol. The DNA was eluted in 30 .mu.l of H20.

[0160] B) Preparation of the Gene of Interest.

[0161] The gene of interest was digested with BamHI and HindIII for two hours 37.degree. C. The digestion reaction was run on an E-Gel.RTM. CloneWell.TM. apparatus (Invitrogen) using 0.8% SYBR Green. The fragment corresponding to the gene of interest was isolated from the second row of wells on the gel.

[0162] C) Ligation Reaction of the Gene to pcDNA.

[0163] The prepared pcDNA (step A) was mixed with the DNA from step B in the presence of T4 ligase and incubated at room temperature for 30 minutes. Following the ligation, the products were transformed into TOP10 cells (Invitrogen; chemically competent strain of E. coli) and the correct clone was picked and stored as a glycerol stock at the -80.degree. C.

4. Expression of RDB1503, RDB1527, and RDB1529

[0164] All the proteins were expressed in CHO cells using FreeStyle.TM. Max Reagent (Invitrogen) following the manufacturer's protocol. Briefly, a day prior to transfection the cells were seeded at 0.5.times.10.sup.6 cells/mL and on the day of transfection they were adjusted to 1.times.10.sup.6 cells/mL as recommended by manufacturer. For a 1 liter transfection, two tubes (A and B) of media (OptiPRO.TM., Invitrogen) were prepared containing about 19 ml, 1 mg of DNA was added to tube A and 1 ml of FreeStyle.TM. Max reagent was added to tube B. Immediately the contents of both tubes were mixed and incubated at room temperature for 15 minutes. After the incubation period the mixture was added slowly to the 1 liter of CHO cells. After transfection the cells were left for 6-to-7 days and then the supernatant was collected.

5. Purification of RDB1527 and RDB1529

[0165] The expressed protein in the supernatant was captured on a protein A column to bind the Fc portion of the fusion protein. After binding the protein, the column was washed with up to 5 column volume of PBS. The protein was eluted from the column by lowering the pH of the running buffer and directly neutralized with Tris buffer pH=7. The purified protein was then dialyzed overnight against PBS.

Example 2. In Vitro Activity of Circularly Permutated IL6 Fusion Proteins

[0166] HEK-Blue.TM. IL-6 cells (Invivogen) are human embryonic kidney cells specifically designed to detect bioactive IL-6 in vitro by monitoring the IL-6-induced expression of a STAT3-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. SEAP can be readily monitored when using the SEAP detection medium QUANTI-Blue.TM. (Invivogen). The human cell line and detection medium were used to test the ability of the circularly permutated IL6 and IL6 fusion proteins constructs RDB1503, RDB1527 and RDB1529 to agonize or antagonize the IL-6-induced SEAP.

[0167] 100 .mu.L of media containing HEK-Blue.TM. IL-6 cells were plated into 96-well microtiter plates to a final concentration of 50,000 cells/well. To measure agonist activity, IL6 and RDB1503 were prepared at initial concentration of 200 pM then serially diluted and added in duplicate test samples to the HEK-Blue.TM. IL-6 cells. To measure antagonist potency, RDB1527 and RDB1529 were prepared at an initial concentration of 3.3 nM then serially diluted and added in duplicate test samples to the HEK-Blue.TM. IL-6 cells in the presence of a constant concentration of IL6 of 12.5 pM. The samples were incubated at 37.degree. C., 5% CO2 from 20-24 hours, then 40 .mu.L of each sample transferred to a new 96 well plate containing 160 .mu.L of QUANTI-Blue.TM. in each well, and incubated at 37.degree. C., 5% CO2. Absorbance readings were taken at 630 nm after 3 hours of incubation.

[0168] Circularly permuted IL-6 (RDB1503) demonstrated agonist activity with a comparable EC.sub.50 to that of IL-6 (FIG. 4). RDB1527 (CP_IL-6_D1_Fc) was able to inhibit IL-6-induced SEAP expression in a dose-dependent fashion with an IC.sub.50 value of 0.22 nM (FIG. 5). In contrast, RDB1529 (unmodified IL-6_D1_Fc) showed no antagonist effect (FIG. 5).

[0169] The following conclusions were drawn: 1) Circular permutation of IL-6 results in no loss in binding affinity; 2) fusion of the D1 domain of gp130 to the C-terminus of wild type IL-6 does not convert IL-6 to a potent antagonist, whereas fusion of the D1 domain of gp130 to the C-terminus of the circularly permuted IL-6 results in potent antagonism of IL-6 mediated signaling.

Example 3. Kinetics Measurements of wtIL6, RDB1527 and RDB1529

[0170] Wild type IL6-Fc (wtIL6), RDB1527 and RDB1529 were immobilized on a Biacore.TM. sensor chip using the human antibody capture kit (GE Healthcare) as per manufacturer's protocol. IL-6R was passed over the surface of the chip in a stepwise model. IL-6R was prepared in 5 concentrations; 3.0 nM, 1.0 nM, 0.33 nM, 0.11 nM, and 0.03 nM. In the First cycle the 0.03 nM concentration of IL-6R was flowed over the bound ligand on the surface of the chip for 180 seconds, after which a blank solution was passed over the surface to allow the IL-6R to dissociate. The same procedure was repeated for an additional four times using an increasing concentration from 0.03 to 3 nM of 6R. The resulting sensorgrams were analyzed with the native instrument software to calculate the binding affinities of the constructs. The binding affinity of RDB1527 to IL-6R was calculated to be 40 pM (FIG. 6C), representing an increase of over 200-fold when compared to the affinity of the control wtIL6 (9 nM, FIG. 6A). The sensorgram for RDB1529 (FIG. 6B) resulted in a poor fit, and thus the calculated binding affinity is not reliable. This may be due to mixed binding between IL-6 and IL-6R and independently D1 and IL-6R.

[0171] Based upon the results it was concluded that the binding affinity of RDB1527 to IL-6R is 40 pM, or greater than 200-fold higher than that of wtIL6. This data, in combination with the potent antagonist activity, strongly suggests that the binding determinants on IL-6 and on D1 are simultaneously binding to IL-6R, and preventing association of the signaling complex, as designed.

Example 4. Design of Circularly Permutated IL1.beta. Fused to Domains D1-D2 of IL-1RI (RDB1538)

[0172] The crystal structure of the heterotrimeric IL-1.beta. signaling complex (4DEP.pdb; FIG. 7A) was utilized to design a circularly permutated variant of IL-1.beta. (RDB1515; SEQ ID NO: 8 (protein) and SEQ ID NO: 9 (nucleic acid)), such that the engineered N terminus can be fused to the C-terminus of the D1-D2 domain of IL-1RI through a short spacer. Briefly, the engineered N and C termini correspond to residues 224 and 223, respectively, in wild-type IL-1.beta., and the native IL-1.beta. termini were joined through a 7 amino acid linker. The newly created N-terminus was fused to domain D1-D2 of RI with a 5 amino acid spacer and a FLAG tag was added to the N-terminus of RI, resulting in RDB1538 (FIG. 7B; SEQ ID NO: 10 (protein) and SEQ ID NO: 11 (nucleic acid)). The resulting fusion protein is designed to be an antagonist of IL-1.beta.-mediated signaling by binding to IL-1RAcP and preventing the full signaling complex to assemble.

Example 5. Design of Circularly Permutated IL-2 Fused to IL-2R.alpha. (RDB1405)

[0173] Upon binding IL-2R.alpha., the binding conformation of IL-2 is stabilized to allow for a high affinity complex to be formed with IL-2R.beta. and .gamma..sub.c. RDB1405 is designed to be a super agonist of IL-2-mediated signaling, particularly in cells lacking IL-2R.alpha., as it would be able to form the high affinity complex without requiring binding to cell-associated IL-2R.alpha.. The crystal structure of the quaternary signaling complex of IL-2 (2B5I.pdb; FIG. 8A) was utilized to design a circularly permutated variant of IL-2, such that the engineered C terminus can be fused to the N-terminus of IL-2R.alpha. through a short spacer. Briefly, the engineered N and C termini correspond to residues 95 and 94, respectively, in wild-type IL-2. The native IL-2 C-terminus was joined to residue 4 of IL-2 through a 2 amino acid linker. Finally, the newly created C-terminus was fused to the N-terminus of IL-2R.alpha. through a 6 amino acid spacer, and the C-terminus of IL-2R.alpha. was fused to human IgG1 Fc, resulting in RDB1405 (FIG. 8B; SEQ ID NO: 12 (protein) and SEQ ID NO: 13 (nucleic acid)).

Example 6. Design of Circularly Permutated Fusion Proteins

[0174] An IL-2 or IL-15 fusion protein with improved selectivity for cells expressing IL-2.beta..gamma. (but not IL-2R.alpha.) over cells expressing IL-2R.alpha..beta..gamma. relative wild-type IL-2 (wild-type IL-2 has a higher preference for cells expressing IL-2R.alpha..beta..gamma.) was designed. By fusing IL-2R.alpha. to IL-2 or IL-15R.alpha. to IL-15, the resulting fusion protein had greater activity on cells lacking the respective alpha chain (IL-2R.alpha. or IL-15R.alpha.) as compared to the native ligand, and preference for cells expressing the respective alpha chain would be reduced. Thus, the ratio of activity, EC.sub.50 (IL-2R.alpha..beta..gamma..sup.+)/EC.sub.50(IL-2R.alpha..sup.-IL-2R.beta.- .gamma..sup.+) would increase for CP-IL-2-IL-2R.alpha. fusion proteins would be less for relative to wild type IL-2. Analogous results would be expected for CP-IL-15-IL-15R.alpha. fusion proteins. Circular permutation of the cytokine is required to appropriately orient the termini in an optimal location for fusion as the native termini are oriented distally to the alpha chains in the signaling complex.

[0175] Results:

[0176] In cells lacking IL-2R.alpha., but expressing IL-2.beta..gamma. (HH cell line).sup.i, the engineered constructs are as effective (in fact, two to five-fold better) as Proleukin in promoting STATS phosphorylation (FIG. 11A, left panel). In contrast, in a cell line expressing the heterotrimeric high affinity receptor complex, IL-2R.alpha..beta..gamma., (CTLL-2 cell line).sup.ii, two of the engineered constructs (RDB1411, RDB1413) are 100- to 300-fold less active than Proleukin as measured by cell proliferation. Overall, the engineered constructs show between 400 and 600-fold greater selectivity for cells lacking IL-2R.alpha., relative to wild-type IL-2, and thus have the potential to deliver an improved therapeutic profile. Although the increase in the ratio of activity was observed, as expected, it was surprising, that the greater effect on the increase in the ratio was the dramatic loss of activity (100- to 300-fold) in cells containing IL-2R.alpha., rather than an enhancement in activity (only 2- to 5-fold) for cells lacking IL-2R.alpha..

