ARMED CHIMERIC RECEPTORS AND METHODS OF USE THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

The instant application claims the benefit of and priority to U.S. Provisional Application No. 63/383,059, filed on November 9, 2022, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML copy, created on Month XX, 20XX, is named XXXXXUS_sequencelisting.xml, and is X, XXX, XXX bytes in size.

BACKGROUND

Cell-based therapy platforms provide promising avenues for treating a variety of diseases. One such promising platform is CAR-T based therapies in the treatment of cancer. Given their promise, improvements in cell-based therapies are needed. 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. For example, 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. However, uncontrolled or unregulated armoring strategies can have negative impacts on treatment, such as off-target effects and toxicity in subjects. Thus, 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.

SUMMARY

Provided herein, among other things, are technologies for use in cell-based therapy platforms that allow for enhanced efficacy, e.g., via boosting cytotoxicity, activating certain immunoregulatory programs (e.g., increasing immune cell infiltration, decreasing regulatory T cell numbers, etc.), reshaping niche environments (e.g., tumor microenvironments), and so forth. Such provided technologies, also referred to as “armoring” technologies, are particularly useful in the context of CAR-T based therapies. In many embodiments, the present disclosure provides a cell-based therapy platform involving regulated armoring of the cell-based therapy, such as regulated secretion of payload effector molecules (e.g., cytokines). Accordingly, the present disclosure provides engineered cells (e.g., immunoresponsive cells) capable of controlled secretion of payload effector molecules, such as cytokines, that enhance the effectiveness of one or more other immunoregulatory therapeutic modalities (e.g., any CAR therapy). In many embodiments, engineered cells provided herein 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. In some embodiments, 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.

Technologies utilized in accordance with the present invention (e.g., engineered nucleic acids, engineered cells, chimeric proteins, etc.) may also be use in the treatment of a disease or disorder. In some embodiments, a disease or disorder is cancer. In some embodiments, 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. For example, 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. For example, 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., Gaspar, D. et al. Front Microbiol. 2013;

4: 294; Chu, H. et al. PLoS One. 2015; 10(5): e0126390, and web site : ap s . unmc . edu/ AP/main . php ) .

In one aspect, the present disclosure provides 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 molecule comprising the first cytokine, 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.

In some embodiments, 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. In some embodiments, the first expression cassette and the second expression cassette are oriented within the engineered nucleic acid in a head-to-head directionality. In some embodiments, the first expression cassette and the second expression cassette are oriented within the engineered nucleic acid in a tail-to-tail directionality. In some embodiments, the second promoter comprises a constitutive promoter, an inducible promoter, or a synthetic promoter. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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 protease, a PCSK7 protease, a matriptase protease, a matriptase-2 protease, an MMP9 protease, and an NS 3 protease. In some embodiments, 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. In some embodiments, 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).

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the post- translational modification tag comprises a lipid-anchor domain. In some embodiments, the lipid- anchor domain is selected from the group consisting of: a GPI lipid-anchor, a myristoylation tag, and a palmitoylation tag. In some embodiments, the cell membrane tethering domain comprises a cell surface receptor, or a cell membrane-bound portion thereof. In some embodiments, the cytokine of the membrane-cleavable chimeric protein is tethered to a cell membrane of the cell.

In some embodiments, the cell further comprises a protease capable of cleaving the protease cleavage site. In some embodiments, the protease is endogenous to the cell. In some embodiments, 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 protease, an MT3-MMP protease, an MT5-MMP protease, a furin protease, a PCSK7 protease, a matriptase protease, a matriptase-2 protease, and an MMP9 protease. In some embodiments, 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.

In some embodiments, the first exogenous polynucleotide sequence further comprises a polynucleotide sequence encoding a secretion signal peptide. In some embodiments, the secretion signal peptide is operably associated with the first cytokine. In some embodiments, the secretion signal peptide is native or non-native to the first cytokine.

In some embodiments, the transcriptional effector domain comprises a transcriptional activator domain. In some embodiments, 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). In some embodiments, the transcriptional effector domain comprises a transcriptional repressor domain. In some embodiments, 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.

In some embodiments, the DNA binding domain comprises a zinc finger (ZF) protein domain. In some embodiments, the ZF protein domain is modular in design and comprises an array of zinc finger motifs. In some embodiments, the ZF protein domain comprises an array of one to ten zinc finger motifs. In some embodiments, the ZF protein domain comprises the amino acid sequence of SEQ ID NO: 320.

In some embodiments, the ACP further comprises a repressible protease and one or more cognate cleavage sites of the repressible protease. In some embodiments, the repressible protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3). In some embodiments, the NS3 protease comprises the amino acid sequence of SEQ ID NO: 321. In some embodiments, the NS3 protease is repressible by a protease inhibitor. In some embodiments, the protease inhibitor is selected from the group consisting of: simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir. In some embodiments, the cognate cleavage site of the repressible protease comprises an NS3 protease cleavage site. In some embodiments, the NS3 protease cleavage site comprises a NS3/NS4A, a NS4A/NS4B, a NS4B/NS5A, or a NS5A/NS5B junction cleavage site. In some embodiments, the one or more cognate cleavage sites of the repressible protease are localized between the DNA binding domain and the transcriptional effector domain. In some embodiments, the ACP-responsive promoter comprises a minimal promoter sequence. In some embodiments, the ACP-responsive promoter comprises one or more zinc finger binding sites.

In some embodiments, 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.

In another aspect, 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. In some embodiments, 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). In some embodiments, the CAR is a GPC3-specific CAR.

In another aspect, 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 IL12p70 fusion protein, 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. In some embodiments, (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 transcriptional effector domain comprises a transcriptional activator domain, wherein the transcriptional activator domain comprises a VPR activation domain or a p65 activation domain.

In another aspect, the present disclosure provides an expression vector comprising the engineered nucleic acid provided herein.

In another aspect, the present disclosure provides 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.

In another aspect, 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. In some embodiments, the cancer comprises a GPC3-expressing cancer. In some embodiments, 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. In some embodiments, the administering comprises systemic administration or intratumoral administration. In some embodiments, the immunoresponsive cell is derived from the subject or is allogeneic with reference to the subject.

In another aspect, 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 wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide.

In some embodiments, 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 comprising the first cytokine, 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.

In some embodiments, 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. In some embodiments, a first expression cassette and a second expression cassette are oriented within an engineered nucleic acid in a head-to-head directionality. In some embodiments, a first expression cassette and a second expression cassette are oriented within an engineered nucleic acid in a tail-to-tail directionality.

In some embodiments, a second promoter comprises a constitutive promoter, an inducible promoter, or a synthetic promoter. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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 protease, a PCSK7 protease, a matriptase protease, a matriptase-2 protease, an MMP9 protease, and an NS 3 protease. In some embodiments, a protease cleavage site is cleavable by an ADAM 17 protease.

In some embodiments, 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.

In some embodiments, 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. In some embodiments, a protease cleavage site comprises the amino acid sequence of PRAEAVKGG (SEQ ID NO: 179). In some embodiments, a protease cleavage site comprises the amino acid sequence of PRAEALKGG (SEQ ID NO: 180). In some embodiments, a protease cleavage site comprises the amino acid sequence of PRAEYSKGG (SEQ ID NO: 181). In some embodiments, 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). In some embodiments, 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).

In some embodiments, 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.

In some embodiments, a cell membrane tethering domain comprises a transmembrane- intracellular domain and/or a transmembrane domain. In some embodiments, 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. In some embodiments, a transmembrane-intracellular domain and/or transmembrane domain is derived from B7-1. In some embodiments, a transmembrane- intracellular domain and/or transmembrane domain comprises the amino acid sequence of SEQ ID NO: 219.

In some embodiments, 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. In some embodiments, a post- translational modification tag comprises a lipid-anchor domain. In some embodiments, the lipid- anchor domain is selected from the group consisting of: a GPI lipid-anchor, a myristoylation tag, and a palmitoylation tag.

In some embodiments, a cell membrane tethering domain comprises a cell surface receptor, or a cell membrane-bound portion thereof.

In some embodiments, a cytokine of a membrane-cleavable chimeric protein is tethered to a cell membrane of the cell.

In some embodiments, an immunoresponsive cell further comprises a protease capable of cleaving a protease cleavage site. In some embodiments, a protease is endogenous to the cell. In some embodiments, 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-MMP protease, an MT3-MMP protease, an MT5-MMP protease, a furin protease, a PCSK7 protease, a matriptase protease, a matriptase-2 protease, and an MMP9 protease. In some embodiments, 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.

In some embodiments, a first exogenous polynucleotide sequence further comprises a polynucleotide sequence encoding a secretion signal peptide. In some embodiments, a secretion signal peptide is operably associated with the first cytokine. In some embodiments, a secretion signal peptide is native to the first cytokine. In some embodiments, a secretion signal peptide is non-native to the first cytokine. In some embodiments, a second exogenous polynucleotide sequence further encodes a membrane-cleavable chimeric protein. In some embodiments, a second expression cassette further comprises a polynucleotide sequence encoding a secretion signal peptide.

In some embodiments, a transcriptional effector domain comprises a transcriptional activator domain. In some embodiments, 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). In some embodiments, a transcriptional activator domain comprises a VPR activation domain. In some embodiments, a VPR activation domain comprises the amino acid sequence of SEQ ID NO: 325.

In some embodiments, a transcriptional effector domain comprises a transcriptional repressor domain. In some embodiments, 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.

In some embodiments, a DNA binding domain comprises a zinc finger (ZF) protein domain. In some embodiments, a ZF protein domain is modular in design and comprises an array of zinc finger motifs. In some embodiments, a ZF protein domain comprises an array of one to ten zinc finger motifs. In some embodiments, a ZF protein domain comprises the amino acid sequence of SEQ ID NO: 320.

In some embodiments, an ACP further comprises a repressible protease and one or more cognate cleavage sites of the repressible protease. In some embodiments, a repressible protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3). In some embodiments, a NS3 protease comprises the amino acid sequence of SEQ ID NO: 321. In some embodiments, a cognate cleavage site of the repressible protease comprises an NS 3 protease cleavage site. In some embodiments, a NS3 protease cleavage site comprises a NS3/NS4A, a NS4A/NS4B, a NS4B/NS5A, or a NS5A/NS5B junction cleavage site. In some embodiments, a NS3 protease is repressible by a protease inhibitor. In some embodiments, a protease inhibitor is selected from the group consisting of: simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir. In some embodiments, a protease inhibitor is grazoprevir (GRZ).

In some embodiments, an ACP further comprises a nuclear localization signal (NLS). In some embodiments, a NLS comprises the amino acid sequence of SEQ ID NO: 296.

In some embodiments, one or more cognate cleavage sites of a repressible protease are localized between a DNA binding domain and a transcriptional effector domain.

In some embodiments, an ACP further comprises a ligand binding domain of estrogen receptor variant ERT2.

In some embodiments, an ACP-responsive promoter comprises a minimal promoter sequence.

In some embodiments, an ACP binding domain sequence comprises one or more zinc finger binding sites.

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: 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 IL12p70 fusion protein, 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.

In some embodiments, 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. In some embodiments, 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.

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: 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.

In some embodiments, 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).

The present disclosure provides for pharmaceutical 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.

In some embodiments, a tumor comprises a GPC3 -expressing tumor. In some embodiments, 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.

In some embodiments, a cancer comprises a GPC3 -expressing cancer. In some embodiments, 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.

In some embodiments, administering comprises systemic administration. In some embodiments, administering comprises intratumoral administration.

In some embodiments, 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). In FIG. 47A, 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. In 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.

DETAILED DESCRIPTION

Immunoresponsive cells are provided for herein.

In a first instance, immunoresponsive cells are engineered to have the following:

(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 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 be expressed as a single polypeptide.

In a second instance, immunoresponsive cells are engineered to have the following:

(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 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

(b) 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 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.

In a third instance, 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 - 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 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). In some embodiments, the ACP is a transcriptional repressor. In some embodiments, 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. Specifically, 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. Without wishing to be bound by theory, the components of the Membrane-Cleavable system present in the membrane-cleavable chimeric protein generally regulate secretion through the below cellular processes:

MT: 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”)

C: Following expression and localization of the chimeric protein into the cell membrane, the protease cleavage site directs cleavage of the chimeric protein such that the effector molecule is released (“secreted”) into the extracellular space.

Generally, 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)

In some aspects, membrane-cleavable chimeric proteins (or engineered nucleic acids encoding the membrane-cleavable chimeric proteins) provided for herein comprise a protein of interest (e.g., any of the effector molecules described herein), a protease cleavage site, and a cell membrane tethering domain.

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. For example, an effector molecule may act as a ligand to increase or decrease enzymatic activity, gene expression, and/or cell signaling. Thus, in some embodiments, an effector molecule modulates (activates or inhibits) different immunomodulatory mechanisms. By directly binding to and modulating a molecule, an effector molecule may also indirectly modulate a second, downstream molecule. In some embodiments, an effector molecule is or comprises an antibody, or functional fragment thereof. In some embodiments, and effector molecule is or comprises a cytokine. In some embodiments, 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) fragments thereof. In some embodiments, an effector molecule is or comprises a chemokine. In some embodiments, 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, CXCL17, CX3CL1, and combinations and active fragments thereof.

In many embodiments, 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).

The term “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. Effector molecules as provided for herein (e.g., effector molecules included in an engineered protein described herein, e.g., a membrane- cleavable chimeric protein) 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. In many embodiments, a disease-immunosuppressive mechanism that is modulated by one or more effector molecules provided herein (e.g., those encoded within engineered nucleic acids described 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).

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.

In some embodiments, 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%). For example, 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%. In some embodiments, 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%. It should be understood that “an increase” in an immuno stimulatory and/or anti-tumor immune response, for example, systemically or in a tumor microenvironment, is relative to the immunostimulatory and/or anti-tumor immune response that would otherwise occur, in the absence of the effector molecule(s).

In some embodiments, 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). For example, 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. In some embodiments, 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 general, it will be understood by a skilled artisan that changes in the foregoing immunostimulatory and/or anti-tumor immune mechanisms may be assessed by any known method in the field.

In some embodiments, 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%). For example, 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%. In some embodiments, 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%. It should be understood that “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).

In some embodiments, 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). For example, 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. In some embodiments, 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). In general, it will be understood by a skilled artisan that changes in the foregoing immunosuppressive mechanisms may be assessed by any known method in the field.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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) inhibits myeloid-derived suppressor cell signaling, activity and/or recruitment, (p) degrades immunosuppressive factors/metabolites, (q) inhibits vascular endothelial growth factor signaling, and/or (r) directly kills tumor cells.

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.).

Table 1. Exemplary Effector Molecules

Table 2. Sequences encoding exemplary effector molecules

The present disclosure provides for, among other things, 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. As discussed herein, one or more provided engineered nucleic acids (e.g., a first, a second, a third, a fourth, etc. engineered nucleic acid) may be present in an engineered cell, e.g., an immunoresponsive cell.

In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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.

As discussed herein, the present disclosure further provides for engineered cells that are capable of expressing one or more immunotherapy or immunomodulatory molecules (e.g., CARs, effector molecules, and/or chimeric proteins as described herein) from one or more engineered nucleic acids, for example, any engineered nucleic acids described herein. In many embodiments, 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.

In some embodiments, the present disclosure provides 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. For example, 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). In a further example, 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. In some embodiments, 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. In many embodiments, 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).

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 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.

Immunoresponsive cells 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.

Immunoresponsive cells 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.

Immunoresponsive cells 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.

Immunoresponsive cells provided for herein 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 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 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.

Secretion Signals and Signal-Anchors

In many embodiments, one or more effector molecules (e.g., cytokines, such as those described herein) 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. In some embodiments, for chimeric proteins having the formula MT - C - S, 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. In some embodiments, for chimeric proteins having the formula S - C - MT, a membrane tethering domain having a reverse signal-anchor sequence (e.g., signal-anchor sequences of certain Type III transmembrane proteins) can be used, generally without a separate secretion signal peptide, that direct newly synthesized proteins destined for membrane localization to the proper protein processing pathways.

In many embodiments, for all membrane-cleavable chimeric proteins described herein, the one or more effector molecules are secretable effector molecules (referred to as “S” in the formula S - C - MT or MT - C - S). In some embodiments, a chimeric protein comprises a secretion signal. In embodiments with two or more chimeric proteins, each chimeric protein can comprise a secretion signal. In embodiments with two or more chimeric proteins, 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.

Table 3. Exemplary Signal Secretion Peptides

Protease Cleavage Site

In many embodiments, 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. Examples of 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, a BACE2 protease cleavage site, a SIP protease cleavage site, an MT1-MMP protease cleavage site, an MT3-MMP protease cleavage site, an MT5-MMP protease cleavage site, a furin protease cleavage site, a PCSK7 protease cleavage site, a matriptase protease cleavage site, a matriptase-2 protease cleavage site, an MMP9 protease cleavage site, or an NS 3 protease cleavage site.

One example of a 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. For a description of 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. 163-206; herein incorporated by reference in its entirety. For example, the sequences of 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 are provided. Representative NS 3 sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. YP_001491553, YP_001469631, YP_001469632, NP_803144, NP.671491, YP_001469634, YP_001469630, YP_001469633, ADA68311, ADA68307, AFP99000, AFP98987, ADA68322, AFP99033, ADA68330, AFP99056, AFP99041, CBF60982, CBF60817, AHH29575, AIZ00747, AIZ00744, AB 136969, ABN05226, KF516075, KF516074, KF516056, AB826684, AB826683, JX171009, JX171008, JX171000, EU847455, EF154714, GU085487, JX171065, JX171063; all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference.

Another example of 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. For example, certain protease cleavage sites capable of being cleaved by ADAM 17 are also capable of cleavage by additional ADAM family proteases, such as ADAM10. Accordingly, 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. For example, 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. In such cases, 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. Accordingly, 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. Exemplary 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.

Table 4A - Potential ADAM17 Protease Cleavage Site Sequences

In some embodiments, 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. In some embodiments, 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). In some embodiments, 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). In some embodiments, 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).

In certain embodiments, 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. For example and without limitation, the cleavage site may be flanked on both the N terminal and C terminal sides by a partial glycine- serine (GS) linker sequence. Upon cleavage, the N terminal partial GS linker, and C terminal partial GS linker, join to form a GS linker sequence, such as SEQ ID NO: 215.

In certain embodiments, 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). In some embodiments, 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.

In certain embodiments, the protease cleavage site is N-terminal to a linker. In certain embodiments, 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 GCTCAGGCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAAT (SEQ ID NO: 292). In some embodiments, 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. In some embodiments, 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. In certain embodiments, SEQ ID NO: 198 is comprised within a linker. In certain embodiments, 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). In some embodiments, 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. In many embodiments, 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). Examples of 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. An exemplary nucleic acid sequence encoding SEQ ID NO: 196 is ACCACCACACCAGCTCCTCGGCCACCAACTCCAGCTCCAACAATTGCCAGCCAGCC TCTGTCTCTGAGGCCCGAAGCTTGTAGACCTGCTGCAGGCGGAGCCGTGCATACAA GAGGACTGGATTTCGCCTGCGAC (SEQ ID NO: 337). In certain embodiments, 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.

In a Membrane-Cleavable system, as provided for herein, following expression and localization of the chimeric protein (e.g., any chimeric protein described herein) into the cell membrane, 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.

In some embodiments, a protease that cleaves the protease cleavage site is a protease specific for that specific protease cleavage site. For example, in the case of a disintegrin and metalloproteinase (“ADAM”) family protease, 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”).

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. In other words, 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. For example, and without wishing to be bound by theory, ADAM 17 is believed to be restricted in its endogenous expression to NK cell and T cells. Thus, 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. In other examples, 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.

A protease that cleaves the protease cleavage site of the chimeric protein can be heterologous to a cell expressing the chimeric protein. For example, 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. For example, the protease can be generally expressed in a specific cellular environment, such as a tumor microenvironment. In such cases, in general, 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. In embodiments having membrane-cleavable chimeric proteins, in general, 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. For example, 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 protease, a matriptase-2 protease, and an MMP9 protease. A protease can be an NS3 protease. A protease can be an ADAM 17 protease.

Proteases can be tumor associated proteases, such as, a cathepsin, a cysteine protease, an aspartyl protease, a serine protease, or a metalloprotease. Specific examples of 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. Proteases can also include, but are not limited to, proteases listed in Table 4B below. Exemplary cognate protease cleavage sites for certain proteases are also listed in Table 4B.

