KR20230114749A - Orthogonal IL-21 receptor/cytokine system - Google Patents

Abstract

Orthogonal IL-21 receptors and orthogonal IL-21 cytokines are described. The IL-21 receptor-cytokine pair is an orthogonal interleukin-21 receptor α chain (“ortho-IL-21Rα”) that has impaired binding to the native interleukin-21 cytokine (“IL-21”), and a binding pathway for the native IL-21Rα. It includes the orthologous IL-21 cytokine (“ortho-IL-21”) that is impaired in binding, and the ortho-IL-21Rα binds ortho-IL-21. The IL-21 receptor-cytokine pair can activate IL-21 signaling. Cells engineered to express the orthogonal IL-21 receptor and methods of using the cells for the treatment of various diseases and disorders are also described.

Description

Orthogonal IL-21 receptor/cytokine system

This application claims priority under 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, which The content of each application is incorporated herein by reference.

Orthogonal IL-21 receptors and orthogonal IL-21 cytokines are described.

Cytokines are powerful natural regulators of immune cell proliferation and differentiation. This ability has attracted great attention to cytokines as a potential therapy, but has also complicated their clinical utility. This was especially true for cytokines that have multiple multicellular targets and thus have a high potential for pleiotropic effects. One example is interleukin-2 ("IL-2"), a potent T-cell mitogen whose anticancer activity is offset by unwanted proliferation of regulatory (suppressor) T cells and a painful vascular leak syndrome. In the specific case of IL-2, protein engineering can be used to solve several clinical problems: for example, removing the ability of cytokines to act preferentially on regulatory T cells. An alternative approach involves generating orthogonally constrained forms of cytokines and receptors. In this regard, U.S. Patent Nos. 10,869,887; Sockolosky JT, Trotta E, Parisi G, et al. Selective targeting of engineered T cells using orthogonal IL-2 cytokine-receptor complexes (“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 each of which are incorporated herein by reference.

Orthogonal cytokine systems are mutations in which cytokines and their receptors lose compatibility with their natural (parental) partners while retaining the ability to interact productively with each other. These orthogonal cytokine:receptor pairs can be said to demonstrate "preemptive" or "private" interactions. The approach of generating orthogonal constrained forms of cytokines and their receptors extends the cytokine's range of activity to therapeutic (i.e., adoptively transferred) cells - these cells are the only cells expressing the engineered receptor and thus consequently respond to the engineered cytokine. It is the only cell that can do this - it has useful value in cell therapy because it provides a way to limit it to.

Interleukin-21 ("IL-21") is another pleiotropic cytokine that acts on a wide range of lymphoid, bone marrow and epithelial cells. IL-21 regulates innate and adaptive immune responses; It not only plays an important role in anticancer and antiviral responses, but also has a great impact on inflammatory responses that promote the progression of autoimmune and inflammatory diseases. In this regard, SPOLSKI, R., Leonard, W., Interleukin-21: a double-edged sword with therapeutical potential ("Interleukin-21: a double-edged sword with therapeutic potential"), Nat Rev Drug Discov 13, 379- 395 (2014). See https://doi.org/10.1038/nrd4296 . The three-dimensional structure of the native human IL-21 cytokine:receptor complex is known. In this regard, Hamming OJ, Kang L, Svensson A, et al. see 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 an integral part of IL-21R. In this regard, J Biol Chem. 2012; 287 (12):9454-9460. doi: 10.1074/jbc. See Ml l 1.311084. The disclosures of these are incorporated herein by reference.

IL-21 is of particular interest because it enhances cytotoxic T cell responses to viruses and tumors and can synergize with other cytokines such as IL-2 or IL-15. IL-21 exerts this effect by promoting the persistence of T cells with a stem cell memory phenotype, which has been linked to beneficial outcomes in the cell therapy setting. IL-21 is currently being evaluated as a cancer treatment in a number of clinical trials. IL-21 also has significant potential utility in chimeric antigen receptor T (“CAR-T”) cell therapy, where it overcomes clinical failures due to expansion, anticancer potency, exhaustion, suppression, and decreased persistence. This can help. Therefore, especially by processing adoptively transferred cells, the desired behavior of IL-21 can be achieved, that is, it is protected from endogenous signaling pathways, does not affect non-target endogenous cells, and also has desirable behaviors that can control one-time administration to patients. The ability to control is urgently needed.

In one aspect, an orthogonal interleukin-21 receptor alpha chain ("ortho-IL-21Rα" or "ortho-IL-2 IRa molecule", or when referring to a particular ortho-IL-2 IRa constructed as provided herein, “RV”, “receptor variant”) is provided, which ortho-IL-2 IRα is an orthogonal interleukin-21 cytokine (“ortho-IL-21” or “ortho-IL-21 molecule”, or as provided herein). Modified amino acids derived from SEQ ID NO: 4 (SEQ ID NO: 4) that bind to a "cytokine variant" ("CV", when referring to a particular ortho-IL-21 composed of the same) but impair binding to native IL-21. contains sequence. In one aspect, the ortho-IL-21Rα is an amino acid residue of SEQ ID NO: 4 that contacts IL-21, a residue adjacent to such contact residue, or elsewhere within IL-21Rα that affects the organization of the IL-21 binding surface. It includes a modified amino acid sequence comprising a substitution of one or more residues of the residues of. In one aspect, ortho-IL-21Rα is an amino acid numbered at the following position relative to SEQ ID NO: 6 (human IL-21α lacking signal peptide is an ectodomain (ectosome)) Substituents, namely: Y10, Q33, Q35, Y36, E38, L39, F67, H68, F69, M70, A71, D72, D73, 174, L94, A96, E97, P126, A127, Y129, M130, K134, SI, SI 90 or Y191, or combinations thereof. In one aspect, the amino acid substitutions are: D72E/Y129F/D73E; M70I/D73E/Q33H; D72E/L94V/Y191F; D72E/E38D/M130L; M70G/Y129F; F69L/M70L/D73I; D72K/D73K; E38K; M70G, or a combination thereof, consisting essentially of, or consisting of them. In one aspect, the amino acid substitution is: M70G/Y 129F (“RV13” which is “receptor variant 13”, or SEQ ID NO: 19 (SEQ ID NO: 19)) or M70G (“RV22” or SEQ ID NO: 27 (SEQ ID NO: 27)), consists essentially of, or consists of them.

In another aspect, ortho-IL-21 is provided, wherein the ortho-IL-21 comprises a modified amino acid sequence derived from SEQ ID NO: 2 that binds ortho-IL-21Rα but is impaired in binding to native IL-21Rα. do. In one aspect, ortho-IL-21 is an amino acid residue of SEQ ID NO: 2 that contacts IL-21α, residues adjacent to such contact residues, or other elements within IL-21 that affect the configuration of the IL-21α binding surface. It includes a modified amino acid sequence comprising a substitution of one or more residues of the residues of In one aspect, ortho-IL-21 comprises amino acid substitutions numbered at the following positions with respect to SEQ ID NO: 2: R5, H6, 18, R9, M10, Q12, L13, K73, K75, R76, P78 , G84 or P104, or combinations thereof. The phrase "numbered with respect to SEQ ID NO: 2" means to disregard epitope tags and signal peptides for numbering purposes. In one aspect, the amino acid substitutions are R5Q, R5D, R5K, R5N, H6L, I8E, R9D, R9K, R9N, R9N, R9Q, R9Q, R9L, M10L, Q12V, L13D, K73D, K73I, K73I, K75D, R76E, R76A , R5Q/R76E, R5Q/R76A, R76A, P78L, G84E or P104A, or combinations thereof, consisting essentially of, or consisting of. In one aspect, the amino acid substitution comprises, consists essentially of, or consists of: H6L/R9K/M10L/P78L.

In one exemplary aspect, the amino acid substitution numbered with respect to SEQ ID NO: 2, ie, H6L; R9K; M10L; and P78L, optionally comprising G84E or P104A, or a combination thereof, and optionally comprising one of K73V or K73I. In one aspect, this engineered human IL-21 polypeptide is SEQ ID NO: 61 (SEQ ID NO: 61) (H6L/R9K/M10L/K73V/P78L/G84E) or SEQ ID NO: 62 (SEQ ID NO: 62) (H6L/ R9K/M10L/K73I/P78L/P104A).

In another aspect, a system for activating intracellular IL-21 signaling is provided, the system comprising: ortho-IL-21Rα that is impaired in binding to native IL-21, wherein the ortho-IL-21Rα is impaired. is a substitution of one or more of the amino acid residues of SEQ ID NO: 4 that contact IL-21, residues adjacent to such contact residues, or residues elsewhere in IL-21Rα that affect the configuration of the IL-21 binding surface. As comprising a modified amino acid sequence derived from SEQ ID NO: 4; It also includes ortho-IL-21 that is impaired in binding to native IL-21Rα, wherein the ortho-IL-21 is an amino acid residue of SEQ ID NO: 2 that contacts IL-21α, and a residue adjacent to the contact residue. , or a modified amino acid sequence derived from SEQ ID NO: 2 comprising substitutions of one or more residues elsewhere in IL-21 that affect the organization of the IL-21α binding surface, wherein the ortho-IL- 21Ra binds to the ortho-IL-21. In one aspect, the cells are mammalian cells, immune cells, stem cells or T cells.

The present invention may be more readily understood with reference to the following drawings.

