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WO2018161026A1 - Compositions d'il-15 et méthodes pour immunothérapie - Google Patents

Compositions d'il-15 et méthodes pour immunothérapie Download PDF

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Publication number
WO2018161026A1
WO2018161026A1 PCT/US2018/020755 US2018020755W WO2018161026A1 WO 2018161026 A1 WO2018161026 A1 WO 2018161026A1 US 2018020755 W US2018020755 W US 2018020755W WO 2018161026 A1 WO2018161026 A1 WO 2018161026A1
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WIPO (PCT)
Prior art keywords
hdhfr
cell
cells
seq
amino acid
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.)
Ceased
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PCT/US2018/020755
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English (en)
Inventor
Vipin Suri
Kutlu Goksu ELPEK
Dan Jun LI
Brian DOLINSKI
Michelle Lynn OLS
Scott Francis HELLER
Nicole KOSMIDER
Celeste RICHARDSON
Steven Mark Shamah
Tucker EZELL
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Obsidian Therapeutics Inc
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Obsidian Therapeutics Inc
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Application filed by Obsidian Therapeutics Inc filed Critical Obsidian Therapeutics Inc
Publication of WO2018161026A1 publication Critical patent/WO2018161026A1/fr
Priority to US16/558,224 priority Critical patent/US11629340B2/en
Anticipated expiration legal-status Critical
Priority to US17/646,212 priority patent/US12104178B2/en
Priority to US18/787,024 priority patent/US20240401004A1/en
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2086—IL-13 to IL-16
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • 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]
    • C07K14/5443—IL-15
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • C12N9/0028—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1)
    • C12N9/003—Dihydrofolate reductase [DHFR] (1.5.1.3)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y105/00—Oxidoreductases acting on the CH-NH group of donors (1.5)
    • C12Y105/01—Oxidoreductases acting on the CH-NH group of donors (1.5) with NAD+ or NADP+ as acceptor (1.5.1)
    • C12Y105/01003—Dihydrofolate reductase (1.5.1.3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to compositions and methods for immunotherapy.
  • compositions comprise destabilizing domains (DDs) which tune protein stability.
  • Cancer immunotherapy aims to eradicate cancer cells by rejuvenating the tumoricidal functions of tumor-reactive immune cells, predominantly T cells.
  • Strategies of cancer immunotherapy including the recent development of checkpoint blockade, adoptive cell transfer (ACT) and cancer vaccines which can increase the anti-tumor immune effector cells have produced remarkable results in several tumors.
  • ACT adoptive cell transfer
  • TME tumor microenvironment
  • immunotherapeutic agent expression is needed in case of ad verse events.
  • adoptive cell therapies may have a very long and an indefinite half-life. Since toxicity can be progressive, a safety switch is desired to eliminate the infused ceils.
  • Systems and methods that can tune the transgenic protein level and expression window with high flexibility can enhance therapeutic benefit, and reduce potential side effects.
  • the present invention provides biocircuit systems to control the expression of immunotherapeutic agents.
  • the biocircuit system comprises a stimulus and at least one effector module that responds to the stimulus.
  • the effector module may include a stimulus response element (SRE) that binds and is responsive to a stimulus and an immunotherapeutic agent operably linked to the SRE.
  • SRE stimulus response element
  • a SRE is a destabilizing domain (DD) which is destabilized in the absence of its specific ligand and can be stabilized by binding to its specific ligand.
  • the present invention provides compositions and methods for immunotherapy.
  • the compositions relate to tunable systems and agents that induce an immune response in a cell or in a subject.
  • the tunable system and agent may be a biocircuit system comprising at least one effector module that is responsive to at least one stimulus.
  • the biocircuit system may be, but is not limited to, a destabilizing domain (DD) biocircuit system, a dimerization biocircuit system, a receptor biocircuit system, and a cell biocircuit system.
  • DD destabilizing domain
  • the composition for inducing an immune response may comprise an effector module.
  • the effector module may comprise a stimulus response element (SRE) operably linked to at least one payioad.
  • the payload may be an immunotherapeutic agent.
  • the immunotherapeutic agent may be selected from, but not limited to a cytokine, a cytokine receptor, a cytokine-cytokine receptor fusion, and any combinations thereof.
  • the SRE of the composition may be responsive to or interact with at least one stimulus.
  • the SRE may comprise a destabilizing domain (DD).
  • the DD may be derived from a parent protein or from a mutant protein having one, two, there, or more amino acid mutations compared to the parent protein.
  • the parent protein may be selected from, but is not limited to, human protein FKBP, comprising the amino acid sequence of SEQ. ID NO. 3; human DHFR (hDHFR), comprising the amino acid sequence of SEQ. ID NO. 2; E. Coli DHFR, comprising the amino acid sequence of SEQ. ID NO. 1; PDE5, comprising the amino acid sequence of SEQ. ID NO. 4; PPAR, gamma comprising the amino acid sequence of SEQ. ID NO. 5; CA2, comprising the amino acid sequence of SEQ. ID NO. 6: or NQ02, comprising the amino acid sequence of SEQ. ID NO. 7.
  • the parent protein is hDHFR and the DD comprises a mutant protein.
  • the mutant protein may comprise a single mutation and may be selected from, but not limited to hDHFR (II 7V), hDHFR (F59S), hDHFR (N65D), hDHFR (K81R), hDHFR (A 107V), hDHFR (Y122I), hDHFR (N127Y), hDHFR (M140I), hDHFR (K 185E), hDHFR (N 186D), and hDHFR (M140I), hDHFR (Amino acid 2-187 of WT; N 127Y), hDHFR (Amino acid 2-187 of WT; I17V), hDHFR (Ammo acid 2-187 of WT; Y122I), and hDHFR (Amino acid 2-187 of WT; K185E).
  • the mutant protein may comprise two mutations and may be selected from, but not limited to, hDHFR (C7R, Y163C), hDHFR (Al 0V, H88Y), hDHFR (Q36K, Y 1221), hDHFR (M53T, R138I), hDHFR (T57A, 172A), hDHFR (E63G, I176F), hDHFR (G21T, ⁇ 1 221 ). hDHFR (L74N, ⁇ 1 221 ).
  • hDHFR (V75F, Y 1221), hDHFR (L94A, T147A), DHFR (V121 A, Y22I) , hDHFR (Y122I, A125F), hDHFR (HI 31R, E144G), hDHF (T137R, F143L), hDHFR (Y178H, E18IG), and hDHFR (Y183H, 185E), hDHFR (E162G, I176F) hDHFR (Amino acid 2-187 of WT; I 17V, YI221), hDHFR (Ammo acid 2- 187 of WT; Y122I, M140I), hDHFR (Ammo acid 2-187 of WT; N 127Y, Y 1221 ).
  • the mutant may comprise three mutations and the mutant may be selected from hDHFR (V9A, S93R, P 150L), hDHFR (I8V, K133E, Y163C), hDHFR (L23S, V121A, Y 157C), hDHFR (K19E, F89L, E181G), hDHFR (Q36F, N65F, ⁇ 1 221 ).
  • hDHFR (G54R, M140V, S168C), hDHFR (VI 10A, V 136M, K 177R), hDHFR (Q36F, Y122I, A 125F), hDHFR (N49D, F59S, D153G), and hDHFR (G21E, I72V, I176T), hDHFR (Ammo acid 2-187 of WT; Q36F, Y122I, A125F), hDHFR (Ammo acid 2-187 of WT; Y 122I, H131R, E144G), hDHFR ( Amino acid 2-187 of WT; E31D, F32M, V I 161), and hDHFR (Amino acid 2-187 of WT; Q 36!
  • the mutant may comprise four or more mutations and the mutant may be selected from hDHFR (V2A, R33G, Q36R, L100P, K185R), hDHFR (Amino acid 2-187 of WT; D22S, F32M, R33S, Q36S, N65S), hDHFR (I17N, L98S, K99R, Ml 12T, E151G, E162G, E172G), hDHFR (G16S, II 7V, F89L, D96G, K123E, Ml 40V, D146G, K 156R), hDHFR (K81R, K99R, L100P, E102G, N 108D, 123R, H128R, D142G, F180L, K185E), hDHFR (R138G, D142G, F143S, K156R, K158E, E162G, V166A, ⁇ 77 ⁇
  • N127R, H128Y, L132P, F135P, I139T, F148S, F149L, I152V, D153A, D169G, VI 70A, I176A, Kl 77R, V182A, Kl 85R, NI86S), and hDHFR (Al 0T, Q13R, N14S, N20D, P24S, N30S, M38T, T40A, K47R, N49S, K56R, 16 IT, 64R, K69R, I72A, R78G, E82G, F89L, D96G, N108D, Ml 12V, W114R, Y122D, K123E, I139V, Q141R, D142G, F148L, E151G, E155G, Y157R, Q171R, Y183C, E184G, K185del, D187 ).
  • the stimulus of the SRE may be Trimethoprim or Methotrexate.
  • the immunotherapeutic agent of the composition may be a cytokine.
  • the cytokine may be an interleukin, an interferon, a tumor necrosis factor, a transforming growth factor B, a CC chemokine, a CXC chemokine, a CX3C chemokine or a growth factor.
  • the interleukin may be a whole or a portion of a IL15 and may comprise the amino acid sequence of SEQ ID NO. 55.
  • the IL15 may be modified.
  • the modifications may comprise fusing SEQ ID NO. 55 to the whole or a portion of, a transmembrane domain.
  • the IL15 may optionally be modified by incorporating a hinge domain.
  • the immunotherapeutic agent may be a cytokine- cytokine receptor fusion polypeptide, in some embodiments, the cytokine-cytokine receptor fusion polypeptide may comprise the whole or a portion of SEQ. ID NO. 55, fused to the whole or a portion of any of SEQ. ID NOs. 211-218 to produce a IL15-IL15 receptor fusion polypeptide.
  • the cytokine-cytokine receptor fusion polypeptide may be modified.
  • the modification may comprise fusing the IL15-IL15 receptor fusion polypeptide to the whole, or a portion, of a transmembrane protein.
  • an optional hinge domain may be incorporated.
  • the SRE of the composition may stabilize the immunotherapeutic agent by a stabilization ratio of 1 or more.
  • the stabilization ratio may comprise the ratio of expression, function or level of the immunotherapeutic agent in the presence of the stimulus to the
  • the SRE of the composition may destabilize the immunotherapeutic agent by a destabilization ratio between 0, and 0.09.
  • the destabilization ratio may comprise the ratio of expression, function or level of an immunotherapeutic agent in the absence of the stimulus specific to the SRE to the expression, function or level of the immunotherapeutic agent that is expressed constitutively, and in the absence of the stimulus specific to the SRE.
  • the present invention also provides polynucleotides comprising the compositions of the invention.
  • the polynucleotides may be a DNA or RNA molecule. In one aspect, the polynucleotides may comprise spatiotemporally selected codons. In one aspect, the
  • polynucleotides of the invention may be a DNA molecule.
  • the polynucleotides may be an RNA molecule.
  • the RNA molecule may be a messenger molecule.
  • the RNA molecule may be chemically modified.
  • the polynucleotides may further comprise, at least one additional feature selected from, but not limited to, a promoter, a linker, a signal peptide, a tag, a cleavage site and a targeting peptide.
  • the present invention also provides vectors comprising polynucleotides described herein.
  • the vector may be a viral vector.
  • the viral vector may be a retroviral vector, a lentivirai vector, a gamma retroviral vector, a recombinant AAV vector, an adeno viral vector, and an oncolytic viral vector.
