JP2023549293A - 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 consists of an orthogonal interleukin-21 receptor a chain ("ortho-IL-21Rα") that has impaired binding to the native interleukin-21 cytokine ("IL-21") and a native It may include an orthogonal IL-21 cytokine (“ortho-IL-21”) with impaired binding to IL-21Ra, where ortho-IL-21Ra binds ortho-IL-21. The IL-21 receptor-cytokine pair can activate IL-21 signaling. Cells engineered to express orthogonal IL-21 receptors and methods for using such cells for the treatment of various diseases and disorders are also described.

Description

Cross-References to Related Applications This application is filed in U.S. Provisional Patent Application No. 63/105,414, filed on October 26, 2020, and U.S. Provisional Patent Application No. 63/212, filed on June 18, 2021. , 547, each of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING The Sequence Listing has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The name of the ASCII copy created on October 25, 2021 is Neptune-IL21-PCT_ST25. txt and has a size of 123,571 bytes.

Cytokines are powerful natural regulators of immune cell proliferation and differentiation. This potency makes cytokines very attractive as potential therapeutic agents, but also complicates their clinical utility. This is particularly true for cytokines that have multiple cellular targets and therefore have a high potential for pleiotropic effects. One example is interleukin-2 (“IL-2”), a robust T cell mitogen whose anticancer activity is offset by undesirable proliferation of regulatory (suppressive) T cells and painful vascular leak syndrome. It is. In the specific case of IL-2, protein engineering can be used to solve some of the clinical problems, such as eliminating the ability of cytokines to preferentially act on regulatory T cells. Another approach involves generating orthogonally constrained forms of cytokines and their receptors. Please see below. US Pat. No. 10,869,887; Sockolosky JT, Trotta E, Parisi G, et al. Selective targeting of engineered T cells using orthogonal IL-2 cytokine-receptor complexes. Science. 2018;359(6379):1037-1042. doi:10.1126/science. aar3246. The disclosures of each of these are incorporated herein by reference in their entirety.

Orthogonal cytokine systems are systems in which cytokines and their receptors are mutated such that they lose compatibility with their native (parent) partners but retain the ability to interact productively with each other. Such orthogonal cytokine:receptor pairs can therefore be said to demonstrate "privileged" or "private" interactions. Approaches that generate orthogonally constrained forms of cytokines and their receptors have value in cell therapy because they provide a way to limit the scope of cytokine activity to only the cells that are being treated (i.e., adoptively transferred). There is. These therapeutic cells are the only cells that express the engineered receptor and, therefore, the only cells that can respond to the engineered cytokine.

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

IL-21 is of particular interest because it enhances cytotoxic T cell responses against viruses and tumors and can act synergistically with other cytokines such as IL-2 or IL-15. IL-21 does this, in part, by promoting the persistence of T cells with a stem cell memory phenotype, which has been associated with beneficial outcomes in the context of cell therapy. IL-21 is currently being evaluated as a cancer therapeutic in multiple clinical trials. IL-21 also has significant potential utility in chimeric antigen receptor T (“CAR-T”) cell therapy, with clinical failure due to poor proliferation, antitumor efficacy, exhaustion, suppression and persistence. can help you overcome. Thus, in particular, by engineering desired behaviors into adoptively transferred cells that are protected from endogenous signaling pathways, do not affect untargeted endogenous cells, and can be controlled when administered to a patient. , there is an urgent need for the ability to modulate the effects of IL-21.

In one aspect, the orthogonal interleukin-21 receptor alpha chain (“ortho-IL-21Rα” or “ortho-IL-21Rα molecule”, or a specific ortho-IL-21Rα chain constructed as provided herein) When referring to 21Rα, “RV” as in “receptor variant”) is provided, and ortho-IL-21Rα is referred to as orthogonal interleukin-21 cytokine (“ortho-IL-21” or “ortho-IL-21 molecule,” or “CV” as in “cytokine variant” when referring to a particular ortho-IL-21 constructed as provided herein, but with no binding to native IL-21. Contains a modified amino acid sequence derived from SEQ ID NO: 4 with impaired binding. In one aspect, ortho-IL-21Rα is located at one or more amino acid residues of SEQ ID NO: 4 that contact IL-21, at residues in the immediate vicinity of such contact residues, or at the steric site of the IL-21 binding surface. Contains modified amino acid sequences that include substitutions of residues elsewhere in IL-21Rα that affect conformation. In one aspect, ortho-IL-21Rα is Y10, Q33, Q35, Y36, E38, L39, F67, numbered relative to SEQ ID NO: 6 (mature form of human IL-21α ectodomain lacking signal peptide). , H68, F69, M70, A71, D72, D73, I74, L94, A96, E97, P126, A127, Y129, M130, K134, S190, Y191, or combinations thereof. In one aspect, the amino acid substitution is D72E/Y129F/D73E; M70I/D73E/Q33H; D72E/L94V/Y191F; D72E/E38D/M130L; M70G/Y129F; , or a combination thereof. In one aspect, the amino acid substitution comprises or consists essentially of M70G/Y129F ("RV13" or SEQ ID NO: 19 in the case of "receptor variant 13") or M70G ("RV22" or SEQ ID NO: 27). or consisting of them.

In another aspect, ortho-IL-21 is provided, wherein the ortho-IL-21 binds to ortho-IL-21Rα, but has a modification derived from SEQ ID NO: 2 that has impaired binding to native IL-21Rα. Contains amino acid sequence. In one aspect, ortho-IL-21 is located at one or more amino acid residues of SEQ ID NO: 2 that contact IL-21Rα, at residues in the immediate vicinity of such contact residues, or at the steric site of the IL-21Rα binding surface. Contains modified amino acid sequences that include substitutions of residues elsewhere in IL-21 that affect conformation. In one aspect, ortho-IL-21 is R5, H6, I8, R9, M10, Q12, L13, K73, K75, R76, P78, G84 or P104, numbered relative to SEQ ID NO:2, or including amino acid substitutions at combinations of positions. The phrase "numbered relative to SEQ ID NO: 2" is meant to ignore any epitope tags and signaling peptides for numbering purposes. In one aspect, the amino acid substitutions are R5Q, R5D, R5E, R5K, R5N, H6L, I8E, R9E, R9D, R9K, R9N, R9Q, M10L, Q12V, L13D, K73V, K73D, K73I, K75D, R76E, R76N, Comprising, consisting essentially of, or consisting of R76A, R5Q/R76E, R5Q/R76A, P78L, G84E or P104A, or combinations thereof. In one aspect, the amino acid substitution comprises, consists essentially of, or consists of H6L/R9K/M10L/P78L.

In an exemplary embodiment, the amino acid substitutions numbered relative to SEQ ID NO:2 include: H6L; R9K; M10L; P78L; optionally G84E or P104A or combinations thereof; one of K73V or K73I. Provided are engineered human IL-21 polypeptides optionally comprising: In one aspect, such engineered human IL-21 polypeptide is SEQ ID NO: 61 (H6L/R9K/M10L/K73V/P78L/G84E) or SEQ ID NO: 62 (H6L/R9K/M10L/K73I/P78L/P104A). ) may be included.

In another aspect, a system is provided for activating IL-21 signaling in a cell, the system comprising one or more of the amino acid residues of SEQ ID NO: 4 that contact IL-21, such a contact residue. A native IL comprising a modified amino acid sequence derived from SEQ ID NO: 4, including substitutions of residues in the immediate vicinity of or elsewhere on IL-21Rα that affect the conformation of the IL-21 binding surface. ortho-IL-21Rα with impaired binding to IL-21Rα and one or more amino acid residues of SEQ ID NO: 2 that contact IL-21Rα, residues in the immediate vicinity of such contact residues, or ortho- Modified amino acid sequences derived from SEQ ID NO: 2 containing substitutions of residues elsewhere in IL-21 that affect the conformation of the IL-21Rα binding surface where IL-21Rα binds to ortho-IL-21. ortho-IL-21 with impaired binding to native IL-21Rα. In one aspect, the cell is a mammalian cell, immune cell, stem cell, or T cell.

The present invention can be more easily understood by referring to the following figures.

A schematic diagram of the orthogonal IL-21 system is provided. The leftmost illustration shows the wild-type receptor and cytokine productively interacting with each other, and the adjacent illustration shows the impaired interaction between ortho-IL-21Rα and the native cytokine. The right-most vignette shows a compromised interaction between ortho-IL-21 and the native (wild-type) receptor, and the adjacent vignette shows two orthogonal molecules (ortho-IL-21Rα and ortho- IL-21).

Provides a schematic diagram of a route for generating an orthogonal IL-21 system.

A single (sub-saturating) concentration of IL-21-TLuc16 was added to 20 candidate ortho-IL-21Rα molecules, all of which were bound to the Streptactin-coated surface of the wells at a saturating concentration. Results of a representative assay tested for binding to the panel are shown. Eight of the 20 candidate ortho-IL-21Rα molecules showed reduced ability to bind IL-21-TLuc16.

Figures 4A-4E show the results of a representative assay in which a range of (subsaturating) concentrations of IL-21-TLuc16 was tested for binding to a panel of candidate ortho-IL-21Rα molecules. The panel included the wild-type receptor as a control and seven of the eight candidate ortho-IL-21Rα molecules identified in FIG. 3. The panel also included 13 additional candidate ortho-IL-21Rα molecules designed based on the results such as those shown in Figure 3. Specifically, substitutions present in 8 candidate ortho-IL-21Rα molecules were introduced alone or in combination into 13 additional candidate ortho-IL-21Rα molecules. Twenty-one candidate ortho-IL-21Rα molecules were added at saturating concentrations to Streptactin-coated wells of a 96-well plate. Figures 4A-4E present luminometric data for individual plates comparing the binding of IL-21-TLuc16 to five candidate ortho-IL-21Rα molecules and a wild-type control IL-21Rα in each case. show. Eleven of the additional 13 candidate ortho-IL-21Rα molecules showed a significantly reduced ability to bind IL-21-TLuc16 compared to the wild-type control (the exceptions being each single receptors RV23, RV24 and RV28 with substitutions M70L, F69L and D73E).