[0177] In cells lacking IL-2R.alpha., but expressing IL-2R.beta..gamma. (HH cell line).sup.i, the engineered constructs are potent activators of STATS phosphorylation, but about 10.times. less potent than wild-type IL-15 (FIG. 12A, left panel). In a cell line expressing the heterotrimeric high affinity receptor complex, IL-2R.alpha..beta..gamma., (CTLL-2 cell line).sup.ii, the engineered constructs (RDB1408, RDB1416) are 2000- to 6000-fold less active than wild-type IL-15 as measured by cell proliferation. Overall, the engineered constructs show between 200 and 600-fold greater selectivity for cells lacking IL-2R.alpha., relative to wild-type IL-15. Although the increase in the ratio of activity was observed, as expected, it was surprising, that the greater effect on the increase in the ratio was the dramatic loss of activity (2000- to 6000-fold) in cells containing IL-2R.alpha. and that a 10-fold loss of activity was observed for cells lacking IL-2R.alpha..

Constructs:



[0178] RDB1409: CP-IL-2(C145S)-FLAG; Circularly permuted IL-2. The C145S mutation is analogous to that in Proleukin (rhIL-2) to improve the physical properties of the protein. The FLAG tag is added for ease in purification.

TABLE-US-00001

[0178] (SEQ ID NO: 24) SKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF SQSIISTLTGGSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF KFYMPKKATELKHLQCLEEELKPLEEVLNLAQGSDYKDDDDK



[0179] RDB1411: CP-IL-2(C145S)-IL-2R.alpha.-Fc; Circularly permuted IL-2 fused to IL-2R.alpha.. The C145S mutation is analogous to that in Proleukin (rhIL-2) to improve the physical properties of the protein. The Fc is added for ease in purification.

TABLE-US-00002

[0179] (SEQ ID NO: 25) SKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF SQSIISTLTGGSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF KFYMPKKATELKHLQCLEEELKPLEEVLNLAQGSGGGSELCDDDPPEIPH ATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCT SSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWEN EATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLI CTGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.



[0180] RDB1413: CP-IL-2(C145S)-IL-2R.alpha.-FLAG; Circularly permuted IL-2 fused to IL-2R.alpha.. The C145S mutation is analogous to that in Proleukin (rhIL-2) to improve the physical properties of the protein. The FLAG tag is added for ease in purification. Construct analogous to RDB1411, replacing Fc with FLAG.

TABLE-US-00003

[0180] (SEQ ID NO: 26) SKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF SQSIISTLTGGSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF KFYMPKKATELKHLQCLEEELKPLEEVLNLAQGSGGGSELCDDDPPEIPH ATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCT SSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWEN EATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLI CTGDYKDDDDK.



[0181] RDB1408: Fc-IL-15R.alpha.(sushi)-CP-IL-15; Circularly permuted IL-15 fused to the sushi domain of IL-15R.alpha.. The Fc is added for ease in purification.

TABLE-US-00004

[0181] (SEQ ID NO: 27) EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSITCPPPMSVEHADIWV KSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRD GGSELEEKNIKEFLQSFVHIVQMFINGGGSNWVNVISDLKKIEDLIQSMH IDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILA NNSLSSNGNVTESGCKEC.



[0182] RDB1416: FLAG-IL-15R.alpha.(sushi)-CP-IL-15; Circularly permuted IL-15 fused to the sushi domain of IL-15R.alpha.. The FLAG tag is added for ease in purification. Construct analogous to RDB1408, replacing Fc with FLAG.

TABLE-US-00005

[0182] (SEQ ID NO: 28) DYKDDDDKGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTS SLTECVLNKATNVAHWTTPSLKCIRDGGSELEEKNIKEFLQSFVHIVQMF INGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFL LELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKEC.

[0183] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

[0184] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. It should also be understood that the embodiments described herein are not mutually exclusive and that features from the various embodiments may be combined in whole or in part in accordance with the invention.

TABLE-US-00006 1) PRT RDB1503 1 QNQWLQDMTT HLILRSFKEF LQSSLRALRQ MSGGSGGGSS ERIDKQIRYI LDGISALRKE TCNKSNMCES SKEALAENNL NLPKMAEKDG CFQSGFNEET CLVKIITGLL EFEVYLEYLQ NRFESSEEQA RAVQMSTKVL IQFLQKKAKN LDAITTPDPT TNASLLTKLQ 2) DNA RDB1503 1 CAGAACCAGT GGCTGCAGGA CATGACCACC CACCTGATCC TGCGGTCCTT CAAAGAGTTC CTGCAGTCCT CCCTGCGGGC CCTGAGACAG ATGAGCGGAG GATCTGGCGG AGGCTCCTCT GAGCGGATCG ACAAGCAGAT CCGGTACATC CTGGACGGCA TCTCCGCCCT GCGGAAAGAG ACATGCAACA AGTCCAACAT GTGCGAGTCC AGCAAAGAGG CCCTGGCCGA GAACAACCTG AACCTGCCCA AGATGGCTGA GAAGGACGGC TGCTTCCAGT CCGGCTTCAA CGAAGAGACT TGCCTGGTCA AGATCATCAC CGGCCTGCTG GAATTTGAGG TGTACCTGGA ATACCTGCAG AACAGATTCG AGTCCTCCGA GGAACAGGCC AGAGCCGTGC AGATGTCCAC CAAGGTGCTG ATCCAGTTTC TGCAGAAGAA GGCCAAGAAC CTGGACGCTA TCACCACCCC CGACCCTACC ACCAATGCCT CCCTGCTGAC CAAGCTGCAG TGATAA 3) PRT IL6 1 MNSFSTSAFG PVAFSLGLLL VLPAAFPAPV PPGEDSKDVA APHRQPLTSS ERIDKQIRYI LDGISALRKE TCNKSNMCES SKEALAENNL NLPKMAEKDG CFQSGFNEET CLVKIITGLL EFEVYLEYLQ NRFESSEEQA RAVQMSTKVL IQFLQKKAKN LDAITTPDPT TNASLLTKLQ AQNQWLQDMT THLILRSFKE FLQSSLRALR QM 4) PRT RDB1527 1 QNQWLQDMTT HLILRSFKEF LQSSLRALRQ MSGGSGGGSS ERIDKQIRYI LDGISALRKE TCNKSNMCES SKEALAENNL NLPKMAEKDG CFQSGFNEET CLVKIITGLL EFEVYLEYLQ NRFESSEEQA RAVQMSTKVL IQFLQKKAKN LDAITTPDPT TNASLLTKLQ ASELLDPCGY ISPESPVVQL HSNFTAVCVL KEKCMDYFHV NANYIVWKTN HFTIPKEQYT IINRTASSVT FTDIASLNIQ LTCNILTFGQ LEQNVYGITI ISGLVPRGSE PKSSDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K 5) DNA RDB1527 1 CAAAACCAGT GGCTCCAGGA TATGACCACC CACCTCATCC TCAGAAGTTT CAAGGAGTTC CTCCAGTCCA GCCTGAGGGC TCTGAGGCAA ATGAGCGGAG GCTCCGGCGG AGGCAGCTCC GAGAGAATCG ACAAGCAGAT CAGGTACATC CTCGATGGCA TCAGCGCCCT CAGAAAAGAA ACCTGTAATA AGAGCAACAT GTGTGAGAGC AGCAAGGAAG CCCTCGCCGA GAACAATCTG AACCTCCCCA AAATGGCTGA GAAGGACGGA TGCTTCCAGA GCGGCTTCAA TGAGGAGACA TGCCTCGTGA AGATCATCAC AGGACTCCTG GAGTTCGAAG TCTACCTGGA GTACCTCCAG AACAGGTTCG AATCCAGCGA GGAACAGGCT AGGGCTGTGC AGATGTCCAC CAAGGTGCTG ATCCAGTTCC TCCAGAAGAA GGCCAAGAAT CTGGATGCCA TCACCACACC CGATCCTACA ACCAACGCCA GCCTGCTGAC CAAGCTCCAG GCCTCCGAAC TCCTGGACCC TTGTGGCTAC ATTTCCCCTG AAAGCCCTGT GGTGCAACTC CACAGCAATT TCACAGCCGT CTGTGTGCTC AAGGAGAAGT GCATGGACTA CTTTCACGTG AATGCTAATT ATATCGTGTG GAAGACAAAC CACTTCACCA TCCCCAAGGA GCAGTATACC ATCATCAACA GGACCGCCTC CAGCGTGACA TTCACCGACA TCGCTTCCCT CAACATTCAG CTGACCTGCA ATATCCTCAC CTTCGGCCAG CTGGAGCAGA ACGTGTACGG AATCACCATC ATTAGCGGCC TCGTCCCTAG AGGCTCCGAA CCCAAGTCCT CCGATAAAAC CCATACCTGC CCCCCTTGCC CTGCTCCCGA ACTCCTCGGC GGCCCCAGCG TGTTTCTCTT CCCTCCCAAG CCCAAAGATA CCCTGATGAT CAGCAGGACA CCCGAAGTCA CCTGCGTGGT GGTCGACGTG TCCCACGAGG ACCCCGAGGT CAAATTCAAC TGGTACGTCG ATGGCGTGGA GGTGCATAAT GCTAAGACCA AGCCCAGGGA GGAGCAGTAC AACTCCACAT ACAGGGTGGT CTCCGTCCTG ACCGTGCTGC ATCAAGACTG GCTGAACGGC AAGGAGTATA AGTGCAAGGT GAGCAATAAA GCCCTCCCCG CCCCTATTGA GAAGACCATT TCCAAGGCCA AGGGCCAGCC TAGAGAACCT CAAGTCTACA CACTCCCCCC CTCCAGGGAG GAGATGACCA AAAATCAGGT CTCCCTGACC TGCCTGGTGA AGGGCTTCTA TCCTAGCGAC ATCGCCGTCG AGTGGGAGAG CAACGGACAG CCCGAGAACA ACTACAAAAC CACACCTCCC GTGCTCGACA GCGACGGCAG CTTCTTCCTG TACTCCAAGC TCACCGTGGA TAAGTCCAGG TGGCAGCAAG GCAACGTGTT TAGCTGCAGC GTGATGCACG AAGCTCTCCA CAACCACTAT ACCCAGAAGT CCCTCAGCCT CAGCCCTGGC AAGTAGTGA 6) PRT RDB1529 1 LTSSERIDKQ IRYILDGISA LRKETCNKSN MCESSKEALA ENNLNLPKMA EKDGCFQSGF NEETCLVKII TGLLEFEVYL EYLQNRFESS EEQARAVQMS TKVLIQFLQK KAKNLDAITT PDPTTNASLL TKLQAQNQWL QDMTTHLILR SFKEFLQSSL RALRQMSELL DPCGYISPES PVVQLHSNFT AVCVLKEKCM DYFHVNANYI VWKTNHFTIP KEQYTIINRT ASSVTFTDIA SLNIQLTCNI LTFGQLEQNV YGITIISGLV PRGSEPKSSD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK 7) DNA RDB1529 1 CTGACCTCCA GCGAAAGGAT CGACAAGCAG ATCAGGTACA TCCTCGACGG CATCTCCGCT CTCAGAAAGG AGACCTGCAA CAAGAGCAAC ATGTGCGAGA GCAGCAAGGA AGCCCTGGCT GAGAACAATC TCAACCTGCC CAAGATGGCC GAAAAGGATG GATGCTTCCA GAGCGGCTTT AACGAGGAGA CCTGCCTCGT GAAGATCATC ACCGGCCTGC TCGAGTTTGA GGTGTATCTC GAGTACCTGC AGAATAGGTT CGAGAGCAGC GAGGAACAGG CTAGAGCTGT CCAGATGTCC ACCAAGGTGC TGATCCAGTT CCTGCAAAAG AAGGCCAAGA ATCTCGACGC TATCACCACC CCTGACCCTA CCACAAACGC CAGCCTGCTG ACCAAGCTGC AGGCCCAAAA CCAATGGCTC CAGGACATGA CAACCCACCT GATCCTGAGG AGCTTCAAGG AGTTCCTCCA ATCCTCCCTC AGGGCCCTGA GACAGATGAG CGAACTGCTC GACCCTTGTG GATACATTAG CCCTGAATCC CCCGTGGTGC AGCTGCATAG CAATTTCACC GCCGTGTGCG TGCTCAAAGA GAAGTGCATG GACTACTTCC ATGTGAACGC CAACTACATC GTGTGGAAGA CCAACCATTT CACCATCCCC AAGGAGCAAT ACACCATCAT CAACAGAACC GCCAGCAGCG TCACATTCAC CGACATCGCC TCCCTGAACA TCCAACTGAC ATGCAACATT CTCACCTTCG GCCAGCTGGA GCAAAATGTG TACGGCATCA CCATCATTAG CGGCCTCGTC CCTAGAGGCT CCGAACCCAA GTCCTCCGAT AAAACCCATA CCTGCCCCCC TTGCCCTGCT CCCGAACTCC TCGGCGGCCC CAGCGTGTTT CTCTTCCCTC CCAAGCCCAA AGATACCCTG ATGATCAGCA GGACACCCGA AGTCACCTGC GTGGTGGTCG ACGTGTCCCA CGAGGACCCC GAGGTCAAAT TCAACTGGTA CGTCGATGGC GTGGAGGTGC ATAATGCTAA GACCAAGCCC AGGGAGGAGC AGTACAACTC CACATACAGG GTGGTCTCCG TCCTGACCGT GCTGCATCAA GACTGGCTGA ACGGCAAGGA GTATAAGTGC AAGGTGAGCA ATAAAGCCCT CCCCGCCCCT ATTGAGAAGA CCATTTCCAA GGCCAAGGGC CAGCCTAGAG AACCTCAAGT CTACACACTC CCCCCCTCCA GGGAGGAGAT GACCAAAAAT CAGGTCTCCC TGACCTGCCT GGTGAAGGGC TTCTATCCTA GCGACATCGC CGTCGAGTGG GAGAGCAACG GACAGCCCGA GAACAACTAC AAAACCACAC CTCCCGTGCT CGACAGCGAC GGCAGCTTCT TCCTGTACTC CAAGCTCACC GTGGATAAGT CCAGGTGGCA GCAAGGCAAC GTGTTTAGCT GCAGCGTGAT GCACGAAGCT CTCCACAACC ACTATACCCA GAAGTCCCTC AGCCTCAGCC CTGGCAAGTA GTGA 8) PRT RDB1515 1 EKNLYLSCVL KDDKPTLQLE SVDPKNYPKK KMEKRFVFNK IEINNKLEFE SAQFPNWYIS TSQAENMPVF LGGTKGGQDI TDFTMQFVSS GGSGGSGAPV RSLNCTLRDS QQKSLVMSGP YELKALHLQG QDMEQQVVFS MSFVQGEESN DKIPVALGLK 9) DNA RDB1515 1