  • the present invention also provides immune cells for adoptive cell transfer (ACT) which may express the compositions of the invention, the polynucleotides described herein.
  • the immune cells may be infected or transfected with the vectors described herein.
  • the immune cells for ACT may be selected from., but not limited to a CD8+ T cell, a CD4+ T cell, a helper T cell, a natural killer (NK) cell, a NKT cell, a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), a memory T cell, a regulator ⁇ ' T (Treg) cell, a cytokine- induced killer (CIK) cell, a dendritic cell, a human embryonic stem cell, a mesenchymal stem cell, a hematopoietic stem cell, or a mixture thereof,
  • the immune cells may be autologous, allogeneic, syngeneic, or xenogeneic in relation to a particular individual subject.
  • the present invention provides methods for reducing a tumor volume or burden in a subject comprising contacting the subject with the immune cells of the invention. Also provided herein, is a method for inducing an anti-tumor immune response in a subject, comprising administering the immune ceils of the system to the subject.
  • the present invention also provides methods for enhancing the expansion and/or survival of immune cells, comprising contacting the immune cells with the compositions of the invention, the polynucleotides of the invention, and/or the vectors of the invention.
  • compositions of the invention are administered to the subject.
  • the polynucleotides of the invention are administered to the subject.
  • the effector module comprises a stimulus response element (SRE) and at least one pay load comprising a pay load of interest (POI).
  • SRE stimulus response element
  • POI pay load of interest
  • the SRE may be a destabilizing domain (DD).
  • the DD is a domain derived from a protein such as FKBP (F 506 binding protein), E. coli DHFR (Dihydrofolate reductase) (ecDHFR), human DHFR (iiDHFR), or any payioad of interest.
  • FKBP F 506 binding protein
  • EcDHFR E. coli DHFR
  • iiDHFR human DHFR
  • the biocircuit system is a DD biocircuit system..
  • Tire payioad may be an immunotherapeutic agent used for cancer immunotherapy such as a cytokine such as 1L 15 or a cytokine -cytokine receptor polypeptides such as IL15/IL15Ra fusion polypeptide, or any agent that can induce an immune response.
  • a cytokine such as 1L 15
  • a cytokine -cytokine receptor polypeptides such as IL15/IL15Ra fusion polypeptide
  • the SRE and payioad may be operably linked through one or more linkers and the positions of components may vary within the effector module.
  • the effector module may further comprise one or more additional features such as linker sequences (with, specific sequences and lengths), cleavage sites, regulatory- elements (that regulate expression of the protein of interest such as microRNA targeting sites), signal sequences that lead the effector module to a specific cellular or subcellular location, penetrating sequences, or tags and biomarkers for tracking the effector module.
  • the DD may stabilize the protein of interest with a stabilization ratio of at least one in the presence of the stimulus. According to the present invention, the DD may destabilize the protein of interest in the absence of ligand with a destabilization ratio between 0, and 0.99.
  • the present invention also provides immunotherapeutic agents which may be a cytokine fused to its cognate cytokine receptor.
  • the immotherapeutic agent of the invention may a fusion polypeptide comprising the whole or a portion of IL15 fused to the whole or a portion of the ILlSRa.
  • Such fusion polypeptides may be membrane associated or secreted.
  • the invention provides isolated biocircuit polypeptides, effector modules, stimulus response elements (SREs) and payloads, as well as polynucleotides encoding any of the foregoing; vectors comprising polynucleotides of the invention; and cells expressing
  • polypeptides polypeptides, polynucleotides and vectors of the invention.
  • the polypeptides, polynucleotides, viral vectors and cells are useful for inducing anti-tumor immune responses in a subject.
  • the vector of the invention is a viral vector.
  • the viral vector may include, but is not limited to a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector.
  • the vector of the invention may be a non-viral vector, such as a nanoparticles and liposomes.
  • the present invention also provides immune cells engineered to include one or more polypeptides, polynucleotides, or vectors of the present invention.
  • the cells may be immune effector cells, including T cells such as cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, natural killer (NK) cells, NK T cells, cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTLs), and tumor infiltrating lymphocytes (TILs).
  • T cells such as cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, natural killer (NK) cells, NK T cells, cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTLs), and tumor infiltrating lymphocytes (TILs).
  • T cells such as cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, natural killer (NK) cells, NK T cells, cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes
  • the present invention also provides methods for inducing immune responses in a subject using the compositions of the invention. Also provided are methods for reducing a tumor burden in a subject using the compositions of the invention.
  • immunotherapeutic agent in cells or subjects may involve the administering effector modules containing an SRE operably linked to an immunotherapeutic agent.
  • the immunotherapeutic agent is IL15 or IL15 fused to IL15Ra.
  • the SRE is derived from FKBP, DHFR, PDE5, PPAR gamma, CA2 and NQ02. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 shows an overview diagram of a biocircuit system of the invention.
  • the bioeireuit comprises a stimulus and at least one effector module responsive to a stimulus, where the response to the stimulus produces a signal or outcome.
  • the effector module comprises at least one stimulus response element (SRE) and one pay!oad.
  • SRE stimulus response element
  • Figure 2 shows representative effector modules carrying one payload.
  • the signal sequence (SS), SRE and payload may be located or positioned in various arrangements without (A to F) or with (G to Z, and AA to DD) a cleavage site.
  • An optional linker may be inserted between each component of the effector module.
  • Figure 3 shows representative effector modules carrying two payloads without a cleavage site.
  • the two payloads may be either directly linked to each other or separated.
  • Figure 4 shows representative effector modules carrying two payloads with a cleavage site.
  • an SS is positioned at the N-terminus of the construct, while other components: SRE, two payloads and the cleavage site may be located at different positions (A to L).
  • the cleavage site is positioned at the N-terminus of the construct (M to X).
  • An optional linker may be inserted between each component of the effector module.
  • Figure 5 shows effector modules of the invention carrying two payloads, where an SRE- is positioned at the N-terminus of the construct (A to L), while SS, two payloads and the cleavage site can be in any configuration.
  • An optional linker may be inserted between each component of the effector module.
  • Figure 6 shows effector modules of the invention carrying two payloads, where either the two payloads (A to F) or one of the two payloads (G to X) is positioned at the N-terminus of the construct (A to L), while SS, SRE and the cleavage site can be in any configuration.
  • An optional linker may be inserted between each component of the effector module.
  • Figure 7 depicts representative configurations of the stimulus and effector module within a biocircuit system.
  • a trans-membrane effector module is activated either by a free stimulus (Figure 7A) or a membrane bound stimulus ( Figure 7B) which binds to SRE.
  • the response to the stimulus causes the cleavage of the intracellular signai/payload, which activates down-stream effector/payload.
  • Figure 8 depicts a dual stimulus-dual presenter biocircuit system, where two bound stimuli (A and B) from two different presenters (e.g., different cells) bind to two different effector modules in a single receiver (e.g., another single cell) simultaneously and create a dual- signal to downstream payloads.
  • Figure 9 depicts a dual stimulus-single presenter biocircuit system, where two bound stimuli (A and B) from the same presenter (e.g., a single cell) bind to two different effector modules in another single cell simultaneously and create a dual-signal.
  • Figure 10 depicts a single-stimulus-bridged receiver biocircuit system.
  • a bound stimulus (A) binds to an effector module in the bridge ceil and creates a signal to activate a payload which is a stimulus (B) for another effector module in the final receiver (e.g., another cell).
  • Figure 1 1 depicts a single stimulus-single receiver biocircuit system, wherein the single receiver contains the two effector modules which are sequentially activated by a single stimulus.
  • Figure 12 depicts a biocircuit system which requires a dual activation.
  • one stimulus must bind the transmembrane effector module first to prime the receiver cell being activated by the other stimulus.
  • the receiver only activates when it senses both stimuli (B).
  • Figure 13A is a western blot of IL15 protein levels in 293T cells.
  • Figure 13B and 13C are histograms depicting surface expression of IL15 and IL15Ra respectively .
  • Figure 13D is a western blot of IL15 and hDHFR in HCT ' l 16 cells.
  • Figure 14 denotes the frequency of IFNgamma positive T cells.
  • Figure 15A depicts IFN gamma production in T cells.
  • Figure 15B depicts T cell expansion with IL15/IL15Ra treatment.
  • Figure 15C is a dot plot depicting the percentage human cells after in vivo cell transfer.
  • Figure 15D is scatter plot depicting CD4+/CD8+ T cells.
  • Figure 16A is a bar graph depicting IL15Ra positive cells with 24 hour TMP treatment.
  • Figure 16B is a bar graph depicting ILlSRa positive cells with 48 hour TMP treatment.
  • 16C is a bar graph depicting ILlSRa positive cells in response to varying concentrations of TMP.
  • Figure 17A is a western blot of ILlSRa protein levels in HCT1 16 cells.
  • ILiS levels in the media measured by ELISA are represented in Figure 17B and levels measured using the
  • Figure I8A represents percentage of human T cells blood with respect to mouse T cells.
  • Figure 18B represents the number of T cells in blood.
  • Figure 18C represents ratio of CD4 to CD8 cells in the blood.
  • Figure 18D represents the percentage of ILlSRa positive CD4 and CDS T cells in the blood.
  • Figure 19 represents the proliferation of cell lines in response to cytokines.
  • Figure 20A represents the tumor growth of HCT116 cells in xenograft assays.
  • Figure 20B represents tumor growth of HCT116 ceils expressing ILlS/TLISRa fusion constructs with ligand treatment.
  • Figure 21 is a bar graph representing the effect of promoters on transgene expression.
  • Figure 22 depicts the kinetics of TL15Ra surface expression on CD4 T cells after TMP treatment.
  • Figure 23 represents a western blot of lL15-iL15Ra protein in HCT116 tumors from mice treated with TMP for 17 days in xenograft assays.
  • Figure 24 is a graph of the results of the MSD assay of TL15 protein levels in HEK293 cells.
  • Figure 2,5 A provides FACS plots showing the expression of membrane bound IL15 after a dose response study of TMP.
  • Figure 25B is two graphs showing the dose and time of exposure of TMP in vitro influences membrane bound IL15 expression.
  • Figures 26A- 26C show the regulation of membrane bound 1L15 using 1L15 (Figure 26A), ILlSRa (Figure 26B), or iL15/IL15Ra double ++ staining (Figure 26C).
  • Figure 26D shows FACS plots of the expression of TL15.
  • Figure 26E is a graph of the regulation of TL15 in blood and
  • Figure 26F is a graph of the plasma TMP levels.
  • Figure 27 represents the regulation of membrane bound IL15 with PO or IP dosing of TMP.
  • Cancer immunotherapy aims at the induction or restoration of the reactivity of the immune system towards cancer.
  • Significant advances in immunotherapy research have led to the development of various strategies which may broadly be classified into active immunotherapy and passive immunotherapy. In general, these strategies may be utilized to directly kill cancer cells or to counter the immunosuppressive tumor microenvironment.
  • Active immunotherapy aims at induction of an endogenous, long -lasting tumor-antigen specific immune response. The response can further be enhanced by non-specific stimulation of immune response modifiers such as cytokines.
  • passive immunotherapy includes approaches where immune effector molecules such as tumor-antigen specific cytotoxic T cells or antibodies are administered to the host. This approach is short lived and requires multiple applications.
  • a major risk involved in immunotherapy is the on-target but off tumor side effects resulting from T-cell activation in response to normal tissue expression of the tumor associated antigen (TAA).
  • TAA tumor associated antigen
  • Immunotherapy may also produce on target, on-tumor toxicities that emerge when tumor cells are killed in response to the immunotherapy.