Figures 5A and 5B depict IL-21-induced STAT3 signaling responses by Ba/F3 cells expressing native IL-21Rα molecules and eight candidate ortho-IL-21Rα molecules identified as shown in Figure 3. shows. The cells had a STAT3-regulated Cypridina noctiluca luciferase reporter transgene. After exposing them to different concentrations of native IL-21 overnight (approximately 20 hours), the supernatant medium was tested for luciferase activity by luminometry using Vargulin as the enzyme substrate. We show that STAT3 signaling in response to IL-21 was dependent on the cytoplasmic tail of the receptor, as expected, including in cells expressing a form of wild-type IL-21Rα lacking its cytoplasmic tail (Δcyt). Indicated.

Figures 6A-6T show native IL-21 and candidate ortho-ILs inducing signaling through the native IL-21Rα molecule and eight candidate ortho-IL-21Rα molecules identified as shown in Figure 3. - Shows the ability of 21 molecules. Introduction of a STAT3-dependent luciferase reporter present in the same transposon used to confer expression of native IL-21Rα molecules or candidate ortho-IL-21Rα molecules using luminometry as described with respect to Figure 5. Luciferase produced by transfected Ba/F3 cells carrying the gene was quantified. After exposing cells to increasing concentrations of candidate ortho-IL-21 molecules overnight (approximately 20 hours), the supernatant culture medium was tested for luciferase activity.

Figures 7A-7D depict native IL-21 molecules and selected candidates from the collection shown in Figure 6 inducing signaling through native IL-21Rα molecules and selected candidate ortho-IL-21Rα molecules. Demonstrates the potential of ortho-IL-21 molecules. Ba/F3 cells (expressing native IL-21Rα molecules or candidate ortho-IL-21 molecules from a transposon, further carrying a STAT3-luciferase reporter transgene) were treated with increasing concentrations of the indicated candidate ortho-IL-21. After exposure to molecules (approximately 20 hours), supernatant medium was collected and tested for luciferase activity by luminometry as in FIGS. 5 and 6.

Figures 8A, 8B and 8C show wild type IL-21Rα (Figure 8A) or candidate ortho-IL-21Rα containing amino acid substitutions M70G/Y129F (SEQ ID NO: 19) or M70I/D73E/Q33H (SEQ ID NO: 12), respectively. Figure 8 shows the results of a representative screening experiment in which a collection of 96 cytokines were tested for their ability to induce STAT3-dependent signaling responses in Ba/F3 cells expressing the molecules RV13 (Figure 8B) or RV6 (Figure 8C). . STAT3 dependence present on the same transposon used to confer expression of native IL-21Rα molecules or candidate ortho-IL-21Rα molecules using luminometry as described with respect to FIGS. 5 and 6. Luciferase produced by transfected Ba/F3 cells with a luciferase reporter transgene was quantified. After exposing cells to increasing concentrations of candidate ortho-IL-21 molecules overnight (approximately 20 hours), the supernatant culture medium was tested for luciferase activity. The dotted lines indicate the response curves for cytokine assembly, and the responses evoked by the five cytokines of interest (wild-type IL-21, negative control CV22, and candidate ortho-IL-21 molecules CV204, CV374, and CV388) are indicated by the solid lines and highlighted with symbols.

Figures 9A and 9B show that native IL-21Rα induces signaling through wild-type IL-21Rα (Figure 9A) and candidate ortho-IL-21Rα RV13 containing amino acid substitutions M70G/Y129F (SEQ ID NO: 19) (Figure 9B). -21 molecule and candidate ortho-IL-21 molecules. STAT3 dependence present on the same transposon used to confer expression of native IL-21Rα molecules or candidate ortho-IL-21Rα molecules using luminometry as described with respect to FIGS. 5 and 6. Luciferase produced by transfected Ba/F3 cells with a luciferase reporter transgene was quantified. After exposing cells to increasing concentrations of candidate ortho-IL-21 molecules overnight (approximately 20 hours), the supernatant culture medium was tested for luciferase activity.

Figures 10A, 10B and 10C show wild-type IL-21Rα (Figure 10A), candidate ortho-IL-21Rα RV13 containing amino acid substitution M70G/Y129F (SEQ ID NO: 19) (Figure 10B), and amino acid substitution M70G (SEQ ID NO: 19). Figure 10C shows the ability of native and candidate ortho-IL-21 molecules to induce signaling through candidate ortho-IL-21Rα RV22 (Figure 10C), including candidate ortho-IL-21Rα RV22 (No. 27). STAT3 dependence present on the same transposon used to confer expression of native IL-21Rα molecules or candidate ortho-IL-21Rα molecules using luminometry as described with respect to FIGS. 5 and 6. Luciferase produced by transfected Ba/F3 cells with a luciferase reporter transgene was quantified. After exposing cells to increasing concentrations of candidate ortho-IL-21 molecules overnight (approximately 20 hours), the supernatant culture medium was tested for luciferase activity.

Orthogonal IL-21 receptors and orthogonal IL-21 cytokines are provided. Orthogonal IL-21 receptor: cytokine pairs may include ortho-IL-21Rα, which is impaired in binding to native IL-21, and ortho-IL-21, which is impaired in binding to native IL-21Rα, and ortho-IL-21, which is impaired in binding to native IL-21Rα, IL-21Rα binds to ortho-IL-21. Orthogonal IL-21 receptor-cytokine pairs can be used to activate signaling responses in cells. The signaling response may be native, normally downstream of the IL-21 receptor, or alternatively dependent on non-native IL-21 receptors and/or other non-native cellular components. Also provided are cells engineered to express orthogonal IL-21 receptors, and methods for using such cells for the treatment of various diseases and disorders.

DEFINITIONS The terms described herein are for purposes of explanation only and should not be construed as limiting the invention. As used in the specification and the appended claims, the singular forms "a,""an," and "the" include plural referents unless the context clearly dictates otherwise.

"Treat," "treating," "treatment," etc. refers to a subject suffering from a disease or disorder, such as cancer, including ameliorating the condition by alleviating or suppressing at least one symptom, slowing the progression of the disease, etc. Refers to any act that provides benefits to

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

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

The term "polynucleotide" refers to oligonucleotides, nucleotides, or fragments of any of these; of genomic or synthetic origin, which may be single-stranded or double-stranded, and which may represent a sense or antisense strand; DNA or RNA (eg, mRNA, rRNA, tRNA); peptide nucleic acids; or any DNA-like or RNA-like material, whether naturally or synthetically derived. The term can also encompass nucleic acids, such as oligonucleotides, containing known analogs of natural nucleotides, as well as nucleic acid-like structures with synthetic backbones.

The term "polypeptide" refers to an oligopeptide, peptide or protein sequence, or a fragment, portion or subunit of any of these, and naturally occurring or synthetic molecules. The term "polypeptide" also includes amino acids joined to each other by peptide bonds or modified peptide bonds, ie, peptide isosteres, and may contain modified amino acids of any kind. The term "polypeptide" also includes peptides and polypeptide fragments, motifs, and the like. A protein or peptide "chain" refers to a separate subunit of a larger protein or protein complex.

"Homologue" means that the corresponding protein (e.g., IL-21Rα), such as from another species, has an overall effect on the human protein, as long as such homologue peptides retain their respective known activities. Substantially homologous at the primary protein (i.e., mature protein) level. There may be varying levels of homology from 35% to 99%.

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

The phrases "conservative modifications" and "conservative sequence modifications" refer to amino acid modifications that do not significantly affect or change the binding properties of the polypeptide containing the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions and deletions. Modifications can be introduced into proteins by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis, or by DNA synthesis. A conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., Contains amino acids such as tyrosine, phenylalanine, tryptophan, and histidine).

Generally, conservatively substituted variants, homologues and analogs of peptides will differ from the disclosed sequence by at least about 35%, at least about 45%, at least about 55%, at least about 65%, at least about 75%, at least about 80%, at least have about 85%, at least about 90%, at least about 95%, or at least about 96%-99% amino acid sequence identity. RostB. Twilight zone of protein sequence alignments. Protein Eng. 1999;12(2):85-94. doi:10.1093/protein/12.2.85.

Identity or homology to such sequences is defined herein as being conservative as part of sequence identity after aligning the sequences and introducing gaps as necessary to achieve maximum percent homology. It is defined as the percentage of amino acid residues in a candidate sequence that are identical to a known peptide, without considering substitutions. N-terminal, C-terminal or internal extensions, deletions or insertions into the peptide sequence shall not be construed as affecting homology.

Gamma chain (γc) cytokine refers to any cytokine whose cognate cytokine receptor complex contains a common cytokine receptor gamma chain (γc). γc cytokines include IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21.

Orthogonal cytokines: receptor pairs that interact effectively with each other (i.e., so that they can be used to initiate physiologically necessary signaling responses in cells), but not with their natural counterparts. refers to a variant of a naturally occurring cytokine:receptor pair that is significantly impaired in its ability to Orthogonal cytokines are naturally occurring cytokines that have been mutated or otherwise modified (“muteins”) or fully synthetic proteins (sometimes called “synthekins”) that act as agonists on orthogonal cytokine receptors. It can be either. Orthogonal cytokines (whether muteins or syntekins) exhibit no agonist activity or only attenuated agonist activity towards wild-type cytokine receptors.

