EP4511054A2 - Systèmes cytokines/récepteurs d'il-21 orthogonaux - Google Patents

Systèmes cytokines/récepteurs d'il-21 orthogonaux

Info

Publication number
EP4511054A2
EP4511054A2 EP23792786.8A EP23792786A EP4511054A2 EP 4511054 A2 EP4511054 A2 EP 4511054A2 EP 23792786 A EP23792786 A EP 23792786A EP 4511054 A2 EP4511054 A2 EP 4511054A2
Authority
EP
European Patent Office
Prior art keywords
engineered human
amino acid
polypeptide
receptor
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23792786.8A
Other languages
German (de)
English (en)
Inventor
Nigel Killeen
Oren BESKE
Benedikt K. VOLLRATH
Sridhar Govindarajan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Neptune Biosciences LLC
Original Assignee
Neptune Biosciences LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Neptune Biosciences LLC filed Critical Neptune Biosciences LLC
Publication of EP4511054A2 publication Critical patent/EP4511054A2/fr
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • An alternative approach involves generating orthogonally constrained forms of cytokines and their receptors. See U.S. Patent No. 10,869,887; Sockolosky JT, TrottaE, 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 disclosure of each of which is incorporated by reference herein in its entirety. [0004]
  • An orthogonal cytokine system is one in which a cytokine and its receptor have been mutated such that they lose compatibility with their native (parental) partners yet retain the capacity to interact productively with one another.
  • Such an orthogonal cytokine:receptor pair can thus be said to demonstrate “privileged” or “private” interactions.
  • the approach of generating orthogonally constrained forms of cytokines and their receptors is of value for cell therapy because it provides a way to limit the scope of a cytokine’ s activity solely to the therapeutic (i.e., adoptively transferred) cells - these being the only cells expressing the engineered receptor and, consequently, the only cells capable of responding to the engineered cytokine.
  • Interleukin-21 (“IL-21”) is another pleiotropic cytokine with actions in a broad range of lymphoid, myeloid, and epithelial cells. IL-21 regulates both innate and adaptive immune responses; it not only has key roles in antitumor and antiviral responses, but also exerts major effects 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 natural human IL-21 cytokine:receptor complex is known.
  • an orthogonal interleukin-21 receptor alpha chain (an “ortho-IL-21 Rot” or “ortho-IL-21Ra molecule,” or when referring to a specific ortho-IL-2 IRa constructed as provided herein, an “RV,” as in “Receptor Variant”) is provided, the ortho-IL-21Ra comprising a modified amino acid sequence derived from SEQ ID NO: 1 that binds to an orthogonal interleukin-21 cytokine (an “ortho-IL-21” or “ortho-IL-21 molecule,” or when referring to a specific ortho-IL-21 constructed as provided herein, a “CV,” as in “Cytokine Variant”) but has impaired binding to native IL-21.
  • RV13 and RV22 may be substituted in the same manner as RV31, i.e., at positions S190, A127, and E38.
  • an ortho-IL-21 or engineered human IL-21 polypeptide (these phrases are used interchangeably herein) is provided, the ortho-IL-21 comprising a modified amino acid sequence derived from SEQ ID NO: 7 that binds to an ortho-IL-21Ra but has impaired binding to native IL-21Ra.
  • the ortho-IL-21 comprises a modified amino acid sequence comprising a substitution of one or more amino acid residues of SEQ ID NO: 7 that contact IL-
  • the ortho-IL-21 comprises an amino acid substitution, numbered relative to SEQ ID NO: 7, at position: H6, R9, MIO, Rl l, 116, Q19, 166, K73, R76, P78, S8O, G84, or P104, and combinations thereof.
  • the phrase “numbered relative to SEQ ID NO: 7” means, for numbering purposes, to disregard any epitope tags and signaling peptides.
  • the amino acid substitution comprises, consists essentially of, or consists of: H6L, R9K, M10L, RI IS, RUT, I16V, Q19F, I66S, K73V, K73L, K73M, K73I, R76K, R76H, P78L, S80K, S80L, G84E, P104I, orP104A, and combinations thereof.
  • the ortho-IL-21 comprises amino acid substitutions, numbered relative to SEQ ID NO: 7: H6L/M10L/P78L. In another aspect, the ortho-IL-21 further comprises amino acid substitution R9K. In another aspect, the ortho-IL-21 further comprises amino acid substitution G84E. In another aspect, the ortho-IL-21 further comprises one of amino acid substitution P104V or P104A. In another aspect, the ortho-IL-21 further comprises one of amino acid substitution K73V or K73I.
  • such an ortho-IL-21 may include SEQ ID NO: 8 (CV374: H6L/R9K/M10L/K73V/P78L/G84E), SEQ ID NO: 9 (CV388: H6L/R9K/M10L/K73I/ P78L/P104A), SEQ ID NO: 10 (CV414: H6L/R9K/M10L/K73I/P78L), or SEQ ID NO: 11 (C V415 : H6L/R9K/M10L/K731/P78L/G84E/P 104V).
  • an engineered human IL-21 polypeptide that comprises amino acid substitutions, numbered relative to SEQ ID NO: 7: H6L/M10L/P78L.
  • the engineered human IL-21 polypeptide further comprises one of amino acid substitution S80P, S80K, or S80V.
  • the engineered human IL-21 polypeptide further comprises amino acid substitution G84E.
  • the engineered human IL-21 polypeptide further comprises one of amino acid substitution Pl 041 or P104A.
  • the engineered human IL-21 polypeptide further comprises one of amino acid substitution K73V, K73L, or K73M.
  • the engineered human IL-21 polypeptide further comprises one of amino acid substitution R76H or R76K. In another aspect, the engineered human IL-21 polypeptide further comprises one of amino acid substitution R11 S or R1 IT. In another aspect, the engineered human IL-21 polypeptide further comprises amino acid substitution I16V. In another aspect, the engineered human IL-21 polypeptide further comprises amino acid substitution Q19F. In another aspect, the engineered human IL-21 polypeptide further comprises amino acid substitution I66S.
  • such an engineered human IL-21 polypeptide may include SEQ ID NO: 12 (CV339: H6L/M10L/K73V/R76K/P104A), SEQ ID NO: 13 (CV425: H6L/M10L/K73L/P78L), SEQ ID NO: 14 (CV431 : H6L/M10L/K73M/R76H/P78L/G84E), SEQ ID NO: 15 (CV458: H6L/M10L/K73L/P78L/S80P/P104A), SEQ ID NO: 82 (CV588:
  • a system for activating IL-21 signaling in a cell comprising: an ortho-IL-21Ra that has impaired binding to native IL-21, the ortho-IL-21Ra comprising a modified amino acid sequence derived from SEQ ID NO: 1 comprising a substitution of one or more of the amino acid residues of SEQ ID NO: 1 that contact IL-21, residues in the immediate vicinity of such contact residues, or residues elsewhere in IL-21Ra that have an influence on the conformation of the IL-21 binding surface; and an ortho-IL-21 that has impaired binding to native lL-21Ra, the ortho-IL-21 comprising a modified amino acid sequence derived from SEQ ID NO: 7 comprising a substitution of one or more amino acid residues of SEQ ID NO: 7 that contact IL-21Ra, residues in the immediate vicinity of such contact residues, or residues elsewhere in IL-21 that have an influence on the conformation of the IL-2 IRa binding surface, wherein the ortho-IL-2 IR