  • the adverse effects include tumor lysis syndrome, cytokine release syndrome and the related macrophage activation syndrome.
  • the present invention provides systems, compositions, immunotherapeutic agents and methods for cancer immunotlierapy. These compositions provide tunable regulation of gene expression and function in immunotherapy.
  • the present invention also provides biocircuit systems, effector modules, stimulus response elements (SREs) and payloads, as well as polynucleotides encoding any of the foregoing.
  • the systems, compositions, immunotherapeutic agents and other components of the invention can be controlled by a separately added stimulus, which provides a significant flexibility to regulate cancer immunotherapy.
  • the systems, compositions and the methods of the present invention may also be combined with therapeutic agents such as chemotherapeutic agents, small molecules, gene therapy, and antibodies.
  • compositions of the invention has the potential to improve the potency and duration of the efficacy of immunotherapies.
  • Reversibly silencing the biological activity of adoptively transferred cells using compositions of the present invention allows maximizing the potential of cell therapy without irretrievably killing and term inating the therapy,
  • the present invention provides methods for fine tuning of immunotherapy after administration to patients. This in turn improves the safety and efficacy of immunotherapy and increases the subject population that may benefit from, immunotherapy.
  • biocircuit systems which comprise, at their core, at least one effector module system.
  • Such effector module systems comprise at least one effector module having associated, or integral therewith, one or more stimulus response elements (SREs).
  • SREs stimulus response elements
  • the overall architecture of a biocircuit system of the invention is illustrated in Figure 1.
  • a stimulus response element (SRE) may be operably linked to a payload construct which could be any protein of interest (POI) (e.g., an immunotherapeutic agent), to form an effector module.
  • POI protein of interest
  • the SRE when activated by a particular stimulus, e.g., a small molecule, can produce a signal or outcome, to regulate transcription and/or protein levels of the linked payload either up or down by perpetuating a stabilizing signal or destabilizing signal, or any other types of regulation.
  • a biocircuit system are taught in co-owned U.S. Provisional Patent Application No. 62/320,864 tiled April 1 1, 2016, 62/466,596 filed March 3, 2017 and the International Publication WO2017/180587 (the contents each of which are herein incorporated by reference in their entirety)-
  • biocircuit systems, effector modules, SREs and components that tune expression levels and activities of any agents used for immunotherapy are provided.
  • a “biocircuit” or “biocircuit system” is defined as a circuit within or useful in biologic systems comprising a stimulus and at least one effector module responsive to a stimulus, where the response to the stimulus produces at least one signal or outcome within, between, as an indicator of, or on a biologic system.
  • Biologic systems are generally understood to be any cell, tissue, organ, organ system or organism, whether animal, plant, fungi, bacterial, or viral.
  • biocircuits may be artificial circuits which employ the stimuli or effector modules taught by the present invention and effect signals or outcomes in acellular environments such as with diagnostic, reporter systems, devices, assays or kits.
  • the artificial circuits may be associated with one or more electronic, magnetic, or radioactive components or parts.
  • a biocircuit system may be a destabilizing domain (DD) biocircuit system, a dimerization biocircuit system, a receptor biocircuit system, and a ceil biocircuit system. Any of these systems may act as a signal to any other of these biocircuit systems.
  • DD destabilizing domain
  • an immunotherapeutic agent may be an antibody and fragments and variants thereof, a cancer specific T cell receptor (TCR) and variants thereof, an anti-tumor specific chimeric antigen receptor (CAR), a chimeric switch receptor, an inhibitor of a co-inhibitory receptor or ligand, an agonist of a co-stimulatory receptor and ligand, a cytokine, chemokine, a cytokine receptor, a chemokine receptor, a soluble growth factor, a metabolic factor, a suicide gene, a homing receptor, or any agent that induces an immune response in a cell and a subject.
  • TCR cancer specific T cell receptor
  • CAR anti-tumor specific chimeric antigen receptor
  • a chimeric switch receptor an inhibitor of a co-inhibitory receptor or ligand, an agonist of a co-stimulatory receptor and ligand, a cytokine, chemokine, a cytokine receptor, a chemokine
  • the biocircuits of the invention include at least one effector module as a component of an effector module system.
  • an "effector module” is a single or multi-component construct or complex comprising at least (a) one or more stimulus response elements (i.e. proteins of interest (POIs).
  • POIs proteins of interest
  • SRE stimulation response element
  • SRE is a component of an effector module which is joined, attached, linked to or associated with one or more payloads of the effector module and in some instances, is responsible for the responsive nature of the effector module to one or more stimuli.
  • the "responsive" nature of an SRE to a stimulus may be characterized by a covending or non-covalent interaction, a direct or indirect association or a structural or chemical reaction to the stimulus.
  • the response of any SRE to a stimulus may be a matter of degree or kind.
  • the response may be a partial response.
  • the response may be a reversible response.
  • the response may ultimately lead to a regulated signal or output.
  • Such output signal may be of a relative nature to the stimulus, e.g., producing a modulatory effect of between 1% and 100% or a factored increase or decrease such as 2-fold, 3-fold, 4-fold, 5 -fold, 10-fold or more.
  • the present invention provides methods for modulating protein expression, function or level.
  • the modulation of protein expression, function or level refers to modulation of expression, function or level by at least about 20%, such as by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20- 40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-1 0%, 30-40%, 30-50%, 30- 60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40- 90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60- 80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80- 100%,
  • the present invention provides methods for modulating protein, expression, function or level by measuring the stabilization ratio and destabiiization ratio.
  • the stabilization ratio may be defined as the ratio of expression, function or level of a protein of interest in response to the stimulus to the expression, function or level of the protein of interest in the absence of the stimulus specific to the SRE.
  • the stabilization ratio is at least 1, such as by at least 1-10, 1-20, 1 -30, 1-40, 1 -50, 1- 60, 1-70, 1-80, 1- 90, 1-100, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-95, 20-100, 30-40, 30-50, 30-60, 30-70, 30- 80, 30-90, 30-95, 30-100, 40-50, 40-60, 40-70, 40-80, 40-90, 40-95, 40-100, 50-60, 50-70, SO- SO, 50-90, 50-95, 50-100, 60-70, 60-80, 60-90, 60-95, 60-100, 70-80, 70-90, 70-95, 70-100, 80- 90, 80-95, 80-100, 90-95, 90-100 or 95-100.
  • the destabiiization ratio may be defined as the ratio of expression, function or level of a protein of interest in the absence of the stimulus specific to the effector module to the expression, function or level of the protein of interest, that is expressed constitutively and in the absence of the stimulus specific to the SRE.
  • constitutively refers to the expression, function or level of a protein of interest that is not linked to an SRE, and is therefore expressed both in the presence and absence of the stimulus.
  • the destabiiization ratio is at least 0, such as by at least 0.1, 0.2, 0.3, 0.4, 0,5, 0.6, 0.7, 0.8, 0.9, or at least, 0-0.1 , 0-0,2, 0 -0.3, 0-0.4, 0-0.5, 0-0.6, 0-0,7, 0-0,8, 0-0.9, 0, 1 -0.2, 0.1 -0.3, 0.1-0.4, 0.1-0.5, 0.1-0.6, 0.1-0.7, 0.1-0.8, 0.1-0,9, 0,2 -0.3, 0.2-0.4, 0.2-0.5, 0.2- 0.6, 0.2-0.7, 0.2-0.8, 0.2-0.9, 0.3-0.4, 0.3-0.5, 0.3-0.6, 0.3-0.7, 0.3-0.8, 0.3-0.9, 0.4-0.5, 0.4-0.6, 0.4-0.7, 0.4-0.8, 0.4-0.9, 0.5-0.6, 0.5-0,7, 0,5-0,8, 0,5-0.9, 0.6-0.7, 0.6-0.8, 0.6-0.8,
  • the SRE of the effector module may be selected from, but is not limited to, a peptide, peptide complex, peptide-protein complex, protein, fusion protein, protein complex, protein- protein complex.
  • the SRE may comprise one or more regions derived from any natural or mutated protein, or antibody.
  • the SRE is an element, when responding to a stimulus, can une intracellular localization, intramolecular activation, and/or degradation of payloads.
  • the stimulu s of the present invention maybe ultrasound stimulation.
  • the SREs of the present invention may derived from mechanosensitive proteins.
  • the SRE of the present invention may be the mechanically sensitive ion channel, Piezo 1.
  • Expression of the payload of interest in such instances is tuned by providing focused ultrasound stimulation .
  • the SREs of the present invention may be derived from calcium biosensors, and the stimulus of the present invention may calcium.
  • the calcium may be generated by the ultrasound induced mechanical stimulation of mechanosensitive ion channels.
  • the ultrasound activation of the ion channel causes a calcium influx thereby generating the stimulus.
  • the mechanosensitive ion channel is Piezo 1 .
  • Mechanosensors may be advantageous to use since they provide spatial control to a specific location in the body.
  • effector modules of the present invention may comprise additional features that facilitate the expression and regulation of the effector module, such as one or more signal sequences (SSs), one or more cleavage and/or processing sites, one or more targeting and/or penetrating peptides, one or more tags, and/or one or more linkers. Additionally, effector modules of the present invention may further comprise other regulatory moieties such as inducible promoters, enhancer sequences, microRNA sites, and/or microRNA targeting sites. Each aspect or tuned modality may bring to the effector module or biocircuit a differentially tuned feature.
  • SSs signal sequences
  • cleavage and/or processing sites one or more targeting and/or penetrating peptides
  • tags one or more tags
  • linkers such as inducible promoters, enhancer sequences, microRNA sites, and/or microRNA targeting sites.
  • Each aspect or tuned modality may bring to the effector module or biocircuit a differentially tuned feature.
  • an SRE may represent a destabilizing domain
  • mutations in the protein payload may alter its cleavage sites or dimerization properties or half-life and the inclusion of one or more microRNA or microRNA binding site may impart cellular detargeting or trafficking features.
  • the present invention embraces biocircuits which are multifactorial in their tenability.
  • Such biocircuits may be engineered to contain one, two, three, four or more tuned features.
  • effector modules of the present invention may include one or more degrons to tune expression.
  • a "degron" refers to a minimal sequence within a protein that is sufficient for the recognition and the degradation by the proteolytic system.
  • An important property of degrons is that they are transferrable, that is, appending a degron to a sequence confers degradation upon the sequence.
  • the degron may be appended to the destabilizing domains, the payload or both . Incorporation of the degron within the effector module of the invention, confers additional protein instability_to the effector module and may be used to minimize basal expression.
  • the degron may be an N- degron, a phospho degron, a heat inducible degron, a photosensitive degron, an oxygen dependent degron.
  • the degron may be an Ornithine decarboxylase degron as described by Takeuchi et al. (Takeuchi J ei al. (2008). Biochem J. 2008 Mar
  • degrons 1 10( 2 ).401 -7. the contents of which are incorporated by reference in their entirety).
  • Other examples of degrons useful in the present invention include degrons described in International patent publication Nos. WO2017004022, WO2016210343, and WO2011062962; the contents of each of which are incorporated by reference in their entirety.
  • each component of the effector module may be located or positioned in various arrangements without (A to F) or with (G to Z, and AA to DD) a cleavage site.
  • An optional linker may be inserted between each component of the effector module.
  • Figures 3 to 6 illustrate representative effector module embodiments comprising two payloads, i.e. two immunotherapeutic agents. In some aspects, more than two
  • immunotherapeutic agents may be included in the effector module under the regulation of the same SRE (e.g., the same DD).
  • the two or more agents may be either directly linked to each other or separated ( Figure 3).
