CN116529257A - Orthogonal IL-21 receptor/cytokine systems - Google Patents

Abstract

Various Orthogonal (orthographic) IL-21 receptors and Orthogonal IL-21 cytokines are described. The IL-21 receptor-cytokine pair can include an orthogonal interleukin 21 receptor a chain that is impaired in binding to native interleukin 21 cytokine ("IL-21") (ortho-IL-21 Rα ") and an orthogonal IL-21 cytokine that is impaired in binding to native IL-21Ra (ortho-IL-21"), wherein ortho-IL-21Ra binds ortho-IL-21. The IL-21 receptor-cytokine pair may activate IL-21 signaling. Also described are various cells engineered to express orthogonal IL-21 receptors, and various methods of using such cells to treat various diseases and conditions.

Description

Orthogonal IL-21 receptor/cytokine system

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 63/105,414 filed on October 26, 2020 and U.S. Provisional Patent Application No. 63/212,547 filed on June 18, 2021, the entire contents of which are incorporated herein by reference.

Sequence Listing

The sequence listing has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. This ASCII copy was created on October 25, 2021, is named Neptune-IL21-PCT_ST25.txt, and is 123,571 bytes in size.

Background Art

Cytokines are potent natural regulators of immune cell proliferation and differentiation. While this potency makes cytokines extremely attractive as potential therapeutics, it also complicates their clinical application. This is particularly true for cytokines that have multiple cellular targets and therefore have a high potential for pleiotropic effects. An example is interleukin-2 (IL-2), a potent T-cell mitogen whose anticancer activity is offset by the deleterious proliferation of regulatory (inhibitory) T cells and painful vascular leak syndrome. In the specific case of IL-2, protein engineering can be used to address some clinical challenges: for example, removing the ability of cytokines to act preferentially on regulatory T cells. Another approach involves generating orthogonally constrained forms of cytokines and their receptors. See U.S. Patent No. 10,869,887; Sockolosky JT, Trotta E, Parisi G, et al., Selective targeting of engineered T cells using orthogonal IL-2 cytokine-receptor complexes, Science, 2018; 359(6379): 1037-1042, doi: 10.1126/science.aar3246, the disclosures of which are incorporated herein by reference in their entirety.

An orthogonal cytokine system is one in which a cytokine and its receptor are mutated such that they lose compatibility with their natural (parental) partners but retain the ability to interact productively with each other. Such orthogonal cytokine:receptor pairs can therefore be said to exhibit "privileged" or "private" interactions. Methods for generating orthogonally constrained forms of cytokines and their receptors are valuable for cell therapy because they provide a means of limiting the scope of cytokine activity to only therapeutic (i.e., adoptively transferred) cells, which are the only cells expressing the engineered receptor and are therefore the only cells capable of responding to the engineered cytokine.

Interleukin-21 (IL-21) is another pleiotropic cytokine that acts on a wide range of lymphocytes, myeloid cells, and epithelial cells. IL-21 regulates both innate and adaptive immune responses; it plays a key role not only in antitumor and antiviral responses, but also in inflammatory responses that promote the development of autoimmune and inflammatory diseases. Spolski, R., Leonard, W., Interleukin-21: a double-edged sword with therapeutic potential, Nat Rev Drug Discov 13, 379–395 (2014), https://doi.org/10.1038/nrd4296. The three-dimensional structure of the native human IL-21 cytokine: receptor complex is known. See Hamming OJ, Kang L, Svensson A, et al., The crystal structure of the interleukin 21 receptor (IL-21R) bound to IL-21 shows that the sugar chain interacting with the WSXWS motif is a component of IL-21R, J Biol Chem., 2012; 287(12): 9454-9460, doi: 10.1074/jbc.M111.311084, the disclosure of which is incorporated herein by reference in its entirety.

IL-21 is of particular interest because it enhances cytotoxic T cell responses to viruses and tumors and can act synergistically with other cytokines such as IL-2 or IL-15. IL-21 does this in part by promoting the persistence of T cells with a stem cell memory phenotype, which has been associated with beneficial outcomes in the cell therapy setting. IL-21 is currently being evaluated as a cancer therapeutic in multiple clinical trials. IL-21 also has important potential uses in chimeric antigen receptor T ("CAR-T") cell therapy, where it can help overcome clinical failures due to low expansion, anti-tumor efficacy, exhaustion, suppression, and persistence. Therefore, there is a pressing need for the ability to regulate the effects of IL-21, particularly by engineering a desired behavior in adoptively transferred cells that is not affected by endogenous signaling pathways, does not affect non-targeted endogenous cells, and can be controlled once administered to the patient.

Summary of the invention

In one aspect, an orthogonal interleukin-21 receptor alpha chain ("ortho-IL-21Rα" or "ortho-IL-21Rα molecule," or when referring to a specific ortho-IL-21Rα constructed as provided herein, "RV," as in "receptor variant") is provided, the ortho-IL-21Rα comprising a modified amino acid sequence derived from SEQ ID NO: 4 that binds to an orthogonal interleukin-21 cytokine ("ortho-IL-21" or "ortho-IL-21 molecule," or when referring to a specific ortho-IL-21 constructed as provided herein, "CV," as in "cytokine variant"), but has impaired binding to native IL-21. In one aspect, ortho-IL-21Rα comprises a modified amino acid sequence comprising a substitution of one of the following: one or more amino acid residues of SEQ ID NO: 4 that contact IL-21; a residue immediately adjacent to such contact residues; or a residue elsewhere in IL-21Rα that has an effect on the conformation of the IL-21 binding surface. In one aspect, ortho-IL-21Rα comprises amino acid substitutions numbered relative to SEQ ID NO: 6 (mature form of human IL-21α extracellular domain lacking a signal peptide) at positions: Y10, Q33, Q35, Y36, E38, L39, F67, H68, F69, M70, A71, D72, D73, I74, L94, A96, E97, P126, A127, Y129, M130, K134, S190, Y191, or a combination thereof. In one aspect, the amino acid substitutions comprise, consist essentially of, or consist of: D72E/Y129F/D73E; M70I/D73E/Q33H; D72E/L94V/Y191F; D72E/E38D/M130L;

M70G/Y129F; F69L/M70L/D73I; D72K/D73K; E38K; or M70G, or a combination thereof. In one aspect, the amino acid substitution comprises, consists essentially of, or consists of: M70G/Y129F ("RV13", such as "receptor variant 13" or SEQ ID NO: 19) or M70G ("RV22" or SEQ ID NO: 27).

In another aspect, an ortho-IL-21 is provided, the ortho-IL-21 comprising a modified amino acid sequence derived from SEQ ID NO: 2, which binds to ortho-IL-21Rα, but has impaired binding to native IL-21Rα. In one aspect, the ortho-IL-21 comprises a modified amino acid sequence comprising a substitution of one of the following: one or more amino acid residues of SEQ ID NO: 2 that contact IL-21Rα; residues immediately adjacent to such contact residues; or residues elsewhere in IL-21 that affect the conformation of the IL-21α binding surface. In one aspect, the ortho-IL-21 comprises amino acid substitutions numbered relative to SEQ ID NO: 2, positions: R5, H6, I8, R9, M10, Q12, L13, K73, K75, R76, P78, G84, P104, or a combination thereof. For numbering purposes, the phrase "numbered relative to SEQ ID NO: 2" means ignoring any epitope tags and signal peptides. In one aspect, the amino acid substitutions comprise, consist essentially of, or consist of R5Q, R5D, R5E, R5K, R5N, H6L, I8E, R9E, R9D, R9K, R9N, R9Q, M10L, Q12V, L13D, K73V, K73D, K73I, K75D, R76E, R76N, R76A, R5Q/R76E, R5Q/R76A, P78L, G84E, P104A, or a combination thereof. In one aspect, the amino acid substitutions comprise, consist essentially of, or consist of H6L/R9K/M10L/P78L.

In exemplary aspects, an engineered human IL-21 polypeptide is provided, comprising amino acid substitutions numbered relative to SEQ ID NO: 2: H6L, R9K, M10L, P78L, and optionally comprising G84E or P104A or a combination thereof; and optionally comprising one of K73V or K73I. In one aspect, such an engineered human IL-21 polypeptide may comprise SEQ ID NO: 61 (H6L/R9K/M10L/K73V/P78L/G84E) or SEQ ID NO: 62 (H6L/R9K/M10L/K73I/P78L/P104A).

In another aspect, a system for activating IL-21 signaling in a cell is provided, the system comprising: an ortho-IL-21Rα with impaired binding to native IL-21, the ortho-IL-21Rα comprising an amino acid sequence derived from a modification of SEQ ID NO: 4, comprising a substitution of one of the following: one or more amino acid residues of SEQ ID NO: 4 that contact IL-21; residues immediately adjacent to such contact residues; or residues elsewhere in IL-21α that affect the conformation of the IL-21 binding surface; and an ortho-IL-21 with impaired binding to native IL-21Rα, the ortho-IL-21 comprising a modified amino acid sequence derived from SEQ ID NO: 2, comprising a substitution of one of the following: one or more amino acid residues of SEQ ID NO: 2 that contact IL-21α; residues immediately adjacent to such contact residues; or residues elsewhere in IL-21 that affect the conformation of the IL-21α binding surface, wherein ortho-IL-21Rα binds to ortho-IL-21. In one aspect, the cell is a mammalian cell, an immune cell, a stem cell, or a T cell.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention may be more readily understood with reference to the following drawings, in which:

Figure 1 provides a schematic diagram of the orthogonal IL-21 system, with the leftmost cartoon showing an effective interaction between a wild-type receptor and a cytokine, while the adjacent cartoon depicts an impaired interaction between ortho-IL-21Rα and a native cytokine, and the rightmost cartoon depicting an impaired interaction between ortho-IL-21 and a native (wild-type) receptor, while the adjacent cartoon shows two orthogonal molecules (ortho-IL-21Rα and ortho-IL-21);

FIG2 provides a schematic diagram of the approach used to generate an orthogonal IL-21 system;

FIG3 shows the results of a representative assay in which a single (sub-saturating) concentration of IL-21-TLuc16 was tested for binding to a panel of 20 candidate ortho-IL-21Rα molecules (all of which had been bound to the streptactin-coated surface of the wells at saturating concentrations), and 8 of the 20 candidate ortho-IL-21Rα molecules showed reduced ability to bind IL-21-TLuc16;

Figures 4A to 4E show the results of a representative assay in which a range of (subsaturating) concentrations of IL-21-TLuc16 were tested for binding to a panel of candidate ortho-IL-21Rα molecules; the panel included the wild-type receptor as a control and seven of the eight candidate ortho-IL-21Rα molecules identified in Figure 3; the panel also included 13 additional candidate ortho-IL-21Rα molecules that were designed based on the results described in Figure 3; specifically, substitutions present in the eight candidate ortho-IL-21Rα molecules were introduced individually or in combination into the additional 13 candidate ortho-IL-21Rα molecules; the 21 candidate ortho-IL-21Rα molecules were introduced into the additional 13 candidate ortho-IL-21Rα molecules. ortho-IL-21Rα molecules were added at saturating concentrations to streptomycin-coated wells of a 96-well plate; Figures 4A to 4E show luminometry data for a single plate, where in each case, the binding of IL-21-TLuc16 to five candidate ortho-IL-21Rα molecules and a wild-type control IL-21Rα were compared; 11 of the additional 13 candidate ortho-IL-21Rα molecules showed a significantly reduced ability to bind to IL-21-TLuc16 compared to the wild-type control (the exceptions were receptors RV23, RV24, and RV28, carrying single substitutions M70L, F69L, and D73E, respectively);

Figures 5A and 5B show the IL-21-induced STAT3 signaling response of Ba/F3 cells expressing native IL-21Rα and eight candidate ortho-IL-21Rα molecules identified as shown in Figure 3; cells carry a STAT3-regulated Cypridina noctiluca luciferase reporter transgene; they were exposed to various concentrations of native IL-21 overnight (approximately 20 hours) with Vargulin as an enzyme substrate before testing the supernatant culture medium for luciferase activity by luminometry; as expected, cells expressing a form of wild-type IL-21Rα lacking its cytoplasmic tail (Δcyt) were included to show that STAT3 signaling in response to IL-21 is dependent on the cytoplasmic tail of the receptor;

