WO2024102943A1 - Récepteurs chimériques armés et leurs méthodes d'utilisation - Google Patents

Récepteurs chimériques armés et leurs méthodes d'utilisation Download PDF

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WO2024102943A1
WO2024102943A1 PCT/US2023/079282 US2023079282W WO2024102943A1 WO 2024102943 A1 WO2024102943 A1 WO 2024102943A1 US 2023079282 W US2023079282 W US 2023079282W WO 2024102943 A1 WO2024102943 A1 WO 2024102943A1
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protease
domain
cell
seq
protein
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PCT/US2023/079282
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English (en)
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Marcela GUZMAN AYALA
Russell Morrison GORDLEY
Michelle Elizabeth Hung
Gary Lee
Timothy Kuan-Ta Lu
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Senti Biosciences, Inc.
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Publication of WO2024102943A1 publication Critical patent/WO2024102943A1/fr

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  • CAR-T based therapies provide promising avenues for treating a variety of diseases.
  • One such promising platform is CAR-T based therapies in the treatment of cancer.
  • An active area of exploration is engineering cell-based therapies to produce and/or secrete effector molecules such as cytokines, a process referred to as armoring, that enhance the cell-based therapy.
  • unarmored CAR-T therapies have poor efficacy in solid tumors and armoring can impact the entire cancer immunity cycle and boost the activity of CAR-T.
  • uncontrolled or unregulated armoring strategies can have negative impacts on treatment, such as off-target effects and toxicity in subjects.
  • additional methods of controlling and regulating the armoring of cell-based therapies such as regulating production and/or secretion of payload effector molecules, are required.
  • CMOS complementary metal-oxide-semiconductor
  • cytokines regulated secretion of payload effector molecules
  • engineered cells e.g., immunoresponsive cells
  • payload effector molecules such as cytokines
  • engineered cells comprise engineered nucleic acids that encode for one or more controlled release effector molecules and a CAR.
  • the present disclosure further provides for mixed cell populations, wherein a first engineered cell comprises an engineered nucleic acid encoding one or more nucleotide sequences encoding an effector molecule and/or CAR, and a second engineered cell comprises a different engineered nucleic acid encoding one or more nucleotide sequences encoding an effector molecule and/or CAR.
  • provided mixed cell populations or compositions comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or more different engineered cells.
  • a disease or disorder is cancer.
  • the present disclosure a combinatorial cell-based immunotherapy involving regulated armoring for the targeted treatment of cancer, such as ovarian cancer, breast cancer, colon cancer, lung cancer, and pancreatic cancer.
  • the present disclosure provides technologies for limiting systemic toxicity of armoring, e.g., armoring of cells comprising a CAR.
  • the immunotherapy provided herein can be tumor- specific and effective while limiting systemic toxicity and/or other off-target effects due to armoring.
  • These therapies deliver proteins of interest, such as immunomodulatory effector molecules, in a regulated manner, including regulation of secretion kinetics, cell state specificity, and cell or tissue specificity.
  • the design of the delivery vehicle is optimized to improve overall function in cell-based therapies, such as cancer therapy, including, but not limited to, optimization of the membrane-cleavage sites, promoters, linkers, signal peptides, delivery methods, combination, regulation, and order of the immunomodulatory effector molecules.
  • Non-limiting examples of effector molecules encompassed by the present disclosure include cytokines, antibodies, chemokines, nucleotides, peptides, enzymes, and oncolytic viruses.
  • cells may be engineered to express and secrete in a regulated manner at least one, two, three or more of the following effector molecules: IL-12, IL-16, IFN-P, IFN-y, IL-2, IL-15, IL-7, IL-36y, IL-18, IL-ip, IL-21, OX40-ligand, CD40L, anti-PD-1 antibodies, anti- PD-L1 antibodies, anti-CTLA-4 antibodies, anti-TGFp antibodies, anti-TNFR2, MIPla (CCL3), MIPip (CCL5), CCL21, CpG oligodeoxynucleotides, and anti-tumor peptides (e.g., anti- microbial peptides having anti-tumor activity, see, e.g.,
  • an immunoresponsive cell comprising: an engineered nucleic acid comprising a first expression cassette comprising an ACP-responsive promoter operably linked to a first exogenous polynucleotide sequence encoding a first cytokine, and a second expression cassette comprising a second promoter operably linked to second exogenous polynucleotide sequence encoding an activation-conditional control polypeptide (ACP), wherein the ACP comprises a synthetic transcription factor comprising a DNA-binding domain and a transcriptional effector domain, wherein the ACP is capable of inducing expression of the first exogenous polynucleotide sequence by binding to the ACP-responsive promoter, wherein the first exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula: S - C - MT or MT - C - S, wherein S comprises a secretable effector
  • the first expression cassette is configured to be transcribed in an opposite orientation relative to transcription of the second expression cassette within the engineered nucleic acid.
  • the first expression cassette and the second expression cassette are oriented within the engineered nucleic acid in a head-to-head directionality.
  • the first expression cassette and the second expression cassette are oriented within the engineered nucleic acid in a tail-to-tail directionality.
  • the second promoter comprises a constitutive promoter, an inducible promoter, or a synthetic promoter.
  • the constitutive promoter is selected from the group consisting of: CAG, HLP, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.
  • the first cytokine is selected from the group consisting of: IL12, an IL12p70 fusion protein, IL18, and IL21. In some embodiments, the first cytokine is the IL12p70 fusion protein. In some embodiments, the IL12p70 fusion protein comprises the amino acid sequence of SEQ ID NO: 293.
  • the protease cleavage site is cleavable by a protease selected from the group consisting of: a Type I transmembrane protease, a Type II transmembrane protease, a GPI anchored protease, an ADAM8 protease, an ADAM9 protease, an ADAM 10 protease, an ADAM 12 protease, an ADAM 15 protease, an ADAM 17 protease, an ADAM19 protease, an ADAM20 protease, an ADAM21 protease, an ADAM28 protease, an ADAM30 protease, an ADAM33 protease, a BACE1 protease, a BACE2 protease, a SIP protease, an MT1-MMP protease, an MT3-MMP protease, an MT5-MMP protease, a furin proteas
  • the protease cleavage site comprises a first region having the amino acid sequence of PRAE (SEQ ID NO: 176). In some embodiments, the protease cleavage site comprises a second region having the amino acid sequence of KGG (SEQ ID NO: 177). In some embodiments, the first region is located N-terminal to the second region. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEX1X2KGG (SEQ ID NO: 178), wherein Xi is A, Y, P, S, or F, and wherein X 2 is V, L, S, I, Y, T, or A.
  • the protease cleavage site comprises the amino acid sequence selected from the group consisting of PRAEAVKGG (SEQ ID NO: 179), PRAEALKGG (SEQ ID NO: 180), PRAEYSKGG (SEQ ID NO: 181), PRAEPIKGG (SEQ ID NO: 182), PRAEAYKGG (SEQ ID NO: 183), PRAESSKGG (SEQ ID NO: 184), PRAEFTKGG (SEQ ID NO: 185), PRAEAAKGG (SEQ ID NO: 186), DEPHYSQRR (SEQ ID NO: 187), PPLGPIFNPG (SEQ ID NO: 188), PLAQAYRSS (SEQ ID NO: 189), TPIDSSFNPD (SEQ ID NO: 190), VTPEPIFSLI (SEQ ID NO: 191), ITQGLAVSTISSFF (SEQ ID NO: 198).
  • PRAEAVKGG SEQ ID NO: 179
  • PRAEALKGG SEQ ID NO: 180
  • the protease cleavage site is comprised within a peptide linker. In some embodiments, the protease cleavage site is N-terminal to a peptide linker. In some embodiments, the peptide linker comprises a glycine- serine (GS) linker. In some embodiments, the cell membrane tethering domain comprises a transmembrane-intracellular domain and/or a transmembrane domain.
  • GS glycine- serine
  • the transmembrane-intracellular domain and/or transmembrane domain is derived from PDGFR-beta, CD8, CD28, CD3zeta-chain, CD4, 4- 1BB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, or BTLA.
  • the cell membrane tethering domain comprises a post- translational modification tag, or motif capable of post-translational modification to modify the chimeric protein to include a post-translational modification tag, wherein the post-translational modification tag is capable of association with a cell membrane.
  • the post- translational modification tag comprises a lipid-anchor domain.
  • the lipid- anchor domain is selected from the group consisting of: a GPI lipid-anchor, a myristoylation tag, and a palmitoylation tag.
  • the cell membrane tethering domain comprises a cell surface receptor, or a cell membrane-bound portion thereof.
  • the cytokine of the membrane-cleavable chimeric protein is tethered to a cell membrane of the cell.
  • the cell further comprises a protease capable of cleaving the protease cleavage site.
  • the protease is endogenous to the cell.
  • the protease is selected from the group consisting of: a Type I transmembrane protease, a Type II transmembrane protease, a GPI anchored protease, an ADAM8 protease, an ADAM9 protease, an ADAM 10 protease, an ADAM 12 protease, an ADAM 15 protease, an ADAM 17 protease, an ADAM 19 protease, an ADAM20 protease, an ADAM21 protease, an ADAM28 protease, an ADAM30 protease, an ADAM33 protease, a BACE1 protease, a BACE2 protease, a SIP protease, an MT1-MMP proteas
  • the protease is expressed on the cell membrane of the cell. In some embodiments, cleavage of the protease cleavage site releases the cytokine of the membrane-cleavable chimeric protein from the cell membrane of the cell.
  • the first exogenous polynucleotide sequence further comprises a polynucleotide sequence encoding a secretion signal peptide.
  • the secretion signal peptide is operably associated with the first cytokine.
  • the secretion signal peptide is native or non-native to the first cytokine.
  • the transcriptional effector domain comprises a transcriptional activator domain.
  • the transcriptional activator domain is selected from the group consisting of: a Herpes Simplex Virus Protein 16 (VP 16) activation domain; an activation domain comprising four tandem copies of VP16, a VP64 activation domain; a p65 activation domain of NFKB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activation domain); a histone acetyltransferase (HAT) core domain of the human E1A- associated protein p300 (p300 HAT core activation domain).
  • VP 16 Herpes Simplex Virus Protein 16
  • Rta Epstein-Barr virus R transactivator
  • HAT histone acetyltransferase
  • the transcriptional effector domain comprises a transcriptional repressor domain.
  • the transcriptional repressor domain is selected from the group consisting of: a Kruppel associated box (KRAB) repression domain; a truncated Kriippel associated box (KRAB) repression domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif of the hairy-related basic helix-loop-helix repressor proteins, the motif is known as a WRPW repression domain; a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain.
  • KRAB Kruppel associated box
  • KRAB truncated Kriippel associated box
  • REST Repressor Element Silencing Transcription Factor
  • the DNA binding domain comprises a zinc finger (ZF) protein domain.
  • ZF protein domain is modular in design and comprises an array of zinc finger motifs.
  • the ZF protein domain comprises an array of one to ten zinc finger motifs.
  • the ZF protein domain comprises the amino acid sequence of SEQ ID NO: 320.
  • the ACP further comprises a repressible protease and one or more cognate cleavage sites of the repressible protease.
  • the repressible protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3).
  • HCV hepatitis C virus
  • NS3 protease comprises the amino acid sequence of SEQ ID NO: 321.
  • the NS3 protease is repressible by a protease inhibitor.
  • the protease inhibitor is selected from the group consisting of: simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir.
  • the cognate cleavage site of the repressible protease comprises an NS3 protease cleavage site.
  • the NS3 protease cleavage site comprises a NS3/NS4A, a NS4A/NS4B, a NS4B/NS5A, or a NS5A/NS5B junction cleavage site.
  • the one or more cognate cleavage sites of the repressible protease are localized between the DNA binding domain and the transcriptional effector domain.
  • the ACP-responsive promoter comprises a minimal promoter sequence. In some embodiments, the ACP-responsive promoter comprises one or more zinc finger binding sites.
  • the ACP-responsive promoter the engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 317 or 318.
  • the present disclosure provides a cell composition comprising a first immunoresponsive cell provided herein, and a second immunoresponsive cell, wherein the second immunoresponsive cell expresses a chimeric antigen receptor.
  • the second immunoresponsive cell comprises: a second engineered nucleic acid comprising an expression cassette comprising a first promoter operably linked to a first, exogenous polynucleotide sequence encoding a first cytokine, and a second expression cassette comprising a second promoter operably linked to a second exogenous polynucleotide sequence encoding the chimeric antigen receptor (CAR).
  • the CAR is a GPC3-specific CAR.
  • the present disclosure provides an engineered nucleic acid comprising a first expression cassette comprising a ACP-responsive promoter operably linked to a first exogenous polynucleotide sequence encoding an IL12p70 fusion protein, and a second expression cassette comprising a second promoter operably linked to a second exogenous polynucleotide sequence encoding an activation-conditional control polypeptide (ACP), wherein the ACP comprises a synthetic transcription factor comprising a DNA-binding domain and a transcriptional effector domain, wherein the ACP is capable of inducing expression of the first exogenous polynucleotide sequence by binding to the ACP-responsive promoter, wherein the first exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula: S - C - MT or MT - C - S wherein S comprises a secretable effector molecule comprising
  • the first expression cassette and the second expression cassette are oriented within the first engineered nucleic acid in a head-to-head directionality, and (b) the transcriptional effector domain comprises a transcriptional activator domain, wherein the transcriptional activator domain comprises a VPR activation domain or a p65 activation domain.
  • the present disclosure provides an expression vector comprising the engineered nucleic acid provided herein.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the immunoresponsive cell, the cell composition, the engineered nucleic acid, or the expression vector provided herein, and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.
  • the present disclosure provides a method of treating a subject having cancer, the method comprising administering a therapeutically effective dose of the immunoresponsive cell, the cell composition, the engineered nucleic acid, the expression vector, or the pharmaceutical composition provided herein.
  • the cancer comprises a GPC3-expressing cancer.
  • the cancer is selected from the group consisting of: hepatocellular carcinoma (HCC), ovarian clear cell carcinoma, melanoma, squamous cell carcinoma of the lung, hepatoblastoma, nephroblastoma (Wilms tumor), and yolk sac tumor.
  • the administering comprises systemic administration or intratumoral administration.
  • the immunoresponsive cell is derived from the subject or is allogeneic with reference to the subject.
  • the present disclosure provides immunoresponsive cells comprising: an engineered nucleic acid comprising a first expression cassette comprising an ACP-responsive promoter operably linked to a first exogenous polynucleotide sequence encoding a first cytokine, and a second expression cassette comprising a second promoter operably linked to a second exogenous polynucleotide sequence encoding an activation-conditional control polypeptide (ACP), wherein the ACP is capable of inducing expression of the first exogenous polynucleotide sequence, wherein the first exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula: S - C - MT or MT - C - S wherein S comprises a secretable effector molecule comprising the first cytokine, C comprises a protease cleavage site, and MT comprises a cell membrane tethering domain, and
  • an ACP-responsive promoter comprises a synthetic promoter. In some embodiments, an ACP-responsive promoter comprises an ACP-binding domain sequence. In some embodiments, an ACP comprises a synthetic transcription factor. In some embodiments, an ACP comprises a DNA-binding domain and a transcriptional effector domain.
  • the present disclosure further provides for immunoresponsive cells comprising: an engineered nucleic acid comprising a first expression cassette comprising a synthetic transcription factor-responsive promoter operably linked to a first exogenous polynucleotide sequence encoding a first cytokine, and a second expression cassette comprising a second promoter operably linked to second exogenous polynucleotide sequence encoding an activationconditional control polypeptide (ACP), wherein the ACP comprises a synthetic transcription factor comprising a DNA-binding domain and a transcriptional effector domain, wherein the ACP is capable of inducing expression of the first exogenous polynucleotide sequence by binding to the ACP-responsive promoter, wherein the first exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula: S - C - MT or MT - C - S wherein S comprises a secretable effector molecule compris
  • a first expression cassette is configured to be transcribed in an opposite orientation relative to transcription of a second expression cassette within an engineered nucleic acid.
  • a first expression cassette and a second expression cassette are oriented within an engineered nucleic acid in a head-to-head directionality.
  • a first expression cassette and a second expression cassette are oriented within an engineered nucleic acid in a tail-to-tail directionality.
  • a second promoter comprises a constitutive promoter, an inducible promoter, or a synthetic promoter.
  • a second promoter is a constitutive promoter selected from the group consisting of: CAG, HLP, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.
  • a first cytokine is selected from the group consisting of: IL12, an IL12p70 fusion protein, IL18, and IL21. In some embodiments, a first cytokine is an IL12p70 fusion protein. In some embodiments, an IL12p70 fusion protein comprises the amino acid sequence of SEQ ID NO: 293.
  • a protease cleavage site is cleavable by a protease selected from the group consisting of: a Type I transmembrane protease, a Type II transmembrane protease, a GPI anchored protease, an ADAM8 protease, an ADAM9 protease, an ADAM 10 protease, an ADAM 12 protease, an ADAM 15 protease, an ADAM 17 protease, an ADAM 19 protease, an ADAM20 protease, an ADAM21 protease, an ADAM28 protease, an ADAM30 protease, an ADAM33 protease, a BACE1 protease, a BACE2 protease, a SIP protease, an MT1-MMP protease, an MT3-MMP protease, an MT5-MMP protease, a furin protea
  • a protease cleavage site comprises a first region having the amino acid sequence of PRAE (SEQ ID NO: 176). In some embodiments, a protease cleavage site comprises a second region having the amino acid sequence of KGG (SEQ ID NO: 177). In some embodiments, a first region is located N-terminal to the second region.
  • a protease cleavage site comprises the amino acid sequence of PRAEX1X2KGG (SEQ ID NO: 178), wherein XI is A, Y, P, S, or F, and wherein X2 is V, L, S, I, Y, T, or A.
  • a protease cleavage site comprises the amino acid sequence of PRAEAVKGG (SEQ ID NO: 179).
  • a protease cleavage site comprises the amino acid sequence of PRAEALKGG (SEQ ID NO: 180).
  • a protease cleavage site comprises the amino acid sequence of PRAEYSKGG (SEQ ID NO: 181).
  • a protease cleavage site comprises the amino acid sequence of PRAEPIKGG (SEQ ID NO: 182). In some embodiments, a protease cleavage site comprises the amino acid sequence of PRAEAYKGG (SEQ ID NO: 183). In some embodiments, a protease cleavage site comprises the amino acid sequence of PRAESSKGG (SEQ ID NO: 184). In some embodiments, a protease cleavage site comprises the amino acid sequence of PRAEFTKGG (SEQ ID NO: 185). In some embodiments, a protease cleavage site comprises the amino acid sequence of PRAEAAKGG (SEQ ID NO: 186).
  • a protease cleavage site comprises the amino acid sequence of DEPHYSQRR (SEQ ID NO: 187). In some embodiments, a protease cleavage site comprises the amino acid sequence of PPLGPIFNPG (SEQ ID NO: 188). In some embodiments, a protease cleavage site comprises the amino acid sequence of PLAQAYRSS (SEQ ID NO: 189). In some embodiments, a protease cleavage site comprises the amino acid sequence of TPIDSSFNPD (SEQ ID NO: 190). In some embodiments, a protease cleavage site comprises the amino acid sequence of VTPEPIFSLI (SEQ ID NO: 191). In some embodiments, a protease cleavage site comprises the amino acid sequence of ITQGLAVSTISSFF (SEQ ID NO: 198).
  • a protease cleavage site is comprised within a peptide linker. In some embodiments, a protease cleavage site is N-terminal to a peptide linker. In some embodiments, a peptide linker comprises a glycine- serine (GS) linker.
  • GS glycine- serine
  • a cell membrane tethering domain comprises a transmembrane- intracellular domain and/or a transmembrane domain.
  • a transmembrane- intracellular domain and/or transmembrane domain is derived from PDGFR-beta, CD8, CD28, CD3zeta-chain, CD4, 4-1BB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, or BTLA.
  • a transmembrane-intracellular domain and/or transmembrane domain is derived from B7-1.
  • a transmembrane- intracellular domain and/or transmembrane domain comprises the amino acid sequence of SEQ ID NO: 219.
  • a cell membrane tethering domain comprises a post-translational modification tag, or motif capable of post-translational modification to modify the chimeric protein to include a post-translational modification tag, wherein the post-translational modification tag is capable of association with a cell membrane.
  • a post- translational modification tag comprises a lipid-anchor domain.
  • the lipid- anchor domain is selected from the group consisting of: a GPI lipid-anchor, a myristoylation tag, and a palmitoylation tag.
  • a cell membrane tethering domain comprises a cell surface receptor, or a cell membrane-bound portion thereof.
  • a cytokine of a membrane-cleavable chimeric protein is tethered to a cell membrane of the cell.
  • an immunoresponsive cell further comprises a protease capable of cleaving a protease cleavage site.
  • a protease is endogenous to the cell.
  • a protease is selected from the group consisting of: a Type I transmembrane protease, a Type II transmembrane protease, a GPI anchored protease, an ADAM8 protease, an ADAM9 protease, an ADAM 10 protease, an ADAM 12 protease, an ADAM 15 protease, an ADAM 17 protease, an ADAM 19 protease, an ADAM20 protease, an ADAM21 protease, an ADAM28 protease, an ADAM30 protease, an ADAM33 protease, a BACE1 protease, a BACE2 protease, a SIP protease, an MT1-
  • a protease is an ADAM 17 protease. In some embodiments, a protease is expressed on the cell membrane of the cell. In some embodiments, a protease cleavage site releases the cytokine of the membrane-cleavable chimeric protein from the cell membrane of the cell.
  • a first exogenous polynucleotide sequence further comprises a polynucleotide sequence encoding a secretion signal peptide.
  • a secretion signal peptide is operably associated with the first cytokine.
  • a secretion signal peptide is native to the first cytokine.
  • a secretion signal peptide is non-native to the first cytokine.
  • a second exogenous polynucleotide sequence further encodes a membrane-cleavable chimeric protein.
  • a second expression cassette further comprises a polynucleotide sequence encoding a secretion signal peptide.
  • a transcriptional effector domain comprises a transcriptional activator domain.
  • a transcriptional activator domain is selected from the group consisting of: a Herpes Simplex Virus Protein 16 (VP16) activation domain; an activation domain comprising four tandem copies of VP 16, a VP64 activation domain; a p65 activation domain of NFKB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activation domain); a histone acetyltransferase (HAT) core domain of the human ElA-associated protein p300 (p300 HAT core activation domain).
  • a transcriptional activator domain comprises a VPR activation domain.
  • a VPR activation domain comprises the amino acid sequence of SEQ ID NO: 325.
  • a transcriptional effector domain comprises a transcriptional repressor domain.
  • a transcriptional repressor domain is selected from the group consisting of: a Kruppel associated box (KRAB) repression domain; a truncated Kruppel associated box (KRAB) repression domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif of the hairy-related basic helix-loop-helix repressor proteins, the motif is known as a WRPW repression domain; a DNA (cytosine-5)- methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain.
  • KRAB Kruppel associated box
  • KRAB truncated Kruppel associated box
  • REST Repressor Element Silencing Transcription Factor
  • a DNA binding domain comprises a zinc finger (ZF) protein domain.
  • ZF protein domain is modular in design and comprises an array of zinc finger motifs.
  • a ZF protein domain comprises an array of one to ten zinc finger motifs.
  • a ZF protein domain comprises the amino acid sequence of SEQ ID NO: 320.
  • an ACP further comprises a repressible protease and one or more cognate cleavage sites of the repressible protease.
  • a repressible protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3).
  • HCV hepatitis C virus
  • NS3 protease comprises the amino acid sequence of SEQ ID NO: 321.
  • a cognate cleavage site of the repressible protease comprises an NS 3 protease cleavage site.
  • a NS3 protease cleavage site comprises a NS3/NS4A, a NS4A/NS4B, a NS4B/NS5A, or a NS5A/NS5B junction cleavage site.
  • a NS3 protease is repressible by a protease inhibitor.
  • a protease inhibitor is selected from the group consisting of: simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir.
  • a protease inhibitor is grazoprevir (GRZ).
  • an ACP further comprises a nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • a NLS comprises the amino acid sequence of SEQ ID NO: 296.
  • one or more cognate cleavage sites of a repressible protease are localized between a DNA binding domain and a transcriptional effector domain.
  • an ACP further comprises a ligand binding domain of estrogen receptor variant ERT2.
  • an ACP-responsive promoter comprises a minimal promoter sequence.
  • an ACP binding domain sequence comprises one or more zinc finger binding sites.
  • an engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 317. In some embodiments, an engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 318.
  • the present disclosure also provides for immunoresponsive cells comprising an engineered nucleic acid comprising the nucleotide sequence of SEQ ID NO: 317.
  • the present disclosure provides for engineered nucleic acids comprising: a first expression cassette comprising an activation-conditional control polypeptide (ACP)-responsive promoter operably linked to a first exogenous polynucleotide sequence encoding an IL12p70 fusion protein, and a second expression cassette comprising a second promoter operably linked to a second exogenous polynucleotide sequence encoding an ACP, wherein the ACP comprises a synthetic transcription factor comprising a DNA-binding domain and a transcriptional effector domain, wherein the ACP is capable of inducing expression of the first exogenous polynucleotide sequence by binding to the ACP-responsive promoter, wherein the first exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula: S - C - MT or MT - C - S wherein S comprises a secretable effector molecule comprising the
  • a first expression cassette and a second expression cassette are oriented within a first engineered nucleic acid in a head-to-head directionality, and a transcriptional effector domain comprises a transcriptional activator domain, wherein the transcriptional activator domain comprises a VPR activation domain.
  • a first expression cassette and the second expression cassette are oriented within the first engineered nucleic acid in a head-to-head directionality, and a transcriptional effector domain comprises a transcriptional activator domain, wherein the transcriptional activator domain comprises a p65 activation domain.
  • an engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 317. In some embodiments, an engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 318.
  • the present disclosure further provides an engineered nucleic acid comprising the nucleotide sequence of SEQ ID NO: 317.
  • the present disclosure provides for an expression vector comprising any engineered nucleic acid described herein.
  • the present disclosure also provides for an immunoresponsive cell comprising an engineered nucleic acid as described herein, or an expression vector as described herein.
  • the present disclosure provides for cell compositions (e.g., mixed cell composition) comprising a first immunoresponsive cell as described herein, and a second immunoresponsive cell, wherein the second immunoresponsive cell expresses a chimeric antigen receptor.
  • cell compositions e.g., mixed cell composition
  • a second immunoresponsive cell comprises: a second engineered nucleic acid comprising an expression cassette comprising a first promoter operably linked to a first, exogenous polynucleotide sequence encoding a first cytokine, and a second expression cassette comprising a second promoter operably linked to a second exogenous polynucleotide sequence encoding the chimeric antigen receptor (CAR).
  • a second engineered nucleic acid comprising an expression cassette comprising a first promoter operably linked to a first, exogenous polynucleotide sequence encoding a first cytokine
  • a second expression cassette comprising a second promoter operably linked to a second exogenous polynucleotide sequence encoding the chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • compositions comprising an immunoresponsive cell as described herein, a cell composition as described herein, an engineered nucleic acid as described herein, or an expression vector as described herein, and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.
  • the present disclosure provides for methods of treating a disease or disorder in a subject in need thereof, the methods comprising administering a therapeutically effective dose of an immunoresponsive cell as described herein, a cell composition as described herein, an engineered nucleic acid as described herein, an expression vector as described herein, or a pharmaceutical composition as described herein.
  • the present disclosure also provides for methods of stimulating a cell-mediated immune response to a tumor cell in a subject, the methods comprising administering to a subject having a tumor a therapeutically effective dose of an immunoresponsive cell as described herein, a cell composition as described herein, an engineered nucleic acid as described herein, an expression vector as described herein, or a pharmaceutical composition as described herein.
  • the present disclosure further provides for methods of reducing tumor volume in a subject, the methods comprising administering to a subject having a tumor a composition comprising an immunoresponsive cell as described herein, a cell composition as described herein, an engineered nucleic acid as described herein, an expression vector as described herein, or a pharmaceutical composition as described herein.
  • the present disclosure provides for methods of providing an anti-tumor immunity in a subject, the methods comprising administering to a subject in need thereof a therapeutically effective dose of an immunoresponsive cell as described herein, a cell composition as described herein, an engineered nucleic acid as described herein, an expression vector as described herein, or a pharmaceutical composition as described herein.
  • a tumor comprises a GPC3 -expressing tumor.
  • a tumor is selected from the group consisting of: hepatocellular carcinoma (HCC), ovarian clear cell carcinoma, melanoma, squamous cell carcinoma of the lung, hepatoblastoma, nephroblastoma (Wilms tumor), and yolk sac tumor.
  • the present disclosure provides for methods of treating a subject having cancer, the methods comprising administering a therapeutically effective dose of an immunoresponsive cell as described herein, a cell composition as described herein, an engineered nucleic acid as described herein, an expression vector as described herein, or a pharmaceutical composition as described herein.
  • a cancer comprises a GPC3 -expressing cancer.
  • a cancer is selected from the group consisting of: hepatocellular carcinoma (HCC), ovarian clear cell carcinoma, melanoma, squamous cell carcinoma of the lung, hepatoblastoma, nephroblastoma (Wilms tumor), and yolk sac tumor.
  • administering comprises systemic administration. In some embodiments, administering comprises intratumoral administration.
  • an immunoresponsive cell as described herein is derived from the subject. In some embodiments, an immunoresponsive cell as described herein is allogeneic with reference to the subject. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIGs. 1A-1D illustrate schematics of a cytokine-CAR bidirectional construct in head-to- head directionality (FIG. 1A), head-to-tail directionality (FIG. IB), tail-to-tail directionality (FIG. 1C), and.an exemplary anti-GPC3 CAR + IL 15 bidirectional construct (FIG. ID).
  • FIG. 2 provides CAR expression plots assessed by flow cytometry for cells transduced with lentivirus encoding a CAR + IL 15 bidirectional construct and cells transduced with a lentivirus encoding the CAR-only (day 7).
  • FIG. 3 provides CAR expression plots assessed by flow cytometry for cells transduced with retrovirus encoding a CAR + IL15 bidirectional construct and cells transduced with a retrovirus encoding the CAR-only (day 7).
  • FIG. 4 provides CAR expression plots assessed by flow cytometry for cells transduced with lentivirus encoding a CAR + IL 15 bidirectional construct and cells transduced with a lentivirus encoding the CAR-only (day 15).
  • FIG. 5 provides CAR expression plots assessed by flow cytometry for cells transduced with retrovirus encoding a CAR + IL15 bidirectional construct and cells transduced with a retrovirus encoding the CAR-only (day 15).
  • FIG. 6 provides IL15 levels assessed by immunoassay for NK cells transduced with lentiviruses encoding CAR + IL 15 bidirectional construct (“Lenti”) or y-retroviruses encoding CAR + IL15 bidirectional constructs (“SinVec”).
  • FIG. 7 provides killing by NK cells transduced with lentiviruses encoding CAR-only or CAR + IL15 bidirectional constructs, as assessed by a co-culture killing assay.
  • FIG. 8 provides killing by NK cells transduced with y-retroviruses encoding CAR-only or CAR + IL15 bidirectional constructs, as assessed by a co-culture killing assay.
  • FIG. 9 illustrates schematics for bidirectionally orientated constructs, including IL12 expression cassettes having mRNA destabilization elements in the 3’ untranslated region.
  • FIG. 10 provides IL12 levels assessed by immunoassay for NK cells transduced with bidirectional constructs including an inducible IL12 expression cassette and an expression cassette encoding a synthetic transcription factor.
  • FIG. 11 illustrates a schematic of bidirectional construct encoding a cleavable release IL15.
  • FIG. 12 provides a summary of IL 15 bicistronic constructs tested and performance in functional assays.
  • FIG. 13A and FIG. 13B provide expression plots as assessed by flow cytometry for NK cells transduced with SB06251, SB06257, and SB06254, for GPC3 CAR and IL15. Two independent replicates are shown (FIG. 13A and FIG. 13B).
  • FIG. 14A and FIG. 14B provides secreted IL15 levels as assessed by immunoassay for NK cells transduced with SB06251, SB06257, and SB06254. Two independent replicates are shown (FIG. 14A and FIG. 14B).
  • FIG. 15A and FIG. 15B provide cell growth of target cell population following coculture with NK cells transduced with SB06251, SB06257, and SB06254. Two independent replicates are shown (FIG. 15A and FIG. 15B).
  • FIG. 16 provides target cell counts in a serial-killing assay when co-cultured with NK cells tranduced with SB06251, SB06257, and SB06254.
  • FIG. 17A and FIG. 17B provide expression plots as assessed by flow cytometry for NK cells transduced with SB06252, SB06258, and SB06255, for GPC3 CAR and IL15. Two independent replicates are shown (FIG. 17A and FIG. 17B).
  • FIG. 18A and FIG. 18B provide secreted IL15 levels as assessed by immunoassay for NK cells tranduced with SB06252, SB06258, and SB06255. Two independent replicates are shown (FIG. 18A and FIG. 18B).
  • FIG. 19A and FIG. 19B provide cell growth of target cell population following coculture with NK cells tranduced with SB06252, SB06258, and SB06255. Two independent replicates are shown (FIG. 19A and FIG. 19B).
  • FIG. 20 provides target cell counts in a serial-killing assay when co-cultured with NK cells transduced with SB06252, SB06258, and SB06255.