1 is a schematic representation of an orthogonal IL-21 system. The picture on the left shows the productive interaction of the wild-type receptor and the cytokine with each other, while the adjacent picture depicts the impaired interaction between ortho-IL-21Rα and the native cytokine. The figure on the right also depicts the impaired interaction between ortho-IL-21 and its native (wild-type) receptor, while the adjacent figure depicts a productive interaction between the two orthogonal molecules (ortho-IL-21Rα and ortho-IL-21). shows
Figure 2 schematically depicts the pathway to create an orthogonal IL-21 system.
Figure 3 is a representative test of a single concentration (subsaturation) of IL-21 TLuc16 for binding to a panel of 20 candidate ortho-IL-21Rα molecules, all of which bound to streptactin-coated surfaces of wells at saturating concentrations. The results of the analysis are shown. Eight of the 20 candidate ortho-IL-21Ra molecules showed reduced binding capacity to IL-21-TUCl6.
4A-4E depict 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 20 candidate ortho-IL-21Ra molecules. The panel included wild-type receptors as controls and 7 of the 8 candidate ortho-IL-21Rα molecules identified in FIG. 3 . The panel also included 13 candidate ortho-IL-21Rα molecules designed based on the results described in Figure 3; Specifically, substituents present in the 8 candidate ortho-IL-21Rα molecules, alone or in combination, were introduced into the 13 additional candidate ortho-IL-21Rα molecules. Twenty-one of these candidate ortho-IL-21Ra molecules were added as saturating concentrations to streptactin coated wells of a 96-well plate. Figures 4A-4E show photometric data for individual plates, in each case binding of IL-21-TRuc16 to five candidate ortho-IL-21Rα molecules and also to the wild-type control IL-21Rα. binding was compared. Of the additional 13 candidate ortho-IL-21Rα molecules, 11 showed a significantly reduced binding capacity to IL-21-TUC16 compared to the wild-type control (receptors RV23 and RV24 carrying single substitutions M70L, F69L and D73E, respectively). , except RV28).
5A and 5B show the IL-21 derived STAT3 signaling response by Ba/F3 cells expressing native IL-21Rα and the eight candidate ortho-IL-2 IRα molecules identified as shown in FIG. 3 . The cells received the STAT3-regulated Cypridina noctiluca luciferase reporter transgene; They were exposed to various concentrations of native IL-21 overnight (approximately 20 hours) before testing the supernatant medium for luciferase activity by luminescence using Vargulin as the enzyme substrate. Inclusion of cells expressing a form of wild-type IL-21Rα lacking the cytoplasmic tail (β) showed that STAT3 signaling in response to IL-21 is, as expected, dependent on the cytoplasmic tail of the receptor.
Figures 6A-6T show the eight candidate ortho-IL-21Rα molecules identified as shown in Figure 3 and the native IL-21 and candidate ortho-IL-21 molecules that induce signaling via native IL-21Rα. show the ability As described with respect to FIG. 5, transfected mice receiving a STAT3-dependent luciferase reporter transgene residing on the same transposon used to confer expression of either native IL-21Rα or the candidate ortho-IL-21Rα molecule. Luminescence was used to quantify luciferase produced by Ba/F3 cells. These cells were exposed to gradually increasing concentrations of the candidate ortho-IL-21 molecule overnight (approximately 20 hours) before testing the supernatant for luciferase activity by luminescence.
7A-7D depict the ability of native IL-21 and candidate ortho-IL-21 molecules selected from the set shown in FIG. 6 to induce signaling via native IL-21Rα and selected candidate ortho-IL-21α molecules. do. Ba/F3 cells (expressing either native IL-21Rα or a candidate ortho-IL-21 molecule from a transposon harboring a STAT3-luciferase reporter transgene), as shown in FIGS. 5 and 6 , remove supernatant medium and They were exposed to gradually increasing concentrations of the indicator candidate ortho-IL-21 molecule (over about 20 hours) before being tested for luciferase activity by luminescence.
Figures 8A, 8B and 8C show a set of 96 cytokines, wild-type IL-21Rα (Figure 8A), or amino acid substitutions M70G/Y129F (SEQ ID NO: 19) or M70I/D73E/Q33H (SEQ ID NO: Results of a representative screening assay testing for the ability to induce a STAT3-dependent signaling response in Ba/F3 cells expressing the candidate ortho-IL-21α molecules RV13 (FIG. 8B) or RV6 (FIG. 8C) with 12). indicate As described with respect to Figures 5 and 6, transfected cells receiving a STAT3-dependent luciferase reporter transgene residing in the same transposon used to confer expression of the native IL-21Rα or candidate ortho-IL-21Rα molecule. Luminescence was used to quantify luciferase produced by Ba/F3 cells. These cells were exposed to gradually increasing concentrations of the candidate ortho-IL-21 molecule overnight (approximately 20 hours) before testing the supernatant for luciferase activity. Dotted lines represent response curves for a set of cytokines, and responses by the five cytokines of interest (wild type IL-21, negative control CV22, candidate ortho-IL-21 molecules CV204, CV374, CV388) are highlighted with solid lines and symbols.
9A and 9B show wild-type IL-21Rα ( FIG. 9A ) and autogenous inducing signaling through candidate ortho-IL-21α molecule RV13 ( FIG. 9B ) with amino acid substitutions M70G/Y129F (SEQ ID NO: 19). Shows the ability of IL-21 and candidate ortho-IL-21 molecules. As described with respect to Figures 5 and 6, transfected cells receiving a STAT3-dependent luciferase reporter transgene residing in the same transposon used to confer expression of the native IL-21Rα or candidate ortho-IL-21Rα molecule. Luminescence was used to quantify luciferase produced by Ba/F3 cells. These cells were exposed to gradually increasing concentrations of the candidate ortho-IL-21 molecule overnight (approximately 20 hours) before testing the supernatant for luciferase activity.
10A, 10B and 10C show wild-type IL-21Rα (FIG. 10A), candidate ortho-IL-21α molecule RV13 with amino acid substitution M70G/Y129F (SEQ ID NO: 19) (FIG. 10B), and amino acid substitution M70G (SEQ ID NO: 19). NO: 27) depicts the ability of native IL-21 and candidate ortho-IL-21 molecules to induce signaling via candidate ortho-IL-21α molecule RV2 ( FIG. 10C ). As described with respect to Figures 5 and 6, transfected cells receiving a STAT3-dependent luciferase reporter transgene residing in the same transposon used to confer expression of the native IL-21Rα or candidate ortho-IL-21Rα molecule. Luminescence was used to quantify luciferase produced by Ba/F3 cells. These cells were exposed to gradually increasing concentrations of the candidate ortho-IL-21 molecule overnight (approximately 20 hours) before testing the supernatant for luciferase activity.

Orthogonal IL-21 receptors and orthogonal IL-21 cytokines are provided. The orthogonal IL-21 receptor:cytokine pair may comprise ortho-IL-21Rα impaired binding to native IL-21, and ortho-IL-21 impaired binding to native IL-21Rα; -IL-21Rα binds ortho-IL-21. An orthogonal IL-21 receptor:cytokine pair can be used to activate signaling responses in cells. The signal response may be intrinsic downstream of the IL-21 receptor or alternatively dependent on the non-native IL-21 receptor and/or other non-native cellular components. The present invention also provides cells engineered to express orthogonal IL-21 receptors, and methods of using such cells in the treatment of various diseases and disorders.

Justice

The terminology used herein is for descriptive purposes and should not be construed as limiting the present invention. As used in the specification and appended claims, singular forms such as "a," "an" and "the" include their plural forms unless expressly stated otherwise.

“Treat,” “treating,” “treatment,” etc. is beneficial to a subject with a disease or disorder, such as cancer, including ameliorating a condition by reducing or suppressing at least one symptom, delaying progression of a disease, etc. refers to any action that provides

Phrases such as “effective amount” and “therapeutically effective amount” refer to that amount of a composition used in the practice of the present invention sufficient to provide effective treatment to 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 changes. WT proteins, polypeptides, antibodies, immunoglobulins, IgGs, etc., correspond to those commonly found in nature and have amino acid sequences or nucleotide sequences that have not been intentionally modified.

The term “polynucleotide” refers to oligonucleotides, nucleotides or fragments of either of these, i.e.: DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded, and which may represent a sensory or semisensory strand (e.g., mRNA, rRNA, tRNA); peptide nucleic acids; or DNA-like or RNA-like material of natural or synthetic origin. The term may also include nucleic acids, such as oligonucleotides containing known analogues of natural nucleotides, and nucleic acid-like structures having synthetic backbones.

The term “polypeptide” refers to an oligopeptide, peptide or protein sequence or fragment, portion or subunit of any of these, as well as a naturally occurring or synthetic molecule. The term "polypeptide" also includes amino acids linked to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres (isosomes), and may contain modified amino acid sequences. The term “polypeptide” also includes peptides and polypeptide fragments, motifs, and the like. A protein or peptide “chain” refers to a distinct subunit of a larger protein or protein complex.

A "homolog" is a protein of interest, such as a protein from another species (e.g., IL-21Rα), that is substantially similar to a human protein at the whole protein (i.e., mature protein) level, as long as the homologous peptide retains its respective known activity. means homologous. There may be varying degrees of homology, from 35% to 99%.

The term "amino acid modification (or modification)" includes amino acid substitutions, insertions or deletions in a polypeptide sequence. “Amino acid substitution” or “substitution” means replacing an amino acid with another amino acid at a specific position in the sequence of a parent polypeptide. For example, substitution R94K refers to a modified polypeptide in which an arginine at position 94 is replaced with a lysine. In the previous example, 94K represents a lysine substitution at position 94. Multiple substitutions are usually separated by slashes or commas. For example, R94K/L78V and [R94K, L78V] refer to a double variant comprising the substituents R94K and L78V. "Amino acid insertion" or "insertion" means the addition of an amino acid at a specific position in a parent polypeptide sequence. For example, insert - 94 specifies an insert 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- specifies deletion of an 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 properties of a polypeptide comprising an 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 DNA synthesis. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Types of amino acid residues with similar side chains have been defined in the art. These families include 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). , non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (eg threonine, valine, isoleucine), and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine) contains amino acids with

Typically, conservative substitution variants, homologs and analogues of a peptide 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% amino acid sequence identity to the disclosed sequences. %, at least about 85%, at least about 90%, at least about 95%, or at least about 96% to 99% (Rost B. Twilight zone of protein sequence alignment ("The Middle Zone of Protein Sequence Alignment"), Protein Eng . 1999;12(2): 85-94. doi: 10.1093/protein/12.2.85).

Identity or homology with respect to such a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to a known peptide after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percent homology, with any conservation Substituents are also not considered part of sequence identity. N-terminal, C-terminal or internal extensions, deletions or insertions in the peptide sequence are not to be construed as affecting homology.

Gamma chain (γc) cytokine refers to any cytokine for which a homologous cytokine receptor complex comprises a common cytokine receptor gamma chain (γc). γc cytokines include IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21.

Orthogonal cytokine:receptor pairs interact effectively with each other (i.e., can be used to initiate signaling responses in cells with physiological consequences), but natural cytokine:receptor pairs have marked impairments in their ability to interact with their natural counterparts. refers to a variant of The orthogonal cytokine can be a mutated or otherwise modified natural cytokine ("mutein") or a wholly synthetic protein that acts as an agonist for orthogonal cytokine receptors (sometimes referred to as "syntekines"). The orthogonal cytokine (whether a mutein or a syntekine) has no agonist activity or exhibits attenuated agonist activity on wild-type cytokine receptors.

Thus, orthogonal cytokine:receptor pairs can be modified by amino acid changes that: (a) lack or reduce binding to the native cytokine or cognate receptor; It may also include (b) a genetically engineered protein pair that binds specifically to an engineered (orthogonal) partner ligand or receptor. Upon binding to orthologous cytokines, orthologous receptors are transduced through native cellular elements to activate signaling that provides biological activity that mimics the native response, but is specific to engineered cells expressing orthologous receptors. Non-native receptors or cellular elements (eg, non-native cytoplasmic domains in receptors) may be involved to modulate the signal response. The orthogonal receptor does not bind to endogenous cytokines, or the binding force for the orthogonal cytokines, including their native counterparts, is attenuated. In some aspects, the affinity of the orthologous cytokine for its orthologous receptor is comparable to the affinity of the native cytokine for its native receptor, e.g., at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least It has an affinity of about 75%, at least about 100%, and can be 2x, 3x, 4x, 5x, 10x or more of the affinity of the native cytokine for its native receptor.

The term "candidate" used in the qualitative analysis of an orthogonal protein (IL-21 or IL-21Rα) refers to the original or progressive state. The assay procedure may exclude qualitative analysis if the candidate protein is found to have the desired avidity and orthogonal functionality.

As used herein, the phrases "binds/does not bind" and "unable to bind" refer to no detectable binding or insignificant binding, i.e., binding affinity that is significantly lower than that of the natural ligand. say "Binding disorder" refers to a lower than normal binding level between a corresponding wild-type component (eg a cytokine and a receptor).

The present invention includes a cytokine system based on IL-21, a member of the γc family of orthogonal cytokines. 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 STAT5-dominant signaling response, while STAT3 dominates the IL-21 response. IL-21 promotes the fitness of T cell differentiation, which correlates with favorable outcomes in adaptive cell therapy ("ACT"), particularly ACT using CAR T cells, and also promotes improved antitumor activity compared to IL-2 in various tumor model systems. shows the character of As a result, IL-21 may prove to be a better adjuvant for ACT than IL-2. Thus, in one aspect, an orthologous IL-21 system is provided: 1) ortho-IL-21 with impaired binding to native IL-21Rα; and 2) ortho-IL-21Rα capable of binding to the ortho-IL-21 while exhibiting impaired binding to native IL-21 (FIG. 1; FIG. 2, Pathway A).

orthogonal interleukin-21 receptor

In one aspect, an orthogonal interleukin-21 receptor is provided. The orthogonal interleukin-21 receptor may include modifying any one of the chains constituting the entire protein. In some aspects ortho-IL-21Rα is provided. The ortho-IL-21Rα contains an altered amino acid sequence derived from wild-type human IL-21Rα (SEQ ID NO: 4—a mature form of human IL-21Rα lacking the signal peptide). Ortho-IL-21Rα binds ortho-IL-21 but has impaired binding to native IL-21. Human IL-21Rα is represented by SEQ ID NO: 3.