  • the SRE may be positioned at the N-terminus of the construct or the C -terminus of the construct, or in the internal location .
  • the two or more immunotherapeutic agents may be the same type. Biocircuits and components utilizing such effector molecules are given in Figures 7-12.
  • biocircuits of the invention may be m odified to reduce their immunogenicity.
  • Immunogenicity is the result of a complex series of responses to a substance that is perceived as foreign and may include the production of neutralizing and non-neutralizing antibodies, formation of immune complexes, complement activation, mast cell activation, inflammation, hypersensitivity responses, and anaphylaxis.
  • protein engineering may be used to reduce the immunogenicity of the compositions of the invention.
  • modifications to reduce immunogenicity may include modifications that reduce binding of the processed peptides derived from the parent sequence to MHC proteins.
  • amino acid modifications may be engineered such that there are no or a minimal of number of immune epitopes that are predicted to bind with high affinity, to any prevalent MHC alleles.
  • MHC binding epitopes of known protein sequences are known in the art and may be used to score epitopes in the compositions of the present invention. Such methods are disclosed in US Patent Publication No. US 200201 19492, US20040230380, and US 20060148009; the contents of each of which are incorporated by reference in their entirety.
  • Epitope identification and subsequent sequence modification may be applied to reduce immunogenicity.
  • the identification of immunogenic epitopes may be achieved either physically or computationally.
  • Physical methods of epitope identification may include, for example, mass spectrometry and tissue culture/cellular techniques.
  • Computational approaches that utilize information obtained on antigen processing, loading and display, structural and/or proteomic data toward identifying non-self-peptides that may result from antigen processing, and thai are likely to have good binding characteristics in the groove of the MHC may also be utilized.
  • One or more mutations may be introduced into the biocircuits of the invention directing the expression of the protein, to maintain its functionality while simultaneously rendering the identified epitope less or non-immunogenic.
  • compositions of the invention may also be useful in the present invention.
  • Compositions of the invention may also be engineered to include non-classical amino acid sidechains to design less immunogenic compositions. Any of the methods discussed in International Patent Publication No. WO2005051975 for reducing immunogenicity may be useful in the present invention (the contents of which are incorporated by reference in their entirety).
  • patients may also be stratified according to the immunogenic peptides presented by their immune cells and may be utilized as a parameter to determine suitable patient cohorts that may therapeutically benefit for the compositions of the invention.
  • reduced immunogenicity may be achieved by limiting immunoproteasome processing.
  • the proteasome is an important cellular protease that is found in two forms: the constitutive proteasome, which is expressed in all cell types and which contains active e.g. catalytic subunits and the immunoproteasome that is expressed in cell of the hematopoietic lineage, and which contains different active subunits termed low molecular weight proteins (LMP) namely LMP-2, LMP- 7 and LMP- 10.
  • LMP low molecular weight proteins
  • Immunoproteasome s exhibit altered peptidase activities and cleavage site preferences that result in more efficient liberation of many MHC class I epitopes.
  • a well described function of the immunoproteasome is to generate peptides with hydrophobic C terminus that can be processed to fit in the groove of MHC class I molecules.
  • Deol P et al . have shown that immunoproteasomes may lead to a frequent cleavage of specific peptide bonds and thereby to a faster appearance of a certain peptide on the surface of the antigen presenting ceils; and enhanced peptide quantities (Deol P et al. (2007) J Immunol 178 (12) 7557-7562; the contents of which are incorporated herein reference in its entirety).
  • immunogenicity of the compositions of the invention may be reduced by modifying the sequence encoding the compositions of the invention to prevent immunoproteasome processing.
  • Biocircuits of the present invention may also be combined with immunoproteasome-selective inhibitors to achieve the same effects. Examples of inhibitors useful in the present invention include UK-101 (B li selective compound), IPSI-001, ONX 0914 (PR-957), and PR-924 (IPS1).
  • biocircuit system s, effector modules, and compositions of the present invention relate to post-translational regulation of protein (payload) function, in particular, anti-tumor immune responses of immunotherapeutic agents.
  • the SRE is a stabilizing/destabilizing domain (DD).
  • DD stabilizing/destabilizing domain
  • the presence, absence or an amount of a small molecule ligand that binds to or interacts with the DD, can, upon such binding or interaction modulate the stability of the payload(s) and consequently the function of the payload.
  • the altered function of the payload may vary, hence providing a "tuning" of the payload function.
  • destabilizing domains described herein or known in the art may be used as SREs in the biocircuit systems of the present invention in association with any of the immunotherapeutic agents (payloads) taught herein.
  • Destabilizing domains are small protein domains that can be appended to a target protein of interest. DDs render the attached protein of interest unstable in the absence of a DD-binding ligand such that the protein is rapidly degraded by the ubiquitin-proteasome system of the cell (Stankunas, K., et al., Moi. Cell, 2003, 12: 1615-1624; Banaszynski. et al..
  • the desired characteristics of the DDs may include, but are not limited to, low protein levels in the absence of a ligand of the DD (i .e. low basal stability), large dynamic range, robust and predictable dose-response behavior, and rapid kinetics of degradation. DDs that bind to a desired ligand but not endogenous molecules may be preferred.
  • FDD fluorescent destabilizing domain
  • DDs also include those described in U.S. Pat. NO. 8,173,792 and U.S. Pat. NO. 8,530,636, the contents of which are each incorporated herein by reference in their entirety.
  • the DDs of the present invention may be derived from, some known sequences that have been approved to be capable of post-translational regulation of proteins.
  • some known sequences that have been approved to be capable of post-translational regulation of proteins.
  • Xiong et al. have demonstrated that the non-catalytic N-terminal domain (54-residues) of ACS? ( 1-ammocydopropane-l-carboxyiate sy nthase) in Arabidopsis, when fused to the ⁇ -glucuronidase (GUS) reporter, can significantly decrease the accumulation of the GUS fusion protein (Xiong et al., ./ Exp. Bot. , 2014, 65(15): 4397-4408).
  • GUS ⁇ -glucuronidase
  • Another non-limiting example is the stability control region (SCR, residues 97-118) of Tropomyosin (Tm), which controls protein stability.
  • SCR Stret Control region
  • a destabilizing mutation LI 10A, and a stabilizing mutation A109L dramatically affect Tropomyosin protein dynamics (Kirwan and Hodges, J. Biol. Chem., 2014, 289: 4356-4366).
  • Such sequences can be screened for ligands that bind them and regulate their stability.
  • the identified sequence and ligand pairs may be used as components of the present invention.
  • the DDs of the present invention may be developed from known proteins. Regions or portions or domains of wild type proteins may be utilized as SREs/DDs in whole or in part. They may be combined or rearranged to create new peptides, proteins, regions or domains of which any may be used as SREs/DDs or the s tarting point for the design of further SREs and/or DDs.
  • Ligands such as small molecules that are well known to bind candidate proteins can be tested for their regulation in protein responses.
  • the small molecules may be clinically approved to be safe and have appropriate pharmaceutical kinetics and distribution.
  • the stimulus is a ligand of a destabilizing domain (DD), for example, a small molecule that binds a destabilizing domain and stabilizes the POI fused to the destabilizing domain.
  • DD destabilizing domain
  • ligands, DDs and SREs of the present invention include without limitation, any of those taught in Tables 2-4 of copending commonly owned US Provisional Patent Application NO, 62/320,864 filed on 4/1 1 /2016, or in US Provisional Application No, 62/466,596 filed March 3, 2017 and the Intemaiional Publication WO2017/180587, the contents of which are incorporated herein by reference in their entirety.
  • Some examples of the proteins that may be used to develop DDs and their ligands are listed in Table 1.
  • DDs of the invention may be FKBP DD or ecDHFR DDs such as those listed in Table 2.
  • the position of the mutated amino acid listed in Table 2 is relative to the ecDHFR (Uniprot ID: P0ABQ4) of SEQ. ID NO. 1 for ecDHFR DDs and relative to FKBP (Unrprot ID: P62942) of SEQ. ID NO. 3 for FKBP DDs.
  • novel DDs derived from E.coli DHFR may comprise amino acids 2-159 of the wild type ecDHFR sequence (SEQ ID NO. 1). This may be referred to as an Mldel mutation.
  • novel DDs derived from ecDHFR may comprise amino acids 2- 159 of the wild type ecDHFR sequence (SEQ ID NO. 1) (also referred to as an Mldel mutation), and may include one, two, three, four, five or more mutations including, but not limited to, Mldel, R12Y, R12H, Y100I, and E129K.
  • novel DDs derived from FKBP may comprise amino acids 2- 107 of the wild type FKBP sequence (SEQ ID NO. 3). This may be referred to as an Mldel mutation.
  • novel DDs derived from FKBP may comprise amino acids 2- 107 of the wild type FBKP sequence (SEQ ID NO. 3) (also referred to as an Mldel mutation), and may include one, two, three, four, five or more mutations including, but not limited to, Mldel, E31G, F36V, R71 G, K105E, and L106P.
  • Inventors of the present invention have tested and identified several candidate human proteins that may be used to develop destabilizing domains. As show in Table 2, these candidates include human DHFR (liDHFR), PDE5 (phosphodiesterase 5), PPAR gamma (peroxisome proliferator-activated receptor gamma), CA2 (Carbonic anhydrase II) and NQ02 (NRH:
  • Candidate destabilizing domain sequence identified from protein domains of these proteins may be mutated to generate libraries of mutants based on the template candidate domain sequence.
  • Mutagenesis strategies used to generate DD libraries may include site-directed mutagenesis e.g. by using structure guided information; or random mutagenesis e.g. using error-prone PCR, or a combination of both.
  • destabilizing domains identified using random mutagenesis may be used to identify structural properties of the candidate DDs that may be required for destabilization, which may then be used to further generate libraries of mutations using site directed mutagenesis.
  • DD mutant libraries may be screened for mutations with altered, preferably higher binding affinity to the ligand, as compared to the wild type protein.
  • DD libraries may also be screened using two or more ligands and DD mutations that are stabilized by some ligands but not others may be preferentially selected.
  • DD mutations that bind preferentially to the ligand compared to a naturally occurring protein may also be selected. Such methods may be used to optimize ligand selection and ligand binding affinity of the DD. Additionally, such approaches can be used to minimize deleterious effects caused by off-target ligand binding.
  • suitable DDs may be identified by screening mutant libraries using barcodes. Such methods may be used to detect, identify and quantify individual mutant clones within the heterogeneous mutant librar '.
  • Each DD mutant within the library may have distinct barcode sequences (with respect to each other).
  • the polynucleotides can also have different barcode sequences with respect to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acid bases.
  • Each DD mutant within the library may also comprise a plurality of barcode sequences. When used in plurality may be used such that each barcode is unique to any other barcode. Alternatively, each barcode used may not be unique, but the combination of barcodes used may create a unique sequence that can be individually tracked.
  • the barcode sequence may be placed upstream of the SRE, downstream of the SRE, or in some instances may be placed within the SRE.
  • DD mutants may be identified by barcodes using sequencing approaches such as Sanger sequencing, and next generation sequencing, but also by polymerase chain reaction and quantitative polymerase chain reaction.
  • polymerase chain reaction primers that amplify a different size product for each barcode may be used to identify each barcode on an agarose gel.
  • each barcode may have a unique quantitative polymerase chain reaction probe sequence that enables targeted amplification of each barcode.
  • DDs of the invention may be derived from human dihydrofoiate reductase (hDHFR).
  • hDHFR is a small (18 kDa) enzyme that catalyzes the reduction of dihydrofoiate and plays a vital role in variety of anabolic pathway.