  • FIG. 21A and FIG. 21B provide expression plots as assessed by flow cytometry for NK cells transduced with bicistronic constructs SB06261, SB6294, and SB6298, for GPC3 CAR and IL15. Two independent replicates are shown (FIG. 21A and FIG. 21B).
  • FIG. 22A and FIG. 22B provide secreted IL15 levels as assessed by immunoassay for NK cells tranduced with SB06261, SB6294, and SB6298. Two independent replicates are shown (FIG. 22A and FIG. 22B).
  • FIG. 23A and FIG. 23B provide cell growth of target cell population following coculture with NK cells tranduced with SB06252, SB06258, and SB06255. Two independent replicates are shown (FIG. 23A and FIG. 23B).
  • FIG. 24A and FIG. 24B provide characterization of cleavable release IL15 bicstronic constructs SB06691, SB06692, and SB06693. Expression plots as assessed by flow cytometry for NK cells transduced with SB06691, SB06692, and SB06693, for GPC3 CAR and IL15, are shown in FIG. 24A. Secreted IL15 levels as assessed by immunoassay for NK cells tranduced with SB06691, SB06692, and SB06693 are shown in FIG. 24B.
  • FIG. 25 illustrates a schematic of a bidirectional construct encoding a cleavable release IL12.
  • FIG. 26 provides a dose-response curve of IL12 secretion for NK cells following treatment with grazoprevir (GRZ).
  • FIG. 27A and FIG. 27B provide in vivo mouse data demonstrating IL12 levels in mouse blood following injectetion with NK cells tranduced with SB04599, SB05042, and SB05058. IL12 levels are shown in FIG. 27A and IL12 fold change is shown in FIG. 27B.
  • FIGs. 28A-28C provide characterization of cells transduced with different constructs expressing the GPC3 CAR and IL15.
  • FIG. 28A shows flow cytometry plots demonstrating expression of GPC3 CAR, membrane bound IL15, and respective copy numbers on NK cells transduced with different GPC3 CAR/IL15 expression constructs.
  • FIG. 28B shows measurement of secreted IL- 15.
  • FIG. 28C shows cell killing of HepG2 as assessed by a serial killing assay.
  • FIG. 29A and FIG. 29B provide additional data of serial killing using transduced NK Cells.
  • FIG. 29A shows serial killing of HepG2 cells.
  • FIG. 29B shows serial killing of HuH-7 cells.
  • FIG. 30A and FIG. 30B provide data assessing transduced NK cell function using rapid expansion (G-Rex).
  • FIG. 30A shows expression of GPC3 CAR, membrane bound IL 15(mIL15), and secreted IL15 (sIL15).
  • FIG. 30B shows serial killing of the transduced NK cells.
  • FIG. 31 provides results from a xenograft tumor model as measured by bioluminescence imaging, in which mice are injected with NK cells.
  • FIG. 32A and FIG. 32B provide the results of a xenograft tumor model in mice that are injected with NK cells and summary.
  • FIG. 32A provides a survival curve of mice treated with NK cells.
  • FIG. 32B provides a summary of the median survival of mice treated with the NK cells.
  • FIG. 33 provides results of a BLI experiment to assess tumor reduction in mice injected with NK cells.
  • FIG. 34 provides a quantification of each condition in terms of BLI measurements that were normalized to day 10.
  • FIG. 35A and FIG. 35B provide results from a xenograft tumor (HepG2) mouse model in which mice were injected three times with NK cells over the course of the study.
  • FIG. 35A provides results of mice that were imaged using BLI.
  • FIG. 35B provides a time course of fold change of BLI over the course of the study.
  • FIG. 36A and FIG. 36B provide the fold change BLI in mice injected with transduced NK cells.
  • FIG. 36A provides results corresponding to measurements performed 13 days after tumor implantation.
  • FIG. 36B provides results corresponding to measurements performed 20 days after tumor implantation.
  • FIG. 37A and FIG. 37B provide results of tumor reduction in a xenograft model.
  • FIG. 37A shows a summary of the BLI Fold change in two different in vivo experiments.
  • FIG. 37B shows a summary of the normalized mean BLI Fold change in two different in vivo experiments, but the treatment groups are separated, and animal are tracked individually.
  • FIG. 38A and FIG. 38B provide results from a xenograft tumor model in which NK cells are injected intratumorally.
  • FIG. 38A provides measurements of tumor volume.
  • FIG. 38B shows a survival curve.
  • FIG. 39A and FIG. 39B provide results for expression of IL- 12 in the presence or absence of grazoprevir.
  • FIG. 39A provides measurements of concentration and fold change 24 hours after induction with grazoprevir.
  • FIG. 39B provides measurements of concentration and fold change 72 hours after induction.
  • FIG. 40 provides results from a mouse that was injected NK cells expressing regulated IL 12 at different concentrations and throughout the experiment.
  • FIG. 41 provides expression (GPC3 CAR and IL15) results of co-transduction with the IL-12 and GPC3 CAR/IL15 constructs into NK cells.
  • FIG. 42A and FIG. 42B provide results of secreted IL 15 and secreted IL 12 expression in the presence or absence of grazoprevir.
  • FIG. 42A provides measurements of secreted IL15 concentration.
  • FIG. 42B provides measurements of secreted IL 12 expression.
  • FIG. 43 provides measurements of secreted IL 15 and secreted IL 12 of NK cells during a serial killing assay.
  • FIGs. 44A-44D provide results of a serial killing assay for different co-transductions in NK cells for cell killing of Huh-7 and HepG2 cells.
  • FIG. 44A provides the serial killing results for NK cells co-transduced with SB05042 + SB06258.
  • FIG. 44B provides the serial killing results for NK cells co-transduced with SB05042 + SB06257.
  • FIG. 44C provides the serial killing results for NK cells co-transduced with SB05042 + SB06294.
  • FIG. 44D provides a combination of the results in FIGs. 44A-C.
  • FIGs. 45A-45D provide results from assessment of the clonal selection of NK cells expressing the GPC3 CAR.
  • FIG. 45A provides results on copies per cell.
  • FIG. 45B provides results of GCP3 CAR expression.
  • FIG. 45C provides results for IL15 expression.
  • FIG. 45D provides measurement of secreted IL 15.
  • FIG. 46A and FIG. 46B provide flow cytometry data of GPC3 CAR and IL 15 expression on selected clones transduced with SB06258.
  • FIG. 46A provides results of selected clones.
  • FIG. 46B provides results of selected clones further transduced with SB05042 (IL12).
  • FIGs. 47A-47D provide results from IL12 expression in the presence or absence of a small molecule, grazoprevir (FIG. 47A and FIG. 47B) or endoxifen (FIG. 47C and FIG. 47D).
  • a dose-response curve is present in transduced natural killer cells derived from 3 different donors.
  • FIG. 47B shows the concentration of IL12 expressed as a function of time at 0.1 f M Grazoprevir.
  • FIG. 47C a dose-response curve is present in transduced natural killer cells that were transduced with 2 different constructs containing a different ERT2 variant.
  • FIG. 47D shows the concentration of IL12 expressed as a function of time at 0.1 nM endoxifen.
  • FIG. 48A and FIG. 48B show characterization of NK cells that are transduced with a GPC3/crIL15 encoding constructs and/or crIL12 encoding constructs and assessing the levels of 3 different NK cell markers.
  • FIG. 48A provides the results from the characterization of natural killer cells that were co-transduced with the GPC3/crIL15 encoding constructs and crIL12 encoding constructs.
  • FIG. 48B provides results for the characterization of a mixture of NK cells in which one population of cells are transduced with the GPC3/crIL15 constructs and another population are transduced with the crIL12 encoding constructs.
  • Immunoresponsive cells are provided for herein.
  • immunoresponsive cells are engineered to have the following:
  • a first engineered nucleic acid comprising a first expression cassette comprising a first promoter operably linked to a first exogenous polynucleotide sequence encoding a first cytokine, and a second expression cassette comprising a second promoter operably linked to a second exogenous polynucleotide sequence encoding a chimeric antigen receptor (CAR) that binds to GPC3; and
  • CAR chimeric antigen receptor
  • a second engineered nucleic acid comprising a third expression cassette comprising an activation-conditional control polypeptide (ACP)- responsive promoter operably linked to a third exogenous polynucleotide sequence encoding a second cytokine, and a fourth expression cassette comprising a fourth promoter operably linked to a fourth exogenous polynucleotide sequence encoding an ACP, wherein the ACP comprises a synthetic transcription factor comprising a DNA-binding domain and a transcriptional effector domain, wherein the ACP is capable of inducing expression of the third exogenous polynucleotide sequence by binding to the ACP-responsive promoter, wherein at least one of the first exogenous polynucleotide sequence and the third exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N- terminal to C-terminal, having the formula: S - C - MT or MT - C - S configured to
  • immunoresponsive cells are engineered to have the following:
  • a first engineered nucleic acid comprising a first expression cassette comprising a first promoter operably linked to a first exogenous polynucleotide sequence encoding a first cytokine and a second exogenous polynucleotide sequence encoding a chimeric antigen receptor (CAR) that binds to GPC3, and a second expression cassette comprising an activation-conditional control polypeptide (ACP)- responsive promoter operably linked to a third exogenous polynucleotide sequence encoding a second cytokine; and
  • ACP activation-conditional control polypeptide
  • a second engineered nucleic acid comprising a third expression cassette comprising a third promoter operably linked to fourth exogenous polynucleotide sequence encoding an ACP
  • the ACP comprises a synthetic transcription factor comprising a DNA-binding domain and a transcriptional effector domain, wherein the ACP is capable of inducing expression of the third exogenous polynucleotide sequence by binding to the ACP-responsive promoter, wherein the ACP comprises a synthetic transcription factor, wherein at least one of the first exogenous polynucleotide sequence and the third exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula: S - C - MT or MT - C - S configured to be expressed as a single polypeptide.
  • S refers to a secretable effector molecule (e.g., a cytokine).
  • C refers to a protease cleavage site.
  • MT refers to a cell membrane tethering domain.
  • immunoresponsive cells are engineered to have an engineered nucleic acid comprising a first expression cassette comprising an ACP-responsive promoter operably linked to a first exogenous polynucleotide sequence encoding a first cytokine, and a second expression cassette comprising a second promoter operably linked to a second exogenous polynucleotide sequence encoding an activation-conditional control polypeptide (ACP), wherein the ACP is capable of inducing expression of the first exogenous polynucleotide sequence, wherein the first exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula:
  • S comprises a secretable effector molecule comprising the first cytokine
  • C comprises a protease cleavage site
  • MT comprises a cell membrane tethering domain, and wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide.
  • the ACP of the immunoresponsive cells includes a synthetic transcription factor.
  • a synthetic transcription factor is a non-naturally occurring protein that includes a DNA-binding domain and a transcriptional effector domain and is capable of modulating (/'. ⁇ ?., activating or repressing) transcription through binding to a cognate promoter recognized by the DNA-binding domain (e.g., a synthetic transcription factor-responsive promoter, such as an ACP-responsive promoter).
  • the ACP is a transcriptional repressor.
  • the ACP is a transcriptional activator.
  • the membrane-cleavable chimeric protein is engineered such that secretion of the effector molecule (e.g., a cytokine) can be regulated in a protease-dependent manner.
  • the membrane-cleavable chimeric protein is engineered such that secretion of the effector molecule can be regulated as part of a “Membrane-Cleavable” system, where incorporation of a protease cleavage site (“C”) and a cell membrane tethering domain (“MT”) allow for regulated secretion of an effector molecule in a protease-dependent manner.
  • C protease cleavage site
  • MT cell membrane tethering domain
  • the components of the Membrane-Cleavable system present in the membrane-cleavable chimeric protein generally regulate secretion through the below cellular processes:
  • the cell membrane tethering domain contains a transmembrane domain (or a transmembrane-intracellular domain) that directs cellular-trafficking of the chimeric protein such that the protein is inserted into, or otherwise associated with, a cell membrane (“tethered”)
  • protease cleavage site directs cleavage of the chimeric protein such that the effector molecule is released (“secreted”) into the extracellular space.
  • the protease cleavage site is protease-specific, including sites engineered to be protease-specific.
  • the protease cleavage site can be selected or engineered to achieve optimal protein expression, cell-type specific cleavage, cell-state specific cleavage, and/or cleavage and release of the payload at desired kinetics (e.g., ratio of membrane-bound to secreted chimeric protein levels)
  • an effector molecule refers to a molecule (e.g., a nucleic acid such as DNA or RNA, or a protein (polypeptide) or peptide) that binds to another molecule and modulates the biological activity of that molecule to which it binds.
  • an effector molecule may act as a ligand to increase or decrease enzymatic activity, gene expression, and/or cell signaling.
  • an effector molecule modulates (activates or inhibits) different immunomodulatory mechanisms.
  • an effector molecule may also indirectly modulate a second, downstream molecule.
  • an effector molecule is or comprises an antibody, or functional fragment thereof.
  • an effector molecule is or comprises a cytokine.
  • an effector molecule comprises a cytokine selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, TNFa, TNFp, IFNa, IFNp, IFNy, G-CSF, GM-CSF, Erythropoietin, TGFp, and combinations and active (or functional)
  • an effector molecule is or comprises a chemokine.
  • an effector molecule comprises a chemokine selected from the group consisting of: CCL1, CCL2, CCL3, CCL3L1, CCL4, CCL4L1, CCR4L2, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, XCL1, XCL2, CXCL1, CXCL2, CXCL3, CXCL4, CXCL4L1, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL
  • an effector molecule is or comprises a cytokine or active fragment thereof (e.g., the secretable effector molecule referred to as “S” in the formula S - C - MT or MT - C - S).
  • modulate encompasses maintenance of a biological activity, inhibition (partial or complete) of a biological activity, and/or stimulation/activation (partial or complete) of a biological activity. In certain contexts, “modulate” may also encompass decreasing or increasing (e.g., enhancing) a biological activity. It is understood that disease-related immunosuppression may be facilitated by many immunosuppressive mechanisms including, but not limited to, attraction of immunosuppressive lymphocytic populations to particular cell environments (e.g., a tumor microenvironment), secretion of immunosuppressive effector molecules (e.g., cytokines) on particular cell types, expression of immunosuppressive cell surface markers on particular cell types, and so forth.
  • immunosuppressive mechanisms including, but not limited to, attraction of immunosuppressive lymphocytic populations to particular cell environments (e.g., a tumor microenvironment), secretion of immunosuppressive effector molecules (e.g., cytokines) on particular cell types, expression of immunosuppressive cell surface
  • Effector molecules as provided for herein may be used to modulate one or more disease-mediated immunosuppressive mechanisms in order to enhance localized or systemic immune response to treat a disease or disorder.
  • a disease-immunosuppressive mechanism that is modulated by one or more effector molecules provided herein is a tumor-mediated immunosuppressive mechanisms.
  • Two or more different effector molecules are considered to “modulate different tumor-mediated immunosuppressive mechanisms” when one effector molecule modulates a tumor-mediated immunosuppressive mechanism (e.g., stimulates T cell signaling) that is different from the tumor-mediated immunosuppressive mechanism modulated by the one or more other effector molecule (e.g., stimulates antigen presentation and/or processing).
  • a tumor-mediated immunosuppressive mechanism e.g., stimulates T cell signaling
  • the one or more other effector molecule e.g., stimulates antigen presentation and/or processing
  • Modulation by an effector molecule may be direct or indirect. Direct modulation occurs when an effector molecule binds to another molecule and modulates activity of that molecule. Indirect modulation occurs when an effector molecule binds to another molecule, modulates activity of that molecule, and as a result of that modulation, the activity of yet another molecule (to which the effector molecule is not bound) is modulated.
  • modulation of a tumor-mediated immunosuppressive mechanism by at least one effector molecule results in an increase in an immuno stimulatory and/or antitumor immune response (e.g., systemically or in the tumor microenvironment) by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%).
  • modulation of a tumor-mediated immunosuppressive mechanism may result in an increase in an immuno stimulatory and/or anti-tumor immune response by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%.
  • modulation of a tumor-mediated immunosuppressive mechanism results in an increase in an immuno stimulatory and/or anti-tumor immune response 10-20%, 10-30%, 10- 40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20-30%, 20-40%, 20- 50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20-200%, 50-60%, 50-70%, 50-80%, 50- 90%, 50-100%, or 50-200%.
  • an increase” in an immuno stimulatory and/or anti-tumor immune response is relative to the immunostimulatory and/or anti-tumor immune response that would otherwise occur, in the absence of the effector molecule(s).
  • modulation of a tumor-mediated immunosuppressive mechanism by at least one effector molecule results in an increase in an immuno stimulatory and/or antitumor immune response (e.g., systemically or in the tumor microenvironment) by at least 2 fold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100 fold).
  • modulation of a tumor-mediated immunosuppressive mechanism may result in an increase in an immuno stimulatory and/or antitumor immune response by at least 3 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, or at least 100 fold.
  • modulation of a tumor-mediated immunosuppressive mechanism results in an increase in an immuno stimulatory and/or antitumor immune response by 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, or 2-100 fold.
  • Non-limiting examples of immuno stimulatory and/or anti-tumor immune mechanisms include T cell signaling, activity and/or recruitment, antigen presentation and/or processing, natural killer cell-mediated cytotoxic signaling, activity and/or recruitment, dendritic cell differentiation and/or maturation, immune cell recruitment, pro-inflammatory macrophage signaling, activity and/or recruitment, stroma degradation, immuno stimulatory metabolite production, stimulator of interferon genes (STING) signaling (which increases the secretion of IFN and Thl polarization, promoting an anti-tumor immune response), and/or Type I interferon signaling.
  • An effector molecule may stimulate at least one (one or more) of the foregoing immuno stimulatory mechanisms, thus resulting in an increase in an immunostimulatory response.
  • Changes in the foregoing immuno stimulatory and/or anti-tumor immune mechanisms may be assessed, for example, using in vitro assays for T cell proliferation or cytotoxicity, in vitro antigen presentation assays, expression assays (e.g., of particular markers), and/or cell secretion assays (e.g., of cytokines).
  • in vitro assays for T cell proliferation or cytotoxicity in vitro antigen presentation assays
  • expression assays e.g., of particular markers
  • cell secretion assays e.g., of cytokines
  • modulation of a tumor-mediated immunosuppressive mechanism by at least one effector molecule results in a decrease in an immunosuppressive response (e.g., systemically or in the tumor microenvironment) by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%).
  • modulation of a tumor-mediated immunosuppressive mechanism may result in a decrease in an immunosuppressive response by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%.
  • modulation of a tumor-mediated immunosuppressive mechanism results in a decrease in an immunosuppressive response 10- 20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20- 30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20-200%, 50-60%, 50- 70%, 50-80%, 50-90%, 50-100%, or 50-200%.
  • a decrease” in an immunosuppressive response for example, systemically or in a tumor microenvironment, is relative to the immunosuppressive response that would otherwise occur, in the absence of the effector molecule(s).
  • modulation of a tumor-mediated immunosuppressive mechanism by at least one effector molecule results in a decrease in an immunosuppressive response (e.g., systemically or in the tumor microenvironment) by at least 2 fold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100 fold).
  • modulation of a tumor-mediated immunosuppressive mechanism may result in a decrease in an immunosuppressive response by at least 3 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, or at least 100 fold.
  • modulation of a tumor-mediated immunosuppressive mechanism results in a decrease in an immunosuppressive response by 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, or 2-100 fold.
  • Non-limiting examples of immunosuppressive mechanisms include negative costimulatory signaling, pro-apoptotic signaling of cytotoxic cells (e.g., T cells and/or NK cells), T regulatory (Treg) cell signaling, tumor checkpoint molecule production/maintenance, myeloid-derived suppressor cell signaling, activity and/or recruitment, immunosuppressive factor/metabolite production, and/or vascular endothelial growth factor signaling.
  • An effector molecule may inhibit at least one (one or more) of the foregoing immunosuppressive mechanisms, thus resulting in a decrease in an immunosuppressive response.
  • Changes in the foregoing immunosuppressive mechanisms may be assessed, for example, by assaying for an increase in T cell proliferation and/or an increase in IFNy production (negative co- stimulatory signaling, T re g cell signaling and/or MDSC); Annexin V/PI flow staining (pro-apoptotic signaling); flow staining for expression, e.g., PDL1 expression (tumor checkpoint molecule production/maintenance); ELISA, LUMINEX®, RNA via qPCR, enzymatic assays, e.g., IDO tryptophan catabolism (immunosuppressive factor/metabolite production); and phosphorylation of PI3K, Akt, p38 (VEGF signaling).
  • assaying for an increase in T cell proliferation and/or an increase in IFNy production negative co- stimulatory signaling, T re g cell signaling and/or MDSC
  • Annexin V/PI flow staining pro-apoptotic signal
  • effector molecules function additively: the effect of two effector molecules, for example, may be equal to the sum of the effect of the two effector molecules functioning separately. In other embodiments, effector molecules function synergistically: the effect of two effector molecules, for example, may be greater than the combined function of the two effector molecules.
  • Effector molecules that modulate tumor-mediated immunosuppressive mechanisms and/or modify tumor microenvironments may be selected from any known cytokine, e.g., cytokines described herein.
  • At least one of the effector molecules stimulates an immuno stimulatory mechanism in the tumor microenvironment and/or inhibits an immunosuppressive mechanism in the tumor microenvironment.
  • At least one of the effector molecules (a) stimulates T cell signaling, activity and/or recruitment, (b) stimulates antigen presentation and/or processing, (c) stimulates natural killer cell-mediated cytotoxic signaling, activity and/or recruitment, (d) stimulates dendritic cell differentiation and/or maturation, (e) stimulates immune cell recruitment, (f) stimulates pro-inflammatory macrophage signaling, activity and/or recruitment or inhibits anti-inflammatory macrophage signaling, activity and/or recruitment, (g) stimulates stroma degradation, (h) stimulates immuno stimulatory metabolite production, (i) stimulates Type I interferon signaling, (j) inhibits negative costimulatory signaling, (k) inhibits pro- apoptotic signaling of anti-tumor immune cells, (1) inhibits T regulatory (T re g) cell signaling, activity and/or recruitment, (m) inhibits tumor checkpoint molecules, (n) stimulates stimulator of interferon genes (STING) signaling, (o) inhibit
  • Non-limiting examples of cytokines are listed in Table 1 and specific sequences encoding exemplary effector molecules are listed in Table 2.
  • Effector molecules can be human, such as those listed in Table 1 or Table 2 or human equivalents of murine effector molecules listed in Table 1 or Table 2.
  • Effector molecules can be human-derived, such as the endogenous human effector molecule or an effector molecule modified and/or optimized for function, e.g., codon optimized to improve expression, modified to improve stability, or modified at its signal sequence (see below).
  • Various programs and algorithms for optimizing function are known to those skilled in the art and can be selected based on the improvement desired, such as codon optimization for a specific species (e.g., human, mouse, bacteria, etc.).
  • engineered nucleic acids comprising nucleotide sequences encoding one or more immunotherapy or immunomodulatory molecules, as described herein, e.g., CARs, effector molecules, and/or chimeric proteins.
  • one or more provided engineered nucleic acids e.g., a first, a second, a third, a fourth, etc. engineered nucleic acid
  • an engineered cell e.g., an immunoresponsive cell.
  • a first engineered nucleic acid can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 307-319, or 326- 329.
  • a first engineered nucleic acid can include a nucleotide sequence having the sequence shown in any one of SEQ ID NOs: 307-319, or 326-329.
  • the first engineered nucleic acid can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 309.
  • the first engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 309.
  • the first engineered nucleic acid can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 326.
  • the first engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 326.
  • the first engineered nucleic acid can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 310.
  • the first engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 310.
  • the first engineered nucleic acid can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 327.
  • the first engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 327.
  • the first engineered nucleic acid can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 314.
  • the first engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 314.
  • the first engineered nucleic acid can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 315.
  • the first engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 315.
  • a second engineered nucleic acid can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 307- 319, or 326-329.
  • a second engineered nucleic acid can include a nucleotide sequence having the sequence shown in any one of SEQ ID NOs: 307-319, or 326- 329.
  • the second engineered nucleic acid can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 317.
  • the second engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 317.
  • the second engineered nucleic acid can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 318.
  • the second engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 318.
  • the first engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 310; and the second engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 317.
  • the first engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 327; and the second engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 317.
  • the first engineered nucleic acid can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 310; and the second engineered nucleic acid can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 317.
  • the first engineered nucleic acid can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 327; and the second engineered nucleic acid can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 317.
  • the first engineered nucleic acid can include a nucleotide sequence having the sequence shown in any one of SEQ ID NOs: 307-319, or 326-329; and the second engineered nucleic acid can include a nucleotide sequence as shown in any one of SEQ ID NOs: 307-319, or 326-329.
  • the first engineered nucleic acid can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 307-319, or 326-329; and the second engineered nucleic acid can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 307-319, or 326-329.
  • an engineered cell is an immunoresponsive cell that is capable of expressing a chimeric antigen receptor (CAR) and/or an effector molecule (e.g., a cytokine or functional fragment thereof) from one or more engineered nucleic acids (e.g., a first, a second, a third, a fourth, a fifth, etc. engineered nucleic acid), such as those described herein.
  • CAR chimeric antigen receptor
  • an effector molecule e.g., a cytokine or functional fragment thereof
  • engineered nucleic acids e.g., a first, a second, a third, a fourth, a fifth, etc. engineered nucleic acid
  • the present disclosure provides a mixed cell population or mixed cell composition
  • a mixed cell population or mixed cell composition comprising: a first engineered cell (e.g., a first immunoresponsive cell) comprising one or more engineered nucleic acids, such as any of those described herein, and a second engineered cell (e.g., a second immunoresponsive cell) comprising one or more engineered nucleic acids, such as any of those described herein, that are different than those present in the first engineered cell.
  • a mixed cell composition may comprise a first engineered cell comprising a first engineered nucleic acid capable of expressing a CAR, and a second engineered cell comprising a second engineered nucleic acid capable of expressing an effector molecule (e.g., a cytokine).
  • a mixed cell composition may comprise a first engineered cell comprising a first engineered nucleic acid capable of expressing a CAR and a first effector molecule, and a second engineered cell comprising a second engineered nucleic acid capable of expressing a second effector molecule.
  • a mixed cell composition may comprise one or more, two or more, three or more, four or more, six or more, seven or more, or eight or more different engineered cells, where each engineered cell comprises one or more engineered nucleic acids that are different relative to other engineered cells within the mixed cell composition.
  • Provided engineered cells, such as immunoresponsive cells may be administered as cell therapy in the treatment of a disease or disorder.
  • a disease or disorder treated by a provided engineered cell therapy is cancer (e.g., hepatocellular carcinoma (HCC), ovarian clear cell carcinoma, melanoma, squamous cell carcinoma of the lung, hepatoblastoma, nephroblastoma (Wilms tumor), and yolk sac tumor).
  • cancer e.g., hepatocellular carcinoma (HCC), ovarian clear cell carcinoma, melanoma, squamous cell carcinoma of the lung, hepatoblastoma, nephroblastoma (Wilms tumor), and yolk sac tumor).
  • Immunoresponsive cells provided for herein can include any one of the engineered nucleic acids described herein. Immunoresponsive cells provided for herein can include combinations of any one of the engineered nucleic acids described herein. Immunoresponsive cells provided for herein can include two or more of any one of the engineered nucleic acids described herein.
  • Immunoresponsive cells provided for herein can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 307-319, or 326-329.
  • Immunoresponsive cells provided for herein can include a nucleotide sequence having the sequence shown in any one of SEQ ID NOs: 307-319, or 326-329.
  • Immunoresponsive cells provided for herein can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 309.
  • Immunoresponsive cells provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 309.
  • Immunoresponsive cells provided for herein can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 326.
  • Immunoresponsive cells provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 326.
  • Immunoresponsive cells provided for herein can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 310.
  • Immunoresponsive cells provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 310.
  • Immunoresponsive cells provided for herein can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 327.
  • Immunoresponsive cells provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 327.
  • Immunoresponsive cells provided for herein can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 314.
  • Immunoresponsive cells provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 314.
  • Immunoresponsive cells provided for herein can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 315.
  • Immunoresponsive cells provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 315.
  • Immunoresponsive cells provided for herein can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 317.
  • Immunoresponsive cells provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 317.
  • Immunoresponsive cells provided for herein can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 318.
  • Immunoresponsive cells provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 318.
  • Immunoresponsive cells can include a first engineered nucleic acid including a nucleotide sequence having the sequence shown in SEQ ID NO: 310; and a second engineered nucleic acid including a nucleotide sequence having the sequence shown in SEQ ID NO: 317.
  • Immunoresponsive cells can include a first engineered nucleic acid including a nucleotide sequence having the sequence shown in SEQ ID NO: 327; and a second engineered nucleic acid including a nucleotide sequence having the sequence shown in SEQ ID NO: 317.
  • Immunoresponsive cells can include a first engineered nucleic acid including a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 310; and a second engineered nucleic acid including a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 317.
  • Immunoresponsive cells can include a first engineered nucleic acid including a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 327; and a second engineered nucleic acid including a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 317.
  • Immunoresponsive cells can include a first engineered nucleic acid including a nucleotide sequence having the sequence shown in any one of SEQ ID NOs: 307- 319, or 326-329; and a second engineered nucleic acid can include a nucleotide sequence as shown in any one of SEQ ID NOs: 307-319, or 326-329.
  • Immunoresponsive cells can include a first engineered nucleic acid including a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 307-319, or 326-329; and a second engineered nucleic acid including a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 307-319, or 326-329.
  • Expression vectors provided for herein can include any one of the engineered nucleic acids described herein. Expression vectors provided for herein can include combinations of any one of the engineered nucleic acids described herein. Expression vectors provided for herein can include two or more of any one of the engineered nucleic acids described herein.
  • Expression vectors provided for herein can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 309.
  • Expression vectors provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 309.
  • Expression vectors provided for herein can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 326.
  • Expression vectors provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 326.
  • Expression vectors provided for herein can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 310.
  • Expression vectors provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 310.
  • Expression vectors provided for herein can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 327.
  • Expression vectors provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 327.
  • Expression vectors provided for herein can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 314.
  • Expression vectors provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 314.
  • Expression vectors provided for herein can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 315.
  • Expression vectors provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 315.
  • Expression vectors provided for herein can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 317.
  • Expression vectors provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 317.
  • Expression vectors provided for herein can include a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 318.
  • Expression vectors provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 318.
  • Expression vectors provided for herein can include a first engineered nucleic acid including a nucleotide sequence having the sequence shown in SEQ ID NO: 310; and a second engineered nucleic acid including a nucleotide sequence having the sequence shown in SEQ ID NO: 317.
  • Expression vectors provided for herein can include a first engineered nucleic acid including a nucleotide sequence having the sequence shown in SEQ ID NO: 327; and a second engineered nucleic acid including a nucleotide sequence having the sequence shown in SEQ ID NO: 317.
  • Expression vectors provided for herein can include a first engineered nucleic acid including a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 310; and a second engineered nucleic acid including a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 317.
  • Expression vectors provided for herein can include a first engineered nucleic acid including a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 327; and a second engineered nucleic acid including a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 317.
  • one or more effector molecules of the membrane-cleavable chimeric proteins provided for herein are secretable effector molecules having a secretion signal peptide (also referred to as a signal peptide or signal sequence) at the chimeric protein’s N-terminus (e.g., an effector molecule’s N- terminus for S - C - MT) that direct newly synthesized proteins destined for secretion or membrane localization (also referred to as membrane insertion) to the proper protein processing pathways.
  • a secretion signal peptide also referred to as a signal peptide or signal sequence
  • a membrane tethering domain may comprise a signal-anchor sequence (e.g., signal-anchor sequences of a Type II transmembrane protein) that direct newly synthesized proteins destined for membrane localization to the proper protein processing pathways.
  • a membrane tethering domain having a reverse signal-anchor sequence e.g., signal-anchor sequences of certain Type III transmembrane proteins
  • the one or more effector molecules are secretable effector molecules (referred to as “S” in the formula S - C - MT or MT - C - S).
  • a chimeric protein comprises a secretion signal.
  • each chimeric protein can comprise a secretion signal.
  • each chimeric protein can comprise a secretion signal such that each effector molecule is capable of secretion from an engineered cell following cleavage of the protease cleavage site.
  • the secretion signal peptide operably associated with an effector molecule can be a native secretion signal peptide (e.g., the secretion signal peptide generally endogenously associated with the given effector molecule, such as a cytokine’s endogenous secretion signal peptide).
  • the secretion signal peptide operably associated with an effector molecule can be a non-native secretion signal peptide native secretion signal peptide.
  • Non-native secretion signal peptides can promote improved expression and function, such as maintained secretion, in particular environments, such as tumor microenvironments.
  • Non-limiting examples of non- native secretion signal peptide are shown in Table 3.
  • membrane-cleavable chimeric proteins described herein contain a protease cleavage site (referred to as “C” in the formula S - C - MT or MT - C - S).
  • a protease cleavage site can be any amino acid sequence motif capable of being cleaved by a protease.