In some aspects, the modified amino acid sequence is any of the amino acid residues of SEQ ID NO: 4 that contact IL-21, residues in the immediate vicinity of the contact residues, or other residues within IL-2 IRα that affect the suitability of the IL-21 binding surface. Substituents of one or more moieties are included. Amino acids in the immediate vicinity are those contained within 1, 2, 3, 4 or 5 amino acids of the contact residues in the primary protein sequence, or amino acid residues similarly adjacent in the tertiary protein structure. In some aspects, the ortho-IL-21Rα comprises an amino acid substitution numbered with respect to SEQ ID NO:6 at the following position. Namely: Y10, Q33, Q35, Y36, E38, L39, F67, H68, F69, M70, D72, D73, 174, L94, A96, E97, P126, A127, Y129, M130, K134, S190 or Y191, or these includes a combination of

One method of generating an orthologous version of IL-21 involves first mutating IL-21Rα to reduce its ability to bind to IL-21. The 20 IL-21Rα amino acids make significant direct contact with IL-21 within a binding surface of 990 Å ² . Any of these 20 residues can impair the receptor's ability to bind normally to IL-21. For example, a methionine residue at position 70 of IL-2 IRα has been identified as a major contributor to the binding interaction. The large hydrophobic side chain of this residue fits into the Il-21 pocket, which is composed mostly of hydrophilic residues (arginine-9, glutamine-12, arginine-76, lysine-73, isoleucine-16), some of which are compatible with IL-21Rα. Reposition to improve contact (in particular, arginine-9 and arginine-76 of IL-21 are in contact with aspartic acid-72 and aspartic acid-73 of IL-21Rα, respectively). Changing methionine-70 to another residue can adjust this rearrangement such that some of these contacts are weakened or lost. Alterations of aspartate-72 or -73 of IL-21Rα are likely to reduce the binding free energy of the interaction (e.g., at positions 9 and 76) such that compensatory changes in IL-21 are required to restore them. .

Helix C of IL-21 has two exchangeable states in the free structure of the cytokine, one in the disordered state and the other in the α-helical conformation. The α-helical conformation is stabilized in the complex of IL-21 and IL-21Rα, as is the first part of the CD loop. Helix C of IL-21 contains the above lysine-73 and arginine-76 in close proximity to the methionine-70 of the IL-21Rα. The CD loop of IL-21 contains lysine-77, proline-79 and serine-80, which together form a pocket for tyrosine-36 of IL-21Rα; In addition, lysine-77 makes ionic contact with glutamic acid-38 of IL-21Rα. Changing the CD loop amino acid sequence to a similar sequence found in IL-4 results in a 10-fold increase in the potency of IL-21 as measured by cellular assays. This observation suggests that significant binding energy is expended in stabilizing helix C of IL-21. In addition, changes in IL-21Rα to glutamate-38 and tyrosine-36 can significantly affect IL-21 binding in a manner that can be reversible due to compensatory changes to IL-21 in the helix C or CD loops. suggests

Discrete avian sequence motifs can be used in the design of orthogonal IL-21 systems. Of the 44 available avian sequences of IL-21, 39 have greatly shortened CD loops (6 residues) compared to human and mouse. This, together with the lack of tyrosine-36 in all 74 available avian IL-21Rα sequences, suggests that helix C of avian IL-21 binds the receptor in a manner distinct from human IL-21. This also provides additional rationale for modifications to the aforementioned binding moieties to construct orthogonal IL-21 systems.

Considering structural aspects, candidate amino acids can be identified that can be modified to alter IL-21Rα in a way that impairs binding to native IL-21. However, these changes must be of a character compatible with compensatory changes in IL-21 (ie, changes in IL-21 that will restore binding to ortho-IL-21Rα). Because the number of compensatory changes required to restore binding may be too great to process or incompatible with cytokine function (e.g., γc binding is lost, cytokines or cytokine receptors are unstable, difficult to express, immune , or pharmacologically problematic), changes in IL-21Rα that result in large fluctuations in compatibility are undesirable.

At least two assays can be used to screen ortho-IL-21Rα for loss of binding to IL-21. One is a direct binding assay that can be performed using purified proteins and sensitive biophysical assay procedures such as surface plasmon resonance (“SPR”) or biolayer interferometry (“BLI”). Another assay is a functional assay involving an appropriate cell line. Ba/F3 cells possess a number of useful properties: (i) do not express endogenous IL-21Rα; (ii) expressing mouse γc, which effectively displaces human γc in signaling using human IL-21Rα and human IL-21; (iii) phosphorylate and proliferate STAT3 in response to IL-21R signaling; and (iv) the availability of STAT3 reporter transgenes (such as transgenes expressing luciferase) as an easy and preferred means for monitoring IL-21 signaling. Alternatively, Jurkat or Molt-3 cells can be selected, which both express γc while expressing minimal or deficient endogenous IL-21Rα, and are also well suited for IL-21R signaling. do. HeLa cells transfected to express γc can also be used.

A method for isolating a candidate ortho-IL-21Rα involves separately expressing the variant in Ba/F3 cells (or one of the alternatives described above) via transfection. Flow cytometry is used to confirm the presence of candidate ortho-IL-2 IRα molecules on the cell surface and epitopes recognized by available antibodies. In one aspect, the candidate ortho-IL-21Rα comprises amino acid substitutions at the following positions: Y10, Q33, Q35, Y36, E38, L39, H68, F69, M70, A71, D72, D73, 174, L94, A96 , E97, P126, A127, Y129, M130, M130, K134, S190 or Y191 or a combination thereof, where the numbers indicate residue positions in the mature IL-21Rα ectodomain (SEQ ID: 6) and the letters represent single letter codes where used indicates amino acid identity, the first letter denotes a wild-type residue and the second letter, if present, a substitution residue). In one aspect, the amino acid substitutions are M70G, M70A, M70I, M70L, Y129F, Y129A, Y129S, Y129H, M130F, Y36H, FMAD(69-73) to LLADI, FYM(128-130) to FHF, D72K, D73K, D72E , D73E, D73I, E38K, E38S, E38D, F67L, F67Y, F69L, S190Y, Y191F, Q35L, H68Q, 174V, L94I, L94V, E97D, A127T, Q33N, Q33H, K134R, M130I, M130L, S19 0T, or any of these includes, consists essentially of, or consists of any combination thereof. In one aspect, the amino acid substitutions are: 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, consisting essentially of, or consisting of them. In one aspect, the amino acid substitutions are: [H68Q,E38D,Q33H], [Y129F,H68Q,Y191F], [Y36F,H68Q,D73E], [Y129F,F67Y,M130L], [L94V,Q33H,M130L], [Y36F ,F67Y,E38D,E38D,M130L], [Y36F,M70I,L94V], [M70I,F67Y,Y191F], [Y129H, M130F], [Y129F], [M130S], [M70G, D73E], [M70G, D73E , Q33H], [M70I, D73E], [M70I, E38K] and [M70I, E38K, D73E], consisting essentially of, or consisting of them. In one aspect, the amino acid substitution comprises, consists essentially of, or consists of M70G/Y129F or M70G.

Ba/F3 cells expressing the candidate ortho-IL-2 IRa can be cultured with native human IL-21 prior to analysis for signal response. In some experiments, time courses can be used to improve assay sensitivity and resolution. Another experiment involves comparing dose-response curves. IL-21 reactivity was monitored by flow cytometry or immunoblotting for Ba/F3 cell proliferation, tyrosine phosphorylation of STAT3, etc., or by using a reporter (luciferase or secreted alkaline) from a STAT3-dependent reporter transgene present in cells. It is detected through the expression of phosphorylation enzyme, etc.). While native IL-21Rα is capable of a robust response to IL-21 using either of these assays, the preferred ortho-form of the receptor will have a reduced or no response. This non-responsive ortho-IL-21Ra molecule is a candidate for the orthogonally restricted IL-21 cytokine-receptor system.

Orthogonal interleukin-21 cytokine

Another aspect is a method derived from wild-type human IL-21 (SEQ ID NO: 2—a mature form of human IL-21 lacking the signal peptide) that binds ortho-IL-21Rα but has impaired binding to native IL-21Rα. Ortho-IL-21 with modified amino acid sequences is provided. Human IL-21 is represented by SEQ ID NO: 1.

In some aspects, ortho-IL-21 binds ortho-IL-21Rα but has impaired binding to native IL-21Rα. In another aspect, the modified amino acid sequence is a substitution of one or more amino acid residues that contact IL-21Rα, residues adjacent to the contact residue, or other residues within IL-21R that affect the suitability of the IL-21Rα binding surface. includes

A method for confirming the orthogonal form of IL-21 can be used. Ten residues of IL-21 participate in polar interactions with IL-21Rα. That is, the 10 residues are as follows: arginine-5, arginine-9, arginine-11, glutamine-12, aspartic acid-15, serine-70, lysine-73, arginine-76, lysine-77, 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 form additional van der Waals contacts. Any of these residues may be substituted to overcome changes present in the candidate ortho-IL-21Rα molecule and restore binding.

The crystal structure of IL-21 bound by IL-21Rα can be used to identify IL-21 residues that are proximal to IL-21Rα processed changes. For example, if changing the methionine-70 of IL-21Rα is sufficient to eliminate IL-21 binding, a compensatory change to a subsequent methionine-70 contacting residue in IL-21 can restore binding. Namely, these residues are: arginine-9, glutamine-12, isoleucine-16, lysine-73 and arginine-76. However, these five IL-21 residues other than methionine-70 contact the following IL-21Rα residues: tyrosine-10, leucine-39, glutamic acid-38, phenylalanine-67, alanine-71, aspartic acid- 72, aspartic acid-73, tyrosine-129, methionine-130 and tyrosine-191. On the other hand, the 10 IL-21Rα residues mentioned above mediate further contact with the following IL-21 residues: arginine-5, isoleucine-8, glutamine-19, serine-70, lysine-77. Thus, taking into account the direct contacts present in the native IL-21:IL-21Rα crystal structure, a single substitution at the methionine-70 position of IL-21Rα would replace 10 IL-21 residues (arginine-5, isoleucine-8, arginine-5, isoleucine-8, arginine -9, glutamine-12, isoleucine-16, glutamine-19, serine-70, lysine-76, lysine-77). Accordingly, it can be inferred that compensation for the substitution (i.e., restoration of IL-21 binding) can be achieved by a change to one or more of these 10 residues. In addition, changes in residues that are not in direct contact with IL-21Rα may result in tuning to a remotely acting conformation such that binding can be restored with a methionine-70 substitution variant of IL-21Rα.

Phage display or yeast display technology allows for screening (selection) of large mutation spaces. This can usually be achieved by constructing a very diverse library of variants in which a small number of residues in a protein change in a random (or quasi-random) combinatorial manner. Next, the library is screened for the desired properties. In the present invention, screening may include identifying members of a library of IL-21 variants capable of binding to a candidate ortho-IL-21Rα. As described in the example above, if changing methionine-70 is sufficient to eliminate the binding of IL-21Rα to native IL-21, the 10 contact residues (arginine-5, isoleucine-8) likely to be affected , arginine-9, glutamine-12, isoleucine-16, glutamine-19, serine-70, lysine-73, arginine-76 and lysine-77) to construct an IL-21 library with randomly combinatorial mutations. there is. The theoretical complexity of such libraries can exceed 10 ¹³ . Typically, creating a library consisting of more than 10 ⁸ - 10 ⁹ variants is risky and practically impossible. Screening of such a library may involve a selection procedure in which IL-21 variants displaying surface-attached phages or yeasts are separated based on their ability to adhere to substrates coated with the candidate ortho-IL-21Rα (wild-type IL-21Rα). except substrates coated with ). These selections are performed as repeat cycles to augment desired properties in IL-21. Through an analytical screening procedure (including SPR or BLI), select products are characterized in detail and those with optimal properties are identified.

In one aspect, the ortho-IL-21 comprises one or more amino acid residues of SEQ ID NO: 2 that are in contact with IL-21Rα, residues adjacent to the contact residue or other elements in IL-21 that affect the compatibility of the IL-21Rα binding surface. Include modified amino acid sequences with substitution of residues. In one aspect, ortho-IL-21 is set to SEQ ID NO: 2 at the following position: R5, H6, 18, R9, MIO, Q12, L13, K73, K75, R76, P78, G84 or P104, or a combination thereof. amino acid substituents numbered relative to In one aspect, the amino acid substitutions are: R5Q, R5D, R5E, R5K, R5N, H6L, I8E, R9D, R9K, R9N, R9Q, M10L, Q12V, L13D, K73D, K73I, K73D, K75D, R76E, R76N, R76A , R5Q/R76E, P78L, G84E or P104A, or combinations thereof, consisting essentially of, or consisting of. In one aspect, the amino acid substitution comprises, consists essentially of, or consists of: H6L/R9K/M10L/P78L.