Figures 6A to 6T show the ability of native IL-21 and candidate ortho-IL-21 molecules to induce signaling through native IL-21Rα and eight candidate ortho-IL-21Rα molecules as shown in Figure 3; as described in Figure 5, luminometry was used to quantify luciferase produced by transfected Ba/F3 cells carrying a STAT3-dependent luciferase reporter gene transgene residing on the same transposon that was used to confer expression of native IL-21Rα or candidate ortho-IL-21Rα molecules; cells were exposed to increasing concentrations of candidate ortho-IL-21 molecules overnight (approximately 20 hours) prior to testing supernatant culture media for luciferase activity;

Figures 7A to 7D show the ability of native IL-21 and selected candidate ortho-IL-21 molecules from the collection shown in Figure 6 to induce signaling through native IL-21Rα and selected candidate ortho-IL-21Rα molecules; Ba/F3 cells (expressing native IL-21Rα or candidate ortho-IL-21 molecules from a transposon that also carries a STAT3-luciferase reporter transgene) were exposed to increasing concentrations of the designated candidate ortho-IL-21 molecules (approximately 20 hours), and the supernatant culture medium was then recovered and tested for luciferase activity by luminometry, as shown in Figures 5 and 6;

Figures 8A, 8B, and 8C show the results of a representative screening experiment in which a panel of 96 cytokines was tested for the ability to induce a STAT3-dependent signaling response in Ba/F3 cells expressing wild-type IL-21Rα (Figure 8A), candidate ortho-IL-21Rα molecules RV13 (Figure 8B), or RV6 (Figure 8C), comprising amino acid substitutions M70G/Y129F (SEQ ID NO: 19) or M70I/D73E/Q33H (SEQ ID NO: 20), respectively. NO: 12); As described in Figures 5 and 6, luminometry was used to quantify luciferase produced by transfected Ba/F3 cells carrying a STAT3-dependent luciferase reporter transgene residing on the same transposon that was used to confer expression of native IL-21Rα or candidate ortho-IL-21Rα molecules; cells were exposed to increasing concentrations of candidate ortho-IL-21 molecules overnight (approximately 20 hours) prior to testing supernatant culture media for luciferase activity; dashed lines show the response curve for the cytokine panel, while the responses induced by five cytokines of interest (wild-type IL-21, negative control CV22, and candidate ortho-IL-21 molecules CV204, CV374, and CV388) are highlighted with solid lines and symbols;

Figures 9A and 9B show the ability of native IL-21 and candidate ortho-IL-21 molecules to induce signaling through wild-type IL-21Rα (Figure 9) and candidate ortho-IL-21RαRV13 comprising amino acid substitutions M70G/Y129F (SEQ ID NO: 19) (Figure 9B); luciferase produced by transfected Ba/F3 cells carrying a STAT3-dependent luciferase reporter gene transgene resident on the same transposon used to confer expression of native IL-21Rα or candidate ortho-IL-21Rα molecules was quantified using luminometry as described in Figures 5 and 6; cells were exposed to increasing concentrations of candidate ortho-IL-21 molecules overnight (approximately 20 hours) prior to testing supernatant culture media for luciferase activity; and

Figures 10A, 10B and 10C show the natural IL-21 and candidate ortho-IL-21 molecules by wild-type IL-21Rα (Figure 10A), candidate ortho-IL-21RαRV13 comprising amino acid substitutions M70G/Y129F (SEQ ID NO: 19) (Figure 10B), and candidate ortho-IL-21RαRV13 comprising amino acid substitutions M70G (SEQ ID NO: 19). NO: 27)'s candidate ortho-IL-21RαRV22 ( FIG. 10C )'s ability to induce signaling; as described in FIGS. 5 and 6 , luminometry was used to quantify luciferase produced by transfected Ba/F3 cells carrying a STAT3-dependent luciferase reporter gene transgene residing on the same transposon that was used to confer expression of native IL-21Rα or candidate ortho-IL-21Rα molecules; cells were exposed to increasing concentrations of candidate ortho-IL-21 molecules overnight (approximately 20 hours) prior to testing the supernatant culture medium for luciferase activity.

Specific implementation plan

A variety of orthogonal IL-21 receptors and orthogonal IL-21 cytokines are provided. An orthogonal IL-21 receptor: cytokine pair can include an ortho-IL-21Rα with impaired binding to natural IL-21 and an ortho-IL-21 with impaired binding to natural IL-21Rα, wherein ortho-IL-21Rα binds to ortho-IL-21. Orthogonal IL-21 receptor-cytokine pairs can be used to activate signaling responses in cells. The signaling response can be a natural signaling response that is typically downstream of the IL-21 receptor, or an alternative signaling response that depends on a non-natural IL-21 receptor and/or other non-natural cell components. A variety of cells engineered to express orthogonal IL-21 receptors, as well as methods of using such cells to treat various diseases and disorders are also provided.

definition:

The terms set forth herein are used for description only and should not be construed as limiting the present invention. As used in the specification and the appended claims, the singular forms "a", "an" and "the" include plural forms thereof unless otherwise specified.

"Treat," "treating," "treatment," and the like refer to any action that provides a benefit to a subject suffering from a disease or disorder such as cancer, including ameliorating the disorder by alleviating or suppressing at least one symptom, slowing the progression of the disease, and the like.

The phrases "effective amount" and "therapeutically effective amount" refer to the amount of a composition used to practice the invention sufficient to provide effective treatment in a subject.

Wild type, "WT" and "native" are used interchangeably herein and refer to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A WT protein, polypeptide, antibody, immunoglobulin, IgG, etc. has an amino acid sequence or nucleotide sequence corresponding to an amino acid sequence or nucleotide sequence commonly found in nature and has not been intentionally modified.

The term "polynucleotide" refers to an oligonucleotide, a nucleotide, or a fragment of any of these; a DNA or RNA of genomic or synthetic origin (e.g., mRNA, rRNA, tRNA), which may be single-stranded or double-stranded, and may represent a sense strand or an antisense strand; a peptide nucleic acid; or any DNA-like or RNA-like material, whether natural or synthetic. This term may also include nucleic acids, such as oligonucleotides, containing known analogs of natural nucleotides, as well as nucleic acid-like structures with synthetic backbones.

The term "polypeptide" refers to an oligopeptide, peptide or protein sequence, or a fragment, part or subunit of any of these, as well as naturally occurring or synthetic molecules. The term "polypeptide" also includes amino acids linked to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain any type of modified amino acids. The term "polypeptide" also includes peptides and polypeptide fragments, motifs, etc. Protein or peptide "chains" refer to different subunits of a larger protein or protein complex.

"Homologs" refers to corresponding proteins (e.g., IL-21Rα), such as proteins from other species, which are substantially homologous to human proteins at the level of whole protein (i.e., mature protein), as long as such homologous peptides retain their respective known activities. There can be different levels of homology from 35% to 99%.

The term "amino acid modification" includes amino acid substitutions, insertions or deletions in a polypeptide sequence. "Amino acid substitution" or "substitution" refers to replacing an amino acid at a specific position in a parent polypeptide sequence with another amino acid. For example, the substitution R94K refers to a modified polypeptide in which the arginine at position 94 is replaced by lysine. For the previous example, 94K means that position 94 is replaced with lysine. Multiple substitutions are usually separated by slashes or commas. For example, R94K/L78V and [R94K,L78V] refer to a double variant comprising the substitutions R94K and L78V. "Amino acid insertion" or "insertion" refers to adding an amino acid at a specific position in a parent polypeptide sequence. For example, insertion -94 refers to an insertion at position 94. "Amino acid deletion" or "deletion" refers to the removal of an amino acid at a specific position in a parent polypeptide sequence. For example, R94- refers to the deletion of arginine at position 94.

The phrases "conservative modifications" and "conservative sequence modifications" refer to amino acid modifications that do not significantly affect or alter the binding characteristics of a polypeptide comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions, and deletions. Modifications can be introduced into proteins by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis, or by DNA synthesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Typically, conservatively substituted variants, homologs and analogs of the peptides have at least about 35%, at least about 45%, at least about 55%, at least about 65%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% to 99% amino acid sequence identity to the disclosed sequences. Rost B. Twilight zone of protein sequence alignments, Protein Eng, 1999; 12(2): 85-94. doi:10.1093/protein/12.2.85.

Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical to the known peptides, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum homology percentage, and without considering any conservative substitutions as part of the sequence identity. N-terminal, C-terminal or internal extensions, deletions or insertions of the peptide sequence should not be construed as affecting homology.

Gamma chain (γc) cytokines refer to any cytokine in which the cognate cytokine receptor complex includes a common cytokine receptor gamma chain (γc). γc cytokines include IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.

An orthogonal cytokine: receptor pair refers to a variant of a natural cytokine: receptor pair that interacts effectively with each other (i.e., such that they can be used to initiate a physiologically relevant signaling response in a cell), but whose ability to interact with their natural counterparts is severely impaired. An orthogonal cytokine can be a mutated or otherwise modified natural cytokine (a "mutein"), or a completely synthetic protein (sometimes referred to as a "synthekine") that acts as an agonist of an orthogonal cytokine receptor. An orthogonal cytokine (whether a mutein or a synthekine) exhibits no or only diminished agonist activity at a wild-type cytokine receptor.

Thus, an orthogonal cytokine: receptor pair can comprise a pair of genetically engineered proteins that are modified by amino acid changes to: (a) lack or reduce binding to a natural cytokine or cognate receptor; (b) specifically bind to a corresponding engineered (orthogonal) ligand or receptor. Upon binding to the orthogonal cytokine, the orthogonal receptor activates signal transduction transduced by the natural cellular element to provide a biological activity that mimics the natural response, but is specific to the engineered cell expressing the orthogonal receptor. Non-natural receptors or cellular elements (e.g., non-natural cytoplasmic domains in a receptor) may be involved to modify the signal response. The orthogonal receptor does not bind to any endogenous cytokine or binds only with reduced affinity, including the natural counterpart of the orthogonal cytokine, while the orthogonal cytokine does not bind to any endogenous receptor or binds only with reduced affinity, including the natural counterpart of the orthogonal receptor. In some aspects, the affinity of the orthogonal cytokine for the orthogonal receptor is comparable to the affinity of the native cytokine for the native receptor, e.g., having at least about 1% of the affinity of the native cytokine receptor for the native receptor, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 100%, and can be higher, e.g., 2×, 3×, 4×, 5×, 10× or more of the affinity of the native cytokine for the native receptor.

When used for the identification of orthogonal proteins (IL-21 or IL-21Rα), the term "candidate" infers a prototype or developmental state. If a candidate protein is found to have the desired degree of binding privilege and orthogonal function, the analysis procedure may disqualify it.

As used herein, the phrases "does not/does not bind" and "cannot bind" refer to no detectable binding, or negligible binding, i.e., having a much lower binding affinity than the natural ligand. "Impaired binding" refers to binding below the normal binding level between the corresponding wild-type components (e.g., cytokines and receptors).

The present invention includes an orthogonal cytokine system based on IL-21, which is a member of the γc cytokine family. IL-21 is structurally related to IL-2 and signals through a dimeric receptor composed of IL-21Rα and γc. The IL-2 receptor promotes a signaling response dominated by STAT5, while STAT3 dominates the IL-21 response. IL-21 promotes a form of T cell differentiation associated with good results of adoptive cell therapy (ACT), especially ACT using CAR T cells, and shows enhanced anti-tumor properties compared to IL-2 in various tumor model systems. Therefore, IL-21 may prove to be a better ACT adjuvant than IL-2. Thus, in one aspect, an IL-21 orthogonal system is provided, comprising: (1) ortho-IL-21 having impaired binding to native IL-21Rα; and (2) ortho-IL-21Rα capable of binding to ortho-IL-21 while exhibiting impaired binding to native IL-21 ( FIG. 1 , FIG. 2 , path A).