Table 4B: Exemplary Proteases with Cognate Cleavage Sites and Inhibitors

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

(MER000894), memapsin-2 (MER005870), renin (MER000917), cathepsin D (MER000911), cathepsin E (MER000944), memapsin-1 (MER005534), napsin A (MER004981), Mername- AA034 peptidase (MER014038), pepsin A4 (MER037290), pepsin A5 (Homo sapiens) (MER037291), hCG1733572 (Homo sapiens)-type putative peptidase (MER107386), napsin B pseudogene (MER004982), CYMP g.p. (Homo sapiens) (MER002929), subfamily A1A unassigned peptidases (MER181559), mouse mammary tumor virus retropepsin (MER048030), rabbit endogenous retrovirus endopeptidase (MER043650), S71-related human endogenous retropepsin (MER001812), RTVL-H-type putative peptidase (MER047117), RTVL-H-type putative peptidase (MER047133), RTVL-H-type putative peptidase (MER047160), RTVL-H- type putative peptidase (MER047206), RTVL-H-type putative peptidase (MER047253), RTVL- H-type putative peptidase (MER047260), RTVL-H-type putative peptidase (MER047291), RTVL-H-type putative peptidase (MER047418), RTVL-H-type putative peptidase (MER047440), RTVL-H-type putative peptidase (MER047479), RTVL-H-type putative peptidase (MER047559), RTVL-H-type putative peptidase (MER047583), RTVL-H-type putative peptidase (MERO 15446), human endogenous retrovirus retropepsin homologue 1 (MER015479), human endogenous retrovirus retropepsin homologue 2 (MER015481), endogenous retrovirus retropepsin pseudogene 1 (Homo sapiens chromosome 14) (MER029977), endogenous retrovirus retropepsin pseudogene 2 (Homo sapiens chromosome 8) (MER029665), endogenous retrovirus retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER002660), endogenous retrovirus retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER030286), endogenous retrovirus retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER047144), endogenous retrovirus retropepsin pseudogene 5 (Homo sapiens chromosome 12) (MER029664), endogenous retrovirus retropepsin pseudogene

6 (Homo sapiens chromosome 7) (MER002094), endogenous retrovirus retropepsin pseudogene

7 (Homo sapiens chromosome 6) (MER029776), endogenous retrovirus retropepsin pseudogene

8 (Homo sapiens chromosome Y) (MER030291), endogenous retrovirus retropepsin pseudogene

9 (Homo sapiens chromosome 19) (MER029680), endogenous retrovirus retropepsin pseudogene 10 (Homo sapiens chromosome 12) (MER002848), endogenous retrovirus retropepsin pseudogene 11 (Homo sapiens chromosome 17) (MER004378), endogenous retrovirus retropepsin pseudogene 12 (Homo sapiens chromosome 11) (MER003344), endogenous retrovirus retropepsin pseudogene 13 (Homo sapiens chromosome 2 and similar) (MER029779), endogenous retrovirus retropepsin pseudogene 14 (Homo sapiens chromosome 2) (MER029778), endogenous retrovirus retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER047158), endogenous retrovirus retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER047332), endogenous retrovirus retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER003182), endogenous retrovirus retropepsin pseudogene 16 (MER047165), endogenous retrovirus retropepsin pseudogene 16 (MER047178), endogenous retrovirus retropepsin pseudogene 16 (MER047200), endogenous retrovirus retropepsin pseudogene 16 (MER047315), endogenous retrovirus retropepsin pseudogene 16 (MER047405), endogenous retrovirus retropepsin pseudogene 16 (MER030292), endogenous retrovirus retropepsin pseudogene 17 (Homo sapiens chromosome 8) (MER005305), endogenous retrovirus retropepsin pseudogene 18 (Homo sapiens chromosome 4) (MER030288), endogenous retrovirus retropepsin pseudogene 19 (Homo sapiens chromosome 16) (MER001740), endogenous retrovirus retropepsin pseudogene 21 (Homo sapiens) (MER047222), endogenous retrovirus retropepsin pseudogene 21 (Homo sapiens) (MER047454), endogenous retrovirus retropepsin pseudogene 21 (Homo sapiens) (MER047477), endogenous retrovirus retropepsin pseudogene 21 (Homo sapiens) (MER004403), endogenous retrovirus retropepsin pseudogene 22 (Homo sapiens chromosome X) (MER030287), subfamily A2A non-peptidase homologues (MER047046), subfamily A2A non-peptidase homologues (MER047052), subfamily A2A non-peptidase homologues

(MER047076), subfamily A2A non-peptidase homologues (MER047080), subfamily A2A non- peptidase homologues (MER047088), subfamily A2A non-peptidase homologues

(MER047089), subfamily A2A non-peptidase homologues (MER047091), subfamily A2A non- peptidase homologues (MER047092), subfamily A2A non-peptidase homologues

(MER047093), subfamily A2A non-peptidase homologues (MER047094), subfamily A2A non- peptidase homologues (MER047097), subfamily A2A non-peptidase homologues (MER047099), subfamily A2A non-peptidase homologues MER047101), subfamily A2A non- peptidase homologues (MER047102), 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), subfamily A2A non- peptidase homologues (MER047114), subfamily A2A non-peptidase homologues

(MER047118), subfamily A2A non-peptidase homologues (MER047121), subfamily A2A non- peptidase homologues (MER047122), subfamily A2A non-peptidase homologues

(MER047126), subfamily A2A non-peptidase homologues (MER047129), subfamily A2A non- peptidase homologues (MER047130), subfamily A2A non-peptidase homologues

(MER047134), subfamily A2A non-peptidase homologues (MER047135), subfamily A2A non- peptidase homologues (MER047137), subfamily A2A non-peptidase homologues

(MER047140), subfamily A2A non-peptidase homologues (MER047141), subfamily A2A non- peptidase homologues (MER047142), subfamily A2A non-peptidase homologues

(MER047148), subfamily A2A non-peptidase homologues (MER047149), subfamily A2A non- peptidase homologues (MER047151), subfamily A2A non-peptidase homologues

(MER047154), subfamily A2A non-peptidase homologues (MER047155), subfamily A2A non- peptidase homologues (MER047156), 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 (MER047171), subfamily A2A non-peptidase homologues (MER047173), subfamily A2A non-peptidase homologues (MER047174), subfamily A2A nonpeptidase homologues (MER047179), subfamily A2A non-peptidase homologues

(MER047183), subfamily A2A non-peptidase homologues (MER047186), subfamily A2A non- peptidase homologues (MER047190), subfamily A2A non-peptidase homologues

(MER047191), subfamily A2A non-peptidase homologues (MER047196), subfamily A2A non- peptidase homologues (MER047198), subfamily A2A non-peptidase homologues

(MER047199), subfamily A2A non-peptidase homologues (MER047201), subfamily A2A non- peptidase homologues (MER047202), subfamily A2A non-peptidase homologues

(MER047203), subfamily A2A non-peptidase homologues (MER047204), 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

(MER047219), subfamily A2A non-peptidase homologues (MER047221), subfamily A2A non- peptidase homologues (MER047224), 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

(MER047239), subfamily A2A non-peptidase homologues (MER047240), subfamily A2A non- peptidase homologues (MER047242), subfamily A2A non-peptidase homologues

(MER047243), subfamily A2A non-peptidase homologues (MER047249), subfamily A2A non- peptidase homologues (MER047251), subfamily A2A non-peptidase homologues

(MER047252), subfamily A2A non-peptidase homologues (MER047254), subfamily A2A non- peptidase homologues (MER047255), 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

(MER047280), subfamily A2A non-peptidase homologues (MER047281), subfamily A2A non- peptidase homologues (MER047282), subfamily A2A non-peptidase homologues

(MER047284), subfamily A2A non-peptidase homologues (MER047285), subfamily A2A non- peptidase homologues (MER047289), subfamily A2A non-peptidase homologues

(MER047290), subfamily A2A non-peptidase homologues (MER047294), subfamily A2A non- peptidase homologues (MER047295), subfamily A2A non-peptidase homologues

(MER047298), subfamily A2A non-peptidase homologues (MER047300), subfamily A2A non- peptidase homologues (MER047302), 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 (MER047310), subfamily A2A non- peptidase homologues (MER047311), subfamily A2A non-peptidase homologues

(MER047314), subfamily A2A non-peptidase homologues (MER047318), subfamily A2A non- peptidase homologues (MER047320), subfamily A2A non-peptidase homologues

(MER047321), subfamily A2A non-peptidase homologues (MER047322), subfamily A2A non- peptidase homologues (MER047326), subfamily A2A non-peptidase homologues

(MER047327), subfamily A2A non-peptidase homologues (MER047330), subfamily A2A non- peptidase homologues (MER047333), subfamily A2A non-peptidase homologues

(MER047362), subfamily A2A non-peptidase homologues (MER047366), subfamily A2A non- peptidase homologues (MER047369), subfamily A2A non-peptidase homologues

(MER047370), subfamily A2A non-peptidase homologues (MER047371), subfamily A2A non- peptidase homologues (MER047375), subfamily A2A non-peptidase homologues

(MER047376), subfamily A2A non-peptidase homologues (MER047381), subfamily A2A non- peptidase homologues (MER047383), subfamily A2A non-peptidase homologues

(MER047384), subfamily A2A non-peptidase homologues (MER047385), subfamily A2A non- peptidase homologues (MER047388), subfamily A2A non-peptidase homologues

(MER047389), subfamily A2A non-peptidase homologues (MER047391), subfamily A2A non- peptidase homologues (MER047394), subfamily A2A non-peptidase homologues

(MER047396), subfamily A2A non-peptidase homologues (MER047400), subfamily A2A non- peptidase homologues (MER047401), subfamily A2A non-peptidase homologues (MER047403), subfamily A2A non-peptidase homologues (MER047406), subfamily A2A non- peptidase homologues (MER047407), subfamily A2A non-peptidase homologues (MER047410), subfamily A2A non-peptidase homologues (MER047411), subfamily A2A nonpeptidase homologues (MER047413), 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

(MER047445), subfamily A2A non-peptidase homologues (MER047449), subfamily A2A non- peptidase homologues (MER047450), 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

(MER047493), subfamily A2A non-peptidase homologues (MER047494), subfamily A2A non- peptidase homologues (MER047495), subfamily A2A non-peptidase homologues

(MER047496), subfamily A2A non-peptidase homologues (MER047497), subfamily A2A non- peptidase homologues (MER047499), subfamily A2A non-peptidase homologues

(MER047502), subfamily A2A non-peptidase homologues (MER047504), subfamily A2A non- peptidase homologues (MER047511), subfamily A2A non-peptidase homologues

(MER047513), subfamily A2A non-peptidase homologues (MER047514), subfamily A2A non- peptidase homologues (MER047515), subfamily A2A non-peptidase homologues

(MER047516), subfamily A2A non-peptidase homologues (MER047520), subfamily A2A non- peptidase homologues (MER047533), subfamily A2A non-peptidase homologues

(MER047537), subfamily A2A non-peptidase homologues (MER047569), subfamily A2A non- peptidase homologues (MER047570), subfamily A2A non-peptidase homologues