Thus, an orthogonal cytokine:receptor pair is one that has been engineered (a) to lack or reduce binding to the native cytokine or cognate receptor, and (b) to an engineered (orthogonal) ligand or receptor counterpart. It may include pairs of genetically engineered proteins that are modified by amino acid changes to specifically bind. Upon binding of orthogonal cytokines, orthogonal receptors activate signaling that is transduced through native cellular elements to mimic the native response, but specific to engineered cells expressing orthogonal receptors. provides biological activity. Non-native receptors or cellular elements (eg, non-native cytoplasmic domains in receptors) can be included to modify the signaling response. The orthogonal receptors do not bind, or bind only with reduced willingness, to any endogenous cytokines, including the native counterparts of the orthogonal cytokines, and the orthogonal cytokines include the native counterparts of the orthogonal receptors. It does not bind to any endogenous receptors or binds only with reduced motivation. In some embodiments, the affinity of the orthogonal cytokine for the orthogonal receptor is comparable to the affinity of the native cytokine for the native receptor, e.g., at least about 1%, at least about 5% of the affinity of the native cytokine receptor pair. , at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 100%, greater than the affinity of the native cytokine for the native receptor, e.g., 2-fold, 3-fold, 4-fold. , with an affinity that may be 5-fold, 10-fold or more.

The term "candidate" when used to qualify an orthogonal protein (IL-21 or IL-21Rα) infers a prototypical or developmental state. The analysis procedure can remove qualification if the candidate protein is found to have the desired degree of binding privilege and orthogonal functionality.

As used herein, the phrases "does not bind/do not bind" and "cannot bind" refer to no detectable binding, or insignificant binding, i.e., less than the binding affinity of the natural ligand. refers to having a much lower binding affinity. "Compromised binding" refers to lower than normal levels of binding between corresponding wild-type components (eg, cytokines and receptors).

The present invention includes an orthogonal cytokine system based on IL-21, a member of the γc family of cytokines. IL-21 is structurally related to IL-2 and signals through a dimeric receptor consisting of IL-21Rα and γc. The IL-2 receptor promotes a STAT5-dominated signaling response, whereas STAT3 dominates the IL-21 response. IL-21 promotes a form of T cell differentiation that correlates with favorable outcomes in adoptive cell therapy (“ACT”), particularly ACT using CAR T cells, and compared to IL-2 in various tumor model systems. It exhibits enhanced anti-tumor properties. As a result, IL-21 may prove to be a better adjuvant for ACT than IL-2. Thus, in one aspect, 1) ortho-IL-21 has impaired binding to native IL-21Rα, and 2) is capable of binding ortho-IL-21 but has impaired binding to native IL-21. An IL-21 orthogonal system containing ortho-IL-21Rα is provided (FIG. 1; FIG. 2, Pathway A).

Orthogonal Interleukin-21 Receptors In one aspect, orthogonal interleukin-21 receptors are provided. Orthogonal interleukin-21 receptors may contain modifications of any of the chains that make up the entire protein. In some embodiments, ortho-IL-21Rα is provided. ortho-IL-21Rα contains a modified amino acid sequence derived from wild-type human IL-21Rα (SEQ ID NO: 4 - the mature form of human IL-21Rα lacking the signal peptide). Ortho-IL-21Rα binds ortho-IL-21 but is impaired in binding to native IL-21. Human IL-21Rα is represented by SEQ ID NO:3.

In some embodiments, the modified amino acid sequence affects the conformation of the amino acid residues of SEQ ID NO: 4 that contact IL-21, the residues in the immediate vicinity of such contact residues, or the IL-21 binding surface. including one or more substitutions of residues elsewhere in IL-21Rα that affect the Amino acids within the immediate vicinity are amino acids within 1, 2, 3, 4 or 5 amino acids of the contacting residue in the primary protein sequence, or similarly nearby amino acid residues in the tertiary protein structure. In some aspects, ortho-IL-21Rα is Y10, Q33, Q35, Y36, E38, L39, F67, H68, F69, M70, A71, D72, D73, numbered relative to SEQ ID NO:6. Includes amino acid substitutions at positions I74, L94, A96, E97, P126, A127, Y129, M130, K134, S190 or Y191, or combinations thereof.

One strategy for creating orthogonal versions of IL-21 involves first mutating IL-21Rα so that its ability to bind to IL-21 is reduced. The 20 IL-21Rα amino acids make significant direct contact with IL-21 within the 990 Å ² binding surface. Mutating any of these 20 residues can impair the receptor's ability to bind IL-21 normally. For example, the methionine residue at position 70 of IL-21Rα has been identified as a major contributor to binding interactions. 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, and isoleucine-16). in order to improve contact with IL-21Rα (in particular, arginine-9 and arginine-76 of IL-21 are combined with aspartate-72 and aspartate-73 of IL-21Rα, respectively). (contact) reposition. By changing methionine-70 to a different residue, this rearrangement can be adjusted so that some of these contacts are attenuated or lost. Altering aspartate-72 or aspartate-73 of IL-21Rα may also lower the binding free energy of interaction, resulting in compensation of IL-21 (e.g., at positions 9 and 76). changes will be required to restore it.

Helix C of IL-21 exists in two interchangeable states in the free structure of the cytokine, one disordered and the other α-helical. The α-helical form, like the first part of the CD loop, is stabilized in the complex of IL-21 and IL-21Rα. Helix C of IL-21 contains the aforementioned lysine-73 and arginine-76 proximal to methionine-70 of IL-21Rα. The CD loop of IL-21 contains lysine-77, proline-79, and serine-80 that collectively form a pocket for tyrosine-36 of IL-21Rα, and lysine-77 also contains glutamic acid of IL-21Rα. -38 comes into ionic contact. Changing the CD loop amino acid sequence to a similar sequence found in IL-4 results in a 10-fold enhancement of IL-21 potency measured using a cellular assay. This observation suggests that significant binding energy is spent stabilizing helix C of IL-21. Furthermore, changes in IL-21Rα to glutamate-38 and tyrosine-36 significantly affect IL-21 binding in a manner that may be reversible by compensatory changes to IL-21 in helix C or CD loops. Suggest getting.

Individual 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 a significantly (6 residues) shortened CD loop compared to human and mouse. Together with the absence of tyrosine-36 in all of the 74 available avian IL-21Rα sequences, this suggests that helix C of avian IL-21 is distinct from helix C of human IL-21. This suggests that it is likely to engage with the receptor. This also provides a further basis for modifications to the above binding residues to construct orthogonal IL-21 systems.

Based on structural considerations, candidate amino acids can be identified that can be modified to alter IL-21Rα such that its binding to native IL-21 is impaired. However, these changes must be of a nature that is not incompatible with compensatory changes in IL-21 (ie, changes in IL-21 that restore binding to ortho-IL-21Rα). Because the number of compensatory changes required to restore binding may present too great an engineering challenge or be incompatible with cytokine function (e.g., due to loss of γc binding or or changes in IL-21Rα that result in major changes in its conformation (because the cytokine receptor becomes unstable, difficult to express, immunogenic, or pharmacologically problematic). is undesirable.

At least two types of assays can be used to screen ortho-IL-21Rα for loss of binding to IL-21. One is a direct binding assay, which is performed using purified proteins and sensitive biophysical analytical procedures such as surface plasmon resonance ("SPR") or biolayer interferometry ("BLI"). be able to. Another assay is a functional assay involving appropriate cell lines. Ba/F3 cells have been successfully used for this purpose because they have several useful traits: (i) they do not express endogenous IL-21Rα; (ii) they do not express human IL-21Rα; and (iii) respond to IL-21R signaling by phosphorylating and proliferating STAT3; and (iv ) Enabling the use of STAT3 reporter transgenes (eg, those expressing luciferase) as an easy and attractive means to monitor IL-21 signaling. Jurkat or Molt-3 cells are alternative choices, both have minimal or absent expression of endogenous IL-21Rα, but express γc and are IL-21R signaling competent. . HeLa cells transfected to express γc can also be used.

A strategy for isolating candidate ortho-IL-21Rα involves expressing the variants individually in Ba/F3 cells (or one of the alternatives just mentioned) by transfection. Flow cytometry is used to confirm the presence of candidate ortho-IL-21Rα molecules on the cell surface and the epitope recognized by available antibodies. In one aspect, candidate ortho-IL-21Rα is Y10, Q33, Q35, Y36, E38, L39, F67, H68, F69, M70, A71, D72, D73, I74, L94, A96, E97, P126, A127, Positions of Y129, M130, K134, S190 or Y191, or combinations thereof, where numbers refer to residue positions in the mature IL-21Rα ectodomain (SEQ ID NO: 6) and letters refer to amino acid positions using single letter codes. Refers to identity, including amino acid substitutions, where the first letter is the wild-type residue and the second letter is the substituted residue, if present. In one aspect, the amino acid substitutions are M70G, M70A, M70I, M70L, Y129F, Y129A, Y129S, Y129H, M130F, M130S, Y36H, Y36F, 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, I74V, L94I, L94V, E97D, A127T, Q33N, Q33H, K134R, M1 30I, M130L, comprising, consisting essentially of, or consisting of S190T or a combination thereof. In one aspect, the amino acid substitution is D72E/Y129F/D73E; M70I/D73E/Q33H; D72E/L94V/Y191F; D72E/E38D/M130L; M70G/Y129F; , or a combination thereof. 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], [D72E, 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]. In one aspect, the amino acid substitution comprises, consists essentially of, or consists of M70G/Y129F or M70G.