Methods for Additional Identification of Orthogonal IL-21 Receptor-Cytokine Pairs

An alternative approach to the identification of ortho-IL-21 molecules involves iterative mutation cycles, which in turn focus on a small number of residues selected from those that make direct intermolecular contacts in the IL-21:IL-21Ra structure. This approach may also include residues that are close to the point of contact and/or likely related by structural characteristics. A broad range of this approach may include any of the residues in close proximity to the entire region in contact with IL-21Rα and any additional residues randomly selected as close in structural features. A more noteworthy type involves residues - including 10 residues likely involved in substitution of the methionine-70 position - that can be directly affected by specific substituents present in the candidate ortho-IL-21Rα. An alternative broad approach is to make substitutions with minimal regard to the structure of IL-21, but instead to partially emphasize substitutions occurring in the IL-21 orthologue of other species. This approach involves single substitutions of individual residues throughout the IL-21 primary sequence.

The iterative process begins with multiple (eg, 10-100) candidate ortho-IL-21 forms. Some types of approaches include double or triple mutations in the initial population, while others evaluate only single-point mutations in IL-21. These candidate ortho-IL-21 molecules are tested for activity using the cellular assay described above (eg, using Ba/F3 cells harboring a STAT3-dependent luciferase reporter transgene). At least two types of cells are used in this assay: cells expressing the candidate ortho-IL-21Rα, and cells expressing wild-type IL-21Rα as a counter screen.

The likelihood of success of the alternative approach corresponds to the number of candidate ortho-IL-21Rα molecules tested. Increasing this number reduces the possibility of inadvertently selecting a candidate ortho-IL-21Rα that cannot (even partially) restore IL-21 binding with a small number (eg, less than 3) of substituents. Increasing this number also increases the possibility of isolating mutually parallel orthologous systems that do not exhibit crossover or crossover with wild-type IL-21 or IL-21Rα.

Data from an initial screening round can be distributed and analyzed with a focus on identifying substituents of IL-21 that, upon isolation, enhance binding to candidate ortho-IL-21Rα molecules and reduce binding to native IL-21Rα. A second round of screening can be performed by combining positive score substituents from the first round into new candidate ortho-IL-21 molecules. These candidate ortho-IL-21 molecules (and, if desired, additional variants with conservative or non-conservative substitutions at positive positions) can be used to improve binding to the candidate ortho-IL-21Rα molecule and to native IL-21Rα. Retest for binding failure.

Additional rounds of screening involving additional substituent combinations until at least one candidate ortho-IL-21 is isolated with the desired properties (no activity towards native IL-21Rα and near normal activity with at least one candidate ortho-IL-21Rα). can be performed.

An alternative screening approach may be facilitated with the use of candidate ortho-IL-21Rα, which in some cases has reduced, but not completely absent, binding to wild-type IL-21. Such binding-reduced ortho-IL-21Rα molecules restore some IL-21 binding activity of a small number (eg, less than 3) of individual substituents in IL-21, thereby restoring the unbound candidate ortho-IL-21Rα molecule (i.e., , a candidate ortho-IL-21Rα molecule lacking binding to wild-type IL-21). Once candidate ortho-IL-21 molecules have been isolated by the above screening procedures, additional screening steps can be performed involving new candidate ortho-IL-21Rα in which additional substituents have been combined with those already present. In some aspects, these additional mutations can completely abolish binding to wild-type IL-21 while retaining the ability to bind ortho-IL-21. This round of receptor mutation can be performed in multiples with subsequent rounds of purifying cytokine mutation.

The binding properties of products following the screening approach can be assayed using purified protein and BLI or SPR. A product that most closely resembles wild-type IL-21 and IL-21Rα in terms of binding rate can be selected as a candidate orthogonal IL-21 system.

In one aspect, the candidate ortho-IL-21 molecule can be processed according to the methods described in one or more of the following documents: U.S. Patent Nos. 8,005,620; 8,635,029; and 8,412,461, also Govindarajan S, Mannervik B, Silverman JA, et al., Mapping of amino acid substitutios conferring herbicide resistance in wheat glutathione transferase ("Mapping of amino acid substituents conferring herbicide resistance in wheat glutathione transferase"), ACS Synth Biol . 2015; 4(3):221-227. doi: 10.1021/sb500242x; Musdal Y, Govindarajan S, Mannervik B, Exloring sequence-function space of a polar glutathion transferase using designed inforation-rich gene variants, Protein Eng Des Sel . 2017: 3(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 gene ("Proteolytic enzyme K using machine learning and synthetic gene"), BMC Biotechnol . 2007; 7:16, Published 2007 Mar 26. doi:10.1186/1472-6750-7-16 (each of which is incorporated herein by reference).

In one aspect, the candidate ortho-IL-21Rα molecule has a modified amino acid sequence derived from SEQ ID NO: 2 as described above, to facilitate purification, to increase the half-life and stability of the ortho-IL-21Rα molecule in vivo, or to a patient. is expressed as a fusion protein between a second amino acid sequence that enhances drug properties that is important for successful administration of the ortho-IL-21 molecule. Appropriate amino acid sequences are known in the art and include, but are not particularly limited to, serum albumin, Fc fragments of IgG, single-chain Fc antibody fragments, ABD035, and the like. Fc fragments can be modified to block or at least greatly limit the formation of homodimers, for example through electrostatic steering mutations.

Orthogonal IL-21 receptor-cytokine system

Another aspect is to provide a system for activating IL-21 signaling in a cell. The system comprises a candidate ortho-IL-21Rα that is impaired in binding to native IL-21, wherein the candidate ortho-IL-21Rα is at an amino acid residue of SEQ ID NO: 4 that contacts IL-21, at the contact residue a variant amino acid sequence derived from SEQ ID NO: 4 with substitutions of one or more residues of adjacent residues or other residues in IL-21Rα that affect the suitability of the IL-21 binding surface; The system also includes ortho-IL-21 that is impaired in binding to native IL-21Rα, wherein the ortho-IL-21 comprises at least one amino acid residue of SEQ ID NO: 2 that contacts IL-21Rα, the contact residue and a modified amino acid derived from SEQ ID NO: 2 having substitutions of residues adjacent to or other residues in IL-21 that affect the compatibility of the IL-21Rα binding surface, wherein the ortho-IL-21Rα is ortho-IL-21Rα. bind to -21

In some aspects, the cell is a T cell. In another aspect, the cell is a CAR T cell. The orthogonal cytokine and orthogonal receptor can be any of the candidate ortho-IL-21 and candidate ortho-IL-21Ra molecules described herein. For example, in one aspect ortho-IL-21Rα comprises an amino acid substitution numbered with respect to SEQ ID NO: 6 comprising, consisting essentially of, or consisting of: M70G/Y129F or M70G. do; Ortho-IL-21 is also: (1) H6L/R9K/M10L/P78L; (2) H6L/R9K/M10L/P78L/K73V; (3) H6L/R9K/R9K/M10L/P78L/K73I; (4) H6L/R9K/M10L/P78I/K73V/G84E; (5) H6L/R9K/M10L/P78L/K73I/G84E; (6) H6L/R9K/M10L/P78L/K73V/P104A; and (7) the numbered amino acid substitutions with respect to SEQ ID NO: 2 that comprise, consist essentially of, or consist of one of H6L/R9K/M10L/P78L/K73I/P104A.

Expression vectors for orthogonal cytokines and receptors

Another aspect is to provide expression vectors comprising nucleic acids encoding any of the candidate orthologous IL-21 proteins (eg, receptors or cytokines) described herein. Orthogonal proteins, such as orthogonal ortho-IL-21 or ortho-IL-21Rα, can be produced recombinantly. Ortho-IL-21Rα can be introduced on an expression vector into cells to be processed. DNA encoding an orthogonal protein can be obtained from a variety of sources designed in processing steps.

Amino acid variants can be prepared by introducing appropriate nucleic acid changes into the nucleic acid coding sequence that encodes the protein. Nucleic acid codons encoding amino acids are known to those skilled in the art. As the selected specific codon, those that can be optimally expressed in the host cell used can be selected. Amino acid variants may represent insertions, substitutions, and directed deletions of residues, etc., as described herein. Combinations of these insertions, substitutions and directed deletions can be made to arrive at the final construct, provided that the final construct contains the desired biological activity as defined herein. In some aspects, the nucleic acid encodes ortho-IL-21Rα as described herein.

Nucleic acids are “operably linked” when they are placed into a functional relationship with another nucleic acid sequence. For example, when the DNA for a signal sequence is represented as a preprotein that participates in the secretion of the polypeptide, it is operably linked to the DNA of the polypeptide; A promoter or enhancer is operably linked with a coding sequence if the promoter or enhancer affects transcription of the sequence; or a ribosome binding site is operably linked with a coding sequence when the ribosome binding site is positioned to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, in both contiguous and read stages. However, some sequences, such as enhancers, do not have to be contiguous to be effective.

A nucleic acid encoding ortho-IL-21 or ortho-IL-21Ra can be expressed by insertion into a replicable vector. Many such vectors are available. The vector components generally include, but are not limited to, an origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. Vectors may include viral vectors, plasmid vectors, integration vectors, transposons, and the like. For example, a suitable transposon/transposase-based polynucleotide vector system is described in US Pat. No. 10,041,077, the contents of which are incorporated herein by reference.

Ortho-IL-21 or ortho-IL-21Rα can be produced recombinantly without modification or as a recombinant of a fusion peptide and a heterologous polypeptide, eg, a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. can be created In general, a signal sequence may be a component of a vector or may be part of a coding sequence inserted into a vector. The selected heterologous signal sequence may be one that is recognized and processed by the host cell (ie, cleaved by a signal peptidase). For mammalian cell expression, native signal sequences may be used, or other mammalian signal sequences may be suitable, such as signal sequences from polypeptides secreted from the same or related species, viral secretion leaders such as the herpes simplex gD signal.

Expression vectors contain a selection gene, commonly referred to as a selectable marker. A selection gene encodes a protein necessary for the survival or growth of a transformed host cell grown in selective culture. Host cells that are not transformed with the vector containing the selection gene will not be able to survive in culture. Representative selection genes are: conferring resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate or tetracycline; (b) compensate for nutrient deficiencies; or (c) encodes a protein that supplies important nutrients that cannot be obtained from complex media.

Expression vectors can contain a promoter that can be recognized by an orderly organism and can be operably linked to an orthogonal protein coding sequence. A promoter can be an untranslated sequence located upstream (5') to the start codon of a structural gene (typically about 100 to 1000 bp) that controls the transcription and translation of a particular nucleic acid sequence to which the promoter is operably linked. These promoters are generally divided into two types: inducible and constitutive promoters. Inducible promoters are promoters that initiate higher levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of nutrients or temperature changes.

For example, transcription from a vector in a mammalian host cell may include polyoma virus, poultry virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus (mouse stem cell virus ), promoters from viral genomes such as hepatitis B virus and simian virus 40 ("SV40"), promoters from heterologous mammalian promoters such as the actin promoter, phosphoglycerite kinase ("PGK"), or immunoglobulin It can be controlled by a promoter, a promoter obtained from a heat shock promoter, etc., provided that these promoters are mutually compatible with the host cell system. The early and late promoters of SV40 are conveniently obtained as SV40 restriction fragments that also contain the SV40 viral origin of replication.

Expression vectors may also include enhancer sequences. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, and act on promoters to enhance transcription. Enhancers are relatively orientation and position independent and have been found at positions 5' and 3' to the direction of the transcription unit within introns (intervening arrangements) and even in the coding sequence itself. A number of enhancer sequences are known in mammalian genes (eg, globin, elastase, albumin, fetal protein, and insulin). However, generally enhancers from eukaryotic viruses will be used. For example, the SV40 enhancer at the rear of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer at the rear of the replication origin, and the adenovirus enhancer may be included. The enhancer can be conjugated to the expression vector at positions 5' and 3' to the coding sequence, but is preferably at a position 5' from the promoter.

Expression vectors used in eukaryotic host cells will also contain sequences necessary for termination of transcription and stabilization of mRNA. Such sequences are generally available from the 5' and 3' untranslated regions of eukaryotic or viral DNA or cDNA.