GAGAAGAACC TCTACCTCTC CTGCGTGCTG AAGGACGACA AGCCCACACT CCAGCTGGAG TCCGTGGACC CCAAGAACTA CCCCAAGAAG AAGATGGAGA AGCGGTTCGT GTTCAACAAG ATCGAGATCA ACAACAAGCT GGAGTTCGAG AGCGCCCAGT TCCCCAACTG GTACATTTCC ACCTCCCAGG CCGAGAACAT GCCCGTCTTT CTGGGCGGAA CCAAGGGCGG CCAGGACATC ACCGACTTCA CCATGCAGTT CGTCTCCAGC GGAGGAAGCG GAGGCAGCGG AGCTCCCGTG AGGAGCCTGA ACTGCACCCT GAGGGACAGC CAGCAGAAGT CCCTGGTGAT GTCCGGACCC TACGAACTGA AGGCCCTCCA TCTGCAAGGA CAGGATATGG AGCAGCAGGT GGTGTTCTCC ATGTCCTTCG TCCAGGGCGA AGAGTCCAAC GACAAGATCC CCGTGGCCCT GGGCCTGAAA TAGTGA 10) PRT RDB1538 1 MDAMKRGLCC VLLLCGAVFV SARDYKDDDD KDKCKEREEK IILVSSANEI DVRPCPLNPN EHKGTITWYK DDSKTPVSTE QASRIHQHKE KLWFVPAKVE DSGHYYCVVR NSSYCLRIKI SAKFVENEPN LCYNAQAIFK QKLPVAGDGG LVCPYMEFFK NENNELPKLQ WYKDCKPLLL DNIHFSGVKD RLIVMNVAEK HRGNYTCHAS YTYLGKQYPI TRVIEFITLE ENSGGSGNKL EFESAQFPNW YISTSQAENM PVFLGGTKGG QDITDFTMQF VSSGGSGGSG APVRSLNCTL RDSQQKSLVM SGPYELKALH LQGQDMEQQV VFSMSFVQGE ESNDKIPVAL GLKEKNLYLS CVLKDDKPTL QLESVDPKNY PKKKMEKRFV FNKIEIN 11) DNA RDB1538 1 ATGGACGCTA TGAAGCGGGG ACTGTGCTGC GTGCTCCTGC TGTGCGGCGC TGTCTTTGTC AGCGCCCGGG ACTATAAGGA CGATGATGAC AAGGACAAGT GCAAGGAGCG GGAGGAGAAG ATCATCCTGG TGAGCTCCGC CAACGAGATT GACGTCCGGC CCTGCCCTCT CAACCCCAAC GAGCATAAGG GCACCATCAC CTGGTACAAA GACGACAGCA AAACACCCGT CTCCACCGAG CAAGCCTCCC GGATTCACCA GCACAAGGAG AAGCTCTGGT TCGTGCCCGC TAAGGTGGAG GATTCCGGAC ACTACTACTG TGTGGTCCGG AACTCCAGCT ACTGCCTGAG GATTAAGATC AGCGCTAAGT TCGTCGAGAA CGAGCCCAAC CTCTGCTACA ATGCCCAGGC CATCTTCAAG CAGAAGCTCC CTGTGGCTGG AGACGGAGGC CTGGTCTGCC CCTACATGGA GTTCTTCAAG AACGAGAATA ACGAGCTGCC TAAGCTGCAG TGGTACAAGG ACTGCAAACC CCTGCTCCTC GACAACATCC ACTTCTCCGG CGTCAAGGAC CGGCTGATCG TCATGAACGT GGCCGAGAAG CACAGGGGCA ACTATACCTG TCACGCCAGC TACACCTACC TGGGAAAGCA GTATCCTATC ACCAGGGTGA TTGAGTTCAT CACACTCGAG GAAAACAGCG GCGGCAGCGG CAACAAGCTG GAGTTCGAGT CCGCCCAGTT TCCTAACTGG TACATCTCCA CAAGCCAGGC CGAGAACATG CCTGTCTTCC TGGGCGGCAC CAAAGGCGGC CAAGATATCA CCGACTTCAC CATGCAGTTT GTGAGCTCCG GAGGCTCCGG AGGAAGCGGA GCTCCTGTGC GGTCCCTGAA TTGCACCCTG CGGGATTCCC AACAGAAGAG CCTGGTGATG TCCGGCCCCT ACGAGCTCAA GGCCCTCCAT CTGCAAGGCC AGGACATGGA GCAGCAGGTG GTCTTCAGCA TGAGCTTCGT GCAGGGAGAG GAGTCCAACG ATAAGATCCC CGTCGCTCTC GGACTCAAGG AGAAGAACCT GTACCTCTCC TGCGTGCTGA AGGACGATAA GCCCACCCTC CAGCTGGAAT CCGTGGACCC CAAGAACTAC CCCAAGAAAA AAATGGAAAA GCGGTTTGTC TTTAACAAGA TCGAGATTAA CTAGTGA 12) PRT RDB1405 1 SKNFHLRPRD LISNINVIVL ELKGSETTFM CEYADETATI VEFLNRWITF CQSIISTLTG GSSSTKKTQL QLEHLLLDLQ MILNGINNYK NPKLTRMLTF KFYMPKKATE LKHLQCLEEE LKPLEEVLNL AQGSGGGSEL CDDDPPEIPH ATFKAMAYKE GTMLNCECKR GFRRIKSGSL YMLCTGNSSH SSWDNQCQCT SSATRNTTKQ VTPQPEEQKE RKTTEMQSPM QPVDQASLPG HCREPPPWEN EATERIYHFV VGQMVYYQCV QGYRALHRGP AESVCKMTHG KTRWTQPQLI CTGGGGSEPK SSDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK 13) DNA RDB1405 1 TCCAAGAACT TCCACCTGAG GCCTCGGGAC CTGATCTCCA ACATCAACGT GATCGTGCTG GAACTGAAGG GCTCCGAGAC AACCTTCATG TGCGAGTACG CCGACGAGAC AGCTACCATC GTGGAATTTC TGAACCGGTG GATCACCTTC TGCCAGTCCA TCATCTCCAC CCTGACCGGC GGCTCCTCCA GCACCAAGAA AACCCAGCTG CAGCTGGAAC ATCTGCTGCT GGACCTGCAG ATGATCCTGA ACGGCATCAA CAACTACAAG AACCCCAAGC TGACCCGGAT GCTGACCTTC AAGTTCTACA TGCCCAAGAA GGCCACCGAA CTGAAACATC TGCAGTGCCT GGAAGAAGAA CTGAAGCCCC TGGAAGAGGT GCTGAACCTG GCTCAGGGAT CTGGCGGCGG ATCTGAGCTG TGCGACGACG ACCCTCCTGA GATCCCTCAC GCCACCTTCA AGGCCATGGC TTACAAAGAG GGCACCATGC TGAACTGCGA GTGCAAGAGA GGCTTCCGGC GGATCAAGTC CGGCTCCCTG TACATGCTGT GCACCGGCAA CTCCAGCCAC TCCTCCTGGG ACAACCAGTG CCAGTGCACC TCCTCTGCCA CCCGGAACAC CACCAAACAA GTGACCCCCC AGCCCGAGGA ACAGAAAGAG CGCAAGACCA CCGAGATGCA GTCCCCCATG CAGCCTGTGG ACCAGGCTTC TCTGCCTGGC CACTGCAGAG AGCCTCCACC TTGGGAGAAC GAGGCTACCG AGAGAATCTA CCACTTCGTC GTGGGCCAGA TGGTGTACTA CCAGTGCGTG CAGGGCTACC GCGCCCTGCA TAGAGGACCT GCTGAGTCCG TGTGCAAGAT GACCCACGGC AAGACCCGGT GGACCCAGCC TCAGCTGATC TGTACAGGCG GCGGAGGCTC CGAGCCTAAG TCCTCCGATA AGACCCACAC CTGTCCCCCC TGTCCTGCCC CTGAACTGCT GGGAGGCCCT TCCGTGTTCC TGTTCCCCCC AAAGCCCAAG GACACCCTGA TGATCTCCCG GACCCCCGAA GTGACCTGCG TGGTGGTGGA TGTGTCCCAC GAGGACCCTG AAGTGAAGTT CAATTGGTAC GTGGACGGCG TGGAAGTGCA CAACGCCAAG ACCAAGCCCA GAGAGGAACA GTACAACTCC ACCTACCGGG TGGTGTCCGT GCTGACCGTG CTGCACCAGG ATTGGCTGAA TGGCAAAGAG TACAAGTGCA AGGTGTCCAA CAAGGCCCTG CCAGCCCCCA TCGAAAAGAC CATCTCCAAG GCCAAGGGCC AGCCCCGGGA ACCCCAGGTG TACACACTGC CCCCTAGCCG GGAAGAGATG ACCAAGAACC AGGTGTCCCT GACCTGTCTC GTGAAGGGCT TCTACCCCTC CGATATCGCC GTGGAATGGG AGTCCAACGG CCAGCCTGAG AACAATTATA AGACCACCCC CCCTGTGCTG GACTCCGACG GCTCATTCTT CCTGTACAGC AAGCTGACAG TGGACAAGTC CCGGTGGCAG CAGGGCAACG TGTTCTCCTG CTCCGTGATG CACGAGGCCC TGCACAACCA CTACACCCAG AAGTCCCTGT CCCTGTCTCC CGGCAAGTGA TGA 14) PRT Linker 1 SGGSGGG 15) PRT Linker 1 GGSGGSG 16) PRT Linker 1 GG 17) PRT Stability sequence 1 MG 18) PRT Stability sequence 1 MGG 19) PRT IL-10 1 MAEVPELASE MMAYYSGNED DLFFEADGPK QMKCSFQDLD LCPLDGGIQL RISDHHYSKG FRQAASVVVA MDKLRKMLVP CPQTFQENDL STFFPFIFEE EPIFFDTWDN EAYVHDAPVR SLNCTLRDSQ QKSLVMSGPY ELKALHLQGQ DMEQQVVFSM SFVQGEESND KIPVALGLKE KNLYLSCVLK DDKPTLQLES VDPKNYPKKK MEKRFVFNKI EINNKLEFES AQFPNWYIST SQAENMPVFL GGTKGGQDIT DFTMQFVSS 20) PRT IL-2 1 MYRMQLLSCI ALSLALVTNS APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT 21) PRT gp130 1 MLTLQTWVVQ ALFIFLTTES TGELLDPCGY ISPESPVVQL HSNFTAVCVL