  • protease cleavage sites include, but are not limited to, a Type 1 transmembrane protease cleavage site, a Type II transmembrane protease cleavage site, a GPI anchored protease cleavage site, an ADAM8 protease cleavage site, an ADAM9 protease cleavage site, an ADAM10 protease cleavage site, an ADAM12 protease cleavage site, an ADAM15 protease cleavage site, an ADAM 17 protease cleavage site, an ADAM 19 protease cleavage site, an ADAM20 protease cleavage site, an ADAM21 protease cleavage site, an ADAM28 protease cleavage site, an ADAM30 protease cleavage site, an ADAM33 protease cleavage site, a BACE1 protease cleavage site,
  • protease cleavage site is a hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease cleavage site, including, but not limited to, a NS3/NS4A, a NS4A/NS4B, a NS4B/NS5A, or a NS5A/NS5B cleavage site.
  • HCV hepatitis C virus
  • NS3 protease and representative sequences of its cleavage sites for various strains of HCV see, e.g., Hepatitis C Viruses: Genomes and Molecular Biology (S.L. Tan ed., Taylor & Francis, 2006), Chapter 6, pp.
  • HCV NS4A/4B protease cleavage site HCV NS5A/5B protease cleavage site
  • C-terminal degron with NS4A/4B protease cleavage site N-terminal degron with HCV NS5A/5B protease cleavage site
  • Representative NS 3 sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos.
  • a protease cleavage site is an ADAM 17- specific protease (also referred to as Tumor Necrosis Factor-a Converting Enzyme [TACE]) cleavage site.
  • An ADAM 17- specific protease cleavage site can be an endogenous sequence of a substrate naturally cleaved by ADAM17.
  • An ADAM 17- specific protease cleavage site can be an engineered sequence capable of being cleaved by ADAM17.
  • An engineered ADAM 17- specific protease cleavage site can be engineered for specific desired properties including, but not limited to, optimal expression of chimeric proteins (e.g., those described herein), specificity for ADAM17, rate-of-cleavage by ADAM17, ratio of secreted and membrane-bound chimeric protein levels, and cleavage in different cell states.
  • a protease cleavage site can be selected for specific cleavage by ADAM17.
  • certain protease cleavage sites capable of being cleaved by ADAM 17 are also capable of cleavage by additional ADAM family proteases, such as ADAM10.
  • an ADAM17-specific protease cleavage site can be selected and/or engineered such that cleavage by other proteases, such as ADAM 10, is reduced or eliminated.
  • a protease cleavage site can be selected for rate-of-cleavage by ADAM 17.
  • it can be desirable to select a protease cleavage site demonstrating a specific rate-of-cleavage by ADAM17, such as reduced cleavage kinetics relative to an endogenous sequence of a substrate naturally cleaved by ADAM17.
  • a specific rate-of-cleavage can be selected to regulate the rate of processing of the chimeric protein, which in turn regulates the rate of release/secretion of the payload effector molecule.
  • an ADAM17-specific protease cleavage site can be selected and/or engineered such that the sequence demonstrates a desired rate-of-cleavage by ADAM 17.
  • a protease cleavage site can be selected for both specific cleavage by ADAM 17 and rate-of-cleavage by ADAM 17.
  • ADAM 17-specific protease cleavage sites including those demonstrating particular specificity and rate-of-cleavage kinetics, are shown in Table 4A below with reference to the site of cleavage (P5-P1: N-terminal; Pl'-P5': C-terminal). Further details of ADAM17 and ADAM10, including expression and protease cleavage sites, are described in Sharma, et al. (J Immunol October 15, 2017, 199 (8) 2865-2872), Pham et al. (Anticancer Res. 2017 Oct;37(10):5507-5513), Caescu et al. (Biochem J. 2009 Oct 23; 424(1): 79-88), and Tucher et al. (J. Proteome Res. 2014, 13, 4, 2205-2214), each herein incorporated by reference for purposes.
  • the protease cleavage site comprises a first region having the amino acid sequence of PRAE (SEQ ID NO: 176). In some embodiments, the protease cleavage site comprises a second region having the amino acid sequence of KGG (SEQ ID NO: 177). In some embodiments, the first region is located N-terminal to the second region. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEX1X2KGG (SEQ ID NO: 178), wherein Xi is A, Y, P, S, or F, and wherein Xiis V, L, S, I, Y, T, or A.
  • the protease cleavage site comprises the amino acid sequence of PRAEAVKGG (SEQ ID NO: 179). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEALKGG (SEQ ID NO: 180). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEYSKGG (SEQ ID NO: 181). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEPIKGG (SEQ ID NO: 182). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEAYKGG (SEQ ID NO: 183).
  • the protease cleavage site comprises the amino acid sequence of PRAESSKGG (SEQ ID NO: 184). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEFTKGG (SEQ ID NO: 185). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEAAKGG (SEQ ID NO: 186). In some embodiments, the protease cleavage site comprises the amino acid sequence of DEPHYSQRR (SEQ ID NO: 187). In some embodiments, the protease cleavage site comprises the amino acid sequence of PPLGPIFNPG (SEQ ID NO: 188).
  • the protease cleavage site comprises the amino acid sequence of PLAQAYRSS (SEQ ID NO: 189). In some embodiments, the protease cleavage site comprises the amino acid sequence of TPIDSSFNPD (SEQ ID NO: 190). In some embodiments, the protease cleavage site comprises the amino acid sequence of VTPEPIFSLI (SEQ ID NO: 191).
  • a cleavage site comprises a linker sequence.
  • a cleavage site may be flanked on the N terminal and/or C terminal sides by a linker sequence.
  • the cleavage site may be flanked on both the N terminal and C terminal sides by a partial glycine- serine (GS) linker sequence.
  • GS partial glycine- serine
  • the cleavage site and linker comprise the amino acid sequence of SGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQ (SEQ ID NO: 287).
  • An exemplary nucleic acid sequence encoding SEQ ID NO: 287 is TCTGGCGGCGGAGGATCTGGCGGAGGTGGAAGCGGAGTTACACCCGAGCCTATCTT CAGCCTGATCGGAGGCGGTAGCGGAGGCGGAGGAAGTGGTGGCGGATCTCTGCAA (SEQ ID NO: 288).
  • nucleic acids encoding SEQ ID NO: 287 may comprise SEQ ID NO: 288, or a nucleic acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 288.
  • the protease cleavage site is N-terminal to a linker.
  • the protease cleavage site and linker comprise the amino acid sequence of PRAEALKGGSGGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 289).
  • An exemplary nucleic acid sequence encoding SEQ ID NO: 289 is CCCAGAGCCGAGGCTCTGAAAGGCGGATCAGGCGGCGGTGGTAGTGGAGGCGGAG GCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAAT (SEQ ID NO: 292).
  • nucleic acids encoding SEQ ID NO: 289 may comprise SEQ ID NO: 292, or a nucleic acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 292.
  • the protease cleavage site comprises the amino acid sequence of ITQGLAVSTISSFF (SEQ ID NO: 198), which is a cleavage site that is native to CD16 and is cleavable by ADAM 17.
  • SEQ ID NO: 198 is comprised within a linker.
  • the linker comprises the amino acid sequence of SGGGGSGGGGSGITQGLAVSTISSFFGGGSGGGGSGGGSLQ (SEQ ID NO: 290).
  • An exemplary nucleic acid sequence encoding SEQ ID NO: 290 is AGCGGCGGAGGTGGTAGCGGAGGCGGAGGATCTGGAATTACACAGGGACTCGCCG TGTCTACAATCTCCAGCTTCTTTGGTGGCGGTAGTGGCGGCGGTGGCAGTGGCGGTG GATCTCTTCAA (SEQ ID NO: 291).
  • nucleic acids encoding SEQ ID NO: 290 may comprise SEQ ID NO: 291, or a nucleic acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 291.
  • the protease cleavage site can be C-terminal of the secretable effector molecule.
  • the protease cleavage site can be N-terminal of the secretable effector molecule.
  • membrane-cleavable chimeric proteins described herein comprise a protease cleavage site that is either: (1) C-terminal of the secretable effector molecule and N-terminal of the cell membrane tethering domain (in other words, the protease cleavage site is in between the secretable effector molecule and the cell membrane tethering domain); or (2) N-terminal of the secretable effector molecule and C-terminal of the cell membrane tethering domain (also between the secretable effector molecule and the cell membrane tethering domain with domain orientation inverted).
  • the protease cleavage site can be connected to the secretable effector molecule by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the effector molecule or protease cleavage site.
  • the protease cleavage site can be connected to the cell membrane tethering domain by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the cell membrane tethering domain or protease cleavage site.
  • a polypeptide linker can be any amino acid sequence that connects a first polypeptide sequence and a second polypeptide sequence.
  • a polypeptide linker can be a flexible linker (e.g., a Gly-Ser-Gly sequence).
  • polypeptide linkers include, but are not limited to, GSG linkers (e.g., [GS] 4 GG [SEQ ID NO: 347]), A(EAAAK) 3 A (SEQ ID NO: 348), and Whitlow linkers (e.g.
  • a “KEGS” linker such as the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 349), an eGK linker such as the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 350), an LR1 linker such as the amino acid sequence SGGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 215), and linkers described in more detail in Issued U.S. Pat. No. 5,990,275 herein incorporated by reference).
  • Additional exemplary polypeptide linkers include SGGGGSGGGGSG (SEQ ID NO: 194), TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 196), and GGGSGGGGSGGGSLQ (SEQ ID NO: 197).
  • Other polypeptide linkers may be selected based on desired properties (e.g., length, flexibility, amino acid composition, etc.) and are known to those skilled in the art.
  • nucleic acid sequence encoding SEQ ID NO: 196 is ACCACCACACCAGCTCCTCGGCCACCAACTCCAGCTCCAACAATTGCCAGCCAGCC TCTGTCTCTGAGGCCCGAAGCTTGTAGACCTGCTGCAGGCGGAGCCGTGCATACAA GAGGACTGGATTTCGCCTGCGAC (SEQ ID NO: 337).
  • a nucleic acid encoding SEQ ID NO: 196 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 337.
  • the protease cleavage site directs cleavage of the chimeric protein such that the effector molecule is released (“secreted”) into the extracellular space of a cell.
  • a protease that cleaves the protease cleavage site is a protease specific for that specific protease cleavage site.
  • the protease that cleaves a specific ADAM protease cleavage site is generally limited to the ADAM protease(s) that specifically recognize the specific ADAM protease cleavage site motif.
  • a protease cleavage site can be selected and/or engineered such that cleavage by undesired proteases is reduced or eliminated.
  • Proteases can be membrane-bound or membrane-associated.
  • Proteases can be secreted, e.g., secreted in a specific cellular environment, such as a tumor microenvironment (“TME”).
  • TEE tumor microenvironment
  • a protease that cleaves the protease cleavage site of the chimeric protein can be expressed in the same cell that expresses the chimeric protein.
  • a protease that cleaves the protease cleavage site of the chimeric protein can be endogenous to a cell expressing the chimeric protein.
  • a cell engineered to express the chimeric protein can endogenously express the protease specific for the protease cleavage site present in the chimeric protein.
  • Endogenous expression of the protease refers to both expression under generally homeostatic conditions (e.g., a cell generally considered to be healthy), and also to differential expression under non-homeostatic conditions (e.g., upregulated expression in a tumor cell).
  • the protease cleavage site can be selected based on the known proteases endogenously expressed by a desired cell population. In such cases, in general, the cleavage of the protease cleavage site (and thus release/secretion of a payload) can be restricted to only those cells of interest due to the cell-restricted protease needing to come in contact with the protease cleavage site of chimeric protein expressed in the same cell.
  • ADAM 17 is believed to be restricted in its endogenous expression to NK cell and T cells.
  • selection of an ADAM17-specific protease cleavage site may restrict the cleavage of the protease cleavage site to NK cell and T cells co-expressing the chimeric protein.
  • a protease cleavage site can be selected for a specific tumor- associated protease known to be expressed in a particular tumor population of interest (e.g., in a specific tumor cell engineered to express the chimeric protein).
  • Protease and/or expression databases can be used to select an appropriate protease cleavage site, such as selecting a protease cleavage site cleaved by a tumor-associated proteases through consulting Oncomine (www.oncomine.org), the European Bioinformatic Institute (www.ebi.ac.uk) in particular (www.ebi.ac.uk/gxa), PMAP (www.proteolysis.org), ExPASy Peptide Cutter (ca.expasy.org/tools/peptide cutter) and PMAP.Cut DB (cutdb.burnham.org), each of which is incorporated by reference for all purposes.
  • Oncomine www.oncomine.org
  • the European Bioinformatic Institute www.ebi.ac.uk
  • PMAP www.proteolysis.org
  • ExPASy Peptide Cutter ca.expasy.org/tools/peptide cutter
  • PMAP.Cut DB cutdb.burnham
  • a protease that cleaves the protease cleavage site of the chimeric protein can be heterologous to a cell expressing the chimeric protein.
  • a cell engineered to express the chimeric protein can also be engineered to express a protease not generally expressed by the cell that is specific for the protease cleavage site present in the chimeric protein.
  • a cell engineered to express both the chimeric protein and the protease can be engineered to express each from separate engineered nucleic acids or from a multicistronic systems (multicistronic and multi-promoter systems are described in greater detail in the Section herein titled “Multicistronic and Multiple Promoter Systems”).
  • Heterologous proteases and their corresponding protease cleavage site can be selected as described above with reference to endogenous proteases.
  • a protease that cleaves the protease cleavage site of the chimeric protein can be expressed on a separate distinct cell than the cell that expresses the chimeric protein.
  • the protease can be generally expressed in a specific cellular environment, such as a tumor microenvironment.
  • the cleavage of the protease cleavage site can be restricted to only those cellular environments of interest (e.g., a tumor microenvironment) due to the environment-restricted protease needing to come in contact with the protease cleavage site.
  • the secretion of the effector molecule can be restricted to only those cellular environments of interest (e.g., a tumor microenvironment) due to the environment-restricted protease needing to come in contact with the protease cleavage site.
  • a protease that cleaves the protease cleavage site of the chimeric protein can be endogenous to the separate distinct cell.
  • a protease that cleaves the protease cleavage site of the chimeric protein can be heterologous to the separate distinct cell.
  • the separate distinct cell can be engineered to express a protease not generally expressed by the separate distinct cell.
  • Proteases include, but are not limited to, a Type 1 transmembrane protease, a Type II transmembrane protease, a GPI anchored protease, an ADAM8 protease, an ADAM9 protease, an ADAM 10 protease, an ADAM 12 protease, an ADAM 15 protease, an ADAM 17 protease, an ADAM19 protease, an ADAM20 protease, an ADAM21 protease, an ADAM28 protease, an ADAM30 protease, an ADAM33 protease, a BACE1 protease, a BACE2 protease, a SIP protease, an MT1-MMP protease, an MT3-MMP protease, an MT5-MMP protease, a furin protease, a PCSK7 protease, a matriptase proteas
  • Proteases can be tumor associated proteases, such as, a cathepsin, a cysteine protease, an aspartyl protease, a serine protease, or a metalloprotease.
  • tumor associated proteases include Cathepsin B, Cathepsin L, Cathepsin S, Cathepsin D, Cathepsin E, Cathepsin A, Cathepsin G, Thrombin, Plasmin, Urokinase, Tissue Plasminogen Activator, Metalloproteinase 1 (MMP1), MMP2, MMP3, MMP4, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP20, MMP21, MMP23, MMP24, MMP25, MMP26, MMP28, ADAM, AD AMTS, CD10 (CALLA), or prostate specific antigen.
  • a protease can be any of the following human proteases (MEROPS peptidase database number provided in parentheses; Rawlings N. D., Morton F. R., Kok, C. Y., Kong, J. & Barrett A. J. (2008) MEROPS: the peptidase database. Nucleic Acids Res. 36 Database issue, D320- 325; herein incorporated by reference for all purposes): pepsin A (MER000885), gastricsin
  • MER047076 subfamily A2A non-peptidase homologues
  • MER047080 subfamily A2A non-peptidase homologues
  • MER047088 subfamily A2A non-peptidase homologues
  • MER047107 subfamily A2A non-peptidase homologues
  • MER047108 subfamily A2A non-peptidase homologues
  • MER047109 subfamily A2A non-peptidase homologues
  • MER047110 subfamily A2A non-peptidase homologues MER047111
  • MER047114 subfamily A2A non-peptidase homologues
  • MER047140 subfamily A2A non-peptidase homologues (MER047141), subfamily A2A non- peptidase homologues (MER047142), subfamily A2A non-peptidase homologues
  • MER047157 subfamily A2A non-peptidase homologues
  • MER047159 subfamily A2A non-peptidase homologues
  • MER047161 subfamily A2A non-peptidase homologues
  • MER047163 subfamily A2A non-peptidase homologues
  • MER047166 subfamily A2A non-peptidase homologues
  • MER047173 subfamily A2A non-peptidase homologues
  • MER047174 subfamily A2A nonpeptidase homologues
  • MER047179 subfamily A2A non-peptidase homologues
  • MER047203 subfamily A2A non-peptidase homologues
  • MER047205 subfamily A2A non-peptidase homologues
  • MER047207 subfamily A2A non-peptidase homologues
  • MER047208 subfamily A2A non-peptidase homologues
  • MER047210 subfamily A2A non-peptidase homologues
  • MER047211 subfamily A2A non-peptidase homologues
  • MER047212 subfamily A2A non-peptidase homologues
  • MER047213 subfamily A2A non-peptidase homologues
  • MER047215 subfamily A2A non-peptidase homologues
  • MER047216 subfamily A2A non-peptidase homologues
  • MER047218 subfamily A2A non-peptidase homologues
  • MER047225 subfamily A2A non-peptidase homologues
  • MER047226 subfamily A2A non-peptidase homologues
  • MER047227 subfamily A2A non-peptidase homologues
  • MER047230 subfamily A2A non-peptidase homologues
  • MER047232 subfamily A2A non-peptidase homologues
  • MER047233 subfamily A2A non-peptidase homologues
  • MER047234 subfamily A2A non-peptidase homologues
  • MER047236 subfamily A2A non-peptidase homologues
  • MER047238 subfamily A2A non-peptidase homologues
  • MER047263 subfamily A2A non-peptidase homologues
  • MER047265 subfamily A2A non-peptidase homologues
  • MER047266 subfamily A2A non-peptidase homologues
  • MER047267 subfamily A2A non-peptidase homologues
  • MER047268 subfamily A2A non-peptidase homologues
  • MER047269 subfamily A2A non-peptidase homologues
  • MER047272 subfamily A2A non-peptidase homologues
  • MER047273 subfamily A2A non-peptidase homologues
  • MER047274 subfamily A2A non-peptidase homologues
  • MER047275 subfamily A2A non-peptidase homologues
  • MER047276 subfamily A2A non-peptidase homologues
  • MER047279 subfamily A2A non-peptidase homologues
  • MER047290 subfamily A2A non-peptidase homologues
  • MER047294 subfamily A2A non-peptidase homologues
  • MER047295 subfamily A2A non-peptidase homologues
  • MER047304 subfamily A2A non-peptidase homologues
  • MER047305 subfamily A2A non-peptidase homologues
  • MER047306 subfamily A2A non-peptidase homologues
  • MER047307 subfamily A2A non-peptidase homologues
  • MER047311 subfamily A2A non-peptidase homologues
  • MER047321 subfamily A2A non-peptidase homologues
  • MER047322 subfamily A2A non-peptidase homologues
  • MER047326 subfamily A2A non-peptidase homologues
  • MER047362 subfamily A2A non-peptidase homologues
  • MER047366 subfamily A2A non-peptidase homologues
  • MER047369 subfamily A2A non-peptidase homologues
  • MER047384 subfamily A2A non-peptidase homologues
  • MER047385 subfamily A2A non-peptidase homologues
  • MER047388 subfamily A2A non-peptidase homologues
  • MER047414 subfamily A2A non-peptidase homologues
  • MER047416 subfamily A2A non-peptidase homologues
  • MER047417 subfamily A2A non-peptidase homologues
  • MER047420 subfamily A2A non-peptidase homologues
  • MER047423 subfamily A2A non-peptidase homologues
  • MER047424 subfamily A2A non-peptidase homologues
  • MER047428 subfamily A2A non-peptidase homologues
  • MER047429 subfamily A2A non-peptidase homologues
  • MER047431 subfamily A2A non-peptidase homologues
  • MER047434 subfamily A2A non-peptidase homologues
  • MER047439 subfamily A2A non-peptidase homologues
  • MER047442 subfamily A2A non-peptidase homologues
  • MER047452 subfamily A2A non-peptidase homologues
  • MER047455 subfamily A2A non-peptidase homologues
  • MER047457 subfamily A2A non-peptidase homologues
  • MER047458 subfamily A2A non-peptidase homologues
  • MER047459 subfamily A2A non-peptidase homologues
  • MER047463 subfamily A2A non-peptidase homologues
  • MER047468 subfamily A2A non-peptidase homologues
  • MER047469 subfamily A2A non-peptidase homologues
  • MER047470 subfamily A2A non-peptidase homologues
  • MER047476 subfamily A2A non-peptidase homologues
  • MER047478 subfamily A2A non-peptidase homologues
  • MER047483 subfamily A2A non-peptidase homologues
  • MER047488 subfamily A2A non-peptidase homologues
  • MER047489 subfamily A2A non-peptidase homologues
  • MER047490 subfamily A2A non-peptidase homologues
  • MER047502 subfamily A2A non-peptidase homologues
  • MER047504 subfamily A2A non-peptidase homologues
  • MER047511 subfamily A2A non-peptidase homologues
  • MER047516 subfamily A2A non-peptidase homologues
  • MER047520 subfamily A2A non-peptidase homologues
  • MER047533 subfamily A2A non-peptidase homologues
  • taspase-1 (MER016969), gamma-glutamyltransferase 5 (mammalian-type) (MER001977), gamma-glutamyltransferase 1 (mammalian-type) (MER001629), gamma-glutamyltransferase 2 (Homo sapiens) (MER001976), gamma-glutamyltransferase-like protein 4 (MER002721).
  • gamma- glutamyltransferase-like protein 3 (MER016970). similar to gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026204).
  • EGF-like module containing mucin-like hormone receptor-like 2 (MER037230). CD97 antigen (human type) (MER037286). EGF-like module containing mucin-like hormone receptor-like 3 (MER037288). EGF-like module containing mucin-like hormone receptor-like 1 (MER037278). EGF-like module containing mucin-like hormone receptor-like 4 (MER037294).
  • EGF LAG seven-pass G-type receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (Mus musculus)-type protein (MER123205).
  • GPR56 Homo sapiens)-type protein (MER122057).
  • latrophilin 2 (MER122199).
  • latrophilin- 1 (MER126380).
  • latrophilin 3 (MER124612).
  • protocadherin Flamingo 2 (MER124239).
  • ETL protein (MER126267).
  • G protein-coupled receptor 112 (MER126114). seven transmembrane helix receptor (MER125448).
  • Gprl 14 protein (MER159320).
  • GPR126 vascular inducible G protein-coupled receptor (MER140015).
  • GPR125 Homo sapiens-type protein (MER159279).
  • GPR116 Homo sapiens-type G-protein coupled receptor (MER159280).
  • GPR128 Homo sapiens-type G-protein coupled receptor (MER162015).
  • GPR133 Homo sapiens-type protein (MER159334) GPR110 G-protein coupled receptor (MER159277), GPR97 protein (MER159322), KPG_006 protein (MER161773) KPG_008 protein (MER161835), KPG_009 protein (MER159335), unassigned homologue (MER166269), GPR113 protein (MER159352), brain-specific angiogenesis inhibitor 2 (MER159746), PIDD auto-processing protein unit 1 (MER020001), PIDD auto-processing protein unit 2 (MER063690), MUC1 self-cleaving mucin (MER074260), dystroglycan (MER054741), proprotein convertase 9 (MER022416), site-1 peptidase (MER001948), furin (MER000375), proprotein convertase 1 (MER000376), proprotein convertase 2 (MER000377), proprotein convertase 4 (MER028255), PACE4 proprotein convertase (
  • proteases can be inactivated by the presence or absence of a specific agent (e.g., that binds to the protease, such as specific small molecule inhibitors). Such proteases can be referred to as a “repressible protease.” Exemplary inhibitors for certain proteases are listed in Table 4B.
  • an NS3 protease can be repressed by a protease inhibitor including, but not limited to, simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir.
  • a protease inhibitor including, but not limited to, simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir.
  • protease activity can be regulated through regulating expression of the protease itself, such as engineering a cell to express a protease using an inducible promoter system (e.g., Tet On/Off systems) or cell-specific promoters (promoters that can be used to express a heterologous protease are described in more detail in the Section herein titled “Promoters”).
  • a protease can also contain a degron, such as any of the degrons described herein, and can be regulated using any of the degron systems described herein.
  • Protease enzymatic activity can also be regulated through selection of a specific protease cleavage site.
  • a protease cleavage site can be selected and/or engineered such that the sequence demonstrates a desired rate-of-cleavage by a desired protease, such as reduced cleavage kinetics relative to an endogenous sequence of a substrate naturally cleaved by the desired protease.
  • a protease cleavage site can be selected and/or engineered such that the sequence demonstrates a desired rate-of-cleavage in a cell-state specific manner.
  • ADAM17 protein levels and localization is known to be influenced by signaling, such as through Protein kinase C (PKC) signaling pathways (e.g., activation by the PKC activator Phorbol-12-myristat-13-acetat [PMA]).
  • PKC Protein kinase C
  • a protease cleavage site can be selected and/or engineered such that cleavage of the protease cleavage site and subsequent release of an effector molecule is increased or decreased, as desired, depending on the protease properties (e.g., expression and/or localization) in a specific cell state.
  • a protease cleavage site (particularly in combination with a specific membrane tethering domain) can be selected and/or engineered for optimal protein expression of the chimeric protein.
  • the membrane-cleavable chimeric proteins provided for herein include a cell-membrane tethering domain (referred to as “MT” in the formula S - C - MT or MT - C - S).
  • the cell-membrane tethering domain can be any amino acid sequence motif capable of directing the chimeric protein to be localized to (e.g., inserted into), or otherwise associated with, the cell membrane of the cell expressing the chimeric protein.
  • the cell-membrane tethering domain can be a transmembrane-intracellular domain.
  • the cell-membrane tethering domain can be a transmembrane domain.
  • the cell-membrane tethering domain can be an integral membrane protein domain (e.g., a transmembrane domain).
  • the cell-membrane tethering domain can be derived from a Type I, Type II, or Type III transmembrane protein.
  • the cell-membrane tethering domain can include post-translational modification tag, or motif capable of post-translational modification to modify the chimeric protein to include a post-translational modification tag, where the post-translational modification tag allows association with a cell membrane.
  • post-translational modification tags include, but are not limited to, lipid-anchor domains (e.g., a GPI lipid-anchor, a myristoylation tag, or palmitoylation tag).
  • cellmembrane tethering domains include, but are not limited to, a transmembrane-intracellular domain and/or transmembrane domain derived from PDGFR-beta, CD8, CD28, CD3zeta-chain, CD4, 4-1BB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, or BTLA.
  • the cell membrane tethering domain can be a cell surface receptor or a cell membrane-bound portion thereof. Sequences of exemplary cell membrane tethering domains are provided in Table 4C.
  • membrane-cleavable chimeric proteins described herein comprise a cell membrane tethering domain that is either: (1) C-terminal of the protease cleavage site and N-terminal of any intracellular domain, if present (in other words, the cell membrane tethering domain is in between the protease cleavage site and, if present, an intracellular domain); or (2) N-terminal of the protease cleavage site and C-terminal of any intracellular domain, if present (also between the protease cleavage site and, if present, an intracellular domain with domain orientation inverted).
  • the degron domain is the terminal cytoplasmic-oriented domain, specifically relative to the cell membrane tethering (in other words, the cell membrane tethering domain is in between the protease cleavage site and the degron).
  • the cell membrane tethering domain can be connected to the protease cleavage site by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of cell membrane tethering domain or protease cleavage site.
  • the cell membrane tethering domain can be connected to an intracellular domain, if present, by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the cell membrane tethering domain or the intracellular domain.
  • the cell membrane tethering domain can be connected to the degron, if present, by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the cell membrane tethering domain or degron.
  • a polypeptide linker can be any amino acid sequence that connects a first polypeptide sequence and a second polypeptide sequence.
  • a polypeptide linker can be a flexible linker (e.g., a Gly- Ser-Gly sequence).
  • polypeptide linkers include, but are not limited to, GSG linkers (e.g., [GS] 4 GG [SEQ ID NO: 347]), A(EAAAK) 3 A (SEQ ID NO: 348), and Whitlow linkers (e.g., a “KEGS” linker such as the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 349), an eGK linker such as the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 350), an LR1 linker such as the amino acid sequence SGGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 215), and linkers described in more detail in Issued U.S. Pat. No.
  • GSG linkers e.g., [GS] 4 GG [SEQ ID NO: 347]
  • A(EAAAK) 3 A SEQ ID NO: 348
  • Whitlow linkers e.g., a “KEGS” linker such as the amino acid sequence
  • Additional polypeptide linkers include SEQ ID NO: 194, SEQ ID NO: 196, and SEQ ID NO: 197.
  • Other polypeptide linkers may be selected based on desired properties (e.g., length, flexibility, amino acid composition etc.) and are known to those skilled in the art.
  • the cell-membrane tethering domain is oriented such that the secreted effector molecule and the protease cleavage site are extracellularly exposed following insertion into, or association with, the cell membrane, such that the protease cleavage site is capable of being cleaved by its respective protease and releasing (“secreting”) the effector molecule into the extracellular space.
  • any of the proteins described herein can include a degron domain including, but not limited to, a cytokine, a CAR, a protease, a transcription factor, a promoter or constituent of a promoter system (e.g., an ACP), and/or any of the membrane-cleavable chimeric protein described herein.
  • the degron domain can be any amino acid sequence motif capable of directing regulated degradation, such as regulated degradation through a ubiquitin- mediated pathway. In the presence of an immunomodulatory drug (IMiD), the degron domain directs ubiquitin-mediated degradation of a degron-fusion protein.
  • IMD immunomodulatory drug
  • the degron domain can be a cereblon (CRBN) polypeptide substrate domain capable of binding CRBN in response to an immunomodulatory drug (IMiD) including, but not limited to, IKZF1, IKZF3, CKla, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787, and ZN827, or a fragment thereof that is capable of drug-inducible binding of CRBN.
  • IMD immunomodulatory drug
  • the CRBN polypeptide substrate domain can be a chimeric fusion product of native CRBN polypeptide sequences, such as a IKZF3/ZFP91/IKZF3 chimeric fusion product having the amino acid sequence of FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRD AL (SEQ ID NO: 175).
  • Degron domains, and in particular CRBN degron systems, are described in more detail in International Application Pub. No. WO2019/089592A1, herein incorporated by reference for all purposes.
  • degron domains include, but are not limited to HCV NS4 degron, PEST (two copies of residues 277-307 of human IKBOI; SEQ ID NO: 161), GRR (residues 352-408 of human pl05; SEQ ID NO: 162), DRR (residues 210-295 of yeast Cdc34; SEQ ID NO: 163), SNS (tandem repeat of SP2 and NB (SP2-NB-SP2 of influenza A or influenza B; e.g., SEQ ID NO: 164), RPB (four copies of residues 1688-1702 of yeast RPB; SEQ ID NO: 165), SPmix (tandem repeat of SP1 and SP2 (SP2-SP1-SP2-SP1-SP2 of influenza A virus M2 protein; SEQ ID NO: 166), NS2 (three copies of residues 79-93 of influenza A virus NS protein; SEQ ID NO: 167), ODC (residues 106-142 of
  • Regulated degradation can be drug-inducible.
  • Drugs capable of mediating/regulating degradation can be small-molecule compounds.
  • Drugs capable of mediating/regulating degradation can include an “immunomodulatory drug” (IMiD).
  • IMDs refer to a class of small-molecule immunomodulatory drugs containing an imide group.
  • Cereblon (CRBN) is known target of IMiDs and binding of an IMiD to CRBN or a CRBN polypeptide substrate domain alters the substrate specificity of the CRBN E3 ubiquitin ligase complex leading to degradation of proteins having a CRBN polypeptide substrate domain (e.g., any of secretable effector molecules or other proteins of interest described herein).
  • imide-containing IMiDs include, but are not limited to, a thalidomide, a lenalidomide, or a pomalidomide.
  • the IMiD can be an FDA- approved drug.
  • Proteins described herein can contain a degron domain (e.g., referred to as “D” in the formula S - C - MT - D or D - MT - C - S for membrane-cleavable chimeric proteins described herein).
  • D degron domain
  • degron/ubiquitin-mediated degradation of the chimeric protein does not occur.
  • the protease cleavage site directs cleavage of the chimeric protein such that the effector molecule is released (“secreted”) into the extracellular space.
  • the degron domain directs ubiquitin-mediated degradation of the chimeric protein such that secretion of the effector molecule is reduced or eliminated.
  • the degron domain is the terminal cytoplasmic-oriented domain, specifically relative to the cell membrane tethering domain, e.g., the most C-terminal domain in the formula S - C - MT - D or the most N-terminal domain in the formula D - MT - C - S .
  • the degron domain can be connected to the cell membrane tethering domain by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the cell membrane tethering domain or the degron domain.
  • a polypeptide linker can be any amino acid sequence that connects a first polypeptide sequence and a second polypeptide sequence.
  • a polypeptide linker can be a flexible linker (e.g., a Gly- Ser-Gly sequence).
  • polypeptide linkers include, but are not limited to, GSG linkers (e.g., [GS] 4 GG [SEQ ID NO: 347]), A(EAAAK) 3 A (SEQ ID NO: 348), and Whitlow linkers (e.g., a “KEGS” linker such as the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 349), an eGK linker such as the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 350), an LR1 linker such as the amino acid sequence SGGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 215), and linkers described in more detail in Issued U.S. Pat. No.