  • Dihydrofoiate reductase (DHFR) is an essential enzyme that converts 7,8-dihydrofolate (DHF) to 5,6,7,8, tetrahydrofolate (THF) in the presence of nicotinamide adenine dihydrogen phosphate (NADPH).
  • Anti -folate drugs such as methotrexate (MTX), a structural analogue of folic acid, which bind to DHFR more strongly than the natural substrate DHF, interferes with folate metabolism, mainly by- inhibition of dihydrofoiate reductase, resulting in the suppression of purine and pyrimidine precursor synthesis.
  • MTX methotrexate
  • DHF dihydrofoiate reductase
  • the DDs of the invention may be hDHFR mutants including the single mutation hDHFR (Y122I), hDHFR (K81R), hDHFR (F59S), hDHFR (T17V), hDHFR ( 65D), hDHFR (A107V), hDHFR (N127Y), hDHFR
  • hDHFR M53T, R138I
  • hDHFR V75F, Y1221)
  • hDHFR A125F, Y 1221
  • hDHFR L74N, Y 1221
  • hDHFR L94A, ⁇ 47 ⁇
  • hDHFR G21T, Y 122I
  • hDHFR V121A, Y1221)
  • hDHFR Q36K, Y 122I
  • hDHFR C7R, Y163C
  • hDHFR Y178H, E18IG
  • hDHFR A10V, H88Y
  • hDHFR T137R, F143L
  • hDHFR E63G, I176F
  • hDHFR T57A, I72A
  • hDHFR H131R, E144G
  • hDHFR Y 183H, K185E
  • triple mutations hDHFR (M53T, R138I)
  • hDHFR V75F, Y1221)
  • V9A, S93R, P150L hDHFR.
  • K19E, F89L, E181G hDHFR
  • G54R, M140V, S168C hDHFR
  • hDHFR L23S, V121A, Y157C
  • hDHFR VI 10A, V136M, K177R
  • hDHFR 49D, F59S, D153G
  • hDHFR N49D, F59S, D153G).
  • the stimulus is a small, molecule that binds to a SRE to post- translationally regulate protein levels.
  • DHFR ligands trimethoprim (TMP) and methotrexate (MTX) are used to stabilize hDHFR mutants.
  • TMP trimethoprim
  • MTX methotrexate
  • the hDHFR based destabilizing domains are listed in Table 3.
  • the position of the mutated amino acid listed in Table 3 is relative to the human DHF (Uniprot ID: POOS 74) of SEQ. ID NO. 2 for human DHFR.
  • '"del means that the mutation is the deletion of the amino acid at that position relative to the wild type sequence.
  • hDHFR (Y122I, A125F) MVGSLNCIVAVSQNMG1GKNGDLPWPPLRNEFRYF 34
  • hDHFR (Y178H, E18IG) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 37
  • hDHFR (I8V, K133E, Y163C) MVGSLNCVVAVSQNMGIGKNGDLPWPPLRNEFRY 40
  • HDHFR (G54R, M140V, MVGSLNC1VA VSQNMG1GKNGDLPWPPLRNEFR YF 44 S168C) QRMTTTSSVEGKQNLVIMR KTWFS1PEKNRPLKG
  • hDHFR (V110A, V136M, MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 45 K177R) QRMT TSSVEGKQNLVIMGKKTWFSIPEKNRPLKG
  • hDHFR Amino acid 2-187 of VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFF 46 WT; Q36F. Y122I, A 125F) RMTTTSSVEGKONL-VIMGKKTWFSIPEKNRPLKGRI
  • hDHFR V2A, R33G, Q36R, MAGSLNCrVAVSQNMGIGKNGDLPWPPLRNEFGYF 125 L100P, K185R
  • RRM'l'l'l'SSVEGKQNLVIMGKKTWFSIPEKNRPLKG V2A, R33G, Q36R, MAGSLNCrVAVSQNMGIGKNGDLPWPPLRNEFGYF 125 L100P, K185R
  • hDHFR (G16S, I17V, F89L, MVGSLNCIVAVSQNMSVGKNGDLPWPPLRNEFRY 126 D96G, K123E, M140V, D146G, FQRM' ⁇ " I S S VEGKQNL VIMGKKTWF SIPEKNRPLK K156R) GRINLVLSRELKEPPQGAHLLSRSLDGALKLTEQPE
  • hDHFR (F35L, R37G, 65A, MVGSLNCrVAVSQNMGIGKNGDLPWPPLRNEFRYL 340 L68S, K69E, R71 G, L80P, QGMTTTSSVEGKQNLVIMGKKTWFSIPEKARPSEG K99G, G117D, L 332P, I139V, G1NLVLSREPKEPPQGAHFLSRSLDDALGLTEQPEL M140I, D 142G, D146G, E173G, ANK VDMVWI VD GS ⁇ S VYKE AMNHPGHPKLF VTR VI D187G) QGFESGTFFPEIDLEKYKLLPEYPGVLSDVQEGKGI
  • KYKFEVYEKNG hDHFR (I17N, L98S, K99R, MVGSLNCIVAVSQNMGNGKNGDLPWPPLRNEFRY 141
  • hDHFR (R.138G, D142G, MVGSLNCIVAVSQNMGIGKNGDLPWPPLR EFRYF 150 F143S, K156R, K158E, E162G, QRMTTTSSVEGKQNLVIMGKKTWFSIPE NRPLKG V166A, K 177E, Y178C, 185E, RI LVLSRELKEPPQGAHFLSRSLDDALKLTEQPEL N186S) ANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTGIM
  • hDHFR (K81R, K99R, L100P, MVGSLNCIVAVSQNMGIGKNGDLPWPPLR EFRYF 174 E102G, N 108D, K123R, QRMTTTSSVEGKQNLVIMGKKTWFSIPE NRPLKG H128R, D142G, F180L, K185E) RI LVLSRELREPPQGAHFLSRSLDDALRPTGQPEL
  • hDHFR N1.4S, P2 S, F35L, MVGSLNCIVAVSQSMGIGKNGDLSWPPLRNE-FRYL 181 M53T, K56E, R92G, S93G, QRMTTTSSVEGKQNLVITGKETWFSIPEKNRPLKGR N127S, H128Y, F135L, F143S, IM.VLSRELKEPPOGAHFLSGGLDDALKLTEQPELA L159P, L160P, E173A, FiSOL) NKVDMVWIVGGSSVYKEAMSYPGHLKLLVTRIMQ
  • hDHFR (V2A, I17V, N30D, MAGSLNCrVAVSQ MGVGKNGDLPWPPLRDGFRY 196 E31G, Q36R, F59S. K69E, FRRMTTTSS ⁇ 3 ⁇ 4GKQ L ⁇ TMGKKTWSSIPEKNRPLEG ⁇ 72 ⁇ , H88Y, F89L, N10SD, RTNLVLSRELKEPPQGAYLLSRSLDDALKLTEOPEL K109E, V110A, 11 15V, Y122D, ADE A GMVWW GG S S VDKE AMNHP GHPKL S VTR IV L132P, F135S, M140V, E144G, QDFGSDAFFPEIDLEKCKLLPEYPGVLSDAQEERGI T147A, Y157C, V170A, KYKFEVYEKSD
  • hDHFR (L28P, N30H, M38V, MVGSLNCIVAVSQNMGIGKNGDLPWPPPRHEFRYF 197 V44A, L68S, N73G, R78G, QRVTTTSSAEGKQNLVIMGKKTWFSIPEKNRPSKG A97T, K99R, A 107T, K109R, RIGLVLSGELKEPPQGAHFLSRSLDDTLRLTEOPELT Di l l N, L 1 4P, F I 35 V.
  • hDHFR (A10T, Q13R, N14S, MVGSLNCIVTVSRSMG1G DGDLSWPPLRSEFRYF 205 N20D, P24S, N30S, M38T, QRTTATSSVEGRQSLVIMGKRTWFSTPERNRPLRGR T40A, K47R, N49S, K56R, A NL VL S GELK GPPQG AHLL SRSLD G A LKLTEQPEL I61T, K64R, K69R, I72A, ADKVDVVRIVGGSSVDEEA HPGHLKLFVTRVM R78G, E82G, F89L, D96G, RGFESDTLFPGIDLGKRKLLPEYPGVLSDVREEKGI N1()8D, M112V, WU4R, KYKLEVCGNN
  • hDHFR Amino acid 2-187 of VGSLNCrVAVSQNMGVGKNGDLPWPPLRNEFRYF 328 WT; I17V, Y122I
  • hDHFR Amino acid 2-187 of VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQ 329 WT: Y122I, M140I
  • EVYEKND hDI-IFR (Amino acid 2-187 of VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQ 330 WT; N327Y, ⁇ 122 ⁇ ) RMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRI
  • hDI-IFR Amino acid 2-187 of VGSLNCrVAVSQNMGIGK GDLPWPPLR EFRYFQ 331 WT; Y122I, H131R, E144G) RMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRI
  • hDI-IFR Amino acid 2-187 of VGSLNCrVAVSQNMGIGKNGSLPWPPLRNEMSYFS 332 WT; D22S, F32M, R33S, Q36S, RMTTTSSVEGKQNLVIMGKKTWFSIPEKSRPLKGRI N65S) NLVLSRELKEPPQGAHFLSRSLDDALKLTEQPELAN
  • hDHFR Amino acid 2-187 of VGSLNCrVAVSONMGIGKNGDLPWPPLRNDMRYF 333 WT; E31D, F32M, VI 161 ORMT TSSVEGKQNLVMGKKTWFSIPEKNRPLKG
  • hDHFR Amino acid 2-187 of VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQ 334 WT; E162G, I176F) RMTTTSSV GKQNLVIMGKKTWFSIPEKNRPLKGRI
  • hDHFR Amino acid 2-187 of VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQ 335 WT; K185E) RMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRI
  • hDHFR Amino acid 2-187 of VGSLNCrVAVSQNMGIGKNGDLPWPPLRNEFRYFQ 336 WT; Y322L A125F
  • RMTTTS S VEG QNL VIMGKKTWFSIPEKNRPLK GRI
  • hDHFR Amino acid 2-187 of VGSLNCrVAVSQNMGIGKNGDLPWPPLRNE-FRYFF 337 WT; Q36F. N65F, Y122I) RJV TTTSSVEGKQNLVINlGKKTWFSIPEKFRPLKGRI
  • hDHFR Amino acid 2-187 of VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQ 338 WT; N127Y
  • RM ' TITS S VEGKQNL VIMGKKTWF SIPEKNRPLKGRI
  • hDHFR Amino acid 2-187 of VGSLNCIVAVSQNMGVGKNGDLPWPPLRNEFRYF 340 WT; I17V
  • hDHFR Amino acid 2-187 of VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQ 341 WT; Y122I
  • DD mutations that do not inhibit ligand binding may be preferentially selected.
  • ligand binding may be improved by mutation of residues in DHFR.
  • Amino acid positions selected for mutation include aspartic acid at position 22 of SEQ ID NO. 2, glutamic acid at position 31 of SEQ ID NO. 2; phenyl alanine at position 32 of SEQ ID NO. 2; arginine at position 33 of SEQ ID NO. 2; glutamine at position 36 of SEQ ID NO. 2; asparagine at position 65 of SEQ ID NO. 2; and valine at position 115 of SEQ ID NO.
  • one or more of the following mutations may be utilized in the DDs of the present invention to improve TMP binding, including but not limited to, D22S, E3 ID, F32M, R33S, Q36S, N65S, and VI 161.
  • the position of the mutated amino acids is relative to the wildtype human DHFR (Uniprot ID: P00374) of SEQ ID NO. 2.