Orthogonal Interleukin 21 Receptor:

In one aspect, an orthogonal interleukin 21 receptor is provided. The orthogonal interleukin 21 receptor may include modifications to any of the chains that make up the entire protein. In some aspects, an ortho-IL-21Rα is provided. The ortho-IL-21Rα includes a modified amino acid sequence derived from wild-type human IL-21Rα (SEQ ID NO: 4, a mature form of human IL-21Rα lacking a signal peptide). The ortho-IL-21Rα binds to ortho-IL-21, but the binding to native IL-21 is impaired. SEQ ID NO: 3 represents human IL-21Rα.

In some aspects, the modified amino acid sequence comprises a substitution of one of the following: one or more amino acid residues of SEQ ID NO: 4 that contact IL-21, residues immediately adjacent to such contact residues, or residues elsewhere in IL-21Rα that have an effect on the conformation of the IL-21 binding surface. Immediately adjacent amino acids are amino acids within 1, 2, 3, 4, or 5 amino acids of the contact residue in the primary protein sequence, or amino acid residues that are similarly adjacent in the tertiary structure of the protein. In some aspects, ortho-IL-21Rα comprises amino acid substitutions, numbered relative to SEQ ID NO: 6, at positions: Y10, Q33, Q35, Y36, E38, L39, F67, H68, F69, M70, A71, D72, D73, I74, L94, A96, E97, P126, A127, Y129, M130, K134, S190, Y191, or a combination thereof.

One approach to creating an orthogonal version of IL-21 involves first mutating IL-21Rα to reduce its ability to bind IL-21. The binding surface of IL-21Rα directly contacts IL-21. Mutating any of these 20 residues could impair the receptor's ability to bind IL-21 normally. For example, the methionine residue at position 70 of IL-21Rα has been identified as a major contributor to the binding interaction. The large hydrophobic side chain of this residue fits into an IL-21 pocket composed primarily of hydrophilic residues (arginine 9, glutamine 12, arginine 76, lysine 73, and isoleucine 16), repositioning some of these residues to improve contacts with IL-21Rα (notably, arginine-9 and arginine-76 of IL-21 contact aspartate-72 and aspartate-73 of IL-21Rα, respectively). Changing methionine-70 to a different residue could adjust this repositioning, thereby weakening or losing some of these contacts. Changing aspartate-72 or -73 of IL-21Rα could also reduce the binding free energy of the interaction, requiring compensatory changes in IL-21 (e.g., at positions 9 and 76) to restore it.

Helix C of IL-21 exists in two interchangeable states (one disordered, the other alpha helical) in the free structure of the cytokine. The alpha helical form is stabilized in the complex of IL-21 with IL-21Rα, as is the first part of the CD loop. Helix C of IL-21 contains the aforementioned lysine-73 and arginine-76, which are close to methionine-70 of IL-21Rα. The CD loop of IL-21 includes lysine-77, proline-79, and serine-80, which together form a pocket for tyrosine-36 of IL-21Rα; lysine-77 also forms an ionic contact with glutamate 38 of IL-21Rα. Changing the CD loop amino acid sequence to a similar sequence found in IL-4 resulted in a tenfold enhancement in IL-21 potency measured using a cell-based assay. This observation suggests that significant binding energy is consumed in stabilizing helix C of IL-21. Furthermore, it suggests that changes in glutamate-38 and tyrosine-36 of IL-21Rα can significantly affect IL-21 binding in a manner that is potentially reversible through compensatory changes in IL-21 in helix C or the CD loop.

Discrete avian sequence motifs can be used to design orthogonal IL-21 systems. Of the 44 available IL-21 avian sequences, 39 had a significantly (6 residues) shortened CD loop compared to human and mouse. Together with the absence of tyrosine 36 in all 74 available avian IL-21Rα sequences, this suggests that helix C of avian IL-21 may bind to its receptor in a manner that is distinct from human IL-21. This also provides additional basis for modifying the above binding residues to construct orthogonal IL-21 systems.

Based on structural considerations, candidate amino acids can be identified that could be modified to alter IL-21Rα in such a way as to impair its binding to native IL-21. However, these changes must have features that are incompatible with compensatory changes in IL-21 (i.e., changes that restore IL-21 binding to ortho-IL-21Rα). Changes in IL-21Rα that result in large changes in its conformation are undesirable because the number of compensatory changes required to restore binding may present too great an engineering challenge or be incompatible with cytokine function (e.g., because γc binding is lost, or because the cytokine or cytokine receptor becomes unstable, difficult to express, immunogenic, or pharmacologically problematic).

There are at least two assays that can be used to screen for loss of binding of ortho-IL-21Rα to IL-21. One is a direct binding assay, which can be performed using purified protein and sensitive biophysical analytical procedures (e.g., surface plasmon resonance (SPR) or biolayer interferometry (BLI). The other assay is a functional assay involving an appropriate cell line. Ba/F3 cells have been successfully used for this purpose because they have a number of useful properties: (i) they do not express endogenous IL-21Rα; (ii) they express mouse γc, which effectively replaces human γc in signaling with human IL-21Rα and human IL-21; (iii) they respond to IL-21R signals by phosphorylating STAT3 and proliferating; and (iv) they allow the use of a STAT3 reporter transgene (e.g., one expressing luciferase) as a simple and attractive means of monitoring IL-21 signaling. Jurkat or Molt-3 cells are alternative choices, both of which exhibit little or no expression of endogenous IL-21Rα, but express γc and have IL-21R signaling capacity. HeLa cells transfected to express γc can also be used.

The method of isolating candidate ortho-IL-21Rα involves expressing the variant alone in Ba/F3 cells (or one of the alternatives just mentioned) by transfection. Flow cytometry is used to confirm the presence of the candidate ortho-IL-21Rα molecule on the cell surface and the presence of an epitope that can be recognized by the antibody. In one aspect, the candidate ortho-IL-21Rα comprises an amino acid substitution at the following positions: Y10, Q33, Q35, Y36, E38, L39, F67, H68, F69, M70, A71, D72, D73, I74, L94, A96, E97, P126, A127, Y129, M130, K134, S190, Y191, or a combination thereof (wherein numbers refer to residue positions in the mature IL-21Ra extracellular domain (SEQ ID NO: 6) and letters refer to amino acid identity using the single-letter code, with the first letter being the wild-type residue and the second letter (if present) being the substituted residue). In one aspect, the amino acid substitutions comprise, consist essentially of, or consist of: M70G, M70A, M70I, M70L, Y129F, Y129A, Y129S, Y129H, M130F, M130S, Y36H, Y36F, FMAD(69-73) to LLADI, FYM(128-130) to FHF, D72K, D 73K, D72E, D73E, D73I, E38K, E38S, E38D, F67L, F67Y, F69L, S190Y, Y191F, Q35L, H68Q, I74V, L94I, L94V, E97D, A127T, Q33N, Q33H, K134R, M130I, M130L, S190T, or a combination thereof. In one aspect, the amino acid substitutions comprise, consist essentially of, or consist of: D72E/Y129F/D73E; M70I/D73E/Q33H; D72E/L94V/Y191F; D72E/E38D/M130L; M70G/Y129F; F69L/M70L/D73I; D72K/D73K; E38K; or M70G, or a combination thereof. In one aspect, the amino acid substitutions comprise, consist essentially of, or consist of: [H68Q, E38D, Q33H], [Y129F, H68Q, Y191F], [Y36F, H68Q, D73E], [Y129F, F67Y, M130L], [L94V, Q33H, M130L], [Y36F, F67Y, E38D], [D72E, E3 In one aspect, the amino acid substitution comprises, consists essentially of, or consists of: M70G/Y129F or M70G.

Ba/F3 cells expressing candidate ortho-IL-21Rα can be incubated with natural human IL-21 before analyzing the signal response. In some experiments, the time course can be used to improve the sensitivity and resolution of the assay. Other experiments involve comparisons of dose-response curves. IL-21 reactivity is detected by monitoring the proliferation of Ba/F3 cells, tyrosine phosphorylation of STAT3, or the expression of reporter genes (e.g., luciferase or secreted alkaline phosphatase) from STAT3-dependent reporter genes present in the cells by flow cytometry or immunoblotting. Although natural IL-21Rα allows a strong response to IL-21 using any of these analytical techniques, the desired receptor orthogonal (ortho) form will have a reduced response or no response. Ortho-IL-21Rα molecules that prove this unresponsive property are candidate molecules for orthogonal restricted IL-21 cytokine receptor systems.

Orthogonal Interleukin 21 Cytokine:

Another aspect provides an ortho-IL-21 with a modified amino acid sequence, derived from wild-type human IL-21 (SEQ ID NO: 2, a mature form of human IL-21, and lacking a signal peptide), which binds to ortho-IL-21Rα, but has impaired binding to native IL-21Rα. Human IL-21 is represented by SEQ ID NO: 1.

In some aspects, ortho-IL-21 binds to ortho-IL-21Rα, but has impaired binding to native IL-21Rα. In further aspects, the modified amino acid sequence comprises a substitution of one of the following: an amino acid residue of SEQ ID NO: 2 that contacts IL-21Rα, a residue immediately adjacent to such contact residue, or a residue elsewhere in IL-21R that affects the conformation of the IL-21α binding surface.

The method can also be used to identify orthogonal forms of IL-21. Ten residues of IL-21 are involved in polar interactions with IL-21Rα: arginine-5, arginine-9, arginine-11, glutamine-12, aspartate-15, serine-70, lysine-73, arginine-76, lysine-77, and serine-80. Of these, arginine-5, arginine-9, arginine-11, glutamine-12, lysine-73, arginine-76, and lysine-77 also form significant van der Waals contacts with IL-21Rα. Isoleucine-8, isoleucine-16, glutamine-19, tyrosine-23, isoleucine-66, valine-69, and proline-79 make additional van der Waals contacts. Substitutions can be made at any of these residues to overcome the changes present in the candidate ortho-IL-21Rα molecule and restore binding.

The crystal structure of IL-21 bound to IL-21Rα can be used to identify the IL-21 residues that are closest to the engineered changes in IL-21Rα. For example, if changing methionine 70 of IL-21Rα is sufficient to eliminate IL-21 binding, then compensatory changes in any of the following methionine 70 contacting residues in IL-21 may restore binding: arginine 9, glutamine-12, isoleucine-16, lysine-73, and arginine-76. However, in addition to methionine 70, these five IL-21 residues also contact the following IL-21Rα residues: tyrosine 10, leucine 39, glutamate 38, phenylalanine 67, alanine 71, aspartate 72, aspartate-73, tyrosine-129, methionine-130, and tyrosine-191. In turn, the 10 IL-21Rα residues just mentioned mediate additional contacts with IL-21 residues: arginine 5, isoleucine 8, glutamine 19, serine 70, and lysine 77. Thus, considering only the direct contacts present in the native IL-21:IL-21Rα crystal structure, a single substitution at methionine-70 in IL-21Rα could affect the interactions mediated by 10 IL-21 residues (arginine-5, isoleucine 8, arginine 9, glutamine 12, isoleucine 16, glutamine 19, serine 70, lysine 73, arginine 76, and lysine 77). This suggests that compensation for the substitution (i.e., restoration of IL-21 binding) could be achieved by altering one or more of these 10 residues. It is also possible that altering residues that do not make direct contacts with IL-21Rα results in conformational adjustments that act at a distance to allow restoration of binding to the methionine 70-substituted variant of IL-21Rα.

Phage display or yeast display technology can allow screening of large mutation space. This is usually achieved by creating a highly diverse variant library, in which a small amount of residues in the protein are changed in a random (or semi-random) combination mode. The library is then screened for desired properties. For the present invention, screening can include identifying members that can be combined with candidate ortho-IL-21Rα in the IL-21 variant library. If, as in the embodiment just described, the replacement at methionine-70 is enough to eliminate the combination of IL-21Rα and natural IL-21, then an IL-21 library can be constructed, which has random combination mutations in 10 potentially affected contact residues (arginine-5, isoleucine-8, arginine-9, glutamine-12, isoleucine-16, glutamine-19, serine-70, lysine-73, arginine peptide-76 and lysine-77). The theoretical complexity of such a library may exceed 10 ¹³ . Generating a library composed of 10 ⁸ to 10 ⁹ or more variants is usually challenging and impractical. The screening of such a library may involve a selection process in which phages or yeasts that display IL-21 variant forms attached to their surfaces are separated based on their ability to attach to a matrix coated with a candidate ortho-IL-21Rα (rather than to a matrix coated with wild-type IL-21Rα). Such selection cycles are repeated to enrich for the desired properties in IL-21. Analytical screening procedures (including SPR or BLI) are used to describe the characteristics of the selected products in detail and determine the products with the best properties.