  • Figure 1 provides a schematic representation of an orthogonal IL-21 system.
  • the cartoon at the extreme left shows the wild-type receptor and cytokine interacting productively with one another, while the adjacent cartoon depicts the impaired interaction between an ortho-IL-2 IRa and the native cytokine.
  • the cartoon at the extreme right depicts the impaired interaction between ortho-IL-21 and the native (wild-type) receptor, while the adjacent cartoon shows a productive interaction between the two orthogonal molecules (ortho-IL-2 IRa and ortho-IL-21).
  • Figure 2 provides a schematic representation of pathways for generating an orthogonal IL-21 system.
  • Figure 3 shows the results of a representative assay in which a single (sub-saturating) concentration of IL-21-TLucl6 was tested for binding to a panel of 20 candidate ortho-IL-21Ra molecules (all of which had been bound to Streptactin-coated surfaces of wells at saturating concentrations), including RV6 and RV13. Eight of the 20 candidate ortho-IL-21Ra molecules showed diminished capacity to bind IL-21-TLucl6.
  • Figures 4A-C show the results of a representative assay in which a range of (subsaturating) concentrations of IL-21-TLucl6 was tested for binding to a panel of candidate ortho- IL-21Ra molecules, including RV6, RV13, RV22, and RV31.
  • the panel included a wild-type receptor as a control.
  • the candidate ortho-IL-2 IRa molecules were added to Streptactin-coated wells of 96-well plates at saturating concentrations.
  • Figures 4A-C show luminometry data for individual plates in which, in each case, the binding of IL-21-TLucl6 to five candidate ortho-IL-
  • 21Ra molecules and a wild-type control IL-21Ra was compared.
  • Several of the candidate ortho- IL-21Ra molecules showed significantly diminished capacity to bind IL-21-TLucl6 relative to the wild-type control.
  • FIGS 5A and 5B show IL-21 -induced STAT3 signaling responses by Ba/F3 cells expressing native IL-2 IRa and eight candidate ortho-IL-2 IRa molecules, including RV6 and RV13.
  • the cells carried a ST AT3 -regulated Cypridina noctiluca luciferase reporter transgene; they were exposed to different concentrations of native IL-21 overnight (approximately 20 hours) before testing the supernatant medium for luciferase activity by luminometry, with Vargulin serving as the enzyme substrate.
  • Cells expressing a form of wild-type IL-2 IRa lacking its cytoplasmic tail (Acyt) were included to show that STAT3 signaling in response to IL-21 depended on the cytoplasmic tail of the receptor, as expected.
  • Figure 6A-6D show the capacity of native IL-21 and selected candidate ortho-IL-21 molecules, including two, for example, that each comprise one amino acid substitution, numbered relative to SEQ ID NO: 7, at R9K (CV9; SEQ ID NO: 16) and K73V (CV14; SEQ ID NO: 17), to induce signaling via native IL-2 IRa and RV 13.
  • luminometry was used to quantify luciferase produced by transfected Ba/F3 cells carrying a STAT3 -dependent luciferase reporter transgene resident on the same transposon used to confer expression of native IL-2 IRa or candidate ortho-IL-2 IRa molecules.
  • Figure 7 shows the capacity of native IL-21 and CV14 to induce signaling via native IL- 21Ra and RV13.
  • Ba/F3 cells expressing native IL-21Ra or CV14 from a transposon that also carried a STAT3 -luciferase reporter transgene
  • Figures 8A, 8B, and 8C show the results of a representative screening experiment in which a collection of 96 cytokines were tested for their capacity to induce STAT3 -dependent signaling responses in Ba/F3 cells expressing wild-type IL-21Ra ( Figure 8A) or the candidate ortho-IL-2 IRa molecules RV13 ( Figure 8B) or RV6 ( Figure 8C).
  • luminometry was used to quantify luciferase produced by transfected Ba/F3 cells carrying a STAT3 -dependent luciferase reporter transgene resident on the same transposon used to confer expression of native IL-21Ra or the candidate ortho-IL-21Ra molecule.
  • the cells were exposed to increasing concentrations of the candidate ortho-IL-21 molecules overnight (approximately 20 hours) before testing the supernatant culture medium for luciferase activity.
  • the dotted lines show response curves for the cytokine collection, while the responses caused by five cytokines of interest (wild-type IL-21, negative control CV22 comprising amino acid substitutions R5Q/R76A (SEQ ID NO: 18), and candidate ortho-IL-21 molecules CV204 comprising amino acid substitutions H6L/M10L/K73V/P78L/P104A (SEQ ID NO: 19), CV374, and CV388 are highlighted with solid lines and symbols.
  • Figures 9A and 9B show the relative capacity of native IL-21, CV22, CV204, CV374, and CV388 to induce signaling via wild-type IL-2 IRa ( Figure 9A) and the candidate ortho-IL- 2 IRa RV13 ( Figure 9B).
  • luminometry was used to quantify luciferase produced by transfected Ba/F3 cells carrying a STAT3 -dependent luciferase reporter transgene resident on the same transposon used to confer expression of native IL-21Ra or RV13. The cells were exposed to increasing concentrations of the candidate ortho-IL-21 molecules overnight (approximately 20 hours) before testing the supernatant culture medium for luciferase activity.
  • Figures 10A, 10B, and 10C show the capacity of native IL-21, CV22, CV204, CV374, and CV388 to induce signaling via wild-type IL-21Ra ( Figure 10A), the candidate ortho-IL-2 IRa RV13 ( Figure 10B), and the candidate ortho-IL-2 IRa RV22 ( Figure 10C) Responses elicited by wild-type IL-21 and 95 variants are represented in each figure, with the five indicated response curves highlighted.
  • luminometry was used to quantify luciferase produced by transfected Ba/F3 cells carrying a STAT3 -dependent luciferase reporter transgene resident on the same transposon used to confer expression of native IL-2 IRa or the candidate ortho-IL-2 IRa molecule.
  • the cells were exposed to increasing concentrations of the candidate ortho-IL-21 molecules overnight (approximately 20 hours) before testing the supernatant culture medium for luciferase activity.
  • Figures 11A and 11B show the capacity of native IL-21 and candidate ortho-IL-21 molecules CV374 and CV415 to induce signaling via wild-type IL-21Ra ( Figure 11A) or the candidate ortho-IL-21Ra molecule RV22 ( Figure 11B). Responses elicited by wild-type TL-21 and 95 variants are represented in each figure, with the three indicated response curves highlighted.
  • Figure 11C shows a comparison of the responses elicited by wild-type IL-21 or the candidate ortho-IL-21 molecule CV415 on cells expressing wild-type IL-21Ra or the candidate ortho-IL- 2 IRa molecule RV22.
  • luminometry was used to quantify luciferase produced by transfected Ba/F3 cells carrying a STAT3 -dependent luciferase reporter transgene resident on the same transposon used to confer expression of native IL-2 IRa or the candidate ortho-IL-2 IRa molecule.