  • GSG linkers e.g., [GS] 4 GG [SEQ ID NO: 347]
  • A(EAAAK) 3 A SEQ ID NO: 348
  • Whitlow linkers e.g., a “KEGS” linker such as the amino acid sequence
  • Additional polypeptide linkers include SEQ ID NO: 194, SEQ ID NO: 196, and SEQ ID NO: 197.
  • Other polypeptide linkers may be selected based on desired properties (e.g., length, flexibility, amino acid composition etc.) and are known to those skilled in the art.
  • the degron is oriented in relation to the cell membrane tethering domain such that the degron is exposed to the cytosol following localization to the cell membrane such that the degron domain is capable of mediating degradation (e.g., exposure to the cytosol and cytosol) and is capable of mediating ubiquitin-mediated degradation.
  • the degron domain can be N-terminal or C-terminal of the protein of interest, e.g., the effector molecule.
  • the degron domain can be connected to the protein of interest by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the protein of interest or the degron domain.
  • a polypeptide linker can be any amino acid sequence that connects a first polypeptide sequence and a second polypeptide sequence.
  • a polypeptide linker can be a flexible linker (e.g., a Gly-Ser-Gly sequence).
  • polypeptide linkers include, but are not limited to, GSG linkers (e.g., [GS]4GG [SEQ ID NO: 347]), A(EAAAK) 3 A (SEQ ID NO: 348), and Whitlow linkers (e.g., a “KEGS” linker such as the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 349), an eGK linker such as the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 350), an LR1 linker such as the amino acid sequence SGGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 215), and linkers described in more detail in Issued U.S. Pat. No.
  • GSG linkers e.g., [GS]4GG [SEQ ID NO: 347]
  • A(EAAAK) 3 A SEQ ID NO: 348
  • Whitlow linkers e.g., a “KEGS” linker such as the amino acid sequence KE
  • Additional polypeptide linkers include SEQ ID NO: 194, SEQ ID NO: 196, and SEQ ID NO: 197.
  • Other polypeptide linkers may be selected based on desired properties (e.g., length, flexibility, amino acid composition etc.) and are known to those skilled in the art.
  • a polypeptide linker can be cleavable, e.g., any of the protease cleavage sites described herein.
  • engineered nucleic acids e.g., an expression cassette
  • at least one protein of the present disclosure such as the cytokines, CARs, ACPs, and/or membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein.
  • engineered nucleic acids e.g., an expression cassette
  • engineered nucleic acids e.g., an expression cassette
  • engineered nucleic acids e.g., an expression cassette
  • encoding two or more proteins such as two or more of the cytokines, CARs, ACPs, and/or membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein.
  • an engineered nucleic acid is or comprises one or more expression cassettes, e.g., any expression cassette described herein.
  • the engineered nucleic acids encode an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a cytokine, a CAR, an ACP, and/or a membrane-cleavable chimeric protein oriented from N- terminal to C-terminal, having the formula: S - C - MT or MT - C - S.
  • S refers to a secretable effector molecule.
  • C refers to a protease cleavage site.
  • MT refers to a cell membrane tethering domain.
  • the promoter is operably linked to the exogenous polynucleotide sequence and the encoded S - C - MT or MT - C - S chimeric protein is configured to be expressed as a single polypeptide.
  • the engineered nucleic acids encode an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a cytokine. In certain embodiments described herein, the engineered nucleic acids encode an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a CAR. In certain embodiments described herein, the engineered nucleic acids encode an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a membrane-cleavable chimeric protein having a protein of interest (e.g., any of the effector molecules described herein). The promoter is operably linked to the exogenous polynucleotide sequence and the encoded membrane-cleavable chimeric protein is configured to be expressed as a single polypeptide.
  • the engineered nucleic acids encode an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a combination of one or more cytokines, one or more CARs, one or more ACPs, and/or one or more membrane-cleavable chimeric proteins, as described herein.
  • the engineered nucleic acids encode an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a cytokine and CAR.
  • the engineered nucleic acids encode an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a cytokine and an ACP.
  • the engineered nucleic acids encode two or more expression cassettes each containing a promoter and an exogenous polynucleotide sequence encoding a cytokine, CAR, ACP, and/or membrane-cleavable chimeric protein described herein. In certain embodiments described herein, the engineered nucleic acids encode two or more expression cassettes each containing a promoter and each separately encoding an exogenous polynucleotide sequence encoding a cytokine and CAR, respectively.
  • the engineered nucleic acids encode two or more expression cassettes each containing a promoter and each separately encoding an exogenous polynucleotide sequence encoding a cytokine and an ACP, respectively.
  • the two or more expression cassettes are oriented in a head-to-tail orientation.
  • the two or more expression cassettes are oriented in a head-to-head orientation.
  • the two or more expression cassettes are oriented in a tail-to-tail orientation.
  • each expression cassette contains its own promoter to drive expression of the polynucleotide sequence encoding a cytokine and/or CAR.
  • the cytokine and CAR are organized as such: 5’-cytokine-CAR-3’ or 5’-CAR-cytokine-3’.
  • an “engineered nucleic acid” is a nucleic acid that does not occur in nature. It should be understood, however, that while an engineered nucleic acid as a whole is not naturally- occurring, it may include nucleotide sequences that occur in nature.
  • an engineered nucleic acid comprises nucleotide sequences from different organisms (e.g., from different species). For example, in some embodiments, an engineered nucleic acid includes a murine nucleotide sequence, a bacterial nucleotide sequence, a human nucleotide sequence, and/or a viral nucleotide sequence.
  • engineered nucleic acids includes recombinant nucleic acids and synthetic nucleic acids.
  • a “recombinant nucleic acid” refers to a molecule that is constructed by joining nucleic acid molecules and, in some embodiments, can replicate in a live cell.
  • a “synthetic nucleic acid” refers to a molecule that is amplified or is synthesized (e.g., chemically, or by other means). Synthetic nucleic acids include those that are chemically modified, or otherwise modified, but can base pair with naturally-occurring nucleic acid molecules. Modifications include, but are not limited to, one or more modified internucleotide linkages and non-natural nucleic acids. Modifications are described in further detail in U.S. Pat. No. 6,673,611 and U.S.
  • Modified internucleotide linkages can be a phosphorodithioate or phosphorothioate linkage.
  • Non-natural nucleic acids can be a locked nucleic acid (LNA), a peptide nucleic acid (PNA), glycol nucleic acid (GNA), a phosphorodiamidate morpholino oligomer (PMO or “morpholino”), and threose nucleic acid (TNA).
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • GNA glycol nucleic acid
  • PMO or “morpholino” a phosphorodiamidate morpholino oligomer
  • TAA threose nucleic acid
  • Non-natural nucleic acids are described in further detail in International Application WO 1998/039352, U.S. Application Pub. No. 2013/0156849, and U.S. Pat. Nos.
  • Engineered nucleic acids of the present disclosure may be encoded by a single molecule (e.g., included in the same plasmid or other vector) or by multiple different molecules (e.g., multiple plasmids or other vectors, including multiple different independently-replicating molecules).
  • Engineered nucleic acids can be an isolated nucleic acid.
  • Isolated nucleic acids include, but are not limited to a cDNA polynucleotide, an RNA polynucleotide, an RNAi oligonucleotide (e.g., siRNAs, miRNAs, antisense oligonucleotides, shRNAs, etc.), an mRNA polynucleotide, a circular plasmid, a linear DNA fragment, a vector, a minicircle, a ssDNA, a bacterial artificial chromosome (BAC), and yeast artificial chromosome (YAC), and an oligonucleotide.
  • a cDNA polynucleotide an RNA polynucleotide
  • an RNAi oligonucleotide e.g., siRNAs, miRNAs, antisense oligonucleotides, shRNAs, etc.
  • an mRNA polynucleotide e.g., a circular plasm
  • Engineered nucleic acids of the present disclosure may be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press).
  • engineered nucleic acid constructs are produced using GIBSON ASSEMBLY® Cloning (see, e.g., Gibson, D.G. et al. Nature Methods, 343-345, 2009; and Gibson, D.G. et al. Nature Methods, 901-903, 2010, each of which is incorporated by reference herein).
  • GIBSON ASSEMBLY® typically uses three enzymatic activities in a single-tube reaction: 5' exonuclease, the Y extension activity of a DNA polymerase and DNA ligase activity.
  • the 5 ' exonuclease activity chews back the 5 ' end sequences and exposes the complementary sequence for annealing.
  • the polymerase activity then fills in the gaps on the annealed regions.
  • a DNA ligase then seals the nick and covalently links the DNA fragments together.
  • the overlapping sequence of adjoining fragments is much longer than those used in Golden Gate Assembly, and therefore results in a higher percentage of correct assemblies.
  • engineered nucleic acid constructs are produced using INFUSION® cloning (Clontech).
  • the engineered nucleic acids encoding the proteins herein encode an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding the protein.
  • an engineered nucleic acid e.g., an engineered nucleic acid comprising an expression cassette
  • comprises a promoter operably linked to a nucleotide sequence e.g., an exogenous polynucleotide sequence
  • a nucleotide sequence e.g., an exogenous polynucleotide sequence
  • the engineered nucleic acid may comprise a promoter operably linked to a nucleotide sequence encoding at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10 distinct proteins.
  • an engineered nucleic acid comprises a promoter operably linked to a nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more distinct proteins.
  • an engineered nucleic acid e.g., an engineered nucleic acid comprising an expression cassette
  • the engineered nucleic acid may comprise a promoter operably linked to a nucleotide sequence encoding at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10 cytokines.
  • an engineered nucleic acid comprises a promoter operably linked to a nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cytokines.
  • an engineered nucleic acid (e.g., an engineered nucleic acid comprising an expression cassette) comprises a promoter operably linked to a nucleotide sequence (e.g., an exogenous polynucleotide sequence) encoding at least 2 membrane-cleavable chimeric proteins.
  • the engineered nucleic acid may comprise a promoter operably linked to a nucleotide sequence encoding at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10 membrane-cleavable chimeric proteins.
  • an engineered nucleic acid comprises a promoter operably linked to a nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more membrane-cleavable chimeric proteins.
  • a “promoter” refers to a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled.
  • a promoter may also contain sub-regions at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. Promoters may be constitutive, inducible, repressible, tissue-specific or any combination thereof.
  • a promoter drives expression or drives transcription of the nucleic acid sequence that it regulates.
  • a promoter is considered to be “operably linked” when it is in a correct functional location and orientation in relation to a nucleic acid sequence it regulates to control (“drive”) transcriptional initiation and/or expression of that sequence.
  • a promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment of a given gene or sequence. Such a promoter can be referred to as “endogenous.”
  • a coding nucleic acid sequence may be positioned under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the encoded sequence in its natural environment.
  • promoters may include promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that are not "naturally occurring" such as, for example, those that contain different elements of different transcriptional regulatory regions and/or mutations that alter expression through methods of genetic engineering that are known in the art.
  • sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including polymerase chain reaction (PCR) (see, e.g., U.S. Pat. No. 4,683,202 and U.S. Pat. No. 5,928,906).
  • PCR polymerase chain reaction
  • Promoters of an engineered nucleic acid may be “inducible promoters,” which refer to promoters that are characterized by regulating (e.g., initiating or activating) transcriptional activity when in the presence of, influenced by or contacted by a signal.
  • the signal may be endogenous or a normally exogenous condition (e.g., light), compound (e.g., chemical or nonchemical compound) or protein (e.g., cytokine) that contacts an inducible promoter in such a way as to be active in regulating transcriptional activity from the inducible promoter.
  • Activation of transcription may involve directly acting on a promoter to drive transcription or indirectly acting on a promoter by inactivation a repressor that is preventing the promoter from driving transcription.
  • deactivation of transcription may involve directly acting on a promoter to prevent transcription or indirectly acting on a promoter by activating a repressor that then acts on the promoter.
  • a promoter may be “responsive to” or “modulated by” a particular local stimulus or state (e.g., a local tumor state or signal).
  • a promoter is “responsive to” or “modulated by” a local tumor state (e.g., inflammation or hypoxia) or signal if in the presence of that state or signal, transcription from the promoter is activated, deactivated, increased, or decreased.
  • the promoter comprises a response element.
  • a “response element” is a short sequence of DNA within a promoter region that binds specific molecules (e.g., transcription factors) that modulate (regulate) gene expression from the promoter.
  • Response elements that may be used in accordance with the present disclosure include, without limitation, a phloretin- adjustable control element (PEACE), a zinc-finger DNA-binding domain (DBD), an interferon- gamma-activated sequence (GAS) (Decker, T. et al. J Interferon Cytokine Res. 1997 Mar;17(3): 121-34, incorporated herein by reference), an interferon- stimulated response element (ISRE) (Han, K. J. et al. J Biol Chem. 2004 Apr 9;279(15): 15652-61, incorporated herein by reference), a NF-kappaB response element (Wang, V. et al. Cell Reports.
  • PEACE phloretin- adjustable control element
  • DBD zinc-finger DNA-binding domain
  • GAS interferon- gamma-activated sequence
  • ISRE interferon- stimulated response element
  • NF-kappaB response element Wang,
  • STAT3 response element Zhang, D. et al. J of Biol Chem. 1996; 271: 9503-9509, incorporated herein by reference.
  • Other response elements are encompassed herein.
  • Response elements can also contain tandem repeats (e.g., consecutive repeats of the same nucleotide sequence encoding the response element) to generally increase sensitivity of the response element to its cognate binding molecule. Tandem repeats can be labeled 2X, 3X, 4X, 5X, etc. to denote the number of repeats present.
  • Non-limiting examples of responsive promoters (also referred to as “inducible promoters”) (e.g., TGF-beta responsive promoters) are listed in Table 5A, which shows the design of the promoter and transcription factor, as well as the effect of the inducer molecule towards the transcription factor (TF) and transgene transcription (T) is shown (B, binding; D, dissociation; n.d., not determined) (A, activation; DA, deactivation; DR, derepression) (see Homer, M. & Weber, W. FEBS Letters 586 (2012) 20784-2096m, and references cited therein).
  • Non-limiting examples of components of inducible promoters include those presented in Table 5B. Table 5A. Examples of Responsive Promoters
  • Non-limiting examples of promoters include the cytomegalovirus (CMV) promoter, the elongation factor 1-alpha (EFla) promoter, the elongation factor (EFS) promoter, the MND promoter (a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer), the phosphoglycerate kinase (PGK) promoter, the spleen focus-forming virus (SFFV) promoter, the simian virus 40 (SV40) promoter, and the ubiquitin C (UbC) promoter (see, e.g., Table 5C).
  • CMV cytomegalovirus
  • EFla elongation factor 1-alpha
  • EFS elongation factor
  • MND promoter a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer
  • PGK phospho
  • the promoter can be a tissue-specific promoter.
  • a tissue-specific promoter directs transcription of a nucleic acid, (e.g., the engineered nucleic acids encoding the proteins herein (e.g., a cytokine, CAR, ACP, and/or membrane-cleavable chimeric protein described herein)) such that expression is limited to a specific cell type, organelle, or tissue.
  • Tissuespecific promoters include, but are not limited to, albumin (liver specific, Pinkert et al., (1987)), lymphoid specific promoters (Calame and Eaton, 1988), particular promoters of T-cell receptors (Winoto and Baltimore, (1989)) and immunoglobulins; Banerji et al., (1983); Queen and Baltimore, 1983), neuron specific promoters (e.g. the neurofilament promoter; Byrne and Ruddle, 1989), pancreas specific promoters (Edlund et al., (1985)) or mammary gland specific promoters (milk whey promoter, U.S. Pat. No. 4,873,316 and European Application Publication No.
  • promoter as well as developmentally regulated promoters such as the murine hox promoters (Kessel and Gruss, Science 249:374-379 (1990)) or the a-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3:537-546 (1989)), the contents of each of which are fully incorporated by reference herein.
  • the promoter can be constitutive in the respective specific cell type, organelle, or tissue.
  • Tissue-specific promoters and/or regulatory elements can also include promoters from the liver fatty acid binding (FAB) protein gene, specific for colon epithelial cells; the insulin gene, specific for pancreatic cells; the transphyretin, alpha.1 -antitrypsin, plasminogen activator inhibitor type 1 (PAI- 1), apolipoprotein Al and LDL receptor genes, specific for liver cells; the myelin basic protein (MBP) gene, specific for oligodendrocytes; the glial fibrillary acidic protein (GFAP) gene, specific for glial cells; OPSIN, specific for targeting to the eye; and the neural-specific enolase (NSE) promoter that is specific for nerve cells.
  • FAB liver fatty acid binding
  • PKI- 1 plasminogen activator inhibitor type 1
  • MBP myelin basic protein
  • GFAP glial fibrillary acidic protein
  • OPSIN specific for targeting to the eye
  • NSE neural-specific enolase
  • tissue-specific promoters include, but are not limited to, the promoter for creatine kinase, which has been used to direct expression in muscle and cardiac tissue and immunoglobulin heavy or light chain promoters for expression in B cells.
  • Other tissue specific promoters include the human smooth muscle alpha-actin promoter.
  • tissue-specific expression elements for the liver include but are not limited to HMG-COA reductase promoter, sterol regulatory element 1, phosphoenol pyruvate carboxy kinase (PEPCK) promoter, human C- reactive protein (CRP) promoter, human glucokinase promoter, cholesterol L 7-alpha hydroylase (CYP-7) promoter, beta- galactosidase alpha-2,6 sialylkansferase promoter, insulin-like growth factor binding protein (IGFBP-I) promoter, aldolase B promoter, human transferrin promoter, and collagen type I promoter.
  • HMG-COA reductase promoter sterol regulatory element 1
  • PEPCK phosphoenol pyruvate carboxy kinase
  • CRP C- reactive protein
  • glucokinase promoter human glucokinase promoter
  • cholesterol L 7-alpha hydroylase (CYP-7) promoter beta
  • Exemplary tissue-specific expression elements for the prostate include but are not limited to the prostatic acid phosphatase (PAP) promoter, prostatic secretory protein of 94 (PSP 94) promoter, prostate specific antigen complex promoter, and human glandular kallikrein gene promoter (hgt-1).
  • Exemplary tissue-specific expression elements for gastric tissue include but are not limited to the human H+/K+-ATPase alpha subunit promoter.
  • Exemplary tissue- specific expression elements for the pancreas include but are not limited to pancreatitis associated protein promoter (PAP), elastase 1 transcriptional enhancer, pancreas specific amylase and elastase enhancer promoter, and pancreatic cholesterol esterase gene promoter.
  • tissue-specific expression elements for the endometrium include, but are not limited to, the uteroglobin promoter.
  • tissue-specific expression elements for adrenal cells include, but are not limited to, cholesterol side-chain cleavage (SCC) promoter.
  • tissue-specific expression elements for the general nervous system include, but are not limited to, gamma enolase (neuron- specific enolase, NSE) promoter.
  • tissuespecific expression elements for the brain include, but are not limited to, the neurofilament heavy chain (NF-H) promoter.
  • NF-H neurofilament heavy chain
  • tissue-specific expression elements for lymphocytes include, but are not limited to, the human CGL-l/granzyme B promoter, the terminal deoxy transferase (TdT), lambda 5, VpreB, and lek (lymphocyte specific tyrosine protein kinase p561ck) promoter, the humans CD2 promoter and its 3 '-transcriptional enhancer, and the human NK and T cell specific activation (NKG5) promoter.
  • tissue- specific expression elements for the colon include, but are not limited to, pp60c-src tyrosine kinase promoter, organspecific neoantigens (OSNs) promoter, and colon specific antigen-P promoter.
  • Tissue-specific expression elements for breast cells are for example, but are not limited to, the human alphalactalbumin promoter.
  • tissue-specific expression elements for the lung include, but are not limited to, the cystic fibrosis transmembrane conductance regulator (CFTR) gene promoter.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • a promoter of the present disclosure is modulated by signals within a tumor microenvironment.
  • a tumor microenvironment is considered to modulate a promoter if, in the presence of the tumor microenvironment, the activity of the promoter is increased or decreased by at least 10%, relative to activity of the promoter in the absence of the tumor microenvironment. In some embodiments, the activity of the promoter is increased or decreased by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, relative to activity of the promoter in the absence of the tumor microenvironment.
  • the activity of the promoter is increased or decreased by 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20-200%, 50-60%, 50-70%, 50-80%, 50-90%, 50-100%, or 50-200%, relative to activity of the promoter in the absence of the tumor microenvironment.
  • the activity of the promoter is increased or decreased by at least 2 fold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100 fold), relative to activity of the promoter in the absence of the tumor microenvironment.
  • the activity of the promoter is increased or decreased by at least 3 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, or at least 100 fold, relative to activity of the promoter in the absence of the tumor microenvironment.
  • the activity of the promoter is increased or decreased by 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, or 2-100 fold, relative to activity of the promoter in the absence of the tumor microenvironment.
  • a promoter of the present disclosure is activated under a hypoxic condition.
  • a “hypoxic condition” is a condition where the body or a region of the body is deprived of adequate oxygen supply at the tissue level. Hypoxic conditions can cause inflammation (e.g., the level of inflammatory cytokines increase under hypoxic conditions).
  • the promoter that is activated under hypoxic condition is operably linked to a nucleotide encoding a protein that decreases the expression of activity of inflammatory cytokines, thus reducing the inflammation caused by the hypoxic condition.
  • the promoter that is activated under hypoxic conditions comprises a hypoxia responsive element (HRE).
  • HRE hypoxia responsive element
  • a “hypoxia responsive element (HRE)” is a response element that responds to hypoxia-inducible factor (HIF).
  • HRE in some embodiments, comprises a consensus motif NCGTG (where N is either A or G).
  • NCGTG consensus motif
  • ACP Activation-Conditional Control Polypeptide
  • a synthetic promoter is a promoter system including an activation-conditional control polypeptide (ACP) binding domain sequence and a promoter sequence.
  • ACP activation-conditional control polypeptide
  • a promoter system is also referred to herein as an “ACP-responsive promoter.”
  • an ACP promoter system includes a first expression cassette encoding an activation-conditional control polypeptide (ACP) and a second expression cassette encoding an ACP-responsive promoter operably linked to an exogenous polynucleotide sequence, such as the exogenous polynucleotide sequence encoding the cytokines, including membrane-cleavable chimeric proteins versions of cytokines, described herein or any other protein of interest (e.g., a protease or CAR).
  • the first expression cassette and second expression cassette are each encoded by a separate engineered nucleic acid. In other embodiments, the first expression cassette and the second expression cassette are encoded by the same engineered nucleic acid.
  • the ACP-responsive promoter can be operably linked to a nucleotide sequence encoding a single protein of interest or multiple proteins of interest.
  • a synthetic promoter comprises the nucleic acid sequence of AATTAACGGGTTTCGTAACAATCGCATGAGGATTCGCAACGCCTTTGAAGCAGTCG ACGCCGAAGTCCCGTCTCAGTAAAGGTTGAAGCAGTCGACGCCGAAGAATCGGACT GCCTTCGTATGAAGCAGTCGACGCCGAAGGTATCAGTCGCCTCGGAATGAAGCAGT CGACGCCGAAGATTCGTAAGAGGCTCACTCTCCCTTACACGGAGTGGATAACTAGT TCTAGAGGGTATATAATGGGGGCCAACGCGTACCGGTGTC (SEQ ID NO: 298).
  • a synthetic promoter comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 298.
  • a synthetic promoter comprises the nucleic acid sequence of CGGGTTTCGTAACAATCGCATGAGGATTCGCAACGCCTTCGGCGTAGCCGATGTCG CGCTCCCGTCTCAGTAAAGGTCGGCGTAGCCGATGTCGCGCAATCGGACTGCCTTCG TACGGCGTAGCCGATGTCGCGCGTATCAGTCGCCTCGGAACGGCGTAGCCGATGTC GCGCATTCGTAAGAGGCTCACTCTCCCTTACACGGAGTGGATAACTAGTTCTAGAG GGTATATAATGGGGGCCA (SEQ ID NO: 299).
  • a synthetic promoter comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 299.
  • the promoters of the ACP promoter system can include any of the promoter sequences described herein (see “Promoters” above).
  • the ACP-responsive promoter can be derived from minP, NFkB response element, CREB response element, NF AT response element, SRF response element 1, SRF response element 2, API response element, TCF-EEF response element promoter fusion, Hypoxia responsive element, SMAD binding element, STAT3 binding site, minCMV, YB_TATA, minTK, inducer molecule responsive promoters, and tandem repeats thereof.
  • the ACP-responsive promoter includes a minimal promoter.
  • the ACP-binding domain can include a transcription factorbinding domain, e.g., a synthetic transcription factor (synTF) -binding domain.
  • the ACP-binding domain includes one or more zinc finger binding sites.
  • the ACP-responsive promoter includes a minimal promoter and the ACP-binding domain includes one or more zinc finger binding sites.
  • the ACP-binding domain can include 1, 2, 3, 4,5 ,6 7, 8, 9, 10, or more zinc finger binding sites.
  • the transcription factor is a zinc-finger-containing transcription factor.
  • the zinc-finger- containing transcription factor is a synthetic transcription factor.
  • the ACP-binding domain includes one or more zinc finger binding sites and the ACP has a DNA- binding zinc finger protein domain (ZF protein domain). In some embodiments, the ACP has a DNA-binding zinc finger protein domain (ZF protein domain) and an effector domain. In some embodiments, the ACP-binding domain includes one or more zinc finger binding sites and the ACP has a DNA-binding zinc finger protein domain (ZF protein domain) and an effector domain. In some embodiments, the ZF protein domain is modular in design and is composed of zinc finger arrays (ZFA). A zinc finger array comprises multiple zinc finger protein motifs that are linked together. Each zinc finger motif binds to a different nucleic acid motif.
  • a ZFA with specificity to any desired nucleic acid sequence e.g., a ZFA with desired specificity to an ACP-binding domain having a specific zinc finger binding site composition and/or configuration.
  • the ZF motifs can be directly adjacent to each other, or separated by a flexible linker sequence.
  • a ZFA is an array, string, or chain of ZF motifs arranged in tandem.
  • a ZFA can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,1 3, 14, or 15 zinc finger motifs.
  • the ZFA can have from 1-10, 1-15, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5- 10, or 5-15 zinc finger motifs.
  • the ZF protein domain can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more ZFAs.
  • the ZF domain can have from 1-10, 1-15, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4- 7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, or 5-15 ZFAs.
  • the ZF protein domain comprises one to ten ZFA(s).
  • the ZF protein domain comprises at least one ZFA.
  • the ZF protein domain comprises at least two ZFAs.
  • the ZF protein domain comprises at least three ZFAs.
  • the ZF protein domain comprises at least four ZFAs.
  • the ZF protein domain comprises at least five ZFAs.
  • the ZF protein domain comprises at least ten ZFAs.
  • the DNA-binding domain comprises a tetracycline (or derivative thereof) repressor (TetR) domain.
  • the ACP can also further include an effector domain, such as a transcriptional effector domain.
  • a transcriptional effector domain can be the effector or activator domain of a transcription factor.
  • Transcription factor activation domains are also known as transactivation domains, and act as scaffold domains for proteins such as transcription coregulators that act to activate or repress transcription of genes.
  • Any suitable transcriptional effector domains can be used in the ACP including, but not limited to, a Herpes Simplex Virus Protein 16 (VP 16) activation domain; an activation domain consisting of four tandem copies of VP16, a VP64 activation domain; a p65 activation domain of NFKB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains, the tripartite activator is known as a VPR activation domain; a histone acetyltransferase (HAT) core domain of the human El A- associated protein p300, known as a p300 HAT core activation domain; a Kruppel associated box (KRAB) repression domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif (SEQ ID NO: 346) of the hairy-related basic
  • the effector domain is s transcription effector domain selected from: a Herpes Simplex Virus Protein 16 (VP 16) activation domain; an activation domain consisting of four tandem copies of VP 16, a VP64 activation domain; a p65 activation domain of NFKB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains, the tripartite activator is known as a VPR activation domain; a histone acetyltransferase (HAT) core domain of the human E1A- associated protein p300, known as a p300 HAT core activation domain; a Kruppel associated box (KRAB) repression domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif (SEQ ID NO: 346) of the hairy-related basic helix-
  • the ACP is a small molecule (e.g., drug) inducible polypeptide.
  • the ACP may be induced by tetracycline (or derivative thereof), and comprises a TetR domain and a VP 16 effector domain.
  • the ACP includes an estrogen receptor variant, such as ERT2, and may be regulated by tamoxifen, or a metabolite thereof (such as 4-hydroxy-tamoxifen [4-OHT], N-desmethyltamoxifen, tamoxifen-N-oxide, or endoxifen), through tamoxifen-controlled nuclear localization.
  • the ACP comprises a nuclear-localization signal (NLS).
  • the NLS comprises the amino acid sequence of MPKKKRKV (SEQ ID NO: 296).
  • An exemplary nucleic acid sequence encoding SEQ ID NO: 296 is ATGCCCAAGAAGAAGCGGAAGGTT (SEQ ID NO: 297) or ATGCCCAAGAAAAAGCGGAAGGTG (SEQ ID NO: 340).
  • a nucleic acid sequence encoding SEQ ID NO: 296 may comprise SEQ ID NO: 297 or SEQ ID NO: 340, or comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 297 or SEQ ID NO: 340.
  • the ACP comprises an estrogen receptor variant such as ERT2.
  • the ACP comprises an estrogen receptor or variant thereof as described herein, e.g., in Table 25.
  • ERT2 comprises a ligand binding domain (ER- LBD) comprising an amino acid sequence corresponding to amino acids 282-595 of SEQ ID NO: 356 (human Estrogen Receptor, UniProt ID No: P03372), comprising amino acid substitutions G400V, M543A, and L544A or amino acid substitutions G400V, M543A, L544A, and V595A, and comprising one or more additional amino acid substitutions to ligand binding residues within a region of SEQ ID NO: 356 selected from positions 343-354, positions 380- 392, positions 404-463, and positions 517-540, and position 547.
  • SEQ ID NO: 356 selected from positions 343-354, positions 380- 392, positions 404-463, and positions 517-540, and position 547.
  • the one or more amino acid substitutions result in: (a) greater sensitivity to a non-endogenous ligand as compared to an endogenous ligand, (b) greater sensitivity to a non-endogenous ligand as compared to an ER-LBD of SEQ ID NO: 357 or SEQ ID NO: 358, and/or (c) greater selectivity to a non-endogenous ligand as compared to an ER-LBD of SEQ ID NO: 357 or SEQ ID NO: 358.
  • the one or more additional amino acid substitutions may result in: (a) greater sensitivity to a non-endogenous ligand as compared to an endogenous ligand, (b) greater sensitivity to a non-endogenous ligand as compared to an ER-LBD of SEQ ID NO: 357 or SEQ ID NO: 358 and/or (c) greater selectivity to a non-endogenous ligand as compared to an ER-LBD of SEQ ID NO: 357 or SEQ ID NO: 358.
  • the one or more additional amino acid substitutions results in greater sensitivity to a non-endogenous ligand as compared to an ER- LBD of SEQ ID NO: 357.
  • the one or more additional amino acid substitutions results in greater sensitivity to a non-endogenous ligand as compared to an ER- LBD of SEQ ID NO: 357. In some embodiments, the one or more additional amino acid substitutions results in greater selectivity to a non-endogenous ligand as compared to an ER- LBD of SEQ ID NO: 357. In some embodiments, the one or more additional amino acid substitutions results in greater selectivity to a non-endogenous ligand as compared to an ER- LBD of SEQ ID NO: 358.
  • Ligand binding residues refers to residues located at the ligand binding pocket of estrogen receptor (ER) or an ER- ligand binding domain, and includes the pocket for binding to an endogenous ligand (e.g., estradiol) and the pocket for binding to a non-endogenous ligand such as 4-OHT. Residues within positions 343-354, positions 380-392 and positions 404-463 corresponding to SEQ ID NO: 356 are involved in binding to both endogenous and non- endogenous ligands.
  • endogenous ligand e.g., estradiol
  • Residues within positions 517-547 are located within a helix referred to as helix 12 and are involved in endogenous ligand binding.
  • a non-endogenous ligand as compared to sensitivity to a non- endogenous ligand means that the modified ER-LBD binds to a non-endogenous ligand (e.g., endoxifen) with a higher affinity as compared to the affinity of its binding to an endogenous ligand (e.g., estradiol).
  • a non-endogenous ligand e.g., endoxifen
  • an endogenous ligand e.g., estradiol
  • Greater sensitivity to a non-endogenous ligand as compared to sensitivity an ER-LBD not including the one or more amino acid substitutions means that the modified ER-LBD binds to a non-endogenous ligand (e.g., endoxifen) with a higher affinity as compared to the affinity of binding of ER-LBD not including the one or more additional amino acid substitutions to the non-endogenous ligand.
  • a non-endogenous ligand e.g., endoxifen
  • the greater sensitivity is at least a 1.5-fold, at least a 2-fold, at least a 3-fold, at least a 4-fold, or at least a 5-fold improvement in binding affinity to a non-endogenous ligand, as compared to binding of an ER-LBD not including the one or more additional amino acid substitutions.
  • greater sensitivity is demonstrated by greater transcriptional modulation (e.g., greater transcriptional activation or greater transcriptional repression) of a chimeric transcription factor including a modified ER- LBD, as compared to a chimeric transcription factor including an ER-LBD that lacks the one or more additional amino acid substitutions.