In one aspect, ortho-IL-21 comprises a modified amino acid sequence comprising a substitution of one or more amino acid residues of SEQ ID NO: 2 that contact IL-21Rα, residues immediately adjacent to such contact residues, or residues elsewhere in IL-21 that affect the conformation of the IL-21α binding surface. In one aspect, ortho-IL-21 comprises amino acid substitutions numbered relative to SEQ ID NO: 2 at positions: R5, H6, I8, R9, M10, Q12, L13, K73, K75, R76, P78, G84, P104, or a combination thereof. In one aspect, the amino acid substitutions comprise, consist essentially of, or consist of R5Q, R5D, R5E, R5K, R5N, H6L, I8E, R9E, R9D, R9K, R9N, R9Q, M10L, Q12V, L13D, K73V, K73D, K73I, K75D, R76E, R76N, R76A, R5Q/R76E, R5Q/R76A, P78L, G84E, P104A, or a combination thereof. In one aspect, the amino acid substitutions comprise, consist essentially of, or consist of H6L/R9K/M10L/P78L.

Other methods for identifying orthogonal IL-21 receptor-cytokine pairs:

Another approach to identifying ortho-IL-21 molecules involves iterative cycles of mutations, again focusing on a small number of residues selected from those that make direct intermolecular contacts in the IL-21:IL-21Rα structure. This approach may also include residues close to contact points and/or residues in potentially relevant structural features. A broad version of this approach could involve residues anywhere near the entire region that makes contact with IL-21Rα and other semi-randomly selected residues in proximal structural features. A more focused version involves residues, such as the ten that are potentially relevant to methionine 70 substitutions, that are likely to be directly affected by the specific substitution(s) present in the candidate ortho-IL-21Rα. Another broad alternative approach is to make substitutions with minimal (if any) consideration of the structure of IL-21, but with a partial emphasis on substitutions that occur in IL-21 orthologs from other species. This approach involves single substitutions of single residues throughout the IL-21 primary sequence.

The iterative process starts with a large number of (e.g., 10-100) candidate ortho-IL-21 forms. In some versions of this method, double or triple mutants are included in the initial collection, but in other versions, only the single point mutation of IL-21 is assessed. The activity of these candidate ortho-IL-21 molecules is tested using the above-mentioned cell assay (e.g., using Ba/F3 cells carrying STAT3-dependent luciferase reporter genes). At least two cells are used in the assay: cells expressing candidate ortho-IL-21R α and cells expressing wild-type IL-21R α as counter-screening.

The probability of success of the substitution approach corresponds to the number of candidate ortho-IL-21Rα molecules examined. Expanding this number can reduce the probability of inadvertently selecting a candidate ortho-IL-21Rα that is not easily restored (even partially) to IL-21 binding by a small number (e.g., less than three) of substitutions. Expanding this number also increases the probability of being able to isolate parallel, mutually orthogonal systems that do not exhibit crosstalk with each other or with wild-type IL-21 or IL-21Rα.

The data from the first round of screening can be deconvoluted and analyzed with a focus on identifying substitutions in IL-21 that promote improved binding to candidate ortho-IL-21Rα molecules in isolation and reduce binding to native IL-21Rα. A second round of screening can be performed in which the positive scoring substitutions from the first round are combined in new candidate ortho-IL-21 molecules. These candidate ortho-IL-21 molecules (and, if deemed necessary, additional variants with conservative or non-conservative substitutions at positive scoring positions) are tested again to improve binding to candidate ortho-IL-21Rα molecules and reduce binding to native IL-21Rα.

Further rounds of screening, including further substitution combinations, can be performed until at least one candidate ortho-IL-21 has been isolated with the desired properties (lack of activity against native IL-21Rα, near normal activity against at least one candidate ortho-IL-21Rα).

In some cases, alternative screening methods can be facilitated by using candidate ortho-IL-21Rα molecules that retain reduced but not completely absent binding to wild-type IL-21. Such candidate ortho-IL-21Rα molecules that reduce binding may prove to be more permissive than non-binding candidate ortho-IL-21Rα molecules (i.e., candidate ortho-IL-21R-α molecules that lack any binding to wild-type IL-21) to restore some IL-21 binding activity by a small number (e.g., less than three) of discrete substitutions in IL-21. Once the candidate ortho-IL-21 molecules are isolated by the above screening procedures, additional screening steps can be performed, which involve new candidate ortho-IL-21Rα molecules in which additional substitutions are compounded with already existing substitutions. In some aspects, these additional mutations can completely eliminate binding to wild-type IL-21 while retaining the ability to bind to ortho-IL-21. Multiple rounds of such receptor mutagenesis and subsequent cytokine mutagenesis can be performed.

The binding properties of the products of the screening method can be analyzed using purified protein and BLI or SPR. The products that are most similar in binding kinetics to wild-type IL-21 and IL-21Rα can be selected as candidate orthogonal IL-21 systems.

In one aspect, candidate ortho-IL-21 molecules can be engineered according to the methods described in one or more of the following: U.S. Pat. Nos. 8,005,620, 8,635,029, and 8,412,461; Govindarajan S, Mannervik B, Silverman JA, et al., Mapping of amino acid substitutions conferring herbicide resistance in wheat glutathione transferase. ACS Synth Biol. 2015; 4(3): 221-227, doi: 10.1021/sb500242x; Musdal Y, Govindarajan S, Mannavik B, Exploring sequence-function space of a poplar glutathione transferase using designed information-rich gene variants, Protein Eng Des Sel., 2017;30(8):543-549.doi:10.1093/protein/gzx045; and Liao J, Warmuth MK, Govindarajan S, et al., Engineering proteinase K using machine learning and synthetic genes, BMC Biotechnol, 2007;7:16, published on March 26, 2007, doi:10.1186/1472-6750-7-16, all of which are incorporated herein by reference in their entirety.

In one aspect, the candidate ortho-IL-21 molecule is expressed in the form of a fusion protein, which is a fusion protein between a modified amino acid sequence derived from SEQ ID NO: 2 as described above and a second amino acid sequence, and facilitates purification, increases the stability and half-life of the ortho-IL-21 molecule in vivo, or improves the drug properties, which are critical for the successful administration of the ortho-IL-21 molecule to patients. Suitable second amino acid sequences are known in the art, including but not limited to serum albumin, the Fc fragment of IgG, a single-chain Fc antibody fragment, ABD035, etc. The Fc fragment can be modified, for example, with electrostatic manipulation mutations, to prevent or at least significantly limit the formation of homodimers.

Orthogonal IL-21 Receptor Cytokine System:

In another aspect, a system for activating IL-21 signaling in a cell is provided. The system includes a candidate ortho-IL-21Rα with impaired binding to native IL-21, the candidate ortho-IL-21Rα comprising a modified amino acid sequence derived from SEQ ID NO: 4, the modified amino acid sequence comprising a substitution of one of the following: one or more amino acid residues of SEQ ID NO: 4 that contact IL-21; residues that are immediately adjacent to such contact residues; or residues elsewhere in IL-21Rα that affect the conformation of the IL-21 binding surface; and an ortho-IL-21 with impaired binding to native IL-21Rα, the ortho-IL-21 comprising a modified amino acid sequence derived from SEQ ID NO: 2, the modified amino acid sequence comprising a substitution of one of the following: one or more amino acid residues of SEQ ID NO: 2 that contact IL-21Rα; residues that are immediately adjacent to such contact residues; or residues elsewhere in IL-21 that affect the conformation of the IL-21Rα binding surface, wherein the ortho-IL-21Rα binds to the ortho-IL-21.

In some aspects, the cell is a T cell. In a further aspect, the cell is a CAR T cell. The orthogonal cytokine and orthogonal receptor can be any candidate ortho-IL-21 and candidate ortho-IL-21Rα molecules described herein. For example, in one aspect, ortho-IL-21Rα includes amino acid substitutions numbered relative to SEQ ID NO: 6, which comprises, consists essentially of, or consists of: M70G/Y129F or M70G; and ortho-IL-21 includes amino acid substitutions numbered relative to SEQ ID NO: 6, which comprises, consists essentially of, or consists of: M70G/Y129F or M70G; and NO:2 amino acid substitutions, which comprise, consist essentially of, or consist of one of: (1) H6L/R9K/M10L/P78L; (2) H6L/R9K/M10L/P78L/K73V; (3) H6L/R9K/M10L/P78L/K73I; (4) H6L/R9K/M10L/P78L/K73V/G84E; (5) H6L/R9K/M10L/P78L/K73I/G84E; (6) H6L/R9K/M10L/P78L/K73V/P104A; and (7) H6L/R9K/M10L/P78L/K73I/P104A.

Orthogonal cytokine and receptor expression vectors:

On the other hand, multiple expression vectors are provided comprising nucleic acids encoding any candidate orthogonal IL-21 protein (e.g., receptor or cytokine) described herein. Orthogonal proteins, such as ortho-IL-21 or ortho-IL-21Rα, can be produced by recombinant methods. Ortho-IL-21Rα can be introduced into cells to be engineered on expression vectors. The DNA encoding orthogonal proteins can be obtained from various sources designed in the engineering process.

Amino acid sequence variants can be prepared by introducing the nucleic acid coding sequence of coded protein by appropriate nucleotide changes. The nucleic acid codons of coded amino acids are well known to those skilled in the art. Selected specific codons can be selected to optimize the expression in the host cell used. Amino acid variants can represent the insertion, substitution and specific deletion of residues as described herein. Any combination of insertion, substitution and specific deletion can be carried out to reach final construct, and condition is that final construct has the required biological activity defined herein. In some aspects, nucleic acid encoding ortho-IL-21R α as described herein.

A nucleic acid is "operably linked" when it is established into a functional relationship with another nucleic acid sequence. For example, if the DNA of the signal sequence is expressed as a precursor protein that participates in the secretion of the polypeptide, the DNA of the signal sequence is operably linked to the DNA of the polypeptide; if the promoter or enhancer affects the transcription of the sequence, the promoter or enhancer is operably linked to the coding sequence; or if the ribosome binding site is positioned to facilitate translation, the ribosome binding site is operably linked to the coding sequence. Generally, "operably linked" means that the linked DNA sequences are continuous, and in the case of a secretory leader sequence, continuous, and in the reading phase. However, some sequences, such as enhancers, do not have to be continuous to be effective.

Nucleic acid encoding ortho-IL-21 or ortho-IL-21Rα can be inserted into a replicable vector to express. Many such vectors are available. Vector components generally include but are not limited to one or more of the following: replication origin, one or more marker genes, enhancer elements, promoters and transcription termination sequences. Vectors may include viral vectors, plasmid vectors, integration vectors, transposons, etc. For example, suitable polynucleotide vector systems based on transposons/transposases are described in U.S. Patent No. 10,041,077, which is incorporated herein by reference in its entirety.

Ortho-IL-21 or ortho-IL-21Rα can be recombinantly produced without modification, or as a fusion polypeptide with a heterologous polypeptide, such as a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of a mature protein or polypeptide. Typically, the signal sequence can be a component of the vector, or it can be a part of the coding sequence inserted into the vector. The selected heterologous signal sequence can be a signal sequence that is recognized and processed (i.e., cut by a signal peptidase) by the host cell. In mammalian cell expression, a natural signal sequence can be used, or other mammalian signal sequences may be suitable, such as signal sequences of secretory polypeptides from the same or related species, and viral secretion leaders, such as herpes simplex gD signals.

Expression vectors usually contain a selection gene, also called a selective marker. The selection gene encodes a protein required for the survival or growth of transformed host cells grown in a selective culture medium. Host cells that are not transformed with a vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that: (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) complement auxotrophic deficiencies; or (c) provide key nutrients that are not available in complex culture media.