  • the cells were exposed to increasing concentrations of the candidate ortho-IL-21 molecules overnight (approximately 20 hours) before testing the supernatant culture medium for luciferase activity.
  • Figures 12A-12C show the capacity of native IL-21 and candidate ortho-IL-21 molecules CV374, CV415, CV425, CV431, CV458, and CV339 to induce signaling via wild-type IL-21Ra ( Figure 12A), the candidate ortho-lL-21Ra molecule RV22 ( Figure 12B), or the candidate ortho- IL-21Ra molecule RV31 ( Figure 12C).
  • Figure 12A shows the capacity of native IL-21 and candidate ortho-IL-21 molecules CV374, CV415, CV425, CV431, CV458, and CV339 to induce signaling via wild-type IL-21Ra
  • Figure 12B shows the candidate ortho-lL-21Ra molecule RV22
  • Figure 12C the candidate ortho- IL-21Ra molecule RV31
  • Figure 12D shows a comparison of the responses elicited by wild-type IL-21 or the indicated candidate ortho-IL-21 variants on cells expressing wild-type IL-21Ra or the candidate ortho-IL-21Ra molecule RV31.
  • Figures 13A-13J show the capacity of native IL-21 (IL-21-WT, Figures 13A and 13F) and candidate ortho-IL-21 molecules CV415 ( Figures 13B and 13G), CV588 ( Figures 13C and 13H), CV617 ( Figures 13D and 131), and CV631 ( Figures 13E and 13J) to induce signaling via wild-type IL-21Roc, the candidate ortho-IL-21Ra molecule RV22, the candidate ortho-IL-21Ra molecule RV31, and four variants of RV31 (M70/D73E) bearing an additional E38T (RV31- E38T), E38H (RV31-E38H), S190F (RV31-S190F), or A127M (RV31-A127M) substitution.
  • IL-21-WT Figures 13A and 13F
  • CV415 Figures 13B and 13G
  • CV588 Figures 13C and 13H
  • CV617 Figures 13D and 131
  • CV631 Figures 13E and
  • Selection genes typically contain a selection gene, also termed a selectable marker.
  • the selection gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium.
  • Typical selection genes encode proteins that: (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media.
  • Transcription from vectors in mammalian host cells may be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus (such as murine stem cell virus), hepatitis-B virus, and Simian Virus 40 (“SV40”), from heterologous mammalian promoters, e.g., the actin promoter, phosphoglycerate kinase (“PGK”), or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • the early and late promoters of SV40 are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.
  • the expression vector may also include an enhancer sequence.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, which act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent, having been found 5’ and 3 ’ to the transcription unit, within an intron, as well as within the coding sequence itself.
  • Many enhancer sequences are known from mammalian genes (e.g., globin, elastase, albumin, a- fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus.
  • Examples may 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.
  • the enhancer may be spliced into the expression vector at a position 5’ or 3’ to the coding sequence but is preferably located at a site 5’ from the promoter.
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5’ and 3’ untranslated regions of eukaryotic or viral DNAs or cDNAs.
  • Expression vectors might also be comprised of inducible regulatory elements for the purpose of controlling expression of a transgene (encoding, for example, ortho-IL-21 or ortho-IL- 21Roc) with small molecules or other stimulatory agents.
  • regulatory elements include, but are not limited to, promoters containing tetracycline operators that render them sensitive to regulation by tetracycline or derivatives thereof (such as doxycycline). Promoters may also be inducibly regulated by CRISPRa (clustered regularly interspaced short palindromic repeats- activation) using fusions of transcriptional effectors and catalytically dead Cas9. Such promoters may in turn be downstream of other control systems such as those involving dimerizers (of an antibody-based and/or chemical nature) or components based on the Notch receptor.
  • cells are provided that have been engineered to express an ortho-IL-21 Roc.
  • the cells may be genetically engineered to include any suitable expression vector described herein.
  • the expression vector comprises a coding sequence that encodes the orthogonal receptor, the coding sequence being operably linked to a promoter active in the desired cell.
  • Various vectors may be used for this purpose, e g., transposons, viral vectors, plasmid vectors, and minicircle vectors, which can be integrated into the target cell genome or can be episomally maintained.
  • the expression vector is a synthetic transposon that can be integrated into the genome by means of a transposase enzyme. Examples of transposon/transposase systems include Sleeping Beauty, PiggyBac, Leapin® from ATUM Bio, and derivatives thereof.
  • the engineered cell may be a host cell for preparing recombinant protein in vitro.
  • Suitable host cells for recombinant expression of orthogonal proteins include prokaryotes, yeast, and higher eukaryote cells, such as various mammalian host cell lines.
  • the engineered cell is a cell intended for therapeutic use.
  • therapeutic engineered cells may include stem cells, e.g., a hematopoietic stem cell, a natural killer (“NK”) cell, or a T cell.
  • the engineered cell is a T cell.
  • T cells refers to mammalian immune effector cells that may be characterized by expression of a CD3 and/or a T cell antigen receptor, which cells may be engineered to express an ortho-IL-2 IRa.
  • the T cells are selected from naive, activated, or post-activation CD8+ T cells; cytotoxic CD8+ T cells; naive, activated, or post-activation CD4+ T cells; helper T cells, e.g., TH1, TH2, TH9, TH11, TH22, and TFH; regulatory T cells, e.g., TRI, natural TReg, and inducible TReg; and memory T cells, e.g., central memory T cells, effector memory T cells, NKT cells, and y5 T cells.
  • Ortho-IL-21 may be used as an adjunct to ACT.
  • T cells may be engineered to express the ortho-IL-2 IRa by gene (cDNA, minigene, or other nucleic acid construct) transfection, transduction, or transposition.
  • Patients receiving the ACT may be treated (and/or pretreated) with the ortho-IL-21 and dosed repeatedly as needed to augment and sustain a desirable T cell presence and responses.
  • Therapeutic cells may also be engineered to express ortho-IL-21. This could be accomplished using any of the methods appropriate for ectopic expression of ortho-IL-21 Rd.
  • the ortho-IL-21 could be expressed in the same or different cells as those that express ortho-IL-2 IRa, allowing for autocrine or paracrine action, respectively.
  • An example of a paracrine arrangement could be CD4+ T cells expressing the ortho-IL-21 and CD8+ T cells expressing the matched ortho- IL-21Ra.