  • a chimeric transcription factor including a modified ER-LBD in a transfection of transduction assay, is capable of inducing at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 35% greater expression of a reporter under control of a chimeric transcription factor-responsive promoter in response to a non-endogenous ligand (e.g., 4-OHT) (as measured by % of cells positive for the reporter, or as measured by geometric mean fluorescent intensity) as compared to the expression of the reporter under the same conditions but with an ER-LBD that lacks the one or more additional amino acid substitutions.
  • a non-endogenous ligand e.g., 4-OHT
  • Greater selectivity to a non-endogenous ligand refers to preferential binding to a non- endogenous ligand (e.g., 4-OHT or endoxifen) as compared to an endogenous ligand (e.g., estradiol).
  • Selectivity may be measured using a selectivity coefficient, which is the equilibrium constant for the reaction of displacement by one ligand (e.g., a non-endogenous ligand) of another ligand (e.g., an endogenous ligand) in a complex with the substrate (e.g., a modified ER- LBD).
  • a competing ligand e.g., an endogenous ligand
  • the initial ligand e.g., a non-endogenous ligand
  • greater selectivity is demonstrated by improved transcriptional modulation of a chimeric transcription factor in the presence of a non-endogenous ligand as compared to transcriptional modulation in the presence of an endogenous ligand.
  • a chimeric transcription factor including a modified ER-LBD in a transfection of transduction assay, is capable of inducing at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 35% greater expression of a reporter under control of a chimeric transcription factor-responsive promoter in response to a non-endogenous ligand (e.g., 4-OHT) (as measured by % of cells positive for the reporter, or as measured by geometric mean fluorescent intensity) as compared to the expression of the reporter under the same conditions but in response to an endogenous ligand (e.g., estradiol).
  • a non-endogenous ligand e.g., 4-OHT
  • an endogenous ligand e.g., estradiol
  • the one or more amino acid substitutions to ligand binding residues include one or more amino acid substitutions within helix 12.
  • Helix 12 of an ER-LBD includes residue positions 533-547 of SEQ ID NO: 356.
  • the one or more amino acid substituions within helix 12 are at one or more positions selected from 538, 536, 539, 540, 547, 534, 533, and 537.
  • Non-endogenous ligand may refer to, for example, a synthetic estrogen receptor binding ligand that is not naturally expressed by an organism that expresses an estrogen receptor.
  • Non-endogenous estrogen receptor binding ligands include, without limitation, tamoxifen and metabolites thereof, such as 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.
  • the one or more additional amino acid substitutions may be at one or more positions of SEQ ID NO: 356 selected from 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 354, 380, 384, 386, 387, 388, 389, 391, 392, 404, 407, 409, 413, 414, 417, 418, 420, 421, 422, 424, 428, 463, 517, 521, 522, 524, 525, 526, 527, 528, 533, 534, 536, 537, 538, 539, 540, and 547.
  • the one or more additional amino acid substitutions include substitutions at one of the above-listed positions, two of the above-listed positions, three of the above-listed positions, four of the above-listed positions, or five of the above-listed positions.
  • the one or more additional amino acids substitutions are selected from one or more of the substitutions listed in Table 23.
  • the one or more additional mutations comprise at least two mutations, at least three mutations, at least four mutations, at least five mutations, at least six mutations, at least seven mutations, or at least eight mutations.
  • the one or more additional mutations comprise two to ten mutations, two to nine mutations, two to eight mutations, two to seven mutations, two to six mutations, two to five mutations, two to four mutations, two to three mutations, three to ten mutations, three to nine mutations, three to eight mutations, three to seven mutations, three to six mutations, three to five mutations, three to four mutations, four to ten mutations, four to nine mutations, four to eight mutations, four to seven mutations, four to six mutations, four to five mutations, five to ten mutations, five to six mutations, three to four mutations, four to ten mutations, four to nine mutations, four to eight mutations, four to seven mutations, four to six mutations, four to five mutations, five to ten mutations, five
  • the one or more additional mutations comprise at least two mutations that are selected from the mutations listed in Table 24.
  • a modified ER-LBD variant having an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a modified ER-LBD as described herein, provided that the variant includes the G400V/MS43A/L544A triple amino acid substitution or the G400V/M543A/L544A/V595A quadruple amino acid substitution, and includes the one or more additional amino acid substitutions that confer greater sensitivity and/or greater selectivity for a non-endogenous ligand (e.g., one or more of the amino acid substitutions shown in Table 23 and Table 24).
  • the ACP is a small molecule (e.g., drug) inducible polypeptide that includes a repressible protease and one or more cognate cleavage sites of the repressible protease.
  • a repressible protease is active (cleaves a cognate cleavage site) in the absence of the specific agent and is inactive (does not cleave a cognate cleavage site) in the presence of the specific agent.
  • the specific agent is a protease inhibitor.
  • the protease inhibitor specifically inhibits a given repressible protease of the present disclosure.
  • the repressible protease can be any of the proteases described herein that is capable of inactivation by the presence or absence of a specific agent (see “Protease Cleavage Site” above for exemplary repressible proteases, cognate cleavage sites, and protease inhibitors).
  • the ACP has a degron domain (see “Degron Systems and Domains” above for exemplary degron sequences).
  • the degron domain can be in any order or position relative to the individual domains of the ACP.
  • the degron domain can be N-terminal of the repressible protease, C-terminal of the repressible protease, N-terminal of the ZF protein domain, C-terminal of the ZF protein domain, N-terminal of the effector domain, or C-terminal of the effector domain.
  • nucleic acids may comprise a sequence in Table 5D, or a nucleic acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence in Table 5D.
  • engineered nucleic acids are configured to produce multiple proteins (e.g., a cytokine, CAR, ACP, membrane-cleavable chimeric protein, and/or combinations thereof).
  • proteins e.g., a cytokine, CAR, ACP, membrane-cleavable chimeric protein, and/or combinations thereof.
  • nucleic acids may be configured to produce 2-20 different proteins.
  • nucleic acids are configured to produce 2-20, 2-19, 2-18, 2-17,
  • nucleic acids are configured to produce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins.
  • engineered nucleic acids can be multicistronic, i.e., more than one separate polypeptide (e.g., multiple proteins, such as a cytokine, CAR, ACP, and/or membrane- cleavable chimeric protein described herein) can be produced from a single mRNA transcript.
  • Engineered nucleic acids can be multicistronic through the use of various linkers, e.g., a polynucleotide sequence encoding a first protein can be linked to a nucleotide sequence encoding a second protein, such as in a first gene:linker: second gene 5’ to 3’ orientation.
  • a linker can encode a 2A ribosome skipping element, such as T2A.
  • 2A ribosome skipping elements include, but are not limited to, E2A, P2A, and F2A.
  • 2A ribosome skipping elements allow production of separate polypeptides encoded by the first and second genes are produced during translation.
  • a linker can encode a cleavable linker polypeptide sequence, such as a Furin cleavage site or a TEV cleavage site, wherein following expression the cleavable linker polypeptide is cleaved such that separate polypeptides encoded by the first and second genes are produced.
  • a cleavable linker can include a polypeptide sequence, such as such a flexible linker (e.g., a Gly-Ser-Gly sequence), that further promotes cleavage.
  • an engineered nucleic acid disclosed herein comprises an E2A/T2A ribosome skipping element.
  • the E2A/T2A ribosome skipping element comprises the amino acid sequence of GSGQCTNYAEEKEAGDVESNPGPGSGEGRGSEETCGDVEENPGP (SEQ ID NO: 281).
  • nucleic acid encoding SEQ ID NO: 281 is GGTAGCGGCCAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATC TAATCCTGGACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACG TGGAGGAAAACCCTGGACCT (SEQ ID NO: 282).
  • a nucleic acid encoding SEQ ID NO: 281 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 282.
  • an engineered nucleic acid disclosed herein comprises an E2A/T2A ribosome skipping element.
  • the E2A/T2A ribosome skipping element comprises the amino acid sequence of QCTNYALLKLAGDVESNPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 283).
  • An exemplary nucleic acid encoding SEQ ID NO: 283 is CAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATCTAATCCTGG ACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACGTGGAGGAA AACCCTGGACCT (SEQ ID NO: 284).
  • a nucleic acid encoding SEQ ID NO: 283 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 284.
  • a linker can encode an Internal Ribosome Entry Site (IRES), such that separate polypeptides encoded by the first and second genes are produced during translation.
  • IRS Internal Ribosome Entry Site
  • a linker can encode a splice acceptor, such as a viral splice acceptor.
  • a linker can be a combination of linkers, such as a Furin-2A linker that can produce separate polypeptides through 2A ribosome skipping followed by further cleavage of the Furin site to allow for complete removal of 2 A residues.
  • a combination of linkers can include a Furin sequence, a flexible linker, and 2A linker.
  • the linker is a Furin-Gly-Ser-Gly-2A fusion polypeptide.
  • a linker of the present disclosure is a Furin-Gly-Ser-Gly-T2A fusion polypeptide.
  • a multicistronic system can use any number or combination of linkers, to express any number of genes or portions thereof (e.g., an engineered nucleic acid can encode a first, a second, and a third protein, each separated by linkers such that separate polypeptides encoded by the first, second, and third proteins are produced).
  • an engineered nucleic acid can encode a first, a second, and a third protein, each separated by linkers such that separate polypeptides encoded by the first, second, and third proteins are produced).
  • Engineered nucleic acids can use multiple promoters to express genes from multiple ORFs, i.e., more than one separate mRNA transcript can be produced from a single engineered nucleic acid.
  • a first promoter can be operably linked to a polynucleotide sequence encoding a first protein
  • a second promoter can be operably linked to a polynucleotide sequence encoding a second protein.
  • any number of promoters can be used to express any number of proteins.
  • at least one of the ORFs expressed from the multiple promoters can be multicistronic.
  • Expression cassettes encoded on the same engineered nucleic acid can be oriented in any manner suitable for expression of the encoded exogenous polynucleotide sequences.
  • Expression cassettes encoded on the same engineered nucleic acid can be oriented in the same direction, i.e., transcription of separate cassettes proceeds in the same direction. Constructs oriented in the same direction can be organized in a head-to-tail format referring to the 5' end (head) of the first gene being adjacent to the 3' end (tail) of the upstream gene.
  • Expression cassettes encoded on the same engineered nucleic acid can be oriented in an opposite direction, i.e., transcription of separate cassettes proceeds in the opposite direction (also referred to herein as “bidirectional”).
  • FIGs. 1A-1C schematically depict a cytokine- CAR bidirectional construct in head-to-head directionality (FIG. 1A), head-to-tail directionality (FIG. IB), and tail-to-tail directionality (FIG. 1C).
  • Linkers can refer to polypeptides that link a first polypeptide sequence and a second polypeptide sequence, the multicistronic linkers described above, or the additional promoters that are operably linked to additional ORFs described above.
  • Exogenous polynucleotide sequences encoded by the expression cassette can include a 3 ’untranslated region (UTR) comprising an mRNA-destabilizing element that is operably linked to the exogenous polynucleotide sequence, such as exogenous polynucleotide sequences encoding a cytokine (e.g., IL12 or IL12p70).
  • the mRNA-destabilizing element comprises an AU-rich element and/or a stem-loop destabilizing element (SLDE).
  • the mRNA-destabilizing element comprises an AU-rich element.
  • the AU-rich element includes at least two overlapping motifs of the sequence ATTTA (SEQ ID NO: 209). In some embodiments, the AU-rich element comprises ATTTATTTATTTATTTATTTA (SEQ ID NO: 210). In some embodiments, the mRNA- destabilizing element comprises a stem-loop destabilizing element (SLDE). In some embodiments, the SLDE comprises CTGTTTAATATTTAAACAG (SEQ ID NO: 211). In some embodiments, the mRNA-destabilizing element comprises at least one AU-rich element and at least one SLDE. “AuSLDE” as used herein refers to an AU-rich element operably linked to a stem-loop destabilizing element (SLDE).
  • An exemplary AuSLDE sequence comprises ATTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAG (SEQ ID NO: 212).
  • the mRNA-destabilizing element comprises a 2X AuSLDE.
  • An exemplary AuSLDE sequence is provided as ATTTATTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAGtgcggtaagcATTTA TTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAG (SEQ ID NO: 213).
  • an engineered nucleic acid described herein comprises an insulator sequence. Such insulator sequences function to prevent inappropriate interactions between adjacent regions of a construct.
  • an insulator sequence comprises the nucleic acid sequence of ACAATGGCTGGCCCATAGTAAATGCCGTGTTAGTGTGTTAGTTGCTGTTCTTCCACG TCAGAAGAGGCACAGACAAATTACCACCAGGTGGCGCTCAGAGTCTGCGGAGGCAT CACAACAGCCCTGAATTTGAATCCTGCTCTGCCACTGCCTAGTTGAGACCTTTTACT ACCTGACTAGCTGAGACATTTACGACATTTACTGGCTCTAGGACTCATTTTATTCAT TTCATTACTTTTTTTTTCTTTGAGACGGAATCTCGCTCT (SEQ ID NO: 300).
  • an insulator sequence comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 300.
  • engineered immunoresponsive cells and methods of producing the engineered immunoresponsive cells, that produce a protein described herein (e.g., a cytokine, CAR, ACP, and/or membrane-cleavable chimeric protein described herein).
  • engineered immunoresponsive cells of the present disclosure may be engineered to express the proteins provided for herein, such as a cytokine, CAR, ACP, and/or the membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein. These cells are referred to herein as “engineered cells.” These cells, which typically contain engineered nucleic acid, do not occur in nature.
  • the cells are engineered to include a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding a protein, for example, a cytokine, CAR, ACP, and/or a membrane-cleavable chimeric protein.
  • a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding a protein, for example, a cytokine, CAR, ACP, and/or a membrane-cleavable chimeric protein.
  • An engineered cell can comprise an engineered nucleic acid integrated into the cell’s genome.
  • An engineered cell can comprise an engineered nucleic acid capable of expression without integrating into the cell’s genome, for example, engineered with a transient expression system such as a plasmid or mRNA.
  • the present disclosure further provides for mixed cell compositions comprising one or more different engineered cells described herein.
  • mixed cell compositions comprising a first engineered cell, a second engineered cell, a third engineered cell, a fourth engineered cell, and so forth, where each said engineered cell comprises one or more engineered nucleic acids that allow for expression of one or more immunotherapy or immunomodulatory proteins (e.g., CARs, effector molecules, and/or chimeric proteins, as described herein).
  • a mixed cell composition comprises a first engineered cell and a second engineered cell.
  • each engineered cell within a mixed cell composition comprises at least one different engineered nucleic acid (e.g., any engineered nucleic acid described herein) compared to other engineered cells within the mixed cell composition.
  • a first engineered cell is capable of expressing a CAR and/or a first effector molecule (e.g., in a membrane-cleavable chimeric protein, as described herein), and a second engineered cell is capable of expressing a second effector molecule (e.g., in a membrane-cleavable chimeric protein, as described herein).
  • a first engineered cell in a mixed cell composition, is capable of expressing at least one effector molecule (e.g., in a membrane-cleavable chimeric protein, as described herein), and a second engineered cell is capable of expressing at least one effector molecule (e.g., in a membrane-cleavable chimeric protein, as described herein).
  • the present disclosure also encompasses additivity and synergy between provided proteins that are produced in an engineered cell (e.g., an immunoresponsive cell), whether produced within the same engineered cell or in different engineered cells, e.g., in a mixed cell composition.
  • cells are engineered to produce at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) proteins, for example at least each of a cytokine, CAR, ACP, and membrane-cleavable chimeric protein.
  • cells are engineered to produce at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) proteins, where the proteins are selected from at least one of a cytokine, a CAR, an ACP, a membrane-cleavable chimeric protein, and combinations thereof.
  • immunoresponsive cells provide herein are engineered to produce at least one membrane-cleavable chimeric protein having a cytokine effector molecule that is not natively produced by the cells, a CAR, and an ACP.
  • immunoresponsive cells provide herein are engineered to produce at least two cytokines, at least one of which is a membrane-cleavable chimeric protein having a cytokine effector molecule, a CAR, and an ACP.
  • cytokine effector molecule a CAR
  • ACP an ACP
  • a cell e.g., an immune cell
  • a cell is engineered to produce multiple proteins.
  • cells may be engineered to produce 2-20 different proteins, such as 2-20 different membrane-cleavable proteins.
  • a cell e.g., an immunoresponsive cell
  • a cell is engineered to produce at least 4 distinct proteins exogenous to the cell.
  • a cell is engineered to produce 4 distinct proteins exogenous to the cell.
  • cells are engineered to produce 2-20, 2- 19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3- 19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-19, 5-18, 5- 17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-19, 6-18, 6-17, 6-16, 6- 15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18,
  • engineered cells comprise one or more engineered nucleic acids encoding a promoter operably linked to a nucleotide sequence encoding a protein (e.g., an expression cassette).
  • cells are engineered to include a plurality of engineered nucleic acids, e.g., at least two engineered nucleic acids, each encoding a promoter operably linked to a nucleotide sequence encoding at least one (e.g., 1, 2 or 3) protein.
  • cells may be engineered to comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10, engineered nucleic acids, each encoding a promoter operably linked to a nucleotide sequence encoding at least one (e.g., 1, 2 or 3) protein.
  • the cells are engineered to comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more engineered nucleic acids, each encoding a promoter operably linked to a nucleotide sequence encoding at least one (e.g., 1, 2 or 3) protein.
  • Engineered cells can comprise an engineered nucleic acid encoding at least one of the linkers described above, such as polypeptides that link a first polypeptide sequence and a second polypeptide sequence, one or more multicistronic linker described above, one or more additional promoters operably linked to additional ORFs, or a combination thereof.
  • a cell e.g., an immune cell
  • a cell is engineered to express a protease.
  • a cell is engineered to express a protease heterologous to a cell.
  • a cell is engineered to express a protease heterologous to a cell expressing a provided protein (e.g., a membrane-cleavable chimeric protein, CAR, effector molecule, etc.), such as a heterologous protease that cleaves the protease cleavage site of a membrane-cleavable chimeric protein.
  • a provided protein e.g., a membrane-cleavable chimeric protein, CAR, effector molecule, etc.
  • engineered cells comprise one or more engineered nucleic acids encoding a promoter operably linked to a nucleotide sequence encoding a protease, such as a heterologous protease.
  • protease such as a heterologous protease.
  • Protease and protease cleavage sites are described in greater detail in the Section herein titled “Protease Cleavage site.”
  • At least one (e.g., 1, 2, 3, 4, 5, or more) protein includes an effector molecule that stimulates at least one immuno stimulatory mechanism in the tumor microenvironment, or inhibits at least one immunosuppressive mechanism in the tumor microenvironment.
  • at least one (e.g., 1, 2, 3, 4, 5, or more) protein includes an effector molecule that inhibits at least one immunosuppressive mechanism in the tumor microenvironment, and at least one protein (e.g., 1, 2, 3, 4, 5, or more) inhibits at least one immunosuppressive mechanism in the tumor microenvironment.
  • At least two (e.g., 2, 3, 4, 5, or more) of the proteins are effector molecules that each stimulate at least one immuno stimulatory mechanism in the tumor microenvironment. In still other embodiments, at least two (e.g., 1, 2, 3, 4, 5, or more) of the proteins are effector molecules that each inhibit at least one immunosuppressive mechanism in the tumor microenvironment.
  • a cell e.g., an immune cell
  • a cell is engineered to produce at least one protein including an effector molecule that stimulates T cell or NK cell signaling, activity and/or recruitment.
  • a cell is engineered to produce at least one protein that includes an effector molecule that stimulates antigen presentation and/or processing.
  • a cell is engineered to produce at least one protein that includes an effector molecule that stimulates natural killer cell-mediated cytotoxic signaling, activity and/or recruitment.
  • a cell is engineered to produce at least one protein that includes an effector molecule that stimulates dendritic cell differentiation and/or maturation.
  • a cell is engineered to produce at least one protein that includes an effector molecule that stimulates immune cell recruitment. In some embodiments, a cell is engineered to produce at least one protein includes an effector molecule that that stimulates Ml macrophage signaling, activity and/or recruitment. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that stimulates Thl polarization. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that stimulates stroma degradation. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that stimulates immuno stimulatory metabolite production.
  • a cell is engineered to produce at least one protein that includes an effector molecule that stimulates Type I interferon signaling. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that inhibits negative costimulatory signaling. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that inhibits pro-apoptotic signaling (e.g., via TRAIL) of anti-tumor immune cells. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that inhibits T regulatory (T re g) cell signaling, activity and/or recruitment.
  • T regulatory T regulatory
  • a cell is engineered to produce at least one protein that includes an effector molecule that inhibits tumor checkpoint molecules. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that activates stimulator of interferon genes (STING) signaling. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that inhibits myeloid-derived suppressor cell signaling, activity and/or recruitment. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that degrades immunosuppressive factors/metabolites.
  • STING stimulator of interferon genes
  • a cell is engineered to produce at least one protein that includes an effector molecule that inhibits vascular endothelial growth factor signaling. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that directly kills tumor cells (e.g., granzyme, perforin, oncolytic viruses, cytolytic peptides and enzymes, anti-tumor antibodies, e.g., that trigger ADCC).
  • tumor cells e.g., granzyme, perforin, oncolytic viruses, cytolytic peptides and enzymes, anti-tumor antibodies, e.g., that trigger ADCC.
  • At least one protein including an effector molecule that: stimulates T cell signaling, activity and/or recruitment, stimulates antigen presentation and/or processing, stimulates natural killer cell-mediated cytotoxic signaling , activity and/or recruitment, stimulates dendritic cell differentiation and/or maturation, stimulates immune cell recruitment, stimulates macrophage signaling, stimulates stroma degradation, stimulates immunostimulatory metabolite production, or stimulates Type I interferon signaling; and at least one protein including an effector molecule that inhibits negative costimulatory signaling, inhibits pro- apoptotic signaling of anti-tumor immune cells, inhibits T regulatory (Treg) cell signaling, activity and/or recruitment, inhibits tumor checkpoint molecules, activates stimulator of interferon genes (STING) signaling, inhibits myeloid-derived suppressor cell signaling, activity and/or recruitment, degrades immunosuppressive factors/metabolites, inhibits vascular endothelial growth factor signaling, or directly kills tumor cells.
  • an immunoresponsive cell is engineered to produce at least one effector molecule cytokine selected from IL15, IL12, an IL12p70 fusion protein, IL18, and IL21. In some embodiments, an immunoresponsive cell is engineered to produce at least two effector molecule cytokines selected from IL15, IL12, an IL12p70 fusion protein, IL18, and IL21. In some embodiments, an immunoresponsive cell is engineered to produce at least two effector molecule cytokines selected from IL15, IL12, an IL12p70 fusion protein, IL18, and IL21.
  • an immunoresponsive cell is engineered to produce at least the effector molecule cytokines IL15 and IL12p70 fusion protein. In some embodiments, an immunoresponsive cell is engineered to produce at least one membrane-cleavable chimeric protein including an effector molecule cytokine selected from IL15, IL12, an IL12p70 fusion protein, IL18, and IL21. In some embodiments, an immunoresponsive cell is engineered to produce at least two membrane-cleavable chimeric protein including effector molecule cytokines selected from IL15, IL12, an IL12p70 fusion protein, IL18, and IL21.
  • the IL 15 comprises the amino acid sequence of NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 285).
  • An exemplary nucleic acid sequence encoding SEQ ID NO: 285 is AATTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCAT GCACATCGACGCCACACTGTACACCGAGAGCGACGTGCACCCTAGCTGTAAAGTGA CCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGAC GCCAGCATCCACGACACCGTGGAAAACCTGATCATCCTGGCCAACAACAGCCTGAG CAGCAACGGCAATGTGACCGAGTCCGGCTGCAAAGAGTGCGAGGAACTGGAAGAG AAGAATATCAAAGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAA CACAAGC (SEQ ID NO: 286).
  • a nucleic acid encoding SEQ ID NO: 285 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 286.
  • the IL12p70 comprises the amino acid sequence of
  • a nucleic acid encoding SEQ ID NO: 293 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 294.
  • a cell e.g., an immune cell or a stem cell
  • cytokines including at least one of the cytokines being in a membrane- cleavable chimeric protein format (e.g., “S” in the formula S - C - MT or MT - C - S).
  • a cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule (e.g., “S” in the formula S - C - MT or MT - C - S) is IL15, IL12, an IL12p70 fusion protein, IL18, or IL21.
  • secretable effector molecule e.g., “S” in the formula S - C - MT or MT - C - S
  • the secretable effector molecule is IL15, IL12, an IL12p70 fusion protein, IL18, or IL21.
  • a cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule (e.g., “S” in the formula S - C - MT or MT - C - S) is IL- 15.
  • a cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule is IL- 15 and the cell is further engineered to produce one or more additional cytokines.
  • a cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule is IL-15 and the cell is further engineered to produce IL12, an IL12p70 fusion protein, IL18, or IL21.
  • a cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule is IL- 15 and the cell is further engineered to produce IL- 12.
  • a cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule is IL- 15 and the cell is further engineered to produce an IL12p70 fusion protein.
  • a cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule (e.g., “S” in the formula S - C - MT or MT - C - S) is IL- 15 and the cell is further engineered to produce one or more additional membrane-cleavable chimeric proteins.
  • the secretable effector molecule e.g., “S” in the formula S - C - MT or MT - C - S
  • the cell is further engineered to produce one or more additional membrane-cleavable chimeric proteins.
  • a cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule (e.g., “S” in the formula S - C - MT or MT - C - S) is IL- 15 and the cell is further engineered to produce one or more additional membrane-cleavable chimeric proteins including IL12, an IL12p70 fusion protein, IL18, and IL21.
  • the secretable effector molecule e.g., “S” in the formula S - C - MT or MT - C - S
  • the cell is further engineered to produce one or more additional membrane-cleavable chimeric proteins including IL12, an IL12p70 fusion protein, IL18, and IL21.
  • a cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule (e.g., “S” in the formula S - C - MT or MT - C - S) is IL 15 and the cell is further engineered to produce an additional membrane-cleavable chimeric proteins including IL12p70.
  • the secretable effector molecule e.g., “S” in the formula S - C - MT or MT - C - S
  • the cell is further engineered to produce an additional membrane-cleavable chimeric proteins including IL12p70.
  • a cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule (e.g., “S” in the formula S - C - MT or MT - C - S) is an IL12p70.
  • a cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule is IL12p70 and the cell is further engineered to produce one or more additional cytokines.
  • a cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule is IL12p70 and the cell is further engineered to produce IL15, IL 18, or IL21.
  • a cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule is IL12p70 and the cell is further engineered to produce IL 15.
  • a cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule (e.g., “S” in the formula S - C - MT or MT - C - S) is IL12p70 and the cell is further engineered to produce one or more additional membrane-cleavable chimeric proteins.
  • the secretable effector molecule e.g., “S” in the formula S - C - MT or MT - C - S
  • the cell is further engineered to produce one or more additional membrane-cleavable chimeric proteins.
  • a cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule (e.g., “S” in the formula S - C - MT or MT - C - S) is IL12p70 and the cell is further engineered to produce one or more additional membrane-cleavable chimeric proteins including IL 15, IL18, and IL21.
  • the secretable effector molecule e.g., “S” in the formula S - C - MT or MT - C - S
  • the cell is further engineered to produce one or more additional membrane-cleavable chimeric proteins including IL 15, IL18, and IL21.
  • a cell is engineered to produce at least one membrane- cleavable chimeric protein where the secretable effector molecule (e.g., “S” in the formula S - C - MT or MT - C - S) is IL12p70 and the cell is further engineered to produce an additional membrane-cleavable chimeric proteins including IL15.
  • the secretable effector molecule e.g., “S” in the formula S - C - MT or MT - C - S
  • the cell is further engineered to produce an additional membrane-cleavable chimeric proteins including IL15.
  • the present disclosure provides for a mixed cell composition
  • a mixed cell composition comprising a first engineered cell that is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule (e.g., “S” in the formula S - C - MT or MT - C - S) is IL- 15, and a second engineered cell that is engineered to produce one or more additional cytokines.
  • a first engineered cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule is IL- 15, and a second engineered cell is engineered to produce IL12, an IL12p70 fusion protein, IL18, or IL21.
  • a first engineered cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule is IL- 15, and a second engineered cell is engineered to produce IL12.
  • a first engineered cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule is IL- 15, and a second engineered cell is engineered to produce an IL12p70 fusion protein.
  • a mixed cell composition comprises a first engineered cell that is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule (e.g., “S” in the formula S - C - MT or MT - C - S) is IL-15, and a second engineered cell that is engineered to produce one or more additional membrane-cleavable chimeric proteins.
  • the secretable effector molecule e.g., “S” in the formula S - C - MT or MT - C - S
  • a first engineered cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule is IL- 15, and a second engineered cell is engineered to produce one or more additional membrane-cleavable chimeric proteins including IL12, an IL12p70 fusion protein, IL18, and IL21.
  • a first engineered cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector is IL 15, and a second engineered cell is engineered to produce an additional membrane-cleavable chimeric proteins including IL12p70.
  • a mixed cell composition comprises a first engineered cell that is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule (e.g., “S” in the formula S - C - MT or MT - C - S) is an IL12p70, and a second engineered cell that is engineered to produce one or more additional cytokines.
  • a first engineered cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule is IL12p70, and a second engineered cell is engineered to produce IL15, IL18, or IL21.
  • a first engineered cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule is IL12p70, and a second engineered cell is engineered to produce IL15.
  • a mixed cell composition comprises a first engineered cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule (e.g., “S” in the formula S - C - MT or MT - C - S) is IL12p70, and a second engineered cell is engineered to produce one or more additional membrane-cleavable chimeric proteins.
  • a first engineered cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule is IL12p70
  • a second engineered cell is engineered to produce one or more additional membrane- cleavable chimeric proteins including IL15, IL18, and IL21.
  • a first engineered cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule is IL12p70, and a second engineered cell is engineered to produce an additional membrane-cleavable chimeric proteins including IL15.
  • a mixed cell composition provided herein can comprise any combination of two or more engineered cells, e.g., any engineered cell described herein.
  • a cell e.g., any engineered cell described herein
  • an immunoresponsive cell is engineered to express a chimeric antigen receptor (CAR) that binds to GPC3.
  • CAR chimeric antigen receptor
  • an immunoresponsive cell is engineered to express an ACP that includes a synthetic transcription factor.
  • a CAR used in accordance with the present disclosure can include an antigen-binding domain, such as an antibody, an antigen-binding fragment of an antibody, a F(ab) fragment, a F(ab') fragment, a single chain variable fragment (scFv), or a single-domain antibody (sdAb).
  • An antigen recognizing receptors can include an scFv.
  • An scFv can include a heavy chain variable domain (VH) and a light chain variable domain (VL), which can be separated by a peptide linker.
  • an scFv can include the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain.
  • the peptide linker is a gly-ser linker.
  • the peptide linker is a (GGGGS)3 linker (SEQ ID NO: 223) comprising the sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 223).
  • nucleic acid sequence encoding SEQ ID NO: 223 is GGCGGCGGAGGATCTGGCGGAGGTGGAAGTGGCGGAGGCGGATCT (SEQ ID NO: 224) or GGCGGCGGAGGAAGCGGAGGCGGAGGATCCGGTGGTGGTGGATCT (SEQ ID NO: 332).
  • a nucleic acid encoding SEQ ID NO: 223 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 224 or SEQ ID NO: 332.
  • a CAR can have one or more intracellular signaling domains, such as a CD3zeta-chain intracellular signaling domain, a CD97 intracellular signaling domain, a CDl la-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD 154 intracellular signaling domain, a CD8 intracellular signaling domain, an 0X40 intracellular signaling domain, a 4- IBB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP 10 intracellular signaling domain, a DAP 12 intracellular signaling domain, a MyD88 intracellular signaling domain, a 2B4 intracellular signaling domain, a CD 16a intracellular signaling domain, a DNAM-1 intracellular signaling domain, a KIR2DS 1
  • a CAR can also comprise a spacer region that links the extracellular antigen-binding domain to the transmembrane domain.
  • the spacer region may be flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition.
  • the spacer region may be a hinge from a human protein.
  • the hinge may be a human Ig (immunoglobulin) hinge, including without limitation an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge.
  • the spacer region may comprise an IgG4 hinge, an IgG2 hinge, an IgD hinge, a CD28 hinge, a KIR2DS2 hinge, an LNGFR hinge, or a PDGFR-beta extracellular linker.
  • the spacer region comprises a sequence from Table 6B. Table 6B.
  • a CAR can have a transmembrane domain, such as a CD8 transmembrane domain, a CD28 transmembrane domain a CD3zeta-chain transmembrane domain, a CD4 transmembrane domain, a 4- IBB transmembrane domain, an 0X40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, a BTLA transmembrane domain, an 0X40 transmembrane domain, a DAP 10 transmembrane domain, a DAP 12 transmembrane domain, a CD 16a transmembrane domain, a DNAM-1 transmembrane domain, a KIR2DS1 transmembrane domain, a KIR3DS 1 transmembrane domain, an NKp44 transme
  • the CAR antigen-binding domain that binds to GPC3 includes a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH includes: a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of KNAMN (SEQ ID NO: 199), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of RIRNKTNNYATYYADSVKA (SEQ ID NO: 200), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of GNSFAY (SEQ ID NO: 201), and wherein the VL includes: a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLLYSSNQKNYLA (SEQ ID NO: 202), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of WASSRES (SEQ ID NO: 203), and
  • the antigen-binding domain that binds to GPC3 includes a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of KNAMN (SEQ ID NO: 199). In some embodiments, the antigenbinding domain that binds to GPC3 includes a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of RIRNKTNNYATYYADSVKA (SEQ ID NO: 200). In some embodiments, the antigen-binding domain that binds to GPC3 includes a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of GNSFAY (SEQ ID NO: 201).