Expression vectors may also be composed of inducible regulatory elements intended to control the expression of a transgene (eg, encoding ortho-IL-21 or ortho-IL-21Rα) using small molecules or other stimulators. there is. Examples of regulatory elements include, but are not limited to, promoters containing tetracycline operators that make them susceptible to regulation by tetracycline or its derivatives (such as doxycycline). Promoters can also be inducibly regulated by CRISPRa (regularly spaced short palindromic repeat-activated) using transcriptional effectors and fusions of catalytically dead Cas9. Such promoters may thus be downstream of other control systems, such as dimer or (antibody-based and/or chemical) Notch receptor-based components.

processing cells

One aspect of the invention provides cells engineered to express ortho-IL-21Rα. The cells can be genetically engineered to contain any suitable expression vector described herein. In some aspects, the expression vector comprises a coding sequence encoding an orthogonal receptor, which coding sequence is operably linked to a promoter active in a desired cell. Various vectors can be used for this purpose, for example, transposons, viral vectors, plasmid vectors, minicircle vectors, etc., which can be integrated into the target cell genome or maintained in episomal form. In one aspect, the expression vector can be integrated into the genome by a transposase enzyme. Examples of such transposome/transposase systems include ATUM Bio's Sleeping Beauty, PiggyBac, Riplin®, and their derivatives.

The engineered cell may be a host cell for use in the production of recombinant proteins in vitro (ie, in vitro). Suitable host cells for recombinant expression of orthogonal proteins include prokaryotic, yeast, higher eukaryotic, eg, various mammalian host cell lines.

In some aspects, processing cells are further modified beyond expression of ortho-IL-21Rα. Modifications suitable for use in engineered cells are known in the art and include expression of CARs, T cell receptors (“TCRs”), 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 engineered cells for therapeutic use may include stem cells, such as hematopoietic stem cells, natural killer cells (“NK”), or T cells. In some aspects, processing cells are T cells. “T” cells refer to mammalian immune effector cells characterized by expression of CD3 and/or the T cell antigen receptor, and are cells that can be engineered to express ortho-IL-21Rα. In some aspects, T cells are naive, activated or post-activated CD8+ T cells; cytotoxic CD8+ T cells; naïve, activated or post-activated CD4+ T cells; helper T cells such as TH1, TH2, TH9, TH11, TH22 and TFH; regulatory T cells such as TRI, natural TReg and inducible TReg; and memory T cells, ie central memory T cells, effector memory T cells, NKT cells, and also ηδ T cells.

Ortho-IL-21Rα can be used as an adjuvant to ACT. T cells can be engineered to express ortho-IL-21Rα, such as by gene (cDNA, minigene, or other nucleic acid construct) transfection, transduction or transformation. Patients receiving ACT can be treated (and/or pre-treated) with ortho-IL-21 and receive repeated doses as needed to increase and maintain desired T cell presence and response.

Therapeutic cells can also be engineered to express ortho-IL-21. This can be achieved using any of the methods suitable for envelope expression of ortho-IL-21Rα. Ortho-IL-21 can be expressed in the same cell as or different from the cell expressing ortho-IL-21Rα, enabling an autocrine or paracrine response, respectively. An example of a paracrine arrangement would be CD4+ T cells expressing ortho-IL-21 and CD8+ T cells expressing matched ortho-IL-21Rα.

In some aspects, ortho-IL-21 can be processed into a membrane-bound form. This can be achieved by fusing a cytokyl to the amino terminus of the IgG4 CH ₂ -CH ₃ component itself fused to the CD4 transmembrane domain, using native IL-21. Other cytokines can be bound to the cell membrane using a method related to this. This membrane binding limits the diffusion of the cytokine and also restricts its action to the immediate vicinity of cells expressing the membrane-bound cytokine. In vivo, this approach can be used to allow ortho-IL-21Rα expressing cells to encounter ortho-IL-21 only when in proximity to certain types of cells and/or locations in the body. In vitro, this approach can promote certain types of selective differentiation protocols (e.g., differentiation of NK cells from stem cells in the presence of K562 [or other] feeder feeder cells expressing membrane-bound IL-21 and CD137L). there is.

Engineered cells can provide pharmaceutical compositions suitable for therapeutic use, eg, pharmaceutical compositions suitable for human treatment. Therapeutic formulations comprising these cells can be formulated for administration using physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of frozen and aqueous solutions. . These cells can be formulated, dosed and administered in accordance with good medical practice.

treatment method

Cells, such as T cells engineered to express one of the ortho-IL-21Rα molecules described herein, can be used to treat a wide range of pathological conditions. Engineered properties in this therapy may allow for beneficial T cell differentiation, tolerance to exhaustion, long-term persistence, immunomemory responses, and inherent safety that can shut down responses in the event of an attack.

A method for improving cellular response by introducing ortho-IL-21Rα of the present invention to process cells from a recipient or donor, and contacting the processed cells with ortho-IL-21 to stimulate ortho-IL-21Rα to provide. The method may include obtaining target cells, such as T cells, hematopoietic stem cells, and the like, which may specifically be isolated from a biological sample or derived in vitro from a progenitor cell source. The cells may be transduced or transfected with an expression vector containing a sequence encoding ortho-IL-21Rα, and this step may be performed in an appropriate culture medium.

In some aspects, engineered T cells are capable of contacting ortho-IL-21 cells in vivo, i.e., by delivering the engineered T cells to a recipient and injecting an effective dose of ortho-IL-21 into the recipient, the engineered T cells are brought into contact with their native environment, e.g., lymph nodes, etc. In another aspect, such contacting is performed ex vivo. In this in vitro aspect, contacting can be performed using soluble ortho-IL-21 with or without a fusion to another protein component, such as an immunoglobulin Fc domain. In another such in vitro aspect, contacting can also be performed by bringing it into contact with other cells expressing secreted or membrane-bound ortho-IL-21.

Another aspect provides a method of treating a subject in need thereof, specifically introducing processed cells expressing ortho-IL-21Rα into the subject and contacting the cells with an effective amount of ortho-IL-21 to activate them. It provides a method comprising the steps of In some aspects the cell is a T cell and in other aspects a CAR-T cell. In another aspect, the cell is a T cell expressing a native or modified TCR. In another aspect, the cells of interest are NK cells. In yet another aspect, the cells are macrophages or other myeloid cells or leukocytes.

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, Fc fragments of IgG, single chain Fc antibody fragments, ABD035 and the like. Fc fragments can block or at least significantly limit the formation of homodimers by modifying, for example, through electrostatic steering or other mutations.

Another aspect is a method of treating a subject in need thereof, specifically comprising introducing into the subject a processed cell expressing ortho-IL-21Rα and also introducing a second processed cell expressing ortho-IL-21. It provides a treatment method for In some aspects the cell is a T cell and in other aspects the cell is a CAR-T cell.

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

In some aspects, the subject being treated has been diagnosed with cancer. "Cancer" and "malignancy" are used synonymously, and refer to the uncontrolled abnormal growth of cells, the ability of infected cells to spread locally or through the bloodstream and lymphatic system to other parts of the body (i.e. metastatic), and also many refers to a number of diseases with characteristic structural and molecular features of "Cancer cell" refers to a cell that has gone through an early, intermediate or advanced stage of a multistep neoplastic process. The characteristics of the early, intermediate, and progressive stages of neoplastic progression were described using microscopic observation. In these three stages of neoplastic progression, cells at each stage generally have abnormal nuclides, specifically transformation, inversion, disappearance, isosomes, monosomies, and exceptional chromosomes. Cancer cells include "hyperproliferative cells", i.e. cells at the beginning of malignant progression, "dysplastic cells", i.e. cells at intermediate stages of neoplastic progression, and "neoplastic (tumor) cells", i.e., advanced stages of neoplastic progression. Cancer includes, for example, sarcoma, breast cancer, lung cancer, brain cancer, bone cancer, liver cancer, kidney cancer, colon cancer, prostate cancer, and the like. In some aspects, engineered cells are used to treat a cancer selected from the group consisting of colorectal cancer, brain cancer, breast cancer, fibrosarcoma, squamous cell cancer, and the like. In some aspects, the cancer is selected from the group consisting of melanoma, breast cancer, colon cancer, lung cancer, and ovarian cancer. In some aspects, the cancer is a metastatic cancer.

In the case of cancer treatment, the treatment method may further include removing (or excising) the cancer. Removal of cancer is performed by a method selected from the group consisting of cryogenicization, thermal ablation, radiation therapy, chemotherapy, radiofrequency ablation, electroporation, alcohol ablation, high-intensity focused ultrasound treatment, photodynamic therapy, administration of monoclonal antibodies, and administration of immunotoxins. can be achieved using

In some aspects, a subject for treatment is diagnosed as having an infection. As used herein, the term "infection" refers to infecting one or more cells of a subject with 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 disorder caused by an infectious agent. Some infectious agents do not cause recognizable symptoms or disease under certain conditions, but have the potential to cause symptoms or disease under altered conditions. The subject method is used for the treatment of chronic pathogenic infections, the types of which are not limited to, for example: infections caused by viruses such as retrovirus, lentivirus, hepadnavirus, herpes virus, varicella virus and human papilloma virus. ; Intracellular bacterial infections such as Mycobacteria, Chlamydia, Erythia, Rickettsia, Brucella, Legionella, Listeria, Coxilla, Neisseria, Salmonella, Yersina, Helicobacter pylori and the like; Also included are infections with intracellular protozoan pathogens such as Plasmodium, Trypanosoma, Giardia, Toxoplasma and Leishmania.

In some aspects, the subject for treatment is diagnosed as having an autoimmune disease. Autoimmune diseases are characterized by T and B lymphocytes that abnormally target self-proteins, self-polypeptides, self-peptides or other self-molecules, resulting in damage and/or malfunction of organs, tissues or cell types in the body . Autoimmune diseases include the following diseases: rheumatoid arthritis, systemic erythematosus, multiple sclerosis, autoimmune hepatitis, insulin-dependent diabetes, and also degenerative diseases such as osteoarthritis, Alzheimer's disease, and macular degeneration.

For effective treatment, one or more cells of a subject may be contacted with ortho-IL-21. When cells are contacted with ortho-IL-21 in vitro, this ortho-IL-21 can be administered to the processing cells at an appropriate dose for a period sufficient to activate a signal from ortho-IL-21Rα, which is autogenous. Cellular structures such as accessory proteins, co-receptors, etc. can be utilized. Any suitable culture medium may be used. Cells activated in this way can be used for desired purposes and in vivo delivery, including experimental purposes related to antigen specificity determination, cytokine profiling, and the like.

For in vivo contact, an effective dose of engineered cells expressing ortho-IL-21Rα is first injected into the recipient in combination with or prior to administration of ortho-IL-21. Dosage and frequency of administration may vary depending on the agent, mode of administration, and the like. In addition, the dosage may vary according to topical administration such as nasal injection or inhalation method or systemic administration such as im (intramuscular), ip (intraperitoneal), or iv (intravenous) injection. Typically, at least about 10 ⁴ processed cells/kg is administered, at a dose of at least about 10 ⁵ processed cells/kg, at least 10 ⁶ processed cells/kg, at least 10 ⁷ processed cells/kg or more.

Example

Example 1: Binding assay for kinetics of candidate ortho-IL-21Rα molecules with impaired binding to native IL-2L

Direct interaction assays (methods that quantify the ability of IL-21Rα to bind native or mutant forms of IL-21) are used in cells for the identification of IL-21Rα variants with impaired IL-21 binding activity (i.e., candidate orthologous variants). It provides an alternative to based assays. The feasibility of this assay is improved based on the fact that the endogenous interaction (IL-21Rα: IL-21) is active (KD ~ 70 pM). One of the many possible binding assays involves luciferase fusion proteins, which is of interest because of its simplicity and potential to accommodate medium or high-throughput formats.

One type of IL-21Rα:IL-21 binding assay involves attaching the receptor ectodomain to a surface, immersing the coated surface in a solution of an IL-21-luciferase fusion protein, and then detecting the binding IL upon addition of the relevant luciferase. -21 is quantified based on the luminescence value. Alternatively, IL-21 can be immobilized and an IL-21Rα-luciferase fusion protein can be used in the solution.

In either type of binding assay, the preferred orientation of the immobilized receptor or cytokine can be achieved through the use of an affinity tag, such as Twin-Strip-Tag II, a high-affinity peptide ligand for the streptin protein. The IL-21Rα ectodomain containing the carboxy-terminal twin-strep-tag II peptide can be efficiently and selectively immobilized to the well surface of a 96-well plate pre-coated with streptactin protein. In this method, immobilized IL-21Rα should be directed to the cytokine binding region away from the plate surface. Likewise, IL-21 can be immobilized in a relevant manner even when attaching an amino or carboxy terminal twin-strep-tag II peptide tag.