  • novel DDs derived from human DHFR may include one, two, three, four, five or more mutations including, but not limited to, Ml del, V2A, C7R, I8V, V9A, A IOT, A10V, Q13R, N14S, G16S, I17N, 117V, 19E, N20D, G21T, G21E, D22S, L23S, P24S, L28P, N30D, N30H, N30S, E31G, E31D, F32M, R33G, R33S, F35L, Q36R, Q36S, Q36K, Q36F, R37G, M38V, M38T, T40A, V44A, K47R, N49S, N49D, M53T, G54R, K56E, K56R, T57A, F59S, 16 IT, K64R, N65A, N65S, N65D, N65F, L68
  • novel DDs derived from human DHFR may comprise amino acids 2-187 of the wild type human DHFR sequence. This may be referred to as an Mldel mutation.
  • novel DDs derived from human DHFR may comprise amino acids 2-187 of the wild type human DHFR sequence (also referred to as an Mldel mutation), and may include one, two, three, four, five or more mutations including, but not limited to, Mldel, V2A, ⁇ " 7R.
  • payloads of the present invention may be immunotherapeutic agents that induce immune responses in an organism.
  • the immunotherapeutic agent may be a cytokine, a cytokine receptor, a cytokine-cytokine receptor fusion polypeptide or any agent that induces an immune response.
  • the immunotherapeutic agent induces an anticancer immune response in a cell, or in a subject.
  • payloads of the present invention may be cytokines, chemokines, growth factors, and soluble proteins produced by immune cells, cancer cells and other cell types, which act as chemical communicators between cells and tissues within the body. These proteins mediate a wide range of physiological functions, from effects on cell growth, differentiation, migration and survival, to a number of effector activities. For example, activated T cells produce a variety of cytokines for cytotoxic function to eliminate tumor cells.
  • payloads of the present invention may be cytokines, and fragments, variants, analogs and derivatives thereof, including but not limited to interleukms, tumor necrosis factors (TNFs), interferons (IFNs), TGF beta and chemokines. It is understood in the art that certain gene and/or protein nomenclature for the same gene or protein may be inclusive or exclusive of punctuation such as a dash "-" or symbolic such as Greek letters.
  • payloads of the present invention may be cytokines that stimulate immune responses. In other embodiments, payloads of the invention may be antagonists of cytokines that negatively impact anti -cancer immune responses.
  • payloads of the present invention may be cytokine receptors, recombinant receptors, variants, analogs and derivatives thereof: or signal components of cytokines.
  • cytokines of the present invention may be utilized to improve expansion, survival, persistence, and potency of immune cells such as CD8+TE , natural killer cells and tumor infiltrating lymphocytes (TIL) cells used for immunotherapy.
  • TIL tumor infiltrating lymphocytes
  • T cells engineered with two or more DD regulated cytokines are utilized to provide kinetic control of T cell activation and tumor microenvironment remodeling.
  • the present invention provides biocircuits and compositions to minimize toxicity related to cytokine therapy. Despite its success in mitigating tumor burden, systemic cytokine therapy often results in the development of severe dose limiting side effects.
  • cytokines of the present invention may be utilized to modulate cytokine expression in the event of adverse effects.
  • cytokines of the present invention may be designed to have prolonged life span or enhanced specificity to minimize toxicity .
  • the payload of the present invention may be an interleukin (IL) cytokine.
  • Interleukins are a class of glycoproteins produced by leukocytes for regulating immune responses.
  • the term "interleukin (IL)” refers to an interleukin polypeptide from any species or source and includes the full-length protein as well as fragments or portions of the protein.
  • the interleukin payload is selected from ILl, ILlalpha (also called hematopoietin-1), ILlbeta (catabolin), IL1 delta, ILlepsilon, ILleta, IL1 zeta, interleukin- 1 family member 1 to 11 (IL1F1 to ILIFI 1), interleukin- 1 homolog 1 to 4 (ILIHI to H i !
  • ILl related protein 1 to 3 (ILIRPI to IL1RP3), IL2, 1L3, IL4, 11,5, IL6, IL7, IL8, IL9, IL10, ILIOC, IL10D, IL11 , TLl l a, ILl lb, IL12, IL13, IL14, IL15, IL16, IL17, IL17A, I11 7B, IL17C, IL17E, IL17F, IL18, IL19, IL20, IL20 like (IL20L), 1121 , IL22, IL23, IL23A, IL23-pl9, IL23-p40, 1L24, 1125, 1L26, 1L27, 1L28A, IL28B, IL29, IL30, IL31, IL32, IL33, IL34, IL35, IL36 alpha, IL36 beta, IL36 gamma,
  • the payload of the present invention may be an interleukin receptor selected from CD121a, CDwl21b, IL2Ra/CD25, IL2Rp7CD122, IL2Ry/CD132, CDwl31, CD 124, CD131, CDwl25, CD126, CD130, CD127, CDw210, IL8RA, ILl IR , CD212, CD213al, CD213a2, IL14R, ILISRa, CDw217, IL18Ra, IL18RP, IL20Ra, and IL20Rp.
  • interleukin receptor selected from CD121a, CDwl21b, IL2Ra/CD25, IL2Rp7CD122, IL2Ry/CD132, CDwl31, CD 124, CD131, CDwl25, CD126, CD130, CD127, CDw210, IL8RA, ILl IR , CD212, CD213al, CD213a2, IL14R,
  • the payload of the invention may comprise whole or a portion of IL15.
  • Interleukin 15 is a potent immune stimulatory cytokine and an essential survival factor for T cells, and Natural Killer cells. Preclinical studies comparing IL2 and IL15, have shown than ILl 5 is associated with less toxicity than IL2.
  • the effector module of the invention may be a DD-IL15 fusion polypeptide.
  • ILl 5 polypeptide may also be modified to increase its binding affinity' for the ILl 5 receptor.
  • the asparagine may be replaced by aspartic acid at position 72 of ILLS (SEQ. ID NO. 2 of US patent publication
  • the ILl 5 constructs of the invention may be placed under the transcriptional control of the CMV promoter (SEQ. ID NO. 49), an EF la promoter (SEQ. ID NO. 50 or SEQ. ID NO. 342) or a PGK promoter (SEQ. ID NO. 51 ).
  • CMV promoter SEQ. ID NO. 49
  • EF la promoter SEQ. ID NO. 50 or SEQ. ID NO. 342
  • PGK promoter SEQ. ID NO. 51
  • Any portion of IL15 that retains one or more functions of full length or mature ILl 5 may be useful in the present invention. Such functions include the promotion of NK cell survival, regulation of NK cell and T ceil activation and proliferation as well as the support of NK cell development from hematopoietic stem cells.
  • the DD-IL15 comprises the amino acid sequences listed in Table 4.
  • the amino acid sequences in Table 4 may comprise a stop codon which is denoted in the table with a at the end of the amino acid
  • DVHPSCKVTAMKCFLLELQVISI PSGDA include the SIHDTVENLIILANNSLSSNGNVTESGCK stop codon ECEELEEKNIKEFLQSFVHIVQMFTNTS* at the end),
  • the transmembrane domain of the present invention may be selected from the group consisting of a CD 8a transmembrane domain, a CD4 transmembrane domain, a CD 28 transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, and a human Igcn Fc region.
  • the transmembrane domain may be a CTLA-4 transmembrane domain comprising the amino acid sequences of SEQ ID NOs. 1-5 of International Patent Publication NO.
  • WO2014100385 and a PD-1 transmembrane domain comprising the amino acid sequences of SEQ ID NOs. 6-8 of International Patent Publication NO. WO2014100385; the contents of each of which are incorporated herein by reference in their entirety.
  • the payioads of the present in vention may comprise an optional hinge region (also called spacer).
  • a hinge sequence is a short sequence of amino acids that facilitates flexibility of the extracellular targeting domain that moves the target binding domain away from the effector cell surface to enable proper cell/cell contact, target binding and effector ceil activation (Patel et ai,, Gene Therapy, 1999; 6: 412-419).
  • the hinge sequence can be any suitable sequence derived or obtained from any suitable molecule.
  • the hinge sequence may be derived from all or part of an immunoglobulin (e.g., IgGl, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls between the CHI and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge, the extracellular regions of type 1 membrane proteins such as CD8a CD4, CD28 and CD7, which may be a wild type sequence or a derivative.
  • Some hinge regions include an immunoglobulin CH3 domain or both a CH3 domain and a CH2 domain.
  • the hinge region may be modified from an IgGl, IgG2, IgG3, or IgG4 that includes one or more amino acid residues, for example, 1, 2, 3, 4 or 5 residues, substituted with an amino acid residue different from that present in an unmodified hinge.
  • Table 5 provides various transmembrane regions that can be linked to the payioads described herein.
  • CD134 (OX40) VAAILGLGLVLGLLGPLAILLALYLL 79
  • CD28 Transmembrane FWVLVVVOGVLACYSLLVTVAFIIFWV 84 domain
  • CD28 Transmembrane FWVLVWGGVLACYSLLVTVAFIIFWVR 361 domain
  • CD28 Transmembrane FWVLVWGGVLACYSI VTVAFIIFWVRSKRS 93 domain and CD28 and CD 3 RLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA
  • RREEYDVLD RRGRDPEMGGKPRRKNPQEGL YNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR CD28 Transmembrane FWVLVWGGVLACYSLLVTVAFIIF VRSKRS 94 domain and CD28, OX40, RLLHSDYMNMTPRRPGPTRKFIYQPYAPPRDFA and CD 3 Zeta intracellular AYRSRDQRLPPDAHKPPGGGSFRTPIQEEQAD domain AHSTLAKIRVKFSRSADAPAYQQGQNQLYNEL
  • CD28 Transmembrane FWVLVWGGVLACYSLLVTVAFIIFWVRRVKF 95 domain and CD3 Zeta SRSADAPAYQQGQNQLYNELNLGRREEYDVL intracellular domain DKRRGRDPEMGGKPRRK PQEGLYNELQKDK
  • CD28 transmembrane-CD3 AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPS 96 zeta signaling domain PLFPGPSKPFWVLWVGGVLACYSLLVTVAFII ("28z”) FWVRSKRSRLLHSDYM MTPRRPGPTR HYQ
  • CDS Transmembrane MALPVTAL-LLPLALLLHAARP 105 domain CDS T tans membrane AAAFVPVTLPAKPTTTPAPRPPTPAPTIASQPLS 106 domain and CD28 signaling LRPEACRPAAGGAVHTRGLDFACDIYIWAPLA domain GTCGVLLLSLVITLYCNHRNRSKRSRLLHSDY
  • FcERI a Transmembrane FFIPLLWILFAVDTGLFISTQQQVTFLLKIKRTR 365 domain GFRLLNPHPKP P
  • FcERI a- Transmembrane M AP AME SPTLL C V ALLFF APD G VL A VPQKPK V 117 domain SLNPPWNRIFKGENVTLTC GNNFFEVSSTKW
  • Hmge region sequences useful in the present invention are provided in Table 6a.