The expression vector may contain a promoter that can be recognized by the host organism and may be operably linked to an orthogonal protein coding sequence. The promoter may be an untranslated sequence (usually within about 100-1000 bp) located upstream (5') of the start codon of the structural gene that controls the transcription and translation of a specific nucleic acid sequence that is operably linked to it. Such promoters are generally divided into two categories: inducible and constitutive. An inducible promoter is a promoter that initiates an increase in the level of DNA transcription under its control in response to some changes in culture conditions, for example, the presence or absence of nutrients or changes in temperature.

Transcription of the vector in mammalian host cells can be controlled by promoters obtained from viral genomes, such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus (e.g., murine stem cell virus), hepatitis B virus, and Simian Virus 40 (SV40), from heterologous mammalian promoters, such as the actin promoter, phosphoglycerate kinase (PGK) or immunoglobulin promoters, from heat shock promoters, provided that these promoters are compatible with the host cell systems. The early and late promoters of SV40 can be conveniently obtained as an SV40 restriction fragment, which also contains the SV40 viral origin of replication.

The expression vector may also include an enhancer sequence. An enhancer is a cis-acting element of DNA, typically about 10-300 bp, that acts on a promoter to increase its transcription. Enhancers are relatively directional and position-independent, having been found within introns and within the coding sequence itself to 5' and 3' of the transcription unit. Many enhancer sequences are known from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, typically, enhancers from eukaryotic cell viruses are used. Examples may include the late SV40 enhancer of the replication origin, the cytomegalovirus early promoter enhancer, the late polyoma enhancer of the replication origin, and the adenovirus enhancer. The enhancer may be spliced into the expression vector at the 5' end or 3' end of the coding sequence, but is preferably located at the 5' end of the promoter.

The expression vector used in eukaryotic host cells will also contain sequences required for termination of transcription and stabilization of the mRNA. Such sequences are usually available from the 5' and 3' untranslated regions of eukaryotic or viral DNA or cDNA.

The expression vector may also include an inducible regulatory element for controlling the expression of transgenics (e.g., encoding ortho-IL-21 or ortho-IL-21Rα) with small molecules or other stimulants. Examples of regulatory elements include, but are not limited to, promoters containing tetracycline operators that make them sensitive to regulation of tetracycline or its derivatives (e.g., doxycycline). Promoters may also be induced by CRISPRa (clustered regularly interspaced short palindromic repeats activated) using a fusion of transcriptional effectors and catalytically dead Cas9. Such a promoter may in turn be downstream of other control systems, such as those involving dimers (based on antibodies and/or chemical properties) or components based on Notch receptors.

Engineered Cell:

One aspect of the present invention provides a variety of cells that have been engineered to express ortho-IL-21Rα. The cells can be genetically engineered to include any suitable expression vector described herein. In some aspects, the expression vector includes a coding sequence encoding an orthogonal receptor, which is operably linked to a promoter active in the desired cell. Various vectors can be used for this purpose, such as transposons, viral vectors, plasmid vectors, and minicircle vectors, which can be integrated into the target cell genome or can be maintained episomally. In one aspect, the expression vector is a synthetic transposon that can be integrated into the genome by a transposase. Examples of transposon/transposase systems include Sleeping Beauty, PiggyBac, ATUM Bio's and its derivatives.

The engineered cell can be a host cell for producing a recombinant protein in vitro. Suitable host cells for recombinant expression of orthogonal proteins include prokaryotes, yeast, and higher eukaryotic cells, such as various mammalian host cell lines.

In some aspects, the engineered cells are further modified beyond the expression of pro-IL-21Rα. Modifications suitable for engineered cells are known in the art, including the expression of CAR, T cell receptor (TCR), or other receptors or receptor derivatives that recognize specific antigens on antigen presenting cells.

In some aspects, engineered cells are cells for therapeutic use. Examples of therapeutic engineered cells may include stem cells, such as hematopoietic stem cells, natural killer (NK) cells, or T cells. In some aspects, engineered cells are T cells. The term "T cell" refers to a mammalian immune effector cell, which may be characterized by the expression of CD3 and/or T cell antigen receptors, and these cells may be engineered to express pro-IL-21Rα. In some aspects, T cells are selected from the initial Activated or activated CD8+ T cells; cytotoxic CD8+ T cells; naive Activated or activated CD4+ T cells; helper T cells, such as TH1, TH2, TH9, TH11, TH22 and TFH; regulatory T cells, such as TR1, natural TReg and inducible TReg; and memory T cells, such as central memory T cells, effector memory T cells and NKT cells, and γζ T cells.

Ortho-IL-21 can be used as an adjunct to ACT. T cells can be engineered to express ortho-IL-21Rα by gene (cDNA, minigene or other nucleic acid construct) transfection, transduction or transposition. Patients undergoing ACT can be treated (and/or pretreated) with ortho-IL-21 and repeatedly dosed as needed to enhance and maintain the desired T cell presence and response.

Therapeutic cells can also be engineered to express ortho-IL-21. This can be achieved using any method suitable for ectopic expression of ortho-IL-21Rα. The ortho-IL-21 can be expressed in the same or different cells as the cells expressing ortho-IL-21Rα, allowing for autocrine or paracrine effects, respectively. An example of a paracrine setting could be CD4+ T cells expressing ortho-IL-21 and CD8+ T cells expressing matching ortho-IL-21Rα.

In certain aspects, ortho-IL-21 can be expressed in a membrane-tethered form. This has been previously achieved with native IL-21 by fusing the cytokine to the amino terminus of an IgG4 CH ₂ -CH ₃ portion, which itself is fused to a CD4 transmembrane domain. Related approaches have been used to attach other cytokines to the cell membrane. This membrane tethering limits the diffusion of the cytokine and restricts its action to the vicinity of cells expressing the membrane-bound cytokine. In vivo, this approach can be used to ensure that cells expressing ortho-IL-21Rα encounter ortho-IL-21 only when they are close to specific types of cells and/or locations in the body. In vitro, this approach can facilitate certain types of selective differentiation protocols (e.g., NK cell differentiation from stem cells in the presence of K562 (or other) feeder cells expressing membrane-bound IL-21 and CD137L).

Engineered cells can be provided in pharmaceutical compositions suitable for therapeutic use, such as for human treatment. Therapeutic preparations comprising such cells can be frozen or prepared for administration in the form of an aqueous solution with a physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Cells can be formulated, dosed and administered in accordance with good medical practice.

Treatment:

A cell, such as a T cell, engineered to express one of the ortho-IL-21Rα molecules described herein can be used to treat a wide range of conditions. The engineered properties in this therapy can have the benefits of T cell differentiation, resistance to exhaustion, long-term persistence, memory response, and in-built safety properties, thereby stopping the response when pathogenic.

Provided are a variety of methods for enhancing cellular responses, the methods being carried out by introducing the ortho-IL-21Rα of the present invention and engineering cells from a recipient or donor, and by contacting the engineered cells with ortho-IL-21 to stimulate ortho-IL-21Rα. The method of the subject matter may include the step of obtaining targeted cells, such as T cells, hematopoietic stem cells, etc., which may be isolated from a biological sample or may be derived in vitro from a progenitor cell source. The cells may be transduced or transfected with an expression vector comprising a sequence encoding ortho-IL-21Rα, and this step may be carried out in any suitable culture medium.

In some aspects, engineered T cells can be contacted with ortho-IL-21 in vivo, i.e., engineered T cells are transferred to a recipient therein, and an effective dose of ortho-IL-21 is injected into the recipient and allowed to contact the engineered T cells in its natural environment, such as in a lymph node, etc. In other aspects, contact is performed in vitro. In such in vitro aspects, contact can be accomplished using soluble ortho-IL-21, which may or may not include fusion with another protein portion, such as an immunoglobulin Fc domain. In further such in vitro aspects, contact can be achieved by encountering other cells expressing secreted or membrane-bound ortho-IL-21.

On the other hand, a method for treating an object in need is provided, including introducing engineered cells expressing ortho-IL-21Rα, and activating cells by contacting them with an effective amount of ortho-IL-21. In some aspects, the cell is a T cell, and in a further aspect, the cell is a CAR-T cell. In another aspect, the cell is a T cell expressing a natural or modified TCR. In another aspect, the cell is a NK cell. In another aspect, the cell is a macrophage, other myeloid cells or a leukocyte.

In some aspects, ortho-IL-21 is delivered as a fusion protein with a heterologous polypeptide. Suitable heterologous polypeptides are known in the art and include serum albumin, the Fc fragment of IgG, a single-chain Fc antibody fragment, ABD035, etc. The Fc fragment can be modified, for example, with electrostatic manipulation or other mutations, to prevent or at least significantly limit the formation of homodimers.

Another aspect provides a method of treating a subject in need thereof, comprising introducing an engineered cell expressing ortho-IL-21Rα, and introducing a second engineered cell expressing ortho-IL-21. In some aspects, the cell is a T cell, and in further aspects, the cell is a CAR-T cell.

A "subject" can be any mammal, and may also be referred to as a "patient." Examples of mammalian subjects include research animals (e.g., mice or rats), domesticated farm animals (e.g., cows, horses, pigs), pets (e.g., dogs, cats), and humans. In some aspects, the subject is a human.

In some aspects, the subject is diagnosed with cancer. "Cancer" and "malignant tumor" are synonyms, referring to a variety of diseases characterized by uncontrolled abnormal proliferation of cells, local spread of affected cells or spread to other parts of the body (i.e., metastasis) through the bloodstream and lymphatic system, as well as any of many characteristic structural and molecular features. "Cancer cell" refers to a cell that undergoes early, intermediate or late multi-stage tumor progression. The characteristics of the early, intermediate and late stages of tumor progression have been described using a microscope. Cancer cells in each of the three stages of tumor progression usually have abnormal karyotypes, including translocation, inversion, deletion, isochromosomes, monosomy and extra chromosomes. Cancer cells include "proliferative cells", i.e., cells in the early stages of malignant progression, "abnormal proliferative cells" (i.e., cells in the intermediate stage of tumor progression) and "tumor cells" (i.e., cells in the late stage of tumor progression). Examples of cancer include sarcoma, breast cancer, lung cancer, brain cancer, bone cancer, liver cancer, kidney cancer, colon cancer and prostate cancer. In some aspects, engineered cells are used to treat cancers selected from colon cancer, brain cancer, breast cancer, fibrosarcoma and squamous cell carcinoma. In certain aspects, the cancer is selected from melanoma, breast cancer, colon cancer, lung cancer, and ovarian cancer. In certain aspects, the cancer being treated is a metastatic cancer.

In the case of cancer treatment, the treatment method may further include the step of ablating the cancer. Ablation of the cancer may be accomplished using a method selected from cryoablation, thermal ablation, radiotherapy, chemotherapy, radiofrequency ablation, electroporation, alcohol ablation, high intensity focused ultrasound, photodynamic therapy, administration of monoclonal antibodies, and administration of immunotoxins.

In some aspects, the subject being treated has been diagnosed with an infection. As used herein, the term "infection" refers to the infection of one or more cells of a subject by an infectious agent. Infectious agents include, but are not limited to, bacteria, viruses, protozoa, and fungi. Intracellular pathogens are of particular interest. An infectious disease is a disease caused by an infectious agent. Some infectious agents do not cause recognizable symptoms or disease under certain conditions, but may cause symptoms or disease under varying conditions. The subject methods can be used to treat chronic pathogen infections, including, but not limited to, viral infections, such as retroviruses, lentiviruses, hepatotropic viruses, herpes viruses, poxviruses, and human papillomaviruses; intracellular bacterial infections, such as Mycobacterium, Chlamydia, Ehrlichia, Rickettsia, Brucella, Legionella, Francisella, Listeria, Coxiella, Neisseria, Salmonella, Yersinia sp, and Helicobacter pylori; and intracellular protozoan pathogens, such as Plasmodium sp, Trypanosoma sp, Giardia sp, Toxoplasma sp, and Leishmania sp.