KEKCMDYFHV NANYIVWKTN HFTIPKEQYT IINRTASSVT FTDIASLNIQ LTCNILTFGQ LEQNVYGITI ISGLPPEKPK NLSCIVNEGK KMRCEWDGGR ETHLETNFTL KSEWATHKFA DCKAKRDTPT SCTVDYSTVY FVNIEVWVEA ENALGKVTSD HINFDPVYKV KPNPPHNLSV INSEELSSIL KLTWTNPSIK SVIILKYNIQ YRTKDASTWS QIPPEDTAST RSSFTVQDLK PFTEYVFRIR CMKEDGKGYW SDWSEEASGI TYEDRPSKAP SFWYKIDPSH TQGYRTVQLV WKTLPPFEAN GKILDYEVTL TRWKSHLQNY TVNATKLTVN LTNDRYLATL TVRNLVGKSD AAVLTIPACD FQATHPVMDL KAFPKDNMLW VEWTTPRESV KKYILEWCVL SDKAPCITDW QQEDGTVHRT YLRGNLAESK CYLITVTPVY ADGPGSPESI KAYLKQAPPS KGPTVRTKKV GKNEAVLEWD QLPVDVQNGF IRNYTIFYRT IIGNETAVNV DSSHTEYTLS SLTSDTLYMV RMAAYTDEGG KDGPEFTFTT PKFAQGEIEA IVVPVCLAFL LTTLLGVLFC FNKRDLIKKH IWPNVPDPSK SHIAQWSPHT PPRHNFNSKD QMYSDGNFTD VSVVEIEAND KKPFPEDLKS LDLFKKEKIN TEGHSSGIGG SSCMSSSRPS ISSSDENESS QNTSSTVQYS TVVHSGYRHQ VPSVQVFSRS ESTQPLLDSE ERPEDLQLVD HVDGGDGILP RQQYFKQNCS QHESSPDISH FERSKQVSSV NEEDFVRLKQ QISDHISQSC GSGQMKMFQE VSAADAFGPG TEGQVERFET VGMEAATDEG MPKSYLPQTV RQGGYMPQ 22) PRT IL-1RI 1 MKVLLRLICF IALLISSLEA DKCKEREEKI ILVSSANEID VRPCPLNPNE HKGTITWYKD DSKTPVSTEQ ASRIHQHKEK LWFVPAKVED SGHYYCVVRN SSYCLRIKIS AKFVENEPNL CYNAQAIFKQ NLPVAGDGGL VCPYMEFFKN ENNELPKLQW YKDCKPLLLD NIHFSGVKDR LIVMNVAEKH RGNYTCHASY TYLGKQYPIT RVIEFITLEE NKPTRPVIVS PANETMEVDL GSQIQLICNV TGQLSDIAYW KWNGSVIDED DPVLGEDYYS VENPANKRRS TLITVLNISE IESRFYKHPF TCFAKNTHGI DAAYIQLIYP VTNFQKHMIG ICVTLTVIIV CSVFIYKIFK IDIVLWYRDS CYDFLPIKAS DGKTYDAYIL YPKTVGEGST SDCDIFVFKV LPEVLEKQCG YKLFIYGRDD YVGEDIVEVI NENVKKSRRL IIILVRETSS FSWLGGSSEE QIAMYNALVQ DGIKVVLLEL EKIQDYEKMP ESIKFIKQKH GAIRWSGDFT QGPQSAKTRF WKNVRYHMPV QRRSPSSKHQ LLSPATKEKL QREAHVPLG 23) PRT IL-2R.alpha. 1 MDSYLLMWGL LTFIMVPGCQ AELCDDDPPE IPHATFKAMA YKEGTMLNCE CKRGFRRIKS GSLYMLCTGN SSHSSWDNQC QCTSSATRNT TKQVTPQPEE QKERKTTEMQ SPMQPVDQAS LPGHCREPPP WENEATERIY HFVVGQMVYY QCVQGYRALH RGPAESVCKM THGKTRWTQP QLICTGEMET SQFPGEEKPQ ASPEGRPESE TSCLVTTTDF QIQTEMAATM ETSIFTTEYQ VAVAGCVFLL ISVLLLSGLT WQRRQRKSRR TI