  • ortho-IL-21 may be expressed in a membrane-tethered form. This has previously been accomplished with natural IL-21 by fusing the cytokine to the amino-terminus of an IgG4 CH2-CH3 moiety that was itself fused to a CD4 transmembrane domain. Related strategies have been employed to tether other cytokines to the membranes of cells. Such membrane tethering limits the diffusion of the cytokine and restricts its action to the immediate vicinity of the cells expressing the membrane-bound cytokine. In vivo, this approach could be exploited to ensure ortho-IL-2 IRa-expressing cells only encounter the ortho-IL-21 when they are proximal to a specific type of cell and/or location in the body. In vitro, the approach may facilitate certain kinds of selective differentiation protocols (e.g., the differentiation of NK cells from stem cells in the presence of K562 [or other] feeder cells expressing membrane-bound IL-21 and CD137L).
  • selective differentiation protocols e.g., the differentiation of
  • Engineered cells may be provided in pharmaceutical compositions suitable for therapeutic use, e.g., for human treatment.
  • Therapeutic formulations comprising such cells can be frozen or prepared for administration with physiologically acceptable carriers, excipients, or stabilizers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of aqueous solutions.
  • the cells may be formulated, dosed, and administered in a fashion consistent with good medical practice.
  • a cell such as a T cell engineered to express one of the ortho-IL-21 Roc molecules described herein may be used to treat a broad range of conditions.
  • Engineered properties in this therapy may allow for beneficial T cell differentiation, resistance to exhaustion, capacity for longterm persistence, anamnestic responses, and in-built safety features allowing for responses to be halted when they become pathogenic.
  • Methods are provided for enhancing cellular responses by engineering cells from a recipient or donor by introduction of an ortho-IL-21Roc and stimulating the ortho-IL-21 Roc by contacting the engineered cell with ortho-IL-21.
  • the subject methods may include a step of obtaining the targeted cells, e.g., T cells, hematopoietic stem cells, etc., which may be isolated from a biological sample or may be derived in vitro from a source of progenitor cells.
  • the cells may be transduced or transfected with an expression vector comprising a sequence encoding the ortho-IL-21 Roc, which step may be performed in any suitable culture medium.
  • the engineered T cells may be contacted with the ortho-IL-21 in vivo, i.e., where the engineered T cells are transferred to a recipient, and an effective dose of the ortho- IL-21 is injected into the recipient and allowed to contact the engineered T cells in their native environment, e.g., in lymph nodes, etc.
  • the contacting is performed in vitro.
  • the contacting may be accomplished using soluble ortho-IL-21 comprised, or not, of a fusion to another protein moiety such as an immunoglobulin Fc domain.
  • the contacting could be accomplished by encounter with other cells expressing secreted or membrane-tethered ortho-IL-21.
  • Another aspect provides a method for treating a subject in need thereof, including introducing an engineered cell expressing an ortho-IL-21 Roc to the subject and activating the cell by contacting it with an effective amount of an ortho-IL-21.
  • the cell is a T cell, while in further aspects the cell is a CAR-T cell.
  • the cell is a T cell expressing a native or modified TCR.
  • the cell is an NK cell.
  • the cell is a macrophage or other myeloid cell or a leukocyte.
  • the ortho-IL-21 is delivered as a fusion protein with a heterologous polypeptide.
  • Suitable heterologous polypeptides include serum albumin, Fc fragments of IgG, single-chain Fc antibody fragments, ABD035, and the like. Fc fragments may be modified, for example, with electrostatic steering or other mutations, to prevent, or at least significantly limit, the formation of homodimers.
  • Another aspect provides a method for treating a subject in need thereof, including introducing an engineered cell expressing an ortho-IL-2 IRa to the subject and introducing a second engineered cell expressing an ortho-IL-21.
  • the cell is a T cell, while in further aspects the cell is a CAR-T cell.
  • a “subject,” can be any mammal and may also be referred to as a “patient.” Examples of mammalian subjects include research animals (e g., a mouse or rat), domesticated farm animals
  • cancer e g., cow, horse, pig
  • pets e.g., dog, cat
  • humans e.g., the subject is a human.
  • the subject being treated has been diagnosed as having cancer.
  • Cancer and “malignancy” are used as synonymous terms and refer to any of a number of diseases that are characterized by uncontrolled, abnormal proliferation of cells, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other parts of the body (i.e., metastasize), as well as any of a number of characteristic structural and molecular features.
  • a “cancer cell” refers to a cell undergoing early, intermediate, or advanced stages of multi-step neoplastic progression.
  • Cancer cells at each of the three stages of neoplastic progression generally have abnormal karyotypes, including translocations, inversion, deletions, isochromosomes, monosomies, and extra chromosomes.
  • Cancer cells include “hyperplastic cells,” that is, cells in the early stages of malignant progression, “dysplastic cells,” that is, cells in the intermediate stages of neoplastic progression, and “neoplastic cells,” that is, cells in the advanced stages of neoplastic progression.
  • Examples of cancers are sarcoma, breast, lung, brain, bone, liver, kidney, colon, and prostate cancer.
  • the engineered cells are used to treat cancer selected from the group consisting of colon cancer, brain cancer, breast cancer, fibrosarcoma, and squamous carcinoma.
  • the cancer is selected from the group consisting of melanoma, breast cancer, colon cancer, lung cancer, and ovarian cancer.
  • the cancer being treated is metastatic cancer.
  • the method of treatment may further include the step of ablating the cancer.
  • Ablating the cancer may be accomplished using a method selected from the group consisting of cryoablation, thermal ablation, radiotherapy, chemotherapy, radiofrequency ablation, electroporation, alcohol ablation, high intensity focused ultrasound, photodynamic therapy, administration of monoclonal antibodies, and administration of immunotoxins.
  • the subject being treated has been diagnosed as having an infection.
  • infection refers to infection of one or more cells of a subject by an infectious agent.
  • Infectious agents include, but are not limited to, bacteria, viruses, protozoans, and fungi. Intracellular pathogens are of particular interest. Infectious diseases are disorders caused by infectious agents. Some infectious agents cause no recognizable symptoms or disease under certain conditions but have the potential to cause symptoms or disease under changed conditions.
  • the subject methods may be used in the treatment of chronic pathogen infections, including but not limited to viral infections, e.g., retrovirus, lentivirus, hepadnavirus, herpes viruses, pox viruses, and human papilloma viruses; intracellular bacterial infections, e.g., Mycobacterium, Chlamydia, Ehrlichia, Rickettsia, Brucella, Legionella, Francisella, Listeria, Coxiella, Neisseria, Salmonella, Yersinia sp, and Helicobacter pylori; and intracellular protozoan pathogens, e.g., Plasmodium sp, Trypanosoma sp, Giardia sp, Toxoplasma sp, and Leishmania sp.