Ba/F3 cells expressing candidate ortho-IL-21Rα can be incubated with native human IL-21 before being analyzed for signaling responses. In some experiments, a time course can be used to improve the sensitivity and resolution of the assay. Other experiments include comparisons of dose response curves. IL-21 responsiveness can be determined by proliferation of Ba/F3 cells, tyrosine phosphorylation of STAT3 by flow cytometry or immunoblotting, or by detection of a reporter (such as luciferase or secreted alkaline phosphatase) from a STAT3-dependent reporter transgene present in the cell. ) is detected by monitoring the expression of While native IL-21Rα allows for a robust response to IL-21 using either of these analytical techniques, the desired ortho-form of the receptor may have a reduced response or a reduced response. do not have. The ortho-IL-21Rα molecule exhibiting this non-responsive property is a candidate for the orthogonally restricted IL-21 cytokine receptor system.

Orthogonal Interleukin-21 Cytokines Another embodiment includes wild-type human IL-21 (SEQ ID NO: 2 - the mature form lacking the signal peptide) that binds ortho-IL-21Rα but is impaired in binding to native IL-21Rα. provides ortho-IL-21 having a modified amino acid sequence derived from human IL-21). Human IL-21 is represented by SEQ ID NO:1.

In some embodiments, ortho-IL-21 binds ortho-IL-21Rα but is impaired in binding to native IL-21Rα. In a further aspect, the modified amino acid sequence comprises one or more amino acid residues of SEQ ID NO: 2 that contact IL-21Rα, residues in the immediate vicinity of such contact residues, or conformations of the IL-21α binding surface. including substitutions of residues elsewhere in IL-21R that affect IL-21R.

Methods are also available for identifying orthogonal forms of IL-21. The 10 residues of IL-21 are IL-21Rα: arginine-5, arginine-9, arginine-11, glutamine-12, aspartic acid-15, serine-70, lysine-73, arginine-76, lysine-77 and Involved in polar interaction with serine-80. Among 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. Substitutions can be made to any of these residues to overcome the changes present in the candidate ortho-IL-21Rα molecule and restore binding.

The crystal structure of IL-21 bound by IL-21Rα can be used to identify IL-21 residues that are proximal to engineered changes to IL-21Rα. For example, if a change in methionine-70 of IL-21Rα is sufficient to abolish IL-21 binding, compensatory changes to any of the following methionine-70 contact residues in IL-21 restore binding. It is possible to make: arginine-9, glutamine-12, isoleucine-16, lysine-73 and arginine-76. However, in addition to methionine-70, these five IL-21 residues contact the following IL-21Rα residues: tyrosine-10, leucine-39, glutamate-38, phenylalanine-67, alanine-71. , aspartate-72, aspartate-73, tyrosine-129, methionine-130 and tyrosine-191. The ten IL-21Rα residues just mentioned then mediate further contacts with the IL-21 residues arginine-5, isoleucine-8, glutamine-19, serine-70 and lysine-77. . Therefore, simply considering the direct contacts present in the native IL-21:IL-21Rα crystal structure, a single substitution at position methionine-70 in IL-21Rα would result in 10 IL-21 residues (arginine- 5, isoleucine-8, arginine-9, glutamine-12, isoleucine-16, glutamine-19, serine-70, lysine-73, arginine-76, and lysine-77). This infers that substitution compensation (ie, restoration of IL-21 binding) may be achieved by changes to one or more of these ten residues. It is also possible that changes to residues that are not in direct contact with IL-21Rα may result in conformational adjustments that act at a distance to allow binding to be restored for methionine-70 substituted variants of IL-21Rα. It is possible.

Phage display or yeast display technology can allow screening of large mutational spaces. This is typically achieved by creating highly diverse libraries of variants in which a small number of residues in the protein are changed in a random (or semi-random) combinatorial manner. The library is then screened for the desired properties. In the case of the present invention, screening may include identifying members of a library of IL-21 variants that are capable of binding candidate ortho-IL-21Rα. If the substitution at methionine-70 is sufficient to abolish binding of IL-21Rα to native IL-21, as in the example just described, there are 10 contact residues that may be affected. ILs with random combination mutations in groups (arginine-5, isoleucine-8, arginine-9, glutamine-12, isoleucine-16, glutamine-19, serine-70, lysine-73, arginine-76 and lysine-77) -21 libraries can be constructed. The theoretical complexity of such a library can exceed 10 ¹³ . Generating libraries comprised of more than 10 ⁸ -10 ⁹ variants is typically difficult and impractical. Screening of such libraries was performed in such a way that phage or yeast displaying variant forms of IL-21 attached to their surface were transferred to matrices coated with candidate ortho-IL-21Rα (coated with wild-type IL-21Rα). may include a selection process in which cells are separated based on their ability to adhere (rather than to a matrix). Iterative cycles of such selection are performed to enrich for desired properties in IL-21. Analytical screening procedures (including SPR or BLI) are used to characterize the products of selection in detail and identify those with optimal properties.

In one aspect, ortho-IL-21 is located at one or more amino acid residues of SEQ ID NO: 2 that contact IL-21Rα, at residues in the immediate vicinity of such contact residues, or at the steric site of the IL-21Rα binding surface. Contains modified amino acid sequences that include substitutions of residues elsewhere in IL-21 that affect conformation. In one aspect, ortho-IL-21 is R5, H6, I8, R9, M10, Q12, L13, K73, K75, R76, P78, G84 or P104, numbered relative to SEQ ID NO:2, or including amino acid substitutions at combinations of positions. In one aspect, the amino acid substitutions are R5Q, R5D, R5E, R5K, R5N, H6L, I8E, R9E, R9D, R9K, R9N, R9Q, M10L, Q12V, L13D, K73V, K73D, K73I, K75D, R76E, R76N, Comprising, consisting essentially of, or consisting of R76A, R5Q/R76E, R5Q/R76A, P78L, G84E or P104A, or combinations thereof. In one aspect, the amino acid substitution comprises, consists essentially of, or consists of H6L/R9K/M10L/P78L.

Additional Methods for Identifying Orthogonal IL-21 Receptor-Cytokine Pairs An alternative approach for identifying ortho-IL-21 molecules involves making direct intermolecular contacts in the IL-21:IL-21Rα structure. It involves iterative mutagenesis cycles refocusing on a small number of selected residues. This approach may also include residues close to the point of contact and/or of potentially related structural features. A broader version of this approach could include any of the proximal residues throughout the contact region with IL-21Rα, as well as additional semi-randomly selected residues in the proximal structural features. A more focused version identifies residues that may be directly affected by specific substitutions present in candidate ortho-IL-21Rα, such as the 10 residues potentially associated with the methionine-70 substitution. including. An alternative broad approach is to consider that substitutions take minimal, if any, account of the structure of IL-21, but instead focus in part on substitutions that occur in IL-21 orthologs from other species. This is done with the This approach involves single substitutions at individual residues throughout the primary sequence of IL-21.

The iterative process begins with a large number (eg, 10-100) of candidate ortho-IL-21 forms. In some versions of this approach, double or triple mutants are included in the initial set, while in other versions only single point mutations in IL-21 are evaluated. These candidate ortho-IL-21 molecules are tested for activity using the cellular assays described above (eg, using Ba/F3 cells with a STAT3-dependent luciferase reporter transgene). A minimum of two types of cells are used in the assay: cells expressing candidate ortho-IL-21Rα and, as a counterscreen, cells expressing wild-type IL-21Rα.

The likelihood of success of an alternative approach corresponds to the number of candidate ortho-IL-21Rα molecules investigated. By expanding this number, it is possible to inadvertently select candidate ortho-IL-21Rα for which IL-21 binding cannot be easily (even partially) restored with a small number (e.g., less than 3) substitutions. The characteristics are reduced. Expanding this number also increases the likelihood of being able to isolate parallel, mutually orthogonal systems that exhibit no crosstalk with each other or with wild-type IL-21 or IL-21Rα.

We deconvolved the data from the first round of screening and focused on identifying substitutions in IL-21 that alone promoted improved binding to candidate ortho-IL-21Rα molecules and decreased binding to native IL-21Rα. can be analyzed. A second round of screening may be performed, in which the positive scoring substitutions from the first round are combined in a new candidate ortho-IL-21 molecule. These candidate ortho-IL-21 molecules (and further variants in which conservative or non-conservative substitutions are made at positive scoring positions, if considered desirable) have improved binding to candidate ortho-IL-21Rα molecules. and tested again for impaired binding to native IL-21Rα.

including combinations of further substitutions until at least one candidate ortho-IL-21 is isolated with the desired properties (lack of activity with native IL-21Rα and near normal activity with at least one candidate ortho-IL-21Rα). Further screening may be performed.

Alternative screening approaches are facilitated in some situations using candidate ortho-IL-21Rα molecules that retain reduced (but not completely absent) binding to wild-type IL-21. obtain. Such a reduced binding candidate ortho-IL-21Rα molecule is more likely than an unbound candidate ortho-IL-21Rα molecule (i.e., a candidate ortho-IL-21Rα molecule lacking any binding to wild-type IL-21). , may prove possible to restore some IL-21 binding activity by a small number (eg, less than 3) of individual substitutions in IL-21. Once candidate ortho-IL-21 molecules have been isolated by the screening procedures outlined above, further screening steps can be performed involving new candidate ortho-IL-21Rα molecules in which additional substitutions are combined with those already present. In some aspects, these additional mutations may completely eliminate binding to wild-type IL-21 while retaining the ability to bind ortho-IL-21. This receptor mutagenesis may be performed multiple times, with subsequent rounds of purified cytokine mutagenesis.