(MER047584), subfamily A2A non-peptidase homologues (MER047603), subfamily A2A non- peptidase homologues (MER047604), subfamily A2A non-peptidase homologues (MER047606), subfamily A2A non-peptidase homologues (MER047609), subfamily A2A non- peptidase homologues (MER047616), subfamily A2A non-peptidase homologues (MER047619), subfamily A2A non-peptidase homologues (MER047648), subfamily A2A non- peptidase homologues (MER047649), subfamily A2A non-peptidase homologues (MER047662), subfamily A2A non-peptidase homologues (MER048004), subfamily A2A non- peptidase homologues (MER048018), subfamily A2A non-peptidase homologues (MER048019), subfamily A2A non-peptidase homologues (MER048023), subfamily A2A non- peptidase homologues (MER048037), subfamily A2A unassigned peptidases (MER047164), subfamily A2A unassigned peptidases (MER047231), subfamily A2A unassigned peptidases (MER047386), skin aspartic protease (MER057097), presenilin 1 (MER005221), presenilin 2 (MER005223), impas 1 peptidase (MER019701), impas 1 peptidase (MER184722), impas 4 peptidase (MER019715), impas 2 peptidase (MER019708), impas 5 peptidase (MER019712), impas 3 peptidase (MER019711), possible family A22 pseudogene (Homo sapiens chromosome 18) (MER029974), possible family A22 pseudogene (Homo sapiens chromosome 11) (MER023159), cathepsin V (MER004437), cathepsin X (MER004508), cathepsin F (MER004980), cathepsin L (MER000622), cathepsin S (MER000633), cathepsin O (MER001690), cathepsin K (MER000644), cathepsin W (MER003756), cathepsin H (MER000629), cathepsin B (MER000686), dipeptidyl-peptidase I (MER001937), bleomycin hydrolase (animal) (MER002481), tubulointerstitial nephritis antigen (MER016137), tubulointerstitial nephritis antigen-related protein (MER021799), cathepsin L-like pseudogene 1 (Homo sapiens) (MER002789), cathepsin B-like pseudogene (chromosome 4, Homo sapiens) (MER029469), cathepsin B-like pseudogene (chromosome 1, Homo sapiens) (MER029457), CTSLL2 g.p. (Homo sapiens) (MER005210), CTSLL3 g.p. (Homo sapiens) (MER005209), calpain-1 (MER000770), calpain-2 (MER000964), calpain-3 (MER001446), calpain-9 (MER004042), calpain-8 (MER021474), calpain-15 (MER004745), calpain-5 (MER002939), calpain-11 (MER005844), calpain-12 (MER029889), calpain-10 (MER013510), calpain-13 (MER020139), calpain-14 (MER029744), Memame-AA253 peptidase (MER005537), calpamodulin (MER000718), hypothetical protein 940251 (MER003201), ubiquitinyl hydrolase- L1 (MER000832), ubiquitinyl hydrolase-L3 (MER000836), ubiquitinyl hydrolase-BAPl (MER003989), ubiquitinyl hydrolase-UCH37 (MER005539), ubiquitin- specific peptidase 5 (MER002066), ubiquitin- specific peptidase 6 (MER000863), ubiquitin- specific peptidase 4 (MER001795), ubiquitin- specific peptidase 8 (MER001884), ubiquitin- specific peptidase 13 (MER002627), ubiquitin- specific peptidase 2 (MER004834), ubiquitin- specific peptidase 11 (MER002693), ubiquitin- specific peptidase 14 (MER002667), ubiquitin- specific peptidase 7 (MER002896), ubiquitin- specific peptidase 9X (MER005877), ubiquitin- specific peptidase 10 (MER004439), ubiquitin- specific peptidase 1 (MER004978), ubiquitin- specific peptidase 12 (MER005454), ubiquitin- specific peptidase 16 (MER005493), ubiquitin- specific peptidase 15 (MER005427), ubiquitin- specific peptidase 17 (MER002900), ubiquitin- specific peptidase 19 (MER005428), ubiquitin- specific peptidase 20 (MER005494), ubiquitin- specific peptidase 3 (MER005513), ubiquitin- specific peptidase 9Y (MER004314), ubiquitin- specific peptidase 18 (MER005641), ubiquitin- specific peptidase 21 (MER006258), ubiquitin- specific peptidase 22 (MER012130), ubiquitin- specific peptidase 33 (MER014335), ubiquitin- specific peptidase 29 (MER012093), ubiquitin- specific peptidase 25 (MER011115), ubiquitin- specific peptidase 36 (MER014033), ubiquitin- specific peptidase 32 (MER014290), ubiquitin- specific peptidase 26 (Homo sapiens-type) (MER014292), ubiquitin- specific peptidase 24 (MER005706), ubiquitinspecific peptidase 42 (MER011852), ubiquitin- specific peptidase 46 (MER014629), ubiquitinspecific peptidase 37 (MER014633), ubiquitin- specific peptidase 28 (MER014634), ubiquitinspecific peptidase 47 (MER014636), ubiquitin- specific peptidase 38 (MER014637), ubiquitinspecific peptidase 44 (MER014638), ubiquitin- specific peptidase 50 (MER030315), ubiquitinspecific peptidase 35 (MER014646), ubiquitin- specific peptidase 30 (MER014649), Memame- AA091 peptidase (MER014743), ubiquitin- specific peptidase 45 (MER030314), ubiquitinspecific peptidase 51 (MER014769), ubiquitin- specific peptidase 34 (MER014780), ubiquitinspecific peptidase 48 (MER064620), ubiquitin- specific peptidase 40 (MER015483), ubiquitinspecific peptidase 41 (MER045268), ubiquitin- specific peptidase 31 (MER015493), Memame- AA129 peptidase (MER016485), ubiquitin- specific peptidase 49 (MER016486), Mername- AA187 peptidase (MER052579), USP17-like peptidase (MER030192), ubiquitin- specific peptidase 54 (MER028714), ubiquitin- specific peptidase 53 (MER027329), ubiquitin- specific endopeptidase 39 [misleading] (MER064621), Mername-AA090 non-peptidase homologue (MER014739), ubiquitin- specific peptidase 43 [misleading] (MER030140), ubiquitin- specific peptidase 52 [misleading] (MER030317), NEK2 pseudogene (MER014736), C19 pseudogene (Homo sapiens: chromosome 5) (MER029972), Memame-AAO88 peptidase (MER014750), autophagin-2 (MERO 13564), autophagin-1 (MERO 13561), autophagin-3 (MERO 14316), autophagin-4 (MER064622), Cezanne deubiquitinylating peptidase (MER029042), Cezanne-2 peptidase (MER029044), tumor necrosis factor alpha-induced protein 3 (MER029050), trabid peptidase (MER029052), VCIP135 deubiquitinating peptidase (MER152304), otubain-1 (MER029056), otubain-2 (MER029061), CylD protein (MER030104), UfSPl peptidase (MER042724), UfSP2 peptidase (MER060306), DUBA deubiquitinylating enzyme (MER086098), KIAA0459 (Homo sapiens)-like protein (MER122467), Otudl protein (MER125457), glycosyltransferase 28 domain containing 1, isoform CRA_c (Homo sapiens)- like (MER123606), hinlL g.p. (Homo sapiens) (MER139816), ataxin-3 (MER099998), ATXN3L putative peptidase (MER115261), Josephin domain containing 1 (Homo sapiens) (MER125334), Josephin domain containing 2 (Homo sapiens) (MER124068), YOD1 peptidase (MER116559), legumain (plant alpha form) (MER044591), legumain (MER001800), glycosylphosphatidylinositol: protein transamidase (MER002479), legumain pseudogene (Homo sapiens) (MER029741), family C13 unassigned peptidases (MER175813), caspase-1 (MER000850), caspase-3 (MER000853), caspase-7 (MER002705), caspase-6 (MER002708), caspase-2 (MER001644), caspase-4 (MER001938), caspase-5 (MER002240), caspase-8 (MER002849), caspase-9 (MER002707), caspase-10 (MER002579), caspase-14 (MER012083), paracaspase (MER019325), Mername-AA143 peptidase (MER021304), Memame-AA186 peptidase (MER020516), putative caspase (Homo sapiens) (MER021463), FLIP protein (MER003026), Memame-AA142 protein (MER021316), caspase-12 pseudogene (Homo sapiens) (MER019698), Mername-AA093 caspase pseudogene (MER014766), subfamily C14A non-peptidase homologues (MER185329), subfamily C14A non-peptidase homologues (MER179956), separase (Homo sapiens-type) (MER011775), separase-like pseudogene (MER014797), SENP1 peptidase (MER011012), SENP3 peptidase (MER011019), SENP6 peptidase (MER011109), SENP2 peptidase (MER012183), SENP5 peptidase (MER014032), SENP7 peptidase (MER014095), SENP8 peptidase (MER016161), SENP4 peptidase (MER005557), pyroglutamyl-peptidase I (chordate) (MER011032), Mername-AA073 peptidase (MER029978), Sonic hedgehog protein (MER002539), Indian hedgehog protein (MER002538), Desert hedgehog protein (MER012170), dipeptidyl-peptidase III (MER004252), Memame- AA164 protein (MER020410), LOC138971 g.p. (Homo sapiens) (MER020074), Atp23 peptidase (MER060642), prenyl peptidase 1 (MER004246), aminopeptidase N (MER000997), aminopeptidase A (MER001012), leukotriene A4 hydrolase (MER001013), pyroglutamyl- peptidase II (MER012221), cytosol alanyl aminopeptidase (MER002746), cystinyl aminopeptidase (MER002060), aminopeptidase B (MER001494), aminopeptidase PILS (MER005331), arginyl aminopeptidase-like 1 (MER012271), leukocyte-derived arginine aminopeptidase (MER002968), aminopeptidase Q (MER052595), aminopeptidase O (MER019730), Tata binding protein associated factor (MER026493), angiotensin-converting enzyme peptidase unit 1 (MER004967), angiotensin-converting enzyme peptidase unit 2 (MER001019), angiotensin-converting enzyme-2 (MER011061), Memame-AA153 protein (MER020514), thimet oligopeptidase (MER001737), neurolysin (MER010991), mitochondrial intermediate peptidase (MER003665), Memame-AA154 protein (MER021317), leishmanolysin- 2 (MER014492), leishmanolysin-3 (MER18OO31), matrix metallopeptidase- 1 (MER001063), matrix metallopeptidase- 8 (MER001084), matrix metallopeptidase-2 (MER001080), matrix metallopeptidase-9 (MER001085), matrix metallopeptidase-3 (MER001068), matrix metallopeptidase-10 (Homo sapiens-type) (MER001072), matrix metallopeptidase- 11 (MER001075), matrix metallopeptidase-7 (MER001092), matrix metallopeptidase- 12 (MER001089), matrix metallopeptidase- 13 (MER001411), membrane-type matrix metallopeptidase- 1 (MER001077), membrane-type matrix metallopeptidase-2 (MER002383), membrane-type matrix metallopeptidase-3 (MER002384), membrane-type matrix metallopeptidase-4 (MER002595), matrix metallopeptidase-20 (MER003021), matrix metallopeptidase- 19 (MER002076), matrix metallopeptidase-23B (MER004766), membranetype matrix metallopeptidase-5 (MER005638), membrane-type matrix metallopeptidase-6 (MER012071), matrix metallopeptidase-21 (MER006101), matrix metallopeptidase-22 (MER014098), matrix metallopeptidase-26 (MER012072), matrix metallopeptidase-28 (MER013587), matrix metallopeptidase-23A (MER037217), macrophage elastase homologue (chromosome 8, Homo sapiens) (MER030035), Mername-AA156 protein (MER021309), matrix metallopeptidase-like 1 (MER045280), subfamily M10A non-peptidase homologues (MER175912), subfamily M10A non-peptidase homologues (MER187997), subfamily M10A non-peptidase homologues (MER187998), subfamily M10A non-peptidase homologues (MER180000), meprin alpha subunit (MER001111), meprin beta subunit (MER005213), procollagen C-peptidase (MER001113), mammalian tolloid-like 1 protein (MER005124), mammalian-type tolloid-like 2 protein (MER005866), ADAMTS9 peptidase (MER012092), AD AMTS 14 peptidase (MER016700), AD AMTS 15 peptidase (MER017029), AD AMTS 16 peptidase (MER015689), AD AMTS 17 peptidase (MER016302), AD AMTS 18 peptidase (MER016090), AD AMTS 19 peptidase (MER015663), ADAM8 peptidase (MER003902), ADAM9 peptidase (MER001140), ADAM10 peptidase (MER002382), ADAM12 peptidase (MER005107), ADAM19 peptidase (MER012241), ADAM15 peptidase (MER002386), ADAM17 peptidase (MER003094), ADAM20 peptidase (MER004725), ADAMDEC1 peptidase (MER000743), ADAMTS3 peptidase (MER005100), ADAMTS4 peptidase (MER005101), AD AMTS 1 peptidase (MER005546), ADAM28 peptidase (Homo sapiens-type) (MER005495), ADAMTS5 peptidase (MER005548), ADAMTS8 peptidase (MER005545), ADAMTS6 peptidase (MER005893), ADAMTS7 peptidase (MER005894), ADAM30 peptidase (MER006268), ADAM21 peptidase (Homo sapiens-type) (MER004726), AD AMTS 10 peptidase (MER014331), AD AMTS 12 peptidase (MER014337), AD AMTS 13 peptidase (MER015450), ADAM33 peptidase (MER015143), ovastacin (MER029996), ADAMTS20 peptidase (Homo sapiens-type) (MER026906), procollagen I N-peptidase (MER004985), ADAM2 protein (MER003090), ADAM6 protein (MER047044), ADAM7 protein (MER005109), ADAM18 protein (MER012230), ADAM32 protein (MER026938), non- peptidase homologue (Homo sapiens chromosome 4) (MER029973), family M12 non-peptidase homologue (Homo sapiens chromosome 16) (MER047654), family M12 non-peptidase homologue (Homo sapiens chromosome 15) (MER047250), ADAM3B protein (Homo sapiens- type) (MER005199), ADAMI 1 protein (MER001146), ADAM22 protein (MER005102), ADAM23 protein (MER005103), ADAM29 protein (MER006267), protein similar to ADAM21 peptidase preproprotein (Homo sapiens) (MER026944), Mername-AA225 peptidase homologue (Homo sapiens) (MER047474), putative ADAM pseudogene (chromosome 4, Homo sapiens) (MER029975), ADAM3A g.p. (Homo sapiens) (MER005200), ADAMI g.p. (Homo sapiens) (MER003912), subfamily M12B non-peptidase homologues (MER188210), subfamily M12B non-peptidase homologues (MER188211), subfamily M12B non-peptidase homologues (MER188212), subfamily M12B non-peptidase homologues (MER188220), neprilysin (MER001050), endothelin-converting enzyme 1 (MER001057), endothelin-converting enzyme 2 (MER004776), DINE peptidase (MER005197), neprilysin-2 (MER013406), Kell blood-group protein (MER001054), PHEX peptidase (MER002062), i-AAA peptidase (MER001246), i-AAA peptidase (MER005755), paraplegin (MER004454), Afg3-like protein 2 (MER005496), Afg3- like protein 1A (MER014306), pappalysin-1 (MER002217), pappalysin-2 (MER014521), farnesylated-protein converting enzyme 1 (MER002646), metalloprotease-related protein- 1 (MER030873), aminopeptidase AMZ2 (MER011907), aminopeptidase AMZ1 (MER058242), carboxypeptidase Al (MER001190), carboxypeptidase A2 (MER001608), carboxypeptidase B (MER001194), carboxypeptidase N (MER001198), carboxypeptidase E (MER001199), carboxypeptidase M (MER001205), carboxypeptidase U (MER001193), carboxypeptidase A3 (MER001187), metallocarboxypeptidase D peptidase unit 1 (MER003781), metallocarboxypeptidase Z (MER003428), metallocarboxypeptidase D peptidase unit 2 (MER004963), carboxypeptidase A4 (MER013421), carboxypeptidase A6 (MER013456), carboxypeptidase A5 (MER017121), metallocarboxypeptidase O (MER016044), cytosolic carboxypeptidase-like protein 5 (MER033174), cytosolic carboxypeptidase 3 (MER033176), cytosolic carboxypeptidase 6 (MER033178), cytosolic carboxypeptidase 1 (MER033179), cytosolic carboxypeptidase 2 (MER037713), metallocarboxypeptidase D non-peptidase unit (MER004964), adipocyte-enhancer binding protein 1 (MER003889), carboxypeptidase-like protein XI (MER013404), carboxypeptidase-like protein X2 (MER078764), cytosolic carboxypeptidase (MER026952), family M14 non-peptidase homologues (MER199530), insulysin (MER001214), mitochondrial processing peptidase beta-subunit (MER004497), nardilysin (MEROO3883), eupitrilysin (MER004877), mitochondrial processing peptidase non- peptidase alpha subunit (MER001413), ubiquinol-cytochrome c reductase core protein I (MER003543), ubiquinol-cytochrome c reductase core protein II (MER003544), ubiquinol- cytochrome c reductase core protein domain 2 (MER043998), insulysin unit 2 (MER046821), nardilysin unit 2 (MER046874), insulysin unit 3 (MER078753), mitochondrial processing peptidase subunit alpha unit 2 (MER124489), nardilysin unit 3 (MER142856), LOC133083 g.p. (Homo sapiens) (MER021876), subfamily M16B non-peptidase homologues (MER188757), leucyl aminopeptidase (animal) (MER003100), Memame-AA040 peptidase (MER003919), leucyl aminopeptidase- 1 (Caenorhabditis-type) (MERO 13416), methionyl aminopeptidase 1 (MER001342), methionyl aminopeptidase 2 (MER001728), aminopeptidase P2 (MER004498), Xaa-Pro dipeptidase (eukaryote) (MER001248), aminopeptidase Pl (MER004321), mitochondrial intermediate cleaving peptidase 55 kDa (MER013463), mitochondrial methionyl aminopeptidase (MER014055), Mername-AA020 peptidase homologue (MER010972), proliferation- association protein 1 (MER005497), chromatin- specific transcription elongation factor 140 kDa subunit (MER026495), proliferation-associated protein 1-like (Homo sapiens chromosome X) (MER029983), Memame-AA226 peptidase homologue (Homo sapiens) (MER056262), Memame-AA227 peptidase homologue (Homo sapiens) (MER047299), subfamily M24A non-peptidase homologues (MER179893), aspartyl aminopeptidase (MER003373), Gly-Xaa carboxypeptidase (MER033182), carnosine dipeptidase II (MER014551), carnosine dipeptidase I (MER015142), Mername-AA161 protein (MER021873), aminoacylase (MER001271), glutamate carboxypeptidase II (MER002104), NAALADASE L peptidase (MER005239), glutamate carboxypeptidase III (MER005238), plasma glutamate carboxypeptidase (MER005244), Mername-AA103 peptidase (MER015091), Fxna peptidase (MER029965), transferrin receptor protein (MER002105), transferrin receptor 2 protein (MER005152), glutaminyl cyclise (MER015095), glutamate carboxypeptidase II (Homo sapiens)-type non-peptidase homologue (MER026971), nicalin (MER044627), membrane dipeptidase (MER001260), membrane-bound dipeptidase-2 (MER013499), membrane-bound dipeptidase-3 (MERO 13496), dihydro-orotase (MER005767), dihydropyrimidinase (MER033266), dihydropyrimidinase related protein- 1 (MER030143), dihydropyrimidinase related protein-2 (MER030155), dihydropyrimidinase related protein-3 (MER030151), dihydropyrimidinase related protein-4 (MER030149), dihydropyrimidinase related protein-5 (MER030136), hypothetical protein like 5730457F11RIK (MER033184), 13OOO19jO8rik protein (MER033186)), guanine aminohydrolase (MER037714), Kael putative peptidase (MER001577), OSGEPLl-like protein (MER013498), S2P peptidase (MER004458), subfamily M23B non-peptidase homologues (MER199845), subfamily M23B non-peptidase homologues (MER199846), subfamily M23B non-peptidase homologues (MER199847), subfamily M23B non-peptidase homologues (MER137320), subfamily M23B non-peptidase homologues (MER201557), subfamily M23B non-peptidase homologues (MER199417), subfamily M23B non-peptidase homologues (MER199418), subfamily M23B non-peptidase homologues (MER199419), subfamily M23B non-peptidase homologues (MER199420), subfamily M23B non-peptidase homologues (MER175932), subfamily M23B non-peptidase homologues (MER199665), Pohl peptidase (MER020382), Jabl/MPN domain metalloenzyme (MER022057), Memame-AA165 peptidase (MER021865), Brcc36 isopeptidase (MER021890), histone H2A deubiquitinase MYSM1 (MER021887), AMSH deubiquitinating peptidase (MER030146), putative peptidase (Homo sapiens chromosome 2) (MER029970), Memame- AA168 protein (MER021886), COP9 signalosome subunit 6 (MER030137), 26S proteasome non-ATPase regulatory subunit 7 (MER030134), eukaryotic translation initiation factor 3 subunit 5 (MERO3O133), IFP38 peptidase homologue (MER030132), subfamily M67A non- peptidase homologues (MER191181), subfamily M67A unassigned peptidases (MER191144), granzyme B (Homo sapiens-type) (MER000168), testisin (MER005212), tryptase beta (MER000136), kallikrein-related peptidase 5 (MER005544), corin (MER005881), kallikrein- related peptidase 12 (MER006038), DESCI peptidase (MER006298), tryptase gamma 1 (MER011036), kallikrein-related peptidase 14 (MERO11O38), hyaluronan-binding peptidase (MER003612), transmembrane peptidase, serine 4 (MER011104), intestinal serine peptidase (rodent) (MER016130), adrenal secretory serine peptidase (MER003734), tryptase delta 1 (Homo sapiens) (MER005948), matriptase-3 (MER029902), marapsin (MER006119), tryptase-6 (MER006118), ovochymase-1 domain 1 (MER099182), transmembrane peptidase, serine 3 (MER005926), kallikrein-related peptidase 15 (MER000064), Mername-AA031 peptidase (MER014054), TMPRSS13 peptidase (MER014226), Mername-AAO38 peptidase (MER062848), Memame-AA204 peptidase (MER029980), cationic trypsin (Homo sapiens- type) (MER000020), elastase-2 (MER000118), mannan-binding lectin-associated serine peptidase-3 (MER031968), cathepsin G (MER000082), myeloblastin (MER000170), granzyme A (MER001379), granzyme M (MER001541), chymase (Homo sapiens-type) (MER000123), tryptase alpha (MER000135), granzyme K (MER001936), granzyme H (MER000166), chymotrypsin B (MER000001), elastase- 1 (MER003733), pancreatic endopeptidase E (MER000149), pancreatic elastase II (MER000146), enteropeptidase (MER002068), chymotrypsin C (MER000761), prostasin (MER002460), kallikrein 1 (MER000093), kallikrein- related peptidase 2 (MER000094), kallikrein-related peptidase 3 (MER000115), mesotrypsin (MER000022), complement component Clr- like peptidase (MER016352), complement factor D (MER000130), complement component activated Clr (MER000238), complement component activated Cis (MER000239), complement component C2a (MER000231), complement factor B (MER000229), mannan-binding lectin-associated serine peptidase 1 (MER000244), complement factor I (MER000228), pancreatic endopeptidase E form B (MER000150), pancreatic elastase IIB (MER000147), coagulation factor Xlla (MER000187), plasma kallikrein (MER000203) coagulation factor Xia (MER000210), coagulation factor IXa (MER000216), coagulation factor Vila (MER000215), coagulation factor Xa (MER000212), thrombin (MEROOO188), protein C (activated) (MER000222), acrosin (MER000078), hepsin (MER000156), hepatocyte growth factor activator (MER000186), mannan-binding lectin-associated serine peptidase 2 (MER002758), u-plasminogen activator (MER000195), t-plasminogen activator (MER000192), plasmin (MER000175), kallikrein-related peptidase 6 (MER002580), neurotrypsin (MER004171), kallikrein-related peptidase 8 (MER005400), kallikrein-related peptidase 10 (MER003645), epitheliasin (MER003736), kallikrein-related peptidase 4 (MER005266), prosemin (MER004214), chymopasin (MER001503), kallikrein-related peptidase 11 (MER004861), kallikrein-related peptidase 11 (MER216142), trypsin-2 type A (MER000021), HtrAl peptidase (Homo sapiens-type) (MER002577), HtrA2 peptidase (MER208413), HtrA2 peptidase (MER004093), HtrA3 peptidase (MER014795), HtrA4 peptidase (MER016351), Tysndl peptidase (MER050461), TMPRSS12 peptidase (MER017085), HAT-like putative peptidase 2 (MER021884), trypsin C (MER021898), kallikrein-related peptidase 7 (MER002001), matriptase (MER003735), kallikrein-related peptidase 13 (MER005269), kallikrein-related peptidase 9 (MER005270), matriptase-2 (MER005278), umbilical vein peptidase (MER005421), LCLP peptidase (MER001900), spinesin (MER014385), marapsin-2 (MER021929), complement factor D-like putative peptidase (MER056164), ovochymase-2 (MER022410), HAT-like 4 peptidase (MER044589), ovochymase 1 domain 1 (MER022412), epidermis-specific SP-like putative peptidase (MER029900), testis serine peptidase 5 (MER029901), Memame-AA258 peptidase (MER000285), polyserase-IA unit 1 (MER030879), polyserase-IA unit 2 (MERO3O88O), testis serine peptidase 2 (human-type) (MER033187), hypothetical acrosin-like peptidase (Homo sapiens) (MER033253), HAT-like 5 peptidase (MER028215), polyserase-3 unit 1 (MER061763), polyserase-3 unit 2 (MER061748), peptidase similar to tryptophan/serine protease (MER056263), polyserase-2 unit 1 (MER061777), Mername-AA123 peptidase (MER021930), HAT-like 2 peptidase (MER099184), hCG2041452- like protein (MER099172), hCG22067 (Homo sapiens) (MER099169), brain-rescue-factor- 1 (Homo sapiens) (MER098873), hCG2041108 (Homo sapiens) (MER099173), polyserase-2 unit 2 (MER061760), polyserase-2 unit 3 (MER065694), Mername-AA201 (peptidase homologue) MER099175, secreted trypsin-like serine peptidase homologue (MER030000), polyserase-IA unit 3 (MER029880), azurocidin (MER000119), haptoglobin-1 (MER000233), haptoglobin- related protein (MER000235), macrophage-stimulating protein (MER001546), hepatocyte growth factor (MER000185), protein Z (MER000227), TESP1 protein (MER047214), LOC136242 protein (MER016132), plasma kallikrein-like protein 4 (MER016346), PRSS35 protein (MER016350), DKFZp586H2123-like protein (MER066474), apolipoprotein (MEROOO183), psi-KLKl pseudogene (Homo sapiens) (MER033287), tryptase pseudogene I (MER015077), tryptase pseudogene II (MER015078), tryptase pseudogene III (MER015079), subfamily S1A unassigned peptidases (MER216982), subfamily S1A unassigned peptidases (MER216148), amidophosphoribosyltransferase precursor (MER003314), glutamine-fructose-6- phosphate transaminase 1 (MER003322), glutamine:fructose-6-phosphate amidotransferase (MER012158), Memame-AA144 protein (MER021319), asparagine synthetase (MER033254), family C44 non-peptidase homologues (MER159286), family C44 unassigned peptidases (MER185625) family C44 unassigned peptidases (MER185626), secernin 1 (MER045376), secernin 2 (MER064573), secernin 3 (MER064582), acid ceramidase precursor (MER100794), N-acylethanolamine acid amidase precursor (MER141667), proteasome catalytic subunit 1 (MER000556), proteasome catalytic subunit 2 (MER002625), proteasome catalytic subunit 3 (MER002149), proteasome catalytic subunit li (MER000552), proteasome catalytic subunit 2i (MER001515), proteasome catalytic subunit 3i (MER000555), proteasome catalytic subunit 5t (MER026203), protein serine kinase cl7 (MER026497), proteasome subunit alpha 6 (MER000557), proteasome subunit alpha 2 (MER000550), proteasome subunit alpha 4 (MER000554), proteasome subunit alpha 7 (MER033250), proteasome subunit alpha 5 (MER000558), proteasome subunit alpha 1 (MER000549), proteasome subunit alpha 3 (MER000553), proteasome subunit XAPC7 (MER004372), proteasome subunit beta 3 (MER001710), proteasome subunit beta 2 (MER002676), proteasome subunit beta 1 (MER000551), proteasome subunit beta 4 (MER001711), Mername-AA230 peptidase homologue (Homo sapiens) (MER047329), Memame-AA231 pseudogene (Homo sapiens) (MER047172), Memame-AA232 pseudogene (Homo sapiens) (MER047316), glycosylasparaginase precursor (MER003299), isoaspartyl dipeptidase (threonine type) (MER031622), 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), similar to gamma- glutamyltransferase 1 precursor (Homo sapiens) (MER026205), Memame-AA211 putative peptidase (MER026207), gamma-glutamyltransferase 6 (MER159283), gamma-glutamyl transpeptidase homologue (chromosome 2, Homo sapiens) (MER037241), polycystin-1 (MER126824), KIAA1879 protein (MER159329), polycystic kidney disease 1-like 3 (MER172554), gamma-glutamyl hydrolase (MER002963), guanine 5 "-monophosphate synthetase (MER043387), carbamoyl-phosphate synthase (Homo sapiens-type) (MER078640), dihydro-orotase (N-terminal unit) (Homo sapiens-type) (MER060647), DJ-1 putative peptidase (MER003390), Memame-AAIOO putative peptidase (MER014802), Mername-AAlOl nonpeptidase homologue (MER014803), KIAA0361 protein (Homo sapiens-type) (MER042827), Fl 134283 protein (Homo sapiens) (MER044553), non-peptidase homologue chromosome 21 open reading frame 33 (Homo sapiens) (MER160094), family C56 non-peptidase homologues (MER177016), family C56 non-peptidase homologues (MER176613), family C56 non- peptidase homologues (MER176918), 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), cadherin 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 l4 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 (MEROOO383), proprotein convertase 5 (MER002578), proprotein convertase 7 (MER002984), tripeptidyl-peptidase II (MER000355), subfamily S8A non- peptidase homologues (MER201339), subfamily S8A non-peptidase homologues (MER191613), subfamily S8A unassigned peptidases (MER191611), subfamily S8A unassigned peptidases (MER191612), subfamily S8A unassigned peptidases (MER191614), tripeptidyl- peptidase I (MER003575), prolyl oligopeptidase (MER000393), dipeptidyl-peptidase IV (eukaryote) (MER000401), acylaminoacyl-peptidase (MER000408), fibroblast activation protein alpha subunit (MER000399), PREPL A protein (MER004227), dipeptidyl-peptidase 8 (MER013484), dipeptidyl-peptidase 9 (MER004923), FLJ1 putative peptidase (MER017240), Mername-AA194 putative peptidase (MER017353), Memame-AA195 putative peptidase (MER017367), Memame-AA196 putative peptidase (MER017368), Mername-AA197 putative peptidase (MER017371), C14orf29 protein (MER033244), hypothetical protein (MER033245), hypothetical esterase/lipase/thioesterase (MER047309), protein bat5 (MER037840), hypothetical protein flj40219 (MER033212), hypothetical protein flj37464 (MER033240), hypothetical protein flj33678 (MER033241), dipeptidylpeptidase homologue DPP6 (MER000403), dipeptidylpeptidase homologue DPP10 (MER005988), protein similar to Mus musculus chromosome 20 open reading frame 135 (MER037845), kynurenine formamidase (MER046020), thyroglobulin precursor (MER011604), acetylcholinesterase (MERO33188), cholinesterase (MER033198), carboxylesterase DI (MER033213), liver carboxylesterase (MER033220), carboxylesterase 3 (MER033224), carboxylesterase 2 (MER033226), bile saltdependent lipase (MER033227), carboxylesterase-related protein (MER033231), neuroligin 3 (MER033232), neuroligin 4, X-linked (MER033235), neuroligin 4, Y-linked (MER033236), esterase D (MER043126), arylacetamide deacetylase (MER033237), KIAA1363-like protein (MER033242), hormone- sensitive lipase (MER033274), neuroligin 1 (MER033280), neuroligin 2 (MER033283), family S9 non-peptidase homologues (MER212939), family S9 non-peptidase homologues (MER211490), subfamily S9C unassigned peptidases (MER192341), family S9 unassigned peptidases (MER209181), family S9 unassigned peptidases (MER200434), family S9 unassigned peptidases (MER209507), family S9 unassigned peptidases (MER209142), serine carboxypeptidase A (MER000430), vitellogenic carboxypeptidase-like protein (MER005492), RISC peptidase (MER010960), family S15 unassigned peptidases (MER199442), family S15 unassigned peptidases (MER200437), family S15 unassigned peptidases (MER212825), lysosomal Pro-Xaa carboxypeptidase (MER000446), dipeptidyl-peptidase II (MER004952), thymus-specific serine peptidase (MER005538), epoxide hydrolase-like putative peptidase (MER031614), Loc328574-like protein (MER033246), abhydrolase domain-containing protein 4 (MER031616), epoxide hydrolase (MER000432), mesoderm specific transcript protein (MER199890), mesoderm specific transcript protein (MER017123), cytosolic epoxide hydrolase (MER029997), cytosolic epoxide hydrolase (MER213866), similar to hypothetical protein FLJ22408 (MER031608), CGI-58 putative peptidase (MER030163), Williams-Beuren syndrome critical region protein 21 epoxide hydrolase (MER031610), epoxide hydrolase (MER031612), hypothetical protein 922408 (epoxide hydrolase) (MER031617), monoglyceride lipase (MER033247), hypothetical protein (MER033249), valacyclovir hydrolase (MER033259), Ccgl -interacting factor b (MER210738), glycosylasparaginase precursor (MER003299), isoaspartyl dipeptidase (threonine type) (MER031622). 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). similar to gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026205). Mername-AA211 putative peptidase (MER026207). gamma- glutamyltransferase 6 (MER159283). gamma-glutamyl transpeptidase homologue (chromosome 2, Homo sapiens) (MER037241). polycystin-1 (MER126824), KIAA1879 protein (MER159329). polycystic kidney disease 1-like 3 (MER172554). gamma-glutamyl hydrolase (MER002963). guanine 5 "-monophosphate synthetase (MER043387). carbamoyl-phosphate synthase (Homo sapiens-type) (MER078640). dihydro-orotase (N-terminal unit) (Homo sapiens- type) (MER060647). DJ-1 putative peptidase (MER003390). Mername-AAIOO putative peptidase (MER014802). Mername-AAlOl non-peptidase homologue (MER014803). KIAA0361 protein (Homo sapiens-type) (MER042827). Fl 134283 protein (Homo sapiens) (MER044553). non-peptidase homologue chromosome 21 open reading frame 33 (Homo sapiens) (MER160094). family C56 non-peptidase homologues (MER177016), family C56 non- peptidase homologues (MER176613). family C56 non-peptidase homologues (MER176918). 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). cadherin 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 (MEROOO383), proprotein convertase 5 (MER002578), proprotein convertase 7 (MER002984), tripeptidyl-peptidase II (MER000355), subfamily S8A non-peptidase homologues (MER201339), subfamily S8A nonpeptidase homologues (MER191613), subfamily S8A unassigned peptidases (MER191611), subfamily S8A unassigned peptidases (MER191612), subfamily S8A unassigned peptidases (MER191614), tripeptidyl-peptidase I (MER003575), prolyl oligopeptidase (MER000393), dipeptidyl-peptidase IV (eukaryote) (MER000401), acylaminoacyl-peptidase (MER000408), fibroblast activation protein alpha subunit (MER000399), PREPL A protein (MER004227), dipeptidyl-peptidase 8 (MER013484), dipeptidyl-peptidase 9 (MER004923), FLU putative peptidase (MER017240), Mername-AA194 putative peptidase (MER017353), Memame-AA195 putative peptidase (MER017367), Memame-AA196 putative peptidase (MER017368), Mername-AA197 putative peptidase (MER017371), C14orf29 protein (MER033244), hypothetical protein (MER033245), hypothetical esterase/lipase/thioesterase (MER047309), protein bat5 (MER037840), hypothetical protein flj40219 (MER033212), hypothetical protein flj37464 (MER033240), hypothetical protein flj33678 (MER033241), dipeptidylpeptidase homologue DPP6 (MER000403), dipeptidylpeptidase homologue DPP 10 (MER005988), protein similar to Mus musculus chromosome 20 open reading frame 135 (MER037845), kynurenine formamidase (MER046020), thyroglobulin precursor (MER011604), acetylcholinesterase (MERO33188), cholinesterase (MER033198), carboxylesterase DI (MER033213), liver carboxylesterase (MER033220), carboxylesterase 3 (MER033224), carboxylesterase 2 (MER033226), bile salt-dependent lipase (MER033227), carboxylesterase-related protein (MER033231), neuroligin 3 (MER033232), neuroligin 4, X-linked (MER033235), neuroligin 4, Y-linked (MER033236), esterase D (MER043126), arylacetamide deacetylase (MER033237), KIAA1363-like protein (MER033242), hormone- sensitive lipase (MER033274), neuroligin 1 (MER033280), neuroligin 2 (MER033283), family S9 non-peptidase homologues (MER212939), family S9 non-peptidase homologues (MER211490), subfamily S9C unassigned peptidases (MER192341), family S9 unassigned peptidases (MER209181), family S9 unassigned peptidases (MER200434), family S9 unassigned peptidases (MER209507), family S9 unassigned peptidases (MER209142), serine carboxypeptidase A (MER000430), vitellogenic carboxypeptidase-like protein (MER005492), RISC peptidase (MER010960), family S15 unassigned peptidases (MER199442), family S15 unassigned peptidases (MER200437), family S15 unassigned peptidases (MER212825), lysosomal Pro-Xaa carboxypeptidase (MER000446), dipeptidyl-peptidase II (MER004952), thymus-specific serine peptidase (MER005538), epoxide hydrolase-like putative peptidase (MER031614), Loc328574-like protein (MER033246), abhydrolase domain-containing protein 4 (MER031616), epoxide hydrolase (MER000432), mesoderm specific transcript protein (MER199890), mesoderm specific transcript protein (MER017123), cytosolic epoxide hydrolase (MER029997), cytosolic epoxide hydrolase (MER213866), similar to hypothetical protein FLJ22408 (MER031608), CGI-58 putative peptidase (MER030163), Williams-Beuren syndrome critical region protein 21 epoxide hydrolase (MER031610), epoxide hydrolase (MER031612), hypothetical protein flj22408 (epoxide hydrolase) (MER031617), monoglyceride lipase (MER033247), hypothetical protein (MER033249), valacyclovir hydrolase (MER033259), Ccgl -interacting factor b (MER210738).