In some aspects, the subject being treated has been diagnosed with an autoimmune disease. Autoimmune diseases are characterized by abnormal targeting of self-proteins, -polypeptides, -peptides or other self-molecules by T and B lymphocytes, resulting in damage and/or dysfunction of organs, tissues or cell types in the body. Autoimmune diseases include diseases such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, autoimmune hepatitis, insulin-dependent diabetes, and degenerative diseases such as osteoarthritis, Alzheimer's disease and macular degeneration.

For treatment, one or more cells of the subject can be contacted with ortho-IL-21. When cells are contacted with ortho-IL-21 in vitro, ortho-IL-21 can be added to engineered cells at a dose and time period sufficient to activate the signal transduction from ortho-IL-21Rα, which can utilize natural cell mechanisms, such as auxiliary proteins, co-receptors, etc. Any suitable culture medium can be used. The cells so activated can be used for any desired purpose, including experimental purposes related to antigen specific determination, cytokine spectrum determination, etc., and for in vivo delivery.

When contact is made in vivo, an effective dose of engineered cells expressing ortho-IL-21Rα is infused into a receptor together with ortho-IL-21 or before ortho-IL-21 is administered. Dosage and frequency can vary according to dosage form, mode of administration, etc. For local administration, such as intranasal, inhalation, etc., or for systemic administration, such as im, ip, iv, etc., dosage can also vary. Typically, administer at least about 10 ⁴ engineered cells/kg, at least about 10 ⁵ engineered cells/kg, at least about 10 ⁶ engineered cells/kg, at least about 10 ⁷ engineered cells/kg, or more.

Example

Example 1: Binding assay for identifying candidate ortho-IL-21Rα molecules with impaired binding to native IL-21.

Direct interaction assays (quantifying the ability of IL-21Rα to bind to native or mutant forms of IL-21) provide an alternative to cell-based assays for identifying variants with impaired IL-21Rα binding activity (i.e., candidate orthogonal variants). The fact that the native interaction (IL-21Rα:IL-21) is avid (KD ~ 70pM) enhances the feasibility of using this assay. Among the many possible binding assay formats, a binding assay involving a luciferase fusion protein is attractive because it is simple and has the potential to be adapted to medium or high throughput formats.

One version of the IL-21Rα:IL-21 binding assay involves attaching the receptor ectodomain to a surface, soaking the coated surface in a solution of the IL-21-luciferase fusion protein, and then quantifying bound IL-21 based on luminescence when the relevant luciferase substrate is added. Alternatively, IL-21 can be immobilized and the IL-21Rα luciferase fusion protein can be used in solution.

In either format of the binding assay, the desired orientation of the immobilized receptor or cytokine can be achieved by using an affinity tag such as Twin-Strep-Tag II, which is a high affinity peptide ligand for the Streptactin protein. The IL-21Rα extracellular domain carrying a carboxyl-terminal Twin-Strep-Tag II peptide can be efficiently and selectively immobilized on the surface of the wells of a 96-well plate that has been pre-coated with the Streptactin protein. In this way, the immobilized IL-21Rα should be oriented away from the plate surface with its cytokine binding domain. Similarly, if IL-21 is also tagged with an amino- or carboxyl-terminal Twin-Strep-Tag II, IL-21 can be immobilized in a relevant manner.

Use the human IL-21Rα extracellular domain (through carboxyl terminal Twin-Strep-Tag II) and fusion protein (SEQ ID NO:5) ("IL-21-TLuc16" of Fig. 3) of dish fixing to set up mensuration, wherein 16KD Turbo-luciferase (Turbo-luciferase) polypeptide (ThermoFisher) is connected (through the peptide containing glycine-serine) to the carboxyl terminal of human IL-21.Use TurboLuc Luciferase One-Step Glow Assay Kit (ThermoFisher) and standard luminescence method to detect the IL-21 of binding.This mensuration can also be set up with the luciferase fused to the amino terminus of IL-21 or with another form of luciferase (for example: NanoLuc; ThermoFisher).

The ability of 20 candidate ortho-IL-21Rα molecules to bind IL-21-TLuc16 was tested. The wild-type human IL-21Rα extracellular domain (mature form lacking the signal peptide) (V0 (SEQ ID NO: 6)) and the 20 candidate ortho-IL-21Rα molecules shown in Table 1 (SEQ ID NO: 7-25 & 63) (substitutions shown in the title and underlined within the sequence) were expressed as secreted proteins in HEK293 cells.

Table 1:

Clarified supernatants from transiently transfected cells were tested for the presence of IL-21Rα using an enzyme-linked immunosorbent assay (ELISA), which included a streptactin-coated plate, a dilution of the supernatant, and detection using a combination of a mouse monoclonal antibody specific for human IL-21Rα, a horseradish peroxidase-coupled rat antibody specific for mouse IgG, and a chromogenic substrate for peroxidase. This ELISA determined the dilution required to ensure saturation of the streptactin-coated wells for each candidate ortho-IL-21Rα molecule. The candidate ortho-IL-21Rα saturated wells were incubated with IL-21-TLuc16 solution at 4°C (or room temperature in some experiments) for 1 hour (or longer in some experiments). The wells were washed before adding the luciferase substrate solution and the luminescence assay.

Figure 3 shows the results of a representative assay in which a single (subsaturating) concentration of IL-21-TLuc16 was tested against a panel of 20 candidate ortho-IL-21Rα molecules (SEQ ID NOs: 7-25 & 63) (all of which were bound to the streptavidin-coated surface of the wells at saturating concentrations). Eight candidate ortho-IL-21Rα molecules showed reduced ability to bind to IL-21-TLuc16. Repeat experiments (involving titrations of IL-21-TLuc16) confirmed the results.

Eight candidate IL-21Rα molecules (receptor variants or RVs) (i.e., RV2, RV6, RV7, RV10, RV13, RV15, RV18, and RV19) that show impaired binding to IL-21-TLuc16 can be tested for their ability to bind to candidate ortho-IL-21, with the ultimate goal of isolating (multiple) cytokine forms that potently bind (with respect to signaling and immunological activity) to the candidate ortho-IL-21Rα but not native IL-21Rα.

An additional 13 candidate ortho-IL-21Rα molecules carrying alternative combinations of amino acid substitutions present in the eight candidate ortho-IL-21Rα molecules were similarly tested for their ability to bind to IL-21-TLuc16. The 13 candidate ortho-IL-21Rα molecules (SEQ ID NOs: 26-38) shown in Table 2 (substitutions shown in the title and underlined within the sequence) were expressed as secreted proteins in HEK 293 cells.

Table 2

As shown in Figures 4A to 4E, 11 of the candidate ortho-IL-21Rα molecules showed reduced ability to bind to IL-21-TLuc16 (i.e., ortho-IL-21Rα molecules RV23, RV24, and RV28 showed binding that was equivalent or nearly equivalent to the wild-type receptor, while all other ortho-IL-21Rα molecules showed significantly more impaired binding).

Example 2: Attenuation of IL-21 mediated signaling revealed by candidate ortho-IL-21Rα molecules using Ba/F3 cell assay and STAT3 responsive reporter construct.

To further test the extent to which the candidate ortho-IL-21Rα candidates were compromised in their ability to bind IL-21, eight candidates from Example 1 and Figure 3 were expressed in a lymphoid cell line, namely Ba/F3 cells (a mouse pre-B cell lymphoma line). Ba/F3 cells are dependent on the cytokine IL-3 for growth, but will also respond strongly to IL-21 if they are first positive for IL-21Rα expression.

Ba/F3 cells with LeapIn mRNA and transposons encoding wild-type or candidate ortho-IL-21Rα were electroporated (RV2: D72E/Y129F/D73E; RV6: M70I/D73E/Q33H; RV7: D72E/L94V/Y191F; RV10: D72E/E38D/M130L; RV13: M70G/Y129F; RV15: F69L/M70L/D73I; RV18: D72K/D73K; RV19: E38K; and Δcyt, a form of IL-21Rα lacking almost all of its cytoplasmic tail). The transposon also carries STAT-3 regulated genes encoding secreted Cypridina noctiluca luciferase, a constitutively expressed cytoplasmic click beetle luciferase, and a constitutively expressed gene encoding puromycin N-acetyltransferase.

For example, the RV13 expression construct is made by ATUM ( www.atum.bio ) by oligonucleotide-dependent DNA synthesis. This plasmid is over 13Kb in size and contains four independent genes in a stretch of DNA flanked first by genomic insulator sequences (one side from the human D4Z4 locus and the other side from the chicken locus encoding β-globin) and then by the transposon inverted terminal repeat sequence. The insulator sequence is intended to protect the genes within the transposon from position effects (i.e., effects that depend on the transposon integration site in the genome) that might reduce or alter expression. The inverted terminal repeat sequence is replaced by ATUM's proprietary Transposase recognition, its mediation transposon is integrated in the genomic DNA.Table 3 has described four kinds of genes (according to the order that they occur in the transposon) that exist in the transposon.Gene and the transposon that comprises them are designed according to the standard molecular biology principle compatible with the conventional construction assembly method used with ATUM.By the 4th gene listed in the table 3 is carried out suitable change, the variant of this carrier of encoding wild-type or other candidate's ortho-IL-21R α molecule is generated.

Table 3

Transposon vectors encoding wild-type IL-21Rα or any candidate ortho-IL-21Rα molecule were co-expressed with the relevant vectors using ATx or ThermoFisher Neon instruments according to the manufacturer's instructions. In vitro transcribed mRNA of the transposase was co-transfected into Ba/F3 cells. Puromycin selection (1 μg/ml or higher) was performed 48 hours after transfection and continued for at least one week after all cells in the untransfected control culture died. Flow cytometry was used to confirm that the cells selected by puromycin showed uniform expression of IL-21Rα.

To measure IL-21 responses, Ba/F3 cells were first incubated for 20-24 hours in RPMI-1640 medium lacking serum and exogenous cytokines. After washing, cells were stimulated with IL-21 (or candidate ortho-IL-21) at different concentrations in a round-bottom 96-well plate (approximately 100,000 cells/well) for 20-24 hours with IL-21 (in the presence of 0.4% [vol/vol] serum) and then assayed for secreted luciferase (from the STAT3-cLuc gene in the transposon), intracellular luciferase (from the constitutively expressed EEF2-eLuc gene in the transposon), or ATP accumulation. The secreted luciferase assay was used to inform STAT3-dependent signaling in the cell, as occurs when IL-21 binds to its receptor. The other two assays (monitoring cytoplasmic eLuc or ATP accumulation) were used to inform cell number (i.e., proliferation).

As expected, if IL-3 is not provided, Ba/F3 cells can not be propagated.When expressing wild-type IL-21R α, they show IL-21 dependency proliferation (measured by ATP or eLuc as above).In the supernatant of the culture processed, there is also Cypridina luciferase (reporter gene regulated from STAT3) accumulation (Fig. 5 A to Fig. 5 B) of IL-21 dependency.The secretion of Cypridina luciferase depends on IL-21R α cytoplasmic domain (because the tailless form [Δ cyt] of IL-21R α can not induce the proliferation response to IL-21; Fig. 5 A); When the tyrosine residue in the cytoplasmic domain is replaced by phenylalanine residues, secretion is also significantly impaired (not shown).

The luciferase activity of Cypridina noctiluca can be easily detected by adding the relevant luciferase substrate (Vargulin) to the supernatant samples from the cells and measuring the luminescence using a luminometer. Figures 5A and 5B show the luciferase activity detected in the form of light emission (relative light units or RLU) after mixing 20 μL of supernatant from each well with 50 μL of VLAR-2 reagent buffer (targeted system) containing the manufacturer's recommended concentration of Vargulin.

The results confirmed the findings of the binding assays described and shown in Example 1 and Figure 3, namely, impaired IL-21-dependent signaling mediated by the eight candidate ortho-IL-21Rα molecules.

Example 3: Screening of candidate ortho-IL-21 molecules: In the expression of candidate ortho-IL- STAT3-dependent signaling responses were tested in cells expressing either IL-21Rα or wild-type IL-21Rα.

