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WO2018161026A1 - Il15 compositions and methods for immunotherapy - Google Patents

Il15 compositions and methods for immunotherapy 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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WO
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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French (fr)
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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Publication date
Application filed by Obsidian Therapeutics Inc filed Critical Obsidian Therapeutics Inc
Publication of WO2018161026A1 publication Critical patent/WO2018161026A1/en
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

The present invention provides biocircuit systems, effector modules and compositions for cancer immunotherapy. Methods for inducing anti-cancer immune responses in a subject are also provided.

Description

ILLS COMPOSITIONS AND METHODS FOR IMMUNOTHERAPY
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to the US Provisional Patent Application No.
62/466,601, filed on March 3, 2017 entitled Compositions and Methods for Immunotherapy; the US Provisional Patent Application No. 62/484,062, filed on April 11 , 2017 entitled IL15 Compositions and Methods for Immunotherapy, and the US Provisional Patent Application No. 62/555,316, filed on September 7, 2017 entitled IL15 Compositions and Methods for
Immunotherapy the contents of each of which are herein incorporated by reference in their entirety.
SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 2095__1205PCT__SL.txt, created on March 2, 2018, which is 650,427 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to compositions and methods for immunotherapy.
Provided in the present invention include polypeptides of biocircuit systems, effector modules, stimulus response elements (SREs) and immunotherapeutic agents, polynucleotides encoding the same, vectors and cells containing the polypeptides and/or polynucleotides for use in cancer immunotherapy. In one embodiment, the compositions comprise destabilizing domains (DDs) which tune protein stability.
BACKGROUND OF THE INVENTION
[0004] 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.
[0005] The impact of host anti-tumor immunity and cancer immunotherapy is impeded by three major hurdles: 1) low number of tumor antigen-specific T cells due to clonal deletion: 2) poor activation of innate immune cells and accumulation of tolerogenic antigen-presenting cells in the tumor microenvironment; and 3) formation of an immunosuppressive tumor
microenvironment. Particularly, in solid tumors the therapeutic efficacy of immunotherapeutic regimens remains unsatisfactory due to lack of an effective an anti-tumor response in the immunosuppressive tumor microenvironment. Tumor cells often induce immune tolerance or suppression and such tolerance is acquired because even truly foreign tumor antigens will become tolerated. Such tolerance is also active and dominant because cancer vaccines and adoptive transfer of pre-activated immune effector cells (e.g., T cells), are subject to suppression by inhibitory factors in the tumor microenvironment (TME).
[0006] In addition, administration of engineered T cells could result in on/off target toxicities as well as a cytokine release syndrome (reviewed by Tey Clin. Trans!. Immunol, 2014, 3: e l 7 10.1038).
[0007] Development of a tunable switch that can turn on or off the transgenic
immunotherapeutic agent expression is needed in case of ad verse events. For example, 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.
[0008] To develop regulatable therapeutic agents for disease therapy, in particular cancer immunotherapy, 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. In one example, 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.
SUMMARY OF THE INVENTION
[0009] 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. These systems are further taught in co- owned U.S. Provisional Patent Application No. 62/320,864 filed April 11, 2016, 62/466,596 tiled March 3, 2017 and the International Publication WO2017/180587 (the contents of each of which are herein incorporated by reference in their entirety).
[0010] In some embodiments, the composition for inducing an immune response may comprise an effector module. In some embodiments, the effector module may comprise a stimulus response element (SRE) operably linked to at least one payioad. In one aspect, the payload may be an immunotherapeutic agent.
[0011] In some embodiments, 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.
[0012] In one aspect, the SRE of the composition may be responsive to or interact with at least one stimulus.
[0013] In some embodiments, 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. In some embodiments, 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.
[0014] In one aspect, 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). In some embodiments, 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 ). hDHFR (Ammo acid 2-187 of WT; E162G, I176F), and hDHFR (Amino acid 2-187 of WT; H131 R, E144G), and hDHFR (Amino acid 2-187 of WT; Y 122I, A125F). In some embodiments, 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! '.. N65F, V 122!}. In some embodiments, 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, ΚΓ77Ε, Y 178C, K185E, N186S), hDHFR (NHS, P24S, F35L, M53T, K56E, R92G, S93G, N127S, H128Y, F135L, F143S, L159P, L160P, E173A, F180L), hDHFR (F35L, R37G, N65A, L68S, K69E, R71G, L80P, K99G, G117D, L132P, II 39V, M140I, D142G, D146G, E173G, D187G), hDHFR (L28P, N30H, M38V, V44A, L68S, N73G, R78G, A97T, K99R, A107T, K109R, D 11 FN, L134P, F135V, T147A, I152V, K158R, E172G, V182A, E184R), hDHFR (V2A, I17V, N30D, E31G, Q36R, F59S, K69E, I72T, H88Y, F89L, N108D, K109E, VI 10A, II 15V, Y122D, L132P, F135S, Ml 40V, E144G, T147A, Y157C, V170A, K174R, N186S), hDHFR (L100P, E102G, Q103R, P104S, E105G, N108D, V113A, Wl 14R, Y 122C, M i 61. 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 ).
Θ015] In one aspect, the stimulus of the SRE may be Trimethoprim or Methotrexate.
[0016] In some embodiments, 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.
[0017] In one aspect, the interleukin may be a whole or a portion of a IL15 and may comprise the amino acid sequence of SEQ ID NO. 55. In one aspect, the IL15 may be modified. In some embodiments, 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. [0018] In one aspect, 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.
[0019] In some embodiments, 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. In one aspect, an optional hinge domain may be incorporated.
[0020] In one aspect, 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
expression, function or level of the immunotherapeutic agent in the absence of the stimulus.
[0021 ] In one aspect, 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.
[0022] The present invention also provides polynucleotides comprising the compositions of the invention.
[0023] In one aspect, 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. In some embodiments, the polynucleotides may be an RNA molecule. In one aspect, the RNA molecule may be a messenger molecule. In some embodiments, the RNA molecule may be chemically modified.
[0024] In some embodiments, 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.
[0025] The present invention also provides vectors comprising polynucleotides described herein. In one aspect, the vector may be a viral vector. In some embodiments, 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.
[0026] The present invention also provides immune cells for adoptive cell transfer (ACT) which may express the compositions of the invention, the polynucleotides described herein. In one aspect, 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,
[0027] In some embodiments, the immune cells may be autologous, allogeneic, syngeneic, or xenogeneic in relation to a particular individual subject.
[0028] 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.
[0029] 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.
[0030] Also provided herein, is a method for inducing an immune response in a subject, administering the compositions of the invention, the polynucleotides of the invention, and/or the immune cells of the invention to the subject.
[0031] In some embodiments, the effector module comprises a stimulus response element (SRE) and at least one pay load comprising a pay load of interest (POI).
[0032] In some embodiments, the SRE may be a destabilizing domain (DD). In some examples, 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. In this context, the biocircuit system is a DD biocircuit system..
[0033] 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. The SRE and payioad may be operably linked through one or more linkers and the positions of components may vary within the effector module.
[0034] In some embodiments, 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. [0035] In some embodiments, 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.
[0036] The present invention also provides immunotherapeutic agents which may be a cytokine fused to its cognate cytokine receptor. In one embodiment 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.
[0037] 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, polynucleotides and vectors of the invention. The polypeptides, polynucleotides, viral vectors and cells are useful for inducing anti-tumor immune responses in a subject.
[0038] In some embodiments, 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.
[0039] In some embodiments, the vector of the invention may be a non-viral vector, such as a nanoparticles and liposomes.
[0040] 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). The engineered cell may be used for adoptive cell transfer for treating a disease (e.g., a cancer). Methods of enhancing the expansion and/or survival of immune cells are also disclosed herein.
[0041 ] 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.
[0042] Provided herein are methods for tuning the expression and function of
immunotherapeutic agent in cells or subjects. Such method may involve the administering effector modules containing an SRE operably linked to an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is IL15 or IL15 fused to IL15Ra. In some embodiments, the SRE is derived from FKBP, DHFR, PDE5, PPAR gamma, CA2 and NQ02. BRIEF DESCRIPTION OF THE DRAWINGS
[0043] Figure 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.
[0044] 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.
[0045] 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.
[0046] Figure 4 shows representative effector modules carrying two payloads with a cleavage site. In one embodiment, 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). In another embodiment, 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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. [0051] 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.
[0052] Figure 10 depicts a single-stimulus-bridged receiver biocircuit system. In this configuration, 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).
[0053] 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.
[0054] Figure 12 depicts a biocircuit system which requires a dual activation. In this embodiment, 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).
[0055] 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.
[0056] Figure 14 denotes the frequency of IFNgamma positive T cells.
[0057] 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.
[0058] 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. Figure
16C is a bar graph depicting ILlSRa positive cells in response to varying concentrations of TMP.
[0059] 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
MSD assay are presented in Figure 17C.
[0060] 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.
[0061] Figure 19 represents the proliferation of cell lines in response to cytokines.
[0062] 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. [0063] Figure 21 is a bar graph representing the effect of promoters on transgene expression.
[0064] Figure 22 depicts the kinetics of TL15Ra surface expression on CD4 T cells after TMP treatment.
[0065] Figure 23 represents a western blot of lL15-iL15Ra protein in HCT116 tumors from mice treated with TMP for 17 days in xenograft assays.
[0066] Figure 24 is a graph of the results of the MSD assay of TL15 protein levels in HEK293 cells.
[0067] 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.
[0068] 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.
[0069] Figure 27 represents the regulation of membrane bound IL15 with PO or IP dosing of TMP.
DETAILED DESCRIPTION OF THE INVENTION
[ΘΘ70] The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are now described. Other features, objects and advantages of the invention will be apparent from the description. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by- one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present description will control.
I. INTRODUCTION
[0071] 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. In contrast, 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.
[0072] Despite significant advances, the efficacy of current immunotherapy strategies is limited by associated toxicities. These are often related to the narrow therapeutic window associated with immunotherapy, which in part, emerges from the need to push therapy dose to the edge of potentially fatal toxicity to get a clinically meaningful treatment effect. Further, dose expands in vivo since adoptively transferred immune cells continue to proliferate within the patient, often unpredictably.
[0073] 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). Clinical trials utilizing T cells expressing T-cell receptor against specific TAA reported skin rash, colitis and hearing loss in response to immunotherapy.
[0074] 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.
Importantly, these adverse effects may occur during the destmction of tumors, and thus even a successful on-tumor immunotlierapy might result in toxicity. Approaches to reguiatably control immunothe apy are thus highly desirable since they have the potential to reduce toxicity and maximize efficacy.
[0075] 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. In one aspect, 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. Further, 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.
[0076] The tunable nature of the systems and 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,
[0077] 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.
II. COMPOSITIONS OF THE INVENTION
[0078] According to the present invention, biocircuit systems are provided 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). The overall architecture of a biocircuit system of the invention is illustrated in Figure 1. In general, 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. 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 much-detailed description of 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)- In accordance with the present invention, biocircuit systems, effector modules, SREs and components that tune expression levels and activities of any agents used for immunotherapy are provided.
[0079] As used herein, 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. It is also understood that 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.
[0080] In accordance with the present invention, 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.
Effector modules and SREs for immunotherapy
[0081] In accordance with the present invention, biocircuit systems, effector modules, SREs, and components that tune expression levels and activities of any agents used for immunotherapy are provided. As non-limiting examples, 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.
[0082] As stated, the biocircuits of the invention include at least one effector module as a component of an effector module system. As used herein, 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). As used herein a "stimulus 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. As used herein, the "responsive" nature of an SRE to a stimulus may be characterized by a covaient or non-covalent interaction, a direct or indirect association or a structural or chemical reaction to the stimulus. Further, 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.
[0083] In some embodiments, the present invention provides methods for modulating protein expression, function or level. In some aspects, 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%, 90-95%, 90-100% or 95-100%.
[0084] In some embodiments, the present invention provides methods for modulating protein, expression, function or level by measuring the stabilization ratio and destabiiization ratio. As used herein, 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. In some aspects, 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. As used herein, 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. As used herein "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. In some aspects, 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, 0.6-0.9,0.7-0.8, 0.7- 0.9 or 0.8-0.9.
[0085] 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. In this aspect, the SRE is an element, when responding to a stimulus, can une intracellular localization, intramolecular activation, and/or degradation of payloads.
[0086] In some embodiments, the stimulu s of the present invention maybe ultrasound stimulation. In some embodiments, the SREs of the present invention may derived from mechanosensitive proteins. In one embodiment, the SRE of the present invention may be the mechanically sensitive ion channel, Piezo 1. [0087] Expression of the payload of interest in such instances is tuned by providing focused ultrasound stimulation . In other embodiments, 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. In one embodiment, 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.
[0088] In some embodiments, 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. For example, an SRE may represent a destabilizing domain, while 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. Consequently, 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.
[0089] In some embodiments, effector modules of the present invention may include one or more degrons to tune expression. As used herein, 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. In some embodiments, 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. In some embodiments, the degron may be an N- degron, a phospho degron, a heat inducible degron, a photosensitive degron, an oxygen dependent degron. As a non-limiting example, the degron may be an Ornithine decarboxylase degron as described by Takeuchi et al. (Takeuchi J ei al. (2008). Biochem J. 2008 Mar
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.
[0090] As shown in Figure 2, representative effector module embodiments comprising one payload, i.e. one immunotherapeutic agent are illustrated. Each components 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.
[0091] 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 (payloads) 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 .
[0092] In some aspects, the two or more immunotherapeutic agents may be the same type. Biocircuits and components utilizing such effector molecules are given in Figures 7-12.
[0093] In some embodiments, 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. Several factors can contribute to protein immunogenicity, including, but not limited to protein sequence, route and frequency of administration and patient population. In a preferred embodiment, protein engineering may be used to reduce the immunogenicity of the compositions of the invention. In some embodiments, modifications to reduce immunogenicity may include modifications that reduce binding of the processed peptides derived from the parent sequence to MHC proteins. For example, 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. Several methods of identifying 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.
[0094] 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.
[0095] In some embodiments, protein modifications engineered into the structure of the compositions of the invention to interfere with antigen processing and peptide loading such as glycosylation and PEGylation, 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).
[0096] In one embodiment, 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. [ΘΘ97] In some embodiments, 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. 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). This study indicates that reduced immunoproteasome processing may be accompanied by reduced immunogenicity. In some embodiments, 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).
1. Destabilizing domains (DDs)
[0098] In some embodiments, 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. In one embodiment, the SRE is a stabilizing/destabilizing domain (DD). 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. Depending on the degree of binding and/or interaction the altered function of the payload may vary, hence providing a "tuning" of the payload function.
[0099] In some embodiments, 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 (DDs) 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.. Cell; 2006, 126(5): 995-1004; reviewed in Banaszynski, L.A., and Wandless, T.J. Chem. Biol. ; 2006, 13: 1 1-21 and Rakhit R et al , Chem Biol. 2014; 21(9): 1238-1252). However, when a specific small molecule ligand binds its intended DD as a ligand binding partner, the instability is reversed and protein function is restored. The conditional nature of DD stability allows a rapid and non-perturbing switch from stable protein to unstable substrate for degradation. Moreover, its dependency on the concentration of its ligand further provides tunable control of degradation rates.
[00100] In some embodiments, 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.
[00101] Several protein domains with destabilizing properties and their paired small molecules have been identified and used to control protein expression, including FKBP/shield-1 system (Egeler et al., J Biol. Chem. 2011, 286(36): 32328-31336; the contents of which are incorporated herein by reference in their entirety), ecDHFR and its ligand trimethoprim (TMP): estrogen receptor domains which can be regulated by several estrogen receptor antagonists (Miyazaki et al., J Am Chem. Soc, 2012, 134(9): 3942-3945; the contents of which are incorporated by reference herein in their entirety); and fluorescent destabilizing domain (FDD) derived from bilirubin-inducible fluorescent protein, UnaG and its cognate ligand bilirubin (BR) ( Navarro et al, ACS Chem Biol., 2016, June 6; the contents of which are incorporated herein by reference in their entirety).
[00102] Known 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.
[00103] In some embodiments, 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. For example, 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 β-glucuronidase (GUS) reporter, can significantly decrease the accumulation of the GUS fusion protein (Xiong et al., ./ Exp. Bot. , 2014, 65(15): 4397-4408). Xiong et al. further demonstrated that both exogenous 1-aminocyclopropane-l-carboxylic acid (ACC) treatment and salt can rescue the levels of accumulation of the ACS N-terminal and GUS fusion protein. The ACS N-terminus mediates the regulation of ACS7 stability through the ubiquitin~26S proteasome pathway.
[00104] Another non-limiting example is the stability control region (SCR, residues 97-118) of Tropomyosin (Tm), which controls protein stability. 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.
[00105] In some embodiments, 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.
[00106] 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. In some embodiments, 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. In some embodiments, 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.
Table 1; Proteins and their binding ligands
Figure imgf000022_0001
[00107] In some embodiments, 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.
Table 2: ecDHFR DDs and FKBP DDs
Figure imgf000023_0001
[00108] In some embodiments, novel DDs derived from E.coli DHFR (ecDHFR) 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.
[00109] In some embodiments, 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.
[00110] In some embodiments, 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.
[00111] In some embodiments, 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.
[00112] 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:
Quinone oxidoreductase 2). Candidate destabilizing domain sequence identified from protein domains of these proteins (as a template) 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. In some embodiments, 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.
[00113] In some embodiments, 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.
[00114] In some embodiments, 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). In other instances, 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. In some embodiments, polymerase chain reaction primers that amplify a different size product for each barcode may be used to identify each barcode on an agarose gel. In other instances, each barcode may have a unique quantitative polymerase chain reaction probe sequence that enables targeted amplification of each barcode. [00115] In some embodiments, 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. Other inhibitors of hDHFR such as folate, TQD, Trimethoprim (TMP), epigallocatechin gailate (EGCG) and ECG (epicatechin gailate) can also bind to hDHFR mutants and regulates its stability in one aspect of the invention, 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
(K185E), hDHFR (N186D), and hDHFR (M140I); double mutations: hDHFR (M53T, R138I), hDHFR (V75F, Y1221), hDHFR (A125F, Y 1221), hDHFR (L74N, Y 1221), hDHFR (L94A, ΊΤ47Α), 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), and hDHFR (Y 183H, K185E); and triple mutations: hDHFR (Q36F, N65F, Y 122I), hDHFR (G21E, 172V, I176T), hDHFR (I8V, K133E, Y163C), hDHFR. (V9A, S93R, P150L), hDHFR. (K19E, F89L, E181G), hDHFR (G54R, M140V, S168C), hDHFR (L23S, V121A, Y157C), hDHFR (VI 10A, V136M, K177R), hDHFR ( 49D, F59S, D153G) and hDHFR (N49D, F59S, D153G).
[00116] In one embodiment, the stimulus is a small, molecule that binds to a SRE to post- translationally regulate protein levels. In one aspect, DHFR ligands: trimethoprim (TMP) and methotrexate (MTX) are used to stabilize hDHFR mutants. 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. In Table 3, '"del" means that the mutation is the deletion of the amino acid at that position relative to the wild type sequence.
Table 3: Human BHFR mntants and novel destabilizing domains
Figure imgf000025_0001
LANKVDMVWIVGGSSVYKEA HPGHLKLFVTRI MQDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKG
IK YKFEVYEKND
HDHFR (F59S) M VG SLNC1VA VSQNMG1GKNGD LPWPPLRNEFR YF 14
QRMTTTSSVEGKQNLVIMGKKTWSSIPEKNRPLKG
RINLVLSRELKEPPQGAHFLSRSLDDALKLTEOPEL
AN VDMVWIVGGSSVYKEAMNHPGHLKLFVTRIM
QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK
YKFEVYEKND
hDHFR (N65D) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 15
QRMT TSSVEGKQNLVIMGKKTWFSIPEKDRPLKG
Ri LVL SRELKEPPQG AHFL SRSLDD ALKLTEQPEL
AN VDMVWIVGGSSVYKEAMNHPGHLKLFVTRIM
QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK
YKFEVYEKND
hDHFR (K8 I ) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 16
QRMTTTSSWGKQNLVnvlGKKTWFSIPEKNRPLKG RINLVL SRELREPPQG AHFL SR SLDD ALKLTEQPEL ANK VDMVWIVGG S S VYKE AMNHPGHLKLF VTRIM ODFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGD YKFEVYEKND
hDHFR (A107V) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 17
QRMTTTSSVEGKQNLVSMGKKTWFSIPEKNRPLKG RINLVL SRELKEPPQG AHFL SRSLDD ALKLTEQPEL VNK VDMVWI VGG S S VYKE AMNHPGHLKLF VTRIM QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK
S . 's Vi .KNi)
hDHFR (Y122I) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 18
QRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKG
RINLVL SRELKEPPQG AHFL SRSLDD ALKLTEQPEL
AN VDMVWIVGGSSVIKEAMNHPGHLKLFVTRIM
QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK
YKFEVYEKND
hDHFR (N127Y) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 19
QRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKG
RINLVL SRELKEPPQG AHFL SRSLDD ALKLTEQPEL
ANKVDMVW1VGGSSVYKEAMYHPGHLKLFVTRIM
QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK
YKFEVYEKND
hDHFR (Mi 401) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 20
ORMT TSSVEGKQNLVIMGKKTWFSIPEKN PLKG
RINLVL SRELKEPPQG AHFL SRSLDD ALKLTEQPEL ANK VDMVWI VGG S S VYKE AMNHPGHLKLF VT II QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK YKFEVYEKND
hDHFR (K 185E) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 21
ORN TTTSSVEGKONLVIMGKKTWFSIPEKNRPLKG RINLVLSRELKEPPQGAHFLSRSLDDALKLTEQPEL ANK VDMVWI VGG S S VYKE AMNHPGHLKLF VTRIM QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK Y F VYEEND
liDHFR (N 186D) MVGSLNCrVAVSQNMGIGKNGDLPWPPLRNEFRYF 22
QRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKG R INL VL SRELKEPPQGAHFL SR SLDD ALKLTEQPEL ANK VDMVWIVGGSS VYKE AMNHPGHLKLFVTRIM QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK F VYEKDD
hDHFR (C7R, Y163C) MVGSLNRIVAVSQNMGIGKNGDLPWPPLRNEFRYF 23
QRMTTTS S VEGKQNL VIM GKKTWF S IPEKNRPLKG RINLVL SRELKEPPQGAHFLSRSLDD ALKLTEQPEL AN VDMVWIVGGSSVYKEAMNHPGHL LFVTRIM QDFESDTFFPEIDLEKYKLLPECPGVLSDVQEEKGTK K E VYEKND
HDHFR (AiOV, H88Y) M VG SLNC1V WSQNMGIGKNGD LPWPPLRNEFR YF 24
QRMTTTS S VEGKQNL VIM GKKTWF S IPEKNRPLKG
R!NLVLSRELKt PQGAYFLSRSLDDALKLTEOPEL
AN VDMVWIVGGSSVYKEAMNHPGHLKLFVTRIM
QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK
YKFEVYEKND
hDHFR (Q36K, Y122I) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 25
KRMTTTSS GKQNLVDviGK TWFSiPEK RPLKG Ri LVL SRELKhPPQG AHFL SRSLDD ALKLTEQPEL AN VDMV WIVG G S S VKE AMNHPGHL LF VTPJM QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK YKFEVYEKND
hDHFR (M53T, R138I) MVGSLNCIVAVSQN GIGKNGDLPWPPLRNEFRYF 26
QRMTTTSSWGKQNLVITGKKTW'TSIPEKNRPLKGR
INLVLSRELKEPPQGAHFLSRSLDDALKLTEQPELA
NK\mrVWWGGSSVYT EA NHPGHL-KLFVTIIMQ
DFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKY
KFEVYEKND
hDHFR (T57A, 172 A) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 27
QR TTTSSVEGKQNLVI GKKAWFSIPEKNRPLKG RANLVLSRELKEPPQGAHFLSRSLDD ALKLTEQPEL ANK VDMVWI VGG S S \ΎΚΕ AMNHPGHLKLF VTRIM QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK
S . 's Vi .KNi)
hDHFR (E63G, 1176F) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 28
QRMTTTSSVEGKQNLVIMGKKTWFSIPGKNRPLKG
RINLVL SRELKEPPQGAHFL SRSLDD ALKLTEQPEL
ANKVDM IVGGSSWKEAMNHPGHLKLFVTRIM
QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGF
KYKFEVYEKND
hDHFR (G21T, Y 122I) MVGSLNCIVAVSQNMGIGKNTDLPWPPLRNEFRYF 29
QRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKG
RINLVL SRELKEPPQGAHFL SRSLDD ALKLTEQPEL,
ANKVDM IVGGSSVTKEAMNHPGHLKLFVTRIM
QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK
YKFEVYEKND
hDHFR (L74N, Y122I) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 30
QRMTTTSSVEGKQNTA'TMGKKTWFSIPEKNRPLKG RINN VL SRELKEPPQGAHFL SR SLDD ALKLTEQPEL ANK VDMVWI VGG S S VIKE AMNHPGHLKLF VTRIM QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK YKFEVYEKND
hDHFR (V75F, Y122I) MVGSLNCrVAVSQNMGIGKNGDLPWPPLRNEFRYF 31
QPJ TTTSSVEGKQNLVDVlGKKTWFSIPEKNRPLKG
RINLFL SREL KEPPQG AHFL SR SLDD ALKLTEQPEL A
NKVDMVWIVGGSSVIKEAMNHPGHLKLFVTRIMQ
DFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKY
KFEVYEKND
liDHFR (L94A, T147A) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 32
QRMTTTSS VEGKQNL VIM GKKTWFSIPEKNRPLKG RINLVLSRELKhPPQGAHFLSRSADD ALKLTEQPEL ANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTRIM QDFESDAFFPEIDLEKYKLLPEYPGVLSDVQEEKGI KYKFEVYEKND
DHFR (V121A, Y22I) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 33
QRMTTTS S VEGKQNL VIM GKKTWF S IPEKNRPLKG RINLVL SRELKEPPQGAHFLSRSLDD ALKLTEQPEL ANKVDMVWIVGGSSAKEAM HPGHLKLFVTRIM QDFESDTFFPE IDLEKYKLLPEYPG VLSDVQEEK G IK
Y KFE VYEKND
hDHFR (Y122I, A125F) MVGSLNCIVAVSQNMG1GKNGDLPWPPLRNEFRYF 34
QRMTTTS S VEGKQNL VIM GKKTWF S IPEKNRPLKG RINLVLSRELKEP QGAHFLSRSLDDALKLTEOPEL AN VDMVWIVGGSSVIKEFMNHPGHLKLFVTRIM QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK YKFE VYEKND
hDHFR (H131R, E144G) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 35
QRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKG
RiNLVL SRELKEPPQG AHFL SRSLDD ALKLTEQPEL AN VDMV WI VG G S S VYKE AMNHPGRLKL F VTRIM QDFGSDTFFPEIDLEKY'KLLPEYPGVLSDVQEEKGI KYKFEVYEKND
hDHFR (TI37R, F143L) MVGSLNCiVAVSQX GIGKNGDLPWPPLRNEFRYF 36
QRMTTTSS GKQNLVMGKKT FSIPEKNRPLKG RINLVL SRELKEPPQG AHFL SRSLDD ALKLTEQPEL ANK VDMVWI VGG S S \ΎΚΕ AMNHPGHLKLF VT RIM QDLESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK YKFE VYEKND
hDHFR (Y178H, E18IG) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 37
QR TTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKG RINLVL SRELKEPPQG AHFL SRSLDD ALKLTEQPEL ANK VDMVWI VGG S S VYKE AMNFiPGHLKLF VTRIM QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK HKFGVYEKND
hDHFR (Y183H, K185E) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 38
QRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKG
RINLVL SRELKEPPQG AHFL SRSLDD ALKLTEQPEL
ANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTRIM
QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK
YKFEVHEEND
hDHFR (V9A, S93R, P150L) MVGSLNCIAAVSQNMGIGKNGDLPWPPLRNEFRYF 39
QRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKG RINLVLSRELKEPPQGAHFLSRRLDD ALKLTEQPEL ANKVDM 1VGGSS KEAMNHPGHLK FVTRIM QDFESDTFFLEIDLEK YKLLPEYPGVL SD VQEEKGIK YKFEVYEKND
hDHFR (I8V, K133E, Y163C) MVGSLNCVVAVSQNMGIGKNGDLPWPPLRNEFRY 40
FQRMTT SSVEGKQNLVIMGKKTWFSIPEKNRPLK GRINL VLSRELKEPPQG AHFL SRSLDD ALKLTEQPE L ANK VDMVWrVGG S S VYKEAMNHPGHLELF VTRI
MQDFE SDTFFPEIDLEKYKLLPECPG VL SD VQEEKG KYKFEVYEKND
hDHFR (L23S, V121A, Y157C) MVGSLNCIVAVSQNMGIGKNGDSPWPPLRNEFRYF 41
QRN TTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKG
RINLVL- SRELKEPPQG. HFL SRSLDD ALKLTEQPEL ANK VDMVWI VGG S S AYKEAMNHPGHLKLF VTRIM QDFESDTFFPEIDLEKCKLLPEYPGVLSD VQEEKGIK YKFEVYEKND
liDHFR ( 39E, F89L, E!SIG) MVGSLNCIVAVSQNMGIGENGDLPWPPLRNEFRYF 42
QRMTTTSSVEGKQNLVIMGKKTW SIPEKNRPLKG RINLVLSRELKhPPQGAHLLSRSLDD ALKLTEQPEL ANK VDMVWIVGGSS VYKE AMNHPGHLKLF VTRIM QDFESDTFFPE IDLEKYKLLPEYPG VLSDVQEEK G IK YKFGVYEKND
hDHFR (Q36F, N65F, Y1221) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 43
FRMTTTSSVEGKQNLVIMGKKTWFSIPEKFRPLKGR INLVLSRELKEPPQGAHFLSRSLDDALKLTEQPELA M VDM IVGGSSVTKEAMNHPGHLKLFVTRI Q
KFEVYEKND
HDHFR (G54R, M140V, MVGSLNC1VA VSQNMG1GKNGDLPWPPLRNEFR YF 44 S168C) QRMTTTSSVEGKQNLVIMR KTWFS1PEKNRPLKG
RINLVLSRELKEPPQGAHFLSRSLDDALKLTEOPEL
AN VDMVWIVGGSSVYKEAMNHPGHLKLFVTRIV
QDFE SDTFFPE ID LEK Y 1 i PEYPGVLCDVQEEKGI
KY FEVYEKND
hDHFR (V110A, V136M, MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 45 K177R) QRMT TSSVEGKQNLVIMGKKTWFSIPEKNRPLKG
Ri LVL SRELKEPPQG AHFL SRSLDD ALKLTEQPEL AN ADM V WI VG G S S VYKE AMNHPGHLKLFMTRI MQDFESDTFFPEIDLE YKLLPEYPGVLSDVQEEKG IRYKFEVYEKND
hDHFR (Amino acid 2-187 of VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFF 46 WT; Q36F. Y122I, A 125F) RMTTTSSVEGKONL-VIMGKKTWFSIPEKNRPLKGRI
NLVLSRELKEPPOGAHFLSRSLDDALKLTEOPELAN KVDMVWIVGGSSV1KEFMNHPGHLKLFVTRMQDF
ESDTFFPEiDLEKY LLPEYPGVLSDVQEEKGI YKF EVYEK D
hDHFR (N49D, F59S, D 153G) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 47
QR TTTSSVEGKQDLV1MGKKTWSSIPEKNRPLKG RINLVL SRELKEPPQG AHFL SRSLDD ALKLTEQPEL ANK VDMVWI VGG S S VYKE AMNHPGHLKLF VTRIM ODFESDTFFPEIGLEKYKLLPEYPGVLSDVQEEKGIK Y KFEVYEKND
hDHFR. (G21E, 172V, I176T) MVGSLNCIVAVSQNMGIGKNEDLPWPPLRNEFRYF 48
QRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKG
RVNLVLSRELKEPPQGAHFLSRSLDDALKLTEQPEL
ANKWMVWIVGGSSVYKEAMNHPGHLKLFVTRIM
QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGT
KYKFEVYEKND
hDHFR. (LiOOP, E102G, M VGS LNCI VA V SQNMGIGKN GDLP WPPLRNEFR YF 116 Q103R, P104S, E105G, N108D, QRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKG V113A, W114R, Y122C, RINLVL SRELKEPPQG AHFL SRSLDD ALKPTGR SGL M126I, N127R, H128Y, L 132P, ADKVDMARIVGGSSVCKEAIRYPGHPKLPVTRTMQ F135P, I139T, F148S, F149L, DFESDTSLPEVALEKYKT 1 PEYPGVLSGAQEEKGAR I152V, D153A, D169G, V170A, YKFEAYERSD
1176 A, K177R, V182A, K185R,
N186S)
hDHFR (V2A, R33G, Q36R, MAGSLNCrVAVSQNMGIGKNGDLPWPPLRNEFGYF 125 L100P, K185R) RRM'l'l'l'SSVEGKQNLVIMGKKTWFSIPEKNRPLKG
RINLVL SRELKEPPQG AHFL SR SLDD ALKPTEQPEL ANK VDMVWI VGG S S VYKE AMNHPGHLKLF VTRIM
QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK YKFEVYERND
hDHFR (G16S, I17V, F89L, MVGSLNCIVAVSQNMSVGKNGDLPWPPLRNEFRY 126 D96G, K123E, M140V, D146G, FQRM' Ί " I S S VEGKQNL VIMGKKTWF SIPEKNRPLK K156R) GRINLVLSRELKEPPQGAHLLSRSLDGALKLTEQPE
LANKVDM IVGGSSVYEEAM>mPGHLKLFVTRI
VODFESGTFFPEIDLERYKLLPEYPGVLSDVOEEKGI
KYKFEVYEKND
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
M U2T, E151 G, E162G, FQRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLK
E372G) GRTNLVLSRELKhH QGAHFLSRSLDDASRLTEQPEL
ANKVDTWIVGGSSVYKEAMNHPGHL-KLFVTR
QDFESDTFFPGIDLEKYKLLPGYPGVLSDVQGEKGI KYKFEVYEKND
hDHFR. (R.138G, D142G, MVGSLNCIVAVSQNMGIGKNGDLPWPPLR EFRYF 150 F143S, K156R, K158E, E162G, QRMTTTSSVEGKQNLVIMGKKTWFSIPE NRPLKG V166A, K 177E, Y178C, 185E, RI LVLSRELKEPPQGAHFLSRSLDDALKLTEQPEL N186S) ANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTGIM
QGSESDTFFPEIDLERYELLPGYPGALSDVQEEKGIE KF EVYEESD
hDHFR. (K81R, K99R, L100P, MVGSLNCIVAVSQNMGIGKNGDLPWPPLR EFRYF 174 E102G, N 108D, K123R, QRMTTTSSVEGKQNLVIMGKKTWFSIPE NRPLKG H128R, D142G, F180L, K185E) RI LVLSRELREPPQGAHFLSRSLDDALRPTGQPEL
ADKVDMVWIVGGSSVYREAM RPGHLKLFVTRIM
QGFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK
Y LE EEND
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
D SESDTFFPEIDLEKYKPPPEYPGVL SD VQE AKGIK Y KLEVYEKND
hDHFR (V2A, I17V, N30D, MAGSLNCrVAVSQ MGVGKNGDLPWPPLRDGFRY 196 E31G, Q36R, F59S. K69E, FRRMTTTSS\¾GKQ L\TMGKKTWSSIPEKNRPLEG Γ72Τ, 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
K174R, N186S)
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. T 147 A, NR NMVVVIVGGSSVYKEAM HPGHLRPVVTRIMQ 1152V, K1.58R, E172G, V182A, DFESDAFFPEVDLEKYRLLPEYPGVLSDVQGEKGIK E184R1 YKFEAYRKND
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
Y122D, 123E, 11 9V, Q141R,
D142G, F148L, E1.51 G, E155G,
Y157R, Q171R, Y183C, E184G,
K1.85deL D lS7N)
hDHFR (Amino acid 2-187 of VGSLNCrVAVSQNMGVGKNGDLPWPPLRNEFRYF 328 WT; I17V, Y122I) QRMT TSSVEGKQNLVIMGKKTWFSIPE NRPLKG
RI LVLSRELKEPPQGAHFLSRSLDDALKLTEQPEL
ANKVDMVWlVGGSSVIKEAMNHPGHLKLFVTRl^l
QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK
YKFEVYEKND
hDHFR (Amino acid 2-187 of VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQ 329 WT: Y122I, M140I) RMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRI
NLVLSRELKEPPQGAHFLSRSLDDALKLTEQPELAN
KVDMVWIVGGSSVIKEAMNHPGHLKLFVTRIIQDF
ESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYKF
EVYEKND hDI-IFR (Amino acid 2-187 of VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQ 330 WT; N327Y, Υ122Γ) RMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRI
NL VL SRELKEPPQG AHFL S R SLDD ALKLTEQPEL A N
KVDNlVWIVGGSSVIKEANlYHPGHLKLFVTRmiOD
FESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYK
FEVYE-KND
hDI-IFR (Amino acid 2-187 of VGSLNCrVAVSQNMGIGK GDLPWPPLR EFRYFQ 331 WT; Y122I, H131R, E144G) RMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRI
N L VL SRELKEPPQG AH FL S R SLDD AL KLTEQPEL AN
KVDMVWIVGGSSVTKEAMNHPGRLKLFVTRIMQDF
GSDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYK
FEVYEKND
hDI-IFR (Amino acid 2-187 of VGSLNCrVAVSQNMGIGKNGSLPWPPLRNEMSYFS 332 WT; D22S, F32M, R33S, Q36S, RMTTTSSVEGKQNLVIMGKKTWFSIPEKSRPLKGRI N65S) NLVLSRELKEPPQGAHFLSRSLDDALKLTEQPELAN
KVDMVWIVGGSSWKEAMNHPGHLKLFVTRMQD
FESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKY
FEVYEKND
hDHFR (Amino acid 2-187 of VGSLNCrVAVSONMGIGKNGDLPWPPLRNDMRYF 333 WT; E31D, F32M, VI 161) ORMT TSSVEGKQNLVMGKKTWFSIPEKNRPLKG
RINLVL SRELKEPPQG AHFL SRSLDD ALKLTEQPEL ANKVDMVWIIGGSSVYKEAMNHPGHLKLFVTRIM QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIK YKFEVYEKND
hDHFR (Amino acid 2-187 of VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQ 334 WT; E162G, I176F) RMTTTSSV GKQNLVIMGKKTWFSIPEKNRPLKGRI
NLVLSRELKEPPQGAHFLSRSLDDALKLTEQPELAN
K\nDMVWIVGGSS\^TCEAMNHPGHLKLFVTRINlQD
FESDTFFPEIDLEKYKLLPGYPGVLSDVQEEKGFKY
KFEVYE-KND
hDHFR (Amino acid 2-187 of VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQ 335 WT; K185E) RMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRI
NLVLSRELKEPPQGAHFLSRSLDDALKLTEQPELAN KVDMVWIVGGSSWKEAMNHPGHLKLFVTRMQD FESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYK FEVYEEND
hDHFR (Amino acid 2-187 of VGSLNCrVAVSQNMGIGKNGDLPWPPLRNEFRYFQ 336 WT; Y322L A125F) RMTTTS S VEG QNL VIMGKKTWFSIPEKNRPLK GRI
NL VL SRELKEPPQG AHFL S R SLDD ALKLTEQPEL A N KVDMVWIVGGSSVIKEFMNHPGHLKLFVTRIMQDF
ESDTFFPE IDLEK YKLLPEYPG VLSDVQEEK G IK YKF EVYEKND
hDHFR (Amino acid 2-187 of VGSLNCrVAVSQNMGIGKNGDLPWPPLRNE-FRYFF 337 WT; Q36F. N65F, Y122I) RJV TTTSSVEGKQNLVINlGKKTWFSIPEKFRPLKGRI
NL'VLSRELKEPPQGAHFLSRSLDDALKLTEQPELAN KVDNlVWIVGGSSVIKEAIVlNHPGHLKLFVTRmiQD FESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYK FEVYEKND
hDHFR (Amino acid 2-187 of VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQ 338 WT; N127Y) RM' TITS S VEGKQNL VIMGKKTWF SIPEKNRPLKGRI
NLVLSRELKEPPQGAHFLSRSLDDALKLTEQPELAN KVDMVW1VGGSSVYKEAM 'HPGHLKLFVTRIMQD FESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYK FEVYEKND hDI-IFR (Amino acid 2-187 of VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQ 339 WT; H 3 31R, E144G) RMTTTSSVEG QNLVIMGKKTWFSIPEK RPLKGRI
NL VL SRELKEPPQG AHFL S R SLDD ALKLTEQPEL A N
KVDMVWIVGGSSVYKEAMNHPGRLKLFVTOIMQD
FGSDTFFPEIDL.EKYKLL.PEYPGVLSDVOEEKGIKY
KFEVYEKND
hDHFR (Amino acid 2-187 of VGSLNCIVAVSQNMGVGKNGDLPWPPLRNEFRYF 340 WT; I17V) QRM' 1 I TS S VEGKQNL VIMGKKTWF S IPEKNRPLKG
Ri LVL SRELKEPPQG AHFL SRSLDD ALKLTEQPEL
ANKVDMVWIVGGSSVYXEAMNHPGHLKIFVTRIM QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGD YKFEVYEK D
hDHFR (Amino acid 2-187 of VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQ 341 WT; Y122I) RMn SSVEGKQ LV GKKTWFS!PEKNRPLKGRI
NL VL S REL KEPPQG AHFL SR SLDD ALKLTEQPEL AN K VDMVWI VGGS S VIKE AM HPG HLKLF VTR LVIQD FESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYK FEVYEKND
liDHFR (E 162G, I 376F) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 506
QR¾ TTTSSVEGKQNLVTJVLGKKTWFSIPEKNRPLKG
RINLVL.SRELKEPP'QGAHFLSRSLDDALKLTEQPEL
ANK VDMVWI VGG S S VYKE AMNHPGHLKLF VTRIM
QDFESDTFFPEIDLEKYKLLPGYPGVLSDVQEEKGF
KYKFEVYEKND
[00117] In some embodiments, DD mutations that do not inhibit ligand binding may be preferentially selected. In some embodiments, 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. 2, In some embodiments, 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.
[00118] In some embodiments, 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, L68S, K69E, K69R, R71G, I72T, I72A, I72V, N73G, L74N, V75F, R78G, L80P, K81 , E82G, H88Y, F89L, R92G, S93G, S93R, L94A, D96G, A97T, L98S, K99G, K99R, L100P, E102G, Q103R, P104S, E105G, A107T, A107V, N108D, K109E, K109R, V110A, Dll IN, M112T, Ml 12V, V113A, W114R, I115V, 1115L, VI 161, Gl 17D, V121A, Y122C, Y122D, Y122I, K123R, K123E, A125F, M126I, N127R,
N127S, N127Y, H128R, H128Y, H131R, L132P, K133E, L134P, F135P, F135L, F135S, F135V, V136M, T137R, R138G, R138I, I139T, 1139V, M1401, M140V, Q141R, D142G, F143S, F143L, E144G, D146G, ΊΊ47Α, F148S, F148L, F149L, P150L, E151G, 1152V, D153A, D153G, E155G, K156R, Y157R, Y157C, K158E, K158R, L159P, L160P, E162G, Y163C, V166A, S168C, D169G, V170A, Q171R, E172G, E173G, E173A, 174R, I176A, I176F, I176T, K177E, K177R, Y178C, Y178H, F180L, E181G, V182A, Y183C, Y183H, E184R, E184G, K185R, K185del, K185E, N186S, N186D, D187G, and D187N.
[00119] In some embodiments, 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.