A fusion protein (SEQ ID NO: 5) linking a 16KD turbo-luciferase polypeptide (ThermoFisher) to the carboxy-terminus of human IL-21 (via a glycine-serine-containing peptide) and plate-anchored human IL-21 The assay was completed using the 21Rα ectodomain (via the carboxy-terminal twin-strep-tag II). Bound IL-21 was detected using the TurboLuc Luciferase One-Step Glow Assay Kit (ThermoFisher) and standard luminometry. This assay can be completed by using luciferase fused to the amino-terminus of IL-21 or an alternative form of luciferase (eg, NanoLuke; ThermoFisher).

Twenty candidate ortho-IL-2 IRa molecules were tested for their binding ability to IL-21-TUCl6. Wild-type human IL-21Rα ectodomain (mature form lacking signal peptide) (V0 (SEQ ID NO:6)) and 20 candidate ortho-IL-21Rα molecules also shown in Table 1 (SEQ ID NOs: 7 to 25 and 63) (substitutes underlined in the header of the sequence) were expressed in HEK 293 cells as secreted proteins.

SEQ ID NO: 6 Human IL-21Rα ectodomain (mature form lacking signal peptide)

CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWS DPVIFQTQSEELKE SEQ ID NO: 7 >RV1 H68Q, E38D, Q33H ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTW H DQYE D LKDEATSCSLHRSAHNATHATYTCHMDVF Q FMADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTW SEWSDPVIFQTQSEELKE SEQ ID NO: 8 >RV2 D72E, Y129F, D73E ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMA EE IFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAF F MLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSE WSDPVIFQTQSEELKE SEQ ID NO: 9 >RV3 Y129F, H68Q, Y191F ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVF Q FMADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAF F MLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSS F QGTWSEWS DPVIFQTQSEELKE SEQ ID NO: 10 >RV4 Y36F, H68Q, D73E ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ F EELKDEATSCSLHRSAHNATHATYTCHMDVF Q FMAD E IFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTW SEWSDPVIFQTQSEELKE SEQ ID NO: 11 >RV5 Y129F, F67Y, M130L ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDV Y HFMADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAF FL LKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWS DPVIFQTQSEELKE SEQ ID NO: 12 >RV6 M70I, D73E, Q33H ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTW H DQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHF I AD E IFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSE WSDPVIFQTQSEELKE SEQ ID NO: 13 >RV7 D72E, L94V, Y191F ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMA E DIFSVNITDQSGNYSQECGSF V LAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSS F QGTWSE WSDPVIFQTQSEELKE SEQ ID NO: 14 >RV8 L94V, Q33H, M130L ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTW H DQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECGSF V LAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFY L LKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTW SEWSDPVIFQTQSEELKE SEQ ID NO: 15 >RV9 Y36F, F67Y, E38D ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ F E D LKDEATSCSLHRSAHNATHATYTCHMDV Y HFMADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTW SEWSDPVIFQTQSEELKE SEQ ID NO: 16 >RV10 D72E, E38D, M130L ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYE D LKDEATSCSLHRSAHNATHATYTCHMDVFHFMA E DIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFY L LKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGT WSEWSDPVIFQTQSEELKE SEQ ID NO: 17 >RV11 Y36F, M70I, L94V ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ F EELKDEATSCSLHRSAHNATHATYTCHMDVFHF I ADDIFSVNITDQSGNYSQECGSF V LAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTW SEWSDPVIFQTQSEELKE SEQ ID NO: 18 >RV12 M70I, F67Y, Y191F ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDV Y HF I ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSS F QGTWSE WSDPVIFQTQSEELKE SEQ ID NO: 19 >RV13 M70G, Y129F ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHF G ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAF F MLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWS DPVIFQTQSEELKE SEQ ID NO: 20 >RV14 Y36H ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQH EELKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSE WSDPVIFQTQSEELKE SEQ ID NO: 21 >RV15 F69L M70L D73I ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFH LL AD I IFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTW SEWSDPVIFQTQSEELKE SEQ ID NO: 22 >RV16 Y129H M130F ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAF HF LKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSE WSDPVIFQTQSEELKE SEQ ID NO: 23 >RV17 Y129F, M130S ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAF FS LKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSE WSDPVIFQTQSEELKE SEQ ID NO: 24 >RV18 D72K, D73K ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMA KK IFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSE WSDPVIFQTQSEELKE SEQ ID NO: 25 >RV19 E38K ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYE K LKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTW SEWSDPVIFQTQSEELKE SEQ ID NO: 63 >RV20 F67L ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDV L HFMADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWS DPVIFQTQSEELKE

Supernatants differentiated from the cells transfected were streptactin-coated plates, dilution of the supernatant, mouse monoclonal antibody specific for human IL-21Rα, horseradish peroxidase-conjugated rat antibody specific for mouse IgG, and an enzyme-linked immunosorbent assay ("ELISA") consisting of detection using a combination of chromogenic substrates for peroxidase. ELISA established the dilution required to ensure saturation of streptactin-coated wells with each candidate ortho-IL-21Rα molecule. Candidate ortho-IL-21Ra saturation wells were incubated with a solution of IL-21-TLuc16 at 4°C (or room temperature for some experiments) for 1 hour. The wells were washed before adding the luciferase substrate (coelenterazine) solution and applying the luminescence method.

Figure 3 shows the results of a representative assay in which a single (subsaturating) concentration of IL-21-TLuc 16 was tested for binding to a panel of 20 candidate ortho-IL-21Rα molecules (SEQ ID NOs: 7 to 25 & 63). (All of these molecules were bound to the streptactin-coated surface at saturating concentrations). Eight of the candidate ortho-IL-21Rα molecules showed reduced ability to bind IL-21-TUC16. A repeat experiment (with IL-21-TLuc16 titration) confirmed the result.

The eight candidate IL-21Rα molecules (“receptor variants” or “RV”) that showed impaired binding to IL-21-TUC16 (i.e., RV2, RV6, `RV7, RV10, RV13, RV15, RV18 and RV19) Candidate ortho-IL-21 with the ultimate goal of isolating the form of the cytokine(s) (with respect to signaling and immune activity) that binds productively to the candidate ortho-IL-21Rα but not to native IL-21Rα. can be tested for its ability to bind

An additional 13 candidate ortho-IL-21Rα molecules containing alternative combinations of amino acid substitutions present in the 8 candidate ortho-IL-21Rα molecules were similarly tested for binding ability to IL-21-TLuc16. Thirteen candidate ortho-IL-21Rα molecules (SEQ ID NOs: 26 to 38) shown in Table 2 (substitutes underlined in the header of the sequence above) were expressed as secreted proteins in HEK293 cells.

SEQ ID NO: 26 >RV21 D72E ectodomain

CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMA E DIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWS DPVIFQTQSEELKE SEQ ID NO: 27 >RV22 M70G ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHF G ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWS DPVIFQTQSEELKE SEQ ID NO: 28 >RV23 M70L ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHF L ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWS DPVIFQTQSEELKE SEQ ID NO: 29 >RV24 F69L ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFH L MADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSE WSDPVIFQTQSEELKE SEQ ID NO: 30 >RV25 D73I ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMAD I IFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSE WSDPVIFQTQSEELKE SEQ ID NO: 31 >RV26 D72K ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMA K DIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWS DPVIFQTQSEELKE SEQ ID NO: 32 >RV27 D73K ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMAD K IFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSE WSDPVIFQTQSEELKE SEQ ID NO: 33 >RV28 D73E ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMAD E IFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSE WSDPVIFQTQSEELKE SEQ ID NO: 34 >RV29 M70G, D73E ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHF G AD E IFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSE WSDPVIFQTQSEELKE SEQ ID NO: 35 >RV30 M70G, D73E, Q33H ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTW H DQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHF G AD E IFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSE WSDPVIFQTQSEELKE SEQ ID NO: 36 >RV31 M70I, D73E ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHF I AD E IFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSE WSDPVIFQTQSEELKE SEQ ID NO: 37 >RV32 M70I, E38K ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYE K LKDEATSCSLHRSAHNATHATYTCHMDVFHF I ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTW SEWSDPVIFQTQSEELKE SEQ ID NO: 38 >RV33 M70I, E38K, D73E ectodomain
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYE K LKDEATSCSLHRSAHNATHATYTCHMDVFHF I AD E IFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGT WSEWSDPVIFQTQSEELKE

As shown in Figures 4A-4E, 11 of the candidate ortho-IL-21Rα molecules showed reduced binding capacity to IL-21-TUCl6 (i.e., the ortho-IL-21Rα molecules RV23, RV24 and RV28 were wild-type). showed equivalent or nearly equivalent binding to that of the receptor, whereas all other ortho-IL-21Rα molecules showed significantly more impaired binding).

Example 2: Attenuation IL-21-Mediated Signaling Mediated by Candidate Ortho-IL-21Rα Molecules Identified Using a Ba/F3 Cell Assay and a STAT3-Response Reporter Construct

To further test the degree of impairment of the binding ability of the candidate ortho-IL-21Rα to IL-21, eight candidates in Example 1 and FIG. ) was expressed in Ba/F3 cells depend on the cytokine IL-3 for growth, but will proliferate robustly in response to IL-21 if initially rendered positive for expression of IL-21Rα.

Ba/F3 cells were electroporated with lipin transposase® mRNA and a transposon encoding wild-type or candidate ortho-IL-21Rα (RV2: D72E/Y129F/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 the cytoplasmic tail). The transposon also housed a constitutively expressed gene encoding a secreted cypridina noctiluca luciferase, a constitutively expressed cytoplasmic click beetle luciferase, and a puromycin N-acetyltransferase.

As an example, RV13-expressing constructs were prepared by oligonucleotide-dependent DNA synthesis by ATUM (www.atum.bio). This plasmid is over 13 Kb in size and contains genetic insulator sequences (from the human D4Z4 locus on one side and on the other). -obtained from the chicken locus encoding globin) is first flanked, and in turn contains four independent genes within a piece of DNA flanked by transposon inverted terminal repeat sequences. The insulator sequence aims to protect the transposon against positional effects that may reduce or alter expression (ie, effects that depend on the transposon integration site in the genome). The inverted terminal repeat sequence is recognized by Lipin® transposase, a product of ATUM, an enzyme that mediates the incorporation of transposons into genomic DNA. The four genes present inside the transposon are listed in Table 3 (in order of occurrence within the transposon). Genes and transposons containing them were designed according to standard molecular biology principles compatible with construct assembly methods routinely used by ATUM. Variants of this vector encoding wild-type or other candidate ortho-IL-21Rα molecules were generated with appropriate changes in the fourth gene listed in Table 3.

promoter open reading frame Terminator (Terminator) relative orientation PGK-Puro Human PGK Streptomyces puromycin -N-acetyl transferase human beta-globin inverted array STAT3-cLuc preceded by six STAT3-binding sites
Minimal promoter from cytomegalovirus immediate early genotype 1 Cypridina noctiluca luciferase human alpha-globin Arrangement EEF2-eLuc Mouse EEF2 Pyrearinus thermityluminance
Luciferase bos taurus growth hormone Arrangement HSV-IL-21R-RV13 Attenuated promoter from herpes simplex virus type 1 Human IL-21Rα RV13 (M70G/Y129F) Oricthoragus cuniculus beta-globin Arrangement

Transposon vectors encoding wild-type IL-21Rα or candidate ortho-IL-21Rα molecules were co-transfected into Ba/F3 cells along with in vitro transcribed mRNA encoding the corresponding Lipin® transposase according to the manufacturer's instructions. Puromycin selection (at least 1 μg/ml) was added 48 hours after transfection and continued for at least one week after all cells in untransfected control cultures had died. Flow cytometry confirmed that the puromycin-selected cells exhibited uniform IL-21Rα expression.

To assay the IL-21 response, Ba/F3 cells were first cultured in RPMI-1640 medium lacking serum and exogenous cytokines. After washing, cells were 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, prior to assaying round bottom 96 -Stimulated with IL-21 (0.4% [vol/vol] serum in the presence of). Analysis of secreted luciferase provided information about STAT3-dependent signaling events, such as the occurrence of IL-21 binding to the receptor. Two other assays (monitoring cytosolic eLuc or ATP accumulation) provided information on cell number (i.e., proliferation).