Protease enzymatic activity can be regulated. For example, certain 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. For example, 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. In another example, 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. For example, 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. As another example, 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. For example, various cell states (e.g., following cellular signaling, such as immune cell activation) can influence the expression and/or localization of certain proteases. As an illustrative example, 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]). Accordingly, 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. As another example, 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.

Cell Membrane Tethering Domain

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). In general, 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. Examples of 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). Examples of 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.

Table 4C.

In many embodiments, 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). In embodiments featuring a degron associated with the chimeric protein, 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). Examples of 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 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.

In general, 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.

Degron Systems and Domains

In some embodiments, 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. In general, 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.

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. 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. Other examples of 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 ornithine decarboxylase; SEQ ID NO: 168), Nek2A, mouse ODC (residues 422-461; SEQ ID NO: 169), mouse ODC_DA (residues 422-461 of mODC including D433A and D434A point mutations), an APC/C degron, a COP1 E3 ligase binding degron motif, a CRL4-Cdt2 binding PIP degron, an actinfilin-binding degron, a KEAP1 binding degron, a KLHL2 and KLHL3 binding degron, an MDM2 binding motif, an N-degron, a hydroxyproline modification in hypoxia signaling, a phytohormone-dependent SCF- LRR-binding degron, an SCF ubiquitin ligase binding phosphodegron, a phytohormone- dependent SCF-LRR-binding degron, a DSGxxS phospho-dependent degron (SEQ ID NO: 345), an Siah binding motif, an SPOP SBC docking motif, or a PCNA binding PIP box.

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). In general, as used herein, IMiDs 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). For degron domains having a CRBN polypeptide substrate domain, examples of 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). In the absence of an IMiD, degron/ubiquitin-mediated degradation of the chimeric protein does not occur. Following expression and localization of the chimeric protein into the cell membrane, the protease cleavage site directs cleavage of the chimeric protein such that the effector molecule is released (“secreted”) into the extracellular space. In the presence of an immunomodulatory drug (IMiD), the degron domain directs ubiquitin-mediated degradation of the chimeric protein such that secretion of the effector molecule is reduced or eliminated. In general, for membrane-cleavable chimeric proteins fused to a degron domain, 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). Examples of 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 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. In general, 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.

For degron-fusion proteins, 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). Examples of 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. 5,990,275 herein incorporated by reference). 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

Provided herein are engineered nucleic acids (e.g., an expression cassette) encoding 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. Provided herein are 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. In some embodiments, an engineered nucleic acid is or comprises one or more expression cassettes, e.g., any expression cassette described herein.

In certain embodiments 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.

In certain embodiments described herein, 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.

In certain embodiments described herein, 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. In certain embodiments described herein, the engineered nucleic acids encode an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a cytokine and CAR. In certain embodiments described herein, the engineered nucleic acids encode an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a cytokine and an ACP.

In certain embodiments described herein, 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. 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 an ACP, respectively. In certain embodiments, the two or more expression cassettes are oriented in a head-to-tail orientation. In certain embodiments, the two or more expression cassettes are oriented in a head-to-head orientation. In certain embodiments, the two or more expression cassettes are oriented in a tail-to-tail orientation. In some cases, each expression cassette contains its own promoter to drive expression of the polynucleotide sequence encoding a cytokine and/or CAR. In certain embodiments, 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. In some embodiments, 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. The term “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. Application Publication 2004/0019001, each of which is incorporated by reference in their entirety. 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). 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. 6,670,461; 5,539,082; 5,185,444, each herein incorporated by reference in their entirety. Recombinant nucleic acids and synthetic nucleic acids also include those molecules that result from the replication of either of the foregoing. 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.

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). In some embodiments, 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. In some embodiments, engineered nucleic acid constructs are produced using INFUSION® cloning (Clontech).

Promoters

In general, in all embodiments described herein, the engineered nucleic acids encoding the proteins herein (e.g., a cytokine, CAR, ACP, and/or membrane-cleavable chimeric protein described herein) encode an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding the protein. In some embodiments, 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 distinct proteins. For example, 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. In some embodiments, 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. In some embodiments, 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 cytokines. For example, 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. In some embodiments, 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. In some embodiments, 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. For example, 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. In some embodiments, 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. Herein, 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.” In some embodiments, 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. Such 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. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, 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). 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. Conversely, 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. In some embodiments, 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. 2012; 2(4): 824-839, incorporated herein by reference), and a 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

Table 5B. Exemplary Components of Inducible 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).

Table 5C. Exemplary Constitutive Promoters

The promoter can be a tissue-specific promoter. In general, 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. 264,166) 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. Further examples of 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. Exemplary 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. 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. Exemplary tissue-specific expression elements for the endometrium include, but are not limited to, the uteroglobin promoter. Exemplary tissue-specific expression elements for adrenal cells include, but are not limited to, cholesterol side-chain cleavage (SCC) promoter. Exemplary tissue- specific expression elements for the general nervous system include, but are not limited to, gamma enolase (neuron- specific enolase, NSE) promoter. Exemplary tissuespecific expression elements for the brain include, but are not limited to, the neurofilament heavy chain (NF-H) promoter. Exemplary 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. Exemplary 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. Exemplary tissue- specific expression elements for the lung include, but are not limited to, the cystic fibrosis transmembrane conductance regulator (CFTR) gene promoter.

In some embodiments, 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. For example, 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.

In some embodiments, 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. For example, 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. In some embodiments, 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.

In some embodiments, 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). In some embodiments, 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. In some embodiments, the promoter that is activated under hypoxic conditions comprises a hypoxia responsive element (HRE). A “hypoxia responsive element (HRE)” is a response element that responds to hypoxia-inducible factor (HIF). The HRE, in some embodiments, comprises a consensus motif NCGTG (where N is either A or G). Activation-Conditional Control Polypeptide (ACP) Promoter Systems

In some embodiments, a synthetic promoter is a promoter system including an activation-conditional control polypeptide (ACP) binding domain sequence and a promoter sequence. Such a system is also referred to herein as an “ACP-responsive promoter.”In general, 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). In some embodiments, 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. In some embodiments, a synthetic promoter comprises the nucleic acid sequence of AATTAACGGGTTTCGTAACAATCGCATGAGGATTCGCAACGCCTTTGAAGCAGTCG ACGCCGAAGTCCCGTCTCAGTAAAGGTTGAAGCAGTCGACGCCGAAGAATCGGACT GCCTTCGTATGAAGCAGTCGACGCCGAAGGTATCAGTCGCCTCGGAATGAAGCAGT CGACGCCGAAGATTCGTAAGAGGCTCACTCTCCCTTACACGGAGTGGATAACTAGT TCTAGAGGGTATATAATGGGGGCCAACGCGTACCGGTGTC (SEQ ID NO: 298). In some embodiments, 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. In some embodiments, a synthetic promoter comprises the nucleic acid sequence of CGGGTTTCGTAACAATCGCATGAGGATTCGCAACGCCTTCGGCGTAGCCGATGTCG CGCTCCCGTCTCAGTAAAGGTCGGCGTAGCCGATGTCGCGCAATCGGACTGCCTTCG TACGGCGTAGCCGATGTCGCGCGTATCAGTCGCCTCGGAACGGCGTAGCCGATGTC GCGCATTCGTAAGAGGCTCACTCTCCCTTACACGGAGTGGATAACTAGTTCTAGAG GGTATATAATGGGGGCCA (SEQ ID NO: 299). In some embodiments, 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, e.g., either a promoter driving expression of the ACP or the promoter sequence of the ACP-responsive promoter, 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. In some embodiments, the ACP-responsive promoter includes a minimal promoter.

In some embodiments, the ACP-binding domain can include a transcription factorbinding domain, e.g., a synthetic transcription factor (synTF) -binding domain. In some embodiments, the ACP-binding domain includes one or more zinc finger binding sites. In some embodiments, 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. In some embodiments, the transcription factor is a zinc-finger-containing transcription factor. In some embodiments, the zinc-finger- containing transcription factor is a synthetic transcription factor. 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). 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. This results in 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. In some embodiments, 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. In some embodiments, the ZF protein domain comprises one to ten ZFA(s). In some embodiments, the ZF protein domain comprises at least one ZFA. In some embodiments, the ZF protein domain comprises at least two ZFAs. In some embodiments, the ZF protein domain comprises at least three ZFAs. In some embodiments, the ZF protein domain comprises at least four ZFAs. In some embodiments, the ZF protein domain comprises at least five ZFAs. In some embodiments, the ZF protein domain comprises at least ten ZFAs.

In some embodiments, 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. For instance, 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 helix-loop-helix repressor proteins, the motif is known as a WRPW repression domain (SEQ ID NO: 346); a DNA (cytosine-5)- methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain, or any combination thereof.

In some embodiments, 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-loop- helix repressor proteins, the motif is known as a WRPW repression domain (SEQ ID NO: 346); a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain. In some embodiments, the ACP is a small molecule (e.g., drug) inducible polypeptide. For example, in some embodiments, the ACP may be induced by tetracycline (or derivative thereof), and comprises a TetR domain and a VP 16 effector domain. In some embodiments, 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. In some embodiments, the ACP comprises a nuclear-localization signal (NLS). In certain embodiments, 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). In some embodiments, 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.

In some instances, the ACP comprises an estrogen receptor variant such as ERT2. In some embodiments, the ACP comprises an estrogen receptor or variant thereof as described herein, e.g., in Table 25. In some embodiments, 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. In some aspects, 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. In some embodiments, 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 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. Residues within positions 517-547 (e.g., residues 517-40 and residue 547) corresponding to SEQ ID NO: 356 are located within a helix referred to as helix 12 and are involved in endogenous ligand binding.

Greater sensitivity to 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).

Greater sensitivity to a non-endogenous ligand as compared to sensitivity an ER-LBD not including the one or more amino acid substitutions (e.g., an ER-LBD comprising the amino acid sequence of SEQ ID NO: 357 or SEQ ID NO: 358) 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, in a transfection of transduction assay, a chimeric transcription factor including a modified ER-LBD 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.

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). The greater the selectivity coefficient, the more a competing ligand (e.g., an endogenous ligand) will displace the initial ligand (e.g., a non-endogenous ligand) from the complex formed with the substrate (e.g., a modified ER-LBD). In some embodiments, 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. In some embodiments, in a transfection of transduction assay, a chimeric transcription factor including a modified ER-LBD 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).

In some aspects, 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. In some embodiments, 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. In some embodiments, 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.

In some aspects, the one or more additional amino acids substitutions are selected from one or more of the substitutions listed in Table 23.

Table 23 In some aspects, 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.. In some aspects, 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 nine mutations, five to eight mutations, five to seven mutations, five to six mutations, six to ten mutations, six to nine mutations, six to eight mutations, six to seven mutations, seven to ten mutations, seven to nine mutations, seven to eight mutations, eight to ten mutations, eight to nine mutations, or nine to ten mutations.

In some aspects, the one or more additional mutations comprise at least two mutations that are selected from the mutations listed in Table 24.

Table 24

In some embodiments, provided herein is 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).

Table 25

In some embodiments, 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. In some embodiments, 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. In some embodiments, the specific agent is a protease inhibitor. In some embodiments, 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).

In some embodiments, 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. For example, 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.

Exemplary sequences of components of ACPs and exemplary ACPs of the present disclosure are provided in Table 5D. In some embodiments, 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.

Table 5D.

Multicistronic and Multiple Promoter Systems

In some embodiments, engineered nucleic acids (e.g., an engineered nucleic acid comprising an expression cassette as provided herein) are configured to produce multiple proteins (e.g., a cytokine, CAR, ACP, membrane-cleavable chimeric protein, and/or combinations thereof). For example, nucleic acids may be configured to produce 2-20 different proteins. In some embodiments, nucleic acids are configured 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, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7- 9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18, 9-

17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12- 19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-

18, 16-17, 17-20, 17-19, 17-18, 18-20, 18-19, or 19-20 proteins. In some embodiments, 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.

In some embodiments, 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. Other 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. In some embodiments, an engineered nucleic acid disclosed herein comprises an E2A/T2A ribosome skipping element. In certain embodiments, the E2A/T2A ribosome skipping element comprises the amino acid sequence of GSGQCTNYAEEKEAGDVESNPGPGSGEGRGSEETCGDVEENPGP (SEQ ID NO: 281). An exemplary nucleic acid encoding SEQ ID NO: 281 is GGTAGCGGCCAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATC TAATCCTGGACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACG TGGAGGAAAACCCTGGACCT (SEQ ID NO: 282). In certain embodiments, 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. In some embodiments, an engineered nucleic acid disclosed herein comprises an E2A/T2A ribosome skipping element. In certain embodiments, 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). In certain embodiments, 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. 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. In some embodiments, a combination of linkers can include a Furin sequence, a flexible linker, and 2A linker. Accordingly, in some embodiments, the linker is a Furin-Gly-Ser-Gly-2A fusion polypeptide. In some embodiments, a linker of the present disclosure is a Furin-Gly-Ser-Gly-T2A fusion polypeptide.