  • viral infections e.g., retrovirus, lentivirus, hepadnavirus, herpes viruses, pox viruses, and human papilloma viruses
  • intracellular bacterial infections
  • the subject being treated has been diagnosed as having an autoimmune disease.
  • Autoimmune diseases are characterized by T and B lymphocytes that aberrantly target self-proteins, -polypeptides, -peptides, or other self-molecules, causing injury and/or malfunction of an organ, tissue, or cell-type within the body.
  • Autoimmune diseases include diseases such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, autoimmune hepatitis, insulin dependent diabetes mellitus, and degenerative diseases such as osteoarthritis, Alzheimer’s disease, and macular degeneration.
  • one or more engineered cells of the subject may be contacted with ortho-IL-21. Where the engineered cells are contacted with the ortho-IL-21 in vitro, the ortho-IL-
  • the engineered cells thus activated may be used for any desired purpose, including experimental purposes relating to determination of antigen specificity, cytokine profiling, and the like, and for delivery in vivo.
  • an effective dose of engineered cells expressing ortho-IL-2 IRa are infused to the recipient, in combination with or prior to administration of the ortho-IL-21.
  • Dosage and frequency may vary depending on the agent, mode of administration, and the like.
  • the dosage may also be varied for localized administration, e.g., intranasal, inhalation, and the like, or for systemic administration, e.g., i.m., i.p. , i.v., and the like.
  • at least about 10 4 engineered cells/kg are administered, at least about 10 5 engineered cells/kg, at least about 10 6 engineered cells/kg, at least about 10 7 engineered cells/kg, or more.
  • Example 1 Binding Assay for Identification of Candidate Ortho-IL-2 IRa Molecules with Impaired Binding to Native IL-21
  • a direct interaction assay quantifying the capacity of IL-2 IRa to bind native or mutant forms of IL-21 provides an alternative to a cell-based assay for the identification of variants of IL-21Ra with compromised IL-21 binding activity (i.e., candidate orthogonal variants).
  • the feasibility of exploiting such an assay is enhanced by the fact that the native interaction (IL- 21Ra:IL-21) is avid (KD ⁇ 70pM).
  • IL- 21Ra:IL-21 is avid (KD ⁇ 70pM).
  • IL-21Ra:IL-21 binding assay involves attaching the receptor ectodomain to a surface, bathing the coated surface in a solution of an IL-21 -luciferase fusion protein, followed by quantitation of bound IL-21 based on luminescence when the relevant luciferase substrate is added.
  • IL-21 may be immobilized, and an IL-21Ra-luciferase fusion protein may be used in solution.
  • a desirable orientation of the immobilized receptor or cytokine can be accomplished through use of an affinity tag such as Twin-Strep-Tag II, which is a high affinity peptide ligand for the Streptactin protein.
  • the IL-21Ra ectodomain bearing a carboxy-terminal Twin-Strep-Tag II peptide can be efficiently and selectively immobilized on the surfaces of wells of 96-well plates that have been pre-coated with Streptactin protein. In this manner, the immobilized IL-21Ra should be oriented with its cytokine-binding domain distal from the plate surface.
  • IL-21 may be immobilized in a related fashion if it, too, bears an amino- or carboxy-terminal Twin-Strep-Tag 11 peptide tag.
  • This assay could also be established with luciferase fused at the amino-terminus of IL-21 or with an alternative form of luciferase (e.g., NanoLuc; ThermoFisher).
  • Twenty candidate ortho-IL-2 IRa molecules were tested for their capacity to bind IL-21- TLucl6.
  • the wild-type human IL-21Ra ectodomain (mature form lacking the signal peptide) (RVO (SEQ ID NO: 6)) and the 20 candidate ortho-IL-21Ra molecule ectodomains (mature form lacking the signal peptide) (SEQ ID NOs shown in Table 1) were expressed in HEK 293 cells as secreted proteins.
  • Clarified supernatant fluids from the transiently transfected cells were tested for the presence of IL-21Ra with an Enzyme-Linked Immunosorbent Assay (“ELISA”) comprised of Streptactin-coated plates, dilutions of the supernatant fluids, and detection using the combination of a mouse monoclonal antibody specific for the human IL-2 IRa, a horseradish peroxidase- conjugated rat antibody specific for mouse IgG, and a chromogenic substrate for the peroxidase.
  • ELISA Enzyme-Linked Immunosorbent Assay
  • Candidate ortho-IL-2 IRa saturated wells were incubated with a solution of IL-21-TLucl6 for 1 h (or longer in some experiments) at 4 °C (or room temperature in some experiments). The wells were washed before addition of the luciferase substrate (Coelenterazine) solution and luminometry.
  • Figure 3 shows the results of a representative assay in which a single (sub-saturating) concentration of IL-21-TLucl6 was tested for binding to the panel of 20 candidate ortho-IL-21Ra molecules (all of which had been bound to the Streptactin-coated surfaces of the wells at saturating concentrations). Eight of the candidate ortho-IL-2 IRa molecules showed diminished capacity to bind IL-21-TLucl6. Repeat experiments (involving titrations of the IL-21-TLucl6) confirmed the results.
  • Additional candidate ortho-IL-2 IRa molecules carrying alternative combinations of the amino acid substitutions present in the eight candidate ortho-IL-2 IRa molecules were similarly tested for their capacity to bind IL-21-TLucl6.
  • Certain of the additional candidate ortho-IL-21Ra molecule ectodomains (mature form lacking the signal peptide) (SEQ ID NOs shown in Table 2) were expressed in HEK 293 cells as secreted proteins.
  • ortho-IL-2 IRa molecules RV23, RV24, and RV28 showed binding that was equivalent or nearly equivalent to that of the wild-type receptor, whereas all the other ortho-IL-2 IRa molecules showed significantly more impaired binding).
  • the eight candidates from Example 1 and Figure 3 were expressed in a lymphoid cell line, namely Ba/F3 cells (a mouse pre-B cell lymphoma line).
  • Ba/F3 cells are dependent on the cytokine IL-3 for growth but will also proliferate robustly in response to IL-21 if first rendered positive for expression of IL-2 IRa.
  • Ba/F3 cells were electroporated with Leapin Transposase® mRNA and transposons encoding wild-type or ortho-IL-2IRa candidates RV2, RV6, RV7, RV10, RVI3, RV15, RV18, and RV19; and Acyt, which is a form of IL-21Rot lacking almost all of its cytoplasmic tail.
  • the transposons also carried a STAT-3 -regulated gene encoding the secreted Cypridina noctiluca luciferase, a constitutively expressed cytosolic click beetle luciferase, and a constitutively expressed gene encoding puromycin N-acetyl transferase.
  • the RV13 -expression construct was prepared by oligonucleotidedependent DNA synthesis by ATUM (www.atum.bio).