[00120] In some embodiments, 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. I8V, V9A, A10T, A10V, Q13R, N14S, G16S, Ϊ17Ν, I17V, K19E, N20D, G2.iT, 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, KIT, K64R, N65A, N65S, N65D, N65F, L68S, K69E, K69R, R71G, I72T, 172 A, I72V, N73G, L74N, V75F, R78G, L80P, K81 , E82G, H88Y, F89L, R92G, S93G, S93R, L94A, D96G, A97T, L98S, K99G, K99R, L100P, E102G, Q103R, P104S, E105G, A107T, A 107V, N108D, K109E, K109R, VI 10A, Dl 1 IN, Ml 12T, Ml 12V, VI 13 A, Wl 14R, II 15V, li 151.. VI 161, G117D, V121A, Y122C, Y122D, Y122I, K123R, K123E, A125F, M126I, N127R, N127S, N127 T, H128R, H128Y, H131R, L132P, K133E, L134P, F135P, F135L, F135S, F135V, V136M, T137R, R138G, R1381, 1139T, 1139V, M1401, M140V, Q141R, D142G, F143S, F143L, E144G, D146G, T147A, F148S, F148L, F149L, P150L, E151G, I152V, D153A, D153G, E155G, K156R, Y157R, Y157C, K158E, K158R, L159P, L160P, E162G, Y163C, VT66A, S168C, D169G, V170A, Q171R, E172G, E173G, E173A, K174R, I176A, I176F, ΙΓ76Τ, ΚΓ77Ε, K177R, Y178C, Y178H, F180L, E181G, V182A, Y183C, Y183H, E184R, E184G, K185R, K185del, K185E, N186S, N186D, D187G, and D187N.
2. Payloads: Immunotherapeutic agents
[00121] In some embodiments, 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. In one embodiment, the immunotherapeutic agent induces an anticancer immune response in a cell, or in a subject.
Cytokines, chemokines and other soluble factors
[00122] In accordance with the present invention, 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.
[00123] In some embodiments, 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.
Whether these are included or excluded herein, the meaning is not meant to be changed as would be understood by one of skill in the art. For example, IL2, IL2 and IL 2 refer to the same interleukin. Likewise, TNFalpha, TNFot, TNF-alpha, TNF-a, TNF alpha and TNF a all refer to the same protein. In some embodiments, 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.
[00124] In some embodiments, payloads of the present invention may be cytokine receptors, recombinant receptors, variants, analogs and derivatives thereof: or signal components of cytokines.
[00125] In some embodiments, 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. In other embodiments, T cells engineered with two or more DD regulated cytokines are utilized to provide kinetic control of T cell activation and tumor microenvironment remodeling. In one aspect, 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. Two factors contribute to the observed toxicity (a) Pleiotropism, wherein cytokines affect different cells types and sometimes produce opposing effects on the same ceils depending on the context (b) Cytokines have short serum half-life and thus need to be administered at high doses to achieve therapeutic effects, which exacerbates the pleiotropic effects. In one aspect, cytokines of the present invention may be utilized to modulate cytokine expression in the event of adverse effects. In some embodiments, cytokines of the present invention may be designed to have prolonged life span or enhanced specificity to minimize toxicity .
[00126] In some embodiments, the payload of the present invention may be an interleukin (IL) cytokine. Interleukins (ILs) are a class of glycoproteins produced by leukocytes for regulating immune responses. As used herein, 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. In some aspects, 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 ! 14 ) 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, IL36RN, IL37, IL37a, IL37b, IL37c, IL37d, IL37e and IL38. In other aspects, 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.
[00127] In one embodiment, 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. In some embodiments, 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. For example, the asparagine may be replaced by aspartic acid at position 72 of ILLS (SEQ. ID NO. 2 of US patent publication
US20140134128A1; the contents of which are incorporated by reference in their entirety). In some embodiments, 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 ). 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. In some aspects, 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 sequence.
Table 4: 1L15 constructs
Description Promoter Amino acid sequence Amino Nucleic
Acid SEQ Acid SEQ ID NO ID NO/
Sequence
1L2 signal - MYRMQLLSC1ALSLALVTNS 52 58-61 sequence
IgE leader - MDWTWILFLVAAATRVHS 219 229, 343,
344
Linker - EFSTEF 5 62-63
Linker - GGSGG 54 64-68
HA Tag YPYDVPDYA 345 346-347
BamHl - GS - GGATCC
P2A GATNFSLLKQAGDVEENPGP 350 351
Cleavable
Peptide
inCherry LSKGEEDNMAUKEFMRFKVHMEGSVNG 352 353
(M IL) " HEFE1 EGEGEGRPYE GTQTAKLK VTKGG
PLPFAWDILSPQFMYGSKAYVKHPADTP D YLKL S FPEGFKWER VMNFED G G VVT V TQDS SLQDGEFTYKVKLRGTNFPSDGP V MQKKTMGWE A S SERMYPED G ALKGEIK QRLKLKDGGHYDAE\¾TTYKAKKPVQL
PGAYNVNIKLDlTSH E-DYnVEQYERAE
GRHSTGGMDELYK
IL15 NWVNVISDLKKIEDLIQSMHIDATLYTES 55 69-71 (each
DVHPSCKVTAMKCFLLELQVISI PSGDA include the SIHDTVENLIILANNSLSSNGNVTESGCK stop codon ECEELEEKNIKEFLQSFVHIVQMFTNTS* at the end),
72, 378- 379
ecDHFR ISLiAALAVDYVIGMENAMPWNLPADLA 9 73, 372, (Amino acid WFKRNTLNKPVIMGRHTWESIGRPLPGR 233
2-159 of KNIILSSOPGTDDRVTWVKSVDEAIAAC (includes WT; R12Y, GDVPEIMVIGGGRVIEQFLPKAOKLYLT stop codon) Y100I) HE) AE VEGDTHFPD YEPDD WE S VF S E FH
DADAQNSHSYCFEH.ERR*
hDHFR VGSLNCIVAVSQNMGIGKNGDLPWPPLR 341 371
(Amino acid NEFRYFQRMTTTSSVEGKQNLVIMGKKT
2-387 of WFSIPEKNRPLKGRINLVLSRELKEPPOG
WT ) AHFLSRSLDDALKLTE-QPELANKVDMV
(Y122I) WIVGGS S VKEAMNHPGHLKLFVTRIMQ
DFESD l'FF'PEIDLEKYKLLPEYPGVLSDV
QEEKGIKYKFEVYEKND
OT-IL15- CMV M^TIMQLL S C I AL SL AL VTNSNWV VI SD 56 74
001 (IL2 LKKKDLIQSMHID ATL YTE SD VHP S CK V
signal TAMKCFLLELQVISLESGDASIHDTVENL
sequence- IE.AN SLSSNGNVTESGCKECEELEEK I
IL 15 -stop) KEFLQ SF VHI VQMFINT S *
Figure imgf000037_0001
[00128] In some instances, whole or a portion of the IL15 is linked to the whole or a portion of one or more transmembrane proteins. In some embodiments, 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. As non-limiting examples, 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.
[00129] In some embodiments, 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. In certain
embodiments, 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.
Table 5: Transmembrane domains
Transmembrane domain Amiuo Acid Sequence SEQ. ID
NO.
CD8 Transmembrane TTTP APRPPTP APTI ASQPLS L PE ACRP A AGO A 76
domain VHTRGLDFACDI
2B4 Transmembrane FLVIIVILSALFLGTLACFCV 358
domain
4-l.BB Transmembrane IISFFLALTSTALLFLLFFLTLRFSWKRGR
domain
4-1BB Transmembrane IISFFLALTSTALLFLLFFLTLRFSVV 78
domain
CD134 (OX40) VAAILGLGLVLGLLGPLAILLALYLL 79
Transmembrane domain CD S 48 Transmembrane and AWGCTFGALVIVTVGGFIFWRKKRKDAKNNE 80 intracellular domain VSFSQ1KPKKSKLIRVENFEAYFKKQQADSNCG
FAEEYEDLKLVGISQPKYAAELAENRGKNRYN N VLP YDI SR VKL S VQTHS TDD YIN ANYMPG YH SKKDFIATQGPLPNTLKDFWRMVWEKNVYAII MLTKCVEQGRTKCEEYWPSKQAQDYGDITVA MTSEIVLPEvVriRDFTVKNIOTSESHPLROFHF TSWPDHGVPDTTDLLINFRYLVRDYMKQSPPE SPILWCSAGVGRTGTFIAIDRLIYQENENTVD VYGIVTDLR IHRPLMVQTEDQYVFLNQCVLD IVRSQKX>SKVT>LrirQNTTA]S TIYENLAPVTTFG KTNGYIA
CD 148 Transmembrane AVFGC IFG AL VI VTVG GFIFW 81 domain
CD2 Transmembrane KE1TNALETWGALGQDI LDIPSFQMSDDIDDI 82 domain KWE TSDKKKIAQFRKEKETFKEKDTYKLFKN
GTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIF DLK1QERVSKPKISWTCINTT TCEVMNGTDPE LNLYQDGKHLKLSQRVITHKWTTSLSA F CT AGNKVSKESSVEPVSCPEKGLD
CD28 Transmembrane and ffiV YPPPYLDNEKSNGTITHVKGKHLCPSPLF 83 intracellular domain PGPSKPFWVXVWGGVLACYSLLVTVAHIFWV
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAP
PRDFAAYRS
CD28 Transmembrane FWVLVVVOGVLACYSLLVTVAFIIFWV 84 domain
CD28 Transmembrane IE VM YPPP YL DNEK S GTIIH VKGKHL CP SPLFP 85 domain GPSKPFWVLWVGGVLACYSLLVTVAF!IFWV
CD28 Transmembrane IFWVXWVGGVTACYSLLVTVAFIIFWVRSKR 86 domain R
CD28 Transmembrane FWVLVWGGVLACYSLLVTVAFIIFWVRSKRS 87 domain RLLHSDYMNMTPRRPGPTRKHYQP
YAPPRDF A A YR S
CD28 Transmembrane MFWVL VWGGVLACY SL L VTVAFIIFWV 88 domain
CD28 Transmembrane FWVLVWGGVLACYSLLVTVAFHFWV 89 domain
CD28 Transmembrane FWVLVWGGVLACYSI VTVAFIIFWVRSKRS 359 domain RLLHSDYMNNITPRRPGPTRKHYQAYAAARDF
AAYRS
CD28 Transmembrane IE VMYPPP YLDNEK SN G T I IH VKG HI , 360 domain CPSPLFPGPS KPFWVLWVG GVLACYSLLV
TVAF I IFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS
CD28 Transmembrane MFWVL VWGG\XACYSGG\ VAFIIFWV 90 domain
CD28 Transmembrane WVLVVVGGVLACYSLLVTVAFIIFWV 1 domain
CD28 Transmembrane FWVLVWGGVLACYSLLVTVAFIIFWVR 361 domain
CD28 Transmembrane PFWVLVVVGGVLACYSLLVTVAFnFWVRSKR 92 domain SRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF
AAYRS
CD28 Transmembrane FWVLVWGGVLACYSI VTVAFIIFWVRSKRS 93 domain and CD28 and CD 3 RLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA
Zeta intracellular domain AYRSRVKFSRSADAPAYQQGQNQLYNELNLG
RREEYDVLD RRGRDPEMGGKPRRKNPQEGL YNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR CD28 Transmembrane FWVLVWGGVLACYSLLVTVAFIIF VRSKRS 94 domain and CD28, OX40, RLLHSDYMNMTPRRPGPTRKFIYQPYAPPRDFA and CD 3 Zeta intracellular AYRSRDQRLPPDAHKPPGGGSFRTPIQEEQAD domain AHSTLAKIRVKFSRSADAPAYQQGQNQLYNEL
NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ
EGLYTSIELOKDKMAEAYSEIGMKGERRRGKGH
DGLYQGLSTATKDTYDALHMQALPPR
CD28 Transmembrane FWVLVWGGVLACYSLLVTVAFIIFWVRRVKF 95 domain and CD3 Zeta SRSADAPAYQQGQNQLYNELNLGRREEYDVL intracellular domain DKRRGRDPEMGGKPRRK PQEGLYNELQKDK
M AEAYSEIG K GERRRGKGHDG LYQGL STAT K DTYD ALHMQ ALPPR
CD28 transmembrane-CD3 AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPS 96 zeta signaling domain PLFPGPSKPFWVLWVGGVLACYSLLVTVAFII ("28z") FWVRSKRSRLLHSDYM MTPRRPGPTR HYQ
PYAPPRDFAAYRSRV FSRSADAPAYQQGQNQ
LYNELNLGRREEYDVLDKRRGRDPEMGGKPR
RKNPQEGLYNELQKDKM AE A YSEl GMKGERR
RGKGHDGLYQGLSTATKDTYDALHMQALPPR
CD3 zeta Transmembrane L C YLLD GILFI YG VILT ALFLR V 97 domain
CD3 zeta Transmembrane KWKALFTAAILQAQLPITEAQSFGLLDPKLC 98 domain YLLDGILFIYGVILTALFL
CD3 zeta Transmembrane L C YLLD GILFIYG VDLT ALFL 99 domai
CD4 Transmembrane ALIVLGGVAGLLLFIGLGIFFCVRC 100 domain
CD4 Transmembrane MALIVLGGVAGLLLFIGLGIFF 101 domain
CD45 Transmembrane and ALIAFLAFLIIVTSIALLWLYKIYDLHKKRSCN 102 intracellular domain LDEQQELVERDDEKQLMNVEPIHADTT T ETYK
RKIADEGRLFLAEFQSIPRVFSKFPIKEARKPFN
ONKNRYVDILPYDYNRVELSEINGDAGSNYIN
A YIDGFKEPR YIAAQGPRI»:T\¾DFWRMIW
EQ ATVIVMVTRCEEGNRNKCAEYWPSMEEG
TRAFGDWVKINQHKRCPDYIIQKLNIVN KE
KATGREVTfflQFTSWPDHG EDPHLLLKLRR
RVNAF SNFFSGPIWHC SAG VGRTGT YIGID AML
EGLEAENKVDVYGYVVKLRRQRCLMVQVEA
QYSLIHQALVEYNQFGB, 1 EVNLSELHPYLH M
KKRDPPSEPSPLEAEFQRLPSYRSWRTQHIG Q
EENKSKNRNSNVIPYDYNRVPLKHELEMSKES
EHDSDESSDDDSDSEEPS YINASFIMSYW PE
VMIAAQGPLKETIGDFWQMIFQRKVKVIVMLT
ELKHGDQEICAQY GEG QTYGDSEVDLKDT
DKSSTYTLRVFELRHSKRKDSRTVYQYQYT
WSVEQLPAEPKELISMIQWKQKLPOKNSSEGN
KHHKSTPLLIHCRDGSQOTGIFCALLNLLESAE
TEEWDIFQWKALRKARPGMVSTFEQYQFLYD
VIASTYPAQNGOVKKN HQEDKIEFD E\7DKV
KODANCVNPLGAPEKLPEAKEQAEGSEPTSGT
EGPEHSV GPASPALNQGS
CD62 L Transme mb rane PLFIPVAVMVTAFSGLAFITWLA 103 domain
CD7 Transmembrane ALP A ALA VI SFLLGL GL G V AC VL A 104 domain
CDS Transmembrane MALPVTAL-LLPLALLLHAARP 105 domain CDS T tans membrane AAAFVPVTLPAKPTTTPAPRPPTPAPTIASQPLS 106 domain and CD28 signaling LRPEACRPAAGGAVHTRGLDFACDIYIWAPLA domain GTCGVLLLSLVITLYCNHRNRSKRSRLLHSDY
MMVUPRRPGPTRKHYQPYAPPRDFAAYRSRFS
VVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC
RFPEEEEGGCELRVKFSRSADAPAYQQGQNQL
YNELNLGRREEYDVLDKRRGRDPEMGGKPRR
KNPQEGLYNELQKDKMAEAYSEIGMKGERRR
GKGFfDGLYQGLSTATKDTYDALHMQALPPR
CDS transmembrane AAATTTPAPRPPTPAPTIASQPLSLRPEACRPAA 107 domain-CD137 (4-1BB) GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL signaling domain and CD3 VTTLYCKRGRKKLLYIFKQPFMRPVQTTQEED zeia signaling domain GCSCRFPFF.F.EGGCELRVKFSRSADAPAYKQG C'BBz") QNQLYNELNLGRREEYDVLDKRRGRDPEMGG
KPRRKNPQEGLYNELQKD MAEAYSEIGMKG
ERRRGK GHDGLYQGL STATKDTYD ALHMQAL
PPR
CD8a Transmembrane FVPVFLPA PTTTPAPRPPTPAPTIASQPLSLRPE 108 domain ACRPAAGGAVHTRGLDFACDTYIWAPLAGTCG
VLLL S L VITL YCNHRN
CDSa Transmembrane rWAPLAGTCGVLLLSLVITLYC 109 domain
CDSa Transmembrane mWAPLAGTCGVLLLSLVITLYC 1 10 domain
CDSa Transmembrane IYIWAPLAGTCG VLLL SL VITL YCR i l l domain
CDSa Transmembrane PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG 362 domain AVHTRGLDFACDlYiWAPLAGTCGVLLLSLVrT
LYCN
CDSa Transmembrane IYI APL AGTCGVLLL SL VITL VCR 1 12 domain
CDSa Transmembrane I YIW APL AGTC G VLLL SL ViT 113 domain
CDSa Transmembrane IYIWAPLAGTCGVT T T .SLVITLY 114 domain
CDSa Transmembrane TPAPRPPTP APTI A SQPL SLRPEACRP AAGG A W 363 domain (NP 001139345.1) TRGLDFACDIYIW APL AGTCGVLLLSL VITL YC
NHRNRRR
CDSb Transmembrane LGLLVAGVLVLLVSLGVAIHLCC 115 domain
DAP10 Transmembrane ILLAGLVAADAVASLLIVGAVFLCARR 364 domain
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
FHNG SL SEETNS SLNIVN AKFED S GEYK CQHQ
QVNE-SEPVYLEVFSDWLLLOASAEWMEGQPL
IT.RCHGWRNWDWKVIYYKDGEALKYWYEN
HNISITNATVEDSGTYYCTGKVWQLDYESEPL
NITVIKAPREKYWLQFFIPLLVVILFAVDTGLFI
STQQQVTFLLKIKRTRKGFRLLNPHPKPNPK N
FcERI b- Transmembrane MDTESNRRA LALPQEPSSVPAFEVLEISPQEV 366 region S S GRLLKS AS SPPLHTWLT VLKKEQEFLGVTQI
LTAMICLCFGTWCSVLDISHIEGDIFSSFKAGY
PFWGAIFFSISGMLSIISERRNATYLVRGSLGAN
TASSIAGGTGITIL-IINLKKSLAYTHTHSCQKFFFT
KCFMASFSTEIV MLFLTILGLGSAVSLTICG
AGEELKGNKVPEDRVYEELN1YSATYSELEDP
GEMSPPIDL FcERI g- Transmembrane MIPAVVLLLLLLVEQAAALGEPQLCYILDAILF 367 region LYGPVLTLLYCRLKTQVRKAATTSYEKSDGVYT
GLSTRNQETYETLKHEKPPQ
FcERla Transmembrane DIFIPLLVVILFAVDTGLFISTQQQVTFLLKIKRT 118 domain RKGFRLLNPHPKPNPKNNR
GITR Transmembrane PLGWLTWLLAVAACVLLLTSAQLGLHIWQL 119 domain
Her?, Transmembrane SII S AV VG ILL VWLG V VFG ILI1 120 domain
Her2 Transmembrane CHPECQPQNGSVTCFGPEADQCVACAHYKDPP 121 domain FCVARCPSGVKPDLSYMPIWKFPDEEGACQPC
PIN CTHS C VDLDDKGCP AEQR A SPLT SII SAW GILLWVLGWFGILI
ICOS Transmembrane FWLPIGCAAFVWCILGCILI 122 domain
IgGlTransmembrane EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDT 123 domain L IARTPEVTCWVDVSHEDPEVKFNWYVDG
VEVHN AKTKPREEQYNSTYR WS VLTVLHQD
WLNG EYKCK V SNK ALP AP ΙΕΚΉ SK AK GQPR
EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGN SCSVMHEALHNHYTQKS
LSLSPGKKD
LAG-3 Transmembrane L L FLIL G VL SLLLL VTG AFGF 368 domain
OX40 Transmembrane VAAILGLGLVLGLLGPLAILL 124 domain
PD-1 Transmembrane VGWGGLLGSLVLLVWVLAVI 369 domain
Transmembrane domain FWALWVAGVLFCYGLLVTVALCVIWT 370 ] Hmge region sequences useful in the present invention are provided in Table 6a.
Table 6¾: Hinge regions
Figure imgf000042_0001
PA PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHT 143
CD8a Hinge
RGLDFACDIY
TTTPAPRPPTi-V\PTTASQPLSLRPFIACRPAAGGA\'I-ITRGLD 144
CD8a Hinge
FACDIYIWAPLAGTCGVLLLSLVITLYC
TTPAPRPPTPAPTIASQPLSL PEACRPAAGGAVHTRGLDF 145
CD8a Hinge
ACD
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD 146
CD8a Hinge FACDIY
DeitaS Hinge LDKTHTCPPCP 147
APVGLVARLADESGHWLRWLPPPETPMTSHIRYEVDVS 148
EpoR Hinge AGNGAGSVQRVEILEGRTECVLSNLRGRTRYTFAVRARM
AEP SF GGFWS A W SEP VSLLTP SD
FCRIIa Hinge GLAVSTISSFFPPGYQ 149
RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 153
QDLWLRDKATFTCFWGSDLKDAHLTWEVAGKVPTGGV
EEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPS
Hinge
LPPORLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCE
VSGFSPPNILLMWLEDQREV TSGFAPARPPPOPGSTTFW
AWSVL.RVPAPPSPOPATYTCWSHEDSRTLLNASRSLEVS
YVTDH
YVTVSSQDPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPK 152 PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYR SVLTVLHQDWLNGKEYKCKV
Hinge SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK QVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FL YSKLT VDK SRWQQGNVF S C S VMHE ALHNH YTQK SL SL SPGKKDPK
KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG 153
Hinge
LDFA
Hinge LEP S CDKTHTCPPCP 154
KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG 155
Hinge
LD
Hinge EPKS CDKTHTCPPCP 156
Hinge ELKTPLGDTHTCPRCP 157
Hinge E PKS CDTPPPCPRCP 158
Hinge ESKYGPPCPSCP 159
Hinge ERKCCVECPPCP 160
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT 161
CV^VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
Hinge (CHITTRWSVLTVLHQDWLNUKE^KCKVSNKGLPSSIEK^SK CKS) AKGQPREPOWTLPPSQEEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW
QEGN VF S C S VMHE ALHNHYTQK SL SL SL GK
ESKYGPPCPPCPGQPREPOVYTLPPSQEEMTKNQVSLTCL 162
Hinge (CH3) VKGFYP SDI A VEWESNGQPENNYKTTPP VLD SD GSFFL Y S
RLTVDKSRWOEGNVFSCSVMHEALHNHYTQKSLSLSLGK
RWPESPKAOASSVPTAQPQAEGSLAKATTAPATTRNTGR 163
GGEEK KEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV
QDLWLRDKATFTCFWGS DLKDAHL'l' E VAGKVPTGG V
EEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPS
IgD Hinge
LPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCE
VSGFSPPN1LLMWLEDQREVNTSGFAPARPPPQPGSTTFW
AWSVLRVPAPPSPQPATYTCWSHEDSRTLLNASRSLEVS
YVTDH
RWPESPKAOASSVPTAQPQAEGSLAKATTAPATTRNTGR 164
IgD Hinge
GGEEK KEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV QDLWLRDKATFTCFWGSDLKDAHLTWEVAGKVPTGGV EEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLHPSL PPQRLMALREP AAQAP VKL SLNLLAS SDPPE AAS WLL CEV S GF SPPNILLMWLEDQRE VNTS GF AP ARPPPQP G STTFW A WSVLRVPAPPSPQPATYTCWSHEDSRTLLNASRSLEVSY VTDH
RWPESPKAQASSVPTAOPOAEGSLAKATTAPATTRNTGR 165 GGEEKK EKEKEEQEERETKTPECPSHTQPLG LLTPAV QDLWLRDKATFTCFWGSDLKDAHLTWEVAGKVPTGGV EEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPS
IgD Hinge
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
ESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEE 166 lgD Hinge
KKKEKEKEEQEERETKTP
RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 167
GGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV
QDLWLRDKATFTCFWGSDLKDAHLTWEVAGKVPTGGV
EEGLLERHSNGSQSQHSRLTLPRSLWNA GTS VTCTLNHPS
IgD Hinge
LPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCE
VSGFSPPNILL WLEDQREVNTSGFAPARPPPQPGSTTFW
AWSVLRVPAPPSPQPATYTCWSHEDSRTLLNASRSLEVS
YVTDH
RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 168 GGEEKKKEKEKEEQEERETKTPECPSHTQPLGWLLTPAV QDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGV EEGLLERHS GSQSQHSRLTLPRSLWNAGTS VTCTLNHPS
IgD Hinge
LPPQRLMALREP AAQAP VKLSLNLLASSDPPEAASWLLCE VSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFW AWSVLRVPAPPSPQPATYTCWSHEDSRTLLNASRSLEVS YVTDH
RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 169 GGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV QDLWLRDKATFTCFWGSDLKDAHLTWEVAGKVPTGGV EEGLLERHSNGSQSQHSRLTLPRSLWNAGTS VTCTLNHPS
IgD Hinge
LPPQRLMALREP AAQAP VKLSLNLLASSDPPEAASWLLCE VSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFW AWSVLRVPAPPSPQPATYTCWSHEDSRTLLNASRSLEVS YVTDH
RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 170 GGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV QDLWLRDKATFTCFWGSDLKDAHLTWEVAGKVPTGGV EEGLLERHSNGSQSQHSRLTLPRSLWNAGTS VTCTLNHPS
IgD Hinge
LPPQRLMALREP AAQAP VKLSLNLLASSDPPEAASWLLCE VSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFW AWSVLRVPAPPSPQPATYTCWSHEDSRTLLNASRSLEVS YVTDH
AEPKSPDKTHTCPPCPAPPVAGPSWLFPPKPKDTLMIART 171
PEWCV ^VSHEDPEVKFNWYVDGW-VHNAKTKPREE
IgG i (CH2CH3) QYNSTYRWSWTVLHQDWLNGKEYKCKVSNKALPAPIE Hinge domain KTISKAKGQPREPQ YTLPPSRDELTKNQVSLTCLVKGFY
P SDI A VEWESNGQPENNYKTTPP VLD SD G SFFL Y SKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD
AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIART 172 PE VTC VD VSHEDPE VKF N WYVD G VE VHN A KTKPREE
IgG l (CH2CH3) QYNSTTR SVLTVLHQDWLNGKEYKCKVSNKALPAPIE Hinge domain KTiSKAKGQPREPQWTLPPSRDELTKNQVSLTCLVXGFY
P SDI A VEWESNGQPENNYKTTPP VLD SD G SFFL Y SKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD IgGi Hinge AEPKSPDKTHTCPPCPKDPK 173
EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTP 175
EWCVVWVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRWSVLTVLHQDWLNGKEVKCKVSNKALPAPIEK
IgGl Hinge
TISKAKGOPREPQVYTLPPSRDELTKNOVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKS
RWQQGN SCSVMHEALHNHYTQKSLSLSPGKKD
IgGl Hinge SVFLFPPKPKDTL 176
EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTP 177
EWCVVVDVSTTF.DPEVKFNWYVDGVEVHNAKTK-PREEO
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK
IgG l Hinge
T1SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
DIAVEWESNGQPEN YKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGN SCSVMHEALHNHYTQKSLSLSPGK
EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTP 178
EWCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK
IgG l Hinge
T1SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNWSCSVMHEALHNHYTQKSLSLSPGKKDPK
VECPPCPAPPVAGPSVFL-FPPKPKDTLMSRTPEVTCVVVD 179 VSHEOPEYKFNWYVDGVEVH AKTKPREEQYNSTYRVV SVLTVLHQDWLNGKE YKCKVSNKGLP S SIEKTISK AKGQP
IgG l Hinge
REPQWTLPPSREEMTKNQVSLTCLVT GFYPSDIAVEWES NGQPEN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK
DPAEPK SPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIA 180
RTPEVTCV VDVSHtDPEVKFNWYVDGVEVHNAK^ PR
IgGl Hinge
EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPA
(CH2CH3
PIEKTISKAKGQPREPQ TLPPSRDELTKNQVSLTCLVKG
domain)
FYPSDIAVEWESNGQPEN YKTTPPVLDSDGSFFLYSKLT
VOKSRWQQGNWSCS HEALHNHYTQKSLSLSPGKK
IgG3 Hinge ELKTPLGDTTHTCPRCP 182
ELKTPLGDTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPC 183
IgG3 Hinge
PRCPEPKSCDTPPPCPRCP
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT 184 C VVVD VSQEDPEVQFN WYVDGVE VHN AKTKPREEQF S
lgG4 (CH2 and TYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSS!EKTISK
CH3) AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYK l i PVLDSDGSFFLYSRLTVDKSRW QEGN VF S C S VMHE ALH H YTQK SL SL SLGKM
ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVT 185
CVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQS
IgG4 (CH2 and TYRWSVLTVLHODWLNGKEYKCKVSNKGLPSSlEK'llSK CHS) AKGQPREPQVYTLPPSQEEMTKNQVSLTCL GFYPSDIA
VEWESNGQPEN Y'KTTPPVLDSDGSFFLYSRLTVDKSRW
QEGNVFSCSVMHE ALHNH YTQKSLSLSLGKM
lgG4 Hinge SPNMVPHAHHAQ 186
GQPREPQVYTLPPSQEEMTKNOVSLTCLVKGFYPSDIAVE 187
IgG4 Hinge WESNGQPENNYKTIPPVLDSDGSFFLYSRLTVDKSRWQE
GNVF S C S VMHE ALHNHYTQKSL SL SL GK
ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEM 188 TXNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
IgG4 Hinge
DSD GSFFL YSRLT VDK SR WQEGNVF S C S VMHE ALHNHYT QKSLSLSLGK
ESKYGPPCPSCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVT 189 CVWDVSQEDPEVQFNWYVDGVEVHQAK^ PREEQFNS
IgG4 Hinge
TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFVPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW
QEGN VF S C S VMHE ALH HYTQK SLSLSLGK
ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVT 190 CVVVDVSQEDPEVOFNWYVDGVEWQAKTKPREEQFNS
TYRWS^TVLHQDWL GKEYKCKVSNKGLPSSIEKTISK
lgG4 Hinge
AK GQPREPQ TLPP SQEEMTKNQ VSLTCL VK GF VP SDI A VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT\T>KSRW
QEGN VF S C S\¾iHE ALHNHYTQK SL SL SLGK
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT 191
CVVVDVSQEDPEVQFNWYVDGVEVHNAK^ PREEQFNS
TYRWS^T\^HQDWLNGKEYKCKVSMCGLPSSIEKTISK
IgG4 Hinge
AKGQPREPQVYTLPPSQEEMTK QVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW
QEGNVFSCSVMHEALHNHYTQKSLSLSLGKM
IgG4 Hinge GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT 192
IgG4 Hinge ESKYGPPCPPCP 193
ESKYGPPCPPCPGQPREPQWTLPPSQEEMTKNQVSLTCL 194
IgG4 Hinge VK GF YP SDI A VEWESNGQPENN YKTTPP VTD SD GSFFL Y S
RLTVDK SR WQEGNVF S C S VMHE ALHNHYTQK SL SL SLGK
ESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVT 195 C VVVD VSQEDPEVQFN WYVDGVE VHN AKTKPREEQF S TYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK
IgG4 Hinge
AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGN VF S C S VMHE ALHNHYTQK SL SL SLGK
IgG4 Hinge YGPPCPPCP 198
IgG4 Hinge KYGPPCPPCP 199
IgG4 Hinge EWKYGPPCPPCP 200
ESKYGPPCPSCPAPEFL-GGPSVFLFPPKPKDTLMISRTPEVT 201
CV^\T)VSQEDPEVQFN VDG WNAKTKPREEQFNS
TYRWS^TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK
lgG4 Hinge
AKGQPREPQWTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDLSRW QEGN VF S C S VMHE ALHNHYTQK SLSLSLGK
IgG4 Hinge and 202
ESKYGPPCPPCPGGGSSGGGSG
Linker
EPKSPDKTHTCPPCPAPPVAGPSWLFPPKPKDTLMLARTP 203
EWCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAP1EK
igGl Hinge
TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
EPKSPDKTHTCPPCPAPPVAGPSWLFPPKPKDTLMLARTP 204
EWCW\T)V^HEDPEVKF>WYVDGVEVHNAKTKPREEQ
YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK
lgGl Hinge
TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[00131] Hinge and transmembrane region sequences useful in the present invention are provided in Table 6b, Table 6b: Hinge and Transmembrane regions
Figure imgf000047_0001
[00132] In some embodiments, the payloads of the present invention may comprise one or more linkers. The linker may be between 1-30 amnio acids long. In this regard, 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. In other embodiments, the linker may be flexible.
ΘΘ133] 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).
[00134] 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. The IL15/IL15Ra complex is more effective in activating 11. 15 signaling, than IL15 by itself. In some embodiments, tlie pavload of the invention may be a whole or a portion of IL15 fused to the whole or a portion of the IL15Ra. Any portion of 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. In one embodiment, 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. Thus, in some embodiments, pavload may include ILlSRa sushi domain. As a non-limiting example, 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). In one embodiment, 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.
Table 7: ILlSRa variants
Description Sequence Amino acid SEQ ID
ILlSRa Isoforni 1 (NCBI MAPRRA GCRTLGLPALLLLLLLRPPATRGITCPPPMSVEH 211 Reference No. NP 002180.1) ADIWVKSYSLYSRERYICNSGF RKAGTSSLTECVLNKAT
NVAHWTTPSLKCIRDPALVHORPAPPSTVTTAGVTPQPES
L SPSGKEP A AS SP S SNNTA ATT AAIVPG SQLMP SKSPSTGT
TEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHS
DT VAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEA
MEAL P VT WGTS SRDEDLENC SHHL
ILl SRa Isoform 2 (NCBI MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEH 212 reference No. NP_751950.2) ADiWVKSYSLYSRERYiCNSGFKRKAGTSSLTECVLNKAT
NVAHWTTPSLKCIKPAASSPSSNNTAATTAAIVPGSQLMP
SKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPG
VYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPL
ASVEMEAMEALPVTWGTSSRDEDLENCSHHL
IL 15Ra Isofonn 3 (NCBI MSVEHADrWVKSYSLYSRERYTCNSGFKRKAGTSSLTECV 213 Reference No. LNKAT VAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVT
NP OO 1230468.1) PQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSP
STGTTEISSHESSHGTPSQTTAKNWELTASASHOPPGVYPQ GHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVE MEAMEALPVTWGTSSRDEDLENCSHHL
ILlSRa Isoform 4 (NCBI MRLAGRQVPEQRSPPPPGLGSARPGSPAVSCGAAAMAPR 214 Reference No. RARGCRTLGLPALLLLLLLRPPATRDARDRLAVLAGRSRI
NP 001243694.1) SESFNHEVQTHEACWLRTNiENCPQCHHHRTSROQAGIT
CPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSL
TECYLNKATNVAHWTTPSLKC!RDPALVHQRPAPPSTVTT
A G VTPQPE SL S P S GKEP A AS SP S SNNT A ATT AAIVPG SQLM
PSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPP
GVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPP
L AS VEME AME ALP VTW GT S S RDED LEN C S HHL
IL l SRa sushi domain CPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSL 215
TECVLNKATNVAHWTTPSLKC
IL 1 SRa (without signal rrCPPPMS\¾HADIWVKSYSLYSRERYICNSGFKRKAGTSS 216 sequence) LTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTW
TAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQL MPSK SP STGTTEIS SHES SHGTPSQTTAK WELTAS A SHQP PGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTP
PLASVEMEAMEALPVTWGTSSRDEDLENCSHHL
IL15Ra (31-205 of Uniprot rTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSS 217 ID: Q1326 I .1) LTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVT
T AG VTPQPE SL SP S GKEP A AS SP S SN T AATT A AI VP GSQL MPSKSPSTGTTE-ISSHESSHGTPSQTTAKNWELTASASHQP
PGVYPQGHSDTT
iLiSRa (31-95 of Q13261.1) ITCPPP SWHADIWV S Y SLY SRERYIC SGFKRKAGTS S 218
LTECVLNI AT VAHWTTPSLKCIR
[00135] IL15/IL15Ra fusion proteins of the present invention may be linked to all or a functional portion of one or more transmembrane proteins. In some embodiments, 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.
[ΘΘ136] 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. In some instances, IL15 or IL15/IL15Ra fusion proteins may be modified to prevent shedding. For example, 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). J Biol Chem;283(7):4189-99; Anthony SM et al. (2015). PLoS One. 10(3): eOl 20274), and International Patent Application Publication Nos. WO2014066527 and WO2009002562 (the contents of each of which are incorporated herein by reference in their entirety.) Tumor necrosis factor-alpha-converting enzyme
(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. Hence, 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.
[00137] 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). foot and mouth disease virus 2A (F2A), cytoplasmic polyhedrosis virus (BmCPV 2A), and ilacherie virus (BmlFV 2A) oi'B. mors, as described by Kim JH et al. (2011). PLoS
One;6(4):e 18556 and Wang Y et al. (2015). Sci Rep;5: 16273. As a non-limiting example, the single peptide may be composed of the following components: [CAR] - [P2A cleavage site] - [IL15Ra/IL15-DD].
[00138] 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,
WO2016123143 and WO2017000913 (the contents of each of which are incorporated herein by- reference in their entirety.) The examples of 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.
[00139] In some aspects, 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. (201 1) Piasmid:65(3):239-45, and Weinstein-Marom H et al. (2016) J Immunother;39(2):60-70; U.S. Patent NOs, 7/435,596; 8,026,097; 8,399,645;
9,605,049; and 9,623,082; US patent publication NOs, US20140328812 and US20160009784; and International patent publication NOs. WO2015174928 and WO2014005072; the contents of each of which are incorporated herein by reference in their entirety. In some embodiments, 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. In some embodiments, DD-IL15 is fused to HLA-A2 transmembrane and cytoplasmic domains via a Gly-Ser linker (e.g.,
G3y4Ser(Gly3Ser)2 (SEQ ID NO. 534); and 8 aa bridge from the HLA-A2 membrane-proximal part, as described by Weinstein-Marom H et al. (2016) J Immunother;39(2): 60-70, the contents of which are incorporated herein by reference in their entirety. The DD-1L15 fusion molecule may further comprise human MHC-I light chain p2-microglobulin leader peptide. In some embodiments, 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
Immiinother;39(2): 60-70, the contents of which are incorporated herein by reference in their entirety. The DD-IL15 fusion molecule may further comprise human MHC-I light chain β2~ microglobulin leader peptide.
[00140] Under inflammatory conditions, such as infections and autoimmune diseases, expression of the immunoproteasome is induced by interferon-gamma and tumor necrosis factor- alpha as described by Ferrington DA and Gregerson DS (2012) Prog Mol Biol Transl
Sci; 109:75-112 and Kimura H et al. (2015) J Immunol Res;2015:541984. The
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. In some embodiments, 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. Conversely, in some embodiments, 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,
[00141] 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. In one embodiment, DD-IL 15/IL 15Ra may be utilized to enhance CD 19 directed T cell therapies in B cell leukemia and lymphomas. In one aspect, IL15 IL15Ra may be used as payload of the invention to reduce the need for pre-conditioning regimens in current CAR-T treatment paradigms.
[00142] 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).
[00143] In some aspects, 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.