As expected, Ba/F3 cells failed to proliferate without IL-3. When expressing wild-type IL-21Rα, IL-21-dose dependent proliferation (by ATP or eLuc assays as described above) was shown. There was also an IL-21-dose dependent accumulation of cypridina luciferase (obtained from a STAT3-regulated reporter gene) in the supernatant of treated cultures ( FIGS. 5A and 5B ). Secretion of cypridina luciferase is dependent on the IL-21Rα cytoplasmic domain (tailless type of IL-21Rα [ cyt] did not induce proliferation in response to IL-21; Figure 5A); Secretion was also severely impaired when tyrosine residues in the cytoplasmic domain were not replaced with phenylalanine residues (not shown).

Cypridina noctiluca activity was readily detected by adding the relevant luciferase substrate (Vargulin) to a supernatant sample obtained from the cells and measuring luminescence using a luminometer. 5A and 5B show the luminescence values (relative light units or luciferase activity detected in the form of RLU).

These results validated the results of the binding assays described and shown in Example 1 and FIG. 3 with respect to the impairment of IL-21-dependent signaling mediated by eight candidate ortho-IL-21Rα molecules.

Example 3: Screening of Candidate Ortho-IL-21 Molecules: Testing for STAT3-Dependent Signal Responses in Cells Expressing Candidate Ortho-IL-21Rα Molecules or Wild-Type IL-21Rα

Wild-type or candidate ortho-IL-21 molecules were prepared from transiently transfected HEK-293 cells according to procedures routinely used by ATUM (www.atum.bio). The expression vector for this accommodated the IL-21 open reading frame downstream of the optimized cytomegalovirus immediate early gene 1 promoter. A signal peptide from the human IL-2 gene was used instead of the native peptide. An epitope tag for detection, quantification, immobilization or purification is fused to the amino- or carboxy-terminus of the IL-21 coding sequence. In a series of vectors (including vectors encoding IL-21 in the form specified by SEQ ID NOs: 39-60 of Table 4), the element fused to the amino terminus is a twin-strep-tag followed by glycine-glycine-glycine-glycine - followed by three sets of serine linker elements, while the element fused to the carboxy terminus consisted of two sets of the same glycine-glycine-glycine-glycine-serine linker elements followed by the N-Myc epitope tag (9E10 recognized by monoclonal antibodies). Vectors in column 2 do not have a tag at the carboxy terminus (including vectors encoding IL-21 in the form specified by SEQ ID NOs: 61 to 62 in Table 4), but instead have the following elements at the amino terminus of IL-21: It is characterized: a twin-strep-tag followed by an N-Myc epitope tag, and three sets of glycine-glycine-glycine-glycine-serine linker components.

Cultures (10 mL) of transfected cells were maintained for about 5 days (until viability dropped below about 50%). The supernatant was harvested, filtered and aliquoted. Quantification of IL-21 present in the supernatant was performed using ELISA (and in some cases biolayer interferometric assays) directed against various epitope tags or specific for IL-21 components.

Selected candidate ortho-IL-21 molecules (mature protein sequences containing an epitope tag but lacking the signal peptide) are shown in Table 4 (SEQ ID NOs: 39-62) (substitutions in the header portion of the sequence are underlined). .

SEQ ID NO: 39 >IL21_CV1_R5Q
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQD Q HMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL* SEQ ID NO: 40 >IL21_CV2_R5D
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQD D HMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL* SEQ ID NO: 41 >IL21_CV3_R5E
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQD E HMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL* SEQ ID NO: 42 >IL21_CV4_R5K
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQD K HMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL* SEQ ID NO: 43 >IL21_CV5_R5N
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQD N HMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL* SEQ ID NO: 44 >QKLISEEDL* SEQ ID NO: 45 >IL21_CV7_R9E
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQDRHMI E MRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL* SEQ ID NO: 46 >IL21_CV8_R9D
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQDRHMI D MRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL* SEQ ID NO: 47 >IL21_CV9_R9K
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQDRHMI K MRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL* SEQ ID NO: 48 >IL21_CV10_R9N
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQDRHMIN MRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL * SEQ ID NO: 49 >IL21_CV11_R9Q
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQDRHMI Q MRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL* SEQ ID NO: 50 >IL21_CV12_Q12V
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQDRHMIRMR V LIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL* SEQ ID NO: 51 >IL21_CV13_L13D
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQDRHMIRMRQ D IDIVDQLKNYVNDLVPEFLAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL* SEQ ID NO: 52 >IL21_CV14_K73V
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIK V LKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL* SEQ ID NO: 53 >IL21_CV15_K73D
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIK D LKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL* SEQ ID NO: 54 >IL21_CV16_K75D
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKL DRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL* SEQ ID NO: 55 >IL21_CV17_R76E
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLK E KPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL* SEQ ID NO: 56 >IL21_CV18_R76N
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLK N KPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL* SEQ ID NO: 57 >IL21_CV19_R76A
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLK A KPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL* SEQ ID NO: 58 >IL21_CV21_R5Q_R76E
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQD Q HMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLK E KPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL* SEQ ID NO: 59 >IL21_CV22_R5Q_R76A
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQD Q HMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLK A KPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSEQKLISEEDL* SEQ ID NO: 60 >IL21_CV204_H6L_M10L_K73V_P78L_P104A
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKGGGGSGGGGSGGGGSQGQDR L MIR L RQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIK V LKRK L PSTNAGRRQKHRLTCPSCDSYEKKP A KEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGSEQKLISEEDL* SEQ ID NO: 61 >IL21_CV374_H6L_R9K_M10L_K73V_P78L_G84E
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKEQKLISEEDLGGGGSGGGGSGGGGSQGQDR L MI KL RQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIK V LKRK L PSTNA E RRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS* SEQ ID NO: 62 >IL21_CV388_H6L_R9K_M10L_K73I_P78L_P104A
APWSHPQFEKGGGSGGGSGGSSAWSHPQFEKEQKLISEEDLGGGGSGGGGSGGGGSQGQDR L MI KL RQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIK I LKRK L PSTNAGRRQKHRLTCPSCDSYEKKP A KEFLERFKSLLQKMIHQHLSSRTHGSEDS*

Ba/F3 cells expressing the wild-type IL-21Rα or eight candidate ortho-IL-21Rα molecules of Example 1 and FIG. 3 were stimulated with the candidate ortho-IL-21 molecule CV1-CV2 (SEQ ID NOs: 39 to 59). did. The signaling response of Ba/F3 cells to the ortho-IL-21 molecule was monitored by 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 ( FIGS. 6A-6J : R5Q, R5D, R9K, R9N, R9K, R9Q Figures 6K-6T: candidate ortho-IL-21 molecules containing Q12V, L13D, K73D, K75D, R76E, R76A/R76A, R5Q/R76A, and wild-type IL-21; and wild-type IL-21). Ba/F3 cells differed depending on the type of ortho-IL-21Rα molecule they expressed (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). These cells were subsequently maintained in serum-free medium for 24 hours prior to overnight culture with candidate ortho-IL-21 molecules (up to 100,000 per well) in round-bottom 96-well plates. As described above, the transposons providing expression of the WT and candidate ortho-IR-21Rα molecules also housed a STAT3-regulated gene encoding a secreted Cypridina noctiluca luciferase. 6A to 6T show 20 μL of supernatant from each well was mixed with 50 μL of VLAR-2 reagent buffer (Targeting Systems) containing Cypridina noctiluca luciferase substrate (Varguline) at the manufacturer's recommended concentration. The luciferase activity detected using the mixture is expressed in the form of a luminescence value (in relative light units).

As shown in Figures 6A-6T, the following cells expressing the ortho-IL-21Rα molecule showed a measurable response to the ortho-IL-21 molecule:

Ortho-IL-21 (cytokine number) Ortho-IL-21Ra (receptor no.) drawing number R5K (CV4) M70I/D73E/Q33H (RV6) 6C R5K (CV4) E38K (RV19) 6I R5Q (CV1) E38K (RV19) 6I R9K (CV9) M70G/Y129F (RV13) 6J Q12V (CV12) M70I/D73E/Q33H (RV6) 6M K73V (CV14) M70I/D73E/Q33H (RV6) 6M K75D (CV16) M70I/D73E/Q33H (RV6) 6M R76A (CV19) F69L/M70L/D73I (RV15) 6P or 6Q Q12V (CV12) E38K (RV19) 6S K75D (CV16) E38K (RV19) 6S R76N (CV18) E38K (RV19) 6S Q12V (CV12) M70G/Y129F (RV13) 6T L13D (CV13) M70G/Y129F (RV13) 6T K73V (CV14) M70G/Y129F (RV13) 6T K75D (CV16) M70G/Y129F (RV13) 6T

Cells expressing IL-21Rα (the wild-type receptor) showed a measurable response to most variant cytokines, and, although of importance, the design of the experiment (which involved only relatively high concentrations of the four variant cytokines) showed wild-type IL-21Rα. The possibility of quantifying the degree of signaling activity relative to 21 was ruled out.

Candidate ortho-IL-21 molecules (i.e., R5K (CV4), Q12V (CV12), R76A (CV19), and K73V (CV14)) selected from the populations shown in Figures 6A-6T and Table 4 are wild-type IL-21Rα or Retested using Ba/F3 cells expressing the candidate ortho-IL-21Rα molecules RV6, RV10, RV13 or RV15 (i.e. M70I/D73E/Q33H, D72E/E38D/M130L, M70G/Y129F or F69L/M70L/M70L /D73I). The results of this experiment are shown in Figures 7A to 7D. As expected, when exposed to wild-type IL-21, cells expressing the candidate ortho-IL-2Rα molecule showed a reduced signal response compared to those expressing wild-type IL-21Rα (symbolized solid lines in each graph). ). As also predicted from the results in Figures 6A-6T, in three cases (cells expressing the candidate ortho IL-21Rα molecule V6, RV19 or RV13), the wild-type IL-21 molecule (CV12, CV19 or CV14) When exposed respectively, the cells showed a stronger response than the response to wild-type IL-21 (open versus closed diamond symbols in Fig. 7A, C and D, respectively). All four candidate ortho-IL-21 molecules were impaired to varying degrees in their ability to elicit a response via wild-type IL-21Rα, with the candidate ortho-IL-21 molecule CV19 being most severely impaired in this respect (open in FIG. 7C ). versus closed circular symbols).

The results of these experiments identified IL-21 substituents implicated in impaired ability via wild-type IL-21Rα, which have - in some cases - low ability to induce signaling through certain candidate ortho-IL-21Rα molecules.

An infolog variant of IL-21, Govindarajan S, Mannervik B, Silverman JA, et al., Mapping of amino acid substitutios conferring herbicide resistance in wheat glutathione transferase Mapping of amino acid substitutions"), ACS Synth Biol . 2015; 4(3):221-227. doi: 10.1021/sb500242x; Musdal Y, Govindarajan S, Mannervik B, Exloring sequence-function space of a polar glutathion transferase using designed inforation-rich gene variants, Protein Eng Des Sel . 2017: 3(8):543-549. It was generated according to the principles disclosed in doi: 10.1093/protein/gzx045 and the like. These epilog variants of IL-21 were screened for their ability to induce signaling in Ba/F3 cells expressing either wild-type IL-21Rα or the candidate ortho-IL-21Rα, as described above. Representative data from these screening experiments are shown in Figures 8A-8C. Experimental results shown in Figures 8A to 8C, one consisting of wild-type IL-21 and one negative control variant (CV22, containing two incompetent substitutions [R5Q/R76A]; SEQ ID NO: 59) , and also from the analysis of a total of 96 cytokines, including the remaining 94 epilog variants, each of which is a candidate ortho-IL-21 molecule. 8A shows STAT3 responses derived from cells expressing wild-type IL-21Rα exposed to a cytokine aggregate, while FIGS. 8B and 8C are produced by cells expressing the candidate ortho-IL-21Rα molecules RV13 and RV16, respectively. show the reaction. RV13 consists of amino acid substitutions M70G and Y129F (SEQ ID NO: 19), while RV6 consists of amino acid substitutions M70I, D73E and Q33H (SEQ ID NO: 12). The highlighted curves in the three figures represent the responses generated by each of the three cell types 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/K73V/P78L/G84E) consisting of), and CV388 (SEQ ID NO: 62, consisting of H6L/R9K/M10L/P78L/P104A).