In general, 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).

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. For example, a first promoter can be operably linked to a polynucleotide sequence encoding a first protein, and a second promoter can be operably linked to a polynucleotide sequence encoding a second protein. In general, any number of promoters can be used to express any number of proteins. In some embodiments, 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”). Expression cassettes encoded on the same engineered nucleic acid oriented in opposite directions can be oriented in a “head-to-head” directionality. As used herein, head-to-head refers to the 5' end (head) of a first gene of a bidirectional construct being adjacent to the 5' end (head) of an upstream gene of the bidirectional construct. Expression cassettes encoded on the same engineered nucleic acid oriented in opposite directions can be oriented in a “tail-to-tail” directionality. As used herein, tail-to-tail refers to the 3' end (tail) of a first gene of a bidirectional construct being adjacent to the 3' end (tail) of an upstream gene of the bidirectional construct. For example, and without limitation, 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,” as used herein 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). In some embodiments, the mRNA-destabilizing element comprises an AU-rich element and/or a stem-loop destabilizing element (SLDE). In some embodiments, the mRNA-destabilizing element comprises an AU-rich element. In some embodiments, 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 ATTTATTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAG (SEQ ID NO: 212). In some embodiments, the mRNA-destabilizing element comprises a 2X AuSLDE. An exemplary AuSLDE sequence is provided as ATTTATTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAGtgcggtaag cATTTA TTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAG (SEQ ID NO: 213). In certain embodiments, an engineered nucleic acid described herein comprises an insulator sequence. Such insulator sequences function to prevent inappropriate interactions between adjacent regions of a construct. In certain embodiments, an insulator sequence comprises the nucleic acid sequence of ACAATGGCTGGCCCATAGTAAATGCCGTGTTAGTGTGTTAGTTGCTGTTCTTCCACG TCAGAAGAGGCACAGACAAATTACCACCAGGTGGCGCTCAGAGTCTGCGGAGGCAT CACAACAGCCCTGAATTTGAATCCTGCTCTGCCACTGCCTAGTTGAGACCTTTTACT ACCTGACTAGCTGAGACATTTACGACATTTACTGGCTCTAGGACTCATTTTATTCAT TTCATTACTTTTTTTTTCTTTGAGACGGAATCTCGCTCT (SEQ ID NO: 300). In certain embodiments, 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 Cells

Provided herein are 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). In general, 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. In some embodiments, 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. 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. For example, provided for herein are 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). In many embodiments, a mixed cell composition comprises a first engineered cell and a second engineered cell. In some embodiments, 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. In many embodiments, in a 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). In many embodiments, in a mixed cell composition, a first engineered cell 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. Such an effector molecule may, for example, complement the function of effector molecules natively produced by the cells.

In some embodiments, a cell (e.g., an immune cell) is engineered to produce multiple proteins. For example, cells may be engineered to produce 2-20 different proteins, such as 2-20 different membrane-cleavable proteins. In some embodiments, a cell (e.g., an immunoresponsive cell) is engineered to produce at least 4 distinct proteins exogenous to the cell. In some embodiments, a cell (e.g., an immunoresponsive cell) is engineered to produce 4 distinct proteins exogenous to the cell. In some embodiments, 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, 7-17, 7-16, 7-15, 7-14, 7-13, 7- 12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9- 20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11- 12, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16- 20, 16-19, 16-18, 16-17, 17-20, 17-19, 17-18, 18-20, 18-19, or 19-20 proteins. In some embodiments, cells are engineered to produce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins.

In some embodiments, 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). In some embodiments, 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. For example, 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. In some embodiments, 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.

In some embodiments, a cell (e.g., an immune cell) is engineered to express a protease. In some embodiments, a cell is engineered to express a protease heterologous to a cell. In some embodiments, 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. In some embodiments, 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 and protease cleavage sites are described in greater detail in the Section herein titled “Protease Cleavage site.”

Also provided herein are engineered cells that are engineered to produce multiple proteins, at least two of which include effector molecules that modulate different tumor- mediated immunosuppressive mechanisms. In some embodiments, 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. In some embodiments, 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. In yet other embodiments, 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.

In some embodiments, a cell (e.g., an immune 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. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that stimulates antigen presentation and/or processing. In some embodiments, 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. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that stimulates dendritic cell differentiation and/or maturation. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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).

In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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.

In certain embodiments, 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). In certain embodiments, 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.

In certain embodiments, the IL12p70 comprises the amino acid sequence of

MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDG I TWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDIL KDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLS AERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPD PPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKT SATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGSGGGSGGGSGGGSRNLPVATP DPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLE LTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMD PKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVT I DRVMSYLNAS (SEQ ID NO: 293). An exemplary nucleic acid sequence encoding SEQ ID NO: 293 is

ATGTGTCACCAGCAGCTGGTCATCAGCTGGTTCAGCCTGGTGTTCCTGGCCTCTCCT CTGGTGGCCATCTGGGAGCTGAAGAAAGACGTGTACGTGGTGGAACTGGACTGGTA TCCCGATGCTCCTGGCGAGATGGTGGTGCTGACCTGCGATACCCCTGAAGAGGACG GCATCACCTGGACACTGGATCAGTCTAGCGAGGTGCTCGGCAGCGGCAAGACCCTG ACCATCCAAGTGAAAGAGTTTGGCGACGCCGGCCAGTACACCTGTCACAAAGGCGG AGAAGTGCTGAGCCACAGCCTGCTGCTGCTCCACAAGAAAGAGGATGGCATTTGGA GCACCGACATCCTGAAGGACCAGAAAGAGCCCAAGAACAAGACCTTCCTGAGATG CGAGGCCAAGAACTACAGCGGCCGGTTCACATGTTGGTGGCTGACCACCATCAGCA CCGACCTGACCTTCAGCGTGAAGTCCAGCAGAGGCAGCAGTGATCCTCAGGGCGTT ACATGTGGCGCCGCTACACTGTCTGCCGAAAGAGTGCGGGGCGACAACAAAGAATA CGAGTACAGCGTGGAATGCCAAGAGGACAGCGCCTGTCCAGCCGCCGAAGAGTCTC TGCCTATCGAAGTGATGGTGGACGCCGTGCACAAGCTGAAGTACGAGAACTACACC TCCAGCTTTTTCATCCGGGACATCATCAAGCCCGATCCTCCAAAGAACCTGCAGCTG AAGCCTCTGAAGAACAGCAGACAGGTGGAAGTGTCCTGGGAGTACCCCGACACCTG GTCTACACCCCACAGCTACTTCAGCCTGACCTTTTGCGTGCAAGTGCAGGGCAAGTC CAAGCGCGAGAAAAAGGACCGGGTGTTCACCGACAAGACCAGCGCCACCGTGATC TGCAGAAAGAACGCCAGCATCAGCGTCAGAGCCCAGGACCGGTACTACAGCAGCTC TTGGAGCGAATGGGCCAGCGTGCCATGTTCTGGCGGAGGAAGCGGTGGCGGATCAG GTGGTGGATCTGGCGGCGGATCTAGAAACCTGCCTGTGGCCACTCCTGATCCTGGC ATGTTCCCTTGTCTGCACCACAGCCAGAACCTGCTGAGAGCCGTGTCCAACATGCTG CAGAAGGCCAGACAGACCCTGGAATTCTACCCCTGCACCAGCGAGGAAATCGACCA CGAGGACATCACCAAGGATAAGACCAGCACCGTGGAAGCCTGCCTGCCTCTGGAAC TGACCAAGAACGAGAGCTGCCTGAACAGCCGGGAAACCAGCTTCATCACCAACGGC TCTTGCCTGGCCAGCAGAAAGACCTCCTTCATGATGGCCCTGTGCCTGAGCAGCATC TACGAGGACCTGAAGATGTACCAGGTGGAATTCAAGACCATGAACGCCAAGCTGCT GATGGACCCCAAGCGGCAGATCTTCCTGGACCAGAATATGCTGGCCGTGATCGACG AGCTGATGCAGGCCCTGAACTTCAACAGCGAGACAGTGCCCCAGAAGTCTAGCCTG GAAGAACCCGACTTCTACAAGACCAAGATCAAGCTGTGCATCCTGCTGCACGCCTT CCGGATCAGAGCCGTGACCATCGACAGAGTGATGAGCTACCTGAACGCCTCT (SEQ ID NO: 294). In certain embodiments, 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.

In many embodiments, a cell (e.g., an immune cell or a stem cell) is engineered to produce two or more 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).

In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, the present disclosure provides for 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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 one or more additional membrane- cleavable chimeric proteins including IL15, IL18, and IL21. In some embodiments, 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) can also be further engineered to express additional proteins in addition to the cytokines and/or the membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein. As provided herein, an immunoresponsive cell is engineered to express a chimeric antigen receptor (CAR) that binds to GPC3. Also as provided herein, an immunoresponsive cell is engineered to express an ACP that includes a synthetic transcription factor.

Any CAR known in the field can be used in accordance with the present disclosure. 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. For example, 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. In certain embodiments, the peptide linker is a gly-ser linker. In certain embodiments, the peptide linker is a (GGGGS)3 linker (SEQ ID NO: 223) comprising the sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 223). An exemplary nucleic acid sequence encoding SEQ ID NO: 223 is GGCGGCGGAGGATCTGGCGGAGGTGGAAGTGGCGGAGGCGGATCT (SEQ ID NO: 224) or GGCGGCGGAGGAAGCGGAGGCGGAGGATCCGGTGGTGGTGGATCT (SEQ ID NO: 332). In certain embodiments, 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 intracellular signaling domain, a KIR3DS 1 intracellular signaling domain, a NKp44 intracellular signaling domain, a NKp46 intracellular signaling domain, a FceRlg intracellular signaling domain, a NKG2D intracellular signaling domain, an EAT-2 intracellular signaling domain, fragments thereof, combinations thereof, or combinations of fragments thereof. In some embodiments, the intracellular signaling domain comprises a sequence from Table 6A.

Table 6A. In some embodiments, 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. In some embodiments, the spacer region may be a hinge from a human protein. For example, 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. In some embodiments, 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. In some embodiments, 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 transmembrane domain, an NKp46 transmembrane domain, an FceRlg transmembrane domain, an NKG2D transmembrane domain, fragments thereof, combinations thereof, or combinations of fragments thereof. A CAR can have a spacer region between the antigen-binding domain and the transmembrane domain. Exemplary transmembrane domain sequences are provided in Table 6C. Table 6C.

In some embodiments, 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 a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQYYNYPLT (SEQ ID NO: 204). In some embodiments, 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). In some embodiments, 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).

In some embodiments, 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 ACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAACAAGAACGCCATGAACTGGGTCC GACAGGCCCCTGGCAAAGGCCTTGAATGGGTCGGACGGATCCGGAACAAGACCAA CAACTACGCCACCTACTACGCCGACAGCGTGAAGGCCAGATTCACCATCAGCCGGG ACGACAGCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAAAACCGAGGACACC GCCGTGTATTATTGCGTGGCCGGCAACAGCTTTGCCTACTGGGGACAGGGAACCCT GGTCACCGTGTCTGCC (SEQ ID NO: 330). In certain embodiments, 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.

In some embodiments, 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 GACATCGTGATGACACAGAGCCCCGATAGCCTGGCCGTGTCTCTGGGAGAAAGAGC CACCATCAACTGCAAGAGCAGCCAGAGCCTGCTGTACTCCAGCAACCAGAAGAACT ACCTGGCCTGGTATCAGCAAAAGCCCGGCCAGCCTCCTAAGCTGCTGATCTATTGG GCCAGCTCCAGAGAAAGCGGCGTGCCCGATAGATTTTCTGGCTCTGGCAGCGGCAC CGACTTCACCCTGACAATTTCTAGCCTGCAAGCCGAGGACGTGGCCGTGTATTACTG CCAGCAGTACTACAACTACCCTCTGACCTTCGGCCAGGGCACCAAGCTGGAAATCA AA (SEQ ID NO: 333) or GACATCGTGATGACACAGAGCCCCGATAGCCTGGCCGTGTCTCTGGGAGAAAGAGC CACCATCAACTGCAAGAGCAGCCAGAGCCTGCTGTACTCCAGCAACCAGAAGAACT ACCTGGCCTGGTATCAGCAAAAGCCCGGCCAGCCTCCTAAGCTGCTGATCTATTGG GCCAGCTCCAGAGAAAGCGGCGTGCCCGATAGATTTTCTGGCTCTGGCAGCGGCAC CGACTTCACCCTGACAATTTCTAGCCTGCAAGCCGAGGACGTGGCCGTGTATTACTG CCAGCAGTACTACAACTACCCTCTGACCTTCGGCCAGGGCACCAAGCTGGAAATCA AG (SEQ ID NO: 336). In certain embodiments, 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.

In many embodiments, 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. In some embodiments, the ACP is a transcriptional repressor. In some embodiments, the ACP is a transcriptional activator.

Engineered Cell Types

Also provided herein are engineered immunoresponsive cells. 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. In many embodiments, immunoresponsive cells are engineered from NK cells. In many embodiments, immunoresponsive cells are engineered from primary, donor derived NK cells.

A cell (e.g., any engineered cell described herein) can be engineered to produce the proteins described herein using methods known to those skilled in the art. For example, cells can be transduced to engineer a tumor. In some embodiments, a cell is transduced using a virus.

In a particular embodiment, the cell is transduced using an oncolytic virus. Examples of 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, an oncolytic replicating retrovirus, an oncolytic rhabdovirus, an oncolytic Seneca Valley virus, an oncolytic sindbis virus, and any variant or derivative thereof.

The virus, including any of the oncolytic viruses described herein, 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, including any of the oncolytic viruses described herein, 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.

Also provided herein are engineered bacterial cells. 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) can be engineered to comprise any of the engineered nucleic acids described herein. Human cells (e.g., immune 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 human cells (e.g., immune cells) engineered to produce one or more of the proteins described herein. In a particular aspect, provided herein are human cells (e.g., immune cells) engineered to produce two or more of the proteins described herein.

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.

Also provided herein are methods that include culturing the engineered cells of the present disclosure. Methods of culturing the engineered cells described herein are known. One skilled in the art will recognize that culturing conditions will depend on the particular engineered cell of interest. One skilled in the art will recognize that culturing conditions will depend on the specific downstream use of the engineered cell, for example, specific culturing conditions for subsequent administration of the engineered cell to a subject.

Methods of Engineering Cells

Also provided herein are 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).

In general, 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. For example, 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. One skilled in the art will appreciate the choice of delivery method can depend on the specific cell type to be engineered.

Viral-Mediated Delivery

Viral vector-based delivery platforms can be used to engineer cells. In general, a viral vector-based delivery platform engineers a cell through introducing (z.e., delivering) into a host cell. For example, 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). 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. Such 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. For example, 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. For example, 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.

In general, 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. Other exemplary 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. (2011) 239(1): 45-61, Sakuman Qt al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3): 603- 18, Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43 (1): 682-690, Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In vivo Gene Delivery, J. Virol. (1998) 72 (12): 9873- 9880).

The sequences may be preceded with one or more sequences targeting a subcellular compartment. Upon introduction (i.e. delivery) into a host cell, infected cells (i.e., an engineered cell) can express the proteins and/or other protein of interest. 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)). 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. Examples of 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, an oncolytic replicating retrovirus, an oncolytic rhabdovirus, an oncolytic Seneca Valley virus, an oncolytic sindbis virus, and any variant or derivative thereof. 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. In general, 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. In general, 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. 7,311,907; 7,262,049; 7,250,299; 7,226,780; 7,220,578; 7,211,247; 7,160,721; 7,078,031; 7,070,993; 7,056,699; 6,955,919, each herein incorporated by reference for all purposes.

The viral vector-based delivery platform can be adenovirus-based. In general, 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. In general, 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. 5585362; 6,083,716, 7,371,570; 7,348,178; 7,323,177; 7,319,033; 7,318,919; and 7,306,793 and International Patent Application WO96/13597, each herein incorporated by reference for all purposes.

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. 4,797,368; 5,436,146; 6,632,670; 6,642,051; 7,078,387; 7,314,912; 6,498,244; 7,906,111; US patent publications US 2003-0138772, US 2007/0036760, and US 2009/0197338; Gao, et al., J. Virol, 78( 12):6381- 6388 (June 2004); Gao, et al, Proc Natl Acad Sci USA, 100(10):6081-6086 (May 13, 2003); and International Patent applications WO 2010/138263 and WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94: 1351 (1994), each herein incorporated by reference for all purposes). Exemplary methods for constructing recombinant AAV vectors are described in more detail in U.S. Pat. No, 5,173,414; Tratschin et ah, Mol. Cell. Biol. 5:3251- 3260 (1985); Tratschin, et ah, Mol. Cell, Biol. 4:2072-2081 (1984); Hermonat &amp; Muzyczka, PNAS 81:64666470 (1984); and Samuiski et ah, J. Virol. 63:03822-3828 (1989), each herein incorporated by reference for all purposes. In general, 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. In particular examples, an AAV-based vector has a capsid protein having an amino acid sequence corresponding to AAV2. In particular examples, 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. In general, 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. Accordingly, production of 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. In general, 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. For example, 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.

Lipid Structure Delivery Systems

Engineered nucleic acids (e.g., any of the engineered nucleic acids described herein) can be introduced into a cell using a lipid-mediated delivery system. In general, a lipid-mediated delivery system uses a structure composed of an outer lipid membrane enveloping an internal compartment. Examples of 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. As used herein, 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. The selection of lipids is 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. 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.) 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.

As used herein, the term “extracellular vesicle” or “EV” refers to a cell-derived vesicle comprising a membrane that encloses an internal space. In general, extracellular vesicles comprise all membrane-bound vesicles that have a smaller diameter than the cell from which they are derived. Generally 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. By way of example and without limitation, 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.

As used herein the term “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.

As used herein, the term “nanovesicle” (also referred to as a “microvesicle”) 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. In general, 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. The production of nanovesicles may, in some instances, result in the destruction of said producer cell. Preferably, 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. The nanovesicle, once it is derived from a producer cell according to said manipulation, may be isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof.

Lipid nanoparticles (LNPs), 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. In an example, 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. In addition, 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. Similarly, 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. In certain examples, 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. In an example, 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. In an example, the droplet generating device can control the size range and size distribution of the LNPs produced. For example, the LNP can have a size ranging from 1 to 1000 nanometers in diameter, e.g., 1, 10, 50, 100, 500, or 1000 nanometers. Following droplet generation, the delivery vehicles encapsulating the cargo/payload (e.g., an engineered nucleic acid and/or viral delivery system) can be further treated or engineered to prepare them for administration.

Nanoparticle Delivery

Nanomaterials can be used to deliver engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). Nanomaterial vehicles, importantly, 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.

Genomic Editing Systems

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. In general, 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. In general, transposon systems integrate a cargo/payload (e.g., an engineered nucleic acid) randomly into a host genome. Examples of 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. Without wishing to be bound by theory, in general, 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. Briefly, following an insult to genomic DNA (typically a double-stranded break), 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. In a natural context, HDR can use the other chromosome present in a cell as a template. In gene editing systems, exogenous polynucleotides are introduced into the cell to be used as a homologous recombination template (HRT or HR template). In general, 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 (e.g., a gene or a portion of a gene) can be incorporated (z.e., “integrated”) into the given genomic locus during templated HDR. Thus, 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).

In some examples, a HR template can be linear. Examples of linear HR templates include, but are not limited to, a linearized plasmid vector, a ssDNA, a synthesized DNA, and a PCR amplified DNA. In particular examples, a HR template can be circular, such as a plasmid. A circular template can include a supercoiled template.

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.

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 (2018), Article number: 1911), herein incorporated by reference for all that it teaches. In general, 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). While the Cas9 system is illustrated here, other CRISPR systems can be used, such as the Cpfl/Casl2 or Casl3 systems. 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.

In general, the components of a CRISPR system interact with each other to form a Ribonucleoprotein (RNP) complex to mediate sequence specific cleavage. In some CRISPR systems, each component can be separately produced and used to form the RNP complex. In some CRISPR systems, 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 a particular example, 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, 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, can be produced in vitro i.e., synthesized and purified) using a variety of RNA production techniques known to those skilled in the art, 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.