  • This plasmid is over 13Kb in size and comprises four independent genes within a piece of DNA that is flanked first by genomic insulator sequences (from the human D4Z4 locus on one side and from the chicken locus encoding 0-globin on the other), then by transposon inverted terminal repeat (“ITR”) sequences.
  • ITR transposon inverted terminal repeat
  • the insulator sequences are intended to protect the genes within the transposon from position effects (i.e., effects dependent on the site of transposon integration in the genome) that might reduce or variegate expression.
  • the ITR sequences are recognized by ATUM’s proprietary Leapin® transposase enzymes that mediate integration of the transposon into genomic DNA.
  • the four genes present inside the transposon are described in Table 3 (in the order they occur within the transposon).
  • the genes and the transposon that contains them were designed according to standard molecular biology principles compatible with the construct assembly methodology used routinely by ATUM. Variants of this vector encoding wild-type or other candidate ortho-IL-2 IRa molecules were generated by making appropriate changes in the fourth gene listed in Table 3.
  • the transposon vector encoding wild-type IL-21Ra or any one of the candidate ortho-IL- 21Ra molecules was co-transfected into Ba/F3 cells together with in vitro-transcribed mRNA encoding the relevant Leapin® transposase using either the MaxCyte ATx or ThermoFisher Neon instruments according to the manufacturer’s instructions. Puromycin selection (I g/ml or higher) was imposed at 48 hours after transfection and continued for at least a week after all the cells in an untransfected control culture had died. Flow cytometry was used to confirm that the puromycin- selected cells showed uniform expression of IL-21Ra.
  • the Ba/F3 cells were first incubated for 20-24 hours in RPML 1640 medium lacking serum and exogenous cytokines. After washing, they were stimulated with IL-21 (in the presence of 0.4% [vol/vol] serum) for a further 20-24 hours with varying concentrations of TL-21 (or candidate ortho-IL-21 ) in round-bottom 96-well plates ( ⁇ 100,000 cells per well) before assaying secreted luciferase (from the STAT3-cLuc gene in the transposon), intracellular luciferase (from the constitutively expressed EEF2-eLuc gene in the transposon), or ATP accumulation.
  • the secreted luciferase assay was used to inform on STAT3 -dependent signaling in the cells, as occurs when IL-21 engages its receptor.
  • the other two assays (monitoring cytoplasmic eLuc or ATP accumulation) were used to inform on cell number (i.e., proliferation).
  • cell number i.e., proliferation.
  • Ba/F3 cells failed to proliferate if they were not provided IL-3. When expressing the wild-type form of IL-21Ra, they showed IL-21 -dose-dependent proliferation (by
  • Cypridina noctiluca luciferase activity was readily detected by adding the relevant luciferase substrate (Vargulin) to samples of supernatant fluids from the cells and measuring light emission using a luminometer.
  • Figures 5A and 5B show luciferase activity detected as light emission (relative light units or RLU) following admixture of 20 pL of the supernatant fluid from each of the wells with 50 pL of VLAR-2 reagent buffer (Targeting Systems) containing Vargulin at the manufacturer’s recommended concentration.
  • Example 3 Screening of Candidate Ortho-IL-21 Molecules: Testing for STAT3 -Dependent Signaling Responses in Cells Expressing RV13 or Wild-Type IL-21Rq
  • Wild-type or candidate ortho-IL-21 molecules were produced from transiently transfected HEK-293 cells according to procedures that are routinely used at ATUM (www.atum.bio).
  • Expression vectors for this purpose carried the IL-21 open reading frame downstream of an optimized cytomegalovirus Immediate Early Gene 1 promoter.
  • a signal peptide from the human IL-2 gene was used in place of the native one.
  • Epitope tags for detection, quantitation, immobilization, or purification were fused to the amino- or carboxy -termini of the IL-21 coding sequence.
  • the element fused to the amino terminus was a Twin- Strep-Tag followed by three copies of a Glycine-Glycine-Glycine-Glycine-Serine linker moiety, while the element fused to the carboxy terminus comprised two copies of the same Glycine- Glycine-Glycine-Gly cine- Serine linker followed by an N-Myc epitope tag (recognized by the 9E10 monoclonal antibody).
  • a second series of vectors featured no tags at the carboxy terminus but had the following element at the amino-terminus of IL-21 : Twin- Strep-Tag followed immediately by the N-Myc epitope tag then three copies of the Glycine-Glycine-Glycine-Glycine-Serine linker moiety.
  • Ba/F3 cells expressing wild-type IL-21Ra or RV13 were stimulated with candidate ortho- IL-21 molecules CV1-CV19, CV21, and CV22 .
  • the signaling responses of the Ba/F3 cells to the ortho-IL-21 molecules were monitored as above using the STAT3-luciferase assay.
  • Ba/F3 cells were exposed to four concentrations (100, 50, 25, and 12.5 ng/mL) of the indicated candidate ortho-IL-21 molecules ( Figures 6A and 6B: candidate ortho-IL-21 molecules CV1-CV11 and wild-type IL-21; Figures 6C and 6D: candidate ortho-IL-21 molecules CV12- CV19, CV21, CV22, and wild-type IL-21).
  • the cells were placed in serum-free medium for 24 hours before a subsequent overnight incubation ( ⁇ 20 hours) with the candidate ortho-IL-21 molecules in round-bottom 96-well plates (-100,000 cells per well).
  • the transposon conferring expression of WT and RV13 also carried a STAT3 -regulated gene encoding the secreted Cypridina noctiluca luciferase.
  • Figures 6A-6D show activity of this luciferase detected as light emission (relative light units) following admixture of 20 pL of the supernatant fluid from each of the wells with 50 pL of VLAR-2 reagent buffer (Targeting Systems) containing the Cypridina noctiluca luciferase substrate (Vargulin) at the manufacturer’s recommended concentration.
  • RV13 made measurable responses to at least CV9 (bearing an R9K substitution) and C14 (bearing a K73V substitution).
  • Figures 8A-8C derived from the analysis of 96 cytokines, one of which comprised the wildtype form of IL-21, another comprised a negative control variant (CV22, which bears two disabling substitutions [R5Q/R76A]), and 94 Infolog variants, each of which was a candidate ortho-IL-21 molecule
  • Figure 8A shows the STAT3 responses elicited in cells expressing wild-type IL-21Ra exposed to the cytokine collection
  • Figures 8B and 8C show responses made by cells expressing the candidate ortho-IL-2 IRa molecules RV13 and RV6, respectively.
  • the highlighted curves in the three figures show responses made by the three kinds of cells to five selected cytokines, namely, wild-type IL-21, CV22, CV204, CV374, and CV388.