Sequence CWU 1

1

281170PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 1Gln Asn Gln Trp Leu Gln Asp Met Thr Thr His Leu Ile Leu Arg Ser1 5 10 15Phe Lys Glu Phe Leu Gln Ser Ser Leu Arg Ala Leu Arg Gln Met Ser 20 25 30Gly Gly Ser Gly Gly Gly Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg 35 40 45Tyr Ile Leu Asp Gly Ile Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys 50 55 60Ser Asn Met Cys Glu Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu65 70 75 80Asn Leu Pro Lys Met Ala Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe 85 90 95Asn Glu Glu Thr Cys Leu Val Lys Ile Ile Thr Gly Leu Leu Glu Phe 100 105 110Glu Val Tyr Leu Glu Tyr Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu 115 120 125Gln Ala Arg Ala Val Gln Met Ser Thr Lys Val Leu Ile Gln Phe Leu 130 135 140Gln Lys Lys Ala Lys Asn Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr145 150 155 160Thr Asn Ala Ser Leu Leu Thr Lys Leu Gln 165 1702516DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 2cagaaccagt ggctgcagga catgaccacc cacctgatcc tgcggtcctt caaagagttc 60ctgcagtcct ccctgcgggc cctgagacag atgagcggag gatctggcgg aggctcctct 120gagcggatcg acaagcagat ccggtacatc ctggacggca tctccgccct gcggaaagag 180acatgcaaca agtccaacat gtgcgagtcc agcaaagagg ccctggccga gaacaacctg 240aacctgccca agatggctga gaaggacggc tgcttccagt ccggcttcaa cgaagagact 300tgcctggtca agatcatcac cggcctgctg gaatttgagg tgtacctgga atacctgcag 360aacagattcg agtcctccga ggaacaggcc agagccgtgc agatgtccac caaggtgctg 420atccagtttc tgcagaagaa ggccaagaac ctggacgcta tcaccacccc cgaccctacc 480accaatgcct ccctgctgac caagctgcag tgataa 5163212PRTHomo sapiens 3Met Asn Ser Phe Ser Thr Ser Ala Phe Gly Pro Val Ala Phe Ser Leu1 5 10 15Gly Leu Leu Leu Val Leu Pro Ala Ala Phe Pro Ala Pro Val Pro Pro 20 25 30Gly Glu Asp Ser Lys Asp Val Ala Ala Pro His Arg Gln Pro Leu Thr 35 40 45Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg Tyr Ile Leu Asp Gly Ile 50 55 60Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys Ser Asn Met Cys Glu Ser65 70 75 80Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro Lys Met Ala 85 90 95Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe Asn Glu Glu Thr Cys Leu 100 105 110Val Lys Ile Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr Leu Glu Tyr 115 120 125Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gln Ala Arg Ala Val Gln 130 135 140Met Ser Thr Lys Val Leu Ile Gln Phe Leu Gln Lys Lys Ala Lys Asn145 150 155 160Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr Thr Asn Ala Ser Leu Leu 165 170 175Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu Gln Asp Met Thr Thr His 180 185 190Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser Leu Arg Ala 195 200 205Leu Arg Gln Met 2104511PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 4Gln Asn Gln Trp Leu Gln Asp Met Thr Thr His Leu Ile Leu Arg Ser1 5 10 15Phe Lys Glu Phe Leu Gln Ser Ser Leu Arg Ala Leu Arg Gln Met Ser 20 25 30Gly Gly Ser Gly Gly Gly Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg 35 40 45Tyr Ile Leu Asp Gly Ile Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys 50 55 60Ser Asn Met Cys Glu Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu65 70 75 80Asn Leu Pro Lys Met Ala Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe 85 90 95Asn Glu Glu Thr Cys Leu Val Lys Ile Ile Thr Gly Leu Leu Glu Phe 100 105 110Glu Val Tyr Leu Glu Tyr Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu 115 120 125Gln Ala Arg Ala Val Gln Met Ser Thr Lys Val Leu Ile Gln Phe Leu 130 135 140Gln Lys Lys Ala Lys Asn Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr145 150 155 160Thr Asn Ala Ser Leu Leu Thr Lys Leu Gln Ala Ser Glu Leu Leu Asp 165 170 175Pro Cys Gly Tyr Ile Ser Pro Glu Ser Pro Val Val Gln Leu His Ser 180 185 190Asn Phe Thr Ala Val Cys Val Leu Lys Glu Lys Cys Met Asp Tyr Phe 195 200 205His Val Asn Ala Asn Tyr Ile Val Trp Lys Thr Asn His Phe Thr Ile 210 215 220Pro Lys Glu Gln Tyr Thr Ile Ile Asn Arg Thr Ala Ser Ser Val Thr225 230 235 240Phe Thr Asp Ile Ala Ser Leu Asn Ile Gln Leu Thr Cys Asn Ile Leu 245 250 255Thr Phe Gly Gln Leu Glu Gln Asn Val Tyr Gly Ile Thr Ile Ile Ser 260 265 270Gly Leu Val Pro Arg Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His 275 280 285Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 290 295 300Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr305 310 315 320Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 325 330 335Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 340 345 350Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 355 360 365Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 370 375 380Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile385 390 395 400Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 405 410 415Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 420 425 430Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 435 440 445Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 450 455 460Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg465 470 475 480Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 485 490 495His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 500 505 51051539DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 5caaaaccagt ggctccagga tatgaccacc cacctcatcc tcagaagttt caaggagttc 60ctccagtcca gcctgagggc tctgaggcaa atgagcggag gctccggcgg aggcagctcc 120gagagaatcg acaagcagat caggtacatc ctcgatggca tcagcgccct cagaaaagaa 180acctgtaata agagcaacat gtgtgagagc agcaaggaag ccctcgccga gaacaatctg 240aacctcccca aaatggctga gaaggacgga tgcttccaga gcggcttcaa tgaggagaca 300tgcctcgtga agatcatcac aggactcctg gagttcgaag tctacctgga gtacctccag 360aacaggttcg aatccagcga ggaacaggct agggctgtgc agatgtccac caaggtgctg 420atccagttcc tccagaagaa ggccaagaat ctggatgcca tcaccacacc cgatcctaca 480accaacgcca gcctgctgac caagctccag gcctccgaac tcctggaccc ttgtggctac 540atttcccctg aaagccctgt ggtgcaactc cacagcaatt tcacagccgt ctgtgtgctc 600aaggagaagt gcatggacta ctttcacgtg aatgctaatt atatcgtgtg gaagacaaac 660cacttcacca tccccaagga gcagtatacc atcatcaaca ggaccgcctc cagcgtgaca 720ttcaccgaca tcgcttccct caacattcag ctgacctgca atatcctcac cttcggccag 780ctggagcaga acgtgtacgg aatcaccatc attagcggcc tcgtccctag aggctccgaa 840cccaagtcct ccgataaaac ccatacctgc cccccttgcc ctgctcccga actcctcggc 900ggccccagcg tgtttctctt ccctcccaag cccaaagata ccctgatgat cagcaggaca 960cccgaagtca cctgcgtggt ggtcgacgtg tcccacgagg accccgaggt caaattcaac 1020tggtacgtcg atggcgtgga ggtgcataat gctaagacca agcccaggga ggagcagtac 1080aactccacat acagggtggt ctccgtcctg accgtgctgc atcaagactg gctgaacggc 1140aaggagtata agtgcaaggt gagcaataaa gccctccccg cccctattga gaagaccatt 1200tccaaggcca agggccagcc tagagaacct caagtctaca cactcccccc ctccagggag 1260gagatgacca aaaatcaggt ctccctgacc tgcctggtga agggcttcta tcctagcgac 1320atcgccgtcg agtgggagag caacggacag cccgagaaca actacaaaac cacacctccc 1380gtgctcgaca gcgacggcag cttcttcctg tactccaagc tcaccgtgga taagtccagg 1440tggcagcaag gcaacgtgtt tagctgcagc gtgatgcacg aagctctcca caaccactat 1500acccagaagt ccctcagcct cagccctggc aagtagtga 15396506PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 6Leu Thr Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg Tyr Ile Leu Asp1 5 10 15Gly Ile Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys Ser Asn Met Cys 20 25 30Glu Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro Lys 35 40 45Met Ala Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe Asn Glu Glu Thr 50 55 60Cys Leu Val Lys Ile Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr Leu65 70 75 80Glu Tyr Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gln Ala Arg Ala 85 90 95Val Gln Met Ser Thr Lys Val Leu Ile Gln Phe Leu Gln Lys Lys Ala 100 105 110Lys Asn Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr Thr Asn Ala Ser 115 120 125Leu Leu Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu Gln Asp Met Thr 130 135 140Thr His Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser Leu145 150 155 160Arg Ala Leu Arg Gln Met Ser Glu Leu Leu Asp Pro Cys Gly Tyr Ile 165 170 175Ser Pro Glu Ser Pro Val Val Gln Leu His Ser Asn Phe Thr Ala Val 180 185 190Cys Val Leu Lys Glu Lys Cys Met Asp Tyr Phe His Val Asn Ala Asn 195 200 205Tyr Ile Val Trp Lys Thr Asn His Phe Thr Ile Pro Lys Glu Gln Tyr 210 215 220Thr Ile Ile Asn Arg Thr Ala Ser Ser Val Thr Phe Thr Asp Ile Ala225 230 235 240Ser Leu Asn Ile Gln Leu Thr Cys Asn Ile Leu Thr Phe Gly Gln Leu 245 250 255Glu Gln Asn Val Tyr Gly Ile Thr Ile Ile Ser Gly Leu Val Pro Arg 260 265 270Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys 275 280 285Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 290 295 300Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys305 310 315 320Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 325 330 335Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 340 345 350Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 355 360 365His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 370 375 380Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly385 390 395 400Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 405 410 415Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 420 425 430Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 435 440 445Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 450 455 460Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn465 470 475 480Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 485 490 495Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 500 50571524DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 7ctgacctcca gcgaaaggat cgacaagcag atcaggtaca tcctcgacgg catctccgct 60ctcagaaagg agacctgcaa caagagcaac atgtgcgaga gcagcaagga agccctggct 120gagaacaatc tcaacctgcc caagatggcc gaaaaggatg gatgcttcca gagcggcttt 180aacgaggaga cctgcctcgt gaagatcatc accggcctgc tcgagtttga ggtgtatctc 240gagtacctgc agaataggtt cgagagcagc gaggaacagg ctagagctgt ccagatgtcc 300accaaggtgc tgatccagtt cctgcaaaag aaggccaaga atctcgacgc tatcaccacc 360cctgacccta ccacaaacgc cagcctgctg accaagctgc aggcccaaaa ccaatggctc 420caggacatga caacccacct gatcctgagg agcttcaagg agttcctcca atcctccctc 480agggccctga gacagatgag cgaactgctc gacccttgtg gatacattag ccctgaatcc 540cccgtggtgc agctgcatag caatttcacc gccgtgtgcg tgctcaaaga gaagtgcatg 600gactacttcc atgtgaacgc caactacatc gtgtggaaga ccaaccattt caccatcccc 660aaggagcaat acaccatcat caacagaacc gccagcagcg tcacattcac cgacatcgcc 720tccctgaaca tccaactgac atgcaacatt ctcaccttcg gccagctgga gcaaaatgtg 780tacggcatca ccatcattag cggcctcgtc cctagaggct ccgaacccaa gtcctccgat 840aaaacccata cctgcccccc ttgccctgct cccgaactcc tcggcggccc cagcgtgttt 900ctcttccctc ccaagcccaa agataccctg atgatcagca ggacacccga agtcacctgc 960gtggtggtcg acgtgtccca cgaggacccc gaggtcaaat tcaactggta cgtcgatggc 1020gtggaggtgc ataatgctaa gaccaagccc agggaggagc agtacaactc cacatacagg 1080gtggtctccg tcctgaccgt gctgcatcaa gactggctga acggcaagga gtataagtgc 1140aaggtgagca ataaagccct ccccgcccct attgagaaga ccatttccaa ggccaagggc 1200cagcctagag aacctcaagt ctacacactc cccccctcca gggaggagat gaccaaaaat 1260caggtctccc tgacctgcct ggtgaagggc ttctatccta gcgacatcgc cgtcgagtgg 1320gagagcaacg gacagcccga gaacaactac aaaaccacac ctcccgtgct cgacagcgac 1380ggcagcttct tcctgtactc caagctcacc gtggataagt ccaggtggca gcaaggcaac 1440gtgtttagct gcagcgtgat gcacgaagct ctccacaacc actataccca gaagtccctc 1500agcctcagcc ctggcaagta gtga 15248160PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 8Glu Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp Lys Pro Thr1 5 10 15Leu Gln Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys Lys Lys Met 20 25 30Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Asn Asn Lys Leu Glu 35 40 45Phe Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ala 50 55 60Glu Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly Gln Asp Ile65 70 75 80Thr Asp Phe Thr Met Gln Phe Val Ser Ser Gly Gly Ser Gly Gly Ser 85 90 95Gly Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp Ser Gln Gln 100 105 110Lys Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala Leu His Leu 115 120 125Gln Gly Gln Asp Met Glu Gln Gln Val Val Phe Ser Met Ser Phe Val 130 135 140Gln Gly Glu Glu Ser Asn Asp Lys Ile Pro Val Ala Leu Gly Leu Lys145 150 155 1609486DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 9gagaagaacc tctacctctc ctgcgtgctg aaggacgaca agcccacact ccagctggag 60tccgtggacc ccaagaacta ccccaagaag aagatggaga agcggttcgt gttcaacaag 120atcgagatca acaacaagct ggagttcgag agcgcccagt tccccaactg gtacatttcc 180acctcccagg ccgagaacat gcccgtcttt ctgggcggaa ccaagggcgg ccaggacatc 240accgacttca ccatgcagtt cgtctccagc ggaggaagcg gaggcagcgg agctcccgtg 300aggagcctga actgcaccct gagggacagc cagcagaagt ccctggtgat gtccggaccc 360tacgaactga aggccctcca tctgcaagga caggatatgg agcagcaggt ggtgttctcc 420atgtccttcg tccagggcga agagtccaac gacaagatcc ccgtggccct gggcctgaaa 480tagtga 48610397PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 10Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1 5 10 15Ala Val Phe Val Ser Ala Arg Asp Tyr Lys Asp Asp Asp Asp Lys Asp 20 25 30Lys Cys Lys Glu Arg Glu Glu Lys Ile Ile Leu Val Ser Ser Ala Asn 35 40 45Glu Ile Asp Val Arg Pro Cys Pro Leu Asn Pro Asn Glu His Lys Gly 50 55 60Thr Ile Thr Trp Tyr Lys Asp Asp Ser Lys Thr Pro Val Ser Thr Glu65 70 75 80Gln Ala Ser Arg Ile His Gln His Lys Glu Lys Leu Trp Phe Val Pro 85 90 95Ala Lys Val Glu Asp Ser Gly His Tyr Tyr Cys Val Val Arg Asn Ser 100