The binding properties of the products of the screening approach can be analyzed using purified proteins and BLI or SPR. Products that most closely resemble wild-type IL-21 and IL-21Rα in their binding kinetics may be selected as candidate orthogonal IL-21 systems.

In one aspect, candidate ortho-IL-21 molecules are described in US Pat. al. Mapping of amino acid substances 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. Exploring sequence-function space of a poplar glutathione transfer using designed information-rich gene variants. Protein Eng Des Sel. 2017;30(8):543-549. doi:10.1093/protein/gzx045; and Liao J, Warmuth MK, Govindarajan S, et al. Engineering proteinase K using machine learning and synthetic genes. BMC Biotechnol. 2007;7:16. Published 2007 Mar 26. doi:10.1186/1472-6750-7-16, each of which is incorporated herein by reference in its entirety.

In one aspect, the candidate ortho-IL-21 molecule comprises a modified amino acid sequence derived from SEQ ID NO: 2, above, to facilitate purification and increase the stability and half-life of the ortho-IL-21 molecule in vivo. , or expressed as a fusion protein between the ortho-IL-21 molecule and a second amino acid sequence that improves the drug properties important to the successful administration of the ortho-IL-21 molecule in a patient. Suitable second amino acid sequences are known in the art and include, but are not limited to, serum albumin, Fc fragments of IgG, single chain Fc antibody fragments, ABD035, and the like. Fc fragments can be modified, eg, using electrostatic steering mutations, to prevent, or at least significantly limit, homodimer formation.

Orthogonal IL-21 Receptor-Cytokine System Another embodiment provides a system for activating IL-21 signaling in a cell. This system uses one or more of the amino acid residues of SEQ ID NO: 4 that make contact with IL-21, residues in the immediate vicinity of such contact residues, or IL-21 that affect the conformation of the IL-21 binding surface. Contacting IL-21Rα with a candidate ortho-IL-21Rα that is impaired in binding to native IL-21, comprising a modified amino acid sequence derived from SEQ ID NO: 4, including substitutions of residues elsewhere in -21Rα. one or more amino acid residues of SEQ ID NO: 2 that bind to ortho-IL-21, residues in the immediate vicinity of such contact residues, or the conformation of the IL-21Rα binding surface where ortho-IL-21Rα binds to ortho-IL-21. ortho-IL-21 with impaired binding to native IL-21Rα, including a modified amino acid sequence derived from SEQ ID NO: 2, including substitutions of residues elsewhere in IL-21 that affect the .

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

Orthogonal Cytokine and Receptor Expression Vectors Another aspect provides expression vectors comprising a nucleic acid encoding any of the candidate orthogonal IL-21 proteins (eg, receptors or cytokines) described herein. Orthogonal proteins such as ortho-IL-21 or ortho-IL-21Rα can be produced by recombinant methods. Ortho-IL-21Rα can be introduced into engineered cells on an expression vector. DNA encoding orthogonal proteins can be obtained from a variety of sources engineered during the engineering process.

Amino acid sequence variants can be prepared by introducing appropriate nucleotide changes into the nucleic acid coding sequence encoding the protein. Nucleic acid codons encoding amino acids are known to those skilled in the art. The particular codons selected can be chosen to optimize expression in the host cell used. Amino acid variants may represent insertions, substitutions, and certain deletions of residues described herein. Any combination of insertions, substitutions and specific deletions may be made to arrive at the final construct, provided that the final construct has the desired biological activity as defined herein. In some embodiments, the nucleic acid encodes ortho-IL-21Rα as described herein.

Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, if the signal sequence DNA is expressed as a pre-protein involved in secretion of the polypeptide, the signal sequence DNA is operably linked to the polypeptide DNA, and if a promoter or enhancer influences transcription of the sequence. , a promoter or enhancer is operably linked to a coding sequence, or a ribosome binding site is operably linked to a coding sequence if 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, contiguous and ready for reading. However, some sequences, such as enhancers, do not need to be contiguous to be effective.

A nucleic acid encoding ortho-IL-21 or ortho-IL-21Rα can be inserted into a replicable vector for expression. Many such vectors are available. Vector components generally include, but are not limited to, one or more of an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Vectors 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, which is incorporated herein by reference in its entirety.

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

Expression vectors typically contain a selection gene, also called a selection marker. A selection gene encodes a protein necessary for the survival or proliferation of transformed host cells grown in a selective medium. Host cells that are not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes are those that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate or tetracycline, (b) complement an auxotrophic deficiency, or (c) are obtained from complex media. encode proteins that provide important nutrients that cannot be

The expression vector can contain a promoter that can be recognized by the host organism and operably linked to the orthogonal protein coding sequence. Promoters are untranslated sequences located upstream (5') of the start codon (generally within about 100-1000 bp) of structural genes that control the transcription and translation of specific nucleic acid sequences to which they are operably linked. obtain. Such promoters typically fall into two classes: inducible and constitutive. An inducible promoter is a promoter that, under the control of the DNA, initiates an increase in the level of transcription from the DNA in response to some change in culture conditions, such as a change in the presence or absence of nutrients or temperature.

Transcription from vectors in mammalian host cells can be performed using, for example, polyomaviruses, fowlpox virus, adenoviruses (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retroviruses (such as mouse stem cell virus). , from the genomes of viruses such as hepatitis B virus and simian virus 40 (“SV40”), from heterologous mammalian promoters such as the actin promoter, phosphoglycerate kinase (“PGK”), or from immunoglobulin promoters, from heat shock promoters; provided that such promoters are compatible with the host cell system. The SV40 early and late promoters are conveniently obtained as an SV40 restriction fragment that also contains 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, that act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent and are found 5' and 3' to transcription units within introns as well as within the coding sequence itself. Many enhancer sequences are known from mammalian genes (eg globin, elastase, albumin, alpha-fetoprotein and insulin). However, typically enhancers from eukaryotic viruses are used. Examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Enhancers can be spliced into the expression vector at positions 5' or 3' to the coding sequence, but are preferably located 5' from the promoter.

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

Expression vectors may also be constructed with inducible regulatory elements for the purpose of controlling the expression of a transgene (e.g., encoding ortho-IL-21 or ortho-IL-21Rα) with small molecules or other stimulatory agents. obtain. Examples of regulatory elements include, but are not limited to, promoters that include a tetracycline operator that renders them susceptible to regulation by tetracycline or its derivatives (such as doxycycline). Promoters can also be inducibly regulated by CRISPRa (activation of clustered regularly interspaced short palindromic repeats) using fusions of transcriptional effectors and catalytically killed Cas9. Such promoters may in turn be downstream of other control systems, such as those involving dimerizing agents (antibody-based and/or chemical in nature) or Notch receptor-based components.

Engineered Cells One aspect of the invention provides cells engineered to express ortho-IL-21Rα. Cells can be genetically engineered to contain any suitable expression vector described herein. In some embodiments, the expression vector includes a coding sequence encoding an orthogonal receptor, and the coding sequence is operably linked to a promoter that is active in the desired cell. A variety of vectors may be used for this purpose, such as transposons, viral vectors, plasmid vectors, and minicircle vectors, which may be integrated into the target cell genome or maintained episomally. good. In one aspect, the expression vector is a synthetic transposon that can be integrated into the genome by a transposase enzyme. Examples of transposon/transposase systems include Sleeping Beauty, PiggyBac, LeapIn® and derivatives thereof from ATUM Bio.

The engineered cell can be a host cell for preparing recombinant proteins in vitro. Suitable host cells for recombinant expression of orthogonal proteins include prokaryotes, yeast, and higher eukaryotes, including various mammalian host cell lines.

In some embodiments, the engineered cells are further modified beyond expression of ortho-IL-21Rα. Modifications suitable for use in engineered cells are known in the art and include CARs, T-cell receptors ("TCRs"), or other receptors or receptors that recognize specific antigens on antigen-presenting cells. Including expression of receptor derivatives.

In some embodiments, the engineered cells are cells intended for therapeutic use. Examples of therapeutically engineered cells include stem cells, such as hematopoietic stem cells, natural killer ("NK") cells, or T cells. In some embodiments, the engineered cell is a T cell. The term "T cell" refers to a mammalian immune effector cell that can be characterized by the expression of CD3 and/or the T cell antigen receptor, and which can be engineered to express ortho-IL-21Rα. In some embodiments, the T cell is a naive CD8+ T cell, an activated CD8+ T cell, or a post-activated CD8+ T cell; a cytotoxic CD8+ T cell; a naive CD4+ T cell, an activated CD4+ T cell, or a post-activated CD4+ T cell; a helper T cell, For example, TH1, TH2, TH9, TH11, TH22 and TFH; regulatory T cells such as TR1, natural TReg and inducible TReg; and memory T cells such as central memory T cells, effector memory T cells, NKT cells and γδ selected from T cells.

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

Therapeutic cells can also be engineered to express ortho-IL-21. This can be accomplished using any method suitable for ectopic expression of ortho-IL-21Rα. Ortho-IL-21 can be expressed in the same or different cells that express ortho-IL-21Rα, allowing autocrine or paracrine effects, respectively. An example of a paracrine configuration may be a CD4+ T cell expressing ortho-IL-21 and a matched CD8+ T cell expressing ortho-IL-21Rα.