Table 8a: IL15/IL15Ra construct regions
Figure imgf000052_0001
IL15 NWV VISDLKKIEDLIQSMHIDATLYTESDVHPSCKVT 55 69-71 (each AMKCFLLELQVISLES GD A SfflDTVE NLIIL ANNSL S SN include the slop G N VTESGCKECEELEEKNIKE FLO SFVHI VQMFI TS * codors at the end), 72, 378- 379
ILlSRa 1TCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAG 216 231 (includes
T S SLTECVL NK AT VAH W 1 ' 1 SLKCIRDP ALVHQRPAP the stop codon
PSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAA at the end), 232,
I GSQLMPSKSPSTGT EISSHESSHGTPSQTTA NWE 380-382
LTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSL
LACYLKSRQTPPLASVE EAMEALPVTWGTSSRDEDL
ENCSHHL*
IL15Ra (31- ITCPPPMSVEHADiWVKSYSLYSRERYIC SGFKRKAG 217 465
205 of TSSLTECVLNKAT VAHWTTPSLKCIRDP ALVHQRPAP
Uniprot ID: P ST VTT A G VTPQPE SL S P S GKEP A AS S P S S T A ATT A A
Q13261.1) lVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWE
LTASASHQPPGVYPQGHSDTT
mCheny MSKGEEDNMAIIKEFMRFK\7HMEGS GHEFEIEGEG 383 384
EGRPYEGTQTAKLKWKGGPLPFAWDILSPQFMYGSK AYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTV TQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWE
ASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTY KAKKPVQLPGAYN rNIKLDiTSHiv!EDYTIVEQYERAEG
RHSTGGMDELYK*
mCherry LSKGEEDNMAIIKEFMRFK\7HMEGSV GHEFE-IEGEGE 352 353
(MIL) ' GRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKA
Y T HPADIPDYLKLSFPEGFKWERVMNFEDGGV\
QDSSLQDGEFIYKVKLRGTOFPSDGPVMQKKTN1GWEA
SSERMYPEDGALKGEIKQRLKLKDGGHYDAE-VKTTYK
AKKPVQLPGAYNVNIKLDITSH EDYTIVEQYERAEGR
HSTGGMDELYK
HA Tag YPYDVPDYA 345 346-347
Flag DYKDDDDK 387 388
BamHI GS - GGATCC
P2A GATNFSLLKQAGDVEENPGP 350 351
Cleavable
Peptide
ecDHFR ISLIAALA\TJYVIGMENA IPWNLPADLAWFKRNTLNK 9 73, 372, 233 (Amino acid PVT IGRHTWESIGRPLPGRKNIILSSQPGTDDRV VKS (includes stop 2-159 of VDEAIAACGDVPEIMVIGGGRVIEQFLPKAQKLYLTHK) codon) WT; R12Y, AEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCF
Y100I) EILERR*
ecDHFR ISLIAALAVDHVIGMENAMPWNLPADLAWFKRNTLNK 10 234 (includes
(Amino acid PVTMGRHTWESIGRPLPGRKNIILSSQPGTODRVTWVKS stop codon), 2-159 of VDEAIAACGDVPEIMVTGGGRVYEQFLPKAQ LYLTH! 395
WT; R12H, DAEVEGDTHFPDYKPDDWESVFSEFHDADAQNSHSYC
E129K) FEILERR*
FKBP GVQVETISPGDGRTFPKRGQTCWHYTGMLGDGKKVD 12 235-238 (each
(E31G, SSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQGAK includes stop
F36V, LTISPDYAYGATGHPGTIPPHATLVFDVELLELE* codors)
R71G,
K105E)
hDHFR VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRM 336 239, 397, 396 (Amino acid TTTSSVEGKQNLVIMGKKTWFSiPEKNRPLKGRINLVLS (includes stop 2-187 of RELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW codon) WT; Y122L IVGGSSVj- E 'MNHPGHi ,KT .FVTRIMQDFESDTFFPE1D
A 125F) LEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND*
hDHFR VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFFRM 337 240, 399, 398
(Amino acid TTTSS GKQNLVEvlGKKTWFSIPEKFRPLKGRINLVTS (includes stop 2-187 of RELKEPPQGAHFLSRSLDDALKLTEQPELA KVDMVW codon) WT; Q36F, I VG GSSVIKEAM HPGHLKLFVTR IMQDFE SDTFFPE ID
N65F, LEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND*
Υ122Γ)
hDHFR VGSLNCIVAVSQNMGIGK GDLPWPPLRNEFRYFQRM 335 400
(Amino acid TTTSSVEGKQNLV1¾1GKKTWFSIPE NRPLKGR1NLVLS
2-187 of RELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW
WT; IVGGSSVYKEAMNHPGHL LFVTRI 1QDFESDTFFPEID
185E) L EK YKL L PEYPG VL S D VQEEKGIK YKFE V YEEND *
hDHFR VGSLNCIVAVSQ MGIGKNGDLPWPPLRNEFRYFQRM 334 401 (includes
(Amino acid TT SSVEGKQNLVIMGKKT'WFSIPEKNRPLKGRINLVLS stop codon), 2-187 of RELKEPPQGAHFLSRSLDDALKLTEQPELA KVDMVW 402
WT; E162G, IVGGSSVY EAM HPGHLKLFWRIMODFESDTFFPEID
I176F) LEKYKLLPGYPGVLSDVQEEKGFKYKFEVYEKND*
hDHFR VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRWQRM 338 406
(Amino acid TT SS\^GKONLmiGKKTWFSIPEKNRPLKGRINLVLS
2-187 of RELKEPPOGAHFLSRSLDDAL LTEOPELA KWMW
WT; IVGGSS^TiEAAfYHPGHLKLF'V'TRrMQDFESDTFFPEID
N127Y) LEKYKLLPEYPGVLSDVQEEKGIKYKFEVYE-KND
hDHFR VGSLNCIVAVSQNMGVGKNGDLPWPPLRNEFRYFQR 340 442
(Amino acid M i i i SS\i ; ikn\! \ i X ! i k'! \\ ! Sl PHK XU l .kijRi N l A'
2-187 of L SRELKEPPQG A HFL SR S LDD ALK LTEQPEL ANK VDMV
WT; 117 V) WlVGGSSWKEAMNHPGHLKLFVTTllMQDFESDTFFPE
IDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
hDHFR VGSLNCIVAVSQN GVGK GDLPWPPLRNEFRYFQR 328 447
(Amino acid MTTTSSVEGKONLVIMG KTWFSIPEKNRPLKGRINLV
2-187 of LSRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMV
WT; 117 V, WWGGSSVKEAMNHPGHLKLFVTRIMQDh fcSDTFFPEi
Y122I) DLEKYKLLPEYPGVLSDVQEEKGIKYKFEWEKND
hDHFR VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRM 339 450
(Amino acid TTTSSVEGKQNLVIMGKKTWFSiPEKNRPLKGRINLVLS
2-187 of RELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW
WT; IVGGSSVY EAMNHPGRLKLF\TTRIMQDFGSDTFFPEI
H131R, DLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
E144G)
Table 8b: IL15/IL15Ra constructs
Description Promoter Amino acid sequences Amino Nucleic Acid
Acid SEQ ID NO/ SEQ ID Sequence
NO
OT-IL15- EFla MD WTWILFL V AAATR VH SNWVN VI SDLKKIE 223 241
006 (IgE DLIQSMHIDATLYTESDVHPSCKVTAMKCFLL
signal ELOVISLESGDASIHDTVENLIILANNSLSSNG
sequence; NVTESGCKECEELEEK IKEFLOSFVHIVOMFI
1L15; NTSSGGGSGGGGSGGGGSGGGGSGGGSLQIT
Unkerl CPPPMS VEHAD! WVKSYSLYSRE YICN SGFK
(SG3- R AGTSSLTECVLNKATNVAHWTTPSLKCIR
(SG4)5- DPALVHQRPAPPSTVTTAGVTPQPESLSPSGK
SG3S); EPAASSPSSNNTAATTAAIVPGSQLMPSKSPST
IL15Ra; GTTEISSHESSHGTPSQTTAKNWELTASASHO
linker2 PPGVYPQGHSDTTVAISTSTVLLCGLSAVSLL
(GGSGG); ACYLKSRQTPPLASVEMEAMEALPVTWGTSS
ecDHFR RDEDLENC SHHL SGIS L I A AL AVDH VIGMENA
(R12H, MPWNLPADLAWFKRNTLNKPVIMGRHTWES
E 129K)) IGRPLPGRKNIIL S SQPGTDDR VTW VK S VDEAI
AACGDVPEIMVIGGGRVYEQFLPKAQKLYL-T
HIDAEVEGDTHFPDYXPDDWESVFSEFHDAD
AQNSHSYCFEILERR* OT-IL15- EF la MDWTWILFLVAAATRVHSNWVNVISDLKKIE 224 242
007 (IgE DLIQSMHIDATLYTESDVHPSCKVTAMKCFLL
signal ELQVISLESGDASIHDTVENLilLANNSLSSNG
sequence; NVTESGCKECEEL-EEKNIKEFLOSFYHIVQMFI
IL15; NTSSGGGSGGGGSGGGGSGGGGSGGGSLQIT
linkerl CPPPMSVEHADIWVKSYSLYSRERYICNSGFK
(SG3- RKAGTSSLTECVLNKATNVAHWTTPSLKCIR
(SG4)5- DP AL VHQRP APP STVTT A G VTPQPESL SP S GK
SG3S); EP A A S SP S SNNT A ATT A Ai VPG SQLMP SK SP ST
ILlSRa; GTTEISSHESSHGTPSQTTAKNWELTASASHQ
linker! PPGVYPQGHSDTTVAISTSTVLLCGLSAVSLL
(GGSGG); ACYLKSRQTPPLASVEMEA EALPVTWGTSS
FKBP RDEDLENCSHHLSGGVOVETISPGDGRTFPKR
(E31G,F36V GQTCWHYTGMLGDGKKVDSSRDRNKPFKF
, R71G, MLGKQEVIRGWEEGVAQMSVGOGAKLTISPD
K105E)) YAYGATGHPGIIPPHATLVFDVELLELE*
OT-IL15- EF Ia MDWT .FLVAAATRVHSNWVNVISDLKKIE 225 243
008 (IgE DLIQSMHIDATLYTESD PSCKVTAMKCFLL
signal ELQVISLESGDASIHDTW LIILAN SLSSNG
sequence- NVTESGCKECEELEEK IKEFLOSFVHIVOMFI
IL15- linker NTSSGGGSGGGGSGGGGSGGGGSGGGSLQIT
(SG3- CPPPMSVEHADIWVKSYSLYSRERYICNSGFK
(SG4)3- RKAGTSSLTECVLNKATNVAHWTTPSLKCIR
SG3-SLQ)- DP AL VHQRP APPSTVTTAGVTPQPESLSPSGK
IL15Ra- EPAASSPSSNNTAATTAAIVPGSQLMPSKSPST
stop) GTTEISSHESSHGTPSQTTAKNWELTASASHQ
PPGVYPQGHSDTTVAISTSTVLLCGLSAVSLL
ACYLKSRQTPPLASVEMEAMEALPVTWGTSS
RDEDLENCSHHL*
OT-IL 15- EFla MD WTWTLFL V A AATR VH SNWVN VI SDLKK IE 226 244 009 (IgE DLIQSMHIDATLYTESDVHPSCKVTAMKCFLL
signal ELQVISLESGDASIHDTVENLIILANNSLSSNG
sequence- NVTESGCKECEELEEK IKEFLQSFVHIVQMFI
IL15- linker NTSSGGGSGGGGSGGGGSGGGGSGGGSLQIT
(SG3- CPPPMSVEHADIWVKSYSLYSRERYICNSGFK
(SG4)3- RKAGTSSLTECVLNKATNVAHWTTPSLKCIR
SG3-SLQ)- DP AL VHQRP APPSTVTTAGVTPQPESLSPSGK
IL15Ra- EPAASSPSSNNTAATTAAIVPGSQLMPSKSPST
iinker (SG)- GTTEISSHESSHGTPSQTTAKNWELTASASHQ
ecDHFR PPGVYPQGHSDTTVAISTSTVLLCGLSAVSLL
(Amino acid ACYLKSRQTPPLASVEMEAMEALPVTWGTSS
2-159 of RDEDLENCSHHLSGISLIAALAVDYV!GMENA
WT; R12Y, MPWNLPADLAWKRNTLNKPVIMGRHTWES
Y100I)- IGRPLPGRKNIIL S SQPGTDDR VTW VKS VDEA I
stop) AACGDVPEI VIGGGRVIEQFLPKAQKLYLTH
ID AE VE GDTHFPD YEPDD WE S VF SEFHD AD A
QNSHSYCFEILERR*
OT-IL15- EFla MDWTWILFLVAAATRVHSNWVNVISDLKKIE 227 245
010 (IgE DLIQSMHIDATLYTESDVHPSCKVTAMKCFLL
signal ELQVISLESGDASIHDTVENLIILANNSLSSNG
sequence- NVTESGCKECEELEEKNIKEFLQSFVHIVQMFI
IL15- linker NTSSGGGSGGGGSGGGGSGGGGSGGGSLQIT
(SG3- CPPPMSVEHADIWVKSYSLYSRERYICNSGFK
(SG4)3- RKAGTSSLTCCVLM ATNVAHWTTPSLKCIR
SG3-SLQ)- DP AL VHQRP APPSTVTTAGVTPQPESLSPSGK
IL15Ra- EPAASSPSS NTAATTAAIVPGSQLMPSKSPST
linker (SG)- GTTEISSHESSHGTPSQTTAKNWELTASASHQ
hDHFR PPGVYPQGHSDTTVAISTSTVLL(:GLSAVSLL
(Y122I, ACYLKSRQTPPLASVEMEA EALPVTWGTSS
RDEDLENCSHHLSGVGSLNCIVAVSQNMGIG A125F)- KNGDLPWPPLRNEFRYFQRMTTTSSVEGKQN
stop) LVTMGKKTWFS1PEKNRPLKGRINLVLSRELK
EPPOGAHFLSRSLDDALKLTEQPELANKVDM
VWIVGGSSVIKEFMNHPGHLKLF^TRIMQDFE
SDTFFPEIDLEKYKLLPEYPGVLSDVOEEKGIK
YKFE VYEKND *
O -IL15- EFla MDWTW!LFLVAAATRVHSNWVNVISDLKKIE 228 246
0i l (lgE DLIQSMHIDATLYTESDVHPSCKVTAMKCFLL
signal ELQVISLESGDASIHDTVENLTILANNSLSSNG
sequence- NVTESGCKECEEL.FFKNTKEFLQSFVHIVQMFI
IL15- linker NTSSGGGSGGGGSGGGGSGGGGSGGGSLQIT
(SG3- CPPPMSVEHADIWVKSYSLYSRERYICNSGFK
(SG4)3- RKAGTSSLTECVLNKATNVAHWTTPSLKCIR
SG3-SLQ)- DP AL VHQRP APP STVTT A G VTPQPESL SP S GK
IL15Ra; EP A A S SP S SNNT A ATT A AT VPG SQLMP SK SP ST
linker (SG)- GTTE-ISSHESSHGTPSQTTAKNWELTASASHQ
hDHFR PPGVYPQGHSDTTVAISTSTVLLCGLSAVSLL
(Amino acid ACYLKSROTPPLASVEMEAMEAL.PVTWGTSS
2-187 of RDEDLENCSHHLSGVGSLNCrVAVSQN GIG
WT; Q36F, KNGDLPWPPLR EFRYFFRMTTTS S VEGKQN
N65F, LVTMGKKTWFSIPEKFRPLKGRrNLVLSRELKE
Y122I)- PPOGAHFLSRSLDDALKLTEQPELA KVDMV
stop) WIVGGSS\TKEAMNHPGHLKLFVTRIMQDFES
DTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKY
KFE VYEKND*
OT-IL15- EFla ¾iD WTWILFL V A AATR VH SN VN VI SDLKK IE 404 405
017 (IgE DLIOSMHIDATLYTESDVHPSCKVTAMKCFLL
signal ELQVISLESGDA SIHDTVENLIILANNSLS SNG
sequence- NVTESGCKECEELEEKNKEFLQSFVHIVOMFI
IL 15- linker NTSSGGGSGGGGSGGGGSGGGGSGGGSLQIT
(SG3- CPPPMSVEHADIWVKSYSLYSRERYICNSGFK
(SG4)3- RKAGTSSLTECVLNKATNVAHWTTPSLKCIR
SG3-SLQ)- DP AL VHQRP APP STVTT AG VTPQPE SL SP S GK
IL15Ra- EPAASSPSSNNTAATTAAIVPGSQLMPSKSPST
linker (SG)- GTTEISSHESSHGTPSQTTAKNWELTASASHQ
hDHFR PPGVYPQGHSDTTVAISTSTVLLCGLSAVSLL
(Amino acid ACYLKSRQTPPLASVEMEAMEALPVTWGTSS
2-187 of RDEDLENCSHHLSGVGSLNCrVAVSQNMGIG
WT; KNGDLPWPPLRNEFRYFQRMTTTSSVEGKQN
K185E)- LVIMGKKTWFSIPEKNRPLKGRINLVLSRELK
stop) EPPQGAHFLSRSLDDALKLTEQPELANKVDM
VWrVGGSSVYKEAMNHPGHLKLFVTRIMQDF
ESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGI
KYKFEVYEEND*
OT-IL15- EFla MD WTWILFL V AAATR VH SNWVN VI SDLKKIE 504 505
018 (IgE DLIQSMHIDATLYTESDVHPSCKVTAMKCFLL
signal ELQVISLESGDASIHDTVENLIILAN SLSSNG
sequence-
1L15- linker NTSSGGGSGGGGSGGGGSGGGGSGGGSLQIT
(SG3- CPPPMS VEHAD! WVKSYSLYSRERYICN SGFK
(SG4)3- RKAGTSSLTECVLNKATNVAHWTTPSLKCIR
SG3-SLQ)- DP AL VHQRP APPSTVTTAGVTPQPESLSPSGK
lL15Ra- EPAASSPSSNNTAATTAAIVPGSQLMPSKSPST
linker (SG)- GTTEISSHESSHGTPSQTTAKNWELTASASHQ
hDHFR PPGVYPQGHSDTTVAISTSTVLLCGLSAVSLL
(Amino acid ACYLKSRQTPPLASVEMEAMEALPVTWGTSS
2-187 of RDEDLENCSHHLSGVGSLNCTVAVSQNMGIG
WT; E162G, KNGDLPWPPLRNEFRYFQRMTTTSSVEGKQ
I176F)-stop) LVTMGKKTWFSIPEKNRPLKGRINLVT.SRELK
EPPQGAHFLSRSLDDALKLTEQPELANKVDM \ Yi VGi iSSYYki WlM ii' ! ! ! kl .l \TRl M<.)i >!
ESDTFFPEE)LE YKLLPGYPGVLSDVQEEKG
FKYKFEVYEKND*
OT-IL 15- EFla MDWTWILFLVAAATRVHSNWVNVISDLKKIE 407 408
038 (IgE DLIQSMHIDATLYTESDVHPSC VTAMKCFLL
leader - ELQVISLESGDA SlilDTVE LIILANN SLSSNG
IL15 - NVTESGCKECEELEEK IKEFLQSFVHIVQMFI
Linker NTSSGGGSGGGGSGGGGSGGGGSGGGSLQ!T
(SG3- CPPPMS VEHAD! WVKSYSLYSRERYICN SGFK
(SG4)3- RKAGTSSLTECVLNKATNVAHWTTPSLKCIR
SG3-SLQ) - DPALWQRPAPPSTVTTAGVTPQPESLSPSGK
IL1 5Ra - EPAASSPSSNNTAATTAAIVPGSQLMPSKSPST
Linker (SG) GTTEISSHESSHGTPSQTTAK WELTASASHQ
- hDI-IFR PPGVYPQGHSDTTVAISTSTVLLCGLSAVSLL
(Amino acid AC YLKSRQTPPL A S VEMEAME ALP VTWGTS S
2-187 of RDEDLENCSHHLSGVGSLNCIVAVSONMGIG
WT; KNGDLP WPPLRNEFR YFQRMTTT S SVE GKQN
N127Y)- LVmiGKKTWFSIPEK RPLKGRINLVLSRELK
stop) EPPQGAHFLSRSLDDALKLTEQPELANKVDM
VWrVGGSSVYKEAMYHPGHLKLFVTRIMQDF
ESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGI
KY FEVYEKND*
OT-1L1.5- EFla MD WTWILFL V A A ATR VH SNW VN VI SDLKKLE 409 410
051 (IgE DLIQSMHIDATLYTESDVHPSCKVTAMKCFLL
leader - ELQVISLESGDASIHDTWNLIILAN SLSSNG
IL15 - NVTESGCKECEELEEKNIKEFLOSFYHIVQMFI
Linker NTSSGGGSGGGGSGGGGSGGGGSGGGSLQYP
(SG3- YDVPDYAITCPPPMS\¾HADIWVKSYSLYSRE
(SG4)3~ RYICNSGFKRKAGTSSLTECVL KATNVAHW
SG3-SLQ) - TTPSLKCIRDPALVHQRPAPPSTVTTAGVTPOP
HA Tag - ESLSPSGKEPAASSPSSNNTAATTAAIVPGSQL
IL15Ra - MPSKSPSTGTTEISSHESSHGTPSQTTAK WEL
stop) TASASHQPPGVYPQGHSDTTVAISTSTVLLCG
LSAVSLLACYLKSRQTPPLASVEMEAMEALP
VTWGTS SRDEDLENC SHHL *
OT-IL15- EFla MD WTWILFL VAA ATR VHSNWVNVISDLKKIE 413 414
053 (IgE DLIOSMHIDATLYTESDVUPSCKVTAMKCFLL
leader - ELQVISLESGDA SIHDTVENLIILANNSLS SNG
IL15 - NVTESGCKECEELEEKNKEFLQSFVHIVOMFI
Linker NTSSGGGSGGGGSGGGGSGGGGSGGGGSGG
(SG3(SG4)5 GGSGGGSITCPPPMSVEHADIWVKSYSLYSRE
SG3S) - RYICNSGFKRK ACTS SLTECVLNKATNVAHW
IL15Ra - TTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQP
Siop) ESLSPSGKEPAASSPSSNNTAATTAAIVPGSQL
MPSKSPSTGTTEISSHESSHGTPSQTTAK WEL
TASASHQPPGVYPQGHSDTTVAISTSTVLLCG
LSAVSLLACYLKSRQTPPLASVEMEAMEALP
VTWGTS SRDEDLENC SHHL *
OT-IL15- EFla MD WTWILFL VAAATR VHSNWVNVISDLKKIE 415 416
054 (IgE DLIQSMHIDATLYTESD PSCKVTAMKCFLL
leader - ELQVISLESGDASIHDTWNLIILANNSLSSNG
IL15 - NVfESGCKECEELEEKNIKEFLOSFVHIVQMFI
Linker NTSSGGGSGGGGSGGGGSGGGGSYPYDWDY
(SG3(SG4)3 ASGGGSrrCPPPMSVEHADIWVKSYSLYSRER
S) - HA Tag YICN SGFKRK AGTS SLTEC VLNK ATN VAHWT
- Linker TPSLKCIRDPALVHQRPAPPSTVTTAGVTPOPE
(SG3S) - SL SP S GKEP A A S SP S SNNT A ATT A AI VPG SQLM
IL15Ra - PSKSPSTGTTEISSHESSHGTPSOTTAKNWELT
Siop) ASASHQPPGVYPQGHSDTTVAISTSTVLLCGL SAVSLLACYLKSRQTPPLASVEMEAMEALPV
TWGTS SRDEDLENCSHHL *
OT-IL15- EFla MDWTWILFLVAAATRVHSNWV VISDLK JE 417 418
055 (IgE DLIQSMHIDATLYTESDVHPSCKVTAMKCFLL
leader - ELQV1SLESGDASIHDTVENLIILANNSLSSNG
1L15 - NVTESGCKECEELEEKNIKEFLQSFVHIVQMFI
Linker (SG) NTSSGITCPPPMSVEHADIWVKSYSLYSRERY1
- IL15Ra - CNSGFKPvKAGTSSLTECVLN ATNVAHWTTP
Stop) SLKCIRDPALVHORPAPPSTVrrAGVrPQPESL
SPSGKEPAASSPSSN TAATTAAIVPGSQLMPS
KSPSTGTTEISSHESSHGTPSQTTAKNWELTAS
ASHQPPGVYPQGHSDTTVAISTSTVLLCGLSA
VSLLA CYLK SRQTPPLA SVEMEAMEALPVTW
GTS SR DE DLENC SHHL *
OT-1L15- EFla MAPRR ARGCRTLGLP ALLL L LLLRPP AT GN 419 420
060 (lL15Ra WVNVISDLKKIEDLIOSMHIDATLYTESDVHP
signal SCKVTAMKCFLLELOVISLESGDASIHDTVEN
peptide - LIIL AN N SL S S GN VTES GCKECEEL E EKNIKE
IL15 -- FLQSFVHIVQMFINTSSGGGSGGGGSGGGGSG
Linker GGGSGGGSLQITCPPPMSVEHADIWVKSYSLY
(SG3- SRERYIC SGFKRKAGTSSLTECVLNKATNVA
(SG4)3- HWTTPSLKCIRDPALVHQRPAPPSTVTTAGVT
SG3-SLQ) - PQPESLSPSUKEPAASSPSSN TAATTAAIVPG
IL15Ra - SQLMPSKSP STGTTEI S SUES SHGTPSQTTAKN
stop) WELTASASHQPPGVYPQGHSDTTVAISTSTVL
LCGLSAVSLLACYLKSRQTPPLASVEMEAME
ALP VTWGT S SRDEDLENC SHHL *
OT-IL 15- EFla MDWTW1LFLVAAATRVHSNWVNVISDL K1E 421 422,
063 (IgE DLIQSMHEDATLYTESDVHPSCKVTAMKCFLL
leader -IL15 ELQVISLESGDA SlilDTVENLIiLANN SLSSNG
- Linker NVTESGCKECEELEEKNIKEFLQSFVHIVO FI
(SG3- NTSSGGGSGGGGSGGGGSGGGGSGGGSLQIT
(SG4)3- CPPPMSVEHADIWVKSYSLYSRERYICNSGFK
SG3-SLQ) - RK ACTS SLTEC VLNK ATN V AHWTTPSLKC 1R
ILlSRa - DPALWQRPAPPSTVTTAGVTPQPESLSPSGK
BamHI (GS) EPAASSPSSNNTAATTAAIVPGSQLMPSKSPST
- sto p) GTTEIS SHES SHGTPSQTT AKNWELTAS A SHQ
PPGVYPQGHSDTTVAISTSTVLLCGLSAVSLL AC YLKSRQTPPL A S VEMEAME ALP VTWGTS S RDEDLENC SHHLGS *
OT-1L15- EFla MD T WIL FL V A A ATR VH S N W VN VI SDLKKIE 225 243
064 and OT- DLIQSMHIDATLYTESDVHPSCKVTAMKCFLL
IL15-071 ELQVISLESGDASIHDTVENLIILAN SLSSNG
(IgE leader - NVTESGCKECEFI■PEKNIKEFLQSFVHIVQMFI
IL15 -- NTSSGGGSGGGGSGGGGSGGGGSGGGSLQIT
Linker CPPPMSVEHADIWVKSYSLYSRERYICNSGFK
(SG3- RKAGTSSLTECVLNKATNVAHWTTPSLKCIR
(SG4)3- DP AL VHQRP APP STVTT A G VTPQPESL SP S GK
SG3-SLQ) - EPAASSPSSNNTAATTAAIVPGSQLMPSKSPST
IL15Ra ~ GTTE-ISSHESSHGTPSQTTAKNWELTASASHQ
stop) PPGVYPQGHSDTTVAISTSTVLLCGLSAVSLL
ACYLKSRQTPPLASVEMEAMEALPVTWGTSS
RDEDLENCSHHL*
OT-IL15- EFla MDWTWILFLVAAATRVHSNWVNVISDLKKIE 226 244
066 (IgE DLIQSMH1DATLYTESDVHPSCKVTAM CFLL
leader -- ELQVISLESGDASlI-iDTVENLIILA N SLSSNG
IL15 -- NVTESGCKECEELEEKNIKEFLQSFVHIVO FI
Linker NTSSGGGSGGGGSGGGGSGGGGSGGGSLOIT
(SG3- CPPPMSVEHADIWVKSYSLYSRERYICNSGFK
(SG4)3- RK ACTS SLTECVLNKATNVAHWTTPSLKCIR SG3-SLQ) - DPALWQRPAPPSTVTTAGVTPQPESLSPSGK
IL15Ra - EP AA S SPS SNNTAATT A AI VPG SQLMP SKSP ST
Linker (SG) GTTEIS SHES SHGTPSQTT AKNWELT A S A SHO
ecDHFR PPGVYPQGHSDTTVAISTSTVLLCGLSAVSLL
(Amino acid AC YLKSRQTPPL A S VEMEAMEALP VTWGTS S
2-159 of RDEDLENCSHHLSGISLIAALAVDYVIGMENA
WT. R 12Y, MPWNLPADLAWFKRNTLNKP\TMGRHTWES
Y100I) - IGRPLPGRKNULSSQPGTDDRVTWVKSYDEAI
stop) AACGDWEmfVIGGGRVIEQFLPKAQKLYLTH
IDAEVEGDTHFPDYEPDDWESVFSEFHDADA
QNSHSYCFEILERR*
OT-IL15- EFla MDWTWILFLVAAATRVHSNWVNVISDLKKIE 424 425
067 (IgE DLIQSMH3DATLYTESDVHPSC VTAMKCFLL
leader - ELQVISLESGDASlHDTYENLIiLA NSLSSNG
IL 5 - NVTESGCKECEELEEKNKEFLQSFVHIVO¾fFI
Linker NTSSGGGSGGGGSGGGGSGGGGSGGGSLQIT
(SG3- CPPPMSVEHADIWVKSYSLYSRERYICNSGFK
(SG4)3- RK ACTS SLTECVLNKAT VAHWTTPSLKC IR
SG3-SLQ) - DPALVHQRPAPPSTVTTAGVTPOPESLSPSGK
IL15Ra ~ EPAASSPSSNNTAATTAAI GSQLMPSKSPST
Linker (SG) GTTEISSHESSHGTPSQTTAKNWELTASASHQ
- ecDHFR PPGVYPQGHSDTTVAISTSTVLLCGLSAVSLL
(Amino acid ACYLKSRQTPPLASVEMEAMEALPVTWGTSS
2-159 of RDEDLENCSHHLSGISLIAALAVDYVIGMENA
WT, R12Y, MPWr>TLPADLAWFKRNTLNKPVIMGRHTWES
Y100I) - IGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAI
stop) AACGDVPE1MV1GGGRVIEQFLPKAO LYLTH
ID AE VE GDTHFPD YEPDD WE S VF SEFHD AD A
QNSHSYCFEILERR*
OT-IL15- EFla MAPRRARGCRTLGLPALLLLLLLRPPATRGN 426 427 068 (ILlSRa WVNVISDLKKIEDLIQSMHIDATLYTESD\¾P
signal SCKVTAMKCFLLELQVISLESGDASIHDTVEN
peptide - LIIL ANN SL S SN GN VTES GCKECEELEEKNIKE
IL15 - FLQSFVHIVQMFINTSSGGGSGGGGSGGGGSG
L inker GGGSGGGSLQITCPPPMSVEHADIWVKSYSLY
(SG3- SRERYICNSGFKRKAGTSSLTECVLNKATNVA
(SG4)3- HWTTPSLKCIRDPALVHQRPAPPSTVTTAGVT
SG3-SLQ) - PQPESLSPSGKEPAASSPSSNNTAATTAAIVPG
ILlSRa - SQ LMPSKSP STGTTEI S SHES SHGTPSQTT A N
stop) WELTAS ASHQPPGVYPQGHSDTTVA! STSTVL
LCGLSAVSLLACYLKSRQTPPLASVEMEAME
ALPVTWGTSSRDEDLENCSHHL*
OT-IL15- EFla MDWTWILFLVAAATRVHSNWVNVISDLKKIE 428 429 069 (IgE DLIQSMHIDATLYTESDVHPSCKVTAMKCFLL
leader - ELQVISLESGDASIHDTVENLIILANNSLSSNG
IL15 - NVTESGCKECEELEEK IKEFLQSFVHIVQMFI
BarnHI (GS) NTSGSITCPPPMSVEHADIWVKSYSLYSRERYI
- ILlSRa - CNSGFKRKAGTSSLTECVLNKATNVAHWTTP
stop) SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESL
SPSGKEPAASSPSSNNTAATTAAIVPGSQLMPS
KSPSTGTTEISSHESSHGTPSQTTAKNWELTAS
ASHQPPGVYPQGHSDTTVAISTSTVLLCGLSA
V SLL AC YLK SRQTP PL A S VEME AME AL P VT W
GTS SRDEDLENC S HHL *
OT-IL15- EFla MAPRRARGCRTLGLPALLLLLLLRPPATRGN 426 430 070 (IL lSRa WVNVISDLKKIEDLIQSMHIDATLYTESDWP
signal SCKVTAMKCFLLELQVISLESGDASIHDTVEN
peptide - LIILANNSLSSNGNVTESGCKECEELEEKNIKE
IL15 - FLQSFVHIVQMFINTSSGGGSGGGGSGGGGSG
Linker GGGSGGGSLQITCPPPMSVEHADIWVKSYSLY (SG3- SRERYICNSGFKRKAGTSSLTECVL KATNVA
(SG4)3- HWTTPSLKCIRDPALVHQRPAPPSTVTTAGVT
SG3-SLQ) - PQPESLSPSGKEPAASSPSS NTAATTAAIVPG
LLl SRa - SQLMPSKSPSTGTTEISSHESSHGTPSQTTAKN
stop) WELTASASHQPPGVYPQGHSDTTVAISTSTVL
LCGLS A VSLLACYLKSRQTPPLA SVEMEAME
ALP VTWGT S SRDEDLENC SHHL *
OT-IL 15- EFla MDWW1LFLVAAATRVHSNWVNVISDI K1E 225 431
072 (IgE DLIQSMHIDATLYTESDVHPSCKVTAMKCFLL
leader -IL15 ELQVISLESGDASlHDTVENLIiLA NSLSSNG
- Linker NVTESGCKECEELEEKNIKEFLQSFVHIVO FI
(SG3- NTSSGGGSGGGGSGGGGSGGGGSGGGSLQIT
(SG4)3- CPPPMSVEHADIWVKSYSLYSRERYICNSGFK
SG3-SLQ) - RK ACTS SLTEC VLNK ATN V AHWTTPSLKC 1R
IL15Ra - DPALVHQRPAPPSTVTTAGVTPOPESLSPSGK
stop) EPAASSPSSNNTAATTAAIVPGSQLMPSKSPST
GTTEIS SHES SHGTPSQTTAKNWELTASA SHQ
PPGVYPQGHSDTTVAISTSTVLLCGLSAVSLL
AC YLKSRQTPPL A SVEMEAME ALP VTWGTS S
RDEDLENC SHHL *
OT-1L1.5- EFla 432 433
089 (IgE MDWTWILFLVAAATRVHSDYKDDDDKNWV
leader - NVTSDLKKTEDLIQSMHIDATLYTESDVHPSCK
FLAG - VTAMKCFI '· ELQVISLESGDA SIHDTVE LIIL
IL15 - ANNSLSSNGNVTESGCKECEELEEKNIKEFLQ
Linker SFVHIVQMFINTSSGGGSGGGGSGGGGSGGG
(SG3- GSGGGSLC>YPYDVPDYAITCPPPMSV¾HADI
(SG4)3- WVKSYSLYSRERYICNSGFKRKAGTSSLTECV
SG3-SLQ) - LNKATNVAH^rT^SLKCIRDPALWQRPAPPS
HA Tag - TVTT A G VTPQPESL SP S GKEP A A S SP S SNNT A A
ILlSRa - TT A AIVPG SQLMP SK SP S TGTTEI S SHE S SHGTP
iinker SQTTAK WELTASASHQPPGVYPQGHSDTTV
(GSG) - AISTSTVLLCGLSAVSLLACYLKSRQTPPLASV
ecDHFR EMEAMEALPVTWGTSSRDEDLENCSHHLGSG
(Amino acid ISLIAALAVDYVTGMENAMPWNLPADLAWFK
2-159 of RNTLN P VTMGRHTWESIGRPLPGRKNIIL S SO
WT. R12Y, PGTDDRVTWVKSVDEAIAACGDVPEIMVIGG
Y100I) - GRVIEQFLPKAQKLYLTHIDAEVEGDTHFPDY
stop) EPDD WE S VF S EFHD AD AQNSH S YC FEIL ERR *
OT-IL 15- EFla MDWTWTLFL-VAAATRVHSNWVNVISDLKKIE 421 422 109 (IgE DLIOSMHIDATLYTESDVHPSCKVTAMKCFLL
leader -IL 15 ELQVISLESGDA SIHDTVENLIILANNSLS SNG
- Linker NVTESGCKECEELEEKNIKEFLQSFWIVQMFI
(SG3- NTSSGGGSGGGGSGGGGSGGGGSGGGSLQIT
(SG4)3- CPPPMSVEHADIWVKSYSLYSRERYICNSGFK
SG3-SLQ) - RKAGTSSLTECVLNKATNVAHWTTPSLKCIR
ILlSRa - DP AL VHQRP APP ST VTT AG VTPQPE SL SP S GK
BamHI (GS) EPAASSPSSNNTAATTAAIVPGSQLMPSKSPST
- stop) GTTEISSHESSHGTPSOTTAKNWELTASASHQ
PPGVYPOGHSDTTVAiSTSTVLLCGLSAVSLL
ACYLKSRQTPPLASVEMEAMEALPVTWGTSS
RDEDLE C SHHLGS *
OT-IL15- EFla MDWTWILFLVAAATRVHSDYKDDDDKNWV 434 435
110 (IgE NVISDLKKIEDLIQSMHIDATLYTESDVHPSCK
leader - VTAMKCFLLELQVISLESGDASIHDTVENLIIL
FLAG - ANNSLSSNGNVTESGCKECEELEEKNIKEFLO
IL15 - SFVHIVQMFINTSSGGGSGGGGSGGGGSGGG
Linker GSGGGSLQYPYDVPDYAITCPPPMSVEHADI
(SG3- WVKSYSLYSRERYICNSGFKRKAGTSSLTECV
(SG4)3- L KATOVAHWTTPSLKCIRDPALVHQRPAPPS SG3-SLQ) - TVTT AG VTPQPESL SP S GKEP AA S SP S SNNT A A
HA Tag - TTAAIVPGSQL PSKSPSTGTTEISSHESSHGTP
ILlSRa - SQTTAKNWELTASASHOPPGVYPQGHSDTTV
BamHI (GS) AISTSTVLLCGLSAVSLLACYLKSRQTPPLASV
- stop) EME AMEALP VTWGTS SRDEDLENC SHHLGS *
OT-IL 15- EFla MDWTWILFLVAAATRVHSDYKDDDDKNWV 436 437 114 (IgE NVISDLKKIEDLIQSMH1DATLYTESDVHPSCK
leader - VTAMKCF1 I F F ,QVIS LESGDA SIHDTVENLIIL
FLAG - ANN SLSSNGNVTESGCKECEELEEKNIKEFLQ
IL15 - SFVHrVQMFINTSSGGGSGGGGSGGGGSGGG
Linker GSGGGSLQYPYDVPDYAITCPPPMSVEHADI
(SG3- WVKSYSLYSRERYICNSGFKRKAGTSSLTECV
(SG4)3- LNKATNVAHWTTPSLKCIRDPALVHQRPAPPS
SG3-SLQ) - TVTT A G VTPQPE SL S P S GKEP A A S SP S SNNT A A
HA Tag - TT AAIVPG SQLMP SK SP S TG' i 1 i hi S SHES SHGTP
IL15Ra - SQTTAKNWELTASASHQPPGVYPQGHSDTTV
Linker AISTSTVLLCGLSAVSLLACYLKSRQTPPLASV
(GSG) - EMEAMEALPVTWGTSSRDEDLENCSHHLGSG
hDHFR VGSLNCrVAVSQNNlGIGKNGDLPWPPLRNEF
(Amino acid RYFQRMTTTSS\/EGKQNL\'TA4GKKTWTSIPE
2-187 of KNRPLKGRINLVLSRELKEPPQGAHFLSRSLD
WT; DALKLTEQPELANKVDMVWIVGGSSVYKEA
K185E) - MNHPGHLKLFVTRMQDFESDTFFPEIDLEKY
stop) KLLPEYPGVLSDVQEEKGIKYKFEVYEEND*
OT-IL15- EFla fDWTWILFLVAAATRVHSDYKDDDDKNWV 438 439 115 (IgE NVISDLKKIEDLIQSMHIDATLYTE-SDVHPSCK
leader - VTAMKCFLLELOVISLESGDASrHDTVENLIIL
FLAG - ANNSLSSNGNVTESGCKECEELEEKNIKEFLO
IL 5 - SF IVQ fFINTSSGGGSGGGGSGGGGSGGG
Linker GSGGGSLQYPYDVPDYAITCPPPMSYEHADI
(SG3- WVKSYSLYSRERYICNSGFKRKAGTSSLTECV
(SG4)3- LNK ATN VAHWTTP SLKCIRDP AL VHQRP APP S
SG3-SLQ) - TVTT AG VTPQPE SL SP S GKEP A A S SP S SNNT A A
HA Tag - TT AAIVPG SQLMP SK SP S TGTTEI S SHES SHGTP
IL15Ra - SOTTAKNWELTASASHQPPGVYPQGHSDTTV
Linker AISTSTVLLCGLSAVSLLACYLKSRQTPPLASV
(GSG) - EMEAMEALPVTWGTSSRDEDLENCSHHLGSG
hDHFR VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEF
(Amino acid YFQRMTTTSSVEGKQNLViMGKKTWFSIPE
2-187 of KNRPLKGRINLVLSRELKEPPQGAHFLSRSLD
WT; E162G, D ALKLTEQPEL ANK VDM VW1 VGG S S VY EA
I176F) - MNHPGHLKLFVTRIMQDFESDTFFPEIDLEKY
stop) KLLPGYPGVLSDVQEEKGFKYKFEVYEKND*
OT-IL15- EFla MDWTWILFLVAAATRVHSDYKDDDDKNWV 440 441
116 (IgE NVISDLKKIEDLIQSMHIDATLYTESDVHPSCK
leader - VTAMKCFLLELQVISLESGDASIHDTVENLIIL
FLAG - IL15 - SFVHIVQMFINTSSGGGSGGGGSGGGGSGGG
L inker GSGGGSLQYPYDVPDYAITCPPPMSVEHADI
(SG3- WVKSYSLYSRERYICNSGFKRKAGTSSLTECV
(SG4)3- LNK ATN VAHWTTP SLKC IRDP AL VHQRP APP S
SG3-SLQ) - TVTTAGVTPQPESLSPSGKEPAASSPSSNNTAA
HA Tag - TTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTP
ILlSRa - SQTTAKNWELTASASHQPPGVYPQGHSDTTV
L inker AISTSTVLLCGLSAVSLLACYLKSRQTPPLASV
(GSG) - EMEAMEALPVTWGTSSRDEDLENCSHHLGSG
hDHFR VGSLNC I V A VSQNMG I G KNGDLPWPPLRNEF
(Amino acid RYFQRMTTTSSVEGKQNLVIMGKKTWFSrPE
2-187 of KNRPLKGRINLVLSRELKEPPQGAHFLSRSLD
WT; D ALKLTEQPEL ANK VDM IVGGS S VYKEA H131R, HPGRLKLFVTRIMQDFGSDTFFPEIDLEKY
E 144G) - KLLPEYPGVLSDVQEEKGIKYKFEVYEKND*
stop)
OT-IL 15- EFla MDWTWILFLVAAATRVHSDYKDDDDKNWV 443 444
117 (IgE NVISDLKKIEDLIQSMHIDATLYTESDVHPSCK
leader - VTAMKCF1 I F F ,QVIS LESGDA SIHDTVENLIIL
FLAG - ANN SLSSNGNVTESGCKECEELEEKNIKEFLQ
1L15 - SFVHIVQMFINTSSGGGSGGGGSGGGGSGGG
Linker G SGGGSLQYPYD VPD Y Al TCPPPMS VEH ADI
(SG3- WVKSYSLYSRERYICNSGFKRKAGTSSLTECV
(SG4)3- LM ATNVAHWTTPSLKCIRDPALVHQRPAPPS
SG3-SLQ) - TVTT A G VTPQPE SL S P S GKEP A A S SP S SNNT A A
HA Tag - TTAArVPGSQLMPSKSPSTGTTEISSHESSHGTP
IL1 5Ra - SOTTAKNWELTASASHQPPGVYPOGHSDTTV
Linker AISTSTVLLCGLSAVSLLACYLKSRQTPPLASV
(GSG) - EMEAMEALPVTWGTSSRDEDLENCSHHLGSG
hDHFR(Am VGSLNCIVAVSQN GVGKNGDLPWPPLRNEF
ino acid 2- RYFQRMTTTSS\/EGKQNL\/IIv4GKKTWFSIPE
187 of WT; KNRPLKGRINLVLSRELKEPPQGAHFLSRSLD
117 V) - D ALKLTEQPEL ANKVDMVWI VGGS S VYKEA
stop) MNHPGHLKLFVTRIMODFESDTFFPEIDLEKY
KLLPEYPGVL SD VQEEKGIKYKFEVYEKND *
OT-1L Is- EFla MDWTWILFLVAAATRVHSDYKDDDDKNWV 445 446 l i e (IgE NViSDLK KiEDLIQSMHrDATLYTESDVHPSCK
leader - VTAMKCFLLELQVISLESGDASIHDTWNLIIL
FLAG - ANNSLSSNGNVTESGCKECEELEFKNIKEFLO
IL 5 - SF IVQ fFINTSSGGGSGGGGSGGGGSGGG
Linker GSGGGSLQYPYDVPDYAITCPPPMSVEHADI
(SG3- WVKSYSLYSRERYICNSGF RKAGTSSLTE-CV
(SG4)3- LNK ATN VAHWTTP SLKCIRDP AL VHQRP APP S
SG3-SLQ) - TVTT AG VTPQPE SL SP S GKEP A A S SP S SNNT A A
HA Tag - TT AAIVPG SQLMP SK SP S TGTTEI S SHES SHGTP
IL15Ra - SOTTAK WELTASASHQPPGVYPQGHSnTTV
Linker AISTSTVLLCGLSAVSLLACYLKSRQTPPLASV
(GSG) - EMEAMEALPVTWGTSSRDEDLENCSHHLGSG
hDHFR VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEF
(Amino acid RYFQRMTTTSSVTiGKQNLWMGKK'RVFSIPE
2-187 of KNRPLKGR1NLVLSRELKEPPQGAHFLSRSLD
WT, D ALKLTEQPEL ANKVDMVWI VGG S S VYKEA
N127Y) - MYHPGHLKLFVTRMQDFESDTFFPEIDLEKY
stop) KLLPEYPGVLSDVQEEKGIKYKFEVYEKND*
OT-IL 15- EFla MDWTWILFLVAAATRVHSDYKDDDDKNWV 448 449 119 (IgE NVISDLKKIEDLIQSMHIDATLYTESDVHPSCK
leader - WAMKCFLLELQVISLESGDASIHDTVENLIIL
FLAG - IL15 - SFVHIVQMFINTSSGGGSGGGGSGGGGSGGG
Linker GSGGGSLQYPYDVPDYAITCPPPMSVEHADI
(SG3- WVKSYSLYSRERYICNSGFKRKAGTSSLTECV
(SG4)3- LNK ATN VAHWTTP SLKC IR DP AL VHQRP APP S
SG3-SLQ) - TVTTAGVTPQPESLSPSGKEPAASSPSSNNTAA
HA Tag - TTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTP
IL15Ra - SQTTAKNWELTASASHQPPGVYPQGHSDTTV
L inker AISTSTVLLCGLSAVSLLACYLKSRQTPPLASV
(GSG) - EMEAMEALPVTWGTSSRDEDLENCSHHLGSG
hDHFR VGSLNCIVAVSQNMGVGKNGDLPWPPLRNEF
(Amino acid RYFQRMTTTSSVEGKQNLVIMGKKTWFSIPE
2-187 of KNRPLKGRINLVLSRELKEPPQGAHFLSRSLD
WT, 117V, DALKLTEQPELANKVDMVWIVGGSSVKEAM
Y 1221) - NHPGHLKLFVTRIMQDFESDTFFPEIDLEKYK
stop) LLPE YP GVL SD VQEEK GIK YKFE VYEKND * OT-JL15- EFla MDWTWILFLVAAATRVHSNWVNVISDLKKIE 459 460
128 (IgE DLIQSMHIDATLYTESDVHPSCKVTAMKCFLL
leader -IL 15 ELQVISLESGDASIHDTVENLIILAN SLSSNG
- Linker NVTESGCKECEELEEKNIKEFLOSFYHrVQMFI
(SG3- NTSSGGGSGGGGSGGGGSGGGGSGGGSLQIT
(SG4)3- CPPPMSVEHADIWVKSYSLYSRERYICNSGFK
SG3-SLQ) - RKAGTSSLTECVL KATNVAHWTTPSLKCIR
IL15Ra- DP AL VHQRP APP STVTT A G VTPQPESL SP S GK
Spacer - EPAASSPSSN TAATTAAI GSQLMPSKSPST
Flagx3 - GTTEISSHESSHGTPSQTTAKNWELTASASHQ
Spacer - PPGVYPQGHSDTTVAISTSTVLLCGLSAVSLL
BamHI (GS) ACYLKSRQTPPLASVEMEA EALPVTWGTSS
- P2A RDEDLENCSHHLSRMDYKDDDDKDYKDDDD
cleavable KDYKDDDDKSRGSGAT FSLLKQAGDVEENP
peptide - GPLSKGEEDNMAIKEFMRFKVHMEGSVNGH
mCherr EFEIEGEGEGRPYEGTQTAKL V KGGPLPFA
(MIL) - WD IL SPQFMYG SKA Y VKHP ADIPD YLKL SFPE
stop) GFKWERVMNFEDGGVVTVTQDSSLODGEFIY
KVKLRGTNFPSDGPVMQKXTMGWEASSERM
YPEDGALKGEIKQRLKLKDGGHYDAEVKTTY
KAK PVQLPGAY V IKLDITSHNIiDYTIVI-iQ
YERAEGRHSTGGMDELYK*
OT-IL15- EFla MDWTWILFLVAAATRVHSNWVNVISDLKKIE 461 462
129 (IgE DLIOSMHIDATLYTESDVHPSCKVTAMKCFLL
leader -IL 15 ELQVISLESGDA SIHDTVENLIILANNSLS SNG
- Linker NVTESGCKECEELEEKNKEFLQSFWIVOMFI
(SG3- NTSSGGGSGGGGSGGGGSGGGGSGGGSLQIT
(SG4)3- CPPPMSVEHADIWVKSYSLYSRERYICNSGFK
SG3-SLQ) - RK ACTS SLTECVLNKATNVAHWTTPSLKCIR
IL15Ra- DP AL VHQRP APP STVTT AG VTPQPE SL SP S GK
BamHI (GS) EPAASSPSSNNTAATTAAIVPGSQLMPSKSPST
- P2A GTTEISSHESSHGTPSQTTAKNWELTASASHQ
cleavable PPGVYPQGHSDTTVAISTSTVLLCGLSAVSLL
peptide - ACYLKSRQTPPL AS VEMEAME ALP VTWGTS S
mCherry RDEDLENCSHHLGSGATNFSLLKQAGDVEEN
(MlL)-stop) PG PLS G E EDNM AliKE FMRFK VHMEGSVNG
HEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPF
AWDILSPQFMYGSKAYVKHPAD1PDYLKLSFP
EGFKWERVNINFEDGGVVTVTQDSSLQDGEFI
YKVKLRGTNFPSDGPVMQKKTMGWEASSER
MYPEDGALKGEIKQRLKLKDGGHYDAEVKT
TYKAKKPVQLPGAYNVNIKLDITSHNEDYTIV
EQ YERAEGRHSTGGMDELYK*
Table 8c: IL15/IL15Ra constructs
Figure imgf000063_0001
WT, Y122I) YFQRMTTTSSVEGKQNLVIM the stop
- stop - GKKTWFSTPEKNRPLKGRINL codorss) spacer - VLSRELKK QGAHFLSRSLD
IRES - DALKLTEQPELA KVDMVWI
spacer - VGGSSVIKEAMNHPGHLKLF
mCherry - VTRIMODFESDTFFPEIDLEKY
stop) KLLPEYPGVLSDVQEEKGIKY
KFEVYEKND* SR* YDSLEIPPL
SLPPP*RYWPKPLGIRPVCVC
LYVIFHHIAVFWQCEGPETWP
CLLDEHS*GSFPSRQRNARSV
ECREGS S S SGSFLKTNN VC SD
PLQAAEPPTWRQVPLRPKAT
CIRYTCKGGTTPVPRCELDSC
GKSQMALLKRIQQGAEGCPE
GTPLYGI* SGASVHMLYMCL
VEVKKTSRPPEPRGRGFPLKN
T I1WPQP* * ARARRITWPSSR
SSCASRCTWRAP*TATSSRSR
ARARAAPTRAPRPPS*R*PRV
APCPSPGTSCPLSSCTAPRPT*
STPPTSPTT* SCPSPR AS SGS A*
*TSRTAAW*P*PRTPPCRTASS
STR*SCAAPTSPPTAP*CRRKP
WAGRPPPSGCTPRTAP*RARS
SRG * S * RT A ATTTLR SR PPTRP
RSPCSCPAPTTSTSSWTSPPTT
RTTPSW STNAPRAATPPAA
WTSCTS
IgE leader - MDWTWILFLVAAATRVHSN 452 453 1L15 - WVNV1SDL 1EDLIOSMHID
linker (GS) ATLYTESDVHPSCKVTAMKC
-- hDHFR FT J ,ET QVISLESGDASfflDTVE
(Amino acid NLIIL AN SL S S N GN VIES GC
2-187 of KECEELEEKNTKEFLQSFVHIV
WT, Y122I) QMFINTSGSVGSL CIVAVSQ
-- stop NMGIGKNGDLPWPPLRNEFR
YFQRMTTTSSVEGKQNLVIM
GKKTWFSIPEKNRPLKGRINL
VLSRELKEPPQGAHFLSRSLD
DALKLTEQPELANKVDMVWI
VGGSSVIKEAMNHPGHLKLF
VTRIMQDFESDTFFPEIDLEKY
KLLPEYPGVLSDVQEEKGIKY
KFEVYEKND*
mCherry - MSKGEEDNMAIIKEFMRFKV 454 455 stop HMEGSVNGHEFEIEGEGEGRP
YEGTQTAKLKVTKGGPLPFA
WDILSPQFMYGSKAYVKHPA
DIPDYLKLSFPEGFKWERVM
NFED GG V VTVTQD S SLQD GE
FIYKVKLRGTNFPSDGPVMQ
KKTMGWEA S S ERMYPEDG A
LKGEIKQRLKLKDGGHYDAE
VKTTYKAKKPVQLPGAYNV
NIKLDITSHNEDYTIVEQYER
AEGRHSTGGMDELYK* OT-1L15- Full EFla MDWTWILFLVAAATRVHSN 523 456
123 and OT- constnicl. W VNVI SDL k J ED L I QSMHID (full),
IL15- ATLYTESDVHPSCKVTAMKC 524-532
127(IgE FLLELQVISLESGDASIHDTYE (various
leader - NLIIL ANNSL S SNGN VTESGC fragment
ILl 5 - KECEELEKKNIKEFLOSFVHIV taking
BamHI (GS) QMFINTSGS *NLDNTTH*RSR into
- stop - PSPSPPPNVTGRSRLE*GRCAF account
spacer - VYMLFSTILPSFGNVRARKPG the stop
IRES - PVFLTSIPRGLSPLAKGMQGL codons)
spacer - LNVVKEAVPLEAS*RQTTSVA
mCheny - TLCRQRNPPPGDRCLCGQKP
stop) RV*DTPAKAAQPQCHVVSWI
V VER VK WL S S S WNKGLKD A
OKVPHCMGSDLGPRCTCFTC
V*SRLK RLGPPNHGDWFL*
KTR**YGHNHDEQGRGG*HG
HHQGVHALQGAHGGLRERP
RVRDRGRGRGPPLRGHPDRQ
AEGDQGWPPALRLGHPVPSV
HVRLQGLREAPRRHPRI ,1 F A
VLPRGLQVGARDELRGRRRG
DRD PG L LP AGRRVHLQGEAA
RHQLPLRRPRNAEEDHGLGG
LLRADVPRGRRPEGRDQAEA
EAEG RRPLRR * GQDHLQGQE
ARAAARRLQRQHQVGHHLP
QRGLHHR GT VRTRRGPPLHR
RHGRAVQV
IgE leader - - MDWTWILFLVAAATRVHSN 457 458
1L15 - W VN VI SDL 1ED L I QSMHID
BamHI (GS) ATLYTESDVHPSCKVTAMKC
-- stop FI J ,FT QVISLESGDASfflDTVE
NLIILANNSLSSNGNVTESGC KECEELEEKNIKEFLQSFVHIV QMFINTSGS*
mCherry - - MSKGEEDNMAIIKEFMRFKV 454 455 stop HMEGSVNGHEFEIEGEGEGRP
YEGTQTAKLKVTKGGPLPFA
WDILSPQFMYGSKAYVKHPA
DIPDYLKL SFPEGFKWERVM
NFED GG WTVTQD S SLQD GE
FIYKVKLRGTNFPSDGPVMQ
KKTMGW ASSERNOTEDGA
LKGEIKQRLKLKD GGHYD AE
VKTTYKAKKPVQLPGAYNV
NIKLDITSHNEDYTIVEQYER
AEGRHSTGGMDELYK*
[00144] In one embodiment, the pavload of the present invention may comprise ILl 8. ILl 8 is a proinflammatory and immune regulatory cytokine that promotes IFN-y production by T and NK cells. ILl 8 belongs to the ILl family. Secreted ILl 8 binds to a heterodimer receptor complex, consisting of ILlS oc and ?-chains and initiates signal transduction. IL18 acts in concert with other cytokines to modulate immune system functions, including induction of IFN-y production, Th l responses, and NK cell activation in response to pathogen products. IL18 showed anticancer effects in several tumors. Administration of recombinant IL18 protein or IL18 transgene induces melanoma or sarcoma regression through the activation of CD4÷ T and/or NK cell- mediated responses (reviewed by Srivastava et al., Curr. Med. Chem., 2.010, 17: 3353-3357). The combination of IL18 with other cytokines, such as IL12 or co-stimulatory molecules (e.g., CD80) increases IL18 anti-tumor effects. For example, IL18 and IL12A/B or CD80 genes have been integrated successfully in the genome of oncolytic viruses, with the aim to trigger synergistically T cell-mediated anti-tumor immune responses (Choi et ai, Gene Ther., 2011, 18: 898-909). IL2/IL18 fusion proteins also display enhanced anti-tumor properties relative to either cytokine alone and low toxicity in preclinical models (Acres et al., Cancer Res., 2005, 65:9536- 9546).