  • RV13 carries two substitutions relative to wild-type IL-21Ra, namely M70G and Y129F, whereas RV22 carries just M70G. These two variant receptors appear to be equivalently compromised in their capacity to bind native IL-21 ( Figure 4A). They also accounted for a similar pattern of reactivity to the collection of IL-21 molecules used in Figures 9A and 9B. Specifically, like RV13 ( Figure 10B), RV22 mediated significantly impaired signaling responses to wild-type IL-21 (and the negative control molecule CV22) but conferred good responses to CV204, CV374, and CV388 ( Figure 10C).
  • candidate ortho-IL-21 molecules were generated. These variants included a majority that were based on CV374 and CV388 but carried alternative substitutions predicted to impact binding to wild-type IL-2 IRa and the candidate ortho-IL-2 IRa molecule RV22, either on the basis of prior screening data (e.g., Figures 8A-8C) or from the published crystal structure of the IL-21 cytokine-receptor complex.
  • candidate ortho-IL-21 variant CV414 resembles CV388, differing only in the absence of a substitution at position 104.
  • candidate ortho-IL-21 variant CV415 resembles CV388 but includes the G84E substitution present in CV374 and P104V.
  • FIG. 11A-11C The new series of candidate ortho-IL-21 molecules were screened for their capacity to induce signaling in Ba/F3 cells expressing wild-type IL-21Ru or RV22 as described. Representative data from one such screening experiment are provided in Figures 11A-11C.
  • Figure 11A shows the STAT3 responses elicited in cells expressing wild-type IL-21Ra exposed to the cytokine collection (with the responses to CV374, CV415, and wild-type IL-21 highlighted).
  • Figure 11B shows the STAT3 responses elicited in cells expressing RV22.
  • Figure 11C shows a comparison of the STAT3 responses elicited by wild-type IL-21 and CV415 on cells expressing either the wild-type lL-21Ra or RV22.
  • Example 4 Screening of Candidate Ortho-IL-21 Molecules: Testing for STAT3 -Dependent Signaling Responses in Cells Expressing RV6, RV31, or Wild-Type IL-21Rq
  • Example 3 The screening strategy described in Example 3 was partially replicated to create a second orthogonal system.
  • this second system is orthogonal not just to the native IL-21 system but also to the system described above involving candidate ortho-IL-21Ra molecules comprised of an M70G substitution (including RV13 and RV22).
  • candidate ortho-IL-21Ra molecules comprised of an M70G substitution (including RV13 and RV22).
  • candidate ortho-IL-2 IRa molecules RV6 and RV31 were selected for the creation of such a mutually orthogonal system.
  • RV6 and RV13 are both comprised of M70I and D73E mutations, but Q33H is also present in RV6.
  • FIG. 8C Data from one such screening experiment are provided in Figure 8C. Additional cytokine variants were generated based on these data and included in the collection of variants that was the basis of the data shown in Figures 11A-11C. The entire collection was then tested for its capacity to induce signaling in BA/F3 cells expressing RV22, RV31, or the wild-type form of IL-21Ra. As shown in Figures 12A-C, cytokine variants CV425 and CV458 were notable because they elicited strong responses in all three kinds of cells.
  • variant CV431 caused a strong response in cells expressing wild-type IL-21Ra or RV31, but it was comparatively less active against cells expressing RV22.
  • variant CV339 was weakly active against all three kinds of cells.
  • Figure 12D provides a comparison between responses by selected cytokines (including CV339, CV425, CV431, and CV458) on cells expressing either the wild-type form of IL-21Ra or RV31.
  • Receptor variants RV31-S190F and RV31-A127M both afforded improved responsiveness to CV588 relative to RV31 ( Figure 13C). A similar improvement in responsiveness was seen with cytokine variants CV617 and CV631 ( Figures 13D and 13E). These receptor variants conferred impaired responses to wild-type IL-21 ( Figure 13A).
  • RV31-E38T and RV31-E38H were both associated with decreased responsiveness to wild-type IL-21 relative to the parent RV31 receptor ( Figures 13A and 13F). Strikingly, both receptor variants retained equivalent responsiveness to CV588, CV617, and CV631 as RV31 ( Figures 13C-E and Figures 13H-I).
  • Cytokine variant CV588 showed a similarly weak capacity to stimulate cells expressing RV22 or wild-type IL-21Ra relative to cells expressing RV31 or RV31-S190F ( Figure 13C).
  • CV415 showed a much-enhanced preference for cells expressing RV22 than for cells expressing RV31 or RV31-S190F ( Figure 13B)
  • the E38T ( Figure 13B) or E38H ( Figure 13G) substitutions improved this preference by impairing responses to CV415.
  • substitutions present in CV588, CV617, and CV631 improve the capacity of variant IL-21 to stimulate cells expressing RV31 while decreasing its capacity to stimulate cells expressing the wild-type receptor. This kind of preference is enhanced by the S190F or A127M substitutions in RV31, while the E38T or E38H substitutions can be used to impair the capacity of the receptor to respond to wild-type IL-21.
  • substitutions provide the basis for a cytokine receptor system that functions in an orthogonal fashion to both the native and CV415-RV22 systems.
  • in vitro assays will have only very limited predictive value of the effects of a therapeutic in vivo: many therapeutic targets are expressed in multiple cell types (often having opposing effects on the response in vivo), or the therapeutic effect of a given target is dependent on other auxiliary cells. In such situations, an in vitro model, which by its very nature is simplistic, is not a particularly good proxy for the much more complicated situation in vivo. This is not the case in the instant application: the target receptor is synthetic and will only be expressed in cells specifically engineered to do so. Given the specificity of the orthogonal cytokine-receptor system, this significantly reduces the complexity, giving an in vitro assay a better predictive value.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des récepteurs de l'IL-21 orthogonaux et des cytokines de l'IL-21 orthogonales. Les paires récepteurs-cytokines de l'IL-21 peuvent comprendre une chaîne α du récepteur de l'interleukine-21 orthogonale (« ortho-IL-21 Rα ») qui présente une liaison altérée à la cytokine native de l'interleukine-21 (« IL-21 ») et une cytokine de l'IL-21 orthogonale (« ortho-IL-21 ») qui présente une liaison altérée à l'IL-21Rα native, l'ortho-IL-21 Rα se liant à l'ortho-IL-21. La paire récepteur-cytokine de l'IL -21 peut activer la signalisation de l'IL-21. L'invention concerne également des cellules modifiées pour exprimer les récepteurs de l'IL-21 orthogonaux, ainsi que des méthodes d'utilisation de ces cellules pour le traitement de maladies et troubles divers.
EP23792786.8A 2022-04-20 2023-04-20 Systèmes cytokines/récepteurs d'il-21 orthogonaux Pending EP4511054A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US202263333085P 2022-04-20 2022-04-20
US202263333090P 2022-04-20 2022-04-20
PCT/US2023/066016 WO2023205738A2 (fr) 2022-04-20 2023-04-20 Systèmes cytokines/récepteurs d'il-21 orthogonaux