In some embodiments, ortho-IL-21 can be expressed in a membrane-tethered form. This has been previously achieved with native IL-21 by fusing the cytokine to the amino terminus of the IgG4 CH ₂ -CH ₃ moiety, which is itself fused to the CD4 transmembrane domain. Related strategies have been used to tether other cytokines to cell membranes. Such membrane tethering limits the diffusion of cytokines and limits their action to the immediate vicinity of cells expressing membrane-bound cytokines. In vivo, this approach is utilized to ensure that ortho-IL-21Rα-expressing cells encounter ortho-IL-21 only when in close proximity to specific cell types and/or locations in the body. be able to. In vitro, this approach promotes certain selective differentiation protocols (e.g., differentiation of NK cells from stem cells in the presence of K562 [or other] feeder cells expressing membrane-bound IL-21 and CD137L). It is possible.

The engineered cells can be provided in pharmaceutical compositions suitable for therapeutic use, eg, treatment of humans. Therapeutic formulations containing such cells can be frozen or prepared for administration in the form of an aqueous solution with physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition , Osol, A. Ed. (1980)). The cells may be formulated, dosed, and administered in a manner consistent with good medical practice.

Methods of Treatment 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 conditions. The engineered properties in this therapy include beneficial T-cell differentiation, resistance to exhaustion, capacity for long-term persistence, anamnestic response, and built-in safety features that allow the response to be shut down when it becomes pathogenic. It can be possible.

By engineering cells from a recipient or donor by introducing ortho-IL-21Rα of the invention and stimulating ortho-IL-21Rα by contacting the engineered cells with ortho-IL-21, cellular responses can be achieved. A method is provided for enhancing. The subject methods can include obtaining target cells, such as T cells, hematopoietic stem cells, etc., which can be isolated from a biological sample or derived in vitro from a progenitor cell source. Cells may be transduced or transfected with an expression vector containing sequences encoding ortho-IL-21Rα, and this step may be performed in any suitable medium.

In some embodiments, the engineered T cells can be contacted with ortho-IL-21 in vivo, i.e., the engineered T cells are transferred to a recipient and an effective amount of ortho-IL-21 is administered. are injected into recipients, allowing them to contact engineered T cells in their native environment, such as lymph nodes. In other embodiments, the contacting is performed in vitro. In such in vitro embodiments, contacting can be accomplished using soluble ortho-IL-21 with or without fusion to another protein moiety, such as an immunoglobulin Fc domain. In further such in vitro embodiments, contact can be achieved by encountering other cells expressing secreted ortho-IL-21 or membrane-tethered ortho-IL-21.

Another embodiment includes introducing an engineered cell expressing ortho-IL-21Rα into a subject and activating the cell by contacting it with an effective amount of ortho-IL-21. Provides a method for treating a subject in need of. In some embodiments, the cell is a T cell, and in further embodiments, the cell is a CAR-T cell. In another aspect, the cell is a T cell expressing a native or modified TCR. In another embodiment, the cells are NK cells. In another embodiment, the cell is a macrophage or other myeloid cell or white blood cell.

In some embodiments, 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 be modified, for example, with electrostatic steering or other mutations, to prevent, or at least significantly limit, homodimer formation.

Another embodiment involves introducing into the subject an engineered cell that expresses ortho-IL-21Rα, and introducing a second engineered cell that expresses ortho-IL-21. A method for treating a subject is provided. In some embodiments, the cell is a T cell, and in further embodiments, the cell is a CAR-T cell.

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

In some embodiments, the subject being treated has been diagnosed with cancer. "Cancer" and "malignant tumor" are used synonymously and refer to the uncontrolled, abnormal growth of cells, the ability of diseased cells to spread locally or to other parts of the body via the bloodstream and lymphatic system (i.e. , metastases), and any of several diseases characterized by any of several characteristic structural and molecular features. "Cancer cell" refers to a cell in an early, intermediate, or advanced stage of a multistage neoplastic progression. Characteristics of early, intermediate and advanced stages of neoplastic progression have been described using microscopy. Cancer cells in each of the three stages of neoplastic progression generally have an abnormal karyotype, including translocations, inversions, deletions, isochromosomes, monosomy, and supernumerary chromosomes. Cancer cells are classified into "hyperplastic cells," i.e., cells in the early stages of malignant progression, "dysplastic cells," i.e., cells in the middle stages of neoplastic progression, and "neoplastic cells," i.e., cells in the advanced stages of neoplastic progression. contains cells. Examples of cancer are sarcoma, breast cancer, lung cancer, brain cancer, bone cancer, liver cancer, kidney cancer, colon cancer and prostate cancer. In some embodiments, the engineered cells are used to treat cancer selected from the group consisting of colon cancer, brain cancer, breast cancer, fibrosarcoma, and squamous cell carcinoma. In some embodiments, the cancer is selected from the group consisting of melanoma, breast cancer, colon cancer, lung cancer, and ovarian cancer. In some embodiments, the cancer being treated is metastatic cancer.

In the case of cancer treatment, the treatment method may further include the step of excising the cancer. Cancer removal can range from cryoablation, thermal ablation, radiotherapy, chemotherapy, radiofrequency ablation, electroporation, alcohol ablation, high-intensity focused ultrasound, photodynamic therapy, administration of monoclonal antibodies, and administration of immunotoxins. This can be achieved using a method selected from the group consisting of:

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

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

To carry out the treatment, one or more cells of the subject can be contacted with ortho-IL-21. When cells are contacted with ortho-IL-21 in vitro, ortho-IL-21 directs signaling from ortho-IL-21Rα, which may utilize native cellular machinery, e.g., accessory proteins, co-receptors, etc. It can be added to the engineered cells at a dose and for a period sufficient to activate them. Any suitable medium may be used. Cells activated in this manner can be used for any desired purpose, including experimental purposes regarding determination of antigen specificity, cytokine profiling, etc., and for in vivo delivery.

If contacting is performed in vivo, an effective amount of engineered cells expressing ortho-IL-21Rα is injected into the recipient in combination with or prior to administration of ortho-IL-21. Dosage amount and frequency may vary depending on the drug, mode of administration, etc. Dosages may also be varied for local administration (eg, intranasal, inhalation, etc.) or systemic administration (eg, intramuscular, intraperitoneal, intravenous, etc.). Generally, at least about 10 ⁴ engineered cells/kg, at least about 10 ⁵ engineered cells/kg, at least about 10 ⁶ engineered cells/kg, at least about 10 ⁷ engineered cells/kg cells/kg or more are administered.

Examples Example 1: Binding Assay for Identification of Candidate Ortho-IL-21Rα Molecules with Impaired Binding to Native IL-21 Direct Interaction Assay (IL-21Rα Binding to Native or Mutant Forms of IL-21) provides an alternative to cell-based assays to identify variants of IL-21Rα with impaired IL-21 binding activity (ie, candidate orthogonal variants). The feasibility of utilizing such an assay is enhanced by the fact that the native interaction (IL-21Rα:IL-21) is robust (KD approximately 70 pM). Among the many possible forms of binding assays, those involving luciferase fusion proteins are attractive because of their simplicity and the possibility of being adapted to medium- or high-throughput formats.

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

In either form of binding assay, the desired orientation of the immobilized receptor or cytokine can be achieved through the use of affinity tags such as Twin-Strep-Tag II, a high affinity peptide ligand for the Streptactin protein. Can be done. The IL-21Rα ectodomain with the carboxy-terminal Twin-Strep-Tag II peptide can be efficiently and selectively immobilized on the surface of the wells of a 96-well plate precoated with Streptactin protein. In this way, the immobilized IL-21Rα should be oriented such that its cytokine binding domain is distal from the plate surface. Similarly, IL-21 can also be immobilized in a related manner if it has an amino- or carboxy-terminal Twin-Strep-Tag II peptide tag.

The assay consisted of a plate-immobilized human IL-21Rα ectodomain (via a carboxy-terminal Twin-Strep-Tag II) and a 16KD Turbo-luciferase polypeptide (ThermoFisher) attached to the carboxy-terminus of human IL-21 (via a glycine-serine-containing peptide). (via) linked fusion protein (SEQ ID NO: 5) (“IL-21-TLuc16” in Figure 3). Bound IL-21 was detected using the TurboLuc Luciferase One-Step Glow Assay Kit (ThermoFisher) and standard luminometry. This assay could also be established using luciferase fused to the amino terminus of IL-21 or alternative forms of luciferase (eg, NanoLuc; ThermoFisher).

Twenty candidate ortho-IL-21Rα molecules were tested for their ability to bind IL-21-TLuc16. Wild-type human IL-21Rα ectodomain (mature form lacking signal peptide) (V0 (SEQ ID NO: 6)) and 20 candidate ortho-IL-21Rα molecules shown in Table 1 (SEQ ID NO: 7-25 & 63) (substitutions in the header) and underlined in the sequence) were expressed as secreted proteins in HEK 293 cells.

The clarified supernatant from the transiently transfected cells was tested for the presence of IL-21Rα using an enzyme-linked immunosorbent assay (“ELISA”) consisting of Streptactin-coated plates, and the supernatant was diluted. and was detected using a combination of a mouse monoclonal antibody specific for human IL-21Rα, a horseradish peroxidase-conjugated rat antibody specific for mouse IgG, and a chromogenic substrate for peroxidase. This ELISA established the necessary dilutions to ensure saturation of Streptactin-coated wells with each of the candidate ortho-IL-21Rα molecules. Candidate ortho-IL-21Rα-saturated wells were incubated with a solution of IL-21-TLuc16 for 1 hour (or longer in some experiments) at 4°C (or room temperature in some experiments). After washing the wells, a luciferase substrate (coelenterazine) solution was added and luminometry was performed.