[00145] IL18 alone, or in combination of IL12 and IL15, activates NK cells. Preclinical studies have demonstrated that adoptively transferred IL12, IL15 and IL18 pre-activated NK cells display enhanced effector function against established tumors in vivo (Ni et al., J Exp Med. 2012, 209: 2351-2365; and Romee et al. Blood. 2012, 120:4751-4760). Human DL12/IL15/IL18 activated NK cells also display memory-like features and secrete more IFN-y in response to cytokines (e.g., low concentration of 1L2). In one embodiment, the effector module of the present invention may be a DD-IL18 fusion polypeptide.
[00146] In one embodiment, the payload of the present invention may comprise 11,21. 11,21 is another pleiotropic type I cytokine that is produced mainly by T cells and natural killer T (NKT) ceils. IL21 has diverse effects on a variety of cell types including but not limited to CD4+ and CD8÷ T cells, B cells, macrophages, monocytes, and dendritic cells (DCs). The functional receptor for IL21 is composed of IL21 receptor (11,21 R) and the common cytokine receptor gamma chain, which is also a subunit of the receptors for IL2, IL4, IL7, 1L9 and IL15. Studies provide compelling evidence that IL21 is a promising immunotherapeutic agent for cancer immunotherapy. IL21 promotes maturation, enhances cytotoxicity, and induces production of IFN-y and perforin by NK cells. These effector functions inhibit the growth of B 16 melanoma (Kasaian et al. Immunity. 2002, 16(4): 559-569; and Brady et al, J Mmunoi.2004, 172(4):2048- 20 8). IL21 together with IL15 expands antigen-specific CD8+ T-ceil numbers and their effector function, resulting in tumor regression (Zeng et al, J Exp Med.2005, 201(1 ): 139-148), IL21 may also be used to rejuvenate multiple immune effector cells in the tumor microenvironment. IL21 may also directly induce apoptosis in certain ty pes of lymphoma such as diffuse large B-ceil lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia cells, via activation of STATS or STAT1 signal pathway. IL21 , alone or in combination with anti-CD20 mAb
(rituximab) can activate NK cell-dependent cytotoxic effects. Interestingly, discovery of the immunosuppressive actions of IL21 suggests that this cytokine is a "double-edged sword"- IL21 stimulation may lead to either the induction or suppression of immune responses. Both stimulatory and suppressive effects of IL21 must be considered when using IL21 -related immunotherapeutic agents. The level of IL21 needs to be tightly controlled by regulatory elements. In one aspect, the effector module of the present invention may be a DD-IL21 fusion polypeptide.
Θ0147] In some embodiments, payloads of the present invention may comprise type I interferons. Type I interferons (IFNs-I) are soluble proteins important for fighting viral infection m humans. IFNs-I include IFN-alpha subtypes (IFN- al, IF - alb, IF - ale), IFN-beta, 1FN- deita subtypes (IFN-deita 1, IFN-delta 2, IFN-delta 8), IF -gamma, IFN -kappa, and IFN- epsilon, IFN-lambda, IFN-omega, IFN-tau and IFN-zeta. IFN-a and TFN-β are the main IFN-I subtypes in immune responses. All subtypes of IFN-I signal through a unique heterodimeric receptor, interferon alpha receptor (IFNAR), composed of 2 subunits, IFNAR1 and IFNAR2. IFNR activation regulates the host response to viral infections and in adaptive immunity. Several signaling cascades can be activated by IFNR, including the Janus activated kinase-signal transducer and activation of transcription (JAK-STAT) pathway, the mitogen activated protein kinase (MAPK) pathway, the phosphoinositide 3-kinase (PI3K) pathway, the v-crk sarcoma vims CTI O oncogene homolog (avian)-like (CRKL) pathway, and NF-κΒ cascade. It has long been established that type I IFNs directly inhibit the proliferation of tumor cells and vims- infected cells, and increase MHC class I expression, enhancing antigen recognition. IFNs-I have also proven to be involved in immune system regulation. IFNs can either directly, through interferon receptor (IFNR), or indirectly by the induction of chemokines and cytokines, regulate the immune system. Type I IFNs enhance NK cell functions and promote survival of NK cells. Type I IFNs also affect monocytes, supporting the differentiation of monocytes into DC with high capacity for antigen presentation, and stimulate macrophage function and differentiation. Several studies also demonstrate that IFNs-I promote CDS4" T cell survival and functions. In some instances, it may be desirable to tune the expression of Type 1 IFNs using biocircuits of the present invention to avoid immunosuppression caused by long-term treatment with IFNs.
Θ0148] New anticancer immunotherapies are being developed that use recombinant type I IFN proteins, type I IFN transgene, type I IFN-encoding vectors and type I IFN-expressing cells. For example, IFN-a has received approval for treatment of several neoplastic diseases, such as melanoma, RCC and multiple myeloma. Though type I IFNs are powerful tools to directly and indirectly modulate the functions of the immune system, side effects of systemic Song-term treatments and lack of sufficiently high efficacy have dampened the interest of IFN-a for clinical use in oncology. It is believed that if IFN levels are tightly regulated at the malignant tissues, type 1 IFNs are likely more efficacious. Approaches for intermittent deliver - are proposed according to the observation that interniittency at an optimized pace may help to avoid signaling desensitizing mechanisms (negative feedback mechanisms) induced by IFNs-I (i.e., because of SOCS 1 induction) in the responding immune cells. In accordance with the present invention, the effector module may comprise a DD-IFN fusion polypeptide. The DD and its ligand control the expression of IFN to induce an antiviral and antitumor immune responses and in the meantime, to minimize the side effects caused by long-term exposure of IFN.
[00149] In some embodiments, payioads of the present invention may comprise members of tumor necrosis factor (TNF) superfamily. The term. "TNF superfamily" as used herein refers to a group of cytokines that can induce apoptosis. Members of TNF family include TNF-alpha, TNF- beta (also known as lymphotoxin-alpha (LT-a)), lymphotoxin-beta (LT-β), CD40L(CD154), CD27L (CD70), CD30L(CD153), FASL(CD178), 4-1BBL (CD137L), OX40L, TRAIL (TNF- related apoptosis inducing ligand), APRIL (a proliferation-inducing ligand), TWEAK,
TRANCE, TALL-1, GITRL, LIGHT and TNFSF1 to TNFSF20 (TNF ligand superfamily member 1 to 20). In one embodiment, the payload of the invention may be TNF-alpha. TNF- alpha can cause cytolysis of tumor cells, and induce cell proliferation differentiation as well. In one aspect, the effector module of the present invention may comprise a DD-TNF alpha fusion polypeptide.
[00150] In some embodiments, payioads of the present invention may comprise inhibitory molecules that block inhibitory cytokines. The inhibitors may be blocking antibodies specific to an inhibitory cytokine, and antagonists against an inhibitory cytokine, or the like.
[00151] In some aspects, payioads of the present invention may comprise an inhibitor of a secondary cytokine IL35. IL35 belongs to the interleukin-12 (IL12) cytokine family, and is a heterodimer composed of the IL27 β chain Ebi3 and the IL12 a chain p35. Secretion of bioactive IL35 has been described only in forkhead box protein 3 (Foxp3) + regulatory T cells (Tregs) (resting and activated Tregs). Unlike other membranes in the family, IL35 appears to function solely in an anti-inflammatory fashion by inhibiting effector T cell proliferation and perhaps other parameters (Collison et a\.. Nature, 2007, 450(7169): 566-569),
[00152] In some embodiments, payioads of the present invention may comprise inhibitors that block the transforming growth factor beta (TGF-β) subtypes (TGF-βΙ, TGF-p2 and TGF-P3). TGF-β is secreted by many cell types, including macrophages and is often complexed with two proteins LT'BP and LAP. Serum proteinases such as plasmin catalyze the release of active TGF-β from the complex from the activated macrophages. It has been shown that an increase in expression of TGF-β correlates with the malignancy of many cancers. The immunosuppressive activity of TGF-β in the tumor microenvironment contributes to oncogenesis.
[00153] In some embodiments, payloads of the present invention may comprise inhibitors of IDO enzyme.
[00154] In some embodiments, payloads of the present invention may comprise chemokines and chemokine receptors. Chemokines are a family of secreted small cytokines, or signaling proteins that can induce directed chemotaxis in nearby responsive cells. Tire chemokine may be a SCY (small cytokine) selected from the group consisting of SCYAl-28 (CCLl-28), SCYBl-16 (CXCLl-16), SCYCl-2 (XCLl-2), SCYD-1 and SCYE-1; or a C chemokine selected from XCL1 and XCL2; or a CC chemokine selected from CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL I O, (CI . 1 1 , CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, ('('1.24. CCL25, CCL26, CCL27 and CCL28; or a CXC chemokine selected from CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCLIO, CXCL11 , CXCL12, CXCL13, CXCL14, CXCL15, CXCL16 and CXCL17; or a CX3C chemokine CX3CL1. In some aspects, the chemokine receptor may be a receptor for the C chemokines including XCR1 ; or a receptor for the CC chemokines including CCRl, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 and CCR10; or a receptor for the CXC chemokines including CXCR1 , CXCR2, CXCR3, CXCR4 and CXCR5; or a CX3C chemokine receptor CX3CR1.
[0015S] In some embodiments, payloads of the present invention may comprise other immunomodulators that play a critical role in immunotherapy, such as GM-CSF (Granulocyte- macrophage colony stimulating factor), erythropoietin (EPO), MIP3a, monocyte chemotactic protein (MCP)-l, intracellular adhesion molecule (ICAM), macrophage colony stimulating factor (M-CSF), Interleukin-1 receptor activating kinase (iRAK-1), lactotransferrin, and granulocyte colony stimulating factor (G-CSF).
[00156] In some embodiments, the payload of the present invention may comprise
Amphiregulin. Amphiregulin (AREG) is an EGF-like growth factor which binds to the EGFR receptor and enhances CD4+ regulatory T cells (T'regs) function. AREG promotes immune suppression in the tumor environment. Thus, in some embodiment, the payloads of the present invention may comprise Amhiregulin to dampen immune response during immunotherapy.
[00157] In some embodiments, payloads of the present in vention may comprise fusion proteins wherein a cytokine, chemokine and/or other soluble factor may be fused to other biological molecules such as antibodies and or ligands for a receptor. Such fusion molecules may increase the half-life of the cytokines, reduce systemic toxicity, and increase local concentration of the cytokines at the tumor site. Fusion proteins containing two or more cytokines, chernokines and or other soluble factors may be utilized to obtain synergistic therapeutic benefits. In one embodiment, payload may be a GM-CSF/IL2 fusion protein.
[00158] In one embodiment, the payloads of the present invention may be cytokines fu sed to TNF alpha ectodornain. Such payloads are produced as membrane associated cytokines fused to the TNF ectodornain . In one embodiment, the cytokine may be shed from the cell surface by the action of membrane associated proteases, and/or proteases in the extracellular space e.g. MMP9. Any of the cytokines described herein may be useful in the present invention. Such cytokine- TNF scaffold constructs may be used to preserve the native sequence of the processed cytokine while preserving regulation.
[00159] In some embodiments, the present invention provides methods for tuning the expression and function of an immunotherapeutic agent by operably linking it to an SRE within the effector module. Tuning of the immunotherapeutic agent may be invitro in cells or in vivo in a subject. In one embodiment, the immunotherapeutic agent is IL15. In another embodiment, the immunotherapeutic agent is a whole or a portion of IL 15 fused to the whole or a portion of IL15Ra. In some embodiments, the SRE is a DD. The immunotherapeutic agent may be stabilized by the stabilization ratio of 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, 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, 90-95, 90-100 or 95-100. In some embodiments the DD destabilizes the immunotherapeutic agen by a destabilization ratio of 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, 0.6-0.9,0.7-0.8, 0.7-0.9 or 0.8-0.9. As used herein, the term "tune" means to adjust, balance or adapt one thing in response to a stimulus or toward a particular outcome. In one non-limiting example, the SREs and/or DDs of the present invention adjust, balance or adapt the function or structure of compositions to which they are appended, attached or associated with in response to particular stimuli and/or environments. In some embodiments, the compositions of the present invention may be used to tune the expression or function of an immunotherapeutic to less than, or equal to or greater than the expression or function achieved by a constitutively expressed construct.
[00160] In some embodiments, ligands that do not affect the activity of the immune cell, and/or the chimeric antigen receptor, in the absence of the SREs may be preferably selected.
[00161] In some embodiments, the IL12 levels secreted by the immune cells of the invention may approximately be comparable to the TL12 levels secreted by human myeloid dendritic cells (mDCl ), when activated with TLR agonists. In one embodiment, the TLR agonist may be the combination of lipopolysaccharide administered with R848.
[00162] In some embodiments, the IFN gamma secreted by IL 12 induced activation of the immune cells is at least 5 fold greater in the presence of ligand, compared to the levels in the absence of ligand.
[00163] In some embodiments, the IFN gamma secreted by IL15 induced activation of the immune cells is at least 10-fold greater in the presence of ligand, compared to the levels in the absence of ligand.
[00164] In some embodiments, regulation of IL12 provides the necessary safety switch. In some embodiments, IL12 secretion recruit and/or activates effector cells in the tumor
microenvironment. In some embodiments, the IL12 regulation provides a benefit to CAR T function without causing toxicity .
[00165] In some embodiments, regulation of IL15-IL15Ra fusion proteins provides a safety switch as compared to constitutively expressed IL15-IL 15Ra. In some embodiments, 11/15- IL15Ra leads to better expansion, and/or persistence of CAR T cells.
3. Additional effector module features
[001 6] The effector module of the present invention may further comprise a signal sequence which regulates the distribution of the payload of interest, a cleavage and/or processing feature which facilitate cleavage of the payload from the effector module construct, a targeting and/or penetrating signal which can regulate the cellular localization of the effector module, a tag, and/or one or more linker sequences which link different components of the effector module.
Signal sequences
[00167] In addition to the SRE (e.g., DD) and payload region, effector modules of the invention may further comprise one or more signal sequences. Signal sequences (sometimes referred to as signal peptides, targeting signals, target peptides, localization sequences, transit peptides, leader sequences or leader peptides) direct proteins (e.g., the effector module of the present invention) to their designated cellular and/or extracellular locations. Protein signal sequences play a central role in the targeting and translocation of nearly all secreted proteins and many integral membrane proteins.
[00168] A signal sequence is a short (5-30 amino acids long) peptide present at tlie N-terminus of the majority of newly synthesized proteins that are destined towards a particular location. Signal sequences can be recognized by signal recognition particles (SRPs) and cleaved using type I and type II signal peptide peptidases. Signal sequences derived from human proteins can be incorporated as a regulatory module of the effector module to direct the effector module to a particular cellular and/or extracellular location. These signal sequences are experimentally verified and can be cleaved (Zhang et al., Protein Sci. 2004, 13:2819-2824).
[00169] In some embodiments, a signal sequence may be, although not necessarily, located at the N-terminus or C-terminus of tlie effector module, and may be, although not necessarily , cleaved off the desired effector module to yield a "mature" pavload, i.e., an immunotherapeutic agent as discussed herein,
[00170] In some examples, a signal sequence may be a secreted signal sequence derived from a naturally secreted protein, and its variant thereof. In some instances, the secreted signal sequences may be cytokine signal sequences such as, but not limited to, IL2 signal sequence comprising amino acid of SEQ. ID NO. 52, encoded by tlie nucleotide of SEQ. ID NO. 58-61 and/or p40 signal sequence comprising the ammo acid sequence of SEQ. ID NO. 247, encoded by the nucleotide of SEQ. ID NO. 248-256.
[00171 ] In some instances, signal sequences directing the payload of interest to the surface membrane of the target cell may be used. Expression of the payload on the surface of the target ceil may be useful to limit the diffusion of tlie payload to non-target in vivo environments, thereby potentially improving the safety profile of the payloads. Additionally, the membrane presentation of the payload may allow for physiologically and qualitative signaling as well as stabilization and recycling of the payload for a longer half-life. Membrane sequences may be the endogenous signal sequence of the N terminal component of the payload of interest. Optionally, it may be desirable to exchange this sequence for a different signal sequence. Signal sequences may be selected based on their compatibility with tlie secretory pathway of the cell type of interest so that the payload is presented on the surface of the T cell. In some embodiments, the signal sequence may be IgE signal sequence comprising amino acid SEQ. ID NO. 219 and nucleotide sequence of SEQ. ID NO. 229, 343, or 344, an ILI 5Ra signal sequence comprising amino acid SEQ. ID NO. 385 and nucleotide sequence of SEQ. ID NO. 386, or CD8a signal sequence (also referred to as CD8a leader) comprising amino acid SEQ. ID NO. 257 and nucleotide sequence of SEQ, ID NO. 258-262. [00172] Other examples of signal sequences include, a variant may be a modified signal sequence discussed in U.S. Pat. NOs. 8, 148, 494; 8,258,102; 9,133,265; 9,279,007; and U.S. patent application publication NO. 20070141666; and International patent application publication NO. WQ1993018181; the contents of each of which are incorporated herein by reference in their entirety.
[00173] In other examples, a signal sequence may be a heterogeneous signal sequence from other organisms such as virus, yeast and bacteria, which can direct an effector module to a particular cellular site, such as a nucleus (e.g., EP 1209450). Other examples may include Aspartic Protease (NSP24) signal sequences from Trichoderma that can increase secretion of fused protein such as enzymes (e.g., U. S. Pat. NO. 8,093,016 to Cervin and Kim), bacterial lipoprotein signal sequences (e.g., PCX application publication NO. WO199109952 to Lau and Ri ux), E.coli enterotoxin II signal peptides (e.g., U.S. Pat. NO. 6,605,697 to Kwon et al.), E.coli secretion signal sequence (e.g., U.S. patent publication NO. US2016090404 to Malley et al , a lipase signal sequence from a methylotrophic yeast (e.g., U.S. Pat. NO. 8,975,041 ), and signal peptides for DNases derived from Coryneform bacteria (e.g., U.S. Pat. NO. 4,965,197); the contents of each of which are incorporated herein by reference in their entirety.
[00174] Signal sequences may also include nuclear localization signals (NLSs), nuclear export signals (NESs), polarized cell tubuio-vesicular structure localization signals (See, e.g., U.S. Pat. NO. 8, 993,742; ("our et al, Nucleic Acids Res. 2003, 31(1): 393-396; the contents of each of which are incorporated herein by reference in their entirety) .extracellular localization signals, signals to subcellular locations (e.g. lysosome, endoplasmic reticulum, golgi, mitochondria, plasma membrane and peroxisomes, etc.) (See, e.g., U.S. Pat. NO. 7,396,811; and Negi et al., Database, 2,015, 1-7; the contents of each of which are incorporated herein by reference in their entirety).
[00175] In some embodiments, signal sequences of the present invention, include without limitation, any of those taught in Table 6 of copending commonly owned U.S. Provisional Patent Application No. 62/320,864 filed on 4/1 1/20.16, or in US Provisional Application No.
62/466,596 filed March 3, 2017 and the International Publication WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.
Cleavage sites
[00176] In some embodiments, the effector module comprises a cleavage and/or processing feature. The effector module of the present invention may include at least one protein cleavage signal/site. The protein cleavage signal/site may be located at the N-ternimus, the C-terminus, at any space between the N- and the C- termini such as, but not limited to, half-way between the N- and C-termini, between the N-terminus and the half-way point, between the half-way point and the C-terminus, and combinations thereof.
[00177] The effector module may include one or more cleavage signal(s)/site(s) of any proteinases. The proteinases may be a serine proteinase, a cysteine proteinase, an endopeptidase, a dipeptidase, a metalloproteinase, a glutamic proteinase, a threonine proteinase and an aspartic proteinase. In some aspects, the cleavage site may be a signal sequence of furin, actinidain, calpain-1, carboxypeptidase A, carboxypeptidase P, carboxypeptidase Y, caspase-1, caspase-2, caspase-3, caspase-4, easpase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, cathepsin B, cathepsin C, cathepsin G, cathepsin H, cathepsin K, cathepsin L, cathepsin S, cathepsin V, clostripain, chymase, chymotrypsin, elastase, endoproteinase, enterokinase, factor Xa, formic acid, granzyme B, Matrix irietallopeptidase-2, Matrix metallopeptidase-3, pepsin, proteinase K, SUMO protease, subtilisin, TEV protease, thermolysin, thrombin, trypsin and TAGZyme.
[00178] In one embodiment, the cleavage site is a furin cleavage site comprising the amino acid sequence SARNRQKRS (SEQ. ID NO. 263), encoded by nucleotide sequence of SEQ. ID NO. 264; or a revised furin cleavage site comprising the amino acid sequence ARNRQKRS (SEQ. ID NO. 265), encoded by nucleotide sequence of SEQ. ID NO. 266: or a modified furin site comprising the amino acid sequence ESRRVRRNKRSK (SEQ. ID NO. 267), encoded by nucleotide sequence of SEQ. ID NO. 268-270.
[00179] In some embodiments, cleavage sites of the present invention, include without limitation, any of those taught in Table 7 of copending commonly owned U.S. Provisional Patent Application No. 62/320,864 filed on 4/11/2016, or in US Provisional Application No.
62/466,596 filed March 3, 2017 and the International Publication WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.
Protein tags
[00180] In some embodiments, the effector module of the invention may comprise a protein tag. The protein tag may be used for detecting and monitoring the process of the effector module. The effector module may include one or more tags such as an epitope tag (e.g., a FLAG or hemagglutinin (HA) tag). A large number of protein tags may be used for the present effector modules. They include, but are not limited to, self-labeling polypeptide tags (e.g., haloalkane dehalogenase (halotag2 or halotag7), A CP tag, clip tag, MCP tag, snap tag), epitope tags (e.g., FLAG, HA, His, and Myc), fluorescent tags (e.g., green fluorescent protein (GFP), red fluorescent protein (RFP), yello fluorescent protein (YFP), and its variants), bioiumine scent tags (e.g. luciferase and its variants), affinity tags (e.g., maltose-binding protein (MBP) tag, glutathione-S-transferase (GST) tag), immunogenic affinity tags (e.g., protein A/G, IRS, AU1 , AU5, glu-glu, KT3, S-tag, HSV, VSV-G, Xpress and V5), and other tags (e.g., biotin (small molecule), StrepTag (StrepII), SBP, biotin carboxyl carrier protein (BCCP), eXact, CBP, CYD, HPC, CBD mtein-chitin binding domain, Trx, NorpA, and NusA.
[00181] In other embodiments, a tag may also be selected from those disclosed in U.S. Pat. NOs. 8,999,897; 8,357,51 1 ; 7,094, 568; 5,011 ,912; 4,851 ,341 ; and 4,703,004; U.S patent application publication NOs. US2013115635 and US2013012687; and International application publication NO. WO2013091661; the contents of each of which are incorporated herein by reference in their entirety.
[00182] In some aspects, a multiplicity of protein tags, either the same or different tags, may be used; each of the tags may be located at the same N- or C-terminus, whereas in other cases these tags may be located at each terminus.
[00183] In some embodiments, protein tags of the present invention, include without limitation, any of those taught in Table 8 of copending commonly owned U.S. Provisional Patent
Application No. 62/320,864 filed on 4/11/2016, or in US Provisional Application No.
62/466,596 filed March 3, 2017 and the International Publication WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.
Targeting peptides
[00184] In some embodiments, the effector module of the invention may further compri se a targeting and/or penetrating peptide. Small targeting and/or penetrating peptides that selectively recognize cell surface markers (e.g. receptors, trans-membrane proteins, and extra-cellular matrix molecules) can be employed to target the effector module to the desired organs, tissues or cells. Short peptides (5-50 amino acid residues) synthesized in vitro and naturally occurring peptides, or analogs, variants, derivatives thereof, may be incorporated into the effector module for homing the effector module to the desired organs, tissues and cells, and/or subcellular locations inside the cells.
[00185] In some embodiments, a targeting sequence and/or penetrating peptide may be included in the effector module to drive the effector module to a target organ, or a tissue, or a cell (e.g., a cancer cell). In other embodiments, a targeting and/or penetrating peptide may direct the effector module to a specific subcellular location inside a cell.
Θ0186] A targeting peptide has any number of amino acids from about 6 to about 30 inclusive. The peptide may have 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. Generally, a targeting peptide may have 25 or fewer amino acids, for example, 20 or fewer, for example 15 or fewer. [00187] Exemplary targeting peptides may include, but are not limited to, those disclosed in the art, e.g., U.S. Pat. NOs, 9,206,231; 9, 110,059; 8,706,219; and 8,772,449; and U.S. application publication NOs. 2016089447; 2016060296; 2016060314; 2016060312; 201606031 1 ;
2016009772; 2016002613; 2015314011 and 2015166621; and International application publication NOs, WO2015179691 and WO2015183044; the contents of each of which are incorporated herein by reference in their entirety.
[00188] In some embodiments, targeting peptides of the present invention, include without limitation, any of those taught in Table 9 of copending commonly owned U.S. Provisional Patent Application No. 62/320,864 filed on 4/1 /2016, or in US Provisional Application No.
62/466,596 filed March 3, 2017 and the International Publication WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.
Linkers
[00189] In some embodiments, the effector module of the invention may further comprise a linker sequence. The linker region selves primarily as a spacer between two or more
polypeptides within the effector module. The "linker" or "spacer", as used herein, refers to a molecule or group of molecules that connects two molecules, or two parts of a molecule such as two domains of a recombinant protein.
[00190] In some embodiments, "Linker" (L) or "linker domain" or "linker region" or "linker module" or "peptide linker" as used herein refers to an oligo- or polypeptide region of from about I to 100 amino acids in length, which links together any of the domains/regions of the effector module (also called peptide linker). The peptide linker may be 1 -40 amino acids in length, or 2-30 amino acids in length, or 20-80 amino acids in length, or 50-100 amino acids in length. Linker length may also be optimized depending on the type of payload utilized and based on the crystal structure of the payload. In some instances, a shorter linker length may be preferably selected. In some aspects, the peptide linker is made up of amino acids linked together by peptide bonds, preferably from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I), Serine (S), Cysteine (C), Threonine (T), Methionine (M), Proline (P), Phenylalanine (F), Tyrosine (Y), Tryptophan (W), Histidine (H), Lysine (K), Arginine (R), Aspartate (D), Glutamic acid (E), Asparagine (N), and Glutamine (Q). One or more of these ammo acids may be glycosylated, as is understood by those in the art. In some aspects, amino acids of a peptide linker may be selected from Alanine (A), Glycine (G), Proline (P), Asparagine (R), Serine (S), Glutamine (Q) and Lysine (K). [00191] In one example, an artificially designed peptide Sinker may preferably be composed of a polymer of flexible residues like Glycine (G) and Serine (S) so that the adjacent protein domains are free to move relative to one another. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not interfere with one another. The choice of a particular linker sequence may concern if it affects biological activity, stability, folding, targeting and/or pharmacokinetic features of the fusion construct. Examples of peptide linkers include, but are not limited to: MH, SG, GGSG (SEQ. ID NO. 271; encoded by the nucleotide sequence SEQ. ID NO. 272), GGSGG (SEQ, ID NO. 54; encoded by any of the nucleotide sequences SEQ, ID NO. 64-68), GGSGGG (SEQ. ID NO. 273; encoded by any of the nucleotide sequences SEQ. ID NO, 274-275), SGGGS (SEQ. ID NO. 276; encoded by the nucleotide sequence SEQ. ID NO. 277, 310, 389), GGSGGGSGG (SEQ. ID NO. 278; encoded by the nucleotide sequence SEQ. ID NO. 279), GGGGG (SEQ. ID NO. 280), GGGGS (SEQ, ID NO. 281) or (GGGGS)n (n=l (SEQ. ID NO. 281), 2 (SEQ. ID NO. 282), 3 (SEQ. ID NO, 283, encoded by SEQ. ID NO. 319-324), 4 (SEQ. ID NO. 284), 5 (SEQ. ID NO. 285), or 6 (SEQ. ID NO. 286)), SSSSG (SEQ. ID NO. 287) or (SSSSG)n (n=l (SEQ. ID NO. 287), 2 (SEQ. ID NO. 288), 3 (SEQ. ID NO. 289), 4 (SEQ. ID NO. 290), 5 (SEQ. ID NO, 291), or 6 (SEQ. ID NO. 292)),
SGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ. ID NO. 220; encoded by the nucleotide sequence SEQ. ID NO. 230, 373-377), EFSTEF (SEQ. ID NO. 53; encoded by any of the nucleotide sequences SEQ, ID NO. 62-63), GKSSGSGSESKS (SEQ, ID NO. 311),
GGSTSGSGKSSEGKG (SEQ. ID NO. 312), GSTSGSGKSSSEGSGSTKG (SEQ, ID NO. 313), GSTSGSGKPGSGEGSTKG (SEQ. ID NO. 314), VDYPYDVPDYALD (SEQ. ID NO. 315; encoded by nucleotide sequence SEQ. ID NO. 316), EGKSSGSGSESKEF (SEQ. ID NO. 317), GGGGSGGGGSGGGGS (SEQ. ID NO. 283; encoded by any of the nucleotide sequences SEQ. ID NO. 319-324), SGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGS (SEQ, ID NO. 309; encoded by SEQ. ID NO. 390), SGGGSGGGGSGGGGSGGGGS (SEQ. ID NO. 318; encoded by SEQ. ID NO. 391), GS (encoded by GG'TTCC), SG (encoded by AGCGGC), or GSG (encoded by GGATCCGGA or GGATCCGGT).
[00192] In other examples, a peptide linker may be made up of a majority of amino acids that are sterically unhindered, such as Glycine (G) and Alanine (A). Exemplary linkers are
polyglycines (such as (G)4 (SEQ ID NO. 535), (G)5 (SEQ ID NO. 280), (G)8 (SEQ ID NO. 536)), poly(GA), and polyalanines. The linkers described herein are exemplary, and linkers that are much longer and which include other residues are contemplated by the present invention. [00193] A linker sequence may be a natural linker derived from a multi-domain protein, A natural linker is a short peptide sequence that separates two different domains or motifs within a protein.
[00194] In some aspects, linkers may be flexible or rigid. In other aspects, linkers may be cleavabie or non- cleavable. As used herein, the terms "cleavable linker domain or region" or "cleavable peptide linker" are used interchangeably. In some embodiments, the linker sequence may be cleaved enzymatically and/or chemically. Examples of enzymes (e.g.,
proteinase/peptidase) useful for cleaving the peptide linker include, but are not limited, to Arg-C proteinase, Asp-N endopeptidase, chymotrypsin, clostripain, enterokinase, Factor Xa, glutamyl endopeptidase, Granzyme B, Achromobacter proteinase I, pepsin, proline endopeptidase, proteinase K, Staphy lococcal peptidase I, thermolysm, thrombin, trypsin, and members of the Caspase family of proteolytic enzymes (e.g. Caspases 1-10). Chemical sensitive cleavage sites may also be included in a linker sequence. Examples of chemical cleavage reagents include, but are not limited to, cyanogen bromide, which cleaves methionine residues; N-chloro succinirnide, iodobenzoic acid or BNPS-skatole (2-(2-nitrophenyisulfenyi)-3-metliyiindole), which cleaves tryptophan residues; dilute acids, which cleave at aspartyl -prolyl bonds; and e aspartic acid- proline acid cleavabie recognition sites (i.e., a cleavable peptide linker comprising one or more D-P dipeptide moieties). The fusion module may include multiple regions encoding peptides of interest separated by one or more cleavable peptide linkers.
[00195] In other embodiments, a cleavable linker may be a "self-cleaving" linker peptide, such as 2A linkers (for example T2A), 2A-iike linkers or functional equivalents thereof and combinations thereof. In some embodiments, the linkers include the picornaviral 2A-3ike linker, CHYSEL sequences of porcine teschovirus (P2A), Tliosea asigna viras (T2A) or combinations, variants and functional equivalents thereof. Other linkers will be apparent to those skilled in the art and may be used in connection with alternate embodiments of the invention.
[00196] As a non-limiting example, the P2A cleavable peptide may be
GATNFSLLKQAGDVEENPGP (SEQ, ID NO. 350; encoded by SEQ. ID NO, 35 ).