  • CDR-H1 heavy chain complementarity determining region 1
  • the antigenbinding domain that binds to GPC3 includes a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of RIRNKTNNYATYYADSVKA (SEQ ID NO: 200).
  • the antigen-binding domain that binds to GPC3 includes a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLLYSSNQKNYLA (SEQ ID NO: 202). In some embodiments, the antigen-binding domain that binds to GPC3 includes a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of WASSRES (SEQ ID NO: 203). In some embodiments, the antigen-binding domain that binds to GPC3 includes a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQYYNYPLT (SEQ ID NO: 204).
  • the antigen-binding domain that binds to GPC3 includes a VH region having an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of EVQLVETGGGMVQPEGSLKLSCAASGFTFNKNAMNWVRQAPGKGLEWVARIRNKTN NYATYYADSVKARFTISRDDSQSMEYEQMNNEKIEDTAMYYCVAGNSFA YWGQGTEVTVSA (SEQ ID NO: 205) or EVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMNWVRQAPGKGLEWVGRIRNKTNN YATYYADSVKARFTISRDDSKNSLYLQMNSLKTEDTAVYYCVAGNSFAYWGQGTLVT VSA (SEQ ID NO: 206).
  • An exemplary nucleic acid sequence encoding SEQ ID NO: 206 is GAAGTGCAGCTGGTGGAATCTGGCGGAGGACTGGTTCAACCTGGCGGCTCTCTGAG ACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAACAAGAACGCCATGAACTGGGTCCG ACAGGCCCCTGGCAAAGGCCTTGAATGGGTCGGACGGATCCGGAACAAGACCAAC AACTACGCCACCTACTACGCCGACAGCGTGAAGGCCAGGTTCACCATCTCCAGAGA TGACAGCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAAAACCGAGGACACCG CCGTGTACTATTGCGTGGCCGGCAATAGCTTTGCCTACTGGGGACAGGGCACCCTG GTTACAGTTTCTGCT (SEQ ID NO: 222) or GAAGTGCAGCTGGTTGAATCAGGTGGCGGCCTGGTTCAACCTGGCGGATCTCTGAG ACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAACAAGAACGCCATGAACTGG
  • a nucleic acid encoding SEQ ID NO: 206 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 222 or SEQ ID NO: 330.
  • the antigen-binding domain that binds to GPC3 includes a VL region having an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of DIVMSQSPSSLVVSIGEKVTMTCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASS RESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYNYPLTFGAGTKLELK (SEQ ID NO: 207), or DIVMTQSPDSEAVSEGERATINCKSSQSEEYSSNQKNYEAWYQQKPGQPPKEEIYWASS RESGVPDRFSGSGSGTDFTETISSEQAEDVAVYYCQQYYNYPETFGQGTKEEIK (SEQ ID NO: 208).
  • An exemplary nucleic acid sequence encoding SEQ ID NO: 208 is GACATCGTGATGACACAGAGCCCCGATAGCCTGGCCGTGTCTCTGGGAGAAAGAGC CACCATCAACTGCAAGAGCAGCCAGAGCCTGCTGTACTCCAGCAACCAGAAGAACT ACCTGGCCTGGTATCAGCAAAAGCCCGGCCAGCCTCCTAAGCTGCTGATCTATTGG GCCAGCTCCAGAGAAAGCGGCGTGCCCGATAGATTTTCTGGCTCTGGCAGCGGCAC CGACTTCACCCTGACAATTTCTAGCCTGCAAGCCGAGGACGTGGCCGTGTACTACTG CCAGCAGTACTACAACTACCCTCTGACCTTCGGCCAGGGCACCAAGCTGGAAATCA AA (SEQ ID NO: 221) or GACATCGTGATGACACAGAGCCCCGATAGCCTGGCCGTGTCTCTGGGAAAGAGC CACCATCAACTGCAAGAGCAGCCAGAGCCTGCTGTACTCCAGCAACCAGAAGAACT ACCTGGCCTGGTA
  • a nucleic acid encoding SEQ ID NO: 208 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 221 or SEQ ID NO: 336.
  • the ACP of the immunoresponsive cells described herein includes a synthetic transcription factor (SynTF).
  • a synthetic transcription factor is a non-naturally occurring protein that includes a DNA-binding domain and a transcriptional effector domain and is capable of modulating (i.e., activating or repressing) transcription through binding to a cognate promoter recognized by the DNA-binding domain. Cognate promoters that bind synthetic transcription factors (SynTFs) can be referred to as synthetic transcription factor- responsive promoters.
  • the ACP is a transcriptional repressor.
  • the ACP is a transcriptional activator.
  • Immunoresponsive cells can be engineered to comprise any of the engineered nucleic acids described herein (e.g., any of the engineered nucleic acids encoding the cytokines, membrane-cleavable chimeric proteins, and/or CARs described herein). Cells can be engineered to possess any of the features of any of the engineered cells described herein. In a particular aspect, provided herein are cells engineered to produce two cytokines and a CAR, where at least one of the cytokines is membrane-cleavable chimeric protein having the formula S - C - MT or MT - C - S described herein.
  • the engineered immunoresponsive cells include, but are not limited to, a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a viral- specific T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage, a monocyte, a dendritic cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, a mesenchymal stromal cell (MSC), an induced pluripotent stem cell (iPSC), and an iPSC-derived cell.
  • CTL
  • a cell e.g., any engineered cell described herein
  • cells can be transduced to engineer a tumor.
  • a cell is transduced using a virus.
  • the cell is transduced using an oncolytic virus.
  • oncolytic viruses include, but are not limited to, an oncolytic herpes simplex virus, an oncolytic adenovirus, an oncolytic measles virus, an oncolytic influenza virus, an oncolytic Indiana vesiculovirus, an oncolytic Newcastle disease virus, an oncolytic vaccinia virus, an oncolytic poliovirus, an oncolytic myxoma virus, an oncolytic reovirus, an oncolytic mumps virus, an oncolytic Maraba virus, an oncolytic rabies virus, an oncolytic rotavirus, an oncolytic hepatitis virus, an oncolytic rubella virus, an oncolytic dengue virus, an oncolytic chikungunya virus, an oncolytic respiratory syncytial virus, an oncolytic lymphocytic choriomeningitis virus, an oncolytic morbillivirus, an oncolytic lentivirus,
  • the virus can be a recombinant virus that encodes one more transgenes encoding one or more proteins, such as any of the engineered nucleic acids described herein.
  • the virus can be a recombinant virus that encodes one more transgenes encoding one or more of the two or more proteins, such as any of the engineered nucleic acids described herein.
  • Bacterial cells can be engineered to comprise any of the engineered nucleic acids described herein. Bacterial cells can be engineered to possess any of the features of any of the engineered cells described herein. In a particular aspect, provided herein are bacterial cells engineered to produce two or more of the proteins described herein. Bacterial cells can be engineered to produce one or more mammalian-derived proteins. Bacterial cells can be engineered to produce two or more mammalian-derived proteins.
  • Examples of bacterial cells include, but are not limited to, Clostridium beijerinckii, Clostridium sporogenes, Clostridium novyi, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, and Salmonella choleraesuis.
  • An engineered cell can be a human cell.
  • An engineered cell can be a human primary cell.
  • An engineered primary cell can be a tumor infiltrating primary cell.
  • An engineered primary cell can be a primary T cell.
  • An engineered primary cell can be a hematopoietic stem cell (HSC).
  • An engineered primary cell can be a natural killer (NK) cell.
  • An engineered primary cell can be any somatic cell.
  • An engineered primary cell can be a MSC.
  • Human cells e.g., immune cells
  • Human cells can be engineered to comprise any of the engineered nucleic acids described herein.
  • Human cells e.g., immune cells
  • Human cells can be engineered to possess any of the features of any of the engineered cells described herein.
  • human cells e.g., immune cells
  • human cells e.g., immune cells
  • human cells e.g., immune cells
  • An engineered cell can be isolated from a subject (autologous), such as a subject known or suspected to have cancer.
  • Cell isolation methods are known to those skilled in the art and include, but are not limited to, sorting techniques based on cell-surface marker expression, such as FACS sorting, positive isolation techniques, and negative isolation, magnetic isolation, and combinations thereof.
  • An engineered cell can be allogenic with reference to the subject being administered a treatment. Allogenic modified cells can be HLA-matched to the subject being administered a treatment.
  • An engineered cell can be a cultured cell, such as an ex vivo cultured cell.
  • An engineered cell can be an ex vivo cultured cell, such as a primary cell isolated from a subject. Cultured cell can be cultured with one or more cytokines.
  • compositions and methods for engineering immunoresponsive cells to produce one or more proteins of interest e.g., the cytokines, CARs, ACPs, and/or membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein).
  • cells are engineered to produce proteins of interest through introduction (z.e., delivery) of polynucleotides encoding the one or more proteins of interest or effector molecules, e.g., the chimeric proteins described herein including the protein of interest or effector molecule, into the cell’s cytosol and/or nucleus.
  • the polynucleotides encoding the one or more chimeric proteins can be any of the engineered nucleic acids encoding the cytokines, CARs, or membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein.
  • Delivery methods include, but are not limited to, viral-mediated delivery, lipid-mediated transfection, nanoparticle delivery, electroporation, sonication, and cell membrane deformation by physical means.
  • delivery method can depend on the specific cell type to be engineered.
  • Viral vector-based delivery platforms can be used to engineer cells.
  • a viral vector-based delivery platform engineers a cell through introducing (z.e., delivering) into a host cell.
  • a viral vector-based delivery platform can engineer a cell through introducing any of the engineered nucleic acids described herein (e.g., any of the exogenous polynucleotide sequences encoding the cytokines, CARs, ACPs, and/or the membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein, and/or any of the expression cassettes described herein containing a promoter and an exogenous polynucleotide sequence encoding the proteins, oriented from N-terminal to C-terminal).
  • the engineered nucleic acids described herein e.g., any of the exogenous polynucleotide sequences encoding the cytokines, CARs, ACPs, and/
  • a viral vector-based delivery platform can be a nucleic acid, and as such, an engineered nucleic acid can also encompass an engineered virally-derived nucleic acid.
  • engineered virally-derived nucleic acids can also be referred to as recombinant viruses or engineered viruses.
  • a viral vector-based delivery platform can encode more than one engineered nucleic acid, gene, or transgene within the same nucleic acid.
  • an engineered virally- derived nucleic acid e.g., a recombinant virus or an engineered virus
  • can encode one or more transgenes including, but not limited to, any of the engineered nucleic acids described herein that encode one or more of the proteins described herein.
  • the one or more transgenes encoding the one or more proteins can be configured to express the one or more proteins and/or other protein of interest.
  • a viral vector-based delivery platform can encode one or more genes in addition to the one or more transgenes e.g.
  • transgenes encoding the one or more proteins and/or other protein of interest such as viral genes needed for viral infectivity and/or viral production (e.g., capsid proteins, envelope proteins, viral polymerases, viral transcriptases, etc.), referred to as cis-acting elements or genes.
  • a viral vector-based delivery platform can comprise more than one viral vector, such as separate viral vectors encoding the engineered nucleic acids, genes, or transgenes described herein, and referred to as trans-acting elements or genes.
  • a helper-dependent viral vector-based delivery platform can provide additional genes needed for viral infectivity and/or viral production on one or more additional separate vectors in addition to the vector encoding the one or more proteins and/or other protein of interest.
  • One viral vector can deliver more than one engineered nucleic acids, such as one vector that delivers engineered nucleic acids that are configured to produce two or more proteins and/or other protein of interest.
  • More than one viral vector can deliver more than one engineered nucleic acids, such as more than one vector that delivers one or more engineered nucleic acid configured to produce one or more proteins and/or other protein of interest.
  • the number of viral vectors used can depend on the packaging capacity of the above mentioned viral vector-based vaccine platforms, and one skilled in the art can select the appropriate number of viral vectors.
  • any of the viral vector-based systems can be used for the in vitro production of molecules, such as the proteins, effector molecules, and/or other protein of interest described herein, or used in vivo and ex vivo gene therapy procedures, e.g., for in vivo delivery of the engineered nucleic acids encoding one or more proteins and/or other protein of interest.
  • the selection of an appropriate viral vector-based system will depend on a variety of factors, such as cargo/payload size, immunogenicity of the viral system, target cell of interest, gene expression strength and timing, and other factors appreciated by one skilled in the art.
  • Viral vector-based delivery platforms can be RNA-based viruses or DNA-based viruses.
  • Exemplary viral vector-based delivery platforms include, but are not limited to, a herpes simplex virus, a adenovirus, a measles virus, an influenza virus, a Indiana vesiculovirus, a Newcastle disease virus, a vaccinia virus, a poliovirus, a myxoma virus, a reovirus, a mumps virus, a Maraba virus, a rabies virus, a rotavirus, a hepatitis virus, a rubella virus, a dengue virus, a chikungunya virus, a respiratory syncytial virus, a lymphocytic choriomeningitis virus, a morbillivirus, a lentivirus, a replicating retrovirus, a rhabdovirus, a Seneca Valley virus, a Sindbis virus, and any variant or derivative thereof.
  • viral vector-based delivery platforms are described in the art, such as vaccinia, fowlpox, self-replicating alphavirus, marabavirus, adenovirus (See, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616 — 629), or lentivirus, including but not limited to second, third or hybrid second/third generation lentivirus and recombinant lentivirus of any generation designed to target specific cell types or receptors (See, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev.
  • the sequences may be preceded with one or more sequences targeting a subcellular compartment.
  • infected cells i.e., an engineered cell
  • infected cells i.e., an engineered cell
  • Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848.
  • Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351:456- 460 (1991)).
  • BCG vectors are described in Stover et al. (Nature 351:456- 460 (1991)).
  • a wide variety of other vectors useful for the introduction (i.e., delivery) of engineered nucleic acids e.g., Salmonella typhi vectors, and the like will be apparent to those skilled in the art from the description herein.
  • the viral vector-based delivery platforms can be a virus that targets a cell, herein referred to as an oncolytic virus.
  • oncolytic viruses include, but are not limited to, an oncolytic herpes simplex virus, an oncolytic adenovirus, an oncolytic measles virus, an oncolytic influenza virus, an oncolytic Indiana vesiculovirus, an oncolytic Newcastle disease virus, an oncolytic vaccinia virus, an oncolytic poliovirus, an oncolytic myxoma virus, an oncolytic reovirus, an oncolytic mumps virus, an oncolytic Maraba virus, an oncolytic rabies virus, an oncolytic rotavirus, an oncolytic hepatitis virus, an oncolytic rubella virus, an oncolytic dengue virus, an oncolytic chikungunya virus, an oncolytic respiratory syncytial virus, an oncolytic lymphocytic choriomeningitis virus, an oncolytic morbill
  • any of the oncolytic viruses described herein can be a recombinant oncolytic virus comprising one more transgenes (e.g., an engineered nucleic acid) encoding one or more proteins and/or other protein of interest.
  • the transgenes encoding the one or more proteins and/or other protein of interest can be configured to express the proteins and/or other protein of interest.
  • the viral vector-based delivery platform can be retrovirus-based.
  • retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence.
  • the minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the one or more engineered nucleic acids (e.g., transgenes encoding the one or more proteins and/or other protein of interest) into the target cell to provide permanent transgene expression.
  • Retroviral-based delivery systems include, but are not limited to, those based upon murine leukemia, virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency vims (SIV), human immuno deficiency vims (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et ah, J. Virol. 66: 1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et ah, J. Virol. 63:2374-2378 (1989); Miller et al, J, Virol. 65:2220-2224 (1991); PCT/US 94/05700).
  • Other retroviral systems include the Phoenix retrovirus system.
  • the viral vector-based delivery platform can be lentivirus-based.
  • lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers.
  • Lentiviral-based delivery platforms can be HIV -based, such as ViraPower systems (ThermoFisher) or pLenti systems (Cell Biolabs).
  • Lentiviral-based delivery platforms can be SIV, or FIV-based.
  • Other exemplary lentivirus-based delivery platforms are described in more detail in U.S. Pat. Nos.
  • the viral vector-based delivery platform can be adenovirus-based.
  • adenoviral based vectors are capable of very high transduction efficiency in many cell types, do not require cell division, achieve high titer and levels of expression, and can be produced in large quantities in a relatively simple system.
  • adenoviruses can be used for transient expression of a transgene within an infected cell since adenoviruses do not typically integrate into a host’s genome.
  • Adenovirus-based delivery platforms are described in more detail in Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:77007704, 1995; Sakamoto et al., H Gene Ther 5: 1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655, each herein incorporated by reference for all purposes.
  • Other exemplary adenovirus-based delivery platforms are described in more detail in U.S. Pat. Nos.
  • the viral vector-based delivery platform can be adeno-associated virus (AAV)-based.
  • Adeno-associated virus (“AAV”) vectors may be used to transduce cells with engineered nucleic acids (e.g., any of the engineered nucleic acids described herein).
  • AAV systems can be used for the in vitro production of proteins of interest, such as the proteins described herein and/or effector molecules, or used in vivo and ex vivo gene therapy procedures, e.g., for in vivo delivery of the engineered nucleic acids encoding one or more proteins and/or other protein of interest (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. Nos.
  • an AAV-based vector comprises a capsid protein having an amino acid sequence corresponding to any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.RhlO, AAV11 and variants thereof.
  • an AAV-based vector has a capsid protein having an amino acid sequence corresponding to AAV2.
  • an AAV-based vector has a capsid protein having an amino acid sequence corresponding to AAV8.
  • AAV vectors can be engineered to have any of the exogenous polynucleotide sequences encoding the proteins described herein, such as the cytokines, CARs, ACPs, and/or membrane- cleavable chimeric proteins described herein having the formula: S - C - MT or MT - C - S.
  • the viral vector-based delivery platform can be a virus-like particle (VLP) platform.
  • VLPs are constructed by producing viral structural proteins and purifying resulting viral particles. Then, following purification, a cargo/payload (e.g., any of the engineered nucleic acids described herein) is encapsulated within the purified particle ex vivo.
  • VLPs maintains separation of the nucleic acids encoding viral structural proteins and the nucleic acids encoding the cargo/payload.
  • the viral structural proteins used in VLP production can be produced in a variety of expression systems, including mammalian, yeast, insect, bacterial, or in vivo translation expression systems.
  • the purified viral particles can be denatured and reformed in the presence of the desired cargo to produce VLPs using methods known to those skilled in the art. Production of VLPs are described in more detail in Seow et al. (Mol Ther. 2009 May; 17(5): 767-777), herein incorporated by reference for all purposes.
  • the viral vector-based delivery platform can be engineered to target (z.e., infect) a range of cells, target a narrow subset of cells, or target a specific cell.
  • the envelope protein chosen for the viral vector-based delivery platform will determine the viral tropism.
  • the virus used in the viral vector-based delivery platform can be pseudotyped to target a specific cell of interest.
  • the viral vector-based delivery platform can be pantropic and infect a range of cells.
  • pantropic viral vector-based delivery platforms can include the VSV-G envelope.
  • the viral vector-based delivery platform can be amphotropic and infect mammalian cells. Accordingly, one skilled in the art can select the appropriate tropism, pseudotype, and/or envelope protein for targeting a desired cell type.
  • Engineered nucleic acids can be introduced into a cell using a lipid-mediated delivery system.
  • a lipid-mediated delivery system uses a structure composed of an outer lipid membrane enveloping an internal compartment.
  • lipid-based structures include, but are not limited to, a lipid-based nanoparticle, a liposome, a micelle, an exosome, a vesicle, an extracellular vesicle, a cell, or a tissue.
  • Lipid structure delivery systems can deliver a cargo/payload (e.g., any of the engineered nucleic acids described herein) in vitro, in vivo, or ex vivo.
  • a lipid-based nanoparticle can include, but is not limited to, a unilamellar liposome, a multilamellar liposome, and a lipid preparation.
  • a “liposome” is a generic term encompassing in vitro preparations of lipid vehicles formed by enclosing a desired cargo, e.g., an engineered nucleic acid, such as any of the engineered nucleic acids described herein, within a lipid shell or a lipid aggregate.
  • Liposomes may be characterized as having vesicular structures with a bilayer membrane, generally comprising a phospholipid, and an inner medium that generally comprises an aqueous composition.
  • Liposomes include, but are not limited to, emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes can be unilamellar liposomes. Liposomes can be multilamellar liposomes. Liposomes can be multivesicular liposomes. Liposomes can be positively charged, negatively charged, or neutrally charged. In certain embodiments, the liposomes are neutral in charge. Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol.
  • lipids are generally guided by consideration of a desired purpose, e.g., criteria for in vivo delivery, such as liposome size, acid lability and stability of the liposomes in the blood stream.
  • criteria for in vivo delivery such as liposome size, acid lability and stability of the liposomes in the blood stream.
  • a variety of methods are available for preparing liposomes, as described in, e.g., Szokan et al., Ann. Rev. Biophys. Bioeng. 9; 467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369, each herein incorporated by reference for all purposes.
  • a multilamellar liposome is generated spontaneously when lipids comprising phospholipids are suspended in an excess of aqueous solution such that multiple lipid layers are separated by an aqueous medium. Water and dissolved solutes are entrapped in closed structures between the lipid bilayers following the lipid components undergoing self-rearrangement.
  • a desired cargo e.g., a polypeptide, a nucleic acid, a small molecule drug, an engineered nucleic acid, such as any of the engineered nucleic acids described herein, a viral vector, a viral-based delivery system, etc.
  • a desired cargo can be encapsulated in the aqueous interior of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the polypeptide/nucleic acid, interspersed within the lipid bilayer of a liposome, entrapped in a liposome, complexed with a liposome, or otherwise associated with the liposome such that it can be delivered to a target entity.
  • Lipophilic molecules or molecules with lipophilic regions may also dissolve in or associate with the lipid bilayer.
  • a liposome used according to the present embodiments can be made by different methods, as would be known to one of ordinary skill in the art. Preparations of liposomes are described in further detail in WO 2016/201323, International Applications PCT/US85/01161 and PCT/US 89/05040, and U.S. Patents 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706; each herein incorporated by reference for all purposes.
  • Liposomes can be cationic liposomes. Examples of cationic liposomes are described in more detail in U.S. Patent No. 5,962,016; 5,030,453; 6,680,068, U.S. Application 2004/0208921, and International Patent Applications W003/015757A1, WO04029213A2, and W002/100435A1, each hereby incorporated by reference in their entirety. Lipid-mediated gene delivery methods are described, for instance, in WO 96/18372; WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Pat. No. 5,279,833 Rose U.S. Pat. No. 5,279,833; W091/06309; and Feigner etal., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987), each herein incorporated by reference for all purposes.
  • Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment following fusion of multivesicular bodies with the plasma membrane.
  • the size of exosomes ranges between 30 and 100 nm in diameter.
  • Their surface consists of a lipid bilayer from the donor cell's cell membrane, and they contain cytosol from the cell that produced the exosome, and exhibit membrane proteins from the parental cell on the surface.
  • Exosomes useful for the delivery of nucleic acids are known to those skilled in the art, e.g., the exosomes described in more detail in U.S. Pat. No. 9,889,210, herein incorporated by reference for all purposes.
  • extracellular vesicle refers to a cell-derived vesicle comprising a membrane that encloses an internal space.
  • extracellular vesicles comprise all membrane-bound vesicles that have a smaller diameter than the cell from which they are derived.
  • extracellular vesicles range in diameter from 20 nm to 1000 nm, and can comprise various macromolecular cargo either within the internal space, displayed on the external surface of the extracellular vesicle, and/or spanning the membrane.
  • the cargo can comprise nucleic acids (e.g., any of the engineered nucleic acids described herein), proteins, carbohydrates, lipids, small molecules, and/or combinations thereof.
  • extracellular vesicles include apoptotic bodies, fragments of cells, vesicles derived from cells by direct or indirect manipulation (e.g., by serial extrusion or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of the late endosome with the plasma membrane).
  • Extracellular vesicles can be derived from a living or dead organism, explanted tissues or organs, and/or cultured cells.
  • exosome refers to a cell-derived small (between 20-300 nm in diameter, more preferably 40-200 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from the cell by direct plasma membrane budding or by fusion of the late endosome with the plasma membrane.
  • the exosome comprises lipid or fatty acid and polypeptide and optionally comprises a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, such as any of the engineered nucleic acids described herein), a sugar (e.g., a simple sugar, polysaccharide, or glycan) or other molecules.
  • the exosome can be derived from a producer cell, and isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof. An exosome is a species of extracellular vesicle. Generally, exosome production/biogenesis does not result in the destruction of the producer cell. Exosomes and preparation of exosomes are described in further detail in WO 2016/201323, which is hereby incorporated by reference in its entirety.
  • nanovesicle refers to a cell-derived small (between 20-250 nm in diameter, more preferably 30-150 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from the cell by direct or indirect manipulation such that said nanovesicle would not be produced by said producer cell without said manipulation.
  • a nanovesicle is a sub-species of an extracellular vesicle.
  • Appropriate manipulations of the producer cell include but are not limited to serial extrusion, treatment with alkaline solutions, sonication, or combinations thereof.
  • populations of nanovesicles are substantially free of vesicles that are derived from producer cells by way of direct budding from the plasma membrane or fusion of the late endosome with the plasma membrane.
  • the nanovesicle comprises lipid or fatty acid and polypeptide, and optionally comprises a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, such as any of the engineered nucleic acids described herein), a sugar (e.g., a simple sugar, polysaccharide, or glycan) or other molecules.
  • a payload e.g., a therapeutic agent
  • a receiver e.g., a targeting moiety
  • a polynucleotide e.g., a nucleic acid, RNA, or DNA, such as any of the engineered nucleic acids described herein
  • a sugar e.g., a simple sugar, polysaccharide, or glycan
  • the nanovesicle once it is derived from a producer cell according to said manipulation, may be isolated
  • Lipid nanoparticles in general, are synthetic lipid structures that rely on the amphiphilic nature of lipids to form membranes and vesicle like structures (Riley 2017). In general, these vesicles deliver cargo/pay loads, such as any of the engineered nucleic acids or viral systems described herein, by absorbing into the membrane of target cells and releasing the cargo into the cytosol. Lipids used in LNP formation can be cationic, anionic, or neutral. The lipids can be synthetic or naturally derived, and in some instances biodegradable.
  • Lipids can include fats, cholesterol, phospholipids, lipid conjugates including, but not limited to, polyethyleneglycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, and fat soluble vitamins.
  • Lipid compositions generally include defined mixtures of materials, such as the cationic, neutral, anionic, and amphipathic lipids. In some instances, specific lipids are included to prevent LNP aggregation, prevent lipid oxidation, or provide functional chemical groups that facilitate attachment of additional moieties. Lipid composition can influence overall LNP size and stability.
  • the lipid composition comprises dilinoleylmethyl- 4- dimethylaminobutyrate (MC3) or MC3-like molecules.
  • MC3 and MC3-like lipid compositions can be formulated to include one or more other lipids, such as a PEG or PEG-conjugated lipid, a sterol, or neutral lipids.
  • LNPs can be further engineered or functionalized to facilitate targeting of specific cell types. Another consideration in LNP design is the balance between targeting efficiency and cytotoxicity.
  • Micelles in general, are spherical synthetic lipid structures that are formed using singlechain lipids, where the single-chain lipid’s hydrophilic head forms an outer layer or membrane and the single-chain lipid’s hydrophobic tails form the micelle center.
  • Micelles typically refer to lipid structures only containing a lipid mono-layer. Micelles are described in more detail in Quader et al. (Mol Ther. 2017 Jul 5; 25(7): 1501-1513), herein incorporated by reference for all purposes.
  • Nucleic-acid vectors such as expression vectors, exposed directly to serum can have several undesirable consequences, including degradation of the nucleic acid by serum nucleases or off-target stimulation of the immune system by the free nucleic acids.
  • viral delivery systems exposed directly to serum can trigger an undesired immune response and/or neutralization of the viral delivery system. Therefore, encapsulation of an engineered nucleic acid and/or viral delivery system can be used to avoid degradation, while also avoiding potential off-target affects.
  • an engineered nucleic acid and/or viral delivery system is fully encapsulated within the delivery vehicle, such as within the aqueous interior of an LNP.
  • Encapsulation of an engineered nucleic acid and/or viral delivery system within an LNP can be carried out by techniques well-known to those skilled in the art, such as microfluidic mixing and droplet generation carried out on a microfluidic droplet generating device.
  • Such devices include, but are not limited to, standard T-junction devices or flow-focusing devices.
  • the desired lipid formulation such as MC3 or MC3-like containing compositions, is provided to the droplet generating device in parallel with an engineered nucleic acid or viral delivery system and any other desired agents, such that the delivery vector and desired agents are fully encapsulated within the interior of the MC3 or MC3-like based LNP.
  • the droplet generating device can control the size range and size distribution of the LNPs produced.
  • the LNP can have a size ranging from 1 to 1000 nanometers in diameter, e.g., 1, 10, 50, 100, 500, or 1000 nanometers.
  • the delivery vehicles encapsulating the cargo/payload e.g., an engineered nucleic acid and/or viral delivery system
  • the cargo/payload can be further treated or engineered to prepare them for administration.
  • Nanomaterials can be used to deliver engineered nucleic acids (e.g., any of the engineered nucleic acids described herein).
  • Nanomaterial vehicles can be made of non-immunogenic materials and generally avoid eliciting immunity to the delivery vector itself. These materials can include, but are not limited to, lipids (as previously described), inorganic nanomaterials, and other polymeric materials. Nanomaterial particles are described in more detail in Riley et al. (Recent Advances in Nanomaterials for Gene Delivery — A Review. Nanomaterials 2017, 7(5), 94), herein incorporated by reference for all purposes.
  • a genomic editing system can be used to engineer a host genome to encode an engineered nucleic acid, such as an engineered nucleic acid encoding the cytokines, CARs, ACPs, and/or membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein.
  • a “genomic editing system” refers to any system for integrating an exogenous gene into a host cell’s genome. Genomic editing systems include, but are not limited to, a transposon system, a nuclease genomic editing system, and a viral vectorbased delivery platform.
  • a transposon system can be used to integrate an engineered nucleic acid, such as the cytokines, CARs, ACPs, and/or membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein, into a host genome.
  • Transposons generally comprise terminal inverted repeats (TIR) that flank a cargo/payload nucleic acid and a transposase.
  • the transposon system can provide the transposon in cis or in trans with the TIR-flanked cargo.
  • a transposon system can be a retrotransposon system or a DNA transposon system.
  • transposon systems integrate a cargo/payload (e.g., an engineered nucleic acid) randomly into a host genome.
  • a cargo/payload e.g., an engineered nucleic acid
  • transposon systems include systems using a transposon of the Tcl/mariner transposon superfamily, such as a Sleeping Beauty transposon system, described in more detail in Hudecek et al. (Crit Rev Biochem Mol Biol. 2017 Aug;52(4):355-380), and U.S. Patent Nos. 6,489,458, 6,613,752 and 7,985,739, each of which is herein incorporated by reference for all purposes.
  • Another example of a transposon system includes a PiggyBac transposon system, described in more detail in U.S. Patent Nos. 6,218,185 and 6,962,810, each of which is herein incorporated by reference for all purposes.
  • a nuclease genomic editing system can be used to engineer a host genome to encode an engineered nucleic acid, such as an engineered nucleic acid encoding the cytokines, CARs, ACPs, and/or the membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein.
  • an engineered nucleic acid such as an engineered nucleic acid encoding the cytokines, CARs, ACPs, and/or the membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein.
  • the nuclease- mediated gene editing systems used to introduce an exogenous gene take advantage of a cell’s natural DNA repair mechanisms, particularly homologous recombination (HR) repair pathways.
  • HR homologous recombination
  • a cell can resolve the insult by using another DNA source that has identical, or substantially identical, sequences at both its 5’ and 3’ ends as a template during DNA synthesis to repair the lesion.
  • HDR can use the other chromosome present in a cell as a template.
  • exogenous polynucleotides are introduced into the cell to be used as a homologous recombination template (HRT or HR template).
  • any additional exogenous sequence not originally found in the chromosome with the lesion that is included between the 5’ and 3’ complimentary ends within the HRT can be incorporated (z.e., “integrated”) into the given genomic locus during templated HDR.
  • a typical HR template for a given genomic locus has a nucleotide sequence identical to a first region of an endogenous genomic target locus, a nucleotide sequence identical to a second region of the endogenous genomic target locus, and a nucleotide sequence encoding a cargo/payload nucleic acid (e.g., any of the engineered nucleic acids described herein, such as any of the engineered nucleic acids encoding the cytokines, CARs, ACPs, and/or membrane- cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein).
  • a cargo/payload nucleic acid e.g., any of the engineered nucleic acids described herein, such as any of the engineered nucleic acids encoding the cytokines, CARs, ACPs, and/or membrane- cleavable chimeric proteins having the formula S - C - MT or
  • a HR template can be linear.