Publications (1)

Publication Number Publication Date
EP4511054A2 true EP4511054A2 (fr) 2025-02-26

Family

ID=88420746

Family Applications (1)

Application Number Title Priority Date Filing Date
EP23792786.8A Pending EP4511054A2 (fr) 2022-04-20 2023-04-20 Systèmes cytokines/récepteurs d'il-21 orthogonaux

Country Status (2)

Country Link
EP (1) EP4511054A2 (fr)
WO (1) WO2023205738A2 (fr)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4029877B1 (fr) * 2017-08-03 2024-01-17 Amgen Inc. Mutéines d'interleukine-21 et méthodes de traitement
CN112566650A (zh) * 2018-06-21 2021-03-26 沙塔克实验室有限公司 异二聚体蛋白及其用途
US20230226203A1 (en) * 2020-06-18 2023-07-20 Proviva Therapeutics (Hong Kong) Limited Activatable procytokines

Also Published As

Publication number Publication date
WO2023205738A2 (fr) 2023-10-26
WO2023205738A3 (fr) 2023-11-30

Similar Documents

Publication Publication Date Title
US11648296B2 (en) IL-2 orthologs and methods of use
Cho et al. Transplantation of mesenchymal stem cells overexpressing RANK-Fc or CXCR4 prevents bone loss in ovariectomized mice
CN110325205A (zh) T细胞调节性多聚体多肽及其使用方法
KR20170045258A (ko) 신호전달 시스템
WO2017123548A1 (fr) Sécrétion de l'ifna spécifique d'une tumeur par des lymphocytes t car permettant de reprogrammer le microenvironnement d'une tumeur solide
WO2019162695A1 (fr) Cellule
US12473335B2 (en) Orthogonal IL-21 receptor/cytokine systems
US20230027899A1 (en) Cd122 with altered icd stat signaling
US8524656B2 (en) GM-CSF and truncated CCL2 conjugates and methods and uses thereof
Qian et al. Long-acting human interleukin 2 bioconjugate modified with fatty acids by sortase A
ES2415890T3 (es) Procedimiento de preparación de proteínas triméricas
JP2017530721A (ja) 近接度に基づく結合パートナーの選択のための方法
WO2020074868A1 (fr) Cellule
EP4511054A2 (fr) Systèmes cytokines/récepteurs d'il-21 orthogonaux
AU2021370625A1 (en) Orthogonal il-21 receptor/cytokine systems
JP2022547866A (ja) 同種異系細胞組成物と使用方法
CN116529257A (zh) 正交il-21受体/细胞因子系统
JP3499528B2 (ja) シグナルシーケンストラップ法
US8841253B2 (en) Viral/bacterial toxin polypeptides and methods of using same
WO2015197639A1 (fr) Déplétion de cellules spécifiques d'un tissu avec deux protéines chimères

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20241118

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC ME MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)