[00197] In some embodiments, the biocircuits of the present invention may include 2A peptides. The 2A peptide is a sequence of about 20 amino acid residues from a virus that is recognized by a protease (2A peptidases) endogenous to the cell. The 2A peptide was identified among picornaviruses, a typical example of which is the Foot-and Mouth disease virus (Robertson BH, et. a!., J Virol 1985, 54:651 -660). 2A-like sequences have also been found in Picornaviridae like equine rhinitis A virus, as well as unrelated viruses such as porcine teschovirus- 1 and the insect Thosea asigna virus (TaV). In such viruses, multiple proteins are derived from a large polyprotein encoded by an open reading frame. The 2 A peptide mediates the co-trans ational cleavage of this polyprotein at a single site that forms the junction between the vims capsid and replication polyprotein domains. The 2A sequences contain the consensus motif D-V/I-E-X-N-P- G-P. These sequences are thought to act co-translationally, preventing the formation of a normal peptide bond between the glycine and last proline, resulting in the ribosome skipping of the next codon (Donnelly ML et al . (2001), J Gen Virol, 82: 1013-1025). After cleavage, the short peptide remains fused to the C -terminus of the protein upstream of the cleavage site, while the proline is added to the N-terminus of the protein downstream of the cleavage site. Of the 2A peptides identified to date, four have been widely used namely FMDV 2A (abbreviated herein as F2A); equine rhinitis A virus (ERAV) 2A (E2A); porcine teschovinis-1 2 A (P2A) and Thoseaasigna virus 2A (T2A). In some embodiments, the 2A peptide sequences useful in the present invention are selected from SEQ ID NO.8-11 of International Patent Publication WO2010042490, the contents of which are incorporated by reference in its entirety.
[00198] The linkers of the present invention may also be non-peptide linkers. For example, alkyl linkers such as— -NH— (CH2) a-C(O)— , wherein a==2-20 can be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., Ci-Ce) lower acyl, halogen (e.g., CI, Br), CN, NH2, phenyl, etc.
[00199] In some aspects, the linker may be an artificial linker from U.S. Pat. NOs. 4,946,778: 5, 525, 491 ; 5,856,456; and International patent publication NOs. WO2012/083424; the contents of each of which are incorporated herein by reference in their entirety.
[00200] In some embodiments, linkers of the present invention, include without limitation, any of those taught in Table 11 of copending commonly owned U.S. 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 International Publication WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.
[00201] In one embodiment, the linker may be a spacer region of one or more nucleotides. Non- limiting examples of spacers are TCTAGATAATACGACTCACTAGAGATCC (SEQ. ID NO. 392), TATGGCCACAACCATG (SEQ. ID NO. 393),
AATCTAGATAATACGACTCACTAGAGATCC (SEQ. ID NO. 394), TCGCGAATG, or TCGCGA.
[00202] In one embodiment, the linker may be a BamHI site. As a non-limiting example, the BamHI site has the amino acid sequence GS and/or the DNA sequence GGATCC.
Embedded stimulus, signals and other regulatory features [00203] In some embodiments, the effector module of the present invention may further comprise one or more microRNAs, microRNA binding sites, promoters and tunable elements. In one embodiment, microRNA may be used in support of the creation of tunable biocircuits. Each aspect or timed modality may bring to the effector module or biocirciut a differentially tuned feature. For example, a destabilizing domain may alter cleavage sites or dimerization properties or half-life of the payload, and the inclusion of one or more microRNA or microRNA binding site may impart cellular detargeting or trafficking features. Consequently, the present invention embraces biocircuits which are multifactorial in their tenability. Such biocircuits and effector modules may be engineered to contain one, two, three, four or more tuned features.
[00204] In some embodiments, micro RNA sequences of the present invention, include without limitation, any of those taught in Table 13 of copending commonly owned U.S. Provisional Patent Application No. 62/320,864 filed on 4/11/2016, or in US Provisional Application No. 62/466,596 filed March 3, 2017 and the International Publication WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.
Polynucleotides
[00205] The term "polynucleotide" or '"nucleic acid molecule" in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides, e.g., linked nucleosides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a β- D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino- a-LNA having a 2'-amino functionalization) or hybrids thereof.
[00206] In some embodiments, polynucleotides of the invention may be a messenger RNA (mRN A) or any nucleic acid molecule and may or may not be chemically modified. In one aspect, the nucleic acid molecule is a mRNA. As used herein, the term "messenger RNA (mRNA)" refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo,
[00207] Traditionally, the basic components of an mRNA molecule include at least a coding region, a 5'UTR, a 3'UTR, a 5' cap and a poly-A tail. Building on this wild type modular structure, the present invention expands the scope of functionality of traditional mRNA molecules by providing payload constructs which maintain a modular organization, but which comprise one or more stmctural and/or chemical modifications or alterations which impart useful properties to the polynucleotide, for example tenability of function. As used herein, a
"structural" feature or modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide without significant chemical modification to the nucleosides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide "ATCG" may be chemically modified to "AT-5meC-G". The same polynucleotide may be structurally modified from "ATCG" to "ATCCCG". Here, the dinucleotide "CC" has been inserted, resulting in a structural modification to the polynucleotide.
[00208] In some embodiments, polynucleotides of the present invention may harbor 5 'UTR sequences which play a role in translation initiation, 5 'UTR sequences may include features such as Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of genes, Kozak sequences have the consensus XCCR(A/G) CC AUG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG) and X is any nucleotide. In one embodiment, the Kozak sequence is ACCGCC. By engineering the features that are typically found in abundantly expressed genes of target cells or tissues, the stability an d protein production of the polynucleotides of the invention can be enhanced.
[002Θ9] Further provided are polynucleotides, which may contain an internal ribosome entry- site (IRES) which play an important role in initiating protein synthesis in the absence of 5' cap structure in the polynucleotide. An IRES may act as the sole ribosome binding site, or may serve as one of the multiple binding sites. Polynucleotides of the invention containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes giving rise to bicistronic and/or multicistronic nucleic acid molecules.
[00210] In some embodiments, polynucleotides encoding biocircuits, effector modules, SREs and payloads of interest such as immunotherapeutic agents may include from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from. 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from. 500 to 1 ,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 1 ,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1 ,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1 ,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1 ,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from. 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000 nucleotides). In some aspects, polynucleotides of the invention may include more than 10,000 nucleotides.
[00211] Regions of the polynucleotides which encode certain features such as cleavage sites, linkers, trafficking signals, tags or other features may range independently from 10-1,000 nucleotides in length (e.g., greater than 20, 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1 ,000 nucleotides),
[00212] In some embodiments, polynucleotides of the present invention may further comprise embedded regulatory moieties such as microRNA binding sites within the 3'UTR of nucleic acid molecules which when bind to microRNA molecules, down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation. Conversely, for the purposes of the polynucleotides of the present invention, microRNA binding sites can be engineered out of (i.e. removed from) sequences in which they naturally occur in order to increase protein expression in specific tissues. For example, miR-142 and miR-146 binding sites may be removed to improve protein expression in the immune cells. In some embodiments, any of the encoded payloads may be may be regulated by an SRE and then combined with one or more regulatory sequences to generate a dual or multi-tuned effector module or biocircuit system.
[0021.3] In some embodiments, polynucleotides of the present invention may encode fragments, variants, derivatives of polypeptides of the inventions. In some aspects, the variant sequence may keep the same or a similar activity. Alternatively, the variant may have an altered activity (e.g., increased or decreased) relative to the start sequence. Generally, variants of a particular polynucleotide or polypeptide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen et al., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res., 1997, 25:3389-3402.)
[00214] In some embodiments, polynucleotides of the present invention may be modified. As used herein, the terms "modified", or as appropriate, "modification" refers to chemical modification with respect to A, G, U (T in DNA) or C nucleotides. Modifications may be on the nucleoside base and/or sugar portion of the nucleosides which comprise the polynucleotide. In some embodiments, multiple modifications are included in the modified nucleic acid or in one or more individual nucleoside or nucleotide. For example, modifications to a nucleoside may include one or more modifications to the nucleobase and the sugar. Modifications to the polynucleotides of the present invention may include any of those taught in, for example, International Publication NO. WO2G13052523, the contents of which are incorporated herein by reference in its entirety.
[00215] As described herein "nucleoside" is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as "nucleobase"). As described herein, "nucleotide" is defined as a nucleoside including a phosphate group.
[00216] In some embodiments, the modification may be on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases "phosphate" and "phosphodiester" are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substitueni. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another interaucleoside linkage. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphat.es, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, aikyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates). Other modifications which may be used are taught in, for example, international Application NO. WO2013052523, the contents of which are incorporated herein by reference in their entirety.
[00217] Chemical modifications and/or substitution of the nucleotides or nucleobases of the polynucleotides of the invention which are useful in the present invention include any modified substitutes known in the art, for example, (±) 1 -(2-Hydroxypropyl)pseudouridine TP, (2R)-l -(2- Hydroxypropyl)pseudouridine TP, l-(4-Methoxy-phenyl)pseudo-UTP,,2'-0-dimetliyladenosine, l,2'-0-dimethylguanosine, 1 ,2'~0~dimethy]mosine, 1-Hexyl-pseudo-UTP, 1- Homoallylpseudouridine TP, 1 -Hydroxymethylpseudouridine TP, 1-iso-propyl-pseudo-UTP, 1- Me-2-thio-pseudo-UTP, I -Me-4-thio-pseudo-UTP, 1 -Me-alpha-thio-pseudo-UTP, 1 -Me-GTP, 2'-Amino-2 ' -deoxy-ATP, 2 ' -Amino-2' -deoxy-CTP, 2 ' -Amino-2 ' -deoxy-GTP, 2'-Amino-2' - deoxy-UTP, 2'-Azido-2'-deoxy-ATP, tubercidine, under modified hydroxywybutosine, uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, wybutosine, wyosine, xanthine, Xanthosine~5 '-TP, xylo-adenosine, zebularine, a-thio-adenosine, a-thio-cytidine, a-thio- guanosine, and/or a-thio-uridine.
[00218] Polynucleotides of the present invention may comprise one or more of the
modifications taught herein. Different sugar modifications, base modifications, nucleotide modifications, and/or interniicleoside linkages (e.g., backbone stractures) may exist at various positions in the polynucleotide of the invention. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased. A modification may also be a 5'' or 3' terminal modification. The polynucleotide may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or m relation to one or more types of nucleotide, i.e. any one or more of A, G, U or C) or any- intervening percentage (e.g., from l% to 20%, from l% to 25%, from l% to 50%, from l% to 60%, from i% to 70%, from l% to 80%, from l % to 90%, from l % to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from. 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from. 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).
[00219] In some embodiments, one or more codons of the polynucleotides of the present invention may be replaced with other codons encoding the native ammo acid sequence to tune the expression of the SREs, through a process referred to as codon selection. mRNA codon, and tR A anticodon pools tend to vary spatiotemporally i.e. among organisms, cell types, sub cellular locations and over time. Thus, the codon selection described herein is a spatiotemporal (ST) codon selection. [00220] In some embodiments of the invention, certain polynucleotide features may be codon optimized. Codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cell by replacing at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons of the native sequence with codons that are most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Codon usage may be measured using the Codon Adaptation Index (CAI) which measures the deviation of a coding polynucleotide sequence from a reference gene set. Codon usage tables are available at the Codon Usage Database (http://www.kazusa.or.jp/codon/) and the CAI can be calculated by EMBOSS CAI program (http://emboss.sourceforge.net/). Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include to match codon frequencies in target and host organisms to ensure proper folding, bias nucleotide content to alter stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein signaling sequences, remove/add post translation modification sites in encoded protein (e.g. giycosyiation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and degradation sites, to adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art, and non-limiting examples include services from. Gene Art (Life Technologies), DNA2.0 (Menlo Park CA), OptimumGene (GenScript,
Piscataway, NJ), algorithms such as but not limited to, DNA Works v3.2.3 and/or proprietary methods. In one embodiment, a polynucleotide sequence or portion thereof is codon optimized using optimization algorithms. Codon options for each amino acid are well-known in the art as are various species table for optimizing for expression in that particular species.
[00221] In some embodiments of the invention, certain polynucleotide features may be codon optimized. For example, a preferred region for codon optimization may be upstream (5') or downstream (3') to a region which encodes a polypeptide. These regions may be incorporated into the polynucleotide before and/or after codon optimization of the payioad encoding region or open reading frame (ORF).
[00222] After optimization (if desired), the polynucleotide components are reconstituted and transformed into a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.
[00223] Spatiotemporal codon selection may impact the expression of the polynucleotides of the invention, since codon composition determines the rate of translation of the mR A species and its stability. For example, since tRNA anticodons to optimized codons are abundant, and thus translation may be enhanced. In contrast, tRNA anticodons to less common codons are fewer and thus translation may proceed at a slower rate. Presnyak et al. have shown that the stability of an mR A species is dependent on the codon content, and higher stability and thus higher protein expression may be achieved by utilizing optimized codons (Presnyak et al. (2015) Cell 160, 1 1 1 1-1 24; the contents of which are incorporated herein by reference in their entirety). Thus, in some embodiments, ST codon selection may include the selection of optimized codons to enhance the expression of the SRES, effector modules and biocircuits of the invention. In other embodiments, Spatiotemporal codon selection may involve the selection of codons that are less commonly used in the genes of the host cell to decrease the expression of the compositions of the invention. The ratio of optimized codons to codons less commonly used in the genes of the host cell may also be varied to tune expression.
[00224] In some embodiments, certain regions of the polynucleotide may be preferred for codon selection. For example, a preferred region for codon selection may be upstream (5') or downstream (3') to a region which encodes a polypeptide. These regions may be incorporated into the polynucleotide before and/or after codon selection of the payload encoding region or open reading frame (ORF).
[00225] The stop codon of the polynucleotides of the present invention may be modified to include sequences and m otifs to alter the expression levels of the SREs, payloads and effector modules of the present invention. Such sequences may be incorporated to induce stop codon readthrough, wherein the stop codon may specify amino acids e.g. selenocysteine or pyrrol ysine. In other instances, stop codons may be skipped altogether to resume translation through an alternate open reading frame. Stop codon read through may be utilized to tune the expression of components of the effector modules at a specific ratio (e.g. as dictated by the stop codon context). Examples of preferred stop codon motifs include UGAN, UAAN, and UAGN, where N is either C or U.
[00226] Polynucleotide modifications and manipulations can be accomplished by methods known in the art such as, but not limited to, site directed mutagenesis and recombinant technology. The resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.
[00227] In some embodiments, polynucleotides of the invention may comprise two or more effector module sequences, or two or more payloads of interest sequences, which are in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times. In these patterns, each Setter, A, B, or C represent a different effector module component.
[00228] In yet another embodiment, polynucleotides of the invention may comprise two or more effector module component sequences with each component havmg one or more SRE sequences (DD sequences), or two or more payload sequences. As a non-limiting example, the sequences may be in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times in each of the regions. As another non-limiting example, the sequences may be in a pattern such as ABABAB or
AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times across the entire polynucleotide. In these patterns, each letter, A, B, or C represent a different sequence or component.
[00229] According to the present invention, polynucleotides encoding distinct biocircuits, effector modules, SREs and payload constructs may be linked together through the 3 '-end using nucleotides which are modified at the 3 '-terminus. Chemical conjugation may be used to control the stoichiometry of delivery into cells. Polynucleotides can be designed to be conjugated to other polynucleotides, dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, (MPEG)2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucl ease , proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell, hormones and hormone receptors, non-peptidic species, such as lipids, lectins,
carbohydrates, vitamins, cofactors, or a drug. As non-limiting examples, they may be conjugates with other immune conjugates.
[00230] In some embodiments, polynucleotides of the present invention may encode a fusion polypeptide comprising a destabilizing domain (DD) and at least one immunotherapeutic agent taught herein. The DD domain may be a FKBP mutant encoded by nucleotide sequence of SEQ. ID NO. 325-327, and/or 235-238, an ecDHFR mutant encoded by nucleotide sequence of SEQ. ID NO. 73, 233, 234, 372 and/or 395, hDHFR mutant encoded by nucleotide sequence of SEQ. ID NO. 239, 240, 293-303 371, 396-403, 406, 442, 447, 450, and/or 469-503.
[00231] In some embodiments, the polynucleotides of the invention may encode effector modules comprising IL15 as the payload comprising the nucleotide sequence of SEQ ID NOs. 74-75, 355-357, or IL15/IL15Ra fusion polypeptide as the payload comprising the nucleotide sequence of SEQ ID NOs. 241-246, 349, 402, 405, 408, 410, 414, 416, 418, 420, 422, 425, 427, 429-431, 433, 435, 437, 439, 441, and/or 444.
Cells
[00232] In accordance with the present invention, ceils genetically modified to express at least one biocircuit, SRE (e, g, DD), effector module and immunotherapeutic agent of the invention, are provided. Cells of the invention may include, without limitation, immune cells, stem cells and tumor cells. In some embodiments, immune cells are immune effector cells, including, but not limiting to, T cells such as CDS'*" T cells and CD4+ T cells (e.g., Thl , Th2, Th l7, Foxp3+ ceils), memory T cells such as T memory stem cells, central T memory cells, and effector memory T cells, terminally differentiated effector T ceils, natural killer (NK) cells, NK T ceils, tumor infiltrating lymphocytes (TILs), cytotoxic T lymphocytes (CTLs), regulator}- T cells (Tregs), and dendritic cells (DCs), other immune cells that can elicit an effector function, or the mixture thereof. T cells may be Ταβ cells and Τγδ cells. In some embodiments, stem cells may be from human embryonic stem ceils, mesenchymal stem cells, and neural stem ceils. In some embodiments, T cells may be depleted endogenous T cell receptors (See US Pat. NOs. 9, 273, 283; 9, 181, 527; and 9,028, 812; the contents of each of which are incorporated herein by reference in their entirety).
[00233] In some embodiments, cells of the invention may be autologous, allogeneic, syngeneic, or xenogeneic in relation to a particular individual subject.
[00234] In some embodiments, cells of the invention may be mammalian cells, particularly human ceils. Cells of the invention may be primary cells or immortalized cell lines.
[00235] In some embodiments, cells of the invention may include expansion factors as payload to trigger proliferation and expansion of the ceils. Exemplar ' payioads include RAS such as KRAS, NRAS, RRAS, RRAS2, MRAS, ERAS, and HRAS, DIRAS such as DIRASl, DIRAS2, and DIRAS3, NKIRAS such as NKIRAS 1, and NKIRAS2, RAL such as RALA, and RALB, RAP such as RAP I A, RAP1B, RAP2A, RAP2B, and RAP2C, RASD such as RASD1 , and RASD2, RASL such as RASL10A, RASL10B, RASL11A, RASL11 B, and RASL12, REM such as REM1, and REM2, GEM, RERG, RERGL, and RRAD.
[00236] Engineered immune cells can be accomplished by transducing a cell compositions with a polypeptide of a biocircuit, an effector module, a SRE and/or a payload of interest (i.e., immunotherapeutic agent), or a polynucleotide encoding said polypeptide, or a vector comprising said polynucleotide. The vector may be a viral vector such as a lentiviral vector, a gamma-retro viral vector, a recombinant AAV, an adenoviral vector and an oncolytic viral vector. In other aspects, non-viral vectors for example, nanoparticles and liposomes may also be used. In some embodiments, immune cells of the invention are genetically modified to express at least one immunotherapeutic agent of the invention which is tunable using a stimulus. In some examples, two, three or more immunotherapeutic agents constructed in the same biocircuit and effector module are introduced into a cell In other examples, two, three, or more biocircuits, effector modules, each of which comprises an immunotherapeutic agent, may be introduced into a cell.
[00237] In some embodiments, immune cells of the invention may be NK cells modified to payloads and effector modules taught herein.
[00238] Natural killer (NK) ceils are members of the innate lymphoid cell family and characterized in humans by expression of the phenotypic marker CD56 (neural cell adhesion molecule) in the absence of CD3 (T-cell co-receptor). NK cells are potent effector cells of the innate immune system which mediate cytotoxic attack without the requirement of prior antigen priming, forming the first line of defense against diseases including cancer malignancies and viral infection.
[00239] Several pre-clinical and clinical trials have demonstrated that adoptive transfer of NK ceils is a promising treatment approach against cancers such as acute myeloid leukemia (Ruggeri et ai., Science; 2002, 295: 2097-2100; and Gelier et ai., Immunotherapy, 2011, 3: 1445-1459). Adoptive transfer of NK cells expressing CAR such as DAP12-Based Activating CAR revealed improved eradication of tumor cells (Topfer et al., J Immunol. 2015; 194:320 -3212), NK cell engineered to express a CS-1 specific CAR also displayed enhanced cytolysis and interferon-γ (lFN-γ) production in multiple myeloma (Chu et ai., Leukemia, 2014, 28(4): 917-927).
[00240] NK cell activation is characterized by an array of receptors with activating and inhibitory functions. The important activation receptors on NK cells include CD94/NKG2C and NKG2D (the C-type lectin-like receptors), and the natural cytotoxicity receptors (NCR) NKp30, NKp44 and NKp46, which recognize ligands on tumor cells or virally infected cells. NK cell inhibition is essentially mediated by interactions of the polymorphic inhibitory killer cell immunoglobulin-like receptors (KIRs) with their cognate human-leukocyte-antigen (HLA) ligands via the alpha- 1 helix of the HLA molecule. The balance between signals that are generated from activating receptors and inhibitor}' receptors mainly determines the immediate cytotoxic activation .
[00241] NK cells may be isolated from peripheral blood mononuclear cells (PBMCs), or derived from human embryonic stem (ES) cells and induced piuripotent stem ceils (iPSCs). The primary NK cells isolated from PBMCs may be further expanded for adoptive immunotherapy. Strategies and protocols useful for the expansion of NK cells may include interleukin 2 (IL2) stimulation and the use of autologous feeder cells, or the use of genetically modified allogeneic feeder cells. In some aspects, NK cells can be selectively expanded with a combination of stimulating ligands including IL15, IL21, 1L2, 41BBL, IL12, IL18, MICA, 2B4, LFA-1, and BCM1/SLAMF2 (e.g., US patent publication NO. US20150190471).
[00242] Immune cells expressing effector modules comprising a CAR and/or other
immunotherapeutic agents can be used as cancer immunotherapy. Hie immunotherapy comprises the cells expressing a CAR and/or other immunotherapeutic agents as an active ingredient, and may further comprise a suitable excipient. Examples of the excipient may include the aforementioned pharmaceutically acceptable excipients, including various cell culture media, and isotonic sodium chloride.
[00243] In some embodiments, cells of the present invention may be utilized as artificial antigen presenting cells to propagate and activate clinical grade T cells or NK cells for immunotherapy. As a non-limiting example, K562 erytfiroleukemia cells may be used for this purpose.
[00244] In some embodiments, cells of the present invention may be dendritic cells that are genetically modified to express the compositions of the invention. Such cells may be used as cancer vaccines.
HI. PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS
[00245] The present invention further provides pharmaceutical compositions comprising one or more biocircuits, effector modules, SREs (e.g., DDs), stimuli and payloads of interest (i.e., immunotherapeutic agents), vectors, cells and other components of the invention, and optionally at least one pharmaceutically acceptable excipient or inert ingredient.
[00246] As used herein the term "pharmaceutical composition" refers to a preparation of biocircuits, SREs, stimuli and payloads of interest (i.e., immunotherapeutic agents), other components, vectors, cells and described herein, or pharmaceutically acceptable salts thereof, optionally with other chemical components such as physiologically suitable carriers and excipients. The pharmaceutical compositions of the invention comprise an effective amount of one or more active compositions of the invention. The preparation of a pharmaceutical composition that contains at least one composition of the present invention and/or an additional active ingredient will be known to those skilled in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference.
[00247] The term '"excipient" or "inert ingredient" refers to an inactive substance added to a pharmaceutical composition and fonnulation to further facilitate administration of an active ingredient. For the purposes of the present disclosure, the phrase "active ingredient" generally refers to any one or more biocircuits, effector modules, SREs, stimuli and pay loads of interest (i.e., immunotherapeutic agents), other components, vectors, and cells to be delivered as described herein. The phrases "pharmaceutically acceptable'1 refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
[00248] In some embodiments, pharmaceutical compositions and formulations are administered to humans, human patients or subjects. Although the descriptions of pharmaceutical
compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, non-human mammals, including agricultural animals such as cattle, horses, chickens and pigs, domestic animals such as cats, dogs, or research animals such as mice, rats, rabbits, dogs and non-human primates. It will be understood that, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
[0Θ249] A pharmaceutical composition and formulation in accordance with tiie invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a con venient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
[00250] The compositions of the present invention may be formulated in any manner suitable for delivery. The formulation may be, but is not limited to, nanoparticles, poly (lactic-co- glycolic acid) (PLGA) microspheres, lipidoids, lipoplex, liposome, polymers, carbohydrates (including simple sugars), cationic lipids and combinations thereof.
[00251] In one embodiment, the formulation is a nanoparticle which may comprise at least one lipid. The lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12- 5, C 12-200, DLin-MC3 -DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG and PEGylated lipids. In another aspect, the lipid may be a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLm-MC 3 -DMA, DLin-KC2-DMA and DODMA. [00252] For polynucleotides of the invention, the formulation may be selected from, any of those taught, for example, in International Application PCTYU S2012/069610, the contents of which are incorporated herein by reference in its entirety.
[ΘΘ253] Relativ e amounts of the active ingredient, the pharmaceutically acceptable excipient or inert ingredient, and/or any additional ingredients in a pharmaceutical composition in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which die composition is to be administered. By¬ way of example, the composition may comprise between 0.1 and 100, e.g., between 0.5 and 50, between 1-30, between 5-80, at least 80 (w/w) active ingredient.
[00254] Efficacy of treatment or amelioration of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of compositions of the present invention, "effective against" for example a cancer, indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statisticaily significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in disease load, reduction in tumor mass or cell numbers, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of cancer.
[0025S] A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10 in a measurable parameter of disease, and preferably at least 20, 30, 40, 50 or more can be indicative of effective treatment. Efficacy for a given composition or fonnulation of the present invention can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change is observed.
IV. APPLICATIONS
[Θ0256] In one aspect of the present invention, methods for reducing a tumor volume or burden are provided. The methods comprise administering a pharmaceutically effective amount of a pharmaceutical composition comprising at least one biocircuit sy stem, effector module, DD, and/or payload of interest (i.e., an immunotherapeutic agent), at least one vector, or cells to a subject having a tumor. The biocircuit system and effector module having any immunotherapeutic agent as described herein may be in forms of a polypeptide, or a polynucleotide such as mRNA, or a viral vector comprising the polynucleotide, or a cell modified to express the biocircuit, effector module, DD, and payload of interest (i.e., immunotherapeutic agent).
[00257] In another aspect of the present invention, methods for inducing an anti-tumor immune response in a subject are provided. The methods comprise administering a pharmaceutically effective amount of a pharmaceutical composition comprising at least one biocircuit system, effector module, DD, and/or payload of interest (i.e., an immunotherapeutic agent), at least one vector, or cells to a subject having a tumor. Hie biocircuit and effector module having any immunotherapeutic agent as described herein may be in forms of a polypeptide, or a polynucleotide such as mRNA, or a viral vector comprising the polynucleotide, or a cell modified to express the biocircuit, effector module, DD, and payload of interest (i.e., immunotherapeutic agent).
[00258] The methods, according to the present invention, may be adoptive ceil transfer (ACT) using genetically engineered cells such as immune effector cells of the invention, cancer vaccines comprising biocircuit systems, effector modules, DDs, payloads of interest (i.e., immunotherapeutic agents) of the inv ention, or compositions that manipulate the tumor immunosuppressive microenvironment, or the combination thereof. These treatments may be further employed with other cancer treatment such as chemotherapy and radiotherapy.
1. Adoptive cell transfer (adoptive immunotherapy)
[00259] In some embodiments, cells which are genetically modified to express at least one biocircuit system, effector module, DD, and/or payload of interest (immunotherapeutic agent) may be used for adoptive cell therapy (ACT). As used herein, Adoptive cell transfer refers to the administration of immune cells (from autologous, allogenic or genetically modified hosts) with direct anticancer activity. ACT has shown promise in clinical application against malignant and infectious disease. For example, T cells genetically engineered to recognize CD 19 have been used to treat follicular B cell lymphoma (Kochenderfer et al., Blood, 2010, 116:4099-4102; and Kochenderfer and Rosenberg, Nat Rev Clin Oncol, 2013, 10(5): 267-276) and ACT using autologous lymphocytes genetically-modified to express anti-tumor T cell receptors has been used to treat metastatic melanoma (Rosenberg and Dudley, Curr. Opin. Immunol. 2009, 21 : 233- 240).
[00260] According to the present invention, the biocircuits and systems may be used in the development and implementation of cell therapies such as adoptive cell therapy. Certain effector modules useful in cell therapy are given in Figures 7-12. The biocircuits, their components, effector modules and their SREs and payloads may be used in cell therapies in APC platforms for stimulating T cells, as a tool to enhance ex vivo APC stimulation, to improve methods of T cell expansion, in ex vivo stimulation with antigen, in TCR/CAR combinations, in the manipulation or regulation of TILs, in allogeneic cell therapy, in combination T cell therapy with other treatment lines (e.g. radiation, cytokines), or to enhance T cells other than TCRs (e.g. by introducing cytokine genes).
[00261] In one embodiment, the biocircuits, their components, effector modules and their SREs and payloads may be used to improve methods of T cell expansion and/or methods of increasing the length of expression of a payload. The methods may include the use of CD3/CD28 dynabeads. The dynabeads used may be expander dynabeads, activator dynabeads or a combination thereof. The dynabeads may be used at a ratio of dynabeads (B) to T cells (T) of 1 : 1 , 1 :2. 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, 2: 1, 2:3, 2:5, 2:7, 2:9, 3: 1, 3:2, 3:4, 3:5, 3:7, 3:8, 3: 10, 4: 1, 4:3, 4:5, 4:7, 4:9, 5: 1 , 5:2, 5:3, 5:4, 5:6, 5: 7, 5:8, 5:9, 6: 1, 6:5, 6:7, 7: 1, 7:2, 7:3, 7.4. 7: 5, 7:6, 7:8, 7:9, 7: 10, 8: 1, 8:3, 8:5, 8:7, 8:9, 9: 1, 9:2, 9:4, 9:5, 9:7, 9:8, 9: 10, 10: 1, 10:3, 10:7, and 10:9. In one embodiment, the B:T ratio is 3: 1. In one embodiment, the B:T ratio is 1 : 1. In one embodiment, the B:T ratio is 1 :2. In one embodiment, the B:T ratio is 1:3. In one embodiment, the B:T ratio is 2: 1. As a non-limiting example, expander dynabeads are used at a B:T ratio of 3 : 1 and activator dynabeads are used at a B:T ratio of 3: 1. As a non-limiting example, expander dynabeads are used at a B:T ratio of 2: 1 and activator dynabeads are used at a B:T ratio of 3 : 1. As a non-limiting example, expander dynabeads are used at a B:T ratio of 1 : 1 and activator dynabeads are used at a B:T ratio of 3: 1.
[00262] In some embodiments, the compositions of the present invention may be utilized to alter TIL (tumor infiltrating lymphocyte) populations in a subject. In one embodiment any of the payloads described herein may be utilized to change the ratio of CD4 positive cells to CD 8 positive populations. In some embodiments, TILs may be sorted ex vivo and engineered to express any of the cytokines described herein. Payloads of the invention e.g. IL15 or 11,15- IL15Ra may be used to expand CD4 and/or CD 8 populations of TILs to enhance TIL mediated immune response.
[00263] Provided herein are methods for use in adoptive cell therapy. The methods involve preconditioning a subject in need thereof, modulating immune cells with SRE, biocircuits and compositions of the present invention, administering to a subject, engineered immune cells expressing compositions of the invention and the successful engraftment of engineered cells within the subject. [00264] In some embodiments, SREs, biocircuits and compositions of the present invention may be used to minimize preconditioning regimens associated with adoptive cell therapy. As used herein '"preconditioning" refers to any therapeutic regimen administered to a subject to improve the outcome of adoptive cell therapy. Preconditioning strategies include, but are not limited to total body irradiation and/or lymphodepieting chemotherapy. Adoptive therapy clinical trials without preconditioning have failed to demonstrate any clinical benefit, indicating its importance in ACT. Yet, preconditioning is associated with significant toxicity and limits the subject cohort that is suitable for ACT. In some instances, immune cells for ACT may be engineered to express payloads of the invention such as IL15 to reduce the need for
preconditioning (Pengram et al. (2012) Blood 119 (18): 4133-41 ; the contents of which are incorporated by reference in their entirety).
[00265] In some embodiments, immune cells for ACT may be dendritic cells, T cells such as CDS'*" T cells and CD4+ T cells, natural killer (NK) cells, NK T cells, Cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes (TILs), lymphokme activated killer (LAK) cells, memory T cells, regulatory T cells (Tregs), helper T cells, cytokine-induced killer (CIK) cells, and any combination thereof. In other embodiments, immune stimulator}' cells for ACT may be generated from embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC). In some embodiments, autologous or allogeneic immune ceils are used for ACT.
[00266] In some embodiments, NK cells engineered to express the present compositions may be used for ACT. NK ceil activation induces perforin/granzyme-dependent apoptosis in target cells. NK cell activation also induces cytokine secretion such as IFN-γ, TNF-a and GM-CSF. These cytokines enhance the phagocytic function of macrophages and their antimicrobial activity, and augment the adaptive immune response via up-regulation of antigen presentation by antigen presenting cells such as dendritic cells (DCs) (Reviewed by Vivier et al ., Nat. Immunol, 2008, 9(5): 503-510).
[00267] NK cells may also be genetically reprogrammed to circumvent NK cell inhibitory signals upon interaction with tumor cells. For example, using CRISPR, ZFN, or TALEN to genetically modify NK cells to silence their inhibitory receptors may enhance the anti-tumor capacity of NK cells.
[00268] Immune cells can be isolated and expanded ex vivo using a vari ety of methods known in the art. For example, methods of isolating and expanding cytotoxic T cells are described in U.S. Pat. NOs. 6,805,861 and 6,531, 451; US Patent Publication NO. US20160348072A1 and International Patent Publication NO. WO2016168595A1: the contents of each of which are incorporated herein by reference in their entirety. Isolation and expansion of NK cells is described in US Patent Publication NO. US20150152387A1 , U.S. Patent NO 7,435, 596: and Oyer, J.L. (2016). Cytotherapy.18(5):653-63; the contents of each of which are incorporated by reference herein in its entirety. Specifically, human primary NK cells may be expanded in the presence of feeder cells e.g. a myeloid ceil line that has been genetically modified to express membrane bound IL15, IL21, IL12 and 4-1BBL.
[00269] In some instances, sub populations of immune cells may be enriched for ACT. Methods for immune cell enrichment are taught in International Patent Publication NO.
WO2015039100A 1. In another example, T cells positive for B and T lymphocyte attenuator marker BTLA) may be used to enrich for T cells that are anti-cancer reactive as described in U.S. Pat. NO. 9,512,401 (the content of which are incoiporated herein by reference in its entirety).
[00270] In some embodiments, immune ceils for ACT may be depleted of select sub populations to enhance T cell expansion. For example, immune cells may be depleted of Foxp3+ T lymphocytes to minimize the ant-tumor immune response using methods taught in US Patent Publication NO. US 20160298081A1; the contents of which are incoiporated by reference herein in their entirety .
[00271] In some embodiments, activation and expansion of T cells for ACT is achieved by a transiently expressed Chimeric Antigen Receptor (CAR) on the cell surface. Such activation methods are taught in International Patent NO. WO2017015427, the content of which are incoiporated herein by reference in their entirety.
[00272] In some embodiments, immune cells may be activated by antigens associated with antigen presenting cells (APCs). In some embodiments, the APCs may be dendritic cells, macrophages or B ceils that antigen specific or nonspecific. The APCs may autologous or homologous in their organ. In some embodiments, the APCs may be artificial antigen presenting cells (aAPCs) such as cell based aAPCs or acellular aAPCs. Cell based aAPCs are may be selected from either genetically modified allogeneic cells such as human erythroleukemia cells or xenogeneic cells such as murine fibroblasts and Drosophila cells. Alternatively, the APCs maybe be acellular wherein the antigens or costimulatory domains are presented on synthetic surfaces such as latex beads, polystyrene beads, lipid vesicles or exosomes.
[00273] In some embodiments, adoptive cell therapy is carried out by autologous transfer, wherein the cells are deri ved from a subject in need of a treatment and the cells, following isolation and processing are administered to the same subject. In other instances, ACT may involve allogenic transfer wherein the cells are isolated and/or prepared from a donor subject other than the recipient subject who ultimately receives cell therapy . The donor and recipient subject may be genetically identical, or similar or may express the same HLA class or subtype. [00274] In some embodiments, the multiple immunotherapeutic agents introduced into the immune cells for ACT (e.g., T cells and NK cells) may be controlled by the same biocircuit system. In one example, a cytokine such as IL15 and a CAR construct such as CD 19 CAR are linked to the same hDHFR destabilizing domain. The expression of IL15 and CD 19 CAR is tuned using IMP simultaneously. In other embodiments, the multiple immunotherapeutic agents introduced into the immune cells for ACT (e.g., T cells and NK cells) may be controlled by different biocircuit systems. In one example, a cytokine such as IL15 and a CAR constmct such as CD 19 CAR are linked to different DDs in two separate effector modules, thereby can be tuned separately using different stimuli .
[00275] Following genetic modulation using SREs, biocircuits and compositions of the invention, cells are administered to the subject in need thereof. Methods for administration of cells for adoptive ceil therapy are known and may be used in connection with the methods and compositions provided herein. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; US Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1 ): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338; the contents of each of which are incorporated herein by reference in their entirety.
[Θ0276] In some embodiments, immune cells for ACT may also be modified to express one or more immunotherapeutic agents which facilitate immune cells activation, infiltration, expansion, survival and anti-tumor functions. The immunotherapeutic agents may be a CAR or TCR specific to a different target molecule; a cytokine or a cytokine receptor; a chimeric switch receptor that converts an inhibitory signal to a stimulator}7 signal; a homing receptor that guides adoptively transferred cells to a target site such as the tumor tissue; an agent that optimizes the metabolism of the immune cell; or a safety switch gene (e.g., a suicide gene) that kills activated T cells when a severe event is observed after adoptive cell transfer or when the transferred immune cells are no-longer needed.
[00277] In some embodiments, immune cells used for adoptive cell transfer can be genetically manipulated to improve their persistence, cytotoxicity, tumor targeting capacity , and ability to home to disease sites in vivo, with the overall aim of further improving upon their capacity to kill tumors in cancer patients. One example is to introduce effector modules of the invention comprising cytokines such as gamma-cytokines (IL15) into immune cells to promote immune ceil proliferation and survival. Transduction of cytokine genes (e.g., IL15) into cells will be able to propagate immune cells without addition of exogenous cytokines and cytokine expressing NK cells have enhanced tumor cytotoxicity.
[00278] NK cells may also be modified to become insensitive to suppressive cytokines such as TGF-β, thereby preserving their cytotoxicity . For example, NK ceils can be genetically modified to express the dominant negative mutant form of TGF-β type II receptor (ΟΝΤβΚΙ!) on their surface that render NK cells resistant to the suppressive effects of TGF-β.
[00279] In some embodiments, biocircuits, their components, SREs or effector modules may be utilized to prevent T cell exhaustion. As used herein, "T cell exhaustion" refers to the stepwise and progressive loss of T cell function caused by chronic T cell activation. T cell exhaustion is a major factor limiting the efficacy of antiviral and antitumor immunotherapies. Exhausted T cells have lo proliferative and cytokine producing capabilities concurrent with high rates of apoptosis and high surface expression of multiple inhibitor}' receptors. T cell activation leading to exhaustion may occur either in the presence or absence of the antigen .
[00280] In some embodiments, effector modules of the present invention, useful for immunotlierapy may be placed under the transcriptional control of the T cell receptor alpha locus constant (T'RAC) locus in the T cells. Eyquem et al. have shown that expression of the CAR from the TRAC locus prevents T cell exhaustion and the accelerated differentiation of T cells caused by excessive T cell activation (Eyquem J. et ai (2017) Nature.543(7643): 113-117; the contents of which are incorporated herein by reference in their entirety).
[00281 ] In some embodiments, payioads of the invention may be used in conjunction with antibodies or fragments that target T cell surface markers associated with T cell exhaustion. T- ceil surface markers associated with T cell exhaustion that may be used include, but are not limited to, ("Π .Λ- Ι . PD-1 , TGIT, LAG-3, 2B4, BTLA, ΊΊΜ3, VISTA, and CD96.
2. Cancer vaccines
[00282] In some embodiments, biocircuits, effector modules, payioads of interest
(immunotherapeutic agents), vectors, cells and compositions of the present invention may be used in conjunction with cancer vaccines. In one aspect, dendritic cells are modified to express the compositions of the invention and used as cancer vaccines.
[00283] In some embodiments, cancer vaccine may comprise peptides and/or proteins derived from tumor associated antigen (TAA). Such strategies may be utilized to evoke an immune response in a subject, which in some instances may be a cytotoxic T lymphocyte (CTL) response. Peptides used for cancer vaccines may also be modified to match the mutation profile of a subject. For example, EGFR derived peptides with mutations matched to the mutations found in the subject in need of therapy have been successfully used in patients with lung cancer (Li F et ai. (2016) Oncoimmunology. Oct 7;5(12): el 238539; the contents of which are incorporated herein by reference in their entirety).
[00284] In one embodiment, cancer vaccines of the present invention may superagonist altered peptide ligands (APL) derived from TAAs. These are mutant peptide ligands deviate from the native peptide sequence by one or more amino acids, which activate specific CTL clones more effectively than native epitopes. These alterations may allow the peptide to bind better to the restricting Class I MHC molecule or interact more favorably with the TCR of a given tumor- specific CTL subset. APLs may be selected using methods taught in US Patent Publication NO. US20160317633 A 1 , the contents of which are incorporated herein by reference in their entirety. 3. Combination treatments
[00285] In some embodiments, it is desirable to combine compositions, vectors and cells of the invention for administration to a subject. Compositions of the invention comprising different immunotherapeutic agents may be used in combination for enhancement of immunotherapy.
[00286] In some embodiments, it is desirable to combine compositions of the invention with adjuvants, that can enhance the potency and longevity of antigen-specific immune responses. Adjuvants used as immunostimulants in combination therapy include biological molecules or delivery carriers that deliver antigens. As non-limiting examples, the compositions of the invention may be combined with biological adjuvants such as cytokines, Toil Like Receptors, bacterial toxins, and/or saponins. In other embodiments, the compositions of the present invention may be combined with delivery carriers. Exemplary delivery carriers include, polymer microspheres, immune stimulating complexes, emulsions (oil-in-water or water-in-oil), aluminum salts, liposomes or virosomes.
[00287] In some embodiments, immune effector cells modified to express biocircuits, effector modules, DDs and payloads of the invention may be combined with the biological adjuvants described herein. Dual regulation of Chimeric Antigen Receptor (CAR) and cytokines and ligands may be used to segregate the kinetic control of target-mediated activation from intrinsic cell T cell expansion. Such dual regulation also minimizes the need for pre-conditioning regimens in patients. As a non-limiting example, DD regulated CAR e.g. CD19 CAR may be combined with cytokines e.g. IL15 to enhance the anti-tumor efficacy of the CAR.
[00288] In some embodiments, immune effector cells modified to express one or more antigen- specific TCRs or CARs may be combined with compositions of the invention comprising immunotherapeutic agents that convert the immunosuppressive tumor microenvironment.
[00289] In one aspect, effector immune cells modified to express payloads of the invention may¬ be combined with CARs specific to different target molecules on the same cell. In another aspect, different immune cells modified to express the CAR construct such as NK cells and T cells may be used in combination with immune cells expressing the pavloads of the invention for a tumor treatment. For instance, a T cell modified to express a CD 19 CAR may be combined with a NK cell modified to express the same CD 19 CAR to treat B cell malignancy to be used in conjunction with T cells expressing 11,15 or IL15-IL15Ra fusion proteins.