  • linear HR templates include, but are not limited to, a linearized plasmid vector, a ssDNA, a synthesized DNA, and a PCR amplified DNA.
  • a HR template can be circular, such as a plasmid.
  • a circular template can include a supercoiled template.
  • HR arms The identical, or substantially identical, sequences found at the 5’ and 3’ ends of the HR template, with respect to the exogenous sequence to be introduced, are generally referred to as arms (HR arms).
  • HR arms can be identical to regions of the endogenous genomic target locus (z.e., 100% identical).
  • HR arms in some examples can be substantially identical to regions of the endogenous genomic target locus. While substantially identical HR arms can be used, it can be advantageous for HR arms to be identical as the efficiency of the HDR pathway may be impacted by HR arms having less than 100% identity.
  • Each HR arm i.e., the 5’ and 3’ HR arms, can be the same size or different sizes. Each HR arm can each be greater than or equal to 50, 100, 200, 300, 400, or 500 bases in length. Although HR arms can, in general, be of any length, practical considerations, such as the impact of HR arm length and overall template size on overall editing efficiency, can also be taken into account.
  • An HR arms can be identical, or substantially identical to, regions of an endogenous genomic target locus immediately adjacent to a cleavage site. Each HR arms can be identical to, or substantially identical to, regions of an endogenous genomic target locus immediately adjacent to a cleavage site.
  • Each HR arms can be identical, or substantially identical to, regions of an endogenous genomic target locus within a certain distance of a cleavage site, such as 1 base-pair, less than or equal to 10 base-pairs, less than or equal to 50 base-pairs, or less than or equal to 100 base-pairs of each other.
  • a nuclease genomic editing system can use a variety of nucleases to cut a target genomic locus, including, but not limited to, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) family nuclease or derivative thereof, a Transcription activator-like effector nuclease (TALEN) or derivative thereof, a zinc-finger nuclease (ZFN) or derivative thereof, and a homing endonuclease (HE) or derivative thereof.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • TALEN Transcription activator-like effector nuclease
  • ZFN zinc-finger nuclease
  • HE homing endonuclease
  • a CRISPR-mediated gene editing system can be used to engineer a host genome to encode an engineered nucleic acid, such as an engineered nucleic acid encoding the cytokines, CARs, ACPs, and/or membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein.
  • CRISPR systems are described in more detail in M. Adli (“The CRISPR tool kit for genome editing and beyond” Nature Communications; volume 9 (2016), Article number: 1911), herein incorporated by reference for all that it teaches.
  • a CRISPR-mediated gene editing system comprises a CRISPR-associated (Cas) nuclease and a RNA(s) that directs cleavage to a particular target sequence.
  • An exemplary CRISPR-mediated gene editing system is the CRISPR/Cas9 systems comprised of a Cas9 nuclease and a RNA(s) that has a CRISPR RNA (crRNA) domain and a trans-activating CRISPR (tracrRNA) domain.
  • the crRNA typically has two RNA domains: a guide RNA sequence (gRNA) that directs specificity through base-pair hybridization to a target sequence (“a defined nucleotide sequence”), e.g., a genomic sequence; and an RNA domain that hybridizes to a tracrRNA.
  • a tracrRNA can interact with and thereby promote recruitment of a nuclease (e.g., Cas9) to a genomic locus.
  • the crRNA and tracrRNA polynucleotides can be separate polynucleotides.
  • the crRNA and tracrRNA polynucleotides can be a single polynucleotide, also referred to as a single guide RNA (sgRNA).
  • Nucleases can include derivatives thereof, such as Cas9 functional mutants, e.g., a Cas9 “nickase” mutant that in general mediates cleavage of only a single strand of a defined nucleotide sequence as opposed to a complete double- stranded break typically produced by Cas9 enzymes.
  • each component can be separately produced and used to form the RNP complex.
  • each component can be separately produced in vitro and contacted (i.e., “complexed”) with each other in vitro to form the RNP complex.
  • the in vitro produced RNP can then be introduced i.e., “delivered”) into a cell’s cytosol and/or nucleus, e.g., a T cell’s cytosol and/or nucleus.
  • the in vitro produced RNP complexes can be delivered to a cell by a variety of means including, but not limited to, electroporation, lipid-mediated transfection, cell membrane deformation by physical means, lipid nanoparticles (LNP), virus like particles (VLP), and sonication.
  • in vitro produced RNP complexes can be delivered to a cell using a Nucleofactor/Nucleofection® electroporation-based delivery system (Lonza®).
  • Other electroporation systems include, but are not limited to, MaxCyte electroporation systems, Miltenyi CliniMACS electroporation systems, Neon electroporation systems, and BTX electroporation systems.
  • CRISPR nucleases e.g., Cas9
  • Cas9 can be produced in vitro (i.e., synthesized and purified) using a variety of protein production techniques known to those skilled in the art.
  • CRISPR system RNAs e.g., an sgRNA
  • RNA production techniques such as in vitro transcription or chemical synthesis.
  • An in vitro produced RNP complex can be complexed at different ratios of nuclease to gRNA.
  • An in vitro produced RNP complex can also be used at different amounts in a CRISPR- mediated editing system. For example, depending on the number of cells desired to be edited, the total RNP amount added can be adjusted, such as a reduction in the amount of RNP complex added when editing a large number of cells in a reaction.
  • each component e.g., Cas9 and an sgRNA
  • each component can be separately encoded by a polynucleotide with each polynucleotide introduced into a cell together or separately.
  • each component can be encoded by a single polynucleotide (i.e., a multi-promoter or multicistronic vector, see description of exemplary multicistronic systems below) and introduced into a cell.
  • a single polynucleotide i.e., a multi-promoter or multicistronic vector, see description of exemplary multicistronic systems below
  • an RNP complex can form within the cell and can then direct site-specific cleavage.
  • RNPs can be engineered to have moieties that promote delivery of the RNP into the nucleus.
  • a Cas9 nuclease can have a nuclear localization signal (NLS) domain such that if a Cas9 RNP complex is delivered into a cell’s cytosol or following translation of Cas9 and subsequent RNP formation, the NLS can promote further trafficking of a Cas9 RNP into the nucleus.
  • NLS nuclear localization signal
  • the engineered cells described herein can be engineered using non-viral methods, e.g., the nuclease and/or CRISPR mediated gene editing systems described herein can be delivered to a cell using non-viral methods.
  • the engineered cells described herein can be engineered using viral methods, e.g., the nuclease and/or CRISPR mediated gene editing systems described herein can be delivered to a cell using viral methods such as adenoviral, retroviral, lentiviral, or any of the other viral-based delivery methods described herein.
  • more than one CRISPR composition can be provided such that each separately target the same gene or general genomic locus at more than target nucleotide sequence.
  • two separate CRISPR compositions can be provided to direct cleavage at two different target nucleotide sequences within a certain distance of each other.
  • more than one CRISPR composition can be provided such that each separately target opposite strands of the same gene or general genomic locus.
  • two separate CRISPR “nickase” compositions can be provided to direct cleavage at the same gene or general genomic locus at opposite strands.
  • TALEN is an engineered site-specific nuclease, which is composed of the DNA- binding domain of TALE (transcription activator-like effectors) and the catalytic domain of restriction endonuclease Fokl.
  • TALE transcription activator-like effectors
  • Fokl restriction endonuclease Fokl
  • engineered nucleic acids e.g., any of the engineered nucleic acids described herein
  • a cell or other target recipient entity such as any of the lipid structures described herein.
  • Electroporation can used to deliver polynucleotides to recipient entities. Electroporation is a method of internalizing a cargo/payload into a target cell or entity’s interior compartment through applying an electrical field to transiently permeabilize the outer membrane or shell of the target cell or entity. In general, the method involves placing cells or target entities between two electrodes in a solution containing a cargo of interest (e.g., any of the engineered nucleic acids described herein). The lipid membrane of the cells is then disrupted, i.e., permeabilized, by applying a transient set voltage that allows the cargo to enter the interior of the entity, such as the cytoplasm of the cell. In the example of cells, at least some, if not a majority, of the cells remain viable.
  • a cargo of interest e.g., any of the engineered nucleic acids described herein.
  • the lipid membrane of the cells is then disrupted, i.e., permeabilized, by applying a transient set voltage that allows the
  • Electroporation conditions e.g., number of cells, concentration of cargo, recovery conditions, voltage, time, capacitance, pulse type, pulse length, volume, cuvette length, electroporation solution composition, etc.
  • Electroporation conditions vary depending on several factors including, but not limited to, the type of cell or other recipient entity, the cargo to be delivered, the efficiency of internalization desired, and the viability desired. Optimization of such criteria are within the scope of those skilled in the art.
  • a variety devices and protocols can be used for electroporation. Examples include, but are not limited to, Neon® Transfection System, MaxCyte® Flow ElectroporationTM, Lonza® NucleofectorTM systems, and Bio-Rad® electroporation systems.
  • engineered nucleic acids e.g., any of the engineered nucleic acids described herein
  • a cell or other target recipient entity include, but are not limited to, sonication, gene gun, hydrodynamic injection, and cell membrane deformation by physical means.
  • compositions and methods for delivering engineered mRNAs in vivo are described in detail in Kowalski et al. (Mol Ther. 2019 Apr 10; 27(4): 710-728) and Kaczmarek et al. (Genome Med. 2017; 9: 60.), each herein incorporated by reference for all purposes.
  • compositions for delivering a cargo/payload (a “delivery vehicle”).
  • the cargo can comprise nucleic acids (e.g., any of the engineered nucleic acids described herein, such as any of the engineered nucleic acids described herein encoding the cytokines, CARs, ACPs, and/or membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein), as described above.
  • the cargo can comprise proteins, carbohydrates, lipids, small molecules, and/or combinations thereof.
  • the delivery vehicle can comprise any composition suitable for delivering a cargo.
  • the delivery vehicle can comprise any composition suitable for delivering a protein (e.g., any of the proteins described herein).
  • the delivery vehicle can be any of the lipid structure delivery systems described herein.
  • a delivery vehicle can be a lipid-based structure including, but not limited to, a lipid-based nanoparticle, a liposome, a micelle, an exosome, a vesicle, an extracellular vesicle, a cell, or a tissue.
  • the delivery vehicle can be any of the nanoparticles described herein, such as nanoparticles comprising lipids (as previously described), inorganic nanomaterials, and other polymeric materials.
  • the delivery vehicle can be capable of delivering the cargo to a cell, such as delivering any of the proteins described herein to a cell.
  • the delivery vehicle can be capable of delivering the cargo to a cell, such as delivering any of the proteins described herein to a cell.
  • the delivery vehicle can be configured to target a specific cell, such as configured with a re-directing antibody to target a specific cell.
  • the delivery vehicle can be capable of delivering the cargo to a cell in vivo.
  • the delivery vehicle can be capable of delivering the cargo to a tissue or tissue environment (e.g., a tumor microenvironment), such as delivering any of the proteins described herein to a tissue or tissue environment in vivo.
  • Delivering a cargo can include secreting the cargo, such as secreting any of the proteins described herein.
  • the delivery vehicle can be capable of secreting the cargo, such as secreting any of the proteins described herein.
  • the delivery vehicle can be capable of secreting the cargo to a tissue or tissue environment (e.g., a tumor microenvironment), such as secreting any of the proteins described herein into a tissue or tissue environment.
  • the delivery vehicle can be configured to target a specific tissue or tissue environment (e.g., a tumor microenvironment), such as configured with a re-directing antibody to target a specific tissue or tissue environment.
  • a subject e.g., a human subject
  • engineered cells including mixed cell compositions, as provided herein to produce in vivo at least one protein of interest produced by the engineered cells (e.g., any of the cytokines, CARs, ACPs, and/or membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein, or the secreted effector molecules provided for herein following protease cleavage of the chimeric protein).
  • a subject e.g., a human subject
  • engineered cells including mixed cell compositions, as provided herein to produce in vivo at least one protein of interest produced by the engineered cells (e.g., any of the cytokines, CARs, ACPs, and/or membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein, or the secreted effector molecules provided for herein following prote
  • methods that include delivering, or administering, to a subject (e.g., a human subject) engineered cells as provided herein to produce in vivo at least two proteins of interest, e.g., at least two of the cytokines, CARs, ACPs, and/or membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein, produced by the engineered cells.
  • a subject e.g., a human subject
  • engineered cells as provided herein to produce in vivo at least two proteins of interest, e.g., at least two of the cytokines, CARs, ACPs, and/or membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein, produced by the engineered cells.
  • methods that include delivering, or administering, to a subject (e.g., a human subject) any of the delivery vehicles described herein, such as any of the delivery vehicles described herein comprising any of the proteins of interest described herein, e.g., any of the cytokines, CARs, ACPs, and/or membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein.
  • a subject e.g., a human subject
  • any of the delivery vehicles described herein comprising any of the proteins of interest described herein, e.g., any of the cytokines, CARs, ACPs, and/or membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein.
  • methods that include delivering, or administering, to a subject (e.g., a human subject) any of the delivery vehicles described herein, such as any of the delivery vehicles described herein comprising two or more proteins of, e.g., at least two of the cytokines, CARs, ACPs, and/or the membrane- cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein.
  • a subject e.g., a human subject
  • any of the delivery vehicles described herein comprising two or more proteins of, e.g., at least two of the cytokines, CARs, ACPs, and/or the membrane- cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein.
  • the engineered cells or delivery vehicles are administered via intravenous, intraperitoneal, intratracheal, subcutaneous, intratumoral, oral, anal, intranasal (e.g., packed in a delivery particle), or arterial (e.g., internal carotid artery) routes.
  • the engineered cells or delivery vehicles may be administered systemically or locally (e.g., to a TME or via intratumoral administration).
  • An engineered cell can be isolated from a subject, such as a subject known or suspected to have cancer.
  • An engineered cell can be allogenic with reference to the subject being administered a treatment. Allogenic modified cells can be HLA- matched to the subject being administered a treatment.
  • Delivery vehicles can be any of the lipid structure delivery systems described herein. Delivery vehicles can be any of the nanoparticles described herein.
  • Engineered cells or delivery vehicles can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • engineered cells or delivery vehicles can be administered in combination with one or more IMiDs described herein.
  • FDA-approved IMiDs can be administered in their approved fashion.
  • engineered cells or delivery vehicles can be administered in combination with a checkpoint inhibitor therapy.
  • checkpoint inhibitors include, but are not limited to, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-PD-L2 antibodies, anti- CTLA-4 antibodies, anti-LAG-3 antibodies, anti-TIM-3 antibodies, anti-TIGIT antibodies, anti- VISTA antibodies, anti-KIR antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti- HVEM antibodies, anti-BTLA antibodies, anti-GAL9 antibodies, anti-A2AR antibodies, antiphosphatidylserine antibodies, anti-CD27 antibodies, anti-TNFa antibodies, anti-TREMl antibodies, and anti-TREM2 antibodies.
  • Illustrative immune checkpoint inhibitors include pembrolizumab (anti-PD-1; MK-3475/Keytruda® - Merck), nivolumamb (anti-PD-1; Opdivo® - BMS), pidilizumab (anti-PD-1 antibody; CT-011 - Teva/CureTech), AMP224 (anti-PD-1; NCI), avelumab (anti-PD-Ll; Bavencio® - Pfizer), durvalumab (anti-PD-Ll; MEDI4736/Imfinzi® - Medimmune/AstraZeneca), atezolizumab (anti-PD-Ll; Tecentriq® - Roche/Genentech), BMS- 936559 (anti-PD-Ll - BMS), tremelimumab (anti-CTLA-4; Medimmune/AstraZeneca), ipilimumab (anti-CTLA-4; Yervoy ®
  • Some methods comprise selecting a subject (or patient population) having a tumor (or cancer) and treating that subject with engineered cells or delivery vehicles that modulate tumor- mediated immunosuppressive mechanisms.
  • the engineered cells or delivery vehicles of the present disclosure may be used, in some instances, to treat cancer, such as ovarian cancer. Other cancers are described herein.
  • the engineered cells may be used to treat bladder tumors, brain tumors, breast tumors, cervical tumors, colorectal tumors, esophageal tumors, gliomas, kidney tumors, liver tumors, lung tumors, melanomas, ovarian tumors, pancreatic tumors, prostate tumors, skin tumors, thyroid tumors, and/or uterine tumors.
  • the engineered cells or delivery vehicles of the present disclosure can be used to treat cancers with tumors located in the peritoneal space of a subject.
  • the methods provided herein also include delivering a preparation of engineered cells or delivery vehicles.
  • a preparation in some embodiments, is a substantially pure preparation, containing, for example, less than 5% (e.g., less than 4%, 3%, 2%, or 1%) of cells other than engineered cells.
  • a preparation may comprise IxlO 5 cells/kg to IxlO 7 cells/kg cells.
  • Preparation of engineered cells or delivery vehicles can include pharmaceutical compositions having one or more pharmaceutically acceptable carriers.
  • preparations of engineered cells or delivery vehicles can include any of the engineered viruses, such as an engineered AAV virus, or any of the engineered viral vectors, such as AAV vector, described herein.
  • the methods provided herein also include delivering a composition in vivo capable of producing the engineered cells described herein, e.g., capable of delivering any of the engineered nucleic acids described herein to a cell in vivo.
  • compositions include any of the viral-mediated delivery platforms, any of the lipid structure delivery systems, any of the nanoparticle delivery systems, any of the genomic editing systems, or any of the other engineering delivery systems described herein capable of engineering a cell in vivo.
  • the methods provided herein also include delivering a composition in vivo capable of producing any of the proteins of interest described herein, e.g., any of the cytokines, CARs, ACPs, and/or membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein.
  • the methods provided herein also include delivering a composition in vivo capable of producing two or more of the proteins of interest described herein.
  • Compositions capable of in vivo production of proteins of interest include, but are not limited to, any of the engineered nucleic acids described herein.
  • Compositions capable of in vivo production proteins of interest can be a naked mRNA or a naked plasmid.
  • HIV-1 protease SEQ ID NO: 1444:
  • Angiotensin converting enzyme (SEQ ID NO: 156):
  • GRR (residues 352-408 of human pl05; SEQ ID NO: 162):
  • SNS tandem repeat of SP2 and NB (SP2-NB-SP2 of influenza A or influenza B; e.g., SEQ ID NO: 1
  • RPB (four copies of residues 1688-1702 of yeast RPB; SEQ ID NO: 165):
  • SPmix tandem repeat of SP1 and SP2 (SP2-SP1-SP2-SP1-SP2 of influenza A virus M2 protein;
  • NS2 (three copies of residues 79-93 of influenza A virus NS protein; SEQ ID NO: 167):
  • LIEEVRHRLKTTENSGSLIEEVRHRLKTTENSGSLIEEVRHRLKTTENSGS ODC (residues 106-142 of ornithine decarboxylase; SEQ ID NO: 168):
  • FPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV mouse ODC (residues 422-461; SEQ ID NO: 169):
  • Embodiment 1 An immunoresponsive cell comprising:
  • a first engineered nucleic acid comprising a first expression cassette comprising a first promoter operably linked to a first, exogenous polynucleotide sequence encoding a first cytokine, and a second expression cassette comprising a second promoter operably linked to a second exogenous polynucleotide sequence encoding a chimeric antigen receptor (CAR) that binds to GPC3; and
  • CAR chimeric antigen receptor
  • a second engineered nucleic acid comprising a third expression cassette comprising an activation-conditional control polypeptide (ACP)-responsive promoter operably linked to a third exogenous polynucleotide sequence encoding a second cytokine, and a fourth expression cassette comprising a fourth promoter operably linked to a fourth exogenous polynucleotide sequence encoding an ACP, wherein the ACP comprises a synthetic transcription factor comprising a DNA-binding domain and a transcriptional effector domain, wherein the ACP is capable of inducing expression of the third exogenous polynucleotide sequence by binding to the ACP-responsive promoter, wherein at least one of the first exogenous polynucleotide sequence and the third exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula:
  • S comprises a secretable effector molecule comprising the first and/or second cytokine
  • C comprises a protease cleavage site
  • MT comprises a cell membrane tethering domain
  • S - C - MT or MT - C - S is configured to be expressed as a single polypeptide.
  • Embodiment 2 The immunoresponsive cell of embodiment 1, wherein the first expression cassette is configured to be transcribed in an opposite orientation relative to transcription of the second expression cassette.
  • Embodiment 3 The immunoresponsive cell of embodiment 2, wherein the first expression cassette and the second expression cassette are oriented within the first engineered nucleic acid in a head-to-head directionality.
  • Embodiment 4 The immunoresponsive cell of embodiment 1, wherein the first expression cassette is configured to be transcribed in a same orientation relative to the transcription of the second expression cassette.
  • Embodiment 5 The immunoresponsive cell of embodiment 4, wherein the first expression cassette and the second expression cassette are oriented within the first engineered nucleic acid in a head-to-tail directionality.
  • Embodiment 6 The immunoresponsive cell of any one of embodiments 1-5, wherein the first promoter comprises a constitutive promoter, an inducible promoter, or a synthetic promoter.
  • Embodiment 7 The immunoresponsive cell of embodiment 6, wherein the first promoter is a constitutive promoter selected from the group consisting of: CAG, HLP, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.
  • the first promoter is a constitutive promoter selected from the group consisting of: CAG, HLP, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.
  • Embodiment 8 The immunoresponsive cell of any one of embodiments 1-7, wherein the second promoter comprises a constitutive promoter, an inducible promoter, or a synthetic promoter.
  • Embodiment 9 The immunoresponsive cell of embodiment 8, wherein the second promoter is a constitutive promoter selected from the group consisting of: CAG, HLP, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.
  • the second promoter is a constitutive promoter selected from the group consisting of: CAG, HLP, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.
  • Embodiment 10 The immunoresponsive cell of any one of embodiments 1-9, wherein the third expression cassette is configured to be transcribed in an opposite orientation relative to transcription of the fourth expression cassette within the second engineered nucleic acid.
  • Embodiment 11 The immunoresponsive cell of any one of embodiments 1-10, wherein the third expression cassette and the fourth expression cassette are oriented within the second engineered nucleic acid in a head-to-head directionality.
  • Embodiment 12 The immunoresponsive cell of any one of embodiments 1-11, wherein the third expression cassette and the fourth expression cassette are oriented within the second engineered nucleic acid in a tail-to-tail directionality.
  • Embodiment 13 The immunoresponsive cell of any one of embodiments 1-11, wherein the fourth promoter comprises a constitutive promoter, an inducible promoter, or a synthetic promoter.
  • Embodiment 14 The immunoresponsive cell of embodiment 13, wherein the fourth promoter is a constitutive promoter selected from the group consisting of: CAG, HLP, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.
  • the fourth promoter is a constitutive promoter selected from the group consisting of: CAG, HLP, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.
  • Embodiment 15 An immunoresponsive cell comprising:
  • a first engineered nucleic acid comprising a first expression cassette comprising a first promoter operably linked to a first exogenous polynucleotide sequence encoding a first cytokine and a second exogenous polynucleotide sequence encoding a chimeric antigen receptor (CAR) that binds to GPC3, and a second expression cassette comprising an activation-conditional control polypeptide (ACP)-responsive promoter operably linked to a third exogenous polynucleotide sequence encoding a second cytokine; and
  • ACP activation-conditional control polypeptide
  • a second engineered nucleic acid comprising a third expression cassette comprising a third promoter operably linked to fourth exogenous polynucleotide sequence encoding an ACP, wherein the ACP comprises a synthetic transcription factor comprising a DNA-binding domain and a transcriptional effector domain, wherein the ACP is capable of inducing expression of the third exogenous polynucleotide sequence by binding to the ACP-responsive promoter, wherein at least one of the first exogenous polynucleotide sequence and the third exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula:
  • S comprises a secretable effector molecule comprising the first and/or second cytokine
  • C comprises a protease cleavage site
  • MT comprises a cell membrane tethering domain, and wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide.
  • Embodiment 16 The immunoresponsive cell of embodiment 15, wherein transcription of the first expression cassette is oriented in the opposite direction relative to transcription of the second expression cassette within the first engineered nucleic acid.
  • Embodiment 17 The immunoresponsive cell of embodiment 16, wherein the first expression cassette and the second expression cassette are oriented within the first engineered nucleic acid in a head-to-head directionality.
  • Embodiment 18 The immunoresponsive cell of embodiment 15, wherein the first expression cassette is configured to be transcribed in a same orientation relative to transcription of the second expression cassette.
  • Embodiment 19 The immunoresponsive cell of embodiment 18, wherein the first expression cassette and the second expression cassette are oriented within the first engineered nucleic acid in a head-to-tail directionality.
  • Embodiment 20 An immunoresponsive cell comprising:
  • a first engineered nucleic acid comprising a first expression cassette comprising a first promoter operably linked to a first exogenous polynucleotide sequence encoding a chimeric antigen receptor (CAR) that binds to GPC3 and a second exogenous polynucleotide sequence encoding a first cytokine; and
  • CAR chimeric antigen receptor
  • a second engineered nucleic acid comprising a second expression cassette comprising an activation-conditional control polypeptide (ACP)-responsive promoter operably linked to a third exogenous polynucleotide sequence encoding a second cytokine, and a third expression cassette comprising a third promoter operably linked to fourth exogenous polynucleotide sequence encoding an ACP, wherein the ACP comprises a synthetic transcription factor comprising a DNA-binding domain and a transcriptional effector domain, wherein the ACP is capable of inducing expression of the third exogenous polynucleotide sequence by binding to the ACP-responsive promoter, wherein at least one of the second exogenous polynucleotide sequence and the third exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula:
  • S comprises a secretable effector molecule comprising the first and/or second cytokine
  • C comprises a protease cleavage site
  • MT comprises a cell membrane tethering domain, and wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide.
  • Embodiment 21 The immunoresponsive cell of embodiment 20, wherein transcription of the second expression cassette is oriented in the opposite direction relative to transcription of the third expression cassette within the first engineered nucleic acid.
  • Embodiment 22 The immunoresponsive cell of embodiment 20 or embodiment 21, wherein the second expression cassette and the third expression cassette are oriented within the second engineered nucleic acid in a head-to-head directionality.
  • Embodiment 23 The immunoresponsive cell of any one of embodiments 15-22, wherein the first promoter comprises a constitutive promoter, an inducible promoter, or a synthetic promoter.
  • Embodiment 24 The immunoresponsive cell of embodiment 23, wherein the first promoter is a constitutive promoter selected from the group consisting of: CAG, HLP, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.
  • the first promoter is a constitutive promoter selected from the group consisting of: CAG, HLP, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.
  • Embodiment 25 The immunoresponsive cell of any one of embodiments 15-24, wherein the first exogenous polynucleotide sequence and the second exogenous polynucleotide sequence are separated by a linker polynucleotide sequence.
  • Embodiment 26 The immunoresponsive cell of embodiment 25, wherein the linker polynucleotide sequence is operably associated with the translation of the first cytokine and the CAR as separate polypeptides.
  • Embodiment 27 The immunoresponsive cell of embodiment 26, wherein the linker polynucleotide sequence encodes one or more 2 A ribosome skipping elements.
  • Embodiment 28 The immunoresponsive cell of embodiment 27, wherein the one or more 2A ribosome skipping elements are each selected from the group consisting of: P2A, T2A, E2A, F2A, and combinations thereof.
  • Embodiment 29 The immunoresponsive cell of embodiment 28, wherein the one or more 2A ribosome skipping elements comprises an E2A/T2A combination.
  • Embodiment 30 The immunoresponsive cell of embodiment 29, wherein the E2A/T2A combination comprises the amino acid sequence of SEQ ID NO: 281.
  • Embodiment 31 The immunoresponsive cell of embodiment 25 or embodiment 26, wherein the linker polynucleotide sequence encodes an Internal Ribosome Entry Site (IRES).
  • IRS Internal Ribosome Entry Site
  • Embodiment 32 The immunoresponsive cell of any one of embodiments 25-31, wherein the linker polynucleotide sequence encodes a cleavable polypeptide.
  • Embodiment 33 The immunoresponsive cell of embodiment 32, wherein the cleavable polypeptide comprises a furin polypeptide sequence.
  • Embodiment 34 The immunoresponsive cell of any one of embodiments 15-33, wherein the third promoter comprises a constitutive promoter, an inducible promoter, or a synthetic promoter.
  • Embodiment 35 The immunoresponsive cell of embodiment 34, wherein the third promoter is a constitutive promoter selected from the group consisting of: CAG, HLP, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.
  • Embodiment 36 The immunoresponsive cell of any one of embodiments 1-35, wherein the first cytokine is IL- 15.
  • Embodiment 37 The immunoresponsive cell of embodiment 36, wherein the IL- 15 comprises the amino acid sequence of SEQ ID NO: 285.
  • Embodiment 38 The immunoresponsive cell of any one of embodiments 1-36, wherein the second cytokine is selected from the group consisting of: IL12, an IL12p70 fusion protein, IL18, and IL21.
  • Embodiment 39 The immunoresponsive cell of embodiment 38, wherein the second cytokine is the IL12p70 fusion protein.
  • Embodiment 40 The immunoresponsive cell of embodiment 39, wherein the IL12p70 fusion protein comprises the amino acid sequence of SEQ ID NO: 293.
  • Embodiment 41 The immunoresponsive cell of any one of embodiments 1-35, wherein the first cytokine is IL12 or an IL12p70 fusion protein.
  • Embodiment 42 The immunoresponsive cell of any one of embodiments 1-36, wherein the second cytokine is selected from the group consisting of: IL 15, IL18, and IL21.
  • Embodiment 43 The immunoresponsive cell of any one of embodiments 1-42, wherein the protease cleavage site is cleavable by a protease selected from the group consisting of: a Type 1 transmembrane protease, a Type II transmembrane protease, a GPI anchored protease, an ADAM8 protease, an ADAM9 protease, an ADAM 10 protease, an ADAM 12 protease, an ADAM 15 protease, an ADAM 17 protease, an ADAM 19 protease, an ADAM20 protease, an ADAM21 protease, an ADAM28 protease, an ADAM30 protease, an ADAM33 protease, a BACE1 protease, a BACE2 protease, a SIP protease, an MT1-MMP protease, an MT3-MMP protease, an MT
  • Embodiment 44 The immunoresponsive cell of embodiment 43, wherein the protease cleavage site is cleavable by an ADAM 17 protease.
  • Embodiment 45 The immunoresponsive cell of any one of embodiments 1-44, wherein the protease cleavage site comprises a first region having the amino acid sequence of PRAE (SEQ ID NO: 176).
  • Embodiment 46 The immunoresponsive cell of any one of embodiments 1-45, wherein the protease cleavage site comprises a second region having the amino acid sequence of KGG (SEQ ID NO: 177).
  • Embodiment 47 The immunoresponsive cell of embodiment 46, wherein the first region is located N-terminal to the second region.
  • Embodiment 48 The immunoresponsive cell of any one of embodiments 1-47, wherein the protease cleavage site comprises the amino acid sequence of PRAEX1X2KGG (SEQ ID NO: 178), wherein Xi is A, Y, P, S, or F, and wherein X2 is V, L, S, I, Y, T, or A.
  • Embodiment 49 The immunoresponsive cell of embodiment 48, wherein the protease cleavage site comprises the amino acid sequence of PRAEAVKGG (SEQ ID NO: 179).
  • Embodiment 50 The immunoresponsive cell of embodiment 48, wherein the protease cleavage site comprises the amino acid sequence of PRAEALKGG (SEQ ID NO: 180).
  • Embodiment 51 The immunoresponsive cell of embodiment 48, wherein the protease cleavage site comprises the amino acid sequence of PRAEYSKGG (SEQ ID NO: 181).
  • Embodiment 52 The immunoresponsive cell of embodiment 48, wherein the protease cleavage site comprises the amino acid sequence of PRAEPIKGG (SEQ ID NO: 182).
  • Embodiment 53 The immunoresponsive cell of embodiment 48, wherein the protease cleavage site comprises the amino acid sequence of PRAEAYKGG (SEQ ID NO: 183).
  • Embodiment 54 The immunoresponsive cell of embodiment 48, wherein the protease cleavage site comprises the amino acid sequence of PRAESSKGG (SEQ ID NO: 184).
  • Embodiment 55 The immunoresponsive cell of embodiment 48, wherein the protease cleavage site comprises the amino acid sequence of PRAEFTKGG (SEQ ID NO: 185).
  • Embodiment 56 The immunoresponsive cell of embodiment 48, wherein the protease cleavage site comprises the amino acid sequence of PRAEAAKGG (SEQ ID NO: 186).
  • Embodiment 57 The immunoresponsive cell of embodiment 48, wherein the protease cleavage site comprises the amino acid sequence of DEPHYSQRR (SEQ ID NO: 187).
  • Embodiment 58 The immunoresponsive cell of embodiment 48, wherein the protease cleavage site comprises the amino acid sequence of PPLGPIFNPG (SEQ ID NO: 188).
  • Embodiment 59 The immunoresponsive cell of embodiment 48, wherein the protease cleavage site comprises the amino acid sequence of PLAQAYRSS (SEQ ID NO: 189).
  • Embodiment 60 The immunoresponsive cell of embodiment 48, wherein the protease cleavage site comprises the amino acid sequence of TPIDSSFNPD (SEQ ID NO: 190).
  • Embodiment 61 The immunoresponsive cell of embodiment 48, wherein the protease cleavage site comprises the amino acid sequence of VTPEPIFSLI (SEQ ID NO: 191).