Figure 3 shows a single (sub-saturating) concentration of IL-21-TLuc16 applied to 20 candidate ortho-IL-21Rα molecules (SEQ ID NOs: 7-25 & 63), all of which were applied to the Streptactin-coated surface of the well at a saturating concentration. Figure 2 shows the results of a representative assay tested for binding to a panel of cells (which were bound) to a panel. Eight of the candidate ortho-IL-21Rα molecules showed reduced ability to bind IL-21-TLuc16. Replicate experiments (including titration of IL-21-TLuc16) confirmed the results.

Eight candidate IL-21Rα molecules (“receptor variants” or “RVs”) showed impaired binding to IL-21-TLuc16 (i.e., RV2, RV6, RV7, RV10, RV13, RV15, RV18, and RV19). ), with the ultimate goal of isolating a form of the cytokine that productively binds (with respect to signal transduction and immunological activity) to candidate ortho-IL-21Rα, but not to native IL-21Rα. , can be tested for their ability to bind to candidate ortho-IL-21.

An additional 13 candidate ortho-IL-21Rα molecules with alternative combinations of amino acid substitutions present in the 8 candidate ortho-IL-21Rα molecules were similarly tested for their ability to bind IL-21-TLuc16. did. The 13 candidate ortho-IL-21Rα molecules shown in Table 2 (SEQ ID NOs: 26-38) (substitutions are indicated by underlining in the header and sequence) were expressed in HEK 293 cells as secreted proteins.

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

Example 2: Attenuated IL-21-mediated signaling mediated by candidate Ortho-IL-21Rα molecules revealed using Ba/F3 cell assays and STAT3-responsive reporter constructs.
To further test the extent to which candidate ortho-IL-21Rα candidates were impaired in their ability to bind IL-21, eight candidates from Example 1 and Figure 3 were cultured in a lymphoid cell line, namely Ba/F3 cells ( was expressed in a mouse pre-B cell lymphoma line). Ba/F3 cells are dependent on the cytokine IL-3 for proliferation, but also proliferate robustly in response to IL-21 if initially made positive for expression of IL-21Rα.

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

As an example, an RV13 expression construct was prepared by oligonucleotide-dependent DNA synthesis by ATUM (www.atum.bio). This plasmid is over 13 Kb in size and contains first the genomic insulator sequence (from the human D4Z4 locus on one side and the chicken locus encoding β-globin on the other side) and then the transposon inverted terminal repeat sequence. Contains four independent genes within a piece of DNA sandwiched between. The insulator sequence is intended to protect the genes within the transposon from positional effects that could reduce or diversify expression (ie, effects that depend on the site of transposon integration in the genome). The inverted terminal repeats are recognized by ATUM's proprietary LeapIn® transposase enzyme, which mediates integration of the transposon into genomic DNA. The four genes present within the transposon are listed in Table 3 (in the order in which they occur within the transposon). Genes and their containing transposons were designed according to standard molecular biology principles that are compatible with construct assembly methodologies routinely used by ATUM. Variants of this vector encoding wild-type or other candidate ortho-IL-21Rα molecules were generated by making appropriate changes to the fourth gene listed in Table 3.

Transposon vectors encoding either wild-type IL-21Rα or candidate ortho-IL-21Rα molecules were transfected with the relevant LeapIn® using either MaxCyte ATx or ThermoFisher Neon instruments according to the manufacturer's instructions. Ba/F3 cells were co-transfected with in vitro transcribed mRNA encoding the transposase. Puromycin selection (>1 μg/ml) was imposed 48 hours after transfection and continued for at least 1 week after all cells in the untransfected control cultures had died. Flow cytometry was used to confirm that puromycin-selected cells showed uniform expression of IL-21Rα.

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

As expected, Ba/F3 cells were unable to proliferate when IL-3 was not provided. When expressing the wild-type form of IL-21Rα, they showed IL-21 dose-dependent proliferation (by ATP or eLuc assays as described above). There was also an IL-21 dose-dependent accumulation of Cypridina luciferase (from the STAT3 regulated reporter gene) in the supernatants from treated cultures (Figures 5A-5B). Cypridina luciferase secretion is dependent on the IL-21Rα cytoplasmic domain (because the tailless form of IL-21Rα [Δcyt] did not induce proliferation in response to IL-21; Figure 5A) ; secretion was also significantly impaired when the tyrosine residue in the cytoplasmic domain was replaced with a phenylalanine residue (not shown).

Cypridina noctiluca luciferase activity was easily detected by adding the relevant luciferase substrate (Vargulin) to a sample of supernatant fluid from the cells and measuring the luminescence using a luminometer. Figures 5A and 5B show luminescence (relative light units) after mixing 20 μL of supernatant from each of the wells with 50 μL of VLAR-2 reagent buffer (Targeting Systems) containing Vargulin at the manufacturer's recommended concentration. In other words, it shows the luciferase activity detected as RLU).

The results confirmed the binding assay findings described and shown in Example 1 and FIG. 3 regarding impaired IL-21-dependent signaling mediated by the eight candidate ortho-IL-21Rα molecules.

Example 3: Screening of candidate Ortho-IL-21 molecules: Testing for STAT3-dependent signaling responses in cells expressing candidate Ortho-IL-21Rα molecules or wild-type IL-21Rα. Wild-type or candidate ortho-IL-21 molecules were produced from transiently transfected HEK-293 cells according to procedures used by researchers. The expression vector for this purpose carried the IL-21 open reading frame downstream of the optimized cytomegalovirus immediate early gene 1 promoter. The signal peptide from the human IL-2 gene was used instead of the native one. Epitope tags for detection, quantification, immobilization or purification were fused to the amino or carboxy terminus of the IL-21 coding sequence. In one series of vectors (including those encoding forms of IL-21 identified in Table 4 as SEQ ID NOS: 39-60), the element fused to the amino terminus is a Twin-Strep-Tag followed by a 3 The element fused to the carboxy terminus contains two copies of the same glycine-glycine-glycine-glycine-serine linker followed by an N-Myc epitope tag ( 9E10 monoclonal antibody). A second series of vectors (including those encoding forms of IL-21 identified in Table 4 as SEQ ID NOS: 61-62) did not feature a tag at the carboxy-terminus, but at the amino-terminus of IL-21. had the following elements: Twin-Strep-Tag, immediately followed by an N-Myc epitope tag, then three copies of a glycine-glycine-glycine-glycine-serine linker moiety.

Cultures (10 mL) of transfected cells were maintained for approximately 5 days (until viability decreased to less than approximately 50%). The supernatant was collected, filtered, and aliquoted. Quantification of IL-21 present in the supernatant was achieved using ELISAs (and in some cases biolayer interferometry assays) directed against various epitope tags or specific for the IL-21 moiety.

Selected candidate ortho-IL-21 molecules (mature protein sequences containing epitope tags but without signal peptide) are shown in Table 4 (SEQ ID NOs: 39-62) (substitutions are indicated by underlining in the header and sequence).

Ba/F3 cells expressing wild-type IL-21Rα or the eight candidate ortho-IL-21Rα molecules from Example 1 and FIG. It stimulated me. The signaling response of Ba/F3 cells to ortho-IL-21 molecules was monitored as described above using the STAT3-luciferase assay.

Ba/F3 cells were treated with four concentrations (100, 50, 25 and 12.5 ng/mL) of the indicated candidate ortho-IL-21 molecules (Figures 6A-6J: R5Q, R5D, R5E, R5K, R5N, Candidate ortho-IL-21 molecules and wild type IL-21 including I8E, R9E, R9D, R9K, R9N, R9Q; Figures 6K-6T: Q12V, L13D, K73V, K73D, K75D, R76E, R76N, R76A, R5Q /R76E, R5Q/R76A, and wild-type IL-21). Ba/F3 cells differed in the morphology of the expressed ortho-IL-21Rα molecules (RV2: D72E/Y129F/D73E; RV6: M70I/D73E/Q33H; RV7: D72E/L94V/Y191F; RV10: D72E/E38D /M130L; RV13:M70G/Y129F; RV15:F69L/M70L/D73I; RV18:D72K/D73K; RV19:E38K; and wild type IL-21Rα). Cells were placed in serum-free medium for 24 hours and then incubated overnight (approximately 20 hours) with candidate ortho-IL-21 molecules (approximately 100,000 cells per well) in round-bottom 96-well plates. As mentioned above, the transposons conferring expression of the WT molecule and the candidate ortho-IL-21Rα molecule also carried the STAT3-regulated gene encoding the secreted Cypridina noctiluca luciferase. 6A-6T, 20 μL of supernatant from each well was mixed with 50 μL of VLAR-2 reagent buffer (Targeting) containing Cypridina noctiluca luciferase substrate (Vargulin) at the manufacturer's recommended concentration. Figure 2 shows the activity of this luciferase detected as luminescence (relative light units) after mixing with the luciferase system.

As shown in Figures 6A-6T, the following cells expressing ortho-IL-21Rα molecules made measurable responses to ortho-IL-21 molecules:

Cells expressing IL-21Rα (wild-type receptor) made measurable responses to most variant cytokines, but importantly, only four (4) variant cytokines were characterized at relatively high concentrations. The design of the experiment (in which the IL-21 was used) precluded the possibility of quantifying the extent of their signaling activity compared to wild-type IL-21.