[00290] In other embodiments, immune cells modified to express pavloads of the invention may be combined with checkpoint blockade agents.
[00291] In some embodiments, immune effector cells modified to expressed biocircuits, effector modules, DDs and pavloads of the invention may be combined with cancer vaccines of the invention.
[Θ0292] In some embodiments, effector modules of the invention may be used in combination with a cytokine other than IL15 and IL15-IL15Ra fusion proteins, such as a Chimeric antigen receptor, or a safety switch, or a metabolic factor, or an effector module comprising a homing receptor.
[00293] In some embodiments, methods of the invention may include combination of the composition s of the invention with other agents effective in the treatment of cancers, infection diseases and other immunodeficient disorders, such as anti-cancer agents. As used herein, the term "anti-cancer agent" refers to any agent which is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.
[00294] In some embodiments, anti-cancer agent or therapy may be a chemotherapeutic agent, or radiotherapy, immunotherapeutic agent, surgery, or any other therapeutic agent which, in combination with the present invention, improves the therapeutic efficacy of treatm ent.
[00295] In one embodiment, an effector module comprising a IL15 or ILlS-ILlSRa fusion proteins may be used in combination with amino pyrimidine derivatives such as the Burkit's tyrosine receptor kinase (BTK) inhibitor using methods taught in International Patent
Application NO. WQ2016164580, the contents of which are incorporated herein by reference in their entirety.
[00296] In some embodiments, compositions of the present invention may be used in combination with immunotlierapeutics otlier than the inventive therapy described herein, such as antibodies specific to some target molecules on the surface of a tumor cell [00297] Exemplary chemotherapies include, without limitation, Acivicin; Aclarubicin;
Acodazole hydrochloride; Acronine; Adozelesin; Aldesleukin; Altretamine; Ambomycin;
Ametantrone acetate; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperrin, Sulindac, Curcumin, alkylating agents including: Nitrogen mustards such as mechlor-ethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas such as carmustine (BC U), lomustine (CCNU), and semustine (methyl-CC U); thylenimines/methylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa),
hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrrolidine analogs such as 5- fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2'- difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6~thioguanine, azathioprine, 2,'-deoxycoformycin
(pentostatin), erythrohyckoxynonyladenine (EHNA), ffudarabine phosphate, and 2- chlorodeoxyadenosine (cladribine, 2- CdA); natural products including antimitotic drugs such as paclitaxei, vinca alkaloids including vinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate; epipodophylotoxins such as etoposide and teniposide; antibiotics, such as actimomycin D, daunomycin (ruhidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycm (mithramycin), mitomycinC, and actinomycin; enzymes such as -[.-asparaginase, cytokines such as interferon (IFN)-gamma, tumor necrosis factor (TNF)- alpha, TNF-beta and GM-CSF, anti-angiogenic factors, such as angiostatin and endostatin, inhibitors of FGF or VEGF such as soluble forms of receptors for angiogenic factors, including soluble VGF/VEGF receptors, platinum coordination complexes such as cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazme derivatives including N- methylhydrazme (MIFf) and procarbazine, adrenocortical suppressants such as mitotane (ο,ρ'-DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fiuoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin- releasing hormone analogs and leuprolide; non-steroidal antiandrogens such as flutamide; kinase inhibitors, histone deacetylase inhibitors, methylation inhibitors, proteasome inhibitors, monoclonal antibodies, oxidants, anti-oxidants, telomerase inhibitors, BH3 mimetics, ubiquitin ligase inhibitors, stat inhibitors and receptor tyrosin kinase inhibitors such as imatinib mesylate (marketed as Gleevac or Giivac) and erlotinib (an EGF receptor inhibitor) now marketed as Tarveca; anti-virals such as oseltamivir phosphate. Amphotericin B, and palivizumab; Sdi 1 mimetics; Semusiine; Senescence derived inhibitor 1; Sparfosic acid: Spicamycin D;
Spiromustine; Spienopentin: Spongistatin 1: Squaiamine: Stipiamide; Stromelysin inhibitors; Sulfinosine; Superactive vasoactive intestinal peptide antagonist; Velaresol; Veramine; Verdins; Verteporfin; Vinorelbine; Vmxaltme; Vitaxin; Vorozole; Zanoterone; Zeniplatin; Zilascorb; and Zinostatin stimalamer; ΡΟ β small -molecule inhibitor, GSK2636771 ; pan-PI3 inhibitor (BKM120); BRAF inhibitors. Veniurafenib (Zeiboraf) and dabrafenib (Tafmiar); or any analog or derivative and variant of the foregoing.
[00298] Radiotherapeutic agents and factors include radiation and waves that induce DNA damage for example, γ-irradiation, X-rays, -irradiation, microwaves, electronic emissions, radioisotopes, and the like. Therapy may be achieved by irradiating the localized tumor site with the above described forms of radiations. It is most likely that these factors effect a broad range of damage DNA, on the precursors of DNA, the replication and repair of DNA, and the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of tim e (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and ty pe of radiation emitted, and the uptake by the neoplastic ceils.
[00299] Other agents may be used in combination with compositions of the invention may also include, but not limited to, agents that affect the upregulation of cell surface receptors and their ligands such as Fas/Fas ligand, DR4 or DR5/TRAIL and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion such as focal adhesion kinase (FAKs) inhibitors and Lovastatin, or agents that increase the sensitivity of the hyper proliferative cells to apoptotic inducers such as the antibody C225,
[00300] The combinations may include administering the compositions of the invention and other agents at the same time or separately. Alternatively, the present immunothe apy may precede or follow the other agent/therapy by intervals ranging from minutes, days, weeks to months.
4. Diseases
[00301] Provided in the present invention is a method of reducing a tumor volume or burden in a subject in need, the method comprising introducing into the subject a composition of the invention. [00302] The present invention also provides methods for treating a cancer in a subject, comprising administering to the subject an effective amount of an immune effector cell genetically modified to express at least one effector module of the invention.
Cancer
Θ0303] Various cancers may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. As used herein, the term "cancer" refers to any of various malignant neoplasms characteri zed by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites and also refers to the pathological condition characterized by such malignant neoplastic growths. Cancers may be tumors or hematological malignancies, and include but are not limited to, all types of Ivmphomas/leukemias, carcinomas and sarcomas, such as those cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon/rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal marrow, tailbone, testicles, thyroid and uterus.
[00304] Types of carcinomas which may be treated with the compositions of the present invention include, but are not limited to, papilloma/carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma, adenoma/adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma, rhabdomyoma, mesothelioma, angioma, osteoma, chondroma, glioma, lymphoma/leukemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinomas, basal cell carcinoma and sinonasal undifferentiated carcinoma.
[00305] Types of carcinomas which may be treated with the compositions of the present invention include, but are not limited to, soft tissue sarcoma such as alveolar soft part sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and Askin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor), malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and chondrosarcoma.
[00306] As a non-limiting example, the carcinoma which may be treated may be Acute granulocytic leukemia, Acute lymphocytic leukemia, Acute myelogenous leukemia,
Adenocarcinoma, Adenosarcoma, Adrenal cancer, Adrenocortical carcinoma, Anal cancer, Anaplastic astrocytoma, Angiosarcoma, Appendix cancer, Astrocytoma, Basal cell carcinoma, B-Cell lymphoma ), Bile duct cancer, Bladder cancer, Bone cancer, Bowel cancer, Brain cancer, Brain stem glioma. Brain tumor, Breast cancer, Carcinoid tumors. Cervical cancer,
Cliolangiocarcmoma, Chondrosarcoma, Chronic lymphocytic leukemia, Chronic myelogenous leukemia, Colon cancer, Colorectal cancer, Craniopharyngioma, Cutaneous lymphoma, Cutaneous melanoma, Diffuse astrocytoma, Ductal carcinoma in situ, Endometrial cancer, Ependymoma, Epithelioid sarcoma, Esophageal cancer, Ewing sarcoma, Extrahepatic bile duct cancer. Eye cancer, Fallopian tube cancer, Fibrosarcoma, Gallbladder cancer, Gastric cancer, Gastrointestinal cancer, Gastrointestinal carcinoid cancer, Gastrointestinal stromal tumors, General, Germ cell tumor, Glioblastoma multiforme, Glioma, Hairy cell leukemia. Head and neck cancer, Hemangioendothelioma, Hodgkin lymphoma, Hodgkin's disease, Hodgkin's lymphoma, Hypopharyngeal cancer, Infiltrating ductal carcinoma, Infiltrating lobular carcinoma, Inflammatory breast cancer, Intestinal Cancer, intrahepatic bile duct cancer, Invasive / infiltrating breast cancer, Islet cell cancer, Jaw cancer, Kaposi sarcoma, Kidney cancer, Laryngeal cancer, Leiomyosarcoma, Leptomeningeal metastases. Leukemia, Lip cancer, Liposarcoma, Liver cancer, Lobular carcinoma in situ, Low-grade astrocytoma, Lung cancer, Ly mph node cancer, Lymphoma, Male breast cancer, Meduilaiy carcinoma, Meduiloblastoma, Melanoma, Meningioma, Merkel cell carcinoma, Mesenchymal chondrosarcoma,
Mesenchymous, Mesothelioma, Metastatic breast cancer, Metastatic melanoma, Metastatic squamous neck cancer, Mixed gliomas, Mouth cancer, Mucinous carcinoma, Mucosal melanoma, Multiple myeloma, Nasal cavity cancer, Nasopharyngeal cancer, Neck cancer, Neuroblastoma, Neuroendocrine tumors, Non -Hodgkin lymphoma, Non-Hodgkin's lymphoma, Non-small cell lung cancer, Oat cell cancer. Ocular cancer, Ocular melanoma,
Oligodendroglioma, Oral cancer, Oral cavity cancer, Oropharyngeal cancer, Osteogenic sarcoma, Osteosarcoma, Ovarian cancer, Ovarian epithelial cancer, Ovarian germ cell tumor, Ovarian primar ' peritoneal carcinoma. Ovarian sex cord stromal tumor, Paget's disease, Pancreatic cancer, Papillary carcinoma, Paranasal sinus cancer, Parathyroid cancer, Pelvic cancer. Penile cancer, Peripheral nerve cancer, Peritoneal cancer, Pharyngeal cancer,
Pheochromocytoma, Pilocytic astrocytoma, Pineal region tumor, Pineoblastoma, Pituitary gland cancer, Primaiy central nervous system lymphoma. Prostate cancer, Rectal cancer, Renal cell cancer, Renal pelvis cancer, Rhabdomyosarcoma, Salivary gland cancer, Sarcoma, Sarcoma, bone, Sarcoma, soft tissue, Sarcoma, uterine. Sinus cancer. Skin cancer, Small cell lung cancer, Small intestine cancer, Soft tissue sarcoma, Spinal cancer, Spinal column cancer, Spinal cord cancer, Spinal tumor. Squamous cell carcinoma, Stomach cancer, Synovial sarcoma, T-cell lymphoma ), Testicular cancer, Throat cancer, Thymoma / thymic carcinoma, Thyroid cancer, Tongue cancer, Tonsil cancer, Transitional cell cancer, Transitional cell cancer, Transitional cell cancer, Triple-negative breast cancer. Tubal cancer, Tubular carcinoma, Ureteral cancer, Ureteral cancer, Urethral cancer, Uterine adenocarcinoma, Uterine cancer. Uterine sarcoma, Vaginal cancer, and Vulvar cancer.
Infectious diseases
[00307] "Infection diseases" he ein refer to diseases caused by any pathogen or agent that infects mammalian cells, preferably human cells and causes a disease condition . Examples thereof include bacteria, yeast, fungi, protozoans, mycoplasma, viruses, prions, and parasites. Examples include those involved in (a) viral diseases such as, for example, diseases resulting from infection by an adenovirus, a herpesvirus (e.g., HSV-I, HSV-II, CMV, or VZV), a poxvirus (e-g-, an orthopoxvirus such as variola or vaccinia, or molluscum contagiosum), a picornavirus (e.g., rhinovirus or enterovirus), an orthomyxovirus (e.g., influenzavirus), a paramyxovirus (e.g., parainfluenza virus, mumps virus, measles virus, and respiratory syncytial virus (RSV)), a coronavirus (e.g., SARS), a papovavirus (e.g., papillomaviruses, such as those that cause genital warts, common warts, or plantar warts), a hepadnavirus (e.g., hepatitis B virus), a flavivims (e.g., hepatitis C virus or Dengue virus), or a retrovirus (e.g., a lentivirus such as HIV); (b) bacterial diseases such as, for example, diseases resulting from infection by bacteria of, for example, the genus Escherichia, Enterobacier, Salmonella, Staphylococcus, Shigella, Listeria, Aerohacter, Helicobacter, Klebsiella, Proteus. Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium,
Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia,
Haemophilus, or Bordetella; (c) other infectious diseases, such chlamydia, fungal diseases including but not limited to candidiasis, aspergillosis, histoplasmosis, cryptococcal meningitis, parasitic diseases including but not limited to malaria, Pneumocystis carnii pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, and trypanosome infection and prions that cause human disease such as Creutzfeldt-Jakob Disease (CJD), variant Creutzfeldt- Jakob Disease (vCJD), Gerstmann-Straussler-Scheinker syndrome, Fatal Familial Insomnia and kura.
[00308] In some embodiment, biocircuits of the invention may be used for the treatment of infectious diseases. Biocircuits of the invention may be introduced in cells suitable for adoptive ceil transfer such as macrophages, dendritic ceils, natural killer cells, and or T cells. Infectious diseases treated by the biocircuits of the invention may be diseases caused by virases, bacteria, fungi, and/or parasites. ILIS-ILiSRa payloads of the invention may be used to increase immune cell proliferation and/or persistence of the immune cells useful in treating infectious diseases. 5. Microbiome
[00309] Alterations in the composition of the microbiome may impact the action of anti-cancer therapies. A diverse community of symbiotic, commensal and pathogenic microorganisms exist m all environmentally exposed sites in the body and is herein referred to as the "Microbiome." Env ronmentally exposed sites of the body that may be inhabited by a microbiome include the skin, nasopharynx, the oral cavity, respiratory tract, gastrointestinal tract, and the reproductive tract.
[00310] In some embodiments, microbiome native or engineered with immunotherapeutic agents may be used to improve the efficacy of the anti-cancer immunotherapies. Methods of using rnicrobiome to improve responsive to immunotherapeutic agents have been described by Sivan et ai. (Sivan A., et al.2015. Science; 350: 1084-9; the contents of which are incorporated herein by reference in their entirety). In other embodiments, the microorganisms may be delivered along with immunotherapeutic compositions of the present invention to improve the efficacy of immunotherapy .
6. Tools and agents for making therapeutics
[00311] Provided in the present invention are tools and agents that may be used in generating immunotherapeutics for reducing a tumor volume/burden or enhancing immune cell persistence m a subject in need. A considerable number of variables are involved in producing a therapeutic agent, such as stnicture of the payload, type of cells, method of gene transfers, method and time of ex vivo expansion, pre- conditioning and the amount and type of tumor burden in the subject. Such parameters may be optimized using tools and agents described herein.
Cell lines
[00312] The present disclosure provides a mammalian cell that has been genetically modified with the compositions of the invention. Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell tines include, but are not limited to Human embryonic kidney cell line 293, fibroblast cell line NIH 3T3, human colorectal carcinoma cell line HCT1 16, ovarian carcinoma cell line SKOV-3, immortalized T cell lines (e.g. Jurkat cells and SupTl cells), lymphoma cell line Raji cells, NALM-6 cells, K562 cells, He La cells, PC 12 ceils, I II . -60 ceils, NK cell lines (e.g. NKL, NK 92 K962, and YTS), and the like. In some instances, the cell is not an immortalized cell line, but instead a cell obtained from an individual and is herein referred to as a primary cell. For example, the cell is a T lymphocyte obtained from an individual. Other examples include, but are not limited to cytotoxic cells, stem cells, peripheral blood mononuclear cells or progenitor cells obtained from an individual. Tracking SREs, biocircuits and cell lines
[00313] In some embodiments, it may be desirable to track the compositions of the invention or the cells modified by the compositions of the invention. Tracking may be achieved by using reporter moieties, which, as used herein, refers to any protein capable of creating a detectable signal, in response to an input. Examples include alkaline phosphatase, β-galactosidase, chloramphenicol acetyltransferase, β-glucuronidase, peroxidase, β-lactamase, catalytic antibodies, biolumine scent proteins e.g. luciferase, and fluorescent proteins such as Green fluorescent protein (GFP).
[00314] Reporter moieties may be used to monitor the response of the DD upon addition of the ligand corresponding to the DD. In other instances, reporter moieties may be used to track cell survival, persistence, cell growth, and/or localization in vitro, in vivo, or ex vivo.
[00315] In some embodiments, the preferred reporter moiety may be luciferase proteins. In one embodiment, the reporter moiety is the Renilla luciferase (SEQ, ID NO. 304, encoded by nucleic acid sequence of SEQ. ID NO. 305), or a firefly luciferase (SEQ. ID NO. 306, encoded by nucleic acid sequence of SEQ. ID NO. 307).
Animal models
[00316] The utility and efficacy of the compositions of the present invention may be tested in vivo animal models, preferably mouse models. Mouse models used to may be syngeneic mouse models wherein mouse ceils are modified with compositions of the invention and tested in mice of the same genetic background. Examples include pMEL-1 and 4T1 mouse models.
Alternatively, xenograft models where human cells such as tumor cells and immune cells are introduced into immunodeficient mice may also be utilized in such studies. Immunodeficient mice used may be CByJ.Cg-Foxnlm/J, 6;\29S1 -RagllmlMomIJ, B6A29$7~RagTm!Mom/J, B6. CB17- r cicW/SzJ, N^
NOD.CB17-Pr ^/SzJ, NOD.Cg-iVi*SCi¾Vffii ¾i J, NOD-,?c¾/ IL2Rgnuii, Nude (nu) mice, SOD mice, NOD mice, RAGI RAG2 mice, NOD-Scid mice, TLlrgyiull mice, b2mww// mice, NOD-yc/ IL2rynull mice, ~NOD-scid-B2mnidl mice, beige mouse, and HLA transgenic mice. Cellular assays
[00317] In some embodiments, the effectiveness of the compositions of the inventions as immunotherapeutic agents may be evaluated using cellular assays. Levels of expression and/or identity of the composi tions of the invention may be determined according to any methods known in the art for identifying proteins and/or quantitating proteins levels. In some
embodiments, such methods may include Western Blotting, flow cytometry, and immunoassays. [00318] Provided herein are methods for functionally characterizing cells expressing SRE, biocircuits and compositions of the invention . In some embodiments, functional characterization is carried out in primary immune cells or immortalized immune cell lines and may be determined by expression of cell surface markers. Examples of cell surface markers for T cells include, but are not limited to, CD 3, CD4, CD 8, CD 14, CD20, CD1 lb, CD16, CD45 and HLA-DR, CD 69, CD28, CD44, IFNgamma, PD1 , TIM3 and LAGS. Examples of cell surface markers for antigen presenting cells include, but are not limited to, MHC class I, MHC Class II, CD40, CD45, B7-1, B7-2, IFN-γ receptor and IL2 receptor, ICAM-1 and/or Fey receptor. Examples of cell surface markers for dendritic cells include, but are not limited to, MHC class I, MHC Class II, B7-2, CD18, CD29, CD31 , CD43, CD44, CD45, CD54, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR and/or Dectin-1 and the like; while in some cases also having the absence of CD2, CD3, CD4, CD 8, CD14, CD15, CD ! 6. CD 19, CD20, CD56, and/or CD57. Examples of cell surface markers for NK cells include, but are not limited to, CCL3, CCL4, CCL5, CCR4, CXCR4, CXCR3, NKG2D, CD71, CD69, CCR5, Phospho JAK/STAT, phospho ER , phospho p38/ MAPK, phospho AKT, phospho STAT3, Granulysm, Granzyme B, Granzyme K, 1L10, 1L22, IFNg, LAP, Perforin, and TNFa,
V. DELIVERY MODALITIES AND/OR VECTORS
Vectors
[00319] The present invention also provides vectors that package polynucleotides of the invention encoding biocircuits, effector modules, SREs (DDs) and payload constructs, and combinations thereof. Vectors of the present invention may also be used to deliver the packaged polynucleotides to a ceil, a local tissue site or a subject. These vectors may be of any kind, including DNA. vectors, RNA vectors, plasmids, viral vectors and particles. Viral vector technology is well known and described in Sambrook et al. (2001, Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Viruses, which are useful as vectors include, but are not limited to lentiviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, herpes simplex viral vectors, retroviral vectors, oncolytic viruses, and the like.
[00320] In general, vectors contain an origin of replication functional in at least one organism, a promoter sequence and convenient restriction endonuclease site, and one or more selectable markers e.g. a drug resistance gene.
[00321] As used herein a promoter is defined as a DNA sequence recognized by transcription machinery of the cell, required to initiate specific transcription of the polynucleotide sequence of the present invention. Vectors can comprise native or non-native promoters operably linked to the polynucleotides of the invention. The promoters selected may be strong, weak, constitutive, inducible, tissue specific, development stage-specific, and/or organism specific. One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of polynucleotide sequence that is operatively linked to it. Another example of a preferred promoter is Elongation Growth Factor- 1. Alpha (EF-1. alpha). Other constitutive promoters may also be used, including, but not limited to simian vims 40 (SV40), mouse mammary tumor viras (MMTV), human immunodeficiency viras (HIV), long terminal repeat (LTR), promoter, an avian leukemia virus promoter, an Epstein-Barr vims immediate early promoter, a Rous sarcoma vims promoter as well as human gene promoters including, but not limited to the phosphogiycerate kinase (PGK) promoter, actin promoter, the myosin promoter, the hemoglobin promoter, the Ubiquitin C (Ubc) promoter, the human U6 small nuclear protein promoter and the creatine kinase promoter. In some instances, inducible promoters such as but not limited to metallothionine promoter, glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter may be used. In some embodiments, the promoter may be selected from the SEQ, ID NO. 49-51 and 342.
[00322] In some embodiments, the optimal promoter may be selected based on its ability to achieve minimal expression of the SREs and payloads of the invention in the absence of the ligand and detectable expression in the presence of the ligand.
[00323] Additional promoter elements e.g. enhancers may be used to regulate the frequency of transcriptional initiation. Such regions may be located 10-100 base pairs upstream or downstream of the start site. In some instances, two or more promoter elements may be used to cooperatively or independently activate transcription.
[00324] In some embodiments, the recombinant expression vector may comprise regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell into which the vector is to be introduced.
1. Len viral vectors
[00325] In some embodiments, lentiviral vectors/particles may be used as vehicles and delivery modalities. Lentivirases are subgroup of the Retroviridae family of viruses, named because reverse transcription of viral RNA genomes to DNA is required before integration into the host genome. As such, the most important features of lentiviral vehicles/particles are the integration of their genetic material into the genome of a target/host cell. Some examples of lenti virus include the Human Immunodeficiency Viruses: HIV-1 and HIV -2, the Simian
Immunodeficiency Vims (SIV), feline immunodeficiency vims (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia vims (EIAV), equine infectious anemia virus, visna-maedi and caprine arthritis encephalitis vims (CAEV).
[00326] Typically, lentiviral particles making up the gene delivery vehicle are replication defective on their own (also referred to as "self-inactivating"). Lentiviruses are able to infect both dividing and non-dividing cells by virtue of the entry mechanism through the intact host nuclear envelope (Naldini L et al., Curr. Opin. Biotechnol, 1998, 9: 457-463), Recombinant lentiviral vehicles/particles have been generated by multiply attenuating the HIV virulence genes, for example, the genes Env, Vif, Vpr, Vpu, Nef and Tat are deleted making the vector biologically safe. Correspondingly, lentiviral vehicles, for example, derived from HIV-l/HIV-2 can mediate the efficient delivery, integration and long-term expression of transgenes into non- dividing ceils. As used herein, the term "'recombinant" refers to a vector or other nucleic acid containing both lentiviral sequences and non-lentiviral retroviral sequences.
[00327] Lentiviral particles may be generated by co-expressing the virus packaging elements and the vector genome itself in a producer cell such as human HEK293T cells. These elements are usually provided in three (in second generation lentiviral systems) or four separate piasmids (in third generation lentiviral systems). The producer cells are co-transfected with piasmids that encode lentiviral components including the core (i.e. structural proteins) and enzymatic components of the virus, and the envelope protein(s) (referred to as the packaging systems), and a plasmid that encodes the genome including a foreign transgene, to be transferred to the target cell, the vehicle itself (also referred to as the transfer vector). In general, the piasmids or vectors are included in a producer cell line. The piasmids/ vectors are introduced via transfection, transduction or infection into the producer cell line. Methods for transfection, transduction or infection are well known by those of skill in the art. As non-limiting example, the packaging and transfer constmcts can be introduced into producer cell lines by calcium phosphate transfection, iipofection or electroporation, generally together with a dominant selectable marker, such as neo, DHFR, Gin synthetase or ADA, followed by selection in the presence of the appropriate drag and isolation of clones.
[00328] The producer cell produces recombinant viral particles that contain the foreign gene, for example, the effector module of the present invention. The recombinant viral particles are recovered from the culture media and titrated by standard methods used by those of skill in the art. The recombinant lentiviral vehicles can be used to infect target cells.
[00329] Cells that can be used to produce high-titer lentiviral particles may include, but are not limited to, HEK293T cells, 293G cells, STAR ceils (Relander et έί., Μοί Ther., 2005, 11 : 452- 459), FreeStyle™ 293 Expression System. (ThermoF'isher, Waitham, MA), and other HEK293T- based producer cell lines (e.g., Stewart et al.. Hum Gene Ther. 2011, 22(3):357-369; Lee et al., Biotechnol Bioeng, 2012, 10996): 1551-1560; Throm et al„ Blood. 2009, 113(21): 5104-51 10; the contents of each of which are incoiporated herein by reference in their entirety).
[00330] In some aspects, the envelope proteins may be heterologous envelop proteins from other viruses, such as the G protein of vesicular stomatitis virus (VSV G) or baculoviral gp64 envelop proteins. The VSV-G glycoprotein may especially be chosen among species classified in the vesiculovirus genus: Carajas virus (CJSV), Chandipura virus (CHPV), Cocal virus (COCV), Isfahan virus (ISFV), Maraba virus (MARAV), Pity virus (PIRYV), Vesicular stomatitis Alagoas virus (VSAV), Vesicular stomatitis Indiana virus (VS3V) and Vesicular stomatitis New Jersey virus (VSNJV) and/or stains provisionally classified in the vesiculovirus genus as Grass- carp rhabdovirus, BeAn 157575 virus (BeAn 157575), Boteke virus (BTKV), Calchaqui virus (CQIV), Eel virus American (EVA), Gray Lodge virus (GLOV), Jiirona virus (JURY), Klamath virus (KLAVj, Kwatta virus (KWA V), La Joya virus (LTV), Malpais Spring virus (MSPV), Mount Elgon bat virus (MEBV), Perinet virus (PERV), Pike fry rhabdovirus (PFRV), Porton virus (PQRV), Radi virus (RAD1V), Spring viremia of carp virus (SVCV), Tupaia virus (TUPV), Ulcerative disease rhabdovirus (UDRV) and Jug Bogdanovac virus (YBV). The gp64 or other baculoviral env protein can be derived from Autographa califdrnica
nucleopolyhedrovirus (AcMNPV), Anagrapha faicifera nuclear polvhedrosis virus, Bombyx won nuclear pol hedrosis virus, Choristoneura fumiferana nucleopolyhedrovirus, Orgyia pseudotsugata single capsid nuclear polyhedrosis virus, Epiphyas postvittana
nucleopolyhedrovirus, Hyphantria cunea nucleopolyhedrovirus, Galleria mellonella nuclear polyhedrosis virus, Dhori virus, Thogoto virus, Antheraea pemyi nucleopolyhedrovirus or Batken virus.
Θ0331] Additional elements provided in lentiviral particles may comprise retroviral LTR (long- terminal repeat) at either 5 ' or 3 " terminus, a retro viral export element, optionally a lentiviral reverse response element (RRE), a promoter or active portion thereof, and a locus control region (LCR) or active portion thereof. Other elements include central polypurine tract (cPPT) sequence to improve transduction efficiency in non-dividing cells, Woodchuck Hepatitis Vims (WHP) Posttranscriptional Regulatory Element (WPRE) which enhances the expression of the transgene, and increases titer. The effector module is linked to the vector.
Θ0332] Methods for generating recombinant lentiviral particles are discussed in the art, for example, U.S. Pat. NOs. 8, 846, 385; 7,745, 179; 7,629,153; 7,575,924; 7, 179, 903; and 6, 808, 905; the contents of each of which are incorporated herein by reference in their entirety. [00333] Lentivirus vectors used may be selected from, but are not limited to pi A X. pLenti, pLenti6, pLJMl, FUGW, pWPXL, pWPI, pLenti CMV puro DEST, pLJMl-EGFP, pULTRA, plnducer20, pHIV-EGFP, pCW57.1, pTRPE, pELPS, pRRL, and pLionll.
[00334] Lentiviral vehicles known in the art may also be used (See, U.S. Pat. NOs. 9, 260, 725; 9,068, 199; 9,023,646; 8,900,858; 8,748, 169; 8,709,799; 8,420,104; 8,329,462; 8,076, 106;
6,013,516; and 5,994, 136; International Patent Publication NO. WO2012079000; the contents of each of which are incorporated herein by reference in their entirety).
2. Retroviral vectors (y-retroviral vectors)
[00335] In some embodiments, retroviral vectors may be used to package and deliver the biocircuits, biocircuit components, effector modules, SREs or payload constructs of the present invention. Retroviral vectors (RVs) allow the permanent integration of a transgene in target cells. In addition to lentiviral vectors based on complex HIV- 1/2, retroviral vectors based on simple gamma-retroviruses have been widely used to deliver therapeutic genes and demonstrated clinically as one of the most efficient and powerful gene delivery systems capable of transducing a broad range of cell types. Example species of Gamma retroviruses include the murine leukemia viruses (MLVs) and the feline leukemia viruses (FeLV).
[00336] In some embodiments, gamma-retroviral vectors derived from a mammalian gamma- retrovirus such as murine leukemia viruses (MLVs), are recombinant. The MLV families of gamma retroviruses include the ecotropic, amphotropic, xenotropic and polytropic subfamilies. Ecotropic viruses are able to infect only murine cells using mCAT-1 receptor. Examples of ecotropic viruses are Moloney MLV and AKV. Amphotropic viruses infect murine, human and other species through the Pit-2 receptor. One example of an amphotropic virus is the 4070A virus. Xenotropic and polytropic viruses utilize the same (Xprl) receptor, but differ in their species tropism . Xenotropic viruses such as NZB-9-1 infect human and other species but not murine species, whereas polytropic viruses such as focus-forming viruses (MCF) infect murine, human and other species.
[00337] Gamma-retroviral vectors may be produced in packaging cells by co-transfecting the cells with several plasrnids including one encoding the retroviral structural and enzymatic (gag- pol) polyprotem, one encoding the envelope (env) protein, and one encoding the vector mRNA comprising polynucleotide encoding the compositions of the present invention that is to be packaged in newly formed viral particles.
[00338] In some aspects, the recombinant gamma-retroviral vectors are pseudotyped with envelope proteins from other viruses. Envelope glycoproteins are incorporated in the outer lipid layer of the viral particles which can increase/alter the cell tropism. Exemplar}' envelop proteins include the gibbon ape leukemia vims envelope protein (GALV) or vesicular stomatitis virus G protein (VSV-G), or Simian endogenous retrovirus envelop protein, or Measles Virus H and F proteins, or Human immunodeficiency virus gpl20 envelope protein, or cecal vesiculovirus envelop protein (See, e.g., U.S. application publication NO. 2012/164118; the contents of which are incorporated herein by reference in its entirety). In other aspects, envelope glycoproteins may be genetically modified to incorporate targeting/binding ligands into gamma-retroviral vectors, binding ligands including, but not limited to, peptide ligands, single chain antibodies and growth factors (Waehler et ah, Nat. Rev. Genet. 2007, 8(8):573-587; the contents of which are incorporated herein by reference in its entirety). These engineered glycoproteins can retarget vectors to cells expressing their corresponding target moieties. In oilier aspects, a "molecular bridge" may be introduced to direct vectors to specific ceils. The molecular bridge has dual specificities: one end can recognize viral glycoproteins, and the other end can bind to the molecular determinant on the target cell. Such molecular bridges, for example ligand-receptor, avidin-biotin, and chemical conjugations, monoclonal antibodies and engineered fusogenic proteins, can direct the attachment of viral vectors to target cells for transduction (Y ang et al., Biotechnol. Bioeng., 2008, 101(2): 357-368; and Maetzig et al, Viruses, 2011, 3, 677-713; the contents of each of which are incorporated herein by reference in their entirety).
[00339] In some embodiments, the recombinant gamma-retroviral vectors are self-inactivating (SIN) gammaretroviral vectors. 'The vectors are replication incompetent. SIN vectors may harbor a deletion within the 3' U3 region initially comprising enhancer/promoter activity. Furthermore, the 5' U3 region may be replaced with strong promoters (needed in the packaging cell line) derived from Cytomegalovirus or RSV, or an internal promoter of choice, and/or an enhancer element. The choice of the internal promoters may be made according to specific requirements of gene expression needed for a particular purpose of the invention.
[00340] In some embodiments, polynucleotides encoding the biocircuit, biocircuit components, effector module, SRE are inserted within the recombinant viral genome. 'The other components of the viral mRNA of a recombinant gamma-retroviral vector may be modified by insertion or removal of naturally occurring sequences (e.g., insertion of an IRES, insertion of a heterologous polynucleotide encoding a polypeptide or inhibitory nucleic acid of interest, shuffling of a more effective prom oter from a different retrovirus or virus in place of the wild-type prom oter and the like). In some examples, the recombinant gamma-retroviral vectors may comprise modified packaging signal, and/or primer binding site (PBS), and/or 5 '-enhancer/promoter elements in the U 3 -region of the 5'- long terminal repeat (LTR), and/or 3'-SIN elements modified in the IB- region of the 3'-LTR. These modifications may increase the titers and the ability of infection.
- I l l - [00341] Gamma retroviral vectors suitable for delivering biocircuit components, effector modules, SREs or payload constructs of the present invention may be selected from those disclosed in U.S. Pat. NOs. 8,828,718; 7,585,676; 7,351,585; U.S. application publication NO. 2007/048285; PCX application publication NOs. WO2010/113037; WO2014/121005;
WO201 /056014; and EP Pat, NOs. EP1757702; HP 1 757703 (the contents of each of which are incorporated herein by reference in their entirety).
3. Adeno-associated viral vectors (AAV)
[00342] In some embodiments, polynucleoti des of present inventi on may be packaged into recombinant adeno-associated viral (rAAV) vectors. Such vectors or viral particles may be designed to utilize any of the known serotype capsids or combinations of serotype capsids. The serotype capsids may include capsids from any identified AAV serotypes and variants thereof, for example, AAVl, AAV2, AAV2G9, AAV3, AAV4, AAV4-4, AAV5, AAV6, AAV 7, AAV8, AAV9, AAV 10, AAVl 1 , AAV 12 and AAVrhl O.
[00343] In one embodiment, the AAV serotype may be or have a sequence as described in United States Publication No. US2G030138772, herein incorporated by reference in its entirety, such as, but not limited to, AAVl (SEQ, ID NO. 6 and 64 of US20030138772), AAV2 (SEQ. ID NO. 7 and 70 of US20030138772), AAV3 (SEQ. ID NO. 8 and 71 of US20030138772), AAV4 (SEQ. ID NO. 63 of US20030138772), AAV5 (SEQ. ID NO. 114 of US20030138772), AAV6 (SEQ, ID NO. 65 of US20030138772), AAV7 (SEQ. ID NO. 1-3 of US20030138772), AAV8 (SEQ, ID NO. 4 and 95 of US20030138772), AAV9 (SEQ. ID NO. 5 and 100 of
US20030138772), AAV10 (SEQ. ID NO. 1 17 of US20030138772), AAVl 1 (SEQ. ID NO. 118 of US20030138772), AAV 12 (SEQ. ID NO. 119 of US20030138772), AAVrhlO (amino acids 1 to 738 of SEQ. ID NO. 81 of US20030138772) or variants thereof. Non-limiting examples of variants include SEQ ID NOs. 9, 27-45, 47-62, 66-69, 73-81, 84-94, 96, 97, 99, 101 -1 13 of US20030138772, the contents of which are herein incorporated by reference in their entirety. Θ0344] In one embodiment, the AAV serotype may have a sequence as described in Pulicherla et ai, {Molecular The rapy, 201 1 , 19(6): 1070-1078), U.S. Pat. NOs, 6, 156,303; 7, 198,951 ; U.S. Patent Publication NOs. US2015/0159173 and US2014/0359799; and International Patent Publication NOs. WO 1998/011244, WO2005/033321 and WO2014/14422; the contents of each of which are incorporated herein by reference in their entirety.
[00345] AAV vectors include not only single stranded vectors but self-complementary AAV vectors (scAAVs). scAAV vectors contain DNA which anneals together to form double stranded vector genome. By skipping second strand synthesis, scAAVs allow for rapid expression in the cell. [00346] The rAAV vectors may be manufactured by standard methods in the art such as by triple transfection, in sf insect cells or in suspension cell cultures of human cells such as HEK293 cells.
[00347] The biocircuits, biocircuit components, effector modules, SREs or payioad constructs may be encoded in one or more viral genomes to be packaged in the AAV capsids taught herein.
[00348] Such vectors or viral genomes may also include, in addition to at least one or two ITRs (inverted terminal repeats), certain regulatory elements necessary for expression from the vector or viral genome. Such regulatory elements are well known in the art and include for example promoters, introns, spacers, stuffer sequences, and the like.
[00349] In some embodiments, more than one effector module or SRE (e.g. DD) may be encoded in a viral genome.
4. Oncolytic viral vector
[00350] In some embodiments, polynucleotides of present invention may be packaged into oncolytic viruses, such as vaccine viruses. Oncolytic vaccine viruses may include viral particles of a thymidine kinase (TK)-deficient, granulocyte macrophage (GM)-colony stimulating factor (CSF)-expressing, replication-competent vaccinia virus vector sufficient to induce oncolysis of ceils in the tumor (e.g., US Pat. NO. 9,226,977).
5. Messenger RNA (mRNA)
[00351] In some embodiments, the effector modules of the invention may be designed as a messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo. Such mRNA molecules may have the structural components or features of any of those taught in international Application number PCT US2013/030062, the contents of which are incorporated herein by reference in its entirety.
[00352] Polynucleotides of the invention may also be designed as taught in, for example, Ribostem Limited in United Kingdom patent application serial number 0316089.2 filed on July 9, 2003 now abandoned, PCX application number PCT/GB2004/002981 filed on July 9, 2004 published as WO2005005622, United States patent application national phase entry serial number 10/563,897 filed on June 8, 2006 published as US20060247195 now abandoned, and European patent application national phase entry serial number EP2004743322 filed on July 9, 2004 published as EP 1646714 now withdrawn; Novozyrnes, Inc. in PCT application number PCT/US2007/88060 filed on December 19, 2007 published as WO2008140615, United States patent application national phase entry serial number 12/520,072 filed on July 2, 2009 published as US20100028943 and European patent application national phase entry serial number EP2007874376 filed on July 7, 2009 published as EP2104739; University of Rochester in PCX application number PCT/US2006/46120 filed on December 4, 2006 published as
WO2007064952 and United States patent application serial number 11/606,995 filed on
December 1, 2006 published as US20070141030; BioNTech AG in European patent application serial number EP2007024312 filed December 14, 2007 now abandoned, PCT application number PCT/EP2008/01059 filed on December 12, 2008 published as WO2009077134, European patent application national phase entry serial number EP2008861423 filed on June 2, 2010 published as EP2240572, United States patent application national phase entry serial number 12/,735,060 filed November 24, 2010 published as US201 10065103, German patent application serial number DE 10 2005 046 490 filed September 28, 2005, PCT application PCT/EP2006/0448 filed September 28, 2006 published as WO2007036366, national phase European patent EP1934345 published March, 21 , 2012 and national phase US patent application serial number 1 1/992,638 filed August 14, 2009 published as 20100129877; Immune Disease Institute Inc. in United States patent application serial number 13/088,009 filed April 15, 2011 published as US20120046346 and PCT application PCT/US20! 1/32679 filed April 15, 2011 published as WO20110130624: Shire Human Genetic Therapeutics in United States patent application serial number 12/957,340 filed on November 20, 2010 published as US20110244026: Sequitur Inc. in PCT application PCT/US 1998/019492 filed on September 18, 1998 published as WO 1999014346; The Scripps Research Institute in PCT application number PCT/US2010/00567 filed on February 24, 2010 published as WO2010098861, and United States patent application national phase entry serial number 13/203,229 filed November 3, 2011 published as US20120053333; Ludwig- Maximillians University in PCX application number PCT/EP2010/004681 filed on July 30, 2010 published as WO201 1012316; Cellscript Inc. in United States patent number 8,039,214 filed June 30, 2008 and granted October 18, 2011, United States patent application serial numbers 12/962,498 filed on December 7, 2010 published as US201 10143436, 12/962,468 filed on December 7, 2010 published as US201 10143397, 13/237,451 filed on September 20, 2011 published as US20120009649, and PCT applications PCT US2010/59305 filed December 7, 2010 published as WG2011071931 and PCT/US2010/59317 filed on December 7, 2010 published as WO2011071936; The Trustees of the University of Pennsylvania in PCX application number PCT/US2006/32372 filed on August 21 , 2006 published as WO2007024708, and United States patent application national phase entry serial number 11/990,646 filed on March 27, 2009 published as US20090286852; Curevac GMBH in German patent application serial numbers DE10 2001 027 283.9 filed June 5, 2001, DEI O 2001 062 480.8 filed December 19, 2001, and DE 20 2006 051 516 filed October 31, 2006 all abandoned, European patent numbers EP1392341 granted March 30, 2005 and EP1458410 granted January 2, 2008, PCX application numbers PCT/EP2002/06180 filed June 5, 2002 published as WO2002098443, PCT/EP2002/14577 filed on December 19, 2002 published as WQ200305140L
PCT/EP2007/09469 filed on December 31 , 2007 published as WO2008052770,
PCT/EP2008/03033 filed on April 16, 2008 published as WO2009127230, PCT/EP2006/004784 filed on May 19, 2005 published as WO2006122828, PCT/EP2008/00081 filed on January 9, 2007 published as WO2008083949, and United States patent application serial numbers 10/729,830 filed on December 5, 2003 published as US20050032730, 10/870,1 10 filed on June 18, 2004 published as US20050059624, 11/914,945 filed on July 7, 2008 published as
US20080267873, 12/446,912 filed on October 27, 2009 published as US2010047261 now abandoned, 12/522,214 filed on January- 4, 2010 published as US20100189729, 12/787,566 filed on May 26, 2010 published as US201 10077287, 12/787,755 filed on May 26, 2010 published as US20100239608, 13/185,119 filed on July 18, 201 1 published as US201 10269950, and
13/106,548 filed on May 12, 2011 published as US20110311472 all of which are herein incorporated by reference in their entirety-.
[00353] In some embodiments, the effector modules may be designed as self-amplifying RNA. "'Self-amplifying RNA" as used herein refers to RNA molecules that can replicate in the host resulting in the increase in the amount of the RNA and the protein encoded by the RNA. Such self-amplifying RNA may have structural features or components of any of those taught in International Patent Application Publication No. WO201 1005799 (the contents of which are incorporated herein by reference in their entirety).