In some CRISPR systems, each component (e.g., Cas9 and an sgRNA) can be separately encoded by a polynucleotide with each polynucleotide introduced into a cell together or separately. In some CRISPR systems, 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. Following expression of each polynucleotide encoded CRISPR component within a cell (e.g., translation of a nuclease and transcription of CRISPR RNAs), an RNP complex can form within the cell and can then direct site-specific cleavage.

Some RNPs can be engineered to have moieties that promote delivery of the RNP into the nucleus. For example, 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.

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. In some CRISPR systems, 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. For example, two separate CRISPR compositions can be provided to direct cleavage at two different target nucleotide sequences within a certain distance of each other. In some CRISPR systems, more than one CRISPR composition can be provided such that each separately target opposite strands of the same gene or general genomic locus. For example, two separate CRISPR “nickase” compositions can be provided to direct cleavage at the same gene or general genomic locus at opposite strands.

In general, the features of a CRISPR-mediated editing system described herein can apply to other nuclease-based genomic editing systems. 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. By changing the amino acids present in the highly variable residue region of the monomers of the DNA binding domain, different artificial TALENs can be created to target various nucleotides sequences. The DNA binding domain subsequently directs the nuclease to the target sequences and creates a doublestranded break. TALEN-based systems are described in more detail in U.S. Ser. No. 12/965,590; U.S. Pat. No. 8,450,471; U.S. Pat. No. 8,440,431; U.S. Pat. No. 8,440,432; U.S. Pat. No. 10,172,880; and U.S. Ser. No. 13/738,381, all of which are incorporated by reference herein in their entirety. ZFN-based editing systems are described in more detail in U.S. Patent Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005/0064474; 2007/0218528; 2005/0267061, all incorporated herein by reference in their entireties for all purposes.

Other Engineering Delivery Systems

Various additional means to introduce engineered nucleic acids (e.g., any of the engineered nucleic acids described herein) into 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. Cells and other entities can be electroporated in vitro, in vivo, or ex vivo. 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.) 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 Electroporation™, Lonza® Nucleofector™ systems, and Bio-Rad® electroporation systems.

Other means for introducing engineered nucleic acids (e.g., any of the engineered nucleic acids described herein) into 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, such as naked plasmids or mRNA, 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.

Delivery Vehicles

Also provided herein are 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. For example, 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. Accordingly, 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.

Methods of Treatment

Further provided herein are methods that include delivering, or administering, to 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). Further provided herein are 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.

Further provided herein are 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. Further provided herein are 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.

In some embodiments, 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. Thus, 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. For example, 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. In another example, engineered cells or delivery vehicles can be administered in combination with a checkpoint inhibitor therapy. Exemplary 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 ® - BMS), lirilumab (anti-KIR; BMS), monalizumab (anti- NKG2A; Innate Pharma/AstraZeneca). In other examples, engineered cells or delivery vehicles can be administered in combination with TGFbeta inhibitors, VEGF inhibitors, or HPGE2. In another example, engineered cells or delivery vehicles can be administered in combination with an anti-CD40 antibody.

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. For example, 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. For example, 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.

In vivo Expression

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. Such 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.

Other Sequences

HIV-1 protease (SEQ ID NO: 144):

PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVR QY

DQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF

Angiotensin converting enzyme (ACE) (SEQ ID NO: 156):

MGAASGRRGPGLLLPLPLLLLLPPQPALALDPGLQPGNFSADEAGAQLFAQSYNSSA EQ

VLFQSVAASWAHDTNITAENARRQEEAALLSQEFAEAWGQKAKELYEPIWQNFTDPQ L

RRIIGAVRTLGSANLPLAKRQQYNALLSNMSRIYSTAKVCLPNKTATCWSLDPDLTN IL

ASSRSYAMLLFAWEGWHNAAGIPLKPLYEDFTALSNEAYKQDGFTDTGAYWRSWYNS

PTFEDDLEHLYQQLEPLYLNLHAFVRRALHRRYGDRYINLRGPIPAHLLGDMWAQSW E

NIYDMVVPFPDKPNLDVTSTMLQQGWNATHMFRVAEEFFTSLELSPMPPEFWEGSML E

KPADGREVVCHASAWDFYNRKDFRIKQCTRVTMDQLSTVHHEMGHIQYYLQYKDLPV

SLRRGANPGFHEAIGDVLALSVSTPEHLHKIGLLDRVTNDTESDINYLLKMALEKIA FLP

FGYLVDQWRWGVFSGRTPPSRYNFDWWYLRTKYQGICPPVTRNETHFDAGAKFHVPN

VTPYIRYFVSFVLQFQFHEALCKEAGYEGPLHQCDIYRSTKAGAKLRKVLQAGSSRP WQ

EVLKDMVGLDALDAQPLLKYFQPVTQWLQEQNQQNGEVLGWPEYQWHPPLPDNYPE

GIDLVTDEAEASKFVEEYDRTSQVVWNEYAEANWNYNTNITTETSKILLQKNMQIAN H

TLKYGTQARKFDVNQLQNTTIKRIIKKVQDLERAALPAQELEEYNKILLDMETTYSV AT

VCHPNGSCLQLEPDLTNVMATSRKYEDLLWAWEGWRDKAGRAILQFYPKYVELINQA

ARLNGYVDAGDSWRSMYETPSLEQDLERLFQELQPLYLNLHAYVRRALHRHYGAQHI

NLEGPIPAHLLGNMWAQTWSNIYDLVVPFPSAPSMDTTEAMLKQGWTPRRMFKEADD

FFTSLGLLPVPPEFWNKSMLEKPTDGREVVCHASAWDFYNGKDFRIKQCTTVNLEDL V

VAHHEMGHIQYFMQYKDLPVALREGANPGFHEAIGDVLALSVSTPKHLHSLNLLSSE G

GSDEHDINFLMKMALDKIAFIPFSYLVDQWRWRVFDGSITKENYNQEWWSLRLKYQG L

CPPVPRTQGDFDPGAKFHIPSSVPYIRYFVSFIIQFQFHEALCQAAGHTGPLHKCDI YQSK

EAGQRLATAMKLGFSRPWPEAMQLITGQPNMSASAMLSYFKPLLDWLRTENELHGEK

LGWPQYNWTPNSARSEGPLPDSGRVSFLGLDLDAQQARVGQWLLLFLGIALLVATLG L

SQRLFSIRHRSLHRHSHGPQFGSEVELRHS DENV NS3pro (NS2B/NS3) >sp|P33478| 1475-2093 (SEQ ID NO: 157):

SGVLWDTPSPPEVERAVLDDGIYRIMQRGLLGRSQVGVGVFQDGVFHTMWHVTRGAV

LMYQGKRLEPSWASVKKDLISYGGGWRFQGSWNTGEEVQVIAVEPGKNPKNVQTAPG

TFKTPEGEVGAIALDFKPGTSGSPIVNREGKIVGLYGNGVVTTSGTYVSAIAQAKAS QEG

PLPEIEDEVFRKRNLTIMDLHPGSGKTRRYLPAIVREAIRRNVRTLILAPTR WAS EMAE

ALKGMPIRYQTTAVKSEHTGKEIVDLMCHATFTMRLLSPVRVPNYNMIIMDEAHFTD P

ASIARRGYISTRVGMGEAAAIFMTATPPGSVEAFPQSNAVIQDEERDIPERSWNSGY EWI

TDFPGKTVWFVPSIKSGNDIANCLRKNGKRVIQLSRKTFDTEYQKTKNNDWDYVVTT D

ISEMGANFRADRVIDPRRCLKPVILKDGPERVILAGPMPVTVASAAQRRGRIGRNQN KE

GDQYVYMGQPLNNDEDHAHWTEAKMLLDNINTPEGIIPALFEPEREKSAAIDGEYRL R

GEARKTFVELMRRGDLPVWLSYKVASEGFQYSDRRWCFDGERNNQVLEENMDVEMW

TKEGERKKLRPRWLDARTYSDPLALREFKEFAAGRR

DENV NS3pro (NS2B/NS3) >sp|P14340|1476-2093 (SEQ ID NO: 158):

AGVLWDVPSPPPVGKAELEDGAYRIKQKGILGYSQIGAGVYKEGTFHTMWHVTRGAV

LMHKGKRIEPSWADVKKDLISYGGGWKLEGEWKEGEEVQVLALEPGKNPRAVQTKPG

LFKTNAGTIGAVSLDFSPGTSGSPIIDKKGKVVGLYGNGVVTRSGAYVSAIAQTEKS IED

NPEIEDDIFRKRKLTIMDLHPGAGKTKRYLPAIVREAIKRGLRTLILAPTRVVAAEM EEA

LRGLPIRYQTPAIRAEHTGREIVDLMCHATFTMRLLSPVRVPNYNLIIMDEAHFTDP ASIA

ARGYISTRVEMGEAAGIFMTATPPGSRDPFPQSNAPIMDEEREIPERSWSSGHEWVT DFK

GKTVWFVPSIKAGNDIAACLRKNGKKVIQLSRKTFDSEYVKTRTNDWDFVVTTDISE M

GANFKAERVIDPRRCMKPVILTDGEERVILAGPMPVTHSSAAQRRGRIGRNPKNEND QY

IYMGEPLENDEDCAHWKEAKMLLDNINTPEGIIPSMFEPEREKVDAIDGEYRLRGEA RK

TFVDLMRRGDLPVWLAYRVAAEGINYADRRWCFDGIKNNQILEENVEVEIWTKEGER

KKLKPRWLDAKIYSDPLALKEFKEFAAGRK

DENV NS3pro (NS2B/NS3) >sp|Q99D35| 1474-2092 (SEQ ID NO: 159):

SGVLWDVPSPPETQKAELEEGVYRIKQQGIFGKTQVGVGVQKEGVFHTMWHVTRGAV

LTHNGKRLEPNWASVKKDLISYGGGWRLSAQWQKGEEVQVIAVEPGKNPKNFQTMPG

IFQTTTGEIGAIALDFKPGTSGSPIINREGKVVGLYGNGVVTKNGGYVSGIAQTNAE PDG

PTPELEEEMFKKRNLTIMDLHPGSGKTRKYLPAIVREAIKRRLRTLILAPTRVVAAE MEE

ALKGLPIRYQTTATKSEHTGREIVDLMCHATFTMRLLSPVRVPNYNLIIMDEAHFTD PAS

IAARGYISTRVGMGEAAAIFMTATPPGTADAFPQSNAPIQDEERDIPERSWNSGNEW ITD

FVGKTVWFVPSIKAGNDIANCLRKNGKKVIQLSRKTFDTEYQKTKLNDWDFVVTTDI SE

MGANFKADRVIDPRRCLKPVILTDGPERVILAGPMPVTVASAAQRRGRVGRNPQKEN D

QYIFMGQPLNKDEDHAHWTEAKMLLDNINTPEGIIPALFEPEREKSAAIDGEYRLKG ESR KTFVELMRRGDLPVWLAHKVASEGIKYTDRKWCFDGERNNQILEENMDVEIWTKEGE

KKKLRPRWLDARTYSDPLALKEFKDFAAGRK

DENV NS3pro (NS2B/NS3) >sp|Q5UCB8|1475-2092 (SEQ ID NO: 160):

SGALWDVPSPAATQKAALSEGVYRIMQRGLFGKTQVGVGIHIEGVFHTMWHVTRGSV I

CHETGRLEPSWADVRNDMISYGGGWRLGDKWDKEEDVQVLAIEPGKNPKHVQTKPGL

FKTLTGEIGAVTLDFKPGTSGSPIINRKGKVIGLYGNGVVTKSGDYVSAITQAERIG EPDY

EVDEDIFRKKRLTIMDLHPGAGKTKRILPSIVREALKRRLRTLILAPTRVVAAEMEE ALR

GLPIRYQTPAVKSEHTGREIVDLMCHATFTTRLLSSTRVPNYNLIVMDEAHFTDPSS VAA

RGYISTRVEMGEAAAIFMTATPPGTTDPFPQSNSPIEDIEREIPERSWNTGFDWITD YQGK

TVWFVPSIKAGNDIANCLRKSGKKVIQLSRKTFDTEYPKTKLTDWDFVVTTDISEMG AN

FRAGRVIDPRRCLKPVILPDGPERVILAGPIPVTPASAAQRRGRIGRNPAQEDDQYV FSG

DPLKNDEDHAHWTEAKMLLDNIYTPEGIIPTLFGPEREKTQAIDGEFRLRGEQRKTF VEL

MRRGDLPVWLSYKVASAGISYKDREWCFTGERNNQILEENMEVEIWTREGEKKKLRP K

WLDARVYADPMALKDFKEFASGRK

PEST (two copies of residues 277-307 of human IKBOI; SEQ ID NO: 161):

LQMLPESEDEESYDTESEFTEFTEDELPYDDGSLQMLPESEDEESYDTESEFTEFTE DELP

YDD

GRR (residues 352-408 of human pl05; SEQ ID NO: 162):

EIKDKEEVQRKRQKLMPNFSDSFGGGSGAGAGGGGMFGSGGGGGGTGSTGPGYSFPH

DRR (residues 210-295 of yeast Cdc34; SEQ ID NO: 163):

IDDENGSVILQDDDYDDGNNHIPFEDDDVYNYNDNDDDDERIEFEDDDDDDDDSIDN D

SVMDRKQPHKAEDESEDVEDVERVSKKD

SNS (tandem repeat of SP2 and NB (SP2-NB-SP2 of influenza A or influenza B; e.g., SEQ ID

NO: 164):

PESMREEYRKEGSKRIKCPDCEPFCNKRGSPESMREEYRKE

RPB (four copies of residues 1688-1702 of yeast RPB; SEQ ID NO: 165):

RSYSPTSPNYSPTSPSGSYSPTSPNYSPTSPSGGSRSYSPTSPNYSPTSPSGSYSPT SPNYSP

TSPSG

SPmix (tandem repeat of SP1 and SP2 (SP2-SP1-SP2-SP1-SP2 of influenza A virus M2 protein;

SEQ ID NO: 166):

PESMREEYRKEGSSLLTEVETPGSPESMREEYRKEGSSLLTEVETPGSPESMREEYR KE

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):

SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV

ADDITIONAL EMBODIMENTS

Provided below are enumerated embodiments describing specific embodiments of the invention:

Embodiment 1: 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 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 wherein

S comprises a secretable effector molecule comprising the first and/or second cytokine,

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 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.

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.

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.

Embodiment 15: 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 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

(b) 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 - C - MT or MT - C - S wherein

S comprises a secretable effector molecule comprising the first and/or second cytokine,

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 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) 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 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 - C - MT or MT - C - S wherein

S comprises a secretable effector molecule comprising the first and/or second cytokine,

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 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. 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).

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 MT5-MMP protease, a furin protease, a PCSK7 protease, a matriptase protease, a matriptase-2 protease, an MMP9 protease, and an NS 3 protease.

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.

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 PCSK7 protease, a matriptase protease, a matriptase-2 protease, and an MMP9 protease.

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.

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 (SEQ ID NO: 203), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQYYNYPLT (SEQ ID NO: 204).

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).

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.

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, a 2B4 intracellular signaling domain, a CD 16a intracellular signaling domain, a DNAM-1 intracellular signaling domain, a KIR2DS 1 intracellular signaling domain, a KIR3DS 1 intracellular signaling domain, a NKp44 intracellular signaling domain, a NKp46 intracellular signaling domain, a FceRlg intracellular signaling domain, a NKG2D intracellular signaling domain, and an EAT-2 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, a KIR2DS 1 transmembrane domain, a KIR3DS 1 transmembrane domain, an NKp44 transmembrane domain, an NKp46 transmembrane domain, an FceRlg transmembrane domain, and an NKG2D 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).

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.

Embodiment 134: The immunoresponsive cell of any one of embodiments 127-133, wherein the DNA binding domain comprises a zinc finger (ZF) protein domain.

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).

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.

Embodiment 145: The immunoresponsive cell of embodiment 144, wherein the protease inhibitor is grazoprevir (GRZ).

Embodiment 146: The immunoresponsive cell of any one of embodiments 1-145, wherein the ACP further comprises a nuclear localization signal (NLS).

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 (iPSC), and an iPSC-derived cell.

Embodiment 164: The immunoresponsive cell of any one of embodiments 1-162, wherein the cell is a Natural Killer (NK) cell.

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 - C - MT or MT - C - S wherein

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 - C - MT or MT - C - S wherein

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 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:

S - C - MT or MT - C - S wherein

S comprises a secretable effector molecule comprising the IL12p70 fusion protein, 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 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.

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.

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 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 wherein S comprises a secretable effector molecule comprising the first and/or second cytokine, 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 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 a secretable effector molecule comprising the IL12p70 fusion protein, 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 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 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 - C - MT or MT - C - S wherein S comprises a secretable effector molecule comprising the first and/or second cytokine, 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 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.

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.

Embodiment 215: The engineered nucleic acid of any one of embodiments 201-213, 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 216: The engineered nucleic acid of any one of embodiments 201-213, 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 217: The engineered nucleic acid of any one of embodiments 201-216, wherein the fourth promoter comprises a constitutive promoter, an inducible promoter, or a synthetic promoter.

Embodiment 218: The engineered nucleic acid of any one of embodiments 201-217, 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.

Embodiment 219: 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 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 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 wherein S comprises a secretable effector molecule comprising the first and/or second cytokine, 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 220: The immunoresponsive cell of embodiment 219, 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 221: The immunoresponsive cell of embodiment 219, 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 222: The immunoresponsive cell of embodiment 219, wherein the first expression cassette is configured to be transcribed in a same orientation relative to transcription of the second expression cassette.

Embodiment 223: The immunoresponsive cell of embodiment 219, 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 224: The immunoresponsive cell of any one of embodiments 219-223, wherein the first promoter comprises a constitutive promoter, an inducible promoter, or a synthetic promoter.

Embodiment 225: The immunoresponsive cell of any one of embodiments 219-224, 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.

Embodiment 226: The immunoresponsive cell of any one of embodiments 219-225, wherein the first exogenous polynucleotide sequence and the second exogenous polynucleotide sequence are separated by a linker polynucleotide sequence.

Embodiment 227: The immunoresponsive cell of embodiment 226, wherein the linker polynucleotide sequence is operably associated with the translation of the first cytokine and the CAR as separate polypeptides.

Embodiment 228: The immunoresponsive cell of embodiment 226 or 227, wherein the linker polynucleotide sequence encodes one or more 2 A ribosome skipping elements. Embodiment 229: The immunoresponsive cell of embodiment 228, the one or more 2A ribosome skipping elements are each selected from the group consisting of: P2A, T2A, E2A, and F2A.

Embodiment 230: The immunoresponsive cell of embodiment 228 or 229, wherein the one or more 2A ribosome skipping elements comprises an E2A/T2A.

Embodiment 231: The immunoresponsive cell of embodiment 230, wherein the E2A/T2A comprises the amino acid sequence of SEQ ID NO: 281.

Embodiment 232: The immunoresponsive cell of any one of embodiments 226-231, wherein the linker polynucleotide sequence encodes an Internal Ribosome Entry Site (IRES).

Embodiment 233: The immunoresponsive cell of any one of embodiments 226-231, wherein the linker polynucleotide sequence encodes a cleavable polypeptide.

Embodiment 234: The immunoresponsive cell of embodiment 233, wherein the cleavable polypeptide comprises a furin polypeptide sequence.

Embodiment 235: The immunoresponsive cell of any one of embodiments 219-234, the third promoter comprises a constitutive promoter, an inducible promoter, or a synthetic promoter.

Embodiment 236: The immunoresponsive cell of any one of embodiments 226-235, 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 237: The immunoresponsive cell of any one of embodiments 219-236, wherein the first cytokine is IL- 15.

Embodiment 238: The immunoresponsive cell of embodiment 237, wherein the IL-15 comprises the amino acid sequence of SEQ ID NO: 285.

Embodiment 239: The immunoresponsive cell of any one of embodiments 219-238, wherein the second cytokine is selected from the group consisting of: IL12, an IL12p70 fusion protein, IL 18, and IL21.

Embodiment 240: The immunoresponsive cell of any one of embodiments 219-239, wherein the second cytokine is the IL12p70 fusion protein.

Embodiment 241: The immunoresponsive cell of embodiment 240, wherein the IL12p70 fusion protein comprises the amino acid sequence of SEQ ID NO: 293.

Embodiment 242: The immunoresponsive cell of any one of embodiments 219-236 or 239- 241, wherein the first cytokine is IL12 or an IL12p70 fusion protein. Embodiment 243: The immunoresponsive cell of any one of embodiments 219-238 or 241- 242, wherein the second cytokine is selected from the group consisting of: IL15, IL18, and IL21.