105 110Ser Tyr Cys Leu Arg Ile Lys Ile Ser Ala Lys Phe Val Glu Asn Glu 115 120 125Pro Asn Leu Cys Tyr Asn Ala Gln Ala Ile Phe Lys Gln Lys Leu Pro 130 135 140Val Ala Gly Asp Gly Gly Leu Val Cys Pro Tyr Met Glu Phe Phe Lys145 150 155 160Asn Glu Asn Asn Glu Leu Pro Lys Leu Gln Trp Tyr Lys Asp Cys Lys 165 170 175Pro Leu Leu Leu Asp Asn Ile His Phe Ser Gly Val Lys Asp Arg Leu 180 185 190Ile Val Met Asn Val Ala Glu Lys His Arg Gly Asn Tyr Thr Cys His 195 200 205Ala Ser Tyr Thr Tyr Leu Gly Lys Gln Tyr Pro Ile Thr Arg Val Ile 210 215 220Glu Phe Ile Thr Leu Glu Glu Asn Ser Gly Gly Ser Gly Asn Lys Leu225 230 235 240Glu Phe Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr Ser Gln 245 250 255Ala Glu Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly Gln Asp 260 265 270Ile Thr Asp Phe Thr Met Gln Phe Val Ser Ser Gly Gly Ser Gly Gly 275 280 285Ser Gly Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp Ser Gln 290 295 300Gln Lys Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala Leu His305 310 315 320Leu Gln Gly Gln Asp Met Glu Gln Gln Val Val Phe Ser Met Ser Phe 325 330 335Val Gln Gly Glu Glu Ser Asn Asp Lys Ile Pro Val Ala Leu Gly Leu 340 345 350Lys Glu Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp Lys Pro 355 360 365Thr Leu Gln Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys Lys Lys 370 375 380Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Asn385 390 395111197DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 11atggacgcta tgaagcgggg actgtgctgc gtgctcctgc tgtgcggcgc tgtctttgtc 60agcgcccggg actataagga cgatgatgac aaggacaagt gcaaggagcg ggaggagaag 120atcatcctgg tgagctccgc caacgagatt gacgtccggc cctgccctct caaccccaac 180gagcataagg gcaccatcac ctggtacaaa gacgacagca aaacacccgt ctccaccgag 240caagcctccc ggattcacca gcacaaggag aagctctggt tcgtgcccgc taaggtggag 300gattccggac actactactg tgtggtccgg aactccagct actgcctgag gattaagatc 360agcgctaagt tcgtcgagaa cgagcccaac ctctgctaca atgcccaggc catcttcaag 420cagaagctcc ctgtggctgg agacggaggc ctggtctgcc cctacatgga gttcttcaag 480aacgagaata acgagctgcc taagctgcag tggtacaagg actgcaaacc cctgctcctc 540gacaacatcc acttctccgg cgtcaaggac cggctgatcg tcatgaacgt ggccgagaag 600cacaggggca actatacctg tcacgccagc tacacctacc tgggaaagca gtatcctatc 660accagggtga ttgagttcat cacactcgag gaaaacagcg gcggcagcgg caacaagctg 720gagttcgagt ccgcccagtt tcctaactgg tacatctcca caagccaggc cgagaacatg 780cctgtcttcc tgggcggcac caaaggcggc caagatatca ccgacttcac catgcagttt 840gtgagctccg gaggctccgg aggaagcgga gctcctgtgc ggtccctgaa ttgcaccctg 900cgggattccc aacagaagag cctggtgatg tccggcccct acgagctcaa ggccctccat 960ctgcaaggcc aggacatgga gcagcaggtg gtcttcagca tgagcttcgt gcagggagag 1020gagtccaacg ataagatccc cgtcgctctc ggactcaagg agaagaacct gtacctctcc 1080tgcgtgctga aggacgataa gcccaccctc cagctggaat ccgtggaccc caagaactac 1140cccaagaaaa aaatggaaaa gcggtttgtc tttaacaaga tcgagattaa ctagtga 119712539PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 12Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn1 5 10 15Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu 20 25 30Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile 35 40 45Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Ser Ser Ser 50 55 60Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln65 70 75 80Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 85 90 95Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 100 105 110His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 115 120 125Asn Leu Ala Gln Gly Ser Gly Gly Gly Ser Glu Leu Cys Asp Asp Asp 130 135 140Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu145 150 155 160Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys 165 170 175Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser 180 185 190Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr 195 200 205Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr 210 215 220Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly225 230 235 240His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile 245 250 255Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly 260 265 270Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr 275 280 285His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly Gly 290 295 300Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro305 310 315 320Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 325 330 335Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 340 345 350Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 355 360 365Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 370 375 380Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val385 390 395 400Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 405 410 415Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 420 425 430Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 435 440 445Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 450 455 460Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu465 470 475 480Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 485 490 495Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 500 505 510Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 515 520 525Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 530 535131623DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 13tccaagaact tccacctgag gcctcgggac ctgatctcca acatcaacgt gatcgtgctg 60gaactgaagg gctccgagac aaccttcatg tgcgagtacg ccgacgagac agctaccatc 120gtggaatttc tgaaccggtg gatcaccttc tgccagtcca tcatctccac cctgaccggc 180ggctcctcca gcaccaagaa aacccagctg cagctggaac atctgctgct ggacctgcag 240atgatcctga acggcatcaa caactacaag aaccccaagc tgacccggat gctgaccttc 300aagttctaca tgcccaagaa ggccaccgaa ctgaaacatc tgcagtgcct ggaagaagaa 360ctgaagcccc tggaagaggt gctgaacctg gctcagggat ctggcggcgg atctgagctg 420tgcgacgacg accctcctga gatccctcac gccaccttca aggccatggc ttacaaagag 480ggcaccatgc tgaactgcga gtgcaagaga ggcttccggc ggatcaagtc cggctccctg 540tacatgctgt gcaccggcaa ctccagccac tcctcctggg acaaccagtg ccagtgcacc 600tcctctgcca cccggaacac caccaaacaa gtgacccccc agcccgagga acagaaagag 660cgcaagacca ccgagatgca gtcccccatg cagcctgtgg accaggcttc tctgcctggc 720cactgcagag agcctccacc ttgggagaac gaggctaccg agagaatcta ccacttcgtc 780gtgggccaga tggtgtacta ccagtgcgtg cagggctacc gcgccctgca tagaggacct 840gctgagtccg tgtgcaagat gacccacggc aagacccggt ggacccagcc tcagctgatc 900tgtacaggcg gcggaggctc cgagcctaag tcctccgata agacccacac ctgtcccccc 960tgtcctgccc ctgaactgct gggaggccct tccgtgttcc tgttcccccc aaagcccaag 1020gacaccctga tgatctcccg gacccccgaa gtgacctgcg tggtggtgga tgtgtcccac 1080gaggaccctg aagtgaagtt caattggtac gtggacggcg tggaagtgca caacgccaag 1140accaagccca gagaggaaca gtacaactcc acctaccggg tggtgtccgt gctgaccgtg 1200ctgcaccagg attggctgaa tggcaaagag tacaagtgca aggtgtccaa caaggccctg 1260ccagccccca tcgaaaagac catctccaag gccaagggcc agccccggga accccaggtg 1320tacacactgc cccctagccg ggaagagatg accaagaacc aggtgtccct gacctgtctc 1380gtgaagggct tctacccctc cgatatcgcc gtggaatggg agtccaacgg ccagcctgag 1440aacaattata agaccacccc ccctgtgctg gactccgacg gctcattctt cctgtacagc 1500aagctgacag tggacaagtc ccggtggcag cagggcaacg tgttctcctg ctccgtgatg 1560cacgaggccc tgcacaacca ctacacccag aagtccctgt ccctgtctcc cggcaagtga 1620tga 1623147PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 14Ser Gly Gly Ser Gly Gly Gly1 5157PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 15Gly Gly Ser Gly Gly Ser Gly1 5162PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 16Gly Gly1172PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 17Met Gly1183PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 18Met Gly Gly119269PRTHomo sapiens 19Met Ala Glu Val Pro Glu Leu Ala Ser Glu Met Met Ala Tyr Tyr Ser1 5 10 15Gly Asn Glu Asp Asp Leu Phe Phe Glu Ala Asp Gly Pro Lys Gln Met 20 25 30Lys Cys Ser Phe Gln Asp Leu Asp Leu Cys Pro Leu Asp Gly Gly Ile 35 40 45Gln Leu Arg Ile Ser Asp His His Tyr Ser Lys Gly Phe Arg Gln Ala 50 55 60Ala Ser Val Val Val Ala Met Asp Lys Leu Arg Lys Met Leu Val Pro65 70 75 80Cys Pro Gln Thr Phe Gln Glu Asn Asp Leu Ser Thr Phe Phe Pro Phe 85 90 95Ile Phe Glu Glu Glu Pro Ile Phe Phe Asp Thr Trp Asp Asn Glu Ala 100 105 110Tyr Val His Asp Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp 115 120 125Ser Gln Gln Lys Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala 130 135 140Leu His Leu Gln Gly Gln Asp Met Glu Gln Gln Val Val Phe Ser Met145 150 155 160Ser Phe Val Gln Gly Glu Glu Ser Asn Asp Lys Ile Pro Val Ala Leu 165 170 175Gly Leu Lys Glu Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp 180 185 190Lys Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys 195 200 205Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Asn Asn 210 215 220Lys Leu Glu Phe Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr225 230 235 240Ser Gln Ala Glu Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly 245 250 255Gln Asp Ile Thr Asp Phe Thr Met Gln Phe Val Ser Ser 260 26520153PRTHomo sapiens 20Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu1 5 10 15Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu65 70 75 80Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 85 90 95Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140Cys Gln Ser Ile Ile Ser Thr Leu Thr145 15021918PRTHomo sapiens 21Met Leu Thr Leu Gln Thr Trp Val Val Gln Ala Leu Phe Ile Phe Leu1 5 10 15Thr Thr Glu Ser Thr Gly Glu Leu Leu Asp Pro Cys Gly Tyr Ile Ser 20 25 30Pro Glu Ser Pro Val Val Gln Leu His Ser Asn Phe Thr Ala Val Cys 35 40 45Val Leu Lys Glu Lys Cys Met Asp Tyr Phe His Val Asn Ala Asn Tyr 50 55 60Ile Val Trp Lys Thr Asn His Phe Thr Ile Pro Lys Glu Gln Tyr Thr65 70 75 80Ile Ile Asn Arg Thr Ala Ser Ser Val Thr Phe Thr Asp Ile Ala Ser 85 90 95Leu Asn Ile Gln Leu Thr Cys Asn Ile Leu Thr Phe Gly Gln Leu Glu 100 105 110Gln Asn Val Tyr Gly Ile Thr Ile Ile Ser Gly Leu Pro Pro Glu Lys 115 120 125Pro Lys Asn Leu Ser Cys Ile Val Asn Glu Gly Lys Lys Met Arg Cys 130 135 140Glu Trp Asp Gly Gly Arg Glu Thr His Leu Glu Thr Asn Phe Thr Leu145 150 155 160Lys Ser Glu Trp Ala Thr His Lys Phe Ala Asp Cys Lys Ala Lys Arg 165 170 175Asp Thr Pro Thr Ser Cys Thr Val Asp Tyr Ser Thr Val Tyr Phe Val 180 185 190Asn Ile Glu Val Trp Val Glu Ala Glu Asn Ala Leu Gly Lys Val Thr 195 200 205Ser Asp His Ile Asn Phe Asp Pro Val Tyr Lys Val Lys Pro Asn Pro 210 215 220Pro His Asn Leu Ser Val Ile Asn Ser Glu Glu Leu Ser Ser Ile Leu225 230 235 240Lys Leu Thr Trp Thr Asn Pro Ser Ile Lys Ser Val Ile Ile Leu Lys 245 250 255Tyr Asn Ile Gln Tyr Arg Thr Lys Asp Ala Ser Thr Trp Ser Gln Ile 260 265 270Pro Pro Glu Asp Thr Ala Ser Thr Arg Ser Ser Phe Thr Val Gln Asp 275 280 285Leu Lys Pro Phe Thr Glu Tyr Val Phe Arg Ile Arg Cys Met Lys Glu 290 295 300Asp Gly Lys Gly Tyr Trp Ser Asp Trp Ser Glu Glu Ala Ser Gly Ile305 310 315 320Thr Tyr Glu Asp Arg Pro Ser Lys Ala Pro Ser Phe Trp Tyr Lys Ile 325 330 335Asp Pro Ser His Thr Gln Gly Tyr Arg Thr Val Gln Leu Val Trp Lys 340 345 350Thr Leu Pro Pro Phe Glu Ala Asn Gly Lys Ile Leu Asp Tyr Glu Val 355 360 365Thr Leu Thr Arg Trp Lys Ser His Leu Gln Asn Tyr Thr Val Asn Ala 370 375 380Thr Lys Leu Thr Val Asn Leu Thr Asn Asp Arg Tyr Leu Ala Thr Leu385 390 395 400Thr Val Arg Asn Leu Val Gly Lys Ser Asp Ala Ala Val Leu Thr Ile 405 410 415Pro Ala Cys Asp Phe Gln Ala Thr His Pro Val Met Asp Leu Lys Ala 420 425 430Phe Pro Lys Asp Asn Met Leu Trp Val Glu Trp Thr Thr Pro Arg Glu 435 440 445Ser Val Lys Lys Tyr Ile Leu Glu Trp Cys Val Leu Ser Asp Lys Ala 450 455 460Pro Cys Ile Thr Asp Trp Gln Gln Glu Asp Gly Thr Val His Arg Thr465 470 475 480Tyr Leu Arg Gly Asn Leu Ala Glu Ser Lys Cys Tyr Leu Ile Thr Val 485 490 495Thr Pro Val Tyr Ala Asp Gly Pro Gly Ser Pro Glu Ser Ile Lys Ala 500 505 510Tyr Leu Lys Gln Ala Pro Pro Ser Lys Gly Pro Thr Val Arg Thr Lys 515 520 525Lys Val Gly Lys Asn Glu Ala Val Leu Glu Trp Asp Gln Leu Pro Val 530 535 540Asp Val Gln Asn Gly Phe Ile Arg Asn Tyr Thr Ile Phe Tyr Arg Thr545 550 555 560Ile Ile Gly Asn Glu Thr Ala Val Asn Val Asp Ser Ser His Thr Glu 565 570 575Tyr Thr Leu Ser Ser Leu Thr Ser Asp Thr Leu Tyr Met Val Arg Met 580 585 590Ala Ala Tyr Thr Asp Glu Gly Gly Lys Asp Gly Pro Glu Phe Thr Phe 595 600 605Thr Thr Pro Lys Phe Ala Gln Gly Glu Ile Glu Ala Ile Val Val Pro 610 615 620Val Cys Leu Ala Phe Leu Leu Thr Thr Leu Leu Gly Val Leu Phe Cys625