VI. DOSING. DELIVERY AND ADMINISTRATIONS
[00354] The compositions of the invention may be delivered to a cell or a subject through one or more routes and modalities. The viral vectors containing one or more effector modules, SREs, immunotherapeutic agents and other components described herein may be used to deliver them to a cell and/or a subject. Other modalities may also be used such as mRNAs, plasmids, and as recombinant proteins.
1. Delivery to cells
[00355] In another aspect of the invention, polynucleotides encoding biocircuits, effector modules, SREs (e.g., DDs), payloads of interest (immunotherapeutic agents) and compositions of the invention and vectors comprising said polynucleotides may be introduced into cells such as immune effector cells. [00356] In one aspect of the invention, polynucleotides encoding biocircuits, effector modules, SREs (e.g., DDs), payloads of mterest (immunotherapeutic agents) and compositions of the invention, may be packaged into viral vectors or integrated into viral genomes allowing transient or stable expression of the polynucleotides. Preferable viral vectors are retroviral vectors including lentiviral vectors. To construct a retroviral vector, a polynucleotide molecule encoding a biocircuit, an effector module, a DD or a payload of interest (i.e. an immunotherapeutic agent) is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. The recombinant viral vector is then introduced into a packaging cell line containing the gag, pol, and env genes, but without the LTR and packaging components. The recombinant retroviral particles are secreted into the culture media, then collected, optionally concentrated, and used for gene transfer. Lentiviral vectors are especially preferred as they are capable of infecting both dividing and non-dividing cells.
[00357] Vectors may also be transferred to cells by non-viral methods by physical methods such as needles, electroporation, sonoporation, hyrdoporation; chemical carriers such as inorganic particles (e.g. calcium phosphate, silica, gold) and/or chemical methods. In some embodiments, synthetic or natural biodegradable agents may be used for delivery such as cationic lipids, lipid nano emulsions, nanoparticles, peptide based vectors, or polymer based vectors.
[Θ0358] In some embodiments, the polypeptides of the invention may be delivered to the cell directly. In one embodiment, the polypeptides of the invention may be delivered using synthetic peptides comprising an endosomal leakage domain (ELD) fused to a cell penetration domain (CLD). The polypeptides of the invention are co introduced into the cell with the ELD-CLD- synthetic peptide. ELDs facilitate the escape of proteins that are trapped in the endosome, into the cytosol. Such domains are derived proteins of microbial and viral origin and have been described in the art. CPDs allow the transport of proteins across the plasma membrane and have also been described in the art. The ELD-CLD fusion proteins synergisticaliy increase the transduction efficiency when compared to the co-transduction with either domain alone. In some embodiments, a histidine rich domain may optionally be added to the shuttle construct as an additional method of allowing the escape of the cargo from the endosome into the cytosol. The shuttle may also include a cysteine residue at the N or C terminus to generate multimers of the fusion peptide. Multimers of the ELD-CLD fusion peptides generated by the addition of cysteine residue to the terminus of the peptide show even greater transduction efficiency when compared to the single fusion peptide constructs. The polypeptides of the invention may also be appended to appropriate localization signals to direct the cargo to the appropriate sub-cellular location e.g. nucleus. In some embodiments any of the ELDs, CLDs or the fusion ELD-CLD synthetic peptides taught in the International Patent Publication, WO2016161516 and
WO2017175072 may be useful in the present invention (the contents of each of which are herein incorporated by reference in their entirety).
2. Dosing
[00359] The present invention provides methods comprising administering any one or more compositions for immunotherapy to a subject in need thereof. These may be administered to a subject using any amount and any route of administration effective for preventing or treating a clinical condition such as cancer, infection diseases and other immunodeficient diseases.
[00360] Compositions in accordance with the invention are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, or prophylactically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, previous or concurrent therapeutic interventions and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
[00361 ] Compositions of the invention may be used in varying doses to avoid T cell energy, prevent cytokine release syndrome and minimize toxicity associated with immunotherapy. For example, low doses of the compositions of the present invention may be used to initially treat patients with high tumor burden, w hile patients with low tumor burden may be treated with high and repeated doses of the compositions of the invention to ensure recognition of a minimal tumor antigen load. In another instance, the compositions of the present invention may be delivered in a pulsatile fashion to reduce tonic T cell signaling and enhance persistence in vivo. In some aspects, toxicity may be minimized by initially using low doses of the compositions of the invention, prior to administering high doses. Dosing may be modified if serum markers such as ferritin, serum C-reactive protein, IL6, IF -γ, and TNF-a are elevated.
3. Administration
[Θ0362] In some embodiments, the compositions for immunotherapy may be administered to cells ex vivo and subsequently administered to the subject. Immune cells can be isolated and expanded ex vivo using a variety of methods known in the art. For example, methods of isolating cytotoxic T cells are described in U.S. Pat. NOs. 6,805,861 and 6,531 , 451; the contents of each of which are incorporated herein by reference in their entirety. Isolation of NK cells is described in U.S. Pat. NOs. 7,435, 596; the contents of which are incorporated by reference herein in its entirety.
[00363] In some embodiments, compositions of the present invention, may be administered by any of the methods of administration taught in the copending commonly owned U.S. Provisional Patent Application No. 62/320,864 filed on 4/1 /2016, or in US Provisional Application No. 62/466,596 filed March 3, 2017 and the International Publication WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.
[00364] In some embodiments, depending upon the nature of the cells, the cells may be introduced into a host organism e.g. a mammal, in a wide variety of ways including by injection, transfusion, infusion, local instillation or implantation. In some aspects, the cells of the invention may be introduced at the site of the tumor. The number of cells that are employed will depend upon a number of circumstances, the purpose for the introduction, the lifetime of the cells, the protocol to be used, for example, the number of administrations, the ability of the cells to multiply, or the like. The cells may be in a physiologically-acceptable medium.
[00365] In some em bodiments, the cells of the invention m ay be administrated in multiple doses to subjects having a disease or condition. The administrations generally effect an improvement in one or more symptoms of cancer or a clinical condition and/or treat or prevent cancer or clinical condition or symptom thereof.
[00366] In some embodiments, the compositions for immunotherapy may be administered in vivo. In some embodiments, polypeptides of the present invention comprising biocircuits, effector molecules, SREs, pay loads of interest (immunotherapeutic agents) and compositions of the invention may be delivered in vivo to the subject. In vivo delivery of immunotherapeutic agents is well described in the art. For example, methods of delivery of cytokines are described in the E.P. Pat. NO. EP0930892 Al, the contents of which are incorporated herein by reference.
Routes of deliver}'
[00367] The pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SRE^s (e.g., DDs), payloads (i.e. immunotherapeutic agents), vectors and cells of the present invention may be administered by any route to achieve a therapeutically effective outcome.
[00368] These include, but are not limited to enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intra-arterial (into an artery), intramuscular (into a muscle), intracranial (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intrasinal infusion, intravitreal, (through the eye), intravenous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), in ear drops, auricular (in or by way of the ear), buccal (directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis, endocervicai, endosinusial, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, iiitra-amniotic, intraarticular, intrabiliary, intrabronchial, intrabursal, intracartilagmous (within a cartilage), intracaudal (within the cauda equine), intracisternal (within the cisterna magna
cerebellomedularis), intracomeal (within the cornea), dental mtracornal, intracoronary (withm the coronary arteries), intracorporus cavemosum (within the dilatable spaces of the corporus cavernosa of the penis), intradiscal (within a disc), intraductal (within a duct of a gland), mtraduodenal (within the duodenum), intradural (within or beneath the dura), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (within the stomach),
intragingival (within the gingivae), intraileal (within the distal portion of the small intestine), intralesionai (within or introduced directly to a localized lesion), intraluminal (within a lumen of a tube), mtraiymphatic (within the lymph), intramedullary (within the marrow cavity of a bone), intrameningeal (within the meninges), intratnyocardial (within the myocardium), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura), mtraprostatic (within the prostate gland), mtrapulmonary (within the lungs or its bronchi), intrasinal (within the nasal or periorbital sinuses), intraspinal (within the vertebral column), intrasynovial (within the synovial cavity of a joint), intratendinous (within a tendon), intratesticular (within the testicle), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thorax), intratubuiar (within the tubules of an organ), intratumor (within a tumor), intratympanic (within the auras media), intravascular (within a vessel or vessels), intraventricular (within a ventricle), iontophoresis (by means of electric current where ions of soluble salts migrate into the tissues of the body), irrigation (to batlie or flush open wounds or body cavities), laryngeal (directly upon the larynx), nasogastric (through the nose and into the stomach), occlusive dressing technique (topical route administration which is then covered by a dressing which occludes the area), ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (across or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis or spinal .
VII. DEFINITIONS
[00369] At various places in the present specification, features or functions of the compositions of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual sub combination of the members of such groups and ranges. The following is a non-limiting list of term definitions.
[00370] Activity: As used herein, the term "activity"' refers to the condition in which things are happening or being done. Compositions of the invention may have activity and this activity may involve one or more biological events. In some embodiments, biological events may include cell signaling events. In some embodiments, biological events may include cell signaling events associated protein interactions with one or more corresponding proteins, receptors, small molecules or any of the biocircuit components described herein.
[00371] Adoptive eel! therapy (ACT): The terms "Adoptive cell therapy" or "Adoptive cell transfer", as used herein, refer to a cell therapy involving in the transfer of cells into a patient, wherein cells may have originated from the patient, or from another individual, and are engineered (altered) before being transferred back into the patient. The therapeutic cells may be derived from the immune system, such as Immune effector cells: CD4+ T cell; CD8+ T cell, Natural Killer cell (NK cell); and B cells and tumor infiltrating lymphocytes (TILs) derived from the resected tumors. Most commonly transferred cells are autologous anti-tumor T cells after ex vivo expansion or manipulation. For example, autologous peripheral blood lymphocytes can be genetically engineered to recognize specific tumor antigens by expressing T-cell receptors (TCR) or chimeric antigen receptor (CAR).
Θ0372] Agent: As used herein, the term "agent" refers to a biological, pharmaceutical, or chemical compound. Non-limiting examples include simple or complex organic or inorganic molecule, a peptide, a protein, an oligonucleotide, an antibody, an antibody derivative, antibody fragment, a receptor, and soluble factor. [00373] Agonist, the term '"agonist" as used herein, refers to a compound that, in combination with a receptor, can produce a cellular response. An agonist may he a ligand that directly binds to the receptor. Alternatively, an agonist may combine with a receptor indirectly by, for example, (a) forming a complex with another molecule that directly binds to the receptor, or (b) otherwise resulting in the modification of another compound so that the other compound directly binds to the receptor. An agonist may be referred to as an agonist of a particular receptor or family of receptors, e.g., agonist of a co-stimulatory receptor.
[00374] Antagonist: the term "antagonist" as used herein refers to any agent that inhibits or reduces the biological activity of the target(s) it binds.
[00375] Antigen: the term "antigen" as used herein is defined as a molecule that provokes an immune response when it is introduced into a subject or produced by a subject such as tumor antigens which arise by the cancer development itself. This immune response may involve either antibody production, or the activation of specific immunologically -competent cells such as cytotoxic T lymphocytes and T helper cells, or both. An antigen can be derived from organisms, subunits of proteins/antigens, killed or inactivated whole cells or lysates. In the context of the invention, the terms "antigens of interest" or "desired antigens" refers to those proteins and/or other biomolecules provided herein that are immunospecifically bound or interact with antibodies of the present invention and/or fragments, mutants, variants, and/or alterations thereof described herein. In some embodiments, antigens of interest may comprise any of the polypeptides or payloads or proteins described herein, or fragments or portions thereof.
[00376] Approximately: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise e v ident from the context (except where such number would exceed 100 of a possible value).
[00377] Associated with: As used herein, the terms "associated with," "conjugated," "linked," "attached," and "tethered," when used with respect to two or more moieties, mean that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serve as linking agents, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. An "association" need not be strictly through direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a hybridization based connectivity sufficiently stable such that the "associated" entities remain physically associated.
[00378] Autologous: the term "autologous" as used herein is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.
[00379] Barcode: the term "barcode" as used herein refers to polynucleotide or amino acid sequence that distinguishes one polynucleotide or amino acid from another.
[00380] Cancer: the term "cancer" as used herein refers a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues ultimately metastasize to distant parts of the body through the lymphatic system or bloodstream.
[00381] Co-stimulatory molecule: As used herein, in accordance with its meaning in immune T cell activation, refers to a group of immune cell surface receptor/ligands which engage between T cells and APCs and generate a stimulatory signal in T cells which combines with the
stimulatory signal in T cells that results from T cell receptor (TCR) recognition of antigen/MHC complex (pMHC) on APCs
[00382] Cytokines: the term "cytokines", as used herein, refers to a family of small soluble factors with pleiotropic functions that are produced by many cell types that can influence and regulate the function of the immune system.
[Θ0383] Delivery: the term "delivery" as used herein refers to the act or manner of delivering a compound, substance, entity, moiety, cargo or payload. A "delivery agent" refers to any agent which facilitates, at least in part, the in vivo delivery of one or more substances (including, but not limited to compounds and/or compositions of the present invention) to a cell, subject or other biological system cells.
[00384] Destabilized: As used herein, the term "destable," "destabilize," "destabilizing region" or "destabilizing domain" means a region or molecule that is less stable than a starting, reference, wild-type or native form of the same region or molecule.
[00385] Engineered: As used herein, embodiments of the invention are "engineered" when they are designed to have a feature or property, whether structural or chemical, that varies from a starting point, wild type or native molecule.
[00386] Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1 ) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end processing): (3) translation of an RNA into a polypeptide or protein; (4) folding of a polypeptide or protein; and (5) post-translational modification of a polypeptide or protein. [00387] Feature: As used herein, a "feature" refers to a characteristic, a property, or a distinctive element,
[00388] Formulation: As used herein, a "formulation" includes at least a compound and/or composition of the present invention and a delivery agent.
[00389] Fragment: A "fragment," as used herein, refers to a portion. For example, fragments of proteins may comprise polypeptides obtained by digesting full-length protein. In some embodiments, a fragment of a protein includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250 or more amino acids. In some embodiments, fragments of an antibody include portions of an antibody.
[00390] Functional: As used herein, a "functional" biological molecule is a biological entity with a structure and in a form in w h ich it exhibits a property and/or activity by which it is characterized.
[00391] Immune cells: the term "an immune cell", as used herein, refers to any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid ceils such as T cells, B ceils and natural killer (NK) cells). Exemplar],' immune system cells include a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a T γδ cell, a Ταβ cell, a regulatory T cell, a natural killer cell, and a dendritic cell. Macrophages and dendritic cells may be referred to as "antigen presenting cells" or "APCs," which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on tlie surface of the APC complexed with a peptide interacts with a TCR on the surface of a T cell.
[00392] Immunotherapy: the term "immunotherapy" as used herein, refers to a type of treatment of a disease by tlie induction or restoration of tlie reactivity of the immune sy stem towards the disease.
[00393] Immunotherapeutic agent: the term "immunotherapeutic agent" as used herein, refers to the treatment of disease by the induction or restoration of the reactivity of the immune system towards tlie disease with a biological, pharmaceutical, or chemical compound.
[00394] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe). [00395] In vivo: As used herein, the term "in vivo" refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).
[00396] Linker: As used herein, a linker refers to a moiety that connects two or more domains, moieties or entities. In one embodiment, a linker may comprise 10 or more atoms. In a further embodiment, a linker may comprise a group of atoms, e.g., 10-1,000 atoms, and can be comprised of the atoms or groups such as, but not limited to, carbon, amino, alkylammo, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. In some embodiments, a linker may comprise one or more nucleic acids comprising one or more nucleotides. In some embodiments, the linker may comprise an amino acid, peptide, polypeptide or protein. In some embodiments, a moiety bound by a linker may include, but is not limited to an atom, a chemical group, a nucleoside, a nucleotide, a nucleobase, a sugar, a nucleic acid, an ammo acid, a peptide, a polypeptide, a protein, a protein complex, a payload (e.g., a therapeutic agent), or a marker (including, but not limited to a chemical, fluorescent, radioactive or bioiummescent marker). The linker can be used for any useful purpose, such as to form multimers or conjugates, as well as to administer a payload, as described herein. Examples of chemical groups that can be incorporated into the linker include, but are not limited to, alkyl, alkenyi, alkynyi, amido, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl, each of which can be optionally substituted, as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or propylene glycol monomelic units, e.g., diethyiene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers, Other examples include, but are not limited to, cleavable moieties within the linker, such as, for example, a disulfide bond (-S-S-) or an azo bond (-N=N-), which can be cleaved using a reducing agent or photolysis. Non-limiting examples of a selectively cleavable bonds include an amido bond which may be cleaved for example by the use of tris(2- carboxyethyl) phosphine (TCEP), or other reducing agents, and/or photolysis, as well as an ester bond which may be cleaved for example by acidic or basic hydrolysis.
[00397] Checkpoint/factor: As used herein, a checkpoint factor is any moiety or molecule whose function acts at the junction of a process. For example, a checkpoint protein, ligand or receptor may function to stall or accelerate the cell cycle.
[00398] Metabolite: Metabolites are the intermediate products of metabolic reactions catalyzed by enzymes that naturally occur within cells. This term is usually used to describe small molecules, fragments of larger biomolecules or processed products.
[00399] Modified: As used herein, the term "modified" refers to a changed state or structure of a molecule or entity as compared with a parent or reference molecule or entity. Molecules may be modified in many ways including chemically, structurally, and functionally. In some embodiments, compounds and/or compositions of the present invention are modified by the introduction of non-natural amino acids.
[00400] Mutation: As used herein, the term '"mutation" refers to a change and/or alteration. In some embodiments, mutations may be changes and/or alterations to proteins (including peptides and polypeptides) and/or nucleic acids (including polynucleic acids). In some embodiments, mutations comprise changes and/or alterations to a protein and/or nucleic acid sequence. Such changes and/or alterations may comprise the addition, substitution and or deletion of one or more amino acids (in the case of proteins and/or peptides) and/or nucleotides (in the case of nucleic acids and or polynucleic acids, e.g., polynucleotides). In some embodiments, wherein mutations comprise the addition and/or substitution of amino acids and/or nucleotides, such additions and/or substitutions may comprise 1 or more amino acid and/or nucleotide residues and may include modified amino acids and/or nucleotides. The resulting construct, molecule or sequence of a mutation, change or alteration may be referred to herein as a mutant.
[00401] Neoantigen: the term '"neoantigen"', as used herein, refers to a tumor antigen that is present in tumor cells but not normal cells and do not induce deletion of their cognate antigen specific T cells in thymus (i.e., central tolerance). These tumor neoantigens may provide a "'foreign" signal, similar to pathogens, to induce an effective immune response needed for cancer immunotherapy. A neoantigen may be restricted to a specific tumor. A neoantigen be a peptide/protem with a missense mutation (missense neoantigen), or a new peptide with long, completely novel stretches of amino acids from novel open reading frames (neoORFs). The neoORFs can be generated in some tumors by out-of-frame insertions or deletions (due to defects in D A mismatch repair causing microsatellite instability), gene-fusion, read-through mutations in stop codons, or translation of improperly spliced RNA (e.g., Saeterdal et al., P'roc Natl Acad Sci USA, 2001, 98: 13255-13260).
[00402] Off target: As used herein, "off target" refers to any unintended effect on any one or more target, gene, cellular transcript, cell, and/or tissue.
[00403] Operably linked: As used herein, the phrase "operably linked" refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties or the like.
[00404] Payload or payload of interest (POP): the terms "payload" and "payload of interest (POI)", as used herein, are used interchangeable. A payload of interest (ΡΟΓ) refers to any protein or compound whose function is to be altered. In the context of the present invention, the POI is a component in the immune system, including both innate and adaptive immune systems. Payloads of interest may be a protein, a fusion construct encoding a fusion protein, or non- coding gene, or variant and fragment thereof, Payload of interest may, when amino acid based, may be referred to as a protein of interest.
[00405] Pharmaceutically acceptable excipients the term "pharmaceutically acceptable excipient," as used herein, refers to any ingredient other than active agents (e.g., as described herein) present in pharmaceutical compositions and having the properties of being substantially nontoxic and non-inflammatory in subjects. In some embodiments, pharmaceutically acceptable excipients are vehicles capable of suspending and/or dissolving active agents. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids,
disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium, carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethyicellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylceliulose, lactose, magnesium stearate, maltitoi, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyi cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (com), stearic acid, sucrose, talc, titanium, dioxide, vitamin A, vitamin E, vitamin C, and xylitol.
[00406] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts of the compounds described herein are forms of the disclosed compounds wherein the acid or base moiety is in its salt form (e.g., as generated by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids: and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisuifate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,
hydrochloride, hydroiodide, 2 -hydroxy -ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts, for example, from non-toxic inorganic or organic acids. In some embodiments, a pharmaceutically acceptable salt is prepared from a parent compound which contains a bask or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropri ate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et a!.. Journal of Pharmaceutical Science, 66, 1 -19 (1977), each of which is incorporated herein by reference in its entirety. Pharmaceutically acceptable solvate: The term "pharmaceutically acceptable solvate," as used herein, refers to a crystalline form of a compound wherein molecules of a suitable solvent are incorporated in the crystal lattice. For example, solvates may be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof. Examples of suitable solvents are ethanol, water (for example, mono-, di~, and tri -hydrates), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N, N'-dimethylformamide (DMF), N, N'-dimethylacetamide (DMAC), 1 ,3-dimethyl- 2-imidazolidinone (DMEU), l,3-dimethyl-3,4,5,6-tetrahydro-2-(lH)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is referred to as a "hydrate." In some embodiments, the solvent incorporated into a solvate is of a type or at a level that is physiologically tolerable to an organism to which the solvate is administered (e.g., in a unit dosage form of a pharmaceutical composition).
[00407] Stable: As used herein "stable" refers to a compound or entity that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and preferably capable of formulation into an efficacious therapeutic agent.
[00408] Stabilized: As used herein, the term "stabilize", "stabilized," "stabilized region" means to make or become stable. In some embodiments, stability is measured relative to an absolute value. In some embodiments, stability is measured relative to a secondaiy status or state or to a reference compound or entity. [00409] Standard CAR: As used herein, the term, "standard CAR" refers to the standard design of a chimeric antigen receptor. The components of a CAR fusion protein including the extracellular scFv fragment, transmembrane domain and one or more intracellular domains are linearly constructed as a single fusion protein.
[00410] Stimulus response element (SRE): the term "stimulus response element (SRE), as used herein, 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. As used herein, the "responsive" nature of an SRE to a stimulus may be characterized by a covalent or non-covalent interaction, a direct or indirect association or a structural or chemical reaction to the stimulus. Further, 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. One non-limiting example of an SRE is a destabilizing domain (DD).
[00411] Subject: As used herein, the term "subject" or "patient" refers to any organism to which a composition in accordance with the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.
[00412] T cell: A T cell is an immune cell that produces T cell receptors (TCRs). T cells can be naive (not exposed to antigen; increased expression of CD62L, CCR7, CD28, CDS, CD 127, and CD45RA, and decreased expression of CD45RO as compared to TCM), memory T cells (TM) (antigen-experienced and long-lived), and effector cells (antigen-experienced, cytotoxic). Tvi can be further divided into subsets of central memory T cells (TCM, increased expression of CD62L, CCR7, CD28, CD 127, CD45RO, and CD95, and decreased expression of CD54RA as compared to naive T cell and effector memory T cells (TEM, decreased expression of CD62L, CCR7, CD28, CD45RA, and increased expression of CD127 as compared to naive T cells or TCM). Effector T cells (TE) refers to antigen-experienced CD8+ cytotoxic T lymphocytes that have decreased expression of CD62L, CCR7, CD28, and are positive for gramzyme and perforin as compared to TCM. Other exemplary T cells include regulator}' T cells, such as CD4+ CD25+ (Foxp3+) regulatory T cells and Tregl7 cells, as well as Trl , Th3, CD8+CD28-, and Qa-1 restricted T cells. [00413] T cell receptor: T cell receptor (TCR) refers to an immunoglobulin superfamily member having a variable antigen binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail, which is capable of specifically binding to an antigen peptide bound to a MHC receptor. A TCR can be found on the surface of a cell or in soluble form and generally is comprised of a heterodimer having a and β chains (also known as TCRa and TCRp,
respectively), or y and δ chains (also known as TCRy and TCR5, respectively). Tire extracellular portion of TCR chains (e.g., a-chain, β-chain) contains two immunoglobulin domains, a variable domain (e.g., a-chain variable domain or Va, β-chain variable domain or Vp) at the N-tenninus, and one constant domain (e.g., a-chain constant domain or Ca and β-chain constant domain or Cp,) adjacent to the cell membrane. Similar to immunoglobulin, the variable domains contain complementary determining regions (CDRs) separated by framework regions (FRs). A TCR is usually associated with the CDS complex to form a TCR complex. As used herein, the term "TC complex" refers to a complex formed by the association of CDS with TCR. For example, a TCR complex can be composed of a CD3y chain, a CD35 chain, two CD3s chains, a homodimer of Οϋ3ζ chains, a TCRa chain, and a ΤΟ¾β chain. Alternatively, a TCR complex can be composed of a CD3y chain, a CD35 chain, two CD3s chains, a homodimer of ΟΧ ζ chains, a TCRy chain, and a TCR5 chain. A "component of a TCR complex," as used herein, refers to a TCR chain (i.e., TCRa, TCRp, TCRy or TCR5), a CD3 chain (i.e., CD3y, CD38, CD3e or CO3Q, or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRa and TCR , a complex of TCRy and TCR5, a complex of CDS ε and CD38, a complex of CDSy and CD3s, or a sub-TCR complex of TCRa, TCR , CD3y, CD35, and two CD3s chains.
[00414] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition, in some embodiments, a therapeutically effective amount is provided in a single dose. In some embodiments, a therapeutically effective amount is administered in a dosage regimen comprising a plurality of doses. Those skilled in the art will appreciate that in some embodiments, a unit dosage form may be considered to comprise a therapeutically effective amount of a particular agent or entity if it comprises an amount that is effective when administered as part of such a dosage regimen.
[00415] Treatment or treating: As used herein, the terms "treatment" or "treating" denote an approach for obtaining a beneficial or desired result including and preferably a beneficial or desired clinical result. Such beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of (or destroying) cancerous cells or other diseased, reducing metastasis of cancerous cells found in cancers, shrinking the size of the tumor, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and/or prolonging survival of individuals,
[00416] Tune: As used herein, the term "tune" means to adjust, balance or adapt one thing in response to a stimulus or toward a particular outcome. In one non-limiting example, the SREs and/or DDs of the present invention adjust, balance or adapt the function or structure of compositions to which they are appended, attached or associated with in response to particular stimuli and/or environments.
EQUIVALENTS AND SCOPE
[00417] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the in vention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.
[00418] In the claims, articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are consi dered sati sfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or the entire group members are present in, employed in or otherwise relevant to a given product or process.
[00419] It is also noted that the term "comprising" is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of is thus also encompassed and disclosed.
[00420] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. [00421] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.
[00422] It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.
EXAMPLES
Example 1. Generation of novel ligand responsive SREs or DDs by mutagenesis screening Study design
[00423] To engineer constructs that display ligand dependent stability, a candidate ligand binding domain (LED) is selected and a cell-based screen using yellow fluorescent protein (YFP) as a reporter for protein stability is designed to identify mutants of the candidate LED possessing the desired characteristics of a destabilizing domain: low protein levels in the absence of a ligand of the LBD, (i.e., low basal stability), large dynamic range, robust and predictable dose-response behavior, and rapid kinetics of degradation (Banaszynski, et al., (2.006) Cell; 126(5): 995-1004). The candidate LBD binds to a desired ligand but not endogenous signaling molecules.
[00424] The candidate LBD sequence (as a template) is first mutated using a combination of nucleotide analog mutagenesis and error-prone PCR, to generate libraries of mutants based on the template candidate domain sequence. The libraries generated are cloned in-frame at either the 5'- or 3 '-ends of the YFP gene, and a retroviral expression system is used to stably transduce the libraries of YFP fusions into NIH3T3 fibroblasts.
[00425] The transduced NIH3T3 cells are subjected to three to four rounds of sorting using fluorescence -activated cell sorting (FACS) to screen the libraries of candidate DDs. Transduced NIH3T3 cells are cultured in the absence of the high affinity ligand of the ligand binding domain (LBD), and cells that exhibit low levels of YFP expression are selected through FACS.
Screening Strategy I
[00426] The selected cell population is cultured in the presence of the high affinity ligand of the ligand binding domain for a period of time (e.g., 24 hours), at which point cells are sorted again by FACS. Cells that exhibit high levels of YFP expression are selected through FACS and the selected cell population is split into two groups and treated again with the high affinity ligand of the ligand binding domain at different concentrations; one group is treated with the lower concentration of the ligand and the other is treated with a high concentration of the ligand, for a period of time (e.g., 24 hours), at which point ceils are sorted again by FACS. Cells expressing mutants that are responsive to lower concentrations of the ligand are isolated.
[00427] The isolated cells responsive to the lower concentration of the ligand are treated with the ligand again and cells exhibiting low fluorescence levels are collected 4 hours following removal of the ligand from the media. This fourth sorting is designed to enrich cells that exhibit fast kinetics of degradation (Iwamoto et al, Chem Biol. 2010 Sep 24; 17(9): 981-988).
Screening Strategy II
[00428] The selected cell population is subject to additional one or more sorts by FACS in the absence of high affinity ligand of LBD and cells that exhibit low levels of YFP expression are selected for further analysis. Cells are treated with high affinity ligand of the ligand binding domain, for a period of time (e.g. 24 hours), and sorted again by FACS. Cells expressing high levels of YFP are selected for through FACS. Cells with high expression of YFP are treated with ligand again and cells exhibiting low fluorescence levels are collected 4 hours following removal of the ligand from the media to enrich cells that exhibit fast kinetics of degradation. Any of the sorting steps may be repeated to identify DDs with ligand dependent stability.
[00429] The cells are recovered after sorting. The identified candidate cells are harvested and the genomic DNA is extracted. The candidate DDs are amplified by PCR. and isolated. The candidate DDs are sequenced and compared to the LBD template to identify the mutations in candidate DDs.
Example 2. DP regulated IL15
[00430] To test ligand dependent IL15 production, i million HEK-293T cells were plated in a 6-weli plate in growth media containing DMEM and 10% FBS and incubated overnight at 37°C at 5% C02. Cells were then transfected with lOOng of OT-IL 15 -001 (constitutive) or OT-IL15- 002 (ecDHFR-IL 15) using Lipofectamine 2000 and incubated for 48 hrs. Following the mcubation, media was exchanged for growth medium with 10μΜ Trimethoprim or vehicle control and further incubated for 24 hrs. Media was collected and the undiluted samples or samples diluted 4, 16, 256, 1024, 4096 or 16384-fold were tested using human IL15 ELISA. The stabilization ratio was defined as fold change in IL15 expression with ligand treatment compared to treatment with DMSO (i .e. in the absence of ligand) with the same construct. Stabilization ratio greater than 1 is desired. Average IL15 ELISA readings and stabilization ratio are presented in Table 9. Table 9: DD-IL15 induction
Figure imgf000135_0001
[00431] The 16-fold, 4-fold diluted, and undiluted media samples showed stabilization ratio greater than 1.5, suggesting a Trimethoprim dependent stabilization of IL15 at these dilutions. Example 3, DD regulated expression of IL15-IL15Ra fusion molecule
[0Θ432] A fusion molecule is generated by fusing membrane bound IL15, IL15 Receptor alpha subunit (IL15Ra) and DDs such as ecDHFR (DD), FKBP (DD), or human DHFR (DD). These fusion molecules were cloned into pLVX-EF 1 a-IRES-Puro vector.
[00433] To test ligand dependent IL15-IL15Ra production, 1 million HEK-293T cells were plated in a 6-well plate in growth media containing DMEM and 10 FBS and incubated overnight at 37°C, 5% C02. Cells were then transfected with lOOng of constitutive IL15-IL15Ra (OT- IL15-008) or DD linked IL15-IL15Ra (OT-IL15-006, OT-IL15-0Q7, OT-IL 15-009, OT-IL15- 010, OT-IL 15-011) using Lipofectamme 2000 and incubated for 24 hrs. Following the incubation, media is exchanged for growth medium with or without 10μΜ Trimethoprim or ΙμΜ Shield- 1 and further incubated for 24 hrs. Cells were harvested and IL15 levels are analyzed via western blotting using human IL15 antibody (Abeam, Cambridge, UK). OT-IL15-009 showed the strong ligand (Trimethoprim) dependent stabilization of IL15, while OT-IL15-006 and OT- 1L15-007 showed modest ligand dependent stabilization of 1L15 (Figure 13A).
[00434] Surface expression of membrane bound IL15-IL15Ra constructs (OT-IL15-006, OT- IL15-007, OT-IL 15-008, OT-IL15-009, OT-IL15-010, OT-IL15-011) was determined by FACS using anti-ILlS and anti-lL15Ra antibodies. HEK293T cells were transfected with IL15-IL15Ra constructs and then treated with suitable ligand (Shield- 1 or Trimethoprim). 48 hours after transfection, cells were analyzed using FACS, As expected, constitutive IL15-IL15Ra construct OT-IL15-008 showed high surface expression of IL15 and IL15Ra both in the presence and absence of ligand. Consistent with the results from the western blot, OT-IL 15-009 showed the strong ligand (Trimethoprim) dependent surface expression of IL15 and ILlSRa (Figure 13B, Figure 13C), [00435] Membrane boimd-lL1.5~lL1.5Ra constructs (OT-IL15-008 to OT IL15-011) were transduced into human colorectal carcinoma cell line, HCT-116 and stable integrants were selected with 2^ig of puromycin. Stably integrated ceils were then incubated for 24 hours in the presence or absence of ΙΟμΜ Trimethoprim or ΙμΜ Methotrexate.
[00436] Surface expression of IL15 -IL15Ra fusion constructs was examined by staining with PE- conjugated lLI5Ra antibody (Cat no. 330207, Biolegend, San Diego, CA). The median fluorescence intensity obtained with the different constructs in the presence or absence of the corresponding ligand is presented in Table 10.
Table 10: Surface expression of IL15-IL15Ra fusion constructs
Figure imgf000136_0001
[00437] The stabilization ratio was calculated as the fold change in GFP intensity in ligand treated samples compared to treatment with DMSO (i.e. in the absence of ligand) with the same constmct. The destabiiization ratio was calculated as the fold change in GFP intensity in the DD regulated constructs compared to the constitutive construct (OT-1L15-008) in the absence of the ligand. Destabiiization ratios less than 1 and stabilization ratios greater than 1 are desired in DDs. The ratios are presented in Table 11.
Table 11 : IL15-IL15Ra destabiiization and stabilization ratios
Figure imgf000136_0002
[00438] Destabiiization ratios less than one was observed with OT-IL 15-006 (ecDHFR (R12H, E129K)) and OT-IL 15 -011 (hDHFR (Q36F, N65F, Y122I)) indicating a strong destabiiization in the absence of ligand. Stabilization ratio greater than 1 was observed with all constructs with TMP treatment and with both OT-IL 15-010 and 1 1 with MTX treatment. These data show that OT-IL 15 -006 and OT-IL 15 -011 are both strongly destabilized in the absence of ligand and strongly stabilized in the presence of ligand. [00439] The expression and ligand-dependent stabilization of IL15-IL15Ra constructs (OT~ IL15-008 to OT-TL15-011) was measured in HCT-116. Cells were mcubated with
10μΜ Trimethoprim or ΙμΜ Methotrexate or DMSO for 24 hours. Following incubation, cells were harvested and cell extracts were prepared. Cell extracts were run on SDS-PAGE and western blotted with anti-IL15 antibody (Catalog No. 7213, Abeam, Cambridge, UK). As shown in Figure 13D, the IL15/IL15Ra constitutive construct (OT-IL15-008) showed ligand independent 11,15 expression while the DD regulated constructs (OT-IL15-009 to OT-IL 15-011) showed ligand dependent IL15 expression. The identity of the IL15 bands was also confirmed by immunoblotting with the anti-human DHFR antibody (Catalog No. 117705, Genetex, Irvine, CA). As shown in Figure 13D, both ILlS-ILISRa fusion constructs (OT-TL 15-010 and 011 ) showed ligand dependent expression of DHFR expression.
[00440] To evaluate the dose dependence of ligand induced stabilization, IL15-IL15Ra fusion constructs namely, OT-IL15-009 (ecDHFR (R12Y, YIOOI)), OT-IL15-010 (hDHFR (Y1221, A 125F)), and OT-IL15-01 1 (hDHFR (Q36F, N65F, Y122I)) were stably transduced into HCT- 116 cells and incubated with increasing concentrations of Trimethoprim for 24 hours. Surface expression of IL15-IL15Ra fusion construct was quantified by FACS using IL15Ra- PE antibody. The median fluorescence intensity with increasing doses of T'MP is represented in Table 12.
Table 12: Surface expression of IL15-IL15Ra
Figure imgf000137_0001
[00441] As shown in Table 12, all three constructs showed a dose dependent increase in median fluorescence intensity indicating a dose dependent increase in surface expression of IL15- IL15Ra fusion upon addition of DD stabilizing ligand. [00442] The time course of ligand dependent stabilization of IL15-IL15Ra fusion constructs was measured in HCT-1 16 cells. Cells were transduced with OT-IL15-009 (ecDHFR (R12Y, Y1001) construct and incubated with 10μΜ Trimethoprim for 0, 12, 16, 24, 48 or 72 hours. Following incubations, surface expression of IL15-IL15Ra fusion construct was quantified by FACS using IL15Ra- PE antibody and compared to parental untransfected cells. The median fluorescence intensity ( MI D over time is represented in Table 13.
Table 13: Time course of IL15-IL15Ra surface expression
Figure imgf000138_0001
[00443] As shown in Table 13, OT-IL15-009 (ecDHFR (R12Y, Y1001) showed a time- dependent increase in median fluorescence intensity indicating that the surface expression of IL15~IL15Ra fusion increased with increased duration of treatment with DD stabilizing ligand. Example 4, In vitro T cell assay development
[00444] The goal of the study was to determine the T cell stimulation regimen needed to maximize T cell persistence and T cell differentiation in vitro, to mimic an in vivo adoptive cell transfer therapy regimen. The study recapitulates the design of the adoptive cell therapy regimen wherein the T cells were initially exposed to the antigen in vitro which results in activation followed by a resting phase and finally in vivo transfer where the T cells encounter the antigen again. T cells were stimulated CD3/CD28 beads or soluble CD3/CD28 on day 0 and the CD3/CD28 stimulus was washed off at the end of 48 hours. Cells were treated with a dose of 1L12 ranging from 0.01- 1000 ng/mL. On day 9, the Thl phenotvpe of the cells was evaluated by examining the frequency of IFNgamma positive CD4+ cells and CD8+ cells. On day 14, cells were divided into two groups- one group received a second CD3/CD28 stimulation and a second group that was not stimulated. On day 16, the Thl phenotvpe was evaluated in both groups using FACS. The results for day 16 are presented in Figure 14. IFN gamma expression was higher in cells that received a CD3/CD28 restimulation on day 14 compared to cells that did not receive second stimulation. This indicates that both antigen restimulation and cytokine exposure are required for the Thl phenotvpe. Further, as little as 0.1 ng/mL of IL12 could cause Thl- skewing and IFN gamma production from T cells in vitro, and higher doses of IL12 further improved this effect.
Example 5. Measuring human T cells responses in vitro and in vivo
[00445] 1L12 promotes the differentiation of naive T cells into Thl cells which results in the secretion of IFN gamma from T cells. Human T cells were treated with IL12 or left untreated and analyzed by flow cytometry for the expression of IFN gamma and T cell marker CD3. Treatment with IL12 resulted in the differentiation of T cells as measured by an increase in the percentage of IFN gamma positive T cells from 0.21 to 22.3 (see inset of Figure 15A).
[00446] To test if membrane bound IL15 IL15Ra fusion protein (OT-IL15-008) can induce human T cell expansion, human T cells were transduced with the construct. T cell proliferation was measured by evaluating forward and side scatter of the T cell population using flow cytometry. Transduction with membrane bound IL15/IL15Ra fusion construct resulted in the expansion of human T cells (58.9) compared to control untransfected cells (37.8) (Figure 15B).
[00447] Tracking T cells following their adoptive transfer is critical to determine their distribution at different sites in the host, their identity and persistence ove time. Human T cells were stimulated with CD3/CD28 beads and incubated with 50U/ml of IL2. Cells were expanded in vitro for 7 days with IL2 supplementation on day 3 and day 5. On day 5, the CD3/CD28 beads were removed and the cells were cultured for two days. On day 7, cells were washed to remove IL2 and 5 million human T cells were injected intravenously into immune compromised, l QD.Cg~Prkdcsad Ii2rgim ! Wi!/SzJ mice. Blood samples were obtained 4, 24, 120 and 168 hours after cell transfer. Mice were euthanized 168 hours after cell transfer and the bone marrow and spleen were harvested. Immune cells were isolated from ail samples and analyzed for the presence of human T cells using CD3 and CD45 cell surface markers. As shown in Figure 15C, the percentage of CDS positive, CD45 positive human T cells in the blood was higher in animals injected with human T ceils, especially at 120 and 168 hours. CD3 positive, CD45 positive human T cells were also detected in the spleen and bone marrow of animals injected with human T cells. As expected no CD3 positive, CD45 positive human T cells were detected in control animals that were not injected with human T cells.
[00448] To determine the identity of the T cells following adoptive transfer, blood samples were collected from mice 48 hours after injection. CD4 and CD 8 T cells were analyzed for surface expression of CD45RA and CD62L. Both markers are highly expressed in naive T cells but are lost as the T cells become antigen exposure. As shown in Figure 15D, human CD4 and CD 8 T ceils showed high surface expression of both markers prior to injecting into mice, but was lost 48 hours after in vivo cell transfer indicating that the human T cells are exposed to the antigen in vivo.
Example 6, Functional analysis of DD regulated IL15-IL15Ra fusion molecule
[00449] Activation via 1L15 can sustain T cell persistence by conferring a survival advantage. In addition, IL15/lL15Ra fusion molecule has been shown to confer a memory phenotype on T cells and increase proliferation of NK cells (Hurton (2016), PNAS, 113: E7788-7797; the contents of which are incorporated herein by reference in their entirety).
[00450] To evaluate signaling by DD regulated IL15-IL15Ra fusion constructs, NK92 cells are incubated with HCT-l 16 cells expressing DD regulated ILlS-ILlSRa fusion constructs. Trans signaling by IL15/lL15Ra is expected to increase STATS phosphorylation in NK92, which is measured by western blotting, and by FACS. Proliferation of NK92 cells is also measured.
[00451] To evaluate the effect of DD regulated IL15-IL15Ra fusion constructs on primary T cells, cells are transduced with the fusion constructs. T cell proliferation in the absence of exogenous IL15 supplementation is measured. The T cell memory phenotype is measured by quantifying CD62L expression by FACS.
[00452] To assess if DD-IL15/IL15R expressing T cells maintain prolonged persistence in vivo, DD modified T cells are injected into mice. Constructs are tagged with luciferase reporter to allow in vivo tracking in mice. Mice are treated with vehicle control or corresponding iigand, Shield- 1, Trimethoprim or Methotrexate depending on the construct utilized and monitored over a period of 40-50 days using bioluminescent imaging (PerkinElmer, Massachusetts). Mice treated with iigand are expected to retain T cells expressing DD-IL15/IL15Ra while T cells in vehicle control treated animals are not expected to persist.