The highlighted cytokines were then retested in independent focus experiments of similar design (with Ba/F3 cells expressing wild-type IL-21Rα (FIG. 9A) or the candidate ortho-IL-21Rα RV13 (FIG. 9B)). As in the screening experiments ( FIGS. 8A-8C ), the negative control cytokine CV22 failed to induce a signal response in cells expressing IL-21Rα in any form. In contrast, wild-type IL-21 induced a strong response expressing wild-type IL-21Rα, but a much weaker response in RV13-expressing cells. CV204 elicited responses in cells expressing either wild-type IL-21Rα ( FIGS. 8A and 9A ) or expressing any of the candidate ortho-IL-21Rα molecules ( FIGS. 8B , 8C and 9B ).

Surprisingly, CV374 and CV388 showed significantly impaired ability to induce signaling in cells expressing wild-type IL-21Rα (FIGS. 8A and 9A), but, like CV204, showed good activity with cells expressing RV13 (FIG. 8A and FIG. 9A). 8B and 9B). Therefore, in this Ba/F3 assay, the ortho-IL-21 molecules CV374 and CV388 showed signaling selectivity of the kind expected for cytokines with orthogonal functionality to native IL-21.

Candidate ortho-IL-21Rα RV13 (SEQ ID NO: 19) has two substitutions for wild-type IL-21Rα, namely M70G and Y129F, whereas candidate ortho IL-21Rα R22 (SEQ ID NO: 27) has only M70G have These two variant receptors appear equally impaired in their ability to bind native IL-21 (FIG. 4B). In addition, they showed a similar pattern of reactivity to the aggregation of IL-21 molecules used in Figures 9A and 9B. Specifically, similar to RV13 (Fig. 10B), RV22 mediated a greatly impaired signal response to wild-type IL-21 (and the negative control molecule CV22), but obtained superior responses to CV204, CV374 and CV388 (Fig. 10C). . As in Figure 9A, CV374 and CV388 showed impaired response with cells expressing wild-type IL-21Rα, whereas CV204 exhibited similar behavior to wild-type IL-21 (Figure 10A). These results mimic key observations from the data in Figures 9A and 9B, and also show that RV22 may be interchangeable with RV13. Since RV22 contains one less substitution than RV13, RV22 may be preferred for therapeutic purposes.

In most settings, in vitro assays will have only a very limited predictive value of in vivo therapeutic effect: multiple therapeutic targets are expressed in multiple cell types (often with effects that oppose the in vivo response), or the therapeutic effect of a given target may differ from other dependent on auxiliary cells. In this situation, in vitro models that are inherently simple to fabricate are not particularly good substitutes for the much more complex situation in vivo. This is not an example of immediate application: the target receptor is synthetic and will only be expressed in cells specially engineered for it. Given the specificity of the orthogonal cytokine-receptor system, this will greatly reduce complexity and provide better predictive value in in vitro assays.

All patents, patent applications and publications and electronically available materials cited herein are incorporated by reference for their entire disclosures. The foregoing detailed description and examples have been given for a clearer understanding. It is understood that there is no limit thereto. Theories may be put forward to explain possible mechanisms by which the compounds may exert their effects, but the inventors are not bound by the theories set forth herein. The present invention is not limited to the details illustrated and described above, and modifications obvious to those skilled in the art will also be included in the invention defined by the claims.

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

<110> Neptune Biosciences LLC <120> Orthogonal IL-21 Receptor/Cytokine Systems <130> I230002P <150> US 63/105,414 <151> 2020-10-26 <150> US 63/212,547 <151> 2021-06-18 <150> PCT/US 2021/056439 <151> 2021-10-25 <160> 63 <170> PatentIn version 3.5 <210> 1 <211> 162 <212> PRT <213> artificial sequence <220> <223> IL-21 <400> 1 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 <210> 2 <211> 133 <212> PRT <213> artificial sequence <220> <223> IL-21 - lack of signal peptide <400> 2 Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile 1 5 10 15 Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu 20 25 30 Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser 35 40 45 Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu 50 55 60 Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser 65 70 75 80 Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys 85 90 95 Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys 100 105 110 Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His 115 120 125 Gly Ser Glu Asp Ser 130 <210> 3 <211> 538 <212> PRT <213> artificial sequence <220> <223> IL-21Ra <400> 3 Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu Gln Gly 1 5 10 15 Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr 20 25 30 Val Ile Cys Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr 35 40 45 Leu Thr Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser 50 55 60 Cys Ser Leu His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr 65 70 75 80 Cys His Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val 85 90 95 Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe 100 105 110 Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val 115 120 125 Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp 130 135 140 Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr 145 150 155 160 Arg Asn Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile 165 170 175 Ser Val Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys 180 185 190 Asp Ser Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser 195 200 205 Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln 210 215 220 Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His Leu Leu Leu 225 230 235 240 Leu Leu Leu Leu Val Ile Val Phe Ile Pro Ala Phe Trp Ser Leu Lys 245 250 255 Thr His Pro Leu Trp Arg Leu Trp Lys Lys Ile Trp Ala Val Pro Ser 260 265 270 Pro Glu Arg Phe Phe Met Pro Leu Tyr Lys Gly Cys Ser Gly Asp Phe 275 280 285 Lys Lys Trp Val Gly Ala Pro Phe Thr Gly Ser Ser Leu Glu Leu Gly 290 295 300 Pro Trp Ser Pro Glu Val Pro Ser Thr Leu Glu Val Tyr Ser Cys His 305 310 315 320 Pro Pro Arg Ser Pro Ala Lys Arg Leu Gln Leu Thr Glu Leu Gln Glu 325 330 335 Pro Ala Glu Leu Val Glu Ser Asp Gly Val Pro Lys Pro Ser Phe Trp 340 345 350 Pro Thr Ala Gln Asn Ser Gly Gly Ser Ala Tyr Ser Glu Glu Arg Asp 355 360 365 Arg Pro Tyr Gly Leu Val Ser Ile Asp Thr Val Thr Val Leu Asp Ala 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> IL-21Ra residue <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> fusion proteins <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> Human IL-21Ra ectodomain <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> RV1 H68Q, E38D, Q33H ectodomain <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> RV2 D72E, Y129F, D73E ectodomain <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> RV3 Y129F, H68Q, Y191F ectodomain <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> RV4 Y36F, H68Q, D73E ectodomain <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> RV5 Y129F, F67Y, M130L ectodomain <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> RV6 M70I, D73E, Q33H ectodomain <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> RV7 D72E, L94V, Y191F ectodomain <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> RV8 L94V, Q33H, M130L ectodomain <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> RV9 Y36F, F67Y, E38D ectodomain <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> RV10 D72E, E38D, M130L ectodomain <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> RV11 Y36F, M70I, L94V ectodomain <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> RV12 M70I, F67Y, Y191F ectodomain <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> RV13 M70G, Y129F ectodomain <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> RV14 Y36H ectodomain <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> RV15 F69L M70L D73I ectodomain <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> RV16 Y129H M130F ectodomain <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> RV17 Y129F, M130S ectodomain <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> RV18 D72K, D73K ectodomain <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> RV19 E38K ectodomain <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> RV21 D72E ectodomain <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> RV22 M70G ectodomain <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> RV23 M70L ectodomain <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> RV24 F69L ectodomain <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> RV25 D73I ectodomain <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> RV26 D72K ectodomain <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> RV27 D73K ectodomain <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> RV28 D73E ectodomain <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> RV29 M70G, D73E ectodomain <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> RV30 M70G, D73E, Q33H ectodomain <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> RV31 M70I, D73E ectodomain <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> RV32 M70I, E38K ectodomain <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> RV33 M70I, E38K, D73E ectodomain <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> IL21_CV1_R5Q <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> IL21_CV2_R5D <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> IL21_CV3_R5E <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> IL21_CV4_R5K <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> IL21_CV5_R5N <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> IL21_CV6_I8E <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> IL21_CV7_R9E <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> IL21_CV8_R9D <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> IL21_CV9_R9K <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> IL21_CV10_R9N <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> IL21_CV11_R9Q <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> IL21_CV12_Q12V <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> IL21_CV13_L13D <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> IL21_CV14_K73V <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> IL21_CV15_K73D <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> IL21_CV16_K75D <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> IL21_CV17_R76E <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> IL21_CV18_R76N <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> IL21_CV19_R76A <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> IL21_CV21_R5Q_R76E <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> IL21_CV22_R5Q_R76A <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> IL21_CV204_H6L_M10L_K73V_P78L_P104A <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> IL21_CV374_H6L_R9K_M10L_K73V_P78L_G84E <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> IL21_CV388_H6L_R9K_M10L_K73I_P78L_P104A <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> RV20 F67L ectodomain <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)

As an engineered human IL-21 polypeptide,
Amino acid substitutions numbered as follows with respect to SEQ ID NO: 2, namely:
H6L;
R9K;
M10L;
contains P78L;
Also optionally comprising G84E or P104A or a combination thereof,
and optionally comprising either K73V or K73I.
According to claim 1,
A polypeptide comprising the amino acid substitution K73V.
According to claim 1,
A polypeptide comprising the amino acid substitution K73I.
According to claim 1,
A polypeptide comprising the amino acid substitution G84E.
According to claim 1,
A polypeptide comprising amino acid substitution P104A.
According to claim 1,
wherein said polypeptide binds to an engineered human CD360 protein ectodomain.
According to claim 6,
The engineered human CD360 protein ectodomain is Y10, Q33, Q3, Y36, E38, L39, F67, H68, F69, M70, A71, D72, D73, I74, L94, A96, E97, P126, A127, Y129, M130 , K134, S190 and Y191 is modified at one or more residues selected from the polypeptide.
According to claim 6,
The polypeptide of claim 1 , wherein the engineered human CD360 protein ectodomain is modified at M70.
According to claim 6,
The polypeptide of claim 1 , wherein the engineered human CD360 protein ectodomain is modified at M70 and Y129.
According to claim 8,
The polypeptide of claim 1 , wherein the engineered human CD360 protein ectodomain comprises the amino acid substitution M70G.
According to claim 9,
The polypeptide of claim 1 , wherein the engineered human CD360 protein ectodomain comprises amino acid substitutions M70G and Y129F.
According to claim 1,
A polypeptide further comprising amino acid substitutions K73V and G84E.
According to claim 1,
A polypeptide further comprising amino acid substitutions K73I and P104A.
As an engineered human IL-21 polypeptide,
With respect to SEQ ID NO: 2, a polypeptide comprising a group of amino acid substitutions numbered as follows: H6L, R9K, M10L, K73V, P78L and G83E.
As an engineered human IL-21 polypeptide,
A polypeptide comprising a group of amino acid substitutions numbered as follows for SEQ ID NO: 2: H6L, R9K, M10L, K73I, P78L and P104A.
A system for the selective activation of intracellular receptors, the system comprising:
(a) an engineered receptor comprising the human CD360 protein ectodomain with an amino acid substitution at M70G; and (b) an engineered human IL-21 polypeptide according to claim 1 .
A system for the selective activation of intracellular receptors, the system comprising:
(a) an engineered receptor comprising the human CD360 protein ectodomain with amino acid substitutions at M70G and Y129E; and (b) an engineered human IL-21 polypeptide according to claim 1 .
According to claim 16,
Wherein the engineered human CD360 receptor is expressed in mammalian cells.
According to claim 17,
Wherein the engineered human CD360 receptor is expressed in mammalian cells.
According to claim 18,
Wherein the mammalian cells are immune cells or stem cells.
According to claim 19,
Wherein the mammalian cells are immune cells or stem cells.
21. The method of claim 20,
Wherein the mammalian cell is an immune cell and the immune cell is a T cell.
According to claim 21,
Wherein the mammalian cell is an immune cell and the immune cell is a T cell.
An engineered human IL-21 polypeptide according to claim 1; and a pharmaceutically acceptable excipient.
A kit comprising the system according to claim 16 .
A kit comprising the system according to claim 17 .
According to claim 1,
Wherein the processed human IL-21 polypeptide is fused to the Fc domain of an IgG or albumin.