  • LPPQRLMALREP AAQAP VKL SLNLL A S SDPPE A A S WLL CE VS GF SPPNILLM V VLEDQRE VNT S GF AP ARPPPQP GSTTFW AWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVS YVTDH
  • LPPQRLMALREP AAQAP VKLSLNLLASSDPPEAASWLLCE VSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFW AWSVLRVPAPPSPQPATYTCWSHEDSRTLLNASRSLEVS YVTDH
  • LPPQRLMALREP AAQAP VKLSLNLLASSDPPEAASWLLCE VSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFW AWSVLRVPAPPSPQPATYTCWSHEDSRTLLNASRSLEVS YVTDH
  • LPPQRLMALREP AAQAP VKLSLNLLASSDPPEAASWLLCE VSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFW AWSVLRVPAPPSPQPATYTCWSHEDSRTLLNASRSLEVS YVTDH
  • IgG i (CH2CH3)
  • IgG l (CH2CH3)
  • QYNSTTR SVLTVLHQDWLNGKEYKCKVSNKALPAPIE Hinge domain KTiSKAKGQPREPQWTLPPSRDELTKNQVSLTCLVXGFY
  • EWCVVVDVSTTF DPEVKFNWYVDGVEVHNAKTK-PREEO
  • IgG4 CH2 and TYRWSVLTVLHODWLNGKEYKCKVSNKGLPSSlEK'llSK CHS
  • Hinge and transmembrane region sequences useful in the present invention are provided in Table 6b, Table 6b: Hinge and Transmembrane regions
  • the payloads of the present invention may comprise one or more linkers.
  • the linker may be between 1-30 amnio acids long.
  • the linker may be I , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length.
  • the linker may be flexible.
  • Tl e effector modules containing DD-IL15 may be designed to be secreted (using e.g. IL2 signal sequence) or membrane bound (using e.g. IgE or CD8a signal sequence).
  • a unique feature of 1LI5 mediated activation is the mechanism of trans-presentation in which IL15 is presented as a complex with the alpha subunit of IL15 receptor (ILlSRa) that binds to and activates membrane bound IL15 beta/gamma receptor, either on the same cell or a different cell.
  • IL15/IL15Ra complex is more effective in activating 11. 15 signaling, than IL15 by itself.
  • tlie pavload of the invention may be a whole or a portion of IL15 fused to the whole or a portion of the IL15Ra.
  • IL15 and ILLSRa that retains one or more functions of full length or mature IL15 or ILlSRa respectively may be useful m the present invention.
  • Such functions include tlie promotion of NK cell survival, regulation of NK cell and T cell activation and proliferation as well as the support of NK cell development from hematopoietic stem cells.
  • the pavload may be ILi5/IL15Ra fusion polypeptide described in US Patent Publication NO. US20160158285A 1 (the contents of which are incorporated herein by reference in their entirety).
  • the IL15 receptor alpha comprises an extracellular domain called the sushi domain which contains most of the structural elements necessary for binding to IL15.
  • pavload may include ILlSRa sushi domain.
  • the IL15Ra sushi domain may comprise the amino acid sequence of SEQ. ID NO. 14 of US20090238791 Al (the contents of which are incorporated herein by reference in their entirety).
  • the payload of the invention maybe the IL15 fused to ILlSRa sushi domain fusion polypeptide described in US Patent Publication NO. US20090238791A1.
  • a portion of ILlSRa useful in the invention may include 31 -205 amino acids or 3.1 -95 ammo acids of the human ILlSRa (Uniprot ID: Q1326.1). Table 7 provides ILlSRa variants useful in the invention.
  • ILlSRa Isoforni 1 NCBI MAPRRA GCRTLGLPALLLLLLLRPPATRGITCPPPMSVEH 211 Reference No. NP 002180.1) ADIWVKSYSLYSRERYICNSGF RKAGTSSLTECVLNKAT
  • ILl SRa Isoform 2 (NCBI MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEH 212 reference No. NP_751950.2) ADiWVKSYSLYSRERYiCNSGFKRKAGTSSLTECVLNKAT
  • IL 15Ra Isofonn 3 NCBI MSVEHADrWVKSYSLYSRERYTCNSGFKRKAGTSSLTECV 213 Reference No. LNKAT VAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVT
  • ILlSRa Isoform 4 NCBI MRLAGRQVPEQRSPPPPGLGSARPGSPAVSCGAAAMAPR 214 Reference No. RARGCRTLGLPALLLLLLLRPPATRDARDRLAVLAGRSRI
  • IL 1 SRa (without signal rrCPPPMS ⁇ 3 ⁇ 4HADIWVKSYSLYSRERYICNSGFKRKAGTSS 216 sequence) LTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTW
  • IL15Ra (31-205 of Uniprot rTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSS 217 ID: Q1326 I .1) LTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVT
  • IL15/IL15Ra fusion proteins of the present invention may be linked to all or a functional portion of one or more transmembrane proteins.
  • an optional hinge region may be included.
  • Tables 5, Table 6a and Table 6b provide various transmembrane regions, hinge and transmembrane with hinge regions that can be linked to the payloads described herein.
  • the whole or a portion of the membrane associated IL 15 or IL 15/IL 15Ra fusion polypeptides of the invention may be shed into the extracellular space. Shedding as used herein refers to the release of membrane associated biomolecules from the membrane to which they are tethered. In some instances, shedding may be induced by the proteolytic cleavage. A soluble form of human IL15Ra arises from proteolytic shedding of the membrane-anchored receptor has been reported by Mortier et al (Mortier E et al. (2004). J Immunol; 173(3): 1681-8; the contents of which are incorporated by reference in their entirety).
  • Shedding may also be induced by- treatment with PMA, ionomycin, and to a lesser extent by ILlb and TNFa.
  • IL15 or IL15/IL15Ra fusion proteins may be modified to prevent shedding.
  • the IL 15 molecule or the IL15Ra molecule may be truncated or mutated to remove presumable cleavage sites.
  • IL15Ra has a cleavage site (PQGHSDTT from the position 168 to 175 of SEQ. ID NO. 216) in the extracellular domain immediately distal to the transmembrane domain of the receptor, as described by Bergamaschi C et al. (2008).
  • TACE/ADAM17 has been implicated as a protease that cleaves between glycine (at the position 170 of Seq. ID NO. 216) and histidine (at the position 171 of SEQ. ID NO. 216) and generates a naturally occurring soluble form of IL15Ra.
  • the same mechanism may be responsible for the IL15-IL15Ra shedding.
  • the cleavage site of IL15Ra may be mutated such that cleavage by an endogenous protease is prevented.
  • the mutation of the cleavage site may be introduced by substitution, insertion or deletion of amino acid residues.
  • the IL15- IL15Ra fusion molecule may be also modified such that the full-length or truncated IL15- IL15Ra fusion molecule is fused to heterologous hinge domains and/or heterologous
  • transmembrane domains As non-limiting examples, variants of IL15Ra described in SEQ. ID NO. 215, 217 and 218 can be utilized. Additionally, the length and sequence of the linkers that connect IL15 and IL15Ra may be modified.
  • DD-IL15/IL15Ra fusion proteins of the present invention may be expressed as a single polypeptide connected to a CAR, as described in International Patent Application Publication No. WO2016210293, the contents of which are incorporated herein by reference in their entirety.
  • a cleavage site may be disposed between the CAR polypeptide and DD-IL15/IL15Ra fusion polypeptide.
  • the example of a cleavage site includes 2A self-cleaving peptides, such as porcine tesehoviras-i 2A (P2A), thoseaasigna virus 2A (T2A), equine rhinitis A vims 2A (E2A).
  • F2A foot and mouth disease virus 2A
  • BmCPV 2A cytoplasmic polyhedrosis virus
  • BmlFV 2A ilacherie virus
  • the single peptide may be composed of the following components: [CAR] - [P2A cleavage site] - [IL15Ra/IL15-DD].
  • DD ⁇ IL15/IL15Ra fusion proteins of the present invention may be conjugated to tumor- associated antigen-recognizing antibodies or binding partners, thereby constructing bivalent- binding fusion molecules or bi-specific T-cell engagers, as described in International Patent Application Publication Nos. WO2015120187, WO2015120180, WO2016123142,
  • tumor-associated antigen-recognizing antibodies or binding partners include a tripeptide Arg-Gly-Asp that binds to ceil surface integrin receptors and thus target integrin-positive tumors, anti-TN glycopeptide antibody and anti-IL13Ra2 antibody.
  • Tire fusion molecules comprising IL15/IL15Ra fusion proteins are designed to provide a second function in conjunction with CARs to improve T cell activation and persistence.
  • DD-IL15 is trans-presented to T cells or NK cells via IL15 fusion molecules that are linked to heterologous transmembrane domains without ILl SRa, as described by Imamura M et al. (2014) Blood;124(7): 1081-1088, Garg TK et al. (2012) Haematologica;97(9): 1348-1356, Shook DR and Campana D (201 ) Tissue Antigens;78(6):409- 415, Cho D and Campana D (2009) Korean J Lab Med;29(2):89-96, Ayello J et al. (2017) Exp Hematol;46:38-47, Qian L et al.
  • 1L15 is fused to CDSalpha signal peptide and CDSalpha transmembrane, as described by International patent publication NO, WO2015174928, SEQ. ID NO. 1 and SEQ. ID NO. 2, the contents of which are incorporated herein by reference in their entirety.
  • DD-IL15 is fused to HLA-A2 transmembrane and cytoplasmic domains via a Gly-Ser linker (e.g.,
  • DD-1L15 fusion molecule may further comprise human MHC-I light chain p2-microglobulin leader peptide.
  • DD-IL15 is fused to i I -2Kb transmembrane and cytoplasmic domains via a Gly- Ser linker (e.g., Gly4Ser(Gly3Ser)2; SEQ. ID NO. 534) and 8 ammo acid bridge from the H-2Kh membrane-proximal part, as described by Weinstein-Marom H el al. (2016). J
  • the DD-IL15 fusion molecule may further comprise human MHC-I light chain ⁇ 2 ⁇ microglobulin leader peptide.
  • immunoproteasome is abundantly expressed in immune cells, such as antigen-presenting cells, and has more efficient proteolytic functions compared to the constitutive proteasome. For instance, while the constitutive proteasome degrades ubiquitinated proteins to maintain cell viability and homeostasis, the primary role of the immunoproteasome is to process antigens for presentation on MHC class I molecules to CD8+ T lymphocytes as well as to process ubiquitinated proteins.
  • DD ⁇ iL15/IL15Ra fusion proteins of the present invention may be designed to evade the induction of the immunoproteasome, such that the potential degradation of DD-IL15/IL15Ra fusion proteins in the presence of ligand is avoided and the expression level of IL15/IL15Ra fusion proteins is maintained.
  • DD ⁇ iL15/IL15Ra fusion proteins of the present invention may be designed to induce the immunoproteasome in antigen-presenting cells, such that the antigen presentation on MHC class I molecules is bolstered and consequently the proliferation and activation of CD8+ T cells is augmented,
  • Regulated IL15/IL15Ra may be used to promote expansion, survival and potency of CD8TEM ceil populations without impacting regulatory T cells, NK cells and TIL cells.
  • DD-IL 15/IL 15Ra may be utilized to enhance CD 19 directed T cell therapies in B cell leukemia and lymphomas.
  • IL15 IL15Ra may be used as payload of the invention to reduce the need for pre-conditioning regimens in current CAR-T treatment paradigms.
  • Tire effector modules containing DD-IL 15 , DD-IL 15/IL 15Ra and/or DD-IL 15/IL 15Ra sushi domain may be designed to be secreted (using e.g. IL2 signal sequence) or membrane bound (using e.g. IgE or CD8a signal sequence).
  • the DD-IL15/IL15Ra comprises the amino acid sequences provided in Table 8a with any combination of components in any order.
  • ILlSRa may be fused to DD by the amino acid sequence SG.
  • Examples of DD-IL 15/IL 15Ra are provided in Table 8b and Table 8c.
  • the amino acid sequences in Tables 8a, 8b and 8c may comprise a stop codon which is denoted in the table with a "*" at the end of the amino acid sequence.