  • Embodiment 62 The immunoresponsive cell of any one of embodiments 1-44, wherein the protease cleavage site comprises the amino acid sequence of ITQGLAVSTISSFF (SEQ ID NO: 198).
  • Embodiment 63 The immunoresponsive cell of any one of embodiments 1-62, wherein the protease cleavage site is comprised within a peptide linker.
  • Embodiment 64 The immunoresponsive cell of any one of embodiments 1-62, wherein the protease cleavage site is N-terminal to a peptide linker.
  • Embodiment 65 The immunoresponsive cell of embodiment 63 or embodiment 64, wherein the peptide linker comprises a glycine-serine (GS) linker.
  • GS glycine-serine
  • Embodiment 66 The immunoresponsive cell of any one of embodiments 1-62, wherein the cell membrane tethering domain comprises a transmembrane-intracellular domain or a transmembrane domain.
  • Embodiment 67 The immunoresponsive cell of embodiment 66, wherein the transmembrane-intracellular domain and/or transmembrane domain is derived from PDGFR-beta, CD8, CD28, CD3zeta-chain, CD4, 4- IBB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, or BTLA.
  • Embodiment 68 The immunoresponsive cell of embodiment 67, wherein the transmembrane-intracellular domain and/or transmembrane domain is derived from B7- 1.
  • Embodiment 69 The immunoresponsive cell of embodiment 68, wherein the transmembrane-intracellular domain and/or transmembrane domain comprises the amino acid sequence of SEQ ID NO: 219.
  • Embodiment 70 The immunoresponsive cell of any one of embodiments 1-67, wherein the cell membrane tethering domain comprises a post-translational modification tag, or motif capable of post-translational modification to modify the chimeric protein to include a post-translational modification tag, wherein the post-translational modification tag is capable of association with a cell membrane.
  • Embodiment 71 The immunoresponsive cell of embodiment 70, wherein the post- translational modification tag comprises a lipid-anchor domain, optionally wherein the lipid-anchor domain is selected from the group consisting of: a GPI lipid-anchor, a myristoylation tag, and a palmitoylation tag.
  • Embodiment 72 The immunoresponsive cell of any one of embodiments 1-71, wherein the cell membrane tethering domain comprises a cell surface receptor, or a cell membranebound portion thereof.
  • Embodiment 73 The immunoresponsive cell of any one of embodiments 1-72, wherein the cytokine of the membrane-cleavable chimeric protein is tethered to a cell membrane of the cell.
  • Embodiment 74 The immunoresponsive cell of any one of embodiments 1-73, wherein the cell further comprises a protease capable of cleaving the protease cleavage site.
  • Embodiment 75 The immunoresponsive cell of embodiment 74, wherein the protease is endogenous to the cell.
  • Embodiment 76 The immunoresponsive cell of embodiment 74, wherein the protease is selected from the group consisting of: a Type 1 transmembrane protease, a Type II transmembrane protease, a GPI anchored protease, an ADAM8 protease, an ADAM9 protease, an ADAM 10 protease, an ADAM 12 protease, an ADAM 15 protease, an ADAM 17 protease, an ADAM 19 protease, an ADAM20 protease, an ADAM21 protease, an ADAM28 protease, an ADAM30 protease, an ADAM33 protease, a BACE1 protease, a BACE2 protease, a SIP protease, an MT1-MMP protease, an MT3-MMP protease, an MT5-MMP protease, a furin protease, a
  • Embodiment 77 The immunoresponsive cell of embodiment 76, wherein the protease is an ADAM 17 protease.
  • Embodiment 78 The immunoresponsive cell of any one of embodiments 74-77, wherein the protease is expressed on the cell membrane of the cell.
  • Embodiment 79 The immunoresponsive cell of embodiment 78, wherein the protease is capable of cleaving the protease cleavage site.
  • Embodiment 80 The immunoresponsive cell of embodiment 79, wherein cleavage of the protease cleavage site releases the cytokine of the membrane-cleavable chimeric protein from the cell membrane of the cell.
  • Embodiment 81 The immunoresponsive cell of any one of embodiments 1-19 and 23-80, wherein the first exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein.
  • Embodiment 82 The immunoresponsive cell of any one of embodiments 15-81, wherein the first exogenous polynucleotide sequence further comprises a polynucleotide sequence encoding a secretion signal peptide.
  • Embodiment 83 The immunoresponsive cell of any one of embodiments 20-80, wherein the second exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein.
  • Embodiment 84 The immunoresponsive cell of any one of embodiments 15-83, wherein the second exogenous polynucleotide sequence further comprises a polynucleotide sequence encoding a secretion signal peptide
  • Embodiment 85 The immunoresponsive cell embodiment 82 or embodiment 84, wherein the secretion signal peptide is derived from a protein selected from the group consisting of: IL-12, Trypsinogen-2, Gaussia Luciferase, CD5, IgKVII, VSV-G, prolactin, serum albumin preproprotein, azurocidin preproprotein, osteonectin (BM40), CD33, IL-6, IL-8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin-El, GROalpha, CXCL12, IL-21, CD8, GMCSFRa, NKG2D, and IgE.
  • a protein selected from the group consisting of: IL-12, Trypsinogen-2, Gaussia Luciferase, CD5, IgKVII, VSV-G, prolactin, serum albumin preproprotein, azurocidin preproprotein, osteonectin (BM40), CD33, IL-6, IL-8,
  • Embodiment 86 The immunoresponsive cell of embodiment 82, wherein the secretion signal peptide is derived from GMCSFRa.
  • Embodiment 87 The immunoresponsive cell of embodiment 86, wherein the secretion signal peptide comprises the amino acid sequence of SEQ ID NO: 216.
  • Embodiment 88 The immunoresponsive cell of embodiment 84, wherein the secretion signal peptide is derived from IgE.
  • Embodiment 89 The immunoresponsive cell of embodiment 88, wherein the secretion signal peptide comprises the amino acid sequence of SEQ ID NO: 218.
  • Embodiment 90 The immunoresponsive cell of any one of embodiments 15-89, wherein the third exogenous polynucleotide sequence further comprises a polynucleotide sequence encoding a secretion signal peptide.
  • Embodiment 91 The immunoresponsive cell of embodiment 90, wherein the secretion signal peptide is operably associated with the second cytokine.
  • Embodiment 92 The immunoresponsive cell of embodiment 82 or embodiment 91, wherein the secretion signal peptide is native to the second cytokine.
  • Embodiment 93 The immunoresponsive cell of embodiment 82 or embodiment 91, wherein the secretion signal peptide is non-native to the second cytokine.
  • Embodiment 94 The immunoresponsive cell of any one of embodiments 20-93, wherein the third exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein.
  • Embodiment 95 The immunoresponsive cell of embodiment 94, wherein the second expression cassette further comprises a polynucleotide sequence encoding a secretion signal peptide.
  • Embodiment 96 The immunoresponsive cell of any one of embodiments 15-95, wherein the secretion signal peptide is operably associated with the first cytokine.
  • Embodiment 97 The immunoresponsive cell of embodiment 96, wherein the secretion signal peptide is native to the first cytokine.
  • Embodiment 98 The immunoresponsive cell of embodiment 96, wherein the secretion signal peptide is non-native to the first cytokine.
  • Embodiment 99 The immunoresponsive cell of any one of embodiments 15-98, wherein the first exogenous polynucleotide sequence encodes a first membrane-cleavable chimeric protein and the third exogenous polynucleotide sequence encodes a second membrane-cleavable chimeric protein.
  • Embodiment 100 The immunoresponsive cell of any one of embodiments 20-98, wherein the second exogenous polynucleotide sequence encodes a first membrane-cleavable chimeric protein and the third exogenous polynucleotide sequence encodes a second membrane-cleavable chimeric protein.
  • Embodiment 101 The immunoresponsive cell of any one of embodiments 1-100, wherein the CAR comprises an antigen-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises: a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of KNAMN (SEQ ID NO: 199), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of RIRNKTNNYATYYADSVKA (SEQ ID NO: 200), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of GNSFAY (SEQ ID NO: 201), and wherein the VL comprises: a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLLYSSNQKNYLA (SEQ ID NO: 202), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of WASSRES
  • Embodiment 102 The immunoresponsive cell of embodiment 101, wherein the VH region comprises an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of EVQLVETGGGMVQPEGSLKLSCAASGFTFNKNAMNWVRQAPGKGLEWVARIR NKTNNYATYYADSVKARFTISRDDSQSMLYLQMNNLKIEDTAMYYCVAGNSFA YWGQGTLVTVSA (SEQ ID NO: 205) or EVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMNWVRQAPGKGLEWVGRIR NKTNNYATYYADSVKARFTISRDDSKNSLYLQMNSLKTEDTAVYYCVAGNSFA YWGQGTLVTVSA (SEQ ID NO: 206).
  • Embodiment 103 The immunoresponsive cell of embodiment 101, wherein the VH region comprises an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 206.
  • Embodiment 104 The immunoresponsive cell of any one of embodiments 101-103, wherein the VL region comprises an amino acid sequence with at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of DIVMSQSPSSLVVSIGEKVTMTCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIY WASSRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYNYPLTFGAGTK LELK (SEQ ID NO: 207), or DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIY WASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNYPLTFGQGTK LEIK (SEQ ID NO: 208).
  • Embodiment 105 The immunoresponsive cell of embodiment 104, wherein the VL region comprises an amino acid sequence with at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 208.
  • Embodiment 106 The immunoresponsive cell of any one of embodiments 101-98, wherein the antigen-binding domain comprises a single chain variable fragment (scFv).
  • scFv single chain variable fragment
  • Embodiment 107 The immunoresponsive cell of any one of embodiments 101-106, wherein the VH and VL are separated by a peptide linker.
  • Embodiment 108 The immunoresponsive cell of embodiment 107, wherein the peptide linker comprises a glycine-serine (GS) linker.
  • GS glycine-serine
  • Embodiment 109 The immunoresponsive cell of embodiment 108, wherein the GS linker comprises the amino acid sequence of (GGGGS)3 (SEQ ID NO: 223).
  • Embodiment 110 The immunoresponsive cell of embodiment 107, wherein the scFv comprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain.
  • Embodiment 111 The immunoresponsive cell of any one of embodiments 1-110, wherein the CAR comprises one or more intracellular signaling domains, and each of the one or more intracellular signaling domains is selected from the group consisting of: a CD3zeta- chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD 11a- CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD 154 intracellular signaling domain, a CD8 intracellular signaling domain, an 0X40 intracellular signaling domain, a 4- IBB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP 10 intracellular signaling domain, a DAP 12 intracellular signaling domain, a MyD88 intracellular signaling domain,
  • Embodiment 112 The immunoresponsive cell of embodiment 111, wherein the one or more intracellular signaling domains comprises an 0X40 intracellular signaling domain.
  • Embodiment 113 The immunoresponsive cell of embodiment 112, wherein the 0X40 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 269.
  • Embodiment 114 The immunoresponsive cell of embodiment 111, wherein the one or more intracellular signaling domains comprises a CD28 intracellular signaling domain.
  • Embodiment 115 The immunoresponsive cell of embodiment 114, wherein the CD28 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 267.
  • Embodiment 116 The immunoresponsive cell of embodiment 111, wherein the one or more intracellular signaling domains comprises a CD3z intracellular signaling domain.
  • Embodiment 117 The immunoresponsive cell of embodiment 116, wherein the CD3z intracellular signaling domain comprises an amino acid sequence of SEQ ID NO: 277 or SEQ ID NO: 279.
  • Embodiment 118 The immunoresponsive cell of any one of embodiments 1-117, wherein the CAR comprises a transmembrane domain, and the transmembrane domain is selected from the group consisting of: a CD8 transmembrane domain, a CD28 transmembrane domain a CD3zeta-chain transmembrane domain, a CD4 transmembrane domain, a 4- 1BB transmembrane domain, an 0X40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, a BTLA transmembrane domain, an 0X40 transmembrane domain, a DAP 10 transmembrane domain, a DAP 12 transmembrane domain, a CD 16a transmembrane domain, a DNAM-1 transmembrane domain,
  • Embodiment 119 The immunoresponsive cell of embodiment 118, wherein the transmembrane domain is an 0X40 transmembrane domain.
  • Embodiment 120 The immunoresponsive cell of embodiment 119, wherein the 0X40 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 244.
  • Embodiment 121 The immunoresponsive cell of embodiment 118, wherein the transmembrane domain is a CD8 transmembrane domain.
  • Embodiment 122 The immunoresponsive cell of embodiment 121, wherein the CD8 transmembrane domain comprises an amino acid sequence of SEQ ID NO: 236 or SEQ ID NO: 242.
  • Embodiment 123 The immunoresponsive cell of any one of embodiments 118-122, wherein the CAR comprises a spacer region between the antigen-binding domain and the transmembrane domain.
  • Embodiment 124 The immunoresponsive cell of embodiment 123, wherein the spacer region is derived from a protein selected from the group consisting of: CD8, CD28, IgG4, IgGl, LNGFR, PDGFR-beta, and MAG.
  • Embodiment 125 The immunoresponsive cell of embodiment 124, wherein the spacer region is a CD8 hinge.
  • Embodiment 126 The immunoresponsive cell of embodiment 125, wherein the CD8 hinge comprises the amino acid sequence of SEQ ID NO: 226 or SEQ ID NO: 228.
  • Embodiment 127 The immunoresponsive cell of any one of embodiments 1-123, wherein the ACP comprises a DNA binding domain and a transcriptional effector domain.
  • Embodiment 128 The immunoresponsive cell of embodiment 127, wherein the transcriptional effector domain comprises a transcriptional activator domain.
  • Embodiment 129 The immunoresponsive cell of embodiment 128, wherein the transcriptional activator domain is selected from the group consisting of: a Herpes Simplex Virus Protein 16 (VP 16) activation domain; an activation domain comprising four tandem copies of VP 16, a VP64 activation domain; a p65 activation domain of NFKB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activation domain); a histone acetyltransferase (HAT) core domain of the human ElA-associated protein p300 (p300 HAT core activation domain).
  • VP 16 Herpes Simplex Virus Protein 16
  • Rta Epstein-Barr virus R transactivator
  • HAT histone acetyltransferase
  • Embodiment 130 The immunoresponsive cell of embodiment 129, wherein the transcriptional activator domain comprises a VPR activation domain.
  • Embodiment 131 The immunoresponsive cell of embodiment 131, wherein the VPR activation domain comprises the amino acid sequence of SEQ ID NO: 325.
  • Embodiment 132 The immunoresponsive cell of embodiment 128, wherein the transcriptional effector domain comprises a transcriptional repressor domain.
  • Embodiment 133 The immunoresponsive cell of embodiment 132, wherein the transcriptional repressor domain is selected from the group consisting of: a Kruppel associated box (KRAB) repression domain; a truncated Kruppel associated box (KRAB) repression domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif of the hairy-related basic helix-loop-helix repressor proteins, the motif is known as a WRPW repression domain; a DNA (cytosine-5)- methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain.
  • KRAB Kruppel associated box
  • KRAB truncated Kruppel associated box
  • REST Repressor Element Silencing Transcription Factor
  • Embodiment 134 The immunoresponsive cell of any one of embodiments 127-133, wherein the DNA binding domain comprises a zinc finger (ZF) protein domain.
  • ZF zinc finger
  • Embodiment 135 The immunoresponsive cell of embodiment 134, wherein the ZF protein domain is modular in design and comprises an array of zinc finger motifs.
  • Embodiment 136 The immunoresponsive cell of embodiment 134, wherein the ZF protein domain comprises an array of one to ten zinc finger motifs.
  • Embodiment 137 The immunoresponsive cell of embodiment 136, wherein the ZF protein domain comprises the amino acid sequence of SEQ ID NO: 320.
  • Embodiment 138 The immunoresponsive cell of any one of embodiments 1-136, wherein the ACP further comprises a repressible protease and one or more cognate cleavage sites of the repressible protease.
  • Embodiment 139 The immunoresponsive cell of embodiment 138, wherein the repressible protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3).
  • HCV hepatitis C virus
  • NS3 nonstructural protein 3
  • Embodiment 140 The immunoresponsive cell of embodiment 139, wherein the NS3 protease comprises the amino acid sequence of SEQ ID NO: 321.
  • Embodiment 141 The immunoresponsive cell of embodiment 138 or embodiment 139, wherein the cognate cleavage site of the repressible protease comprises an NS 3 protease cleavage site.
  • Embodiment 142 The immunoresponsive cell of embodiment 141, wherein the NS3 protease cleavage site comprises a NS3/NS4A, a NS4A/NS4B, a NS4B/NS5A, or a NS5A/NS5B junction cleavage site.
  • Embodiment 143 The immunoresponsive cell of any one of embodiments 139-142, wherein the NS 3 protease is repressible by a protease inhibitor.
  • Embodiment 144 The immunoresponsive cell of embodiment 143, wherein the protease inhibitor is selected from the group consisting of: simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir.
  • the protease inhibitor is selected from the group consisting of: simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir.
  • Embodiment 145 The immunoresponsive cell of embodiment 144, wherein the protease inhibitor is grazoprevir (GRZ).
  • GRZ grazoprevir
  • Embodiment 146 The immunoresponsive cell of any one of embodiments 1-145, wherein the ACP further comprises a nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • Embodiment 147 The immunoresponsive cell of embodiment 146, wherein the NLS comprises the amino acid sequence of SEQ ID NO: 296.
  • Embodiment 148 The immunoresponsive cell of any one of embodiments 138-144, wherein the one or more cognate cleavage sites of the repressible protease are localized between the DNA binding domain and the transcriptional effector domain.
  • Embodiment 149 The immunoresponsive cell of any one of embodiments 1-148, wherein the ACP further comprises a ligand binding domain of estrogen receptor variant ERT2.
  • Embodiment 150 The immunoresponsive cell of any one of embodiments 1-149, wherein the ACP-responsive promoter is a synthetic promoter.
  • Embodiment 151 The immunoresponsive cell of any one of embodiments 1-150, wherein the ACP-responsive promoter comprises an ACP binding domain sequence and a minimal promoter sequence.
  • Embodiment 152 The immunoresponsive cell of embodiment 151, wherein the ACP binding domain sequence comprises one or more zinc finger binding sites.
  • Embodiment 153 The immunoresponsive cell of any one of embodiments 1,15, or 20, wherein the first engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 309.
  • Embodiment 154 The immunoresponsive cell of any one of embodiments 1,15, or 20, wherein the first engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 326.
  • Embodiment 155 The immunoresponsive cell of any one of embodiments 1,15, or 20, wherein the first engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 310.
  • Embodiment 156 The immunoresponsive cell of any one of embodiments 1,15, or 20, wherein the first engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 327.
  • Embodiment 157 The immunoresponsive cell of any one of embodiments 1,15, or 20, wherein the first engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 314.
  • Embodiment 158 The immunoresponsive cell of any one of embodiments 1,15, or 20, wherein the first engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 315.
  • Embodiment 159 The immunoresponsive cell of any one of embodiments 1-11 or 20-152, wherein the second engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 317.
  • Embodiment 160 The immunoresponsive cell of any one of embodiments 1-11 or 20-152, wherein the second engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 318.
  • Embodiment 161 An immunoresponsive cell comprising: a) a first engineered nucleic acid comprising the nucleotide sequence of SEQ ID NO: 310; and b) a second engineered nucleic acid comprising the nucleotide sequence of SEQ ID NO: 317.
  • Embodiment 162 An immunoresponsive cell comprising: a) a first engineered nucleic acid comprising the nucleotide sequence of SEQ ID NO: 327; and c) a second engineered nucleic acid comprising the nucleotide sequence of SEQ ID NO: 317.
  • Embodiment 163 The immunoresponsive cell of any one of embodiments 1-162, wherein the cell is selected from the group consisting of: a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a viral- specific T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage, a monocyte, a dendritic cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, a mesenchymal stromal cell (MSC), an induced pluripotent stem cell (
  • Embodiment 164 The immunoresponsive cell of any one of embodiments 1-162, wherein the cell is a Natural Killer (NK) cell.
  • NK Natural Killer
  • Embodiment 165 The immunoresponsive cell of embodiment 163 or embodiment 164, wherein the cell is autologous.
  • Embodiment 166 The immunoresponsive cell of embodiment 163 of embodiment 164, wherein the cell is allogeneic.
  • Embodiment 167 An engineered nucleic acid comprising: a first expression cassette comprising a first promoter operably linked to a first, exogenous polynucleotide sequence encoding IL15, and a second expression cassette comprising a second promoter operably linked to a second exogenous polynucleotide sequence encoding a chimeric antigen receptor (CAR) that binds to GPC3, wherein the first exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula:
  • S comprises a secretable effector molecule comprising the IL15, C comprises a protease cleavage site, and MT comprises a cell membrane tethering domain, and wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide.
  • Embodiment 168 The engineered nucleic acid of embodiment 167, wherein a) the first expression cassette and the second expression cassette are oriented within the first engineered nucleic acid in a head-to-tail directionality, b) the first exogenous polynucleotide sequence and the second exogenous polynucleotide sequence are separated by a linker polynucleotide sequence comprising an E2A/T2A ribosome skipping element, and c) the CAR that binds to GPC3 comprises a CD28 intracellular signaling domain or an 0X40 intracellular signaling domain.
  • Embodiment 169 An engineered nucleic acid comprising: a first expression cassette comprising a first promoter operably linked to a first, exogenous polynucleotide sequence encoding a chimeric antigen receptor (CAR) that binds to GPC3 and a second exogenous polynucleotide sequence encoding IL15, wherein the first exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula:
  • S comprises a secretable effector molecule comprising the IL15
  • C comprises a protease cleavage site
  • MT comprises a cell membrane tethering domain, and wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide.
  • Embodiment 170 The engineered nucleic acid of embodiment 169, wherein a) the first exogenous polynucleotide sequence and the second exogenous polynucleotide sequence are separated by a linker polynucleotide sequence comprising an E2A/T2A ribosome skipping element, and b) the CAR that binds to GPC3 comprises a CD28 intracellular signaling domain or an 0X40 intracellular signaling domain.
  • Embodiment 171 The engineered nucleic acid of any one of embodiments 167-170, wherein the engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 309.
  • Embodiment 172 The engineered nucleic acid of any one of embodiments 167-170, wherein the engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 326.
  • Embodiment 173 The engineered nucleic acid of any one of embodiments 167-170,, wherein the engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 310.
  • Embodiment 174 The engineered nucleic acid of any one of embodiments 167-170,, wherein the engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 327.
  • Embodiment 175 The engineered nucleic acid of any one of embodiments 167-170,, wherein the engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 314.
  • Embodiment 176 The engineered nucleic acid of any one of embodiments 167-170,, wherein the engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 315.
  • Embodiment 177 An engineered nucleic acid comprising the nucleotide sequence of SEQ ID NO: 310.
  • Embodiment 178 An engineered nucleic acid comprising the nucleotide sequence of SEQ ID NO: 327.
  • Embodiment 179 An engineered nucleic acid comprising: a first expression cassette comprising an activation-conditional control polypeptide (ACP)-responsive promoter operably linked to a first exogenous polynucleotide sequence encoding an IL12p70 fusion protein, and a second expression cassette comprising a second promoter operably linked to a second exogenous polynucleotide sequence encoding an ACP, wherein the ACP comprises a synthetic transcription factor comprising a DNA-binding domain and a transcriptional effector domain, wherein the ACP is capable of inducing expression of the first exogenous polynucleotide sequence by binding to the ACP-responsive promoter, wherein the first exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula:
  • ACP activation-conditional control polypeptide
  • S comprises a secretable effector molecule comprising the IL12p70 fusion protein
  • C comprises a protease cleavage site
  • MT comprises a cell membrane tethering domain, and wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide.
  • Embodiment 180 The engineered nucleic acid of embodiment 179, wherein a) the first expression cassette and the second expression cassette are oriented within the first engineered nucleic acid in a head-to-head directionality, and b) the ACP comprises a DNA binding domain and a transcriptional effector domain, wherein the transcriptional activator domain comprises a VPR activation domain.
  • Embodiment 181 The engineered nucleic acid of embodiment 179 or 180, wherein the engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 317.
  • Embodiment 182 The engineered nucleic acid of embodiment 179 or 180, wherein the engineered nucleic acid comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 318.
  • Embodiment 183 An engineered nucleic acid comprising the nucleotide sequence of SEQ ID NO: 317.
  • Embodiment 184 An expression vector comprising the engineered nucleic acid of any one of embodiments 167-183.
  • Embodiment 185 An immunoresponsive cell comprising the engineered nucleic acid of any one of embodiments 167-183 or the expression vector of embodiment 184.
  • Embodiment 186 A pharmaceutical composition comprising the immunoresponsive cell of any one of embodiments 1-166 or 185, the engineered nucleic acid of any one of embodiments 167-183, or the expression vector of embodiment 184, and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.
  • Embodiment 187 A method of treating a subject in need thereof, the method comprising administering a therapeutically effective dose of any of the immunoresponsive cells of any one of embodiments 1-166 or 185 ,the engineered nucleic acid of any one of embodiments 167-183, the expression vector of embodiment 184, or the pharmaceutical composition of embodiment 186.
  • Embodiment 188 A method of stimulating a cell-mediated immune response to a tumor cell in a subject, the method comprising administering to a subject having a tumor a therapeutically effective dose of any of the immunoresponsive cells of any one of embodiments 1-166 or 185 ,the engineered nucleic acid of any one of embodiments 167- 183, the expression vector of embodiment 184, or the pharmaceutical composition of embodiment 186.
  • Embodiment 189 A method of reducing tumor volume in a subject, the method comprising administering to a subject having a tumor a composition comprising any of the immunoresponsive cells of any one of embodiments 1-166 or 185 ,the engineered nucleic acid of any one of embodiments 167-183, the expression vector of embodiment 184, or the pharmaceutical composition of embodiment 186.
  • Embodiment 190 A method of providing an anti-tumor immunity in a subject, the method comprising administering to a subject in need thereof a therapeutically effective dose of any of the immunoresponsive cells of any one of embodiments 1-166 or 185 ,the engineered nucleic acid of any one of embodiments 167-183, the expression vector of embodiment 184, or the pharmaceutical composition of embodiment 186.
  • Embodiment 191 The method of any one of embodiments 188-190, wherein the tumor comprises a GPC3-expressing tumor.
  • Embodiment 192 The method of any one of embodiments 188-191, wherein the tumor is selected from the group consisting of: hepatocellular carcinoma (HCC), ovarian clear cell carcinoma, melanoma, squamous cell carcinoma of the lung, hepatoblastoma, nephroblastoma (Wilms tumor), and yolk sac tumor.
  • HCC hepatocellular carcinoma
  • ovarian clear cell carcinoma melanoma
  • squamous cell carcinoma of the lung hepatoblastoma
  • nephroblastoma nephroblastoma
  • yolk sac tumor hepatocellular carcinoma
  • Embodiment 193 A method of treating a subject having cancer, the method comprising administering a therapeutically effective dose of any of the immunoresponsive cells of any one of embodiments 1-166 or 185 ,the engineered nucleic acid of any one of embodiments 167-183, the expression vector of embodiment 184, or the pharmaceutical composition of embodiment 186.
  • Embodiment 194 The method of embodiment 193, wherein the cancer comprises a GPC3- expressing cancer.
  • Embodiment 195 The method of embodiment 193 or embodiment 194, wherein the cancer is selected from the group consisting of: hepatocellular carcinoma (HCC), ovarian clear cell carcinoma, melanoma, squamous cell carcinoma of the lung, hepatoblastoma, nephroblastoma (Wilms tumor), and yolk sac tumor.
  • HCC hepatocellular carcinoma
  • ovarian clear cell carcinoma melanoma
  • squamous cell carcinoma of the lung hepatoblastoma
  • nephroblastoma nephroblastoma
  • yolk sac tumor hepatocellular carcinoma
  • Embodiment 196 The method of any one of embodiments 187-195, wherein the administering comprises systemic administration.
  • Embodiment 197 The method of any one of embodiments 187-195, wherein the administering comprises intratumoral administration.
  • Embodiment 198 The method of any one of embodiments 187-197, wherein the immunoresponsive cell is derived from the subject.
  • Embodiment 199 The method of any one of embodiments 187-198, wherein the immunoresponsive cell is allogeneic with reference to the subject.
  • Embodiment 200 An immunoresponsive cell comprising: (a) a first engineered nucleic acid comprising a first expression cassette comprising a first promoter operably linked to a first, exogenous polynucleotide sequence encoding a first cytokine, and a second expression cassette comprising a second promoter operably linked to a second exogenous polynucleotide sequence encoding a chimeric antigen receptor (CAR) that binds to GPC3; and (b) a second engineered nucleic acid comprising a third expression cassette comprising an activation-conditional control polypeptide (ACP)-responsive promoter operably linked to a third exogenous polynucleotide sequence encoding a second cytokine, and a fourth expression cassette comprising a fourth promoter operably linked to a fourth exogenous polynucleotide sequence encoding an ACP, wherein the ACP comprises a synthetic transcription factor comprising a DNA-binding domain and a transcription
  • Embodiment 201 An engineered nucleic acid comprising: a first expression cassette comprising a first promoter operably linked to a first, exogenous polynucleotide sequence encoding IL15, and a second expression cassette comprising a second promoter operably linked to a second exogenous polynucleotide sequence encoding a chimeric antigen receptor (CAR) that binds to GPC3, wherein the first exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula: S - C - MT or MT - C - S wherein S comprises a secretable effector molecule comprising the IL 15, C comprises a protease cleavage site, and MT comprises a cell membrane tethering domain, and wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide.
  • Embodiment 202 An engineered nucleic acid comprising: a first expression cassette comprising an activation-conditional control polypeptide (ACP)-responsive promoter operably linked to a first exogenous polynucleotide sequence encoding an IL12p70 fusion protein, and a second expression cassette comprising a second promoter operably linked to a second exogenous polynucleotide sequence encoding an activationconditional control polypeptide (ACP), wherein the ACP comprises a synthetic transcription factor comprising a DNA-binding domain and a transcriptional effector domain, wherein the ACP is capable of inducing expression of the first exogenous polynucleotide sequence by binding to the ACP-responsive promoter, wherein the first exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula: S - C - MT or MT - C - S wherein S comprises
  • Embodiment 203 The engineered nucleic acid of embodiment 201 or 202, wherein the first expression cassette is configured to be transcribed in an opposite orientation relative to transcription of the second expression cassette.
  • Embodiment 204 The engineered nucleic acid of embodiment 201 or 202, wherein the first expression cassette and the second expression cassette are oriented within the first engineered nucleic acid in a head-to-head directionality.
  • Embodiment 205 The engineered nucleic acid of embodiment 201 or 202, wherein the first expression cassette is configured to be transcribed in a same orientation relative to the transcription of the second expression cassette.
  • Embodiment 206 The engineered nucleic acid of embodiment 201 or 202, wherein the first expression cassette and the second expression cassette are oriented within the first engineered nucleic acid in a head-to-tail directionality.
  • Embodiment 207 An engineered nucleic acid comprising: (a) a first engineered nucleic acid comprising a first expression cassette comprising a first promoter operably linked to a first exogenous polynucleotide sequence encoding a chimeric antigen receptor (CAR) that binds to GPC3 and a second exogenous polynucleotide sequence encoding a first cytokine; and (b) a second engineered nucleic acid comprising a second expression cassette comprising an activation-conditional control polypeptide (ACP)-responsive promoter operably linked to a third exogenous polynucleotide sequence encoding a second cytokine, and a third expression cassette comprising a third promoter operably linked to fourth ex
  • Embodiment 208 The engineered nucleic acid of embodiment 207, wherein transcription of the second expression cassette is oriented in the opposite direction relative to transcription of the third expression cassette within the first engineered nucleic acid.
  • Embodiment 209 The engineered nucleic acid of embodiment 207 or 208, wherein the second expression cassette and the third expression cassette are oriented within the second engineered nucleic acid in a head-to-head directionality.
  • Embodiment 210 The engineered nucleic acid of any one of embodiments 201-209, wherein the first promoter comprises a constitutive promoter, an inducible promoter, or a synthetic promoter.
  • Embodiment 211 The engineered nucleic acid of any one of embodiments 201-210, wherein the first promoter is a constitutive promoter selected from the group consisting of: CAG, HLP, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.
  • the first promoter is a constitutive promoter selected from the group consisting of: CAG, HLP, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb,
  • Embodiment 212 The engineered nucleic acid of any one of embodiments 201-211, wherein the second promoter comprises a constitutive promoter, an inducible promoter, or a synthetic promoter.
  • Embodiment 213 The engineered nucleic acid of any one of embodiments 201-212, wherein the second promoter is a constitutive promoter selected from the group consisting of: CAG, HLP, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.
  • Embodiment 214 The engineered nucleic acid of any one of embodiments 201-213, wherein the third expression cassette is configured to be transcribed in an opposite orientation relative to transcription of the fourth expression cassette within the second engineered nucleic acid.

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Abstract

La présente invention concerne des cellules immunoréactives modifiées pour exprimer des cytokines, des récepteurs chimériques et des systèmes de facteur de transcription synthétique. La présente invention concerne, en outre, des acides nucléiques, des cellules et des méthodes associées.
PCT/US2023/079282 2022-11-09 2023-11-09 Récepteurs chimériques armés et leurs méthodes d'utilisation WO2024102943A1 (fr)

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