Candidate ortho-IL-21 molecules (i.e., R5K (CV4), Q12V (CV12), R76A (CV19) and K73V (CV14)) selected from the collections represented in Figures 6A-6T and Table 4 were Ba expressing type IL-21Rα or candidate ortho-IL-21Rα molecules RV6, RV10, RV13 or RV15 (i.e., M70I/D73E/Q33H, D72E/E38D/M130L, M70G/Y129F, or F69L/M70L/D73I, respectively) /F3 cells were used. The results of this experiment are shown in FIGS. 7A-7D. As expected, when exposed to wild-type IL-21, cells expressing candidate ortho-IL-21Rα molecules have reduced signaling responses compared to cells expressing wild-type IL-21Rα. (solid line with filled symbols in each graph). Furthermore, as predicted by the results in Figures 6A-6T, in three cases (cells expressing candidate ortho-IL-21Rα molecules RV6, RV19 or RV13), cells expressed candidate ortho-IL-21Rα molecules ( CV12, CV19, or CV14, respectively) gave a stronger response than wild-type IL-21 (open and filled diamond symbols in Figures 7A, 7C, and 7D, respectively). All four candidate ortho-IL-21 molecules are impaired to varying degrees in their ability to elicit responses through wild-type IL-21Rα, and candidate ortho-IL-21 molecule CV19 is impaired in this regard. was most severely impaired (open vs. filled symbols in Figure 7C).

The results of these experiments indicate that IL-21 substitutions associated with impairing the ability to signal through wild-type IL-21Rα, and in some cases impairing the ability to signal through specific candidate ortho-IL-21Rα molecules. identified as having a low level of ability to induce.

Infolog variants of IL-21 were described by Govindarajan S, Mannervik B, Silverman JA, et al. Mapping of amino acid substances conferring herbicide resistance in wheat glutathione transferase. ACS Synth Biol. 2015;4(3):221-227. doi:10.1021/sb500242x and Musdal Y, Govindarajan S, Mannervik B. Exploring sequence-function space of a poplar glutathione transfer using designed information-rich gene variants. Protein Eng Des Sel. 2017;30(8):543-549. It was produced according to the principle described in doi:10.1093/protein/gzx045. These Infolog variants of IL-21 were screened for their ability to induce signal transduction in Ba/F3 cells expressing wild-type IL-21Rα molecules or candidate ortho-IL-21Rα molecules, as described. Representative data from one such screening experiment is provided in Figures 8A-8C. The experimental results shown in Figures 8A-8C are derived from the analysis of 96 cytokines, one containing the wild-type form of IL-21 and one negative control variant (two nullified Each of the 94 Infolog variants was a candidate ortho-IL-21 molecule, including CV22 with substitutions [R5Q/R76A]; SEQ ID NO: 59). Figure 8A shows the STAT3 response elicited in cells expressing wild-type IL-21Rα exposed to cytokine assembly, while Figures 8B and 8C express candidate ortho-IL-21Rα molecules RV13 and RV6, respectively. shows the response made by the cell. RV13 is composed of amino acid substitutions M70G and Y129F (SEQ ID NO: 19), and RV6 is composed of amino acid substitutions M70I, D73E and Q33H (SEQ ID NO: 12). The highlighted curves in the three figures represent five selected cytokines, namely wild-type IL-21, CV22, CV204 (SEQ ID NO: 60, composed of H6L/M10L/K73V/P78L/P104A), CV374 ( SEQ ID NO: 61, consisting of H6L/R9K/M10L/K73V/P78L/G84E) and CV388 (SEQ ID NO: 62, consisting of H6L/R9K/M10L/K73I/P78L/P104A) using three types of cells. shows the response given.

The highlighted cytokines were then retested in independent, similarly designed experiments focused on Ba/F3 cells expressing wild-type IL-21Rα (Figure 9A) or candidate ortho-IL-21Rα RV13 ( (including FIG. 9B). Similar to the screening experiments (FIGS. 8A-8C), CV22, the negative control cytokine, was unable to elicit a signaling response in cells expressing either form of IL-21Rα. In contrast, wild-type IL-21 induced a strong response in cells expressing the wild-type form of 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 candidate ortho-IL-21Rα molecules (FIGS. 8B, 8C, and 9B).

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

Candidate ortho-IL-21Rα RV13 (SEQ ID NO: 19) has two substitutions compared to wild-type IL-21Rα, namely M70G and Y129F, whereas candidate ortho-IL-21Rα RV22 (SEQ ID NO: 27) has only M70G. has. These two variant receptors appear to be equally impaired in their ability to bind native IL-21 (Figure 4B). These also explained similar reactivity patterns for the collection of IL-21 molecules used in Figures 9A and 9B. Specifically, similar to RV13 (Figure 10B), RV22 mediated a significantly impaired signaling response to wild-type IL-21 (and the negative control molecule CV22), but not to CV204, CV374 and CV388. A good response was given (FIG. 10C). As in Figure 9A, both CV374 and CV388 showed impaired responses in cells expressing wild-type IL-21Rα, whereas CV204 behaved similarly to wild-type IL-21 (Figure 10A). These results reproduce important observations made from the data in FIGS. 9A and 9B while also showing that RV22 can be interchangeable with RV13. Because RV22 has one fewer substitution than RV13, RV22 may be preferred for therapeutic purposes.

In most situations, in vitro assays have only very limited predictive value of the efficacy of treatments in vivo: many therapeutic targets are expressed in multiple cell types (often (with opposing effects on the response of cells), or the therapeutic effect of a given target is dependent on other accessory cells. In such situations, in vitro models, which are extremely simplistic in nature, are not particularly good surrogates for the much more complex situation in vivo. This is not the case in this application: the target receptor is synthetic and expressed only in cells specifically engineered to do so. Given the specificity of orthogonal cytokine-receptor systems, this significantly reduces complexity and gives better predictive value to in vitro assays.

The complete disclosures of all patents, patent applications, and publications, and electronically available materials cited herein are incorporated by reference. The above detailed description and examples are given for clarity of understanding only. No unnecessary restrictions should be understood from it. Although theories describing possible mechanisms by which the compounds are effective may be presented, we are not bound by the theories described herein. The invention is not limited to the precise details shown and described; variations obvious to those skilled in the art are included within the invention as defined by the claims.

Sequence Listing 1-63 <223> Synthesis

Claims (27)

Amino acid substitutions numbered for SEQ ID NO:2:
H6L;
R9K;
M10L;
P78L;
including;
optionally comprising G84E or P104A or a combination thereof;
optionally comprising one of K73V or K73I;
Engineered human IL-21 polypeptide. 2. The engineered human IL-21 polypeptide of claim 1, comprising the amino acid substitution K73V. 2. The engineered human IL-21 polypeptide of claim 1, comprising the amino acid substitution K73I. 2. The engineered human IL-21 polypeptide of claim 1, comprising the amino acid substitution G84E. 2. The engineered human IL-21 polypeptide of claim 1, comprising the amino acid substitution P104A. 2. The engineered human IL-21 polypeptide of claim 1, which binds to an engineered human CD360 protein ectodomain. The engineered human CD360 protein ectodomain is Y10, Q33, Q35, Y36, E38, L39, F67, H68, F69, M70, A71, D72, D73, I74, L94, A96, E97, P126, A127, Y129. 7. The engineered human IL-21 polypeptide of claim 6, wherein the engineered human IL-21 polypeptide is modified at one or more residues selected from , M130, K134, S190 and Y191. 7. The engineered human IL-21 polypeptide of claim 6, wherein the engineered human CD360 protein ectodomain is modified at M70. 7. The engineered human IL-21 polypeptide of claim 6, wherein the engineered human CD360 protein ectodomain is modified at M70 and Y129. 9. The engineered human IL-21 polypeptide of claim 8, wherein the engineered human CD360 protein ectodomain comprises the amino acid substitution M70G. 10. The engineered human IL-21 polypeptide of claim 9, wherein the engineered human CD360 protein ectodomain comprises amino acid substitutions M70G and Y129F. 2. The engineered human IL-21 polypeptide of claim 1, comprising amino acid substitutions K73V and G84E. 2. The engineered human IL-21 polypeptide of claim 1, comprising amino acid substitutions K73I and P104A. An engineered human IL-21 polypeptide comprising a set of amino acid substitutions numbered relative to SEQ ID NO: 2: H6L, R9K, M10L, K73V, P78L and G84E. An engineered human IL-21 polypeptide comprising a set of amino acid substitutions numbered relative to SEQ ID NO: 2: H6L, R9K, M10L, K73I, P78L and P104A. Selection of a receptor in a cell comprising (a) an engineered receptor comprising a human CD360 protein ectodomain comprising an amino acid substitution in M70G, and (b) an engineered human IL-21 polypeptide of claim 1. System for activating. (a) an engineered receptor comprising a human CD360 protein ectodomain comprising amino acid substitutions in M70G and Y129F; and (b) a receptor in a cell comprising an engineered human IL-21 polypeptide of claim 1. System for selective activation of. 17. The system of claim 16, wherein the engineered human CD360 receptor is expressed by a mammalian cell. 18. The system of claim 17, wherein the engineered human CD360 receptor is expressed by a mammalian cell. 19. The system of claim 18, wherein the mammalian cell is an immune cell or a stem cell. 20. The system of claim 19, wherein the mammalian cell is an immune cell or a stem cell. 21. The system of claim 20, wherein the mammalian cell is an immune cell and the immune cell is a T cell. 22. The system of claim 21, wherein the mammalian cell is an immune cell and the immune cell is a T cell. A pharmaceutical composition comprising the engineered human IL-21 polypeptide of 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. 2. The engineered human IL-21 polypeptide of claim 1, wherein the engineered human IL-21 polypeptide is fused to an IgG Fc domain or albumin.