630 635 640Phe Asn Lys Arg Asp Leu Ile Lys Lys His Ile Trp Pro Asn Val Pro 645 650 655Asp Pro Ser Lys Ser His Ile Ala Gln Trp Ser Pro His Thr Pro Pro 660 665 670Arg His Asn Phe Asn Ser Lys Asp Gln Met Tyr Ser Asp Gly Asn Phe 675 680 685Thr Asp Val Ser Val Val Glu Ile Glu Ala Asn Asp Lys Lys Pro Phe 690 695 700Pro Glu Asp Leu Lys Ser Leu Asp Leu Phe Lys Lys Glu Lys Ile Asn705 710 715 720Thr Glu Gly His Ser Ser Gly Ile Gly Gly Ser Ser Cys Met Ser Ser 725 730 735Ser Arg Pro Ser Ile Ser Ser Ser Asp Glu Asn Glu Ser Ser Gln Asn 740 745 750Thr Ser Ser Thr Val Gln Tyr Ser Thr Val Val His Ser Gly Tyr Arg 755 760 765His Gln Val Pro Ser Val Gln Val Phe Ser Arg Ser Glu Ser Thr Gln 770 775 780Pro Leu Leu Asp Ser Glu Glu Arg Pro Glu Asp Leu Gln Leu Val Asp785 790 795 800His Val Asp Gly Gly Asp Gly Ile Leu Pro Arg Gln Gln Tyr Phe Lys 805 810 815Gln Asn Cys Ser Gln His Glu Ser Ser Pro Asp Ile Ser His Phe Glu 820 825 830Arg Ser Lys Gln Val Ser Ser Val Asn Glu Glu Asp Phe Val Arg Leu 835 840 845Lys Gln Gln Ile Ser Asp His Ile Ser Gln Ser Cys Gly Ser Gly Gln 850 855 860Met Lys Met Phe Gln Glu Val Ser Ala Ala Asp Ala Phe Gly Pro Gly865 870 875 880Thr Glu Gly Gln Val Glu Arg Phe Glu Thr Val Gly Met Glu Ala Ala 885 890 895Thr Asp Glu Gly Met Pro Lys Ser Tyr Leu Pro Gln Thr Val Arg Gln 900 905 910Gly Gly Tyr Met Pro Gln 91522569PRTHomo sapiens 22Met Lys Val Leu Leu Arg Leu Ile Cys Phe Ile Ala Leu Leu Ile Ser1 5 10 15Ser Leu Glu Ala Asp Lys Cys Lys Glu Arg Glu Glu Lys Ile Ile Leu 20 25 30Val Ser Ser Ala Asn Glu Ile Asp Val Arg Pro Cys Pro Leu Asn Pro 35 40 45Asn Glu His Lys Gly Thr Ile Thr Trp Tyr Lys Asp Asp Ser Lys Thr 50 55 60Pro Val Ser Thr Glu Gln Ala Ser Arg Ile His Gln His Lys Glu Lys65 70 75 80Leu Trp Phe Val Pro Ala Lys Val Glu Asp Ser Gly His Tyr Tyr Cys 85 90 95Val Val Arg Asn Ser Ser Tyr Cys Leu Arg Ile Lys Ile Ser Ala Lys 100 105 110Phe Val Glu Asn Glu Pro Asn Leu Cys Tyr Asn Ala Gln Ala Ile Phe 115 120 125Lys Gln Asn Leu Pro Val Ala Gly Asp Gly Gly Leu Val Cys Pro Tyr 130 135 140Met Glu Phe Phe Lys Asn Glu Asn Asn Glu Leu Pro Lys Leu Gln Trp145 150 155 160Tyr Lys Asp Cys Lys Pro Leu Leu Leu Asp Asn Ile His Phe Ser Gly 165 170 175Val Lys Asp Arg Leu Ile Val Met Asn Val Ala Glu Lys His Arg Gly 180 185 190Asn Tyr Thr Cys His Ala Ser Tyr Thr Tyr Leu Gly Lys Gln Tyr Pro 195 200 205Ile Thr Arg Val Ile Glu Phe Ile Thr Leu Glu Glu Asn Lys Pro Thr 210 215 220Arg Pro Val Ile Val Ser Pro Ala Asn Glu Thr Met Glu Val Asp Leu225 230 235 240Gly Ser Gln Ile Gln Leu Ile Cys Asn Val Thr Gly Gln Leu Ser Asp 245 250 255Ile Ala Tyr Trp Lys Trp Asn Gly Ser Val Ile Asp Glu Asp Asp Pro 260 265 270Val Leu Gly Glu Asp Tyr Tyr Ser Val Glu Asn Pro Ala Asn Lys Arg 275 280 285Arg Ser Thr Leu Ile Thr Val Leu Asn Ile Ser Glu Ile Glu Ser Arg 290 295 300Phe Tyr Lys His Pro Phe Thr Cys Phe Ala Lys Asn Thr His Gly Ile305 310 315 320Asp Ala Ala Tyr Ile Gln Leu Ile Tyr Pro Val Thr Asn Phe Gln Lys 325 330 335His Met Ile Gly Ile Cys Val Thr Leu Thr Val Ile Ile Val Cys Ser 340 345 350Val Phe Ile Tyr Lys Ile Phe Lys Ile Asp Ile Val Leu Trp Tyr Arg 355 360 365Asp Ser Cys Tyr Asp Phe Leu Pro Ile Lys Ala Ser Asp Gly Lys Thr 370 375 380Tyr Asp Ala Tyr Ile Leu Tyr Pro Lys Thr Val Gly Glu Gly Ser Thr385 390 395 400Ser Asp Cys Asp Ile Phe Val Phe Lys Val Leu Pro Glu Val Leu Glu 405 410 415Lys Gln Cys Gly Tyr Lys Leu Phe Ile Tyr Gly Arg Asp Asp Tyr Val 420 425 430Gly Glu Asp Ile Val Glu Val Ile Asn Glu Asn Val Lys Lys Ser Arg 435 440 445Arg Leu Ile Ile Ile Leu Val Arg Glu Thr Ser Ser Phe Ser Trp Leu 450 455 460Gly Gly Ser Ser Glu Glu Gln Ile Ala Met Tyr Asn Ala Leu Val Gln465 470 475 480Asp Gly Ile Lys Val Val Leu Leu Glu Leu Glu Lys Ile Gln Asp Tyr 485 490 495Glu Lys Met Pro Glu Ser Ile Lys Phe Ile Lys Gln Lys His Gly Ala 500 505 510Ile Arg Trp Ser Gly Asp Phe Thr Gln Gly Pro Gln Ser Ala Lys Thr 515 520 525Arg Phe Trp Lys Asn Val Arg Tyr His Met Pro Val Gln Arg Arg Ser 530 535 540Pro Ser Ser Lys His Gln Leu Leu Ser Pro Ala Thr Lys Glu Lys Leu545 550 555 560Gln Arg Glu Ala His Val Pro Leu Gly 56523272PRTHomo sapiens 23Met Asp Ser Tyr Leu Leu Met Trp Gly Leu Leu Thr Phe Ile Met Val1 5 10 15Pro Gly Cys Gln Ala Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro 20 25 30His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn 35 40 45Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr 50 55 60Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys65 70 75 80Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro 85 90 95Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro 100 105 110Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro 115 120 125Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val 130 135 140Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His145 150 155 160Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His Gly Lys Thr Arg 165 170 175Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln 180 185 190Phe Pro Gly Glu Glu Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu 195 200 205Ser Glu Thr Ser Cys Leu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr 210 215 220Glu Met Ala Ala Thr Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln225 230 235 240Val Ala Val Ala Gly Cys Val Phe Leu Leu Ile Ser Val Leu Leu Leu 245 250 255Ser Gly Leu Thr Trp Gln Arg Arg Gln Arg Lys Ser Arg Arg Thr Ile 260 265 27024142PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 24Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn1 5 10 15Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu 20 25 30Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile 35 40 45Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Ser Ser Ser 50 55 60Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln65 70 75 80Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 85 90 95Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 100 105 110His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 115 120 125Asn Leu Ala Gln Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys 130 135 14025539PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 25Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn1 5 10 15Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu 20 25 30Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile 35 40 45Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Ser Ser Ser 50 55 60Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln65 70 75 80Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 85 90 95Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 100 105 110His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 115 120 125Asn Leu Ala Gln Gly Ser Gly Gly Gly Ser Glu Leu Cys Asp Asp Asp 130 135 140Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu145 150 155 160Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys 165 170 175Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser 180 185 190Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr 195 200 205Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr 210 215 220Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly225 230 235 240His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile 245 250 255Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly 260 265 270Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr 275 280 285His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly Gly 290 295 300Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro305 310 315 320Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 325 330 335Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 340 345 350Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 355 360 365Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 370 375 380Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val385 390 395 400Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 405 410 415Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 420 425 430Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 435 440 445Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 450 455 460Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu465 470 475 480Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 485 490 495Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 500 505 510Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 515 520 525Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 530 53526311PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 26Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn1 5 10 15Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu 20 25 30Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile 35 40 45Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Ser Ser Ser 50 55 60Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln65 70 75 80Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 85 90 95Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 100 105 110His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 115 120 125Asn Leu Ala Gln Gly Ser Gly Gly Gly Ser Glu Leu Cys Asp Asp Asp 130 135 140Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu145 150 155 160Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys 165 170 175Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser 180 185 190Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr 195 200 205Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr 210 215 220Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly225 230 235 240His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile 245 250 255Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly 260 265 270Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr 275 280 285His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly Asp 290 295 300Tyr Lys Asp Asp Asp Asp Lys305 31027418PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 27Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala1 5 10 15Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln65 70 75 80Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 130 135 140Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser145 150 155 160Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220Ser Leu Ser Leu Ser Pro Gly Lys Gly Ser Ile Thr Cys Pro Pro Pro225 230 235 240Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr 245 250 255Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly 260 265 270Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala 275 280 285His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Gly Gly Ser Glu 290 295 300Leu Glu Glu Lys Asn Ile Lys Glu