Wild-type or candidate ortho-IL-21 molecules were produced from transiently transfected HEK-293 cells according to the routinely used procedures of ATUM ( www.atum.bio ). The expression vectors used for this purpose carried the IL-21 open reading frame downstream of the optimized cytomegalovirus immediate early gene 1 promoter. The signal peptide from the human IL-2 gene was used instead of the native signal peptide. Epitope tags for detection, quantification, immobilization or purification were fused to the amino or carboxyl terminus of the IL-21 coding sequence. In a series of vectors (including those encoding the forms of IL-21 specified by SEQ ID NOs: 39 to 60 in Table 4), the element fused to the amino terminus was a Twin-Strep-Tag tag followed by three copies of a glycine-glycine-glycine-glycine-serine linker portion, while the element fused to the carboxyl terminus contained two copies of the same glycine-glycine-glycine-glycine-serine linker followed by an N-Myc epitope tag (recognized by the 9E10 monoclonal antibody). The second series of vectors (including those encoding the forms of IL-21 specified by SEQ ID NOs: 61-62 in Table 4) have no tags at the carboxyl terminus, but have the following elements at the amino terminus of IL-21: a Twin-Strep-Tag tag, followed by an N-Myc epitope tag, and then three copies of a glycine-glycine-glycine-glycine-serine linker portion.

Cultures of transfected cells (10 mL) were maintained for approximately five days (until viability dropped below approximately 50%). The supernatant was collected, filtered, and aliquoted. Quantification of IL-21 present in the supernatant was performed using ELISA (in some cases biolayer interferometry) specific for various epitope tags or for IL-21 moieties.

Selected candidate ortho-IL-21 molecules (mature protein sequence including epitope tag but without signal peptide) are shown in Table 4 (SEQ ID NOs: 39 to 62) (substitutions shown in titles and underlined within sequences).

Table 4

Ba/F3 cells expressing wild-type IL-21Rα or the eight candidate ortho-IL-21Rα molecules from Example 1 and Figure 3 were treated with candidate ortho-IL-21 molecules CV1-CV22 (SEQ ID Nos: 39 to 59). The signaling response of Ba/F3 cells to ortho-IL-21 molecules was monitored using the STAT3-luciferase assay as described above.

Ba/F3 cells were exposed to four concentrations (100, 50, 25 and 12.5 ng/mL) of the indicated candidate ortho-IL-21 molecules ( Figures 6A to 6J : candidate ortho-IL-21 molecules containing R5Q, R5D, R5E, R5K, R5N, I8E, R9E, R9D, R9K, R9N, R9Q and wild-type IL-21; Figures 6K to 6T : candidate ortho-IL-21 molecules containing Q12V, L13D, K73V, K73D, K75D, R76E, R76N, R76A, R5Q/R76E, R5Q/R76A and wild-type IL-21). Ba/F3 cells express different forms of ortho-IL-21Rα molecules (RV2: D72E/Y129F/D73E; RV6: M70I/D73E/Q33H; RV7: D72E/L94V/Y191F; RV10: D72E/E38D/M130L; RV13: M70G/Y129F; RV15: F69L/M70L/D73I; RV18: D72K/D73K; RV19: E38K；And wild-type IL-21Rα.Cells were placed in serum-free medium for 24 hours, then incubated overnight (about 20 hours) (about 100000 cells per hole) with candidate's ortho-IL-21 molecules in round-bottom 96-well plates.As described above, the transposon that gives WT and candidate's ortho-IL-21Rα molecule expression also carries the gene regulated by STAT3, and the Cypridina luciferase (Cypridina noctiluca luciferase) of gene encoding secretion.Fig. 6 A to Fig. 6 T show after 20 μ L supernatants from each hole are mixed with 50 μ L VLAR-2 reagent buffers (targeting system), the activity of this luciferase detected in the form of luminescence (relative light unit), and the reagent buffer contains the Cypridina luciferase substrate (Vargulin) of manufacturer's recommended concentration.

As shown in Figures 6A to 6T, the following cells expressing ortho-IL-21R molecules produced a measurable response to ortho-IL-21 molecules:

Table 5

Cells expressing IL-21Rα (the wild-type receptor) mounted measurable responses to most of the variant cytokines, although importantly, the design of the experiments (with only four variant cytokines at relatively high concentrations) precluded quantification of the extent of their signaling activity relative to wild-type IL-21.

The candidate ortho-IL-21 molecules selected from the set shown in Figures 6A to 6T and Table 4 (i.e., R5K (CV4), Q12V (CV12), R76A (CV19), and K73V (CV14)) were retested with Ba/F3 cells expressing wild-type IL-21Rα or candidate ortho-IL-21Rα molecules RV6, RV10, RV13, or RV15 (i.e., M70I/D73E/Q33H, D72E/E38D/M130L, M70G/Y129F, or F69L/M70L/D73I, respectively). The results of the experiment are shown in Figures 7A to 7D. As expected, when exposed to wild-type IL-21, the signal response produced by cells expressing candidate ortho-IL-21Rα molecules was weakened compared to molecules expressing wild-type IL-21Rα (solid lines with filled symbols in each figure). In addition, as predicted by the results in Figures 6A to 6T, in three cases (for cells expressing candidate ortho-IL-21Rα molecules, RV6, RV19 or RV13), cells produced stronger responses when exposed to candidate ortho-IL-21 molecules (CV12, CV19 or CV14, respectively) than when exposed to wild-type IL-21 (open diamond symbols and closed diamond symbols in Figures 7A, 7C and 7D, respectively). The ability of all four candidate ortho-IL-21 molecules to induce responses through wild-type IL-21Rα was impaired to varying degrees, with candidate ortho-IL-21 molecule CV19 being the most severely impaired in this regard (open circle symbols and closed circle symbols in Figure 7C).

Results from these experiments identified IL-21 substitutions associated with impaired ability to signal through wild-type IL-21Rα and, in some cases, low levels of ability to induce signaling through specific candidate ortho-IL-21Rα molecules.

Infolog variants of IL-21 were generated according to the principles described by Govindarajan S, Mannervik B, Silverman JA, et al., Mapping of amino acid substitutions conferring herbicide resistance in wheat glutathione transferase, ACS Synth Biol., 2015;4(3):221-227, doi:10.1021/sb500242x; and Musdal Y, Govindarajan S, Mannervik B., Exploring sequence-function space of a poplar glutathione transferase using designed information-rich gene variants, Protein Eng. Des Sel., 2017; 30(8): 543-549, doi: 10.1093/protein/gzx045. These Infolog variants of IL-21 were screened to determine their ability to induce signal transduction in Ba/F3 cells expressing wild-type IL-21Rα or candidate ortho-IL-21Rα molecules, as described above. Figures 8A to 8C provide representative data from such screening experiments. The experimental results shown in Figures 8A to 8C are derived from the analysis of 96 cytokines, one of which includes wild-type IL-21, and the other includes a negative control variant (CV22, which has two disabled substitutions [R5Q/R76A]; SEQ ID NO: 59) and 94 Infolog variants, each of which is a candidate ortho-IL-21 molecule. Figure 8A shows the STAT3 response elicited in cells expressing wild-type IL-21Rα exposed to a cytokine panel, while Figures 8B and 8C show the responses generated by cells expressing candidate ortho-IL-21Rα molecules RV13 and RV6, respectively. RV13 consists of amino acid substitutions M70G and Y129F (SEQ ID NO: 19), while RV6 consists of amino acid substitutions R70I, D73E, and Q33H (SEQ ID NO: 12). The highlighted curves in the three figures show the responses of the three cells to five selected cytokines, namely wild-type IL-21, CV22, CV204 (SEQ ID NO: 60, consisting of H6L/M10L/K73V/P78L/P104A), CV374 (SEQ ID NO: 61, consisting of H6L/R9K/M10L/K73 V/P78L/G84E) and CV388 (SEQ ID NO: 62, consisting of H6L-R9K/M10L/K73I/P78L/P104A).

The highlighted cytokines were subsequently retested in independent focused experiments of similar design (involving Ba/F3 cells expressing wild-type IL-21Rα (Figure 9A) or candidate ortho-IL-21RαRV13 (Figure 9B)). As with the screening experiments (Figures 8A to 8C), the negative control cytokine CV22 failed to elicit a signaling response in cells expressing either IL-21Rα. In contrast, wild-type IL-21 induced a strong response in cells expressing wild-type IL-21Rα, but induced a much weaker response in cells expressing RV13. CV204 elicited a response in cells expressing wild-type IL-21Rα (Figures 8A and 9A) or either candidate ortho-IL-21Rα molecule (Figures 8B, 8C, and 9B).

Strikingly, CV374 and CV388 were significantly impaired in their ability to induce signaling in cells expressing wild-type IL-21Rα (Figures 8A and 9A), but, like CV204, showed good activity against cells expressing RV13 (Figures 8B and 9B). Thus, in this Ba/F3 assay, the ortho-IL-21 molecules CV374 and CV388 displayed the signaling selectivity expected for cytokines with orthogonal functions to native IL-21.

Candidate ortho-IL-21RαRV13 (SEQ ID NO: 19) carries two substitutions relative to wild-type IL-21Rα, namely M70G and Y129F, while candidate ortho-IL-21RαRV22 (SEQ ID NO: 27) carries only M70G. These two variant receptors appear to be equally impaired in their ability to bind natural IL-21 (Fig. 4B). They also explain a similar response pattern to the IL-21 molecule set used in Fig. 9A and Fig. 9B. Specifically, like RV13 (Fig. 10B), RV22 mediated a significantly impaired signal response to wild-type IL-21 (and negative control molecule CV22), but produced a good response to CV204, CV374 and CV388 (Fig. 10C). As shown in Fig. 9A, both CV374 and CV388 showed impaired responses to cells expressing wild-type IL-21Rα, while CV204 behaved similarly to wild-type IL-21 (Fig. 10A). These results replicate the key observations from the data in Figures 9A and 9B, while also demonstrating that RV22 is interchangeable with RV13. Since RV22 carries one less substitution than RV13, RV22 may be advantageous for therapeutic purposes.

In most cases, in vitro assays have very limited predictive value for in vivo therapeutic efficacy: many therapeutic targets are expressed in multiple cell types (often with opposing effects on in vivo responses), or the therapeutic efficacy of a given target depends on other accessory cells. In such cases, in vitro models are inherently simplistic and are not particularly good substitutes for the more complex situation in vivo. This is not the case in the present application: the target receptor is synthetic and will only be expressed in cells specifically designed for this purpose. Given the specificity of the orthogonal cytokine receptor system, this significantly reduces complexity, giving the in vitro assays a much better predictive value.

The complete disclosures of all patents, patent applications and publications cited herein and electronically available materials are incorporated by reference. The foregoing detailed description and examples are provided only for clear understanding. No unnecessary limitations should be understood therefrom. Although theories describing the effective possible mechanisms of the compounds can be proposed, the inventors are not bound by the theories described herein. The present invention is not limited to the exact details shown and described, because variations apparent to those skilled in the art will be included in the present invention defined by the claims.