Embodiment 244: The immunoresponsive cell of any one of embodiments 219-243, wherein the protease cleavage site is selected from the group consisting of: a Type I 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 ADAM 10 protease cleavage site, an ADAM 12 protease cleavage site, an ADAM 15 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, a BACE2 protease cleavage site, a SIP protease cleavage site, an MT1-MMP protease cleavage site, an MT3-MMP protease cleavage site, an MT5-MMP protease cleavage site, a furin protease cleavage site, a PCSK7 protease cleavage site, a matriptase protease cleavage site, a matriptase-2 protease cleavage site, an MMP9 protease cleavage site, and an NS3 protease cleavage site.

Embodiment 245: The immunoresponsive cell of any one of embodiments 219-244, 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 MT5-MMP protease, a furin protease, a PCSK7 protease, a matriptase protease, a matriptase-2 protease, an MMP9 protease, and an NS 3 protease.

Embodiment 246: The immunoresponsive cell of any one of embodiments 219-245, wherein the protease cleavage site is cleavable by an ADAM 17 protease.

Embodiment 247: The immunoresponsive cell of any one of embodiments 219-246, wherein the protease cleavage site comprises a first region having the amino acid sequence of PRAE (SEQ ID NO: 176).

Embodiment 248: The immunoresponsive cell of any one of embodiments 219-247, wherein the protease cleavage site comprises a second region having the amino acid sequence of KGG (SEQ ID NO: 177). Embodiment 249: The immunoresponsive cell of embodiment 248, wherein the first region is located N-terminal to the second region.

Embodiment 250: The immunoresponsive cell of any one of embodiments 219-249, 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 251: The immunoresponsive cell of any one of embodiments 219-250, wherein the protease cleavage site comprises the amino acid sequence of PRAEAVKGG (SEQ ID NO: 179).

Embodiment 252: The immunoresponsive cell of any one of embodiments 219-250, wherein the protease cleavage site comprises the amino acid sequence of PRAEALKGG (SEQ ID NO: 180).

Embodiment 253: The immunoresponsive cell of any one of embodiments 219-250, wherein the protease cleavage site comprises the amino acid sequence of PRAEYSKGG (SEQ ID NO: 181).

Embodiment 254: The immunoresponsive cell of any one of embodiments 219-250, wherein the protease cleavage site comprises the amino acid sequence of PRAEPIKGG (SEQ ID NO: 182).

Embodiment 255: The immunoresponsive cell of any one of embodiments 219-250, wherein the protease cleavage site comprises the amino acid sequence of PRAEAYKGG (SEQ ID NO: 183).

Embodiment 256: The immunoresponsive cell of any one of embodiments 219-250, wherein the protease cleavage site comprises the amino acid sequence of PRAESSKGG (SEQ ID NO: 184).

Embodiment 257: The immunoresponsive cell of any one of embodiments 219-250, wherein the protease cleavage site comprises the amino acid sequence of PRAEFTKGG (SEQ ID NO: 185).

Embodiment 258: The immunoresponsive cell of any one of embodiments 219-250, wherein the protease cleavage site comprises the amino acid sequence of PRAEAAKGG (SEQ ID NO: 186).

Embodiment 259: The immunoresponsive cell of any one of embodiments 219-250, wherein the protease cleavage site comprises the amino acid sequence of DEPHYSQRR (SEQ ID NO: 187).

Embodiment 260: The immunoresponsive cell of any one of embodiments 219-250, wherein the protease cleavage site comprises the amino acid sequence of PPLGPIFNPG (SEQ ID NO: 188). Embodiment 261: The immunoresponsive cell of any one of embodiments 219-250, wherein the protease cleavage site comprises the amino acid sequence of PLAQAYRSS (SEQ ID NO: 189).

Embodiment 262: The immunoresponsive cell of any one of embodiments 219-250, wherein the protease cleavage site comprises the amino acid sequence of TPIDSSFNPD (SEQ ID NO: 190).

Embodiment 263: The immunoresponsive cell of any one of embodiments 219-250, wherein the protease cleavage site comprises the amino acid sequence of VTPEPIFSLI (SEQ ID NO: 191).

Embodiment 264: The immunoresponsive cell of any one of embodiments 219-250, wherein the protease cleavage site comprises the amino acid sequence of ITQGLAVSTISSFF (SEQ ID NO: 198).

Embodiment 265: The immunoresponsive cell of any one of embodiments 219-264, wherein the protease cleavage site is comprised within a peptide linker.

Embodiment 266: The immunoresponsive cell of embodiment 265, wherein the protease cleavage site is N-terminal to a peptide linker.

Embodiment 267: The immunoresponsive cell of any one of embodiments 265-266, wherein the peptide linker comprises a glycine-serine (GS) linker.

Embodiment 268: The immunoresponsive cell of any one of embodiments 219-267, wherein the cell membrane tethering domain comprises a transmembrane-intracellular domain or a transmembrane domain.

Embodiment 269: The immunoresponsive cell of embodiment 268, wherein 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.

Embodiment 270: The immunoresponsive cell of any one of embodiments 268-269, wherein the transmembrane-intracellular domain and/or transmembrane domain is derived from B7-1.

Embodiment 271: The immunoresponsive cell of any one of embodiments 268-270, wherein the transmembrane-intracellular domain and/or transmembrane domain comprises the amino acid sequence of SEQ ID NO: 219.

Embodiment 272: The immunoresponsive cell of any one of embodiments 219-271, wherein the cell membrane tethering domain comprises a cell surface receptor, or a cell membrane-bound portion thereof. Embodiment 273: The immunoresponsive cell of any one of embodiments 219-272, 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 274: The immunoresponsive cell of embodiment 273, 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 275: The immunoresponsive cell of any one of embodiments 219-274, wherein when expressed in a cell, the secretable effector molecule (e.g., any of the cytokines described herein) is tethered to a cell membrane of the cell.

Embodiment 276: The immunoresponsive cell of any one of embodiments 219-275, wherein when expressed in a cell expressing a protease capable of cleaving the protease cleavage site, the secretable effector molecule is released from the cell membrane.

Embodiment 277: The immunoresponsive cell of embodiment 276, the protease is expressed on the cell membrane of the cell.

Embodiment 278: The immunoresponsive cell of embodiment 276 or 277, wherein the protease expressed on the cell membrane is endogenous to the cell.

Embodiment 279: The immunoresponsive cell of any one of embodiments 276-278, wherein 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 protease, an MT3-MMP protease, an MT5-MMP protease, a furin protease, a PCSK7 protease, a matriptase protease, a matriptase-2 protease, and an MMP9 protease.

Embodiment 280: The immunoresponsive cell of any one of embodiments 276-279, wherein the protease is an ADAM 17 protease.

Embodiment 281: The immunoresponsive cell of any one of embodiments 276-280, wherein the protease expressed on the cell membrane is heterologous to the cell.

Embodiment 282: The immunoresponsive cell of any one of embodiments 276-281, wherein the protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3). Embodiment 283: The immunoresponsive cell of any one of embodiments 219-282, wherein the protease cleavage site comprises an NS3 protease cleavage site.

Embodiment 284: The immunoresponsive cell of any one of embodiments 219-283, wherein the NS3 protease cleavage site comprises a NS3/NS4A, a NS4A/NS4B, a NS4B/NS5A, or a NS5A/NS5B junction cleavage site.

Embodiment 285: The immunoresponsive cell of any one of embodiments 219-284, wherein the protease can be repressed by a protease inhibitor.

Embodiment 286: The immunoresponsive cell of embodiment 285, wherein the protease inhibitor is selected from the group consisting of: simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir.

Embodiment 287: The immunoresponsive cell of any one of embodiments 219-286, wherein the expression and/or localization of the protease is capable of regulation.

Embodiment 288: The immunoresponsive cell of embodiment 287, wherein the expression and/or localization is regulated by a cell state of the cell.

Embodiment 289: The immunoresponsive cell of any one of embodiments 219-288, wherein the first exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein.

Embodiment 290: The immunoresponsive cell of any one of embodiments 219-289, wherein the first exogenous polynucleotide sequence further comprises a polynucleotide sequence encoding a secretion signal peptide.

Embodiment 291: The immunoresponsive cell of any one of embodiments 219-290, wherein the second exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein.

Embodiment 292: The immunoresponsive cell of any one of embodiments 219-291, wherein the second exogenous polynucleotide sequence further comprises a polynucleotide sequence encoding a secretion signal peptide.

Embodiment 293: The immunoresponsive cell of embodiment 292, 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.

Embodiment 294: The immunoresponsive cell of embodiments 292 or 293, wherein the secretion signal peptide is derived from GMCSFRa. Embodiment 295: The immunoresponsive cell of any one of embodiments 292-294, wherein the secretion signal peptide comprises the amino acid sequence of SEQ ID NO: 216.

Embodiment 296: The immunoresponsive cell of any one of embodiments 219-295, wherein the secretion signal peptide is derived from IgE.

Embodiment 297: The immunoresponsive cell of any one of embodiments 219-296, wherein the secretion signal peptide comprises the amino acid sequence of SEQ ID NO: 218.

Embodiment 298: The immunoresponsive cell of any one of embodiments 219-297, wherein the third exogenous polynucleotide sequence further comprises a polynucleotide sequence encoding a secretion signal peptide.

Embodiment 299: The immunoresponsive cell of any embodiment 298, wherein the secretion signal peptide is operably associated with the second cytokine.

Embodiment 300: The immunoresponsive cell of embodiment 298 or 299, wherein the secretion signal peptide is native to the second cytokine.

Embodiment 301: The immunoresponsive cell of embodiment 298 or 299, wherein the secretion signal peptide is non-native to the second cytokine.

Embodiment 302: The immunoresponsive cell of any one of embodiments 219-301, wherein the third exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein.

Embodiment 303: The immunoresponsive cell of any one of embodiments 219-302, wherein the first expression cassette further comprises a polynucleotide sequence encoding a secretion signal peptide.

Embodiment 304: The immunoresponsive cell of embodiment 303, wherein the secretion signal peptide is operably associated with the first cytokine.

Embodiment 305: The immunoresponsive cell of embodiment 303 or 304, wherein the secretion signal peptide is native to the first cytokine.

Embodiment 306: The immunoresponsive cell of embodiment 303 or 304, wherein the secretion signal peptide is non-native to the first cytokine.

Embodiment 307: The immunoresponsive cell of any one of embodiments 219-306, 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 308: The immunoresponsive cell of any one of embodiments 219-307, 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 309: The immunoresponsive cell of any one of embodiments 219-308, wherein the engineered nucleic acid is a single- stranded or double- stranded nucleic acid selected from the group consisting of: a DNA, cDNA, an RNA, an mRNA, and a naked plasmid.

Embodiment 310: The immunoresponsive cell of any one of embodiments 219-309, wherein the exogenous polynucleotide sequences encoded by the expression cassette further comprise a 3 ’untranslated region (UTR) comprising an mRNA-destabilizing element that is operably linked to the exogenous polynucleotide sequence.

Embodiment 311: The immunoresponsive cell of embodiment 310, wherein the mRNA- destabilizing element comprises an AU-rich element and/or a stem-loop destabilizing element (SLDE).

Embodiment 312: The immunoresponsive cell of embodiment 310 or 311, wherein the mRNA-destabilizing element comprises an AU-rich element.

Embodiment 313: The immunoresponsive cell of embodiment 312, wherein the AU-rich element includes at least two overlapping motifs of the sequence ATTTA (SEQ ID NO: 209).

Embodiment 314: The immunoresponsive cell of embodiment 312, wherein the AU-rich element comprises ATTTATTTATTTATTTATTTA (SEQ ID NO: 210).

Embodiment 315: The immunoresponsive cell of embodiment 310 or 311, wherein the mRNA-destabilizing element comprises a stem-loop destabilizing element (SLDE).

Embodiment 316: The immunoresponsive cell of embodiment 315, wherein the SLDE comprises CTGTTTAATATTTAAACAG (SEQ ID NO: 211).

Embodiment 317: The immunoresponsive cell of embodiment 310 or 311, wherein the mRNA-destabilizing element comprises at least one AU-rich element and at least one SLDE.

Embodiment 318: The immunoresponsive cell of embodiment 317, wherein the AuSLDE sequence comprises ATTTATTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAG (SEQ ID NO: 212).

Embodiment 319: The immunoresponsive cell of embodiment 310 or 311, wherein the mRNA-destabilizing element comprises a 2X AuSLDE.

Embodiment 320: The immunoresponsive cell of embodiment 319, wherein the 2X AuSLDE sequence is provided as ATTTATTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAGtgcggtaag cA TTTATTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAG (SEQ ID NO: 213).

Embodiment 321: The immunoresponsive cell of any one of embodiments 219-320, 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 (SEQ ID NO: 203), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQYYNYPLT (SEQ ID NO: 204).

Embodiment 322: The immunoresponsive cell of embodiment 321, 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 323: The immunoresponsive cell of embodiment 321, 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 324: The immunoresponsive cell of any one of embodiments 321-323, 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 EEIK (SEQ ID NO: 208).

Embodiment 325: The immunoresponsive cell of any one of embodiments 321-323, wherein the VE 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 326: The immunoresponsive cell of any one of embodiments 321-325, wherein the antigen-binding domain comprises a single chain variable fragment (scFv).

Embodiment 327: The immunoresponsive cell of any one of embodiments 321-326, wherein the VH and VL are separated by a peptide linker.

Embodiment 328: The immunoresponsive cell of any one of embodiments 321-327, 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 329: The immunoresponsive cell of embodiment 328, wherein the peptide linker comprises a glycine-serine (GS) linker.

Embodiment 330: The immunoresponsive cell of embodiment 329, wherein the GS linker comprises the amino acid sequence of (GGGGS)3 (SEQ ID NO: 223).

Embodiment 331: The immunoresponsive cell of any one of embodiments 321-330, 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, a 2B4 intracellular signaling domain, a CD 16a intracellular signaling domain, a DNAM-1 intracellular signaling domain, a KIR2DS 1 intracellular signaling domain, a KIR3DS 1 intracellular signaling domain, a NKp44 intracellular signaling domain, a NKp46 intracellular signaling domain, a FceRlg intracellular signaling domain, a NKG2D intracellular signaling domain, and an EAT-2 intracellular signaling domain.

Embodiment 332: The immunoresponsive cell of embodiment 331, wherein the one or more intracellular signaling domains comprises an 0X40 intracellular signaling domain.

Embodiment 333: The immunoresponsive cell of embodiment 331 or 332, wherein the 0X40 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 269.

Embodiment 334: The immunoresponsive cell of any one of embodiments 331-333, wherein the one or more intracellular signaling domains comprises a CD28 intracellular signaling domain.

Embodiment 335: The immunoresponsive cell of any one of embodiments 321-334, wherein the CD28 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 267.

Embodiment 336: The immunoresponsive cell of any one of embodiments 321-335, wherein the one or more intracellular signaling domains comprises a CD3z intracellular signaling domain.

Embodiment 337: The immunoresponsive cell of any one of embodiments 321-336, wherein the CD3z intracellular signaling domain comprises an amino acid sequence of SEQ ID NO: 277 or SEQ ID NO: 279.

Embodiment 338: The immunoresponsive cell of any one of embodiments 321-337, 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, a KIR2DS 1 transmembrane domain, a KIR3DS 1 transmembrane domain, an NKp44 transmembrane domain, an NKp46 transmembrane domain, an FceRlg transmembrane domain, and an NKG2D transmembrane domain.

Embodiment 339: The immunoresponsive cell of embodiment 338, wherein the transmembrane domain is an 0X40 transmembrane domain.

Embodiment 340: The immunoresponsive cell of embodiment 338 or 339, wherein the 0X40 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 244. Embodiment 341: The immunoresponsive cell of embodiment 338, wherein the transmembrane domain is a CD8 transmembrane domain.

Embodiment 342: The immunoresponsive cell of embodiment 338 or 341, wherein the CD8 transmembrane domain comprises an amino acid sequence of SEQ ID NO: 236 or SEQ ID NO: 242.

Embodiment 343: The immunoresponsive cell of any one of embodiments 321-342, wherein the CAR comprises a spacer region between the antigen-binding domain and the transmembrane domain.

Embodiment 344: The immunoresponsive cell of embodiment 343, 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 345: The immunoresponsive cell of embodiment 343 or 344, wherein the spacer region is a CD8 hinge.

Embodiment 346: The immunoresponsive cell of embodiment 345, wherein the CD8 hinge comprises the amino acid sequence of SEQ ID NO: 226 or SEQ ID NO: 228.

Embodiment 347: The immunoresponsive cell of any one of embodiments 219-346, wherein the ACP comprises a DNA binding domain and a transcriptional effector domain.

Embodiment 348: The immunoresponsive cell of embodiment 347, wherein the transcriptional effector domain comprises a transcriptional activator domain.

Embodiment 349: The immunoresponsive cell of embodiment 348, 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).

Embodiment 350: The immunoresponsive cell of embodiment 348 or 349, wherein the transcriptional activator domain comprises a VPR activation domain.

Embodiment 351: The immunoresponsive cell of embodiment 350, wherein the VPR activation domain comprises the amino acid sequence of SEQ ID NO: 325.

Embodiment 352: The immunoresponsive cell of embodiment 347, wherein the transcriptional effector domain comprises a transcriptional repressor domain.

Embodiment 353: The immunoresponsive cell of embodiment 352, wherein 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 (SEQ ID NO: 346) of the hairy-related basic helix- loop-helix repressor proteins, the motif is known as a WRPW repression domain (SEQ ID NO: 346); a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain.

Embodiment 354: The immunoresponsive cell of embodiment 347, wherein the DNA binding domain comprises a zinc finger (ZF) protein domain.

Embodiment 355: The immunoresponsive cell of embodiment 354, wherein the ZF protein domain is modular in design and comprises an array of zinc finger motifs.

Embodiment 356: The immunoresponsive cell of embodiment 354 or 355, wherein the ZF protein domain comprises an array of one to ten zinc finger motifs.

Embodiment 357: The immunoresponsive cell of any one of embodiments 354-356, wherein the ZF protein domain comprises the amino acid sequence of SEQ ID NO: 320.

Embodiment 358: The immunoresponsive cell of any one of embodiments 219-357, wherein the ACP further comprises a repressible protease and one or more cognate cleavage sites of the repressible protease.

Embodiment 359: The immunoresponsive cell of embodiment 358, wherein the repressible protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3).

Embodiment 360: The immunoresponsive cell of embodiment 359, wherein the NS3 protease comprises the amino acid sequence of SEQ ID NO: 321.

Embodiment 361: The immunoresponsive cell of any one of embodiments 358-360, wherein the cognate cleavage site of the repressible protease comprises an NS3 protease cleavage site.

Embodiment 362: The immunoresponsive cell of embodiment 361, wherein the NS3 protease cleavage site comprises a NS3/NS4A, a NS4A/NS4B, a NS4B/NS5A, or a NS5A/NS5B junction cleavage site.

Embodiment 363: The immunoresponsive cell of any one of embodiments 359-362, wherein the NS 3 protease is repressible by a protease inhibitor.

Embodiment 364: The immunoresponsive cell of embodiment 363, wherein the protease inhibitor is selected from the group consisting of: simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir.

Embodiment 365: The immunoresponsive cell of embodiment 363 or 364, wherein the protease inhibitor is grazoprevir (GRZ). Embodiment 366: The immunoresponsive cell of any one of embodiments 219-365, wherein the ACP further comprises a nuclear localization signal (NLS).

Embodiment 367: The immunoresponsive cell of embodiment 366, wherein the NLS comprises the amino acid sequence of SEQ ID NO: 296.

Embodiment 368: The immunoresponsive cell of any one of embodiments 358-367, 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 369: The immunoresponsive cell of any one of embodiments 219-368, wherein the ACP further comprises a ligand binding domain of estrogen receptor variant ERT2.

Embodiment 370: The immunoresponsive cell of any one of embodiments 219-369, wherein the ACP-responsive promoter is a synthetic promoter.

Embodiment 371: The immunoresponsive cell of any one of embodiments 219-370, wherein the ACP-responsive promoter comprises an ACP binding domain sequence and a minimal promoter sequence.

Embodiment 372: The immunoresponsive cell of any one of embodiments 219-371, wherein the ACP binding domain sequence comprises one or more zinc finger binding sites.

Embodiment 373: The immunoresponsive cell of any one of embodiments 219-372, 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 374: The immunoresponsive cell of any one of embodiments 219-372, 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 375: The immunoresponsive cell of any one of embodiments 219-372, 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 376: The immunoresponsive cell of any one of embodiments 219-372, 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 377: The immunoresponsive cell of any one of embodiments 219-372, 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 378: The immunoresponsive cell of any one of embodiments 219-372, wherein the first 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 379: The immunoresponsive cell of any one of embodiments 219-372, 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 380: The immunoresponsive cell of any one of embodiments 219-372, 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 381: 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 382: An immunoresponsive cell comprising: (a) a first engineered nucleic acid comprising the nucleotide sequence of SEQ ID NO: 327; and (b) a second engineered nucleic acid comprising the nucleotide sequence of SEQ ID NO: 317.