Example 7. Evaluation of antitumor response of DD regulated payloads in syngeneic mouse models
[00453] The efficacy of cancer immunotherapy in organisms with intact immune ceils is evaluated using syngeneic mouse models e.g. pMEL-1 and 4T1 mouse models. Immune cells such as T cells and NK cells are isolated from syngeneic mice and transduced with DD regulated payloads such as DD-IL15, and DDlL15-IL15Ra. Cells are then injected into mice bearing subcutaneous sy ngeneic tumors and treated with varying concentrations of iigand, Shield- 1, Trimethoprim or Methotrexate, depending on the DD used. Mice treated with immune cells transduced with DD regulated payload are expected to have a reduced tumor burden when compared to control animals. Example 8. Optimizing workflow for discovery of DD-regiiiated immunotherapeutk agents
[00454] To identify DD-IL15/ILI 5Ra constructs suitable for immunotherapy, constructs are introduced into cell lines e.g. HEK293T cells and Jurkat cells. The expression of the construct in the presence or absence of the corresponding ligand is tested. Constructs which sho low basal expression in the absence of ligand and ligand-dose responsive expression are selected for further analysis. If no DD-IL15/IL15Ra constructs show ligand-dependent expression, then constructs are redesigned and the experiment is repeated till a regulatable construct is identified. Next, the ligand dependent regulation of the DD-IL15/IL15Ra constructs is tested in vitro in primary T cells. If the constructs show low basal expression in the absence of the ligand and ligand dose responsive expression, they are subject to in vivo
pharmacokinetics/pharmacodynamics (PK/PD) proof of concept experiments. Otherwise, the constructs are redesigned and the new constructs are subject to similar analysis. The
constitutively expressing IL15/IL15Ra constructs are transduced into T cells and IL15-IL15Ra expression is measured in parallel to the regulated construct. If no expression is detected in vitro, efforts are refocused on testing DD-IL15/IL15Ra constructs in vitro in T ceils. In contrast, if the constitutive constructs show expression, then the expression of IL15/IL15Ra is measured in vivo.
[00455] To test in vivo PK/PD, mice are injected with T ceils expressing DD-IL15/IL15Ra constructs and the test group is dosed with the ligand corresponding to the DD, while the control group is dosed with the appropriate vehicle control. Constructs that display ligand-dependent expression of IL15/ILI 5Ra are selected for in vivo functional proof of concept experiments. Parallel experiments are also conducted using the constitutive IL15/IL15Ra constructs. If constitutive IL15/IL15Ra expression is detected in vivo, then the constructs are selected for functional experiments. If no expression is detected in vivo, then constructs are redesigned.
[00456] The functional analysis in vivo is performed by testing if the constitutive and DD regulated IL15/lL15Ra constructs confer a survival advantage to the expressing T cells in a constitutive or ligand dependent manner respectively. If yes, then in vivo proof of concept is achieved and constructs suitable for immunotherapy are identified. If none of the DD regulated constructs show T cell persistence, then alternate dosing regimens are explored. If the constitutive IL15/IL15Ra constructs do not confer proliferative advantage, then efforts are focused on identifying DD-IL15/IL15Ra constructs that show in vivo expression in T cells.
[00457] The effect of ligands specific to the SREs of the invention on immune cell proliferation was measured to identify concentrations of the ligand that did not inhibit T cell growth or survival. T cells derived from two different donors were stimulated with CD3/CD28 and treated with TMP at doses ranging from 0.04 μΜ to 160 μΜ or DMSO. The percentage of divided cells within the CD4 and the CD8 populations of T cells was measured using FACS. Concentrations of TMP ranging from 0.04 μΜ to 40 μΜ showed no effect on the percentage of divided cells within the CDS and CD4 populations, while 160 μΜ concentration of TMP resulted in an 70- 90% reduction in the percentage of divided cells. Thus, the optimal concentration of TMP for T cell based experiments was determined to be less than 160 μΜ.
Example 10. Regulated expression of IL15-IL15Ra in T cells
[00458] DD regulated IL15-IL15Ra constructs such as OT-IL15-009 or constitutively expressed constructs such as OT-IL15-008 were transduced into T cells such as primary T cells or SupTi cells. The transduction was carried out at two different lentivirus concentrations, 5 μΐ and 20 μ] for the DD regulated construct using Lentiboost™ (Sirion Biotech, Germany). 4 days after transduction, cells were treated with 10μΜ TMP or DMSO control for 24 and 48 hours. Samples were analyzed with an and IL15Ra antibody using FACS. Additional controls samples such as cells treated with Lentiboost only, untransduced cells treated with DMSO or TMP, and Isotype controls were included in the FACS analysis. The FACS results are depicted in Figure 16A for 24 hours of TMP treatment and in Figure 16B for 48 hours of TMP treatment. In both figures, DMSO-A and TMP-A represent cells treated with 5 μΐ of lentivirus and DMSO- B and TMP-B represent cells treated with 20 μΐ of lentivirus. Treatment of T ceils expressing OT-IL 15-009 with TMP for 24 hours resulted in an increase in the expression of IL15Ra in T cells with both doses of lentivirus used. Additionally, very low levels of ILISRa were detected in the DMSO treated samples under the same conditions as well as in the untransduced T cells. As expected, the constitutively expressed construct, OT-IL 15-008 showed high expression of ILISRa. TMP dependent expression of OT-IL15-009 was not observed in SupTl cells (Figure 16A). Similar results were observed for both T cells and SupTl cells at 48 hours (Figure I6B). These results show that tight regulation of IL15-IL15Ra constructs can be achieved in primary T cells.
[00459] The percentage of cells expressing IL15, IL15Ra or both on the cell surface is presented in Table 14.
Table 14: Surface expression of IL15 and ILI SRa
Figure imgf000142_0001
ILlSRa positive 13.0 15.8
1L1.5 positive 0.29 0.60
[00460] As shown in Table 14, the percentage of cells with detectable surface expression of IL15 and IL15Ra was less than 5% with both constructs. Further, the percentage of cells with surface expression of ILlSRa was much higher than the percentage of cells with detectable surface expression of 1L15.
[00461] The effect of increasing doses of TMP on 11. 1 1··. a expression in T cells was measured usmg the OT-iL 15-009 construct. T cells were treated with a range of doses of TMP starting from 0.156 μΜ to 160 μΜ for 24 hours. IL15Ra expression was measured using FACS. As shown in Figure 16C, the percentage of ILlSRa expressing T cells with OT-IL15-009 cells was detected even at the lowest concentration of TMP and percentage of ILlSRa positive cells at the lowest concentration was higher than the untreated control . An increase in the percentage of ILl SRa cells with increasing doses of TMP was observed.
Example 11. TMP dose responsive expression of IL15-IL15Ra
[00462] IL15-IL15Ra fusion constructs, OT-IL15-008, OT-IL15-009, and OT-IL15-010 were stably expressed in HCTl 16 cells and treated with increasing doses of TMP ranging from 10μΜ, 33μΜ, and ΙΟΟμΜ TMP for 24 hours. Cell lysates were immunoblotted with anti ILlSRa antibody. As shown in Figure 17A, IL lSRa expression of OT-IL 15-009 was virtually undetectable in the absence of 'TMP, and addition of increasing doses of TMP resulted in an increase in ILlSRa levels. Modest increase in ILlSRa expression was observed with OT-IL 15- 010 construct with the addition of TMP. As expected, the constitutive construct, OT-IL15-008 showed strong expression of ILlSRa both in the presence and absence of iigand.
Example 12. IL1S shedding
[Θ0463] To test if IL15 is shed into the media, supernatant from HCTl 16 cells expressing ILLS- IL15Ra fusion constructs was subject to immunoassays such as ELISA and MSD (Rockville, Maiyland). In Figure 17B, OD indicates optical density. IL15 was measured in the media of cells expressing IL15-lL15Ra fusion constructs. OT-IL15-009 construct demonstrated a TMP dose dependent increase in IL15 levels detected in the media. The level of IL15 in the media detected with the constitutive construct OT-IL15-008 was much higher than the levels detected with the DD regulated constructs (Figure 17B). As shown in Figure I7C, a dose dependent increase in ILLS levels was observed with both the DD regulated constructs using the MSD immunoassay. The detection of membrane bound IL15-IL15Ra fusion constructs in the supernatant suggests that IL15 constructs are likely shed from the cell surface.
The effect of constitutive ly expressed IL15-IL15Ra fusion constructs on T cell persistence was measured in NSG mice. T cells were transduced with OT-IL15-008 and 4 million cells T cells were injected intravenously into NSG mice (number of mice =3). As a control, additional mice were injected with untransduced T cells. Blood samples were obtained from mice at 2, 3, 4, 5 and 6 weeks and analyzed by FACS for the presence of CD8 and'or CD4 positive human T ceils expressing 1L15 and ILlSRa. The percentage of human T ceils in the blood was calculated as the percentage of total T cells i.e. human T cells (measured using anti human CD45 antibody) and the mouse T cells and endothelial cells (measured using the anti- mouse CD45 antibody). As shown in Figure 18 A, the percentage of T cells in the blood at 2 weeks was greater in mice injected with T cells expressing OT-IL15-008 compared to control mice that were injected with untransduced T cells. This observed increase in T cells decreased over 3,4, and 5 weeks, and the final percentage of T cells at 6 weeks was comparable between the two cohorts. At 6 weeks, one of the mice injected with OT-IL15-008 transduced T cells showed a higher percentage of human T cells in the blood, which may represent a graft versus host disease response. Thus, at 2 weeks, the frequency of human T cells in the blood is increased in die blood of mice injected with OT-IL15-008 transduced T cells.
[00465] The number of T cells in the blood during the experiment was measured by comparing the number of human T cells in 50 uL of mouse blood was measured in the blood samples by FACS using anti human CD45 antibody as a marker for human T cells and anti-murine CDS antibody as a marker for murine endothelial cells. As shown in Figure 18B, the number of human T cells in the blood increased at 2 weeks in mice injected with OT-IL15-008 transduced T cells, as compared to mice injected with untransduced T cells. The differential between the two cohorts was diminished at 3 weeks and 4 weeks. At 6 weeks, one of the mice injected with OT- IL 15-008 transduced T cells showed a higher number of human T ceils in the blood. Thus, at 2 weeks, the frequency and number of human T cells in the blood is increased in the blood of mice injected with OT-IL15-008 transduced T cells supporting the role of IL15-IL15Ra fusion proteins in T cell persistence. The increased T cell frequency and number observed at 6 weeks in one of the mice may be due to graft versus host disease.
[00466] The effect of OT-1L15-008 expression on the CD4 and CDS subset of T cells was measured prior to injecting into mice (Week 0) and 2 weeks after injection. As shown in Figure 18C, the ratio of CD8 and CD4 cells was 1 : 1 prior to injecting into mice. However, at 2 weeks, the proportion of CD 8 positive cells was much higher than the CD4 positive cells in the transduced cells, indicating that OT-IL15-008 causes a preferential expansion of CD8 positive cells. The expression of the OT-IL15-008 construct within the CD4 and CD8 subsets was measured using anti ILlSRa antibody. As shown in Figure 18D, prior to injections, 25 % of the OT-IL15-008 transduced CD4 T cells and CD8 T cells expressed ILlSRa. At week 2, the percentage of ILlSRa positive CD4 and CD8 T cells increased to 80% indicating a preferential expansion of T cells transduced with OT-IL15-008. As expected, untransduced control T cells were negative for ILl SRa expression.
[00467] Immune cells such as Natural Killer cells depend on cytokines such as IL15 for their proliferation and survival. This dependence on cytokines can he used to test the functionality of DD regulated or constitutively expressed cytokines and cytokine fusion proteins.
[00468] The dependency of the NK-92 cells on cytokines for activation was tested. Ceils were initially cultured for 3 days with IL2, following which, cells were washed twice and cultured in media without 1L2 for 7 hours. The cells were cultured for 18 hours in the presence of varying concentrations of ILLS (100 ng/ml, 20 ng/ml, 4 ng/ml, 0.8 ng/ml, 0.16 ng/mi, 0.032 ng/ml, 0.0064 ng/ml and 0.001.28 ng/ml). NK-92 cell activation in response to ILLS treatment was evaluated by FACS analysis using a panel of markers whose increased expression is associated with NK activation. These include NKG2D, CD71, CD69; chemokme receptors such as CCR5, CXCR4, and CXCR3, Perforin, Granzyme B and Interferon gamma (IFNg). Prior to FACS analysis for IFNg, cells were cultured for 4 hours with Brefeldin A. NK cells respond to external stimuli such as cytokines in their environment through the phosphorylation of proteins
J AK/STAT, ERK, and p38/MAPK pathways which are important for cell activation, signaling and differentiation pathways. The phosphorylation of AKT, STAT3 and STATS in response to cytokine addition was measured by FACS. Since phosphorylation events are transient NK-92 ceils were treated with the cytokines for 15 or 60 minutes, prior to the analysis. The fold change in mean fluorescence intensities compared to untreated are presented in Table 15.
Table 15: NK cell activation markers
Figure imgf000145_0001
0.8 1.18 2.30 1 .16 1.09 1 .76 2, 17 1 .92
0.16 0.99 1.41 1.04 1.03 1.44 2.08 2.41
0.032 1.05 1.14 0.92 0.92 1.23 1.67 0.95
0.0064 1.1 1 1.39 0.79 0.78 1 .03 1,26 0.85
0.00128 1.07 1 .15 0.85 0,88 0.99 1.04 1, 19
[00469] Treatment with IL15 resulted in an increase in the expression of CD69, CXCR4, Perforin, Granzyme B, and IFNg. The effect of IL15 on these markers was dose dependent with a higher dose of 1L15 resulting in a corresponding upregulation of markers. Phosphorylation of STAT5 was increased both at 15 and 60 minutes after the addition of IL2 or IL15. Taken together, these results show that cytokines can activate NK cells.
[00470] The dependency of immune cells on cytokines for proliferation was tested in NK-92 ceils and T ceil lines Jurkat cells and SupTl cells. Cells were seeded at 40,000 per well and cultured in the presence of IL2 or ILLS for 3 days. As shown in Figure 19, the number of NK-92 cells decreased with time in the untreated cells. However, treatment with either IL2 or IL15 increased cell numbers. In contrast, the cell numbers obtained with cytokine treatment in SupTl and Jurkat cells was comparable to the untreated control. Thus, NK-92 cells depend on cytokines for survival.
[00471] The functional capability of IL15-IL15Ra constructs was tested in vivo in xenograft assays. The experiment was designed to test the hypothesis that pulsatile expression of IL 15- IL15Ra fusion proteins from cancer cell lines such as HCTl 16, in mice may stimulate the mouse's NK cells to attack the tumor leading to tumor growth inhibition, 5 million HCTl 16 cells expressing constitutive I I. 15-11. 1 Ra construct (OT-IL15-008) or DD regulated construct (OT- 1L15-009) were implanted subcutaneously into nude mice with 50% matrigel. Tumors were allowed to develop over a period of 22 days. On day 8, mice expressing OT-BL 15-009 constructs were separated from the rest of the animals and split into three groups. Group ! received vehicle control for 7 days, while group2 received 100 mg/kg of Trimethoprim, while the third group did not receive any drug treatment. As shown in Figure 20A, mice injected with parental HCTl 16 cells (Gl) were able to form tumors. Mice injected with OT-IL15-008 expressing cells (G2) formed very small tumors in comparison, injecting both the parental (GSR) and the constitutive construct (G5L) in opposite flanks of the same mouse did not affect the tumor growth capacity of either cohort.
[00472] Mice injected with OT-IL15-009 expressing cells were left untreated, or treated with vehicle control or TMP. As shown in Figure 20B, mice treated with TMP had smaller tumors than mice of the other two groups, suggesting that pulsatile expression of IL15-IL15Ra construct may contribute to anti -tumor activity.
The expression of SREs expressed in a vector can be driven by either the retroviral long terminal repeat (LTR) or by cellular or viral promoters located upstream of the SRE. The activity of the promoter may vary with the cell type and thus promoter selection must be optimized for each cell type. To identify optimal promoters for T cells, AcGFP (SEQ. ID NO. 308) was cloned into pLVX. IRES Puro construct with a CMV or an EFla promoter. Patient derived T cells and Sup Tl cells were transduced with the constructs and GFP expression was measured at day 3 and day 5 after transduction using FACS. As shown in Figure 21, both the CMV promoter and the EF la can drive the expression of GFP in SupTl cells and T cells. The percentage of GFP positive T cells was higher when the GFP expression was driven by CMV promoter compared to an EFla promoter, both at 3 days and 6 days after transduction. In contrast, the percentage of GFP positive Sup Tl cells make a Tl cells was much higher when GFP expression was driven by the EFla promoter when compared to the CMV promoter. Thus, the optimum promoter suitable for expression differs based on the ceil type.
Example 17, EBV tumor antigen mediated TCR re-stimulation in vivo
[00474] Human T cells engineered to express DD regulated 1LI5 are not antigen specific which limits their functional analysis in mice. However, functionality of T cells in vivo requires their restimulation which occurs upon engagement with the antigen. This requirement for antigen mediated restimulation can be mimicked experimentally in mice using the Epstein Barr Virus (EBV) antigen. Approximately 90% adults have a current or a previous EBV infection.
Additionally, the major histocompatibility group HLA-A02 has been associated with the decreased risk of developing EBV positive Hodgkin's lymphoma, suggesting that the CTL peptide epitopes that promote EBV clearance are presented by HLA-A02. Tumor cell lines that are HLA-A02 positive e.g. Raji cells are used for in vivo studies. Primary human T cells obtained from various donors are expanded with CD3/CD28 dynabeads. To test reactivity of T cells to the EBV antigen, EBV positive Raji cells and EBV negative Ramos cells are used. The involvement of HLA-A02 in antigen recognition is tested using anti-HLA antibodies with both cell. Cell killing assays are performed by incubating T cells with fluorescently labelled Raji cells or Ramos cells and the ability of the donor T cells to preferentially kill Raji cells is evaluated. The activation of T cells in response to interaction with EBV antigen is measured by culturing mitomycin treated Raji or Ramos ceils with fluorescently labelled T cells expressing IL12 or with exogenously supplemented IL12. The activation and proliferation status of T cells is examined by measuring expression of IL2, IFNg, CD 107a, Granzyme, Perforin. Since most humans have been exposed to EBV, the donor T cells in most instances are expected to be immunoreactive to Raji cells but not Ramos cells. It is likely that T cells reactive to Raji cells will be positive for markers of T cell activation such as IL2, Granzyme and Perforin.
Example 18. Kinetics of !ieand-dependent stabilization of DD-IL15- ILlSRa
[00475] To test DD regulated constructs in T cells, OT-IL15-009 construct was transduced into human donor T cells. As additional controls, cells were transduced with constitutive construct OT-IL15-008 or left untransduced. Ceils were then treated with ΙΟΟμΜ TMP and ILl SRa expression was measured by FACS using anti-IL15Ra antibodies and measured against forward scatter. The percentage of IL15Ra positive ceils increased from 1.68 % in the untreated ceils to 16.6 % in the TMP treated cells, indicating ligand dependent regulation. Expression of IL15Ra was also compared to untransduced cells in the presence or absence of TMP as well as in control cells constitutive!}' expressing ILl S-ILl SRa. In this experiment, it was observed thai the percentage of ILlSRa positive cells increased from 1% to 16% in the T ceils expressing OT- IL 15-009 construct when treated with TMP. This expression level was comparable to cells transduced with the constitutive construct. Untransduced T cells did not show 'expression of ILlSRa both in the presence and absence of ligand.
[00476] The on/off kinetics of ligand-dependent stabilization of DD-IL15-IL15Ra was measured in CD4 positive T cells. T cells were transduced with the ecDHF DD-IL15-IL15Ra fusion construct (OT-IL15-009) and treated with ΙΟΟμΜ ecDHFR ligand Trimethoprim (TMP) or vehicle control, DMSO. At multiple time points (i.e., 1, 2, 4, 6, 8, 15, 22 and 24 hrs) after TMP treatment, the transduced T cells were collected and analyzed for ILlSRa surface expression using anti-IL15Ra antibodies by flow cytometry. Untransduced T cells were used as a negative control. The T cells were sorted into CD4 positive and CDS positive populations and the percentage of ILlSRa positive CD4 positive T cells was analyzed. Table 16 shows the kinetics of surface expression of ILlSRa on CD4 T cells after TMP treatment. The stabilization ratio was calculated as the fold change in ILlSRa expression in ligand treated samples compared to treatment with DMSO (i.e. in the absence of ligand) with the same construct. The
de stabilization ratio was caicuiated as the fold change in ILlSRa levels in the DD regulated constructs compared to the constitutive construct (OT-IL15-008) in the absence of the ligand. Destabi!ization ratios less than 1 and stabilization ratios greater than 1 are desired in DDs. Table 16: TMP dose response
Figure imgf000149_0001
[00477] Among the CD4 positive T cells transduced with the OT-IL 15-009 construct, the proportion of cells with surface expression of ILlSRa remained similar for both TMP treated and DMSO treated cells until 2 hrs after TMP treatment, and was comparable to that of untransduced cells. However, 4 hrs after TMP treatment, the cells transduced with the OT-DL 15-009 construct and treated with TMP exhibited an increased proportion of cells with surface expression of ILlSRa. This trend was observed until 22 hours after treatment with TMP. The CD4 positive T cells with surface-expressed ILlSRa cells constituted ~1% of untransduced cells, indicating that the proportion of cells that expressed endogenous ILlSRa is low. In summary, the stabilization ratio showed a time dependent increase while the destabilization ratio remained at or below 0.1 reflecting low basal expression, reflecting the tunability of the DD regulated 11,15 construct. Example 19. Kinetics of ligand-dependent stabilization of DD-IL15- ILlSRa
[00478] The on/off kinetics of ligand-dependent stabilization of DD-IL15-IL15Ra was measured in CD4 positive T cells. T cells were activated with CD3/CD28 beads at 3: 1 bead to T cell ratio in 24-well plates for 24 hrs. Len virus was added to wells in the presence of
LentiBoost reagent, and cells were incubated for another 24 hrs and washed. Cells were resuspended in fresh media, and media was added every 2-3 days to expand and maintain cells at 0.5-lxl0b/ml. After 7 days of expansion, T cells transduced with the ecDHFR DD-IL15-IL15Ra fusion construct (OT-1L15-009) were treated with ΙΟΟμΜ ecDHFR ligand Trimethoprim (TMP) or vehicle control, DMSO. At multiple time points (i.e., 1, 2, 4, 6, 8, 15, 22 and 24 hrs) after TMP treatment, the transduced T cells were collected and analyzed for ILlSRa surface expression using anti-ILlSRa antibodies by flow cytometry. Untransduced T cells were used as a negative control. The T cells were sorted into CD4 positive and CD 8 positive populations and the percentage of ILlSRa positive CD4 positive T cells was analyzed. Figure 22 shows the kinetics of surface expression of IL lSRa on CD4 T cells after TMP treatment. Among the CD4 positive T cells transduced with the OT-IL 15-009 construct, the proportion of cells with surface expression of IL15Ra remained similar for both TMP treated and DMSO treated cells until 2 hrs after TMP treatment and was comparable to that of untransduced cells. However, from 4 hrs after TMP treatment, the cells transduced with the OT-IL 15 -009 construct and treated with TMP exhibited an increased proportion of cells with surface expression of IL15Ra. This trend was observed until 22 hours after treatment with TMP. The CD4 positive T cells with surface- expressed IL15Ra cells constituted ~1 % of untransduced cells, indicating that the proportion of cells that expressed endogenous IL15Ra is low.
Example 20. Ligand-dependent stabilization of DD-IL15-IL1.5Ra fusion molecules in vivo
[00479] To examine whether ligand treatment induces stabilization of the DD-IL15-IL15Ra fusion molecules in vivo, HCTl 16 cells transduced with the OT-IL 15-009 construct were implanted subcutaneously in BALB/c nude mice and treated with TMP. TMP was orally administered to mice at a dose of 100 mg/kg, twice a day for 1 1 days after implantation, followed by administration of TMP at the dose of 300 mg/kg, twice a day for 6 days. As a negative control, separate mice implanted with HCTl 16 cells transduced with the OT-IL 15 -009 construct were treated with the vehicle twice a day for 17 days. At 4 hrs after the last dosing of TMP or the vehicle control, tumors were han'ested from the mice and analyzed for the levels of IL15-IL15Ra fusion molecules by western blotting. As shown in Figure 23, HCTl 16 tumors han'ested from mice treated with TMP exhibited elevated levels of IL15-IL15Ra expression, compared to tumors treated with the vehicle. The GAPDH level was analyzed as a loading control. These data show that administration of ligand enabled stabilization of the DD-IL15- IL15Ra fusion molecule in vivo.
[00480] Consistent with the efficacy of TMP-dependent IL15-IL15Ra stabilization in vivo, elevated levels of TMP (399.38 ng/'g tumor) were observed in HCTl 16 tumors harvested from mice treated with TMP for 17 days. The levels of TMP associated with HCTl 16 tumors were considerably higher than those observed in mouse plasma at day 3 (15.67 ng/ml plasma) and at day 17 (99.5 ng/ml plasma), indicating that the orally administered TMP was successfully delivered to and accumulated in HCTl 16 tumors implanted in mice.
Example 21. Shedding resistant IL15-IL15Ra constructs
[00481] To maintain the efficiency of the trans-presentation of IL15 via the IL15-IL15Ra fusion molecule, the IL15-IL15Ra shedding needs to be prevented. For this purpose, new DD-IL15- IL15Ra and constitutive IL15-IL15Ra constructs are designed through a variety of modifications on the IL15-IL15Ra fusion molecule. For example, the IL15 molecule or the IL15Ra molecule is truncated or mutated to remove presumable cleavage sites. IL15Ra lias 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). J Biol Chem ;283(7):4189-99; Anthony SM et al. (2015). PLoS One. 10(3):
eO 120274), and International Patent Application Publication Nos. WO2014066527 and
WO2009002562 (the contents of each of which are incorporated herein by reference in their entirety). Tumor necrosis factor-alpha-converting enzyme (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 can be responsible for the IL15-IL15Ra shedding. Hence, the cleavage site of IL15Ra is mutated such that cleavage by an endogenous protease is prevented. The mutation of the cleavage site is introduced by substitution, insertion or deletion of amino acid residues. The IL15-IL15Ra fusion molecule is 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 are modified.
[00482] To confirm that the modifications on the IL15-IL15Ra fusion molecule prevent shedding, the new DD-IL15-IL15Ra or constitutive IL15-IL15Ra constructs are introduced into HCT-1 16 cells. Surface expression of IL15 and IL15Ra on the HCT-116 cells is examined by flow cytometry using a ti-ILI 5 and IL15Ra antibodies to assess surface IL15-IL15Ra shedding. The presence or absence of IL15 in the cell culture supernatant is also analyzed by MSD assay. As a functional assay based on the sensitivity of NK cell activation by shed IL15 in tumor supernatant, the transwell assay is conducted using HCT-116 cells transduced with new DD- IL15~IL15Ra or constitutive IL15-IL15Ra expressing constructs and NK cells. The new DD- IL15-IL15Ra-expressing constructs that do not induce activation of NK cells in the presence of ligand and the new constitutive IL15-IL15Ra-expressing constracts that do not induce activation of NK cells are chosen for use in future experiments.
Example 22. Regulated expression of IL15-IL15Ra fusion molecule with C-terminal DP
[00483] A fusion molecule is generated by fusing membrane bound IL15, IL15 Receptor alpha subunit (ILiSRa) and a human DHFR (DD). These fusion molecules were cloned into pLVX- EF 1 a-IRES-Puro vector.
[00484] To test ligand dependent IL15-IL15Ra production, 1 million HEK-293T cells were plated in a 6-well plate in growth media containing DMEM and 10 FBS and incubated overnight at 37°C, 5% C02, Cells were then transfected with lOOng of constitutive IL15-IL15Ra (OT- IL15-G08) or DD linked IL15-IL15Ra (OT-IL15-037 or OT-IL 15-040) using Lipofectamine
2000 and incubated for 24 hrs. Following the incubation, media is exchanged for growth medium with or without 50μΜ Trimethoprim (TMP) and further incubated for 48 hrs. Ceils were harvested and IL15 levels are analyzed via western blotting using human IL15 antibody (Abeam, Cambridge, UK). The molecular weight of ILlSRa in OT-IL 15 -037 and OT-IL15-040 appeared to be the same as OT-IL15-008.
ΘΘ485] To test if ILl 5 is shed into the media, supernatant from HEK293 cells expressing ILl 5- IL15Ra fusion constructs was subject to immunoassays such as MSD (Rockvilie, Maryland). 48 hours after transfection, cells were analyzed and, as expected, constitutive IL15-IL15Ra construct OT-TL15-008 showed high surface expression of IL15 in the presence and absence of ligand. OT-IL 15-037 and OT-IL 15-040 showed the ligand (Trimethoprim) dependent surface expression of IL15 and ILlSRa (Figure 24). The detection of membrane bound IL15-IL15Ra fusion constracts in the supernatant suggests that ILl 5 constructs are likely shed from the cell surface.
>] In order to determine if the dose and time of exposure to TMP in vitro influenced membrane bound IL l 5 expression, an in vitro dose response study was conducted with T ceils expressing OT-IL 15-073. For this purpose, T cells were activated with CD3/CD28 beads at 3: 1 bead to T cell ratio in 24-well plates for 24 hrs. Lenti virus was added to wells. After 24 hrs, fresh media was added every 2-3 days to expand cells while maintaining cells at 0.5-lxl06/ml. On day 11 of expansion. T cells treated with TMP starting at 100 uM, lOx dilutions and 9 points were analyzed after 2 hours in culture (washed 3x after TMP addition, fresh media added without TMP for 22 hours), 6 hours in culture, or 24 hours in culture and the results are shown in Figure 25A. As shown in Figure 25B and Table 17, this study showed that TMP ligand regulates membrane bound ILl 5 expression and the dose and time of exposure to TMP in vitro influences membrane bound ILl 5 expression .
Table 17. Membrane Bound IL15 Expression
Figure imgf000152_0001
Example 24. Regulated membrane bound IL15 expression in vivo
[00487] To evaluate regulation of membrane bound IL15 in vivo, 2 constructs were selected for evaluation in vivo. Four group of T cells were used for this study and are outlined in Table 18. In Table 18, "N" represents the number of mice in each group.
Table 18. T Cell Groups
Figure imgf000153_0001
[00488] The T cells which were to be used as part of the in vivo study were evaluated 6 days post transduction, day of implant (day 9 post transduction) and 13 day s post transduction and the cells in Groups 2-4 showed expression of the constructs.
[00489] T cells outlined in Table 18 were administered to mice by intravenous administration (3.9 x 106 cells per mouse implanted). On day 3 the mice were dosed with 500 mg/kg of TMP 3 times (4 hours between doses) and bled 2 hours after each dose. The mice were again bled on day 4, 24 hours after the first TMP dose.
[00490] Figures 26A-26C show the expression of membrane bound ILLS, 2, 6, 10, and 24 hours after the first TMP dose, using IL15 staining (Figure 26A), IL15Ra staining (Figure 26B), and IL15/IL15Ra double ++ staining (Figure 26C). Figure 26D are FACS plots for each mouse 10 hours after the first TMP dose. Figure 26E shows the expression of membrane bound IL15 in blood 2, 6, 10, and 24 hours after the first TMP dose and Figure 26F shows the plasma TMP levels 2, 6, 10, and 24 hours after the first TMP dose.
Example 25. Effect of long term intraperitoneal (IP) or oral (PO) TMP dosing on T cell function
[00491] In this study, T cells transduced with OT-IL 15-071 or OT-IL 15-073 (no lentiBoost) were administered intravenously to mice (15 x 106 per mouse). 6 study groups were evaluated for this study: (1) untransduced, (2) OT-1L15-071 T cells, (3) OT-IL15-073 PO vehicle, (4) OT- IL15-073 PO TMP 500 mg/kg), (5) OT-IL15-073 IP vehicle, and (6) OT-IL15-073 IP TMP 300 mg/kg. Tire study design is shown in Table 19. PO dosing is 500 mg/kg TMP in 0.1M citrate and IP dosing is 300 mg/kg TMP lactate in water. Table 1 , Study Design
Figure imgf000154_0001
[00492] The regulated expression in blood was analyzed 6 hours and 24 hours after the first dose, and 6 hours after the 5th dose.
[00493] OT-1L15-071 showed expression of membrane bound ILl 5 and the untransduced control did not show any expression.
[00494] Regulation of membrane bound ILl 5 was seen with repeat PO and IP dosing. As seen in Figure 27, regulated expression of membrane bound ILl 5 was detected 6 hours after the first dose on day 0, and 6 hours after dosing on day 5 ( 126 hrs) with both PO and IP dosing. There was no increase in expression in mice treated with vehicle.
[00495] While the present invention has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be constmed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the invention.
[00496] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, section headings, the materials, methods, and examples are illustrative only and not intended to be limiting.

Claims

1. A composition for inducing an immune response in a cell or a subject comprising an effector module, said effector module comprising a stimulus response element (SRE) operably Hnked to at least one payload, wherein said payload comprises an immunotherapeutic agent.
2. The composition of claim 1 , wherein the immunotherapeutic agent is selected from the group consisting of a cytokine, a cytokine receptor, a cytokine-cytokine receptor fusion, and combinations thereof.
3. The composition of claim 2, wherein the SRE is responsive to or interacts with at least one stimulus.
4. The composition of claim 3, wherein the SRE comprises a destabilizing domain (DD).
5. The composition of claim 4, wherein the DD is derived from a parent protein or a mutant protein having one, two, three or more amino acid mutations compared to said parent protein, wherein the parent protein is selected from ;
(a) human protein FKBP, comprising the amino acid sequence of SEQ. ID NO. 3;
(b) human DHFR (hDHFR), comprising the amino acid sequence of SEQ. ID NO. 2;
(c) E. Coli DHFR, comprising the amino acid sequence of SEQ. ID NO. 1
(d) PDE5, comprising the amino acid sequence of SEQ. ID NO. 4:
(e) PPAR, gamma comprising the amino acid sequence of SEQ. ID NO. 5;
(f) CA2, comprising the amino acid sequence of SEQ. ID NO. 6; or
(g) NQ02, comprising the amino acid sequence of SEQ. ID NO. 7.
6. The composition of claim 5, wherein the parent protein is hDHFR and the DD comprises a mutant protein having:
(a) a single mutation selected from hDHFR (117V), hDHFR (F59S), hDHFR (N65D), hDHFR (K81 R), hDHFR (A107V), hDHFR (Y122I), hDHFR (N 127Y), hDHFR (M140I), hDHFR (K185E), hDHFR ( 186D), hDHFR (M140I), hDHFR (Amino acid 2- 187 of WT; N127Y), hDHFR (Ammo acid 2-187 of WT; II 7V), hDHFR (Ammo acid 2- 187 of WT: Y122I), and hDHFR (Amino acid 2-187 of WT; K185E); (b) a double mutation selected from hDHFR (C7R, Y163C), hDHFR (AIOV, H88Y), hDHFR (Q36K, Y122I), hDHFR (M53T, R138I), hDHFR (T57A, I72A), hDHFR (E63G, I176F), hDHFR (G21T, Y122I), hDHFR (L74N, Y122I), hDHFR (V75F, Y 122I ).
hDHFR (L94A, T147A), DHFR (V121A, Y22I) , hDHFR (Y 122L A125F), hDHFR (H131R, E144G), hDHFR (TT37R, F143L), hDHFR (Y i 781 i. E18IG), hDHFR (Y 183H, Kl 85E), hDHFR (E162G, I176F) hDHFR (Amino acid 2-187 of WT; 117 , Y122I), hDHFR (Amino acid 2-187 of WT; Y122I, M140I), hDHFR (Ammo acid 2-187 of WT; N127Y, V 1220. hDHFR (Amino acid 2-187 of WT; E162G, 1176F), and hDHFR (Ammo acid 2-187 of WT; HI3 R, E144G), and hDHFR (Amino acid 2-187 of WT; Y122I, A125F);
(c) a triple mutation selected from hDHFR (V9A, S93R, P150L), hDHFR (I8V, K133E, Y 163C), hDHFR (L23S, V121A, Y157C), hDHFR (K 19E, F89L, E181G), hDHFR (Q36F, N65F, Y122I), hDHFR (G54R, Ml 40V, S 168C), hDHFR (VHOA, V136M, K177R), hDHFR (Q36F, Y122I, A125F), hDHFR (N49D, F59S, D153G), hDHFR (G21E, I72V, 1176T), hDHFR (Ammo acid 2-187 of WT; Q36F, Y1221, A125F), hDHFR (Amino acid 2-187 of WT; Y 122L H131R, E144G), hDHFR (Am ino acid 2-187 of WT; E3 ID, F32M, VI 161), and hDHFR (Ammo acid 2-187 of WT; Q36F, N65F, Y 122I); or
(d) a quadruple or higher mutation selected from hDHFR (V2A, R33G, Q36R, LI OOP, Kl 85R), hDHFR (Amino acid 2-187 of WT; D22S, F32M, R33S, Q36S, N65S), hDHFR (I17N, L98S, K99R, M112T, E151G, E162G, E172G), hDHFR (G16S, 117V, F89L, D96G, K123E, M140V, D146G, K156R), hDHFR (K81R, K99R, L100P, E102G, N108D, K123R, H128R, D142G, F180L, K185E), hDHFR (R138G, D142G, F143S, K156R, K158E, E162G, V166A, K177E, Y178C, K185E, N186S), hDHFR (NWS, P24S, F35L, M53T, K56E, R92G, S93G, N 127S, H128Y, F135L, F143S, L159P, L160P, E173A, I I 801.). hDHFR (F35L, R37G, N65A, L68S, K69E, R71G, L80P, K99G,
Gl 17D, L132P, I139V, Ml 401, D142G, D146G, E173G, D187G), hDHFR (L28P, N30H, M38V, V44A, L68S, N73G, R78G, A97T, K99R, A107T, K109R, D 111N, L134P, F135V, T147A, 1152V, K158R, E172G, V182A, E184R), hDHFR (V2A, I17V, N30D, E31G, Q36R, F59S, K69E, I72T, H88Y, F89L, N108D, K109E, VI 10A, II 15V, YI22D, L132P, F135S, M140V, E144G, T147A, Y157C, V170A, K174R, N186S), hDHFR (L100P, E102G, Q103R, P104S, E105G, N108D, VI 13A, Wl 14R, Y122C, M126I, N127R, H128Y, L132P, F135P, I139T, F148S, F149L, 1152V, D153A, D169G, V170A, I176A, K I 77R . V182A, K185R, N186S), and hDHFR (A 10T, Q13R, N14S, N20D, P24S, N30S, M38T, T40A, K47R, N49S, K56R, 16 IT, K64R, K69R, I72A, R78G, E82G, F89L, D96G, N108D, Ml 12V, Wl 14R, Y122D, K123E, T139V, Q141R, D142G, F148L, E151G, E155G, Y 157R, Q171R, Y183C, E184G, K185del, D187N).
7. The composition of claim 6, wherein the stimulus Trimethoprim. (TMP) or Methotrexate (MTX).
8. The composition of claim 2, wherein the immunotherapeutic agent is a cytokine.
9. The composition of claim 8, wherein the cytokine is 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.
10. The composition of claim 9, wherein the interleukin is the whole or a portion of IL15 comprising the ammo acid sequence of SEQ. ID NO. 55.
11. The composition of claim 10, wherein SEQ. ID NO. 55 is modified.
12. The composition of claim 11, wherein SEQ. ID NO. 55 modification comprises:
(a) fusing SEQ. ID NO. 55 to the whole, or a portion, of a transmembrane protein, and
(b) optionally, incorporating a hinge domain.
13. The composition of claim. 2, wherein the immunotherapeutic agent is a cytokine-cytokine receptor fusion polypeptide.
14. The composition of claim 13, wherein the cytokine-cytokine receptor fusion polypeptide comprises the whole or a portion of SEQ. ID NO. 55, fused to the whole or a portion of any of SEQ. ID NOs. 21 1-218 to produce a IL15-IL15 receptor fusion polypeptide.
15. The composition of claim. 14, wherein the cytokine-cytokine receptor fusion polypeptide is modified.
16. The composition of claim 15, wherein cytokine -cytokine receptor fusion polypeptide modification comprises: (a) fusing the IL15-IL15 receptor fusion polypeptide to the whole, or a portion, of a transmembrane protein, and
(b) optionally, incorporating hinge domain.
17. The composition of claim 1, wherein the effector module is selected from a IL15-DD comprising the amino acid sequence of either of SEQ. ID Os. 56, 57, 348, 354, or 356 and an IL15/IL15Ra fusion-DD comprising the amino acid sequence of any of SEQ. ID NOs. 223-228, 404, 407, 413, 415, 417, 419, 421, 424, 426, 428, 432, 434, 436, 438, 440, 443, 445, 448, 452, 457, 459, 461, 504, 507, or 523.
18. The composition of claim 17, wherein the SRE stabilizes the immunotherapeutic agent by a stabilization ratio of 1 or more, wherein the stabilization ratio comprises the ratio of expression, function or level of the immunotherapeutic agent in the presence of the stimulus to the expression, function or level of the immunotherapeutic agent in the absence of the stimulus.
19. The composition of any of claims 17-18, wherein the SRE destabilizes the
immunotherapeutic agent by a destabilization ratio between 0, and 0.09, wherein the de stabilization ratio comprises 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.
20. A polynucleotide encoding any of the compositions of claims 1-19.
21. The polynucleotide of claim 20, which is a DNA molecule, or an RNA molecule.
22. The polynucleotide of claim 21, wherein the polynucleotide comprises spatiotemporally selected codons.
23. The polynucleotide of claim 21 which is a DNA molecule.
24. The polynucleotide of claim 21, wherein the polynucleotide is an RNA molecule and said RNA molecule is a messenger RNA.
25. The polynucleotide of claim 24, wherein the polynucleotide is chemically modified ,
26. The polynucleotide of claim 23 or 24, wherein the polynucleotide further comprises at least one additional feature selected from a promoter, a linker, a signal peptide, a tag, a cleavage site and a targeting peptide.
27. A vector comprising a polynucleotide of claim 20.
28. The vector of claim 27, wherein the vector is a viral vector, or a plasmid.
29. The vector of claim 28, wherein the viral vector is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, a gamma retroviral vector, a recombinant AAV vector, an adeno viral vector, and an oncolytic viral vector.
30. An immune cell for adoptive cell transfer (ACT) which expresses any of the compositions of any of claims 1-19, the polynucleotides of any of claims 20-26 and/or is infected or transfected with the vector of any of claims 27-29.
31. The immune cell of claim 30, selected from 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 regulatory T (Treg) cell, a cytokine-induced killer (CIK) ceil, a dendritic cell, a human embryonic stem cell, a mesenchymal stem cell, a hematopoietic stem cell, or a mixture thereof.
32. The immune cell of claim 30, which is autologous, allogeneic, syngeneic, or xenogeneic in relation to a particular individual subject.
33. A method of reducing a tumor volume or burden in a subject in need thereof, comprising contacting said subject with the immune cells of any of claims 30-32.
34. A method of inducing an anti-tumor immune response in a subject comprising administering to the subject an effective amount of the immune cells of any of claims 30-32.
35. A method of enhancing the expansion and/or survival of immune cells comprising contacting the immune cells with the compositions of any of claims 1 -19, the polynucleotides of any of claims 20-26 and/or the vector of any of claims 27-29, wherein the SRE responds to a stimulus and regulates the expression and function of the immunotherapeutic agent, thereby enhancing the expansion and/or survival of the immune cells.
36. The method of claim 35, wherein the contacted immune cell is selected from 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 regulatory 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.
37. A method of inducing an immune response in a subject comprising administering to the subject an effective amount of any of the compositions of claims 1-19, any of the
polynucleotides of claims 20-26, any of the vectors of claims 27-29, or any of the immune ceils of claims 30-32.
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