Sequence Listing

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370 375 380

Glu Gly Pro Cys Thr Trp Pro Cys Ser Cys Glu Asp Asp Gly Tyr Pro

385 390 395 400

Ala Leu Asp Leu Asp Ala Gly Leu Glu Pro Ser Pro Gly Leu Glu Asp

405 410 415

Pro Leu Leu Asp Ala Gly Thr Thr Val Leu Ser Cys Gly Cys Val Ser

420 425 430

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

435 440 445

Leu Lys Pro Pro Leu Ala Asp Gly Glu Asp Trp Ala Gly Gly Leu Pro

450 455 460

Trp Gly Gly Arg Ser Pro Gly Gly Val Ser Glu Ser Glu Ala Gly Ser

465 470 475 480

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

485 490 495

Ser Asp Cys Ser Ser Pro Val Glu Cys Asp Phe Thr Ser Pro Gly Asp

500 505 510

Glu Gly Pro Pro Arg Ser Tyr Leu Arg Gln Trp Val Val Ile Pro Pro

515 520 525

Pro Leu Ser Ser Pro Gly Pro Gln Ala Ser

530 535

<210> 4

<211> 519

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 4

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu Gly Trp Asn Pro His Leu Leu Leu Leu Leu Leu

210 215 220

Leu Val Ile Val Phe Ile Pro Ala Phe Trp Ser Leu Lys Thr His Pro

225 230 235 240

Leu Trp Arg Leu Trp Lys Lys Ile Trp Ala Val Pro Ser Pro Glu Arg

245 250 255

Phe Phe Met Pro Leu Tyr Lys Gly Cys Ser Gly Asp Phe Lys Lys Trp

260 265 270

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

275 280 285

Pro Glu Val Pro Ser Thr Leu Glu Val Tyr Ser Cys His Pro Pro Arg

290 295 300

Ser Pro Ala Lys Arg Leu Gln Leu Thr Glu Leu Gln Glu Pro Ala Glu

305 310 315 320

Leu Val Glu Ser Asp Gly Val Pro Lys Pro Ser Phe Trp Pro Thr Ala

325 330 335

Gln Asn Ser Gly Gly Ser Ala Tyr Ser Glu Glu Arg Asp Arg Pro Tyr

340 345 350

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

355 360 365

Cys Thr Trp Pro Cys Ser Cys Glu Asp Asp Gly Tyr Pro Ala Leu Asp

370 375 380

Leu Asp Ala Gly Leu Glu Pro Ser Pro Gly Leu Glu Asp Pro Leu Leu

385 390 395 400

Asp Ala Gly Thr Thr Val Leu Ser Cys Gly Cys Val Ser Ala Gly Ser

405 410 415

Pro Gly Leu Gly Gly Pro Leu Gly Ser Leu Leu Asp Arg Leu Lys Pro

420 425 430

Pro Leu Ala Asp Gly Glu Asp Trp Ala Gly Gly Leu Pro Trp Gly Gly

435 440 445

Arg Ser Pro Gly Gly Val Ser Glu Ser Glu Ala Gly Ser Pro Leu Ala

450 455 460

Gly Leu Asp Met Asp Thr Phe Asp Ser Gly Phe Val Gly Ser Asp Cys

465 470 475 480

Ser Ser Pro Val Glu Cys Asp Phe Thr Ser Pro Gly Asp Glu Gly Pro

485 490 495

Pro Arg Ser Tyr Leu Arg Gln Trp Val Val Ile Pro Pro Pro Leu Ser

500 505 510

Ser Pro Gly Pro Gln Ala Ser

515

<210> 5

<211> 340

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 5

Met Arg Ser Ser Pro Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met

1 5 10 15

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

20 25 30

Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln

35 40 45

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

50 55 60

Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln

65 70 75 80

Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile

85 90 95

Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala

100 105 110

Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr

115 120 125

Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu

130 135 140

Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu

145 150 155 160

Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

165 170 175

Ser Met Glu Ala Glu Ala Glu Arg Gly Lys Leu Pro Gly Lys Lys Leu

180 185 190

Pro Leu Glu Val Leu Ile Glu Leu Glu Ala Asn Ala Arg Lys Ala Gly

195 200 205

Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser Lys Ile Lys Cys Thr Ala

210 215 220

Lys Met Lys Lys Tyr Ile Pro Gly Arg Cys Ala Asp Tyr Gly Gly Asp

225 230 235 240

Lys Lys Thr Gly Gln Ala Gly Ile Val Gly Ala Ile Val Asp Ile Pro

245 250 255

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

260 265 270

Gln Val Asp Arg Cys Ala Asp Cys Thr Thr Gly Cys Leu Lys Gly Leu

275 280 285

Ala Asn Val Lys Cys Ser Asp Leu Leu Lys Lys Trp Leu Pro Gly Arg

290 295 300

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

305 310 315 320

Gly Leu Ala Gly Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

325 330 335

Gly Gly Gly Ser

340

<210> 6

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 6

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 7

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 7

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

His Asp Gln Tyr Glu Asp Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe Gln Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 8

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 8

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Met Ala Glu Glu Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Phe Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 9

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 9

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe Gln Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Phe Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 10

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 10

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

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

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe Gln Phe Met Ala Asp Glu Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 11

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 11

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Tyr His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Phe Leu Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 12

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 12

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

His Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

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

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 13

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 13

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Met Ala Glu Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Val Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 14

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 14

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

His Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Val Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Leu Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 15

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 15

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

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

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Tyr His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 16

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 16

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Asp Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Met Ala Glu Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Leu Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 17

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 17

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

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

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Ile Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Val Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 18

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 18

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Tyr His Phe Ile Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 19

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 19

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Gly Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Phe Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 20

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 20

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln His Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 21

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 21

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Leu Leu Ala Asp Ile Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 22

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 22

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

His Phe Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 23

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 23

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 24

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 24

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Met Ala Lys Lys Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 25

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 25

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Lys Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 26

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 26

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Met Ala Glu Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 27

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 27

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Gly Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 28

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 28

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Leu Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 29

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 29

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Leu Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 30

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 30

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Met Ala Asp Ile Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 31

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 31

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Met Ala Lys Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 32

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 32

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Met Ala Asp Lys Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 33

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 33

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Met Ala Asp Glu Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 34

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 34

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

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

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 35

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 35

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

His Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

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

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 36

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 36

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

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

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 37

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 37

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Lys Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Ile Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 38

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 38

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Lys Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

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

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 39

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 39

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Gln His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 40

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 40

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Asp His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 41

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 41

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Glu His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 42

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 42

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Lys His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 43

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 43

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Asn His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 44

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 44

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Arg His Met Glu Arg Met Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 45

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 45

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Arg His Met Ile Glu Met Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 46

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 46

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Arg His Met Ile Asp Met Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 47

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 47

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Arg His Met Ile Lys Met Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 48

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 48

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Arg His Met Ile Asn Met Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 49

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 49

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Arg His Met Ile Gln Met Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 50

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 50

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Arg His Met Ile Arg Met Arg Val Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 51

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 51

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Arg His Met Ile Arg Met Arg Gln Asp Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 52

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 52

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

Ile Asn Val Ser Ile Lys Val Leu Lys Arg Lys Pro Pro Ser Thr Asn

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 53

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 53

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

Ile Asn Val Ser Ile Lys Asp Leu Lys Arg Lys Pro Pro Ser Thr Asn

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 54

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 54

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

Ile Asn Val Ser Ile Lys Lys Leu Asp Arg Lys Pro Pro Ser Thr Asn

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 55

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 55

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

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

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 56

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 56

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

Ile Asn Val Ser Ile Lys Lys Leu Lys Asn Lys Pro Pro Ser Thr Asn

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 57

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 57

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

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

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 58

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 58

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Gln His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

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

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 59

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 59

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Gln His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

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

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 60

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 60

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly

35 40 45

Gln Asp Arg Leu Met Ile Arg Leu Arg Gln Leu Ile Asp Ile Val Asp

50 55 60

Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala

65 70 75 80

Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe

85 90 95

Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile

100 105 110

Ile Asn Val Ser Ile Lys Val Leu Lys Arg Lys Leu Pro Ser Thr Asn

115 120 125

Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser

130 135 140

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

145 150 155 160

Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser

165 170 175

Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys

180 185 190

Leu Ile Ser Glu Glu Asp Leu

195

<210> 61

<211> 189

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 61

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu

20 25 30

Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly Gly Gly Gly Ser Gly Gly

35 40 45

Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly Gln Asp Arg Leu Met Ile

50 55 60

Lys Leu Arg Gln Leu Ile Asp Ile Val Asp Gln Leu Lys Asn Tyr Val

65 70 75 80

Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp Val Glu Thr

85 90 95

Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys

100 105 110

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

115 120 125

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

130 135 140

His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro

145 150 155 160

Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys Met Ile His

165 170 175

Gln His Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser

180 185

<210> 62

<211> 189

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 62

Ala Pro Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu

20 25 30

Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly Gly Gly Gly Ser Gly Gly

35 40 45

Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly Gln Asp Arg Leu Met Ile

50 55 60

Lys Leu Arg Gln Leu Ile Asp Ile Val Asp Gln Leu Lys Asn Tyr Val

65 70 75 80

Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp Val Glu Thr

85 90 95

Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys

100 105 110

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

115 120 125

Ile Leu Lys Arg Lys Leu Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys

130 135 140

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

145 150 155 160

Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys Met Ile His

165 170 175

Gln His Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser

180 185

<210> 63

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 63

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Leu His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

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

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

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

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

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

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

Claims (27)

1. An engineered human IL-21 polypeptide comprising a plurality of amino acid substitutions and having a sequence corresponding to SEQ ID NO:2 numbering:

H6L；

R9K；

M10L；

P78L；

and optionally comprises G84E or P104A or a combination thereof;

and optionally one of K73V or K73I.

2. The engineered human IL-21 polypeptide of claim 1, wherein: the engineered human IL-21 polypeptide comprises the amino acid substitution K73V.

3. The engineered human IL-21 polypeptide of claim 1, wherein: the engineered human IL-21 polypeptide comprises the amino acid substitution K73I.

4. The engineered human IL-21 polypeptide of claim 1, wherein: the engineered human IL-21 polypeptide comprises an amino acid substitution G84E.

5. The engineered human IL-21 polypeptide of claim 1, wherein: the engineered human IL-21 polypeptide comprises the amino acid substitution P104A.

6. The engineered human IL-21 polypeptide of claim 1, wherein: the polypeptides bind to the extracellular domain of an engineered human CD360 protein.

7. The engineered human IL-21 polypeptide of claim 6, wherein: the engineered human CD360 protein ectodomain is modified at one or more residues selected from the group consisting of Y10, Q33, Q35, Y36, E38, L39, F67, H68, F69, M70, a71, D72, D73, I74, L94, a96, E97, P126, a127, Y129, M130, K134, S190, and Y191.

8. The engineered human IL-21 polypeptide of claim 6, wherein: the engineered human CD360 protein ectodomain is modified at M70.

9. The engineered human IL-21 polypeptide of claim 6, wherein: the engineered human CD360 protein ectodomain is modified at M70 and Y129.

10. The engineered human IL-21 polypeptide of claim 8, wherein: the engineered human CD360 protein extracellular domain comprises an amino acid substitution M70G.

11. The engineered human IL-21 polypeptide of claim 9, wherein: the engineered human CD360 protein extracellular domain comprises the amino acid substitutions M70G and Y129F.

12. The engineered human IL-21 polypeptide of claim 1, wherein: the engineered human IL-21 polypeptide comprises amino acid substitutions K73V and G84E.

13. The engineered human IL-21 polypeptide of claim 1, wherein: the engineered human IL-21 polypeptide comprises amino acid substitutions K73I and P104A.

14. An engineered human IL-21 polypeptide, wherein the engineered human IL-21 polypeptide comprises: a set of amino acid substitutions relative to SEQ ID NO:2 numbering: H6L, R9K, M10L, K73V, P78L and G84E.

15. An engineered human IL-21 polypeptide, wherein the engineered human IL-21 polypeptide comprises: a set of amino acid substitutions relative to SEQ ID NO:2 numbering: H6L, R9K, M10L, K73I, P78L and P104A.

16. A system for selectively activating a receptor in a cell, the system comprising:

(a) An engineered receptor comprising a human CD360 protein ectodomain, said human CD360 protein ectodomain comprising an amino acid substitution at M70G; and (b) the engineered human IL-21 polypeptide of claim 1.

17. A system for selectively activating a receptor in a cell, the system comprising:

(a) An engineered receptor comprising a human CD360 protein ectodomain, said human CD360 protein ectodomain comprising amino acid substitutions at M70G and Y129F; and (b) the engineered human IL-21 polypeptide of claim 1.

18. The system as recited in claim 16, wherein: the engineered human CD360 receptor is expressed by mammalian cells.

19. The system of claim 17, wherein: the engineered human CD360 receptor is expressed by mammalian cells.

20. The system of claim 18, wherein: the mammalian cells are immune cells or stem cells.

21. The system of claim 19, wherein: the mammalian cells are immune cells or stem cells.

22. The system as recited in claim 20, wherein: the mammalian cells are immune cells, and the immune cells are T cells.

23. The system of claim 21, wherein: the mammalian cells are immune cells, and the immune cells are T cells.

24. A pharmaceutical composition, comprising: the engineered human IL-21 polypeptide of claim 1; and a pharmaceutically acceptable excipient.

25. A kit comprising the system of claim 16.

26. A kit comprising the system of claim 17.

27. The engineered human IL-21 polypeptide of claim 1, wherein: the engineered human IL-21 polypeptide is fused to an Fc domain of IgG or albumin.