Phe Leu Gln Ser Phe Val His Ile305 310 315 320Val Gln Met Phe Ile Asn Gly Gly Gly Ser Asn Trp Val Asn Val Ile 325 330 335Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp 340 345 350Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr 355 360 365Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser 370 375 380Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala385 390 395 400Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 405 410 415Glu Cys28194PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 28Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Ile Thr Cys Pro Pro Pro1 5 10 15Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr 20 25 30Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly 35 40 45Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala 50 55 60His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Gly Gly Ser Glu65 70 75 80Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile 85 90 95Val Gln Met Phe Ile Asn Gly Gly Gly Ser Asn Trp Val Asn Val Ile 100 105 110Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp 115 120 125Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr 130 135 140Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser145 150 155 160Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala 165 170 175Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 180 185 190Glu Cys

Claims

1-15. (canceled)

16. A fusion polypeptide comprising a first polypeptide fusion partner linked to a modified ligand corresponding to all or a portion of a native ligand of a target receptor, wherein the modified ligand has been circularly permuted to create a new N-terminus and a new C-terminus as compared to the native ligand, wherein the new C-terminus and the new N-terminus of the modified ligand do not disrupt any binding domain of the modified ligand for the target receptor, wherein the modified ligand is circularly permuted IL-2, the fusion partner is IL-2R.alpha. and the target receptor is IL-2R.beta..gamma., wherein the fusion polypeptide is optionally further fused to the Fc region of an antibody.

17. The fusion polypeptide of claim 16, wherein the modified ligand is a circularly permuted IL-2 having a C145S mutation.

18. A pharmaceutical composition comprising the fusion polypeptide of claim 16.

19. A pharmaceutical composition comprising the fusion polypeptide of claim 17.

20. A method of selectively agonizing IL-2R.beta..gamma. on a cell comprising contacting the cell with a fusion polypeptide of claim 16.

21. The method of claim 20, wherein the fusion polypeptide is contacted with the cell extracorporeally.

22. A method of selectively agonizing IL-2R.beta..gamma. on a cell comprising contacting the cell with a fusion polypeptide of claim 17.

23. The method of claim 22, wherein the fusion polypeptide is contacted with the cell extracorporeally.

24. The fusion polypeptide of claim 16, wherein the fusion polypeptide is further fused to the Fc region of an antibody.