  • ecDHFR ISLIAALA ⁇ TJYVIGMENA IPWNLPADLAWFKRNTLNK 9 73, 372, 233 (Amino acid PVT IGRHTWESIGRPLPGRKNIILSSQPGTDDRV VKS (includes stop 2-159 of VDEAIAACGDVPEIMVIGGGRVIEQFLPKAQKLYLTHK) codon) WT; R12Y, AEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCF
  • hDHFR VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRM 336 239, 397, 396 (Amino acid TTTSSVEGKQNLVIMGKKTWFSiPEKNRPLKGRINLVLS (includes stop 2-187 of RELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW codon) WT; Y122L IVGGSSVj- E 'MNHPGHi ,KT .FVTRIMQDFESDTFFPE1D
  • linker2 PPGVYPQGHSDTTVAISTSTVLLCGLSAVSLL

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Abstract

La présente invention concerne des systèmes de circuits biologiques, des modules effecteurs et des compositions pour l'immunothérapie anticancéreuse. L'invention concerne également des méthodes pour induire une réponse immunitaire anticancéreuse chez un sujet.
PCT/US2018/020755 2017-03-03 2018-03-02 Compositions d'il-15 et méthodes pour immunothérapie Ceased WO2018161026A1 (fr)

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US10785574B2 (en) 2017-12-14 2020-09-22 Flodesign Sonics, Inc. Acoustic transducer driver and controller
WO2020252405A1 (fr) * 2019-06-12 2020-12-17 Obsidian Therapeutics, Inc. Compositions de ca2 et procédés de régulation ajustable
WO2021040736A1 (fr) 2019-08-30 2021-03-04 Obsidian Therapeutics, Inc. Compositions à base de car cd19 tandem et méthodes d'immunothérapie
WO2021046451A1 (fr) * 2019-09-06 2021-03-11 Obsidian Therapeutics, Inc. Compositions et méthodes de régulation de protéine accordable dhfr
WO2021050789A1 (fr) * 2019-09-10 2021-03-18 Obsidian Therapeutics, Inc. Protéines de fusion de ca2-il15 pour une régulation accordable
WO2021058973A1 (fr) * 2019-09-25 2021-04-01 Prostate cancer research centre Peptides de fusion d'il-15 utilisés pour traiter le cancer
US10973917B2 (en) 2016-05-18 2021-04-13 Modernatx, Inc. MRNA combination therapy for the treatment of cancer
US10975368B2 (en) 2014-01-08 2021-04-13 Flodesign Sonics, Inc. Acoustophoresis device with dual acoustophoretic chamber
US10993967B2 (en) 2018-10-17 2021-05-04 Senti Biosciences, Inc. Combinatorial cancer immunotherapy
CN112795543A (zh) * 2021-02-04 2021-05-14 华中农业大学 杂交瘤细胞株及其分泌的抗草鱼IL-15Rα单克隆抗体和应用
CN113308487A (zh) * 2021-05-28 2021-08-27 山东省淡水渔业研究院（山东省淡水渔业监测中心） 多种草鱼重组细胞因子及其制备方法和在协同增强草鱼免疫功能中的应用
CN113604437A (zh) * 2021-08-13 2021-11-05 青岛华赛伯曼医学细胞生物有限公司 过表达ccr2的免疫细胞及其应用
US11214789B2 (en) 2016-05-03 2022-01-04 Flodesign Sonics, Inc. Concentration and washing of particles with acoustics
US11241485B2 (en) 2017-06-12 2022-02-08 Obsidian Therapeutics, Inc. PDE5 compositions and methods for immunotherapy
WO2022060904A1 (fr) * 2020-09-16 2022-03-24 Obsidian Therapeutics, Inc. Compositions et procédés pour l'expression de récepteurs de lymphocytes t avec cd40l régulé par petites molécules dans les lymphocytes t
US11377651B2 (en) 2016-10-19 2022-07-05 Flodesign Sonics, Inc. Cell therapy processes utilizing acoustophoresis
US11419898B2 (en) 2018-10-17 2022-08-23 Senti Biosciences, Inc. Combinatorial cancer immunotherapy
US11446332B2 (en) 2017-04-13 2022-09-20 Senti Biosciences, Inc. Combinatorial cancer immunotherapy
EP4087861A1 (fr) * 2020-01-08 2022-11-16 Obsidian Therapeutics, Inc. Compositions et procédés pour la régulation accordable de la transcription
US20220409741A1 (en) * 2019-09-02 2022-12-29 Kyungpook National University Industry-Academic Cooperation Foundation Composition for preventing or treating cancer, containing il-2 surface expression-extracellular vesicles as active ingredient
US11708572B2 (en) 2015-04-29 2023-07-25 Flodesign Sonics, Inc. Acoustic cell separation techniques and processes
CN116536375A (zh) * 2023-06-28 2023-08-04 北京大学深圳研究生院 merocyclophanes类化合物的化学-酶法合成方法及其应用
WO2023212655A1 (fr) * 2022-04-28 2023-11-02 Immatics US, Inc. Polypeptides d'il-12, polypeptides d'il-15, polypeptides d'il-18, polypeptides de cd8, compositions et leurs procédés d'utilisation
WO2023212697A1 (fr) * 2022-04-28 2023-11-02 Immatics US, Inc. Il-15 liée à une membrane, polypeptides de cd8, cellules, compositions et leurs procédés d'utilisation
US12006345B2 (en) 2019-02-21 2024-06-11 Xencor, Inc. Untargeted and targeted IL-10 Fc-fusion proteins
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CN120209100A (zh) * 2025-03-28 2025-06-27 河北金要生物科技有限公司 一种表达mIL21和4-1BBL的病毒样颗粒及其在体外扩增NK细胞中的应用

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US10975368B2 (en) 2014-01-08 2021-04-13 Flodesign Sonics, Inc. Acoustophoresis device with dual acoustophoretic chamber
US11708572B2 (en) 2015-04-29 2023-07-25 Flodesign Sonics, Inc. Acoustic cell separation techniques and processes
US11214789B2 (en) 2016-05-03 2022-01-04 Flodesign Sonics, Inc. Concentration and washing of particles with acoustics
US10973917B2 (en) 2016-05-18 2021-04-13 Modernatx, Inc. MRNA combination therapy for the treatment of cancer
US11660341B2 (en) 2016-05-18 2023-05-30 Modernatx, Inc. mRNA combination therapy for the treatment of cancer
US11377651B2 (en) 2016-10-19 2022-07-05 Flodesign Sonics, Inc. Cell therapy processes utilizing acoustophoresis
US12104178B2 (en) 2017-03-03 2024-10-01 Obsidian Therapeutics, Inc. DHFR tunable protein regulation
US11446332B2 (en) 2017-04-13 2022-09-20 Senti Biosciences, Inc. Combinatorial cancer immunotherapy
US11241485B2 (en) 2017-06-12 2022-02-08 Obsidian Therapeutics, Inc. PDE5 compositions and methods for immunotherapy
US11666642B2 (en) 2017-06-12 2023-06-06 Obsidian Therapeutics, Inc. PDE5 compositions and methods for immunotherapy
US10785574B2 (en) 2017-12-14 2020-09-22 Flodesign Sonics, Inc. Acoustic transducer driver and controller
WO2019152663A1 (fr) * 2018-02-01 2019-08-08 Nkmax Co., Ltd. Procédé de production de cellules tueuses naturelles et composition pour le traitement du cancer
IL276365B1 (en) * 2018-02-01 2023-06-01 Nkmax Co Ltd A method for the production of natural killer cells and a preparation for cancer treatment
US11066644B2 (en) 2018-02-01 2021-07-20 Nkmax Co., Ltd. Method of producing natural killer cells and composition for treating cancer
US10590385B2 (en) 2018-02-01 2020-03-17 Nkmax Co., Ltd. Method of producing natural killer cells and composition for treating cancer
IL276365B2 (en) * 2018-02-01 2023-10-01 Nkmax Co Ltd A method for the production of natural killer cells and a preparation for cancer treatment
US12098388B2 (en) 2018-02-01 2024-09-24 Nkmax Co., Ltd. Method of producing natural killer cells and composition for treating cancer
US10993967B2 (en) 2018-10-17 2021-05-04 Senti Biosciences, Inc. Combinatorial cancer immunotherapy
US11419898B2 (en) 2018-10-17 2022-08-23 Senti Biosciences, Inc. Combinatorial cancer immunotherapy
US12006345B2 (en) 2019-02-21 2024-06-11 Xencor, Inc. Untargeted and targeted IL-10 Fc-fusion proteins
CN114450308A (zh) * 2019-06-12 2022-05-06 黑曜石疗法公司 用于调节性调控的ca2组合物和方法
JP2022537670A (ja) * 2019-06-12 2022-08-29 オブシディアン セラピューティクス， インコーポレイテッド Ｃａ２の組成物および調整可能な制御方法
WO2020252405A1 (fr) * 2019-06-12 2020-12-17 Obsidian Therapeutics, Inc. Compositions de ca2 et procédés de régulation ajustable
WO2021040736A1 (fr) 2019-08-30 2021-03-04 Obsidian Therapeutics, Inc. Compositions à base de car cd19 tandem et méthodes d'immunothérapie
US20220409741A1 (en) * 2019-09-02 2022-12-29 Kyungpook National University Industry-Academic Cooperation Foundation Composition for preventing or treating cancer, containing il-2 surface expression-extracellular vesicles as active ingredient
WO2021046451A1 (fr) * 2019-09-06 2021-03-11 Obsidian Therapeutics, Inc. Compositions et méthodes de régulation de protéine accordable dhfr
CN114651003A (zh) * 2019-09-10 2022-06-21 黑曜石疗法公司 用于可调调节的ca2-il15融合蛋白
JP2022547154A (ja) * 2019-09-10 2022-11-10 オブシディアン セラピューティクス， インコーポレイテッド 調節可能な制御のためのｃａ２－ｉｌ１５融合タンパク質
WO2021050789A1 (fr) * 2019-09-10 2021-03-18 Obsidian Therapeutics, Inc. Protéines de fusion de ca2-il15 pour une régulation accordable
JP7692897B2 (ja) 2019-09-10 2025-06-16 オブシディアン セラピューティクス， インコーポレイテッド 調節可能な制御のためのｃａ２－ｉｌ１５融合タンパク質
US11058725B2 (en) 2019-09-10 2021-07-13 Obsidian Therapeutics, Inc. CA2 compositions and methods for tunable regulation
WO2021058973A1 (fr) * 2019-09-25 2021-04-01 Prostate cancer research centre Peptides de fusion d'il-15 utilisés pour traiter le cancer
EP4087861A1 (fr) * 2020-01-08 2022-11-16 Obsidian Therapeutics, Inc. Compositions et procédés pour la régulation accordable de la transcription
WO2022060904A1 (fr) * 2020-09-16 2022-03-24 Obsidian Therapeutics, Inc. Compositions et procédés pour l'expression de récepteurs de lymphocytes t avec cd40l régulé par petites molécules dans les lymphocytes t
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CN113604437A (zh) * 2021-08-13 2021-11-05 青岛华赛伯曼医学细胞生物有限公司 过表达ccr2的免疫细胞及其应用
WO2023212697A1 (fr) * 2022-04-28 2023-11-02 Immatics US, Inc. Il-15 liée à une membrane, polypeptides de cd8, cellules, compositions et leurs procédés d'utilisation
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CN116536375B (zh) * 2023-06-28 2023-11-10 北京大学深圳研究生院 merocyclophanes类化合物的化学-酶法合成方法及其应用
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