Embodiment 383: The immunoresponsive cell of embodiment 381 or 382, 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 tumorinfiltrating 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.

Embodiment 384: The immunoresponsive cell of any one of embodiments 381-383, wherein the cell is a Natural Killer (NK) cell.

Embodiment 385: The immunoresponsive cell of any one of embodiments 381-384, wherein the cell is autologous.

Embodiment 386: The immunoresponsive cell of any one of embodiments 381-385, wherein the cell is allogeneic. Embodiment 387: 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 388: The engineered nucleic acid of embodiment 387, 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 389: 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 - 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 390: The engineered nucleic acid of embodiment 389, 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 391: The engineered nucleic acid of any one of embodiments 387-390, 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 392: The engineered nucleic acid of any one of embodiments 387-390, 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 393: The engineered nucleic acid of any one of embodiments 387-390, 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 394: The engineered nucleic acid of any one of embodiments 387-390, 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 395: The engineered nucleic acid of any one of embodiments 387-390, 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 396: The engineered nucleic acid of any one of embodiments 387-390, 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 397: An engineered nucleic acid comprising the nucleotide sequence of SEQ ID NO: 310.

Embodiment 398: An engineered nucleic acid comprising the nucleotide sequence of SEQ ID NO: 327.

Embodiment 399: 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: S - C - MT or MT - C - S wherein S comprises a secretable effector molecule comprising the IL12p70 fusion protein, 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 400: The engineered nucleic acid of embodiment 399, 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 401: The engineered nucleic acid of embodiment 399 or 400, 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 402: The engineered nucleic acid of any one of embodiments 399-401, 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 403: An engineered nucleic acid comprising the nucleotide sequence of SEQ ID NO: 317.

Embodiment 404: An expression vector comprising any one of the engineered nucleic acids described herein.

Embodiment 405: An immunoresponsive cell comprising the engineered nucleic acid or expression vector of any one of the above aspects.

Embodiment 406: A pharmaceutical composition comprising any of the immunoresponsive cells described herein, any one of the engineered nucleic acids described herein, and/or any one of the expression vectors described herein and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.

Embodiment 407: A method of treating a subject in need thereof, the method comprising administering a therapeutically effective dose of any of the immunoresponsive cells described herein, any one of the engineered nucleic acids described herein, any one of the expression vectors described herein, and/or pharmaceutical compositions described herein.

Embodiment 408: 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 described herein, any one of the engineered nucleic acids described herein, any one of the expression vectors described herein, and/or pharmaceutical compositions described herein.

Embodiment 409: 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 described herein, any one of the engineered nucleic acids described herein, any one of the expression vectors described herein, and/or pharmaceutical compositions described herein.

Embodiment 410: 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 described herein, any one of the engineered nucleic acids described herein, any one of the expression vectors described herein, and/or pharmaceutical compositions described herein.

Embodiment 411: In some aspects, the tumor comprises a GPC3-expressing tumor.

Embodiment 412: In some aspects, 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.

Embodiment 413: A method of treating a subject having cancer, the method comprising administering a therapeutically effective dose of any of the immunoresponsive cells described herein, any one of the engineered nucleic acids described herein, any one of the expression vectors described herein, and/or pharmaceutical compositions described herein.

Embodiment 414: The method of embodiment 413, wherein the cancer comprises a GPC3- expressing cancer.

Embodiment 415: The method of embodiment 413 or 414, 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.

Embodiment 416: The method of any one of embodiments 413-415, wherein the administering comprises systemic administration.

Embodiment 417: The method of any one of embodiments 413-416, wherein the administering comprises intratumoral administration.

Embodiment 418: The method of any one of embodiments 413-417, wherein the immunoresponsive cell is derived from the subject. Embodiment 419: The method of any one of embodiments 413-418, wherein the immunoresponsive cell is allogeneic with reference to the subject.

Embodiment 420: 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 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 wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide.

Embodiment 421: The immunoresponsive cell of embodiment 420, wherein the ACP- responsive promoter comprises a synthetic promoter.

Embodiment 422: The immunoresponsive cell of embodiment 420 or embodiment 421, wherein the ACP-responsive promoter comprises an ACP-binding domain sequence.

Embodiment 423: The immunoresponsive cell of any one of embodiments 420-422, wherein the ACP comprises a synthetic transcription factor.

Embodiment 424: The immunoresponsive cell of any one of embodiments 420-423, wherein the ACP comprises a DNA-binding domain and a transcriptional effector domain.

Embodiment 425: An immunoresponsive cell comprising: 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 a first cytokine, and a second expression cassette comprising a second promoter operably linked to 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 first cytokine, 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 426: The immunoresponsive cell of any one of embodiments 420-425, wherein 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.

Embodiment 427: The immunoresponsive cell of any one of embodiments 420-425, wherein the first expression cassette and the second expression cassette are oriented within the engineered nucleic acid in a head-to-head directionality.

Embodiment 428: The immunoresponsive cell of any one of embodiments 420-425, wherein the first expression cassette and the second expression cassette are oriented within the engineered nucleic acid in a tail-to-tail directionality.

Embodiment 429: The immunoresponsive cell of any one of embodiments 420-428, wherein the second promoter comprises a constitutive promoter, an inducible promoter, or a synthetic promoter.

Embodiment 430: The immunoresponsive cell of embodiment 429, 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 431: The immunoresponsive cell of any one of embodiments 420-430, wherein the first cytokine is selected from the group consisting of: IL12, an IL12p70 fusion protein, IL 18, and IL21.

Embodiment 432: The immunoresponsive cell of embodiment 431, wherein the first cytokine is the IL12p70 fusion protein.

Embodiment 433: The immunoresponsive cell of embodiment 432, wherein the IL12p70 fusion protein comprises the amino acid sequence of SEQ ID NO: 293.

Embodiment 434: The immunoresponsive cell of any one of embodiments 420-433, wherein 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 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 PCSK7 protease, a matriptase protease, a matriptase-2 protease, an MMP9 protease, and an NS 3 protease.

Embodiment 435: The immunoresponsive cell of embodiment 434, wherein the protease cleavage site is cleavable by an ADAM 17 protease.

Embodiment 436: The immunoresponsive cell of any one of embodiments 420-435, wherein the protease cleavage site comprises a first region having the amino acid sequence of PRAE (SEQ ID NO: 176).

Embodiment 437: The immunoresponsive cell of any one of embodiments 420-436, wherein the protease cleavage site comprises a second region having the amino acid sequence of KGG (SEQ ID NO: 177).

Embodiment 438: The immunoresponsive cell of embodiment 437, wherein the first region is located N-terminal to the second region.

Embodiment 439: The immunoresponsive cell of any one of embodiments 420-438, 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 440: The immunoresponsive cell of embodiment 439, wherein the protease cleavage site comprises the amino acid sequence of PRAEAVKGG (SEQ ID NO: 179).

Embodiment 441: The immunoresponsive cell of embodiment 439, wherein the protease cleavage site comprises the amino acid sequence of PRAEALKGG (SEQ ID NO: 180).

Embodiment 442: The immunoresponsive cell of embodiment 439, wherein the protease cleavage site comprises the amino acid sequence of PRAEYSKGG (SEQ ID NO: 181).

Embodiment 443: The immunoresponsive cell of embodiment 439, wherein the protease cleavage site comprises the amino acid sequence of PRAEPIKGG (SEQ ID NO: 182).

Embodiment 444: The immunoresponsive cell of embodiment 439, wherein the protease cleavage site comprises the amino acid sequence of PRAEAYKGG (SEQ ID NO: 183).

Embodiment 445: The immunoresponsive cell of embodiment 439, wherein the protease cleavage site comprises the amino acid sequence of PRAESSKGG (SEQ ID NO: 184).

Embodiment 446: The immunoresponsive cell of embodiment 439, wherein the protease cleavage site comprises the amino acid sequence of PRAEFTKGG (SEQ ID NO: 185).

Embodiment 447: The immunoresponsive cell of embodiment 439, wherein the protease cleavage site comprises the amino acid sequence of PRAEAAKGG (SEQ ID NO: 186).

Embodiment 448: The immunoresponsive cell of any one of embodiments 420-438, wherein the protease cleavage site comprises the amino acid sequence of DEPHYSQRR (SEQ ID NO: 187). Embodiment 449: The immunoresponsive cell of any one of embodiments 420-438, wherein the protease cleavage site comprises the amino acid sequence of PPLGPIFNPG (SEQ ID NO: 188).

Embodiment 450: The immunoresponsive cell of any one of embodiments 420-438, wherein the protease cleavage site comprises the amino acid sequence of PLAQAYRSS (SEQ ID NO: 189).

Embodiment 451: The immunoresponsive cell of any one of embodiments 420-438, wherein the protease cleavage site comprises the amino acid sequence of TPIDSSFNPD (SEQ ID NO: 190).

Embodiment 452: The immunoresponsive cell of any one of embodiments 420-438, wherein the protease cleavage site comprises the amino acid sequence of VTPEPIFSLI (SEQ ID NO: 191).

Embodiment 453: The immunoresponsive cell of any one of embodiments 420-438, wherein the protease cleavage site comprises the amino acid sequence of ITQGLAVSTISSFF (SEQ ID NO: 198).

Embodiment 454: The immunoresponsive cell of any one of embodiments 420-453, wherein the protease cleavage site is comprised within a peptide linker.

Embodiment 455: The immunoresponsive cell of any one of embodiments 420-453, wherein the protease cleavage site is N-terminal to a peptide linker.

Embodiment 456: The immunoresponsive cell of embodiment 454 or embodiment 455, wherein the peptide linker comprises a glycine-serine (GS) linker.

Embodiment 457: The immunoresponsive cell of any one of embodiments 420-455, wherein the cell membrane tethering domain comprises a transmembrane-intracellular domain and/or a transmembrane domain.

Embodiment 458: The immunoresponsive cell of embodiment 457, wherein 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.

Embodiment 459: The immunoresponsive cell of embodiment 458, wherein the transmembrane-intracellular domain and/or transmembrane domain is derived from B7- 1.

Embodiment 460: The immunoresponsive cell of embodiment 459, wherein the transmembrane-intracellular domain and/or transmembrane domain comprises the amino acid sequence of SEQ ID NO: 219. Embodiment 461: The immunoresponsive cell of any one of embodiments 420-460, 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 462: The immunoresponsive cell of embodiment 461, 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 463: The immunoresponsive cell of any one of embodiments 420-462, wherein the cell membrane tethering domain comprises a cell surface receptor, or a cell membrane-bound portion thereof.

Embodiment 464: The immunoresponsive cell of any one of embodiments 420-463, wherein the cytokine of the membrane-cleavable chimeric protein is tethered to a cell membrane of the cell.

Embodiment 465: The immunoresponsive cell of any one of embodiments 420-464, wherein the cell further comprises a protease capable of cleaving the protease cleavage site.

Embodiment 466: The immunoresponsive cell of embodiment 465, wherein the protease is endogenous to the cell.

Embodiment 467: The immunoresponsive cell of embodiment 465 or embodiment 466, wherein 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 protease, an MT3-MMP protease, an MT5-MMP protease, a furin protease, a PCSK7 protease, a matriptase protease, a matriptase-2 protease, and an MMP9 protease.

Embodiment 468: The immunoresponsive cell of embodiment 467, wherein the protease is an ADAM 17 protease.

Embodiment 469: The immunoresponsive cell of any one of embodiments 465-468, wherein the protease is expressed on the cell membrane of the cell.

Embodiment 470: The immunoresponsive cell of any one of embodiments 420-469, wherein cleavage of the protease cleavage site releases the cytokine of the membrane-cleavable chimeric protein from the cell membrane of the cell. Embodiment 471: The immunoresponsive cell of any one of embodiments 420-470, wherein the first exogenous polynucleotide sequence further comprises a polynucleotide sequence encoding a secretion signal peptide.

Embodiment 472: The immunoresponsive cell of embodiment 471, wherein the secretion signal peptide is operably associated with the first cytokine.

Embodiment 473: The immunoresponsive cell of embodiment 471 or embodiment 472, wherein the secretion signal peptide is native to the first cytokine.

Embodiment 474: The immunoresponsive cell of embodiment 471 or embodiment 472, wherein the secretion signal peptide is non-native to the first cytokine.

Embodiment 475: The immunoresponsive cell of any one of embodiments 420-474, wherein the second exogenous polynucleotide sequence further encodes a membrane-cleavable chimeric protein.

Embodiment 476: The immunoresponsive cell of embodiment 475, wherein the second expression cassette further comprises a polynucleotide sequence encoding a secretion signal peptide.

Embodiment 477: The immunoresponsive cell of any one of embodiments 420-476, wherein the transcriptional effector domain comprises a transcriptional activator domain.

Embodiment 478: The immunoresponsive cell of embodiment 477, 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).

Embodiment 479: The immunoresponsive cell of embodiment 478, wherein the transcriptional activator domain comprises a VPR activation domain.

Embodiment 480: The immunoresponsive cell of embodiment 479, wherein the VPR activation domain comprises the amino acid sequence of SEQ ID NO: 325.

Embodiment 481: The immunoresponsive cell of any one of embodiments 420-480, wherein the transcriptional effector domain comprises a transcriptional repressor domain.

Embodiment 482: The immunoresponsive cell of embodiment 481, 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.

Embodiment 483: The immunoresponsive cell of any one of embodiments 420-482, wherein the DNA binding domain comprises a zinc finger (ZF) protein domain.

Embodiment 484: The immunoresponsive cell of embodiment 483, wherein the ZF protein domain is modular in design and comprises an array of zinc finger motifs.

Embodiment 485: The immunoresponsive cell of embodiment 483 or embodiment 484, wherein the ZF protein domain comprises an array of one to ten zinc finger motifs.

Embodiment 486: The immunoresponsive cell of embodiment 485, wherein the ZF protein domain comprises the amino acid sequence of SEQ ID NO: 320.

Embodiment 487: The immunoresponsive cell of any one of embodiments 420-486, wherein the ACP further comprises a repressible protease and one or more cognate cleavage sites of the repressible protease.

Embodiment 488: The immunoresponsive cell of embodiment 487, wherein the repressible protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3).

Embodiment 489: The immunoresponsive cell of embodiment 488, wherein the NS3 protease comprises the amino acid sequence of SEQ ID NO: 321.

Embodiment 490: The immunoresponsive cell of embodiment 487 or embodiment 488, wherein the cognate cleavage site of the repressible protease comprises an NS 3 protease cleavage site.

Embodiment 491: The immunoresponsive cell of embodiment 490, wherein the NS3 protease cleavage site comprises a NS3/NS4A, a NS4A/NS4B, a NS4B/NS5A, or a NS5A/NS5B junction cleavage site.

Embodiment 492: The immunoresponsive cell of any one of embodiments 488-491, wherein the NS 3 protease is repressible by a protease inhibitor.

Embodiment 493: The immunoresponsive cell of embodiment 492, wherein the protease inhibitor is selected from the group consisting of: simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir.

Embodiment 494: The immunoresponsive cell of embodiment 493, wherein the protease inhibitor is grazoprevir (GRZ).

Embodiment 495: The immunoresponsive cell of any one of embodiments 420-494, wherein the ACP further comprises a nuclear localization signal (NLS). Embodiment 496: The immunoresponsive cell of embodiment 495, wherein the NLS comprises the amino acid sequence of SEQ ID NO: 296.

Embodiment 497: The immunoresponsive cell of any one of embodiments 490-496, 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 498: The immunoresponsive cell of any one of embodiments 420-497, wherein the ACP further comprises a ligand binding domain of estrogen receptor variant ERT2.

Embodiment 499: The immunoresponsive cell of any one of embodiments 420-498, wherein the ACP-responsive promoter comprises a minimal promoter sequence.

Embodiment 500: The immunoresponsive cell of any one of embodiments 420-499, wherein the ACP binding domain sequence comprises one or more zinc finger binding sites.

Embodiment 501: The immunoresponsive cell of any one of embodiments 420-431 or 133- 500, 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 502: The immunoresponsive cell of any one of embodiments 420-431 or 133- 500, 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 503: An immunoresponsive cell comprising:

(a) an engineered nucleic acid comprising the nucleotide sequence of SEQ ID NO: 317.

Embodiment 504: 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: S - C - MT or MT - C - S, wherein S comprises a secretable effector molecule comprising the IL12p70 fusion protein, 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 505: The engineered nucleic acid of embodiment 504, 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 transcriptional effector domain comprises a transcriptional activator domain, wherein the transcriptional activator domain comprises a VPR activation domain.

Embodiment 506: The engineered nucleic acid of embodiment 505, 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 transcriptional effector domain comprises a transcriptional activator domain, wherein the transcriptional activator domain comprises a p65 activation domain.

Embodiment 507: The engineered nucleic acid of embodiment 506, 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 508: The engineered nucleic acid of embodiment 506, 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 509: An engineered nucleic acid comprising the nucleotide sequence of SEQ ID NO: 317.

Embodiment 510: An expression vector comprising the engineered nucleic acid of any one of embodiments 85-90.

Embodiment 511: An immunoresponsive cell comprising the engineered nucleic acid of any one of embodiments 504-509 or the expression vector of embodiment 510.

Embodiment 512: A cell composition comprising a first immunoresponsive cell of any one of embodiments 420-509, and a second immunoresponsive cell, wherein the second immunoresponsive cell expresses a chimeric antigen receptor.

Embodiment 513: The cell composition of embodiment 512, wherein 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). Embodiment 514: A pharmaceutical composition comprising the immunoresponsive cell of any one of embodiments 420-503, or 511, the cell composition of embodiment 512 or 513, the engineered nucleic acid of any one of embodiments 504-509, or the expression vector of embodiment 510, and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.

Embodiment 515: A method of treating a disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective dose of the immunoresponsive cell of any one of embodiments 420-503, or 511, the cell composition of embodiment 512 or 513, the engineered nucleic acid of any one of embodiments 504- 509, the expression vector of embodiment 510, or the pharmaceutical composition of embodiment 514.

Embodiment 516: 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 the immunoresponsive cell of any one of embodiments 420-503, or 511, the cell composition of embodiment 512 or 513, the engineered nucleic acid of any one of embodiments 504-509, the expression vector of embodiment 510, or the pharmaceutical composition of embodiment 514.

Embodiment 517: A method of reducing tumor volume in a subject, the method comprising administering to a subject having a tumor a composition comprising the immunoresponsive cell of any one of embodiments 420-503, or 511, the cell composition of embodiment 512 or 913 the engineered nucleic acid of any one of embodiments 504- 509, the expression vector of embodiment 510, or the pharmaceutical composition of embodiment 514.

Embodiment 518: 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 420-503, or 511, the cell composition of embodiment 512 or 513, the engineered nucleic acid of any one of embodiments 504-509, the expression vector of embodiment 510, or the pharmaceutical composition of embodiment 514.

Embodiment 519: The method of any one of embodiments 515-518, wherein the tumor comprises a GPC3-expressing tumor.

Embodiment 520: The method of any one of embodiments 515-519, 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. Embodiment 521: A method of treating a subject having cancer, the method comprising administering a therapeutically effective dose of the immunoresponsive cell of any one of embodiments 420-503, or 511, the cell composition of embodiment 512 or 913 the engineered nucleic acid of any one of embodiments 504-509, the expression vector of embodiment 510, or the pharmaceutical composition of embodiment 514.

Embodiment 522: The method of embodiment 521, wherein the cancer comprises a GPC3- expressing cancer.

Embodiment 523: The method of embodiment 521 or embodiment 522, 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.

Embodiment 524: The method of any one of embodiments 515-523, wherein the administering comprises systemic administration.

Embodiment 525: The method of any one of embodiments 515-524, wherein the administering comprises intratumoral administration.

Embodiment 526: The method of any one of embodiments 515-525, wherein the immunoresponsive cell is derived from the subject.

Embodiment 527: The method of any one of embodiments 515-525, wherein the immunoresponsive cell is allogeneic with reference to the subject.

EXAMPLES

Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. For example, the experiments described and performed below demonstrate the general utility of engineering cells to secrete pay loads (e.g., effector molecules) and delivering those cells to induce an immunogenic response against tumors.

Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

Example 1: Expression and Function of an anti-GPC3 CAR + IL15 Bidirectional Construct

Protein expression, cellular activation, and killing activity of cells transduced with anti- GPC3 CAR + IL 15 bidirectional constructs were assessed. A cartoon diagram of the bidirectional orientation of the constructs is shown in FIGs. 1A-1D.

Materials and Methods Primary, donor-derived NK cells were transduced (50,000 to 100,000 cells/transduction) in a non-TC treated retronectin coated plate with lentivirus (at a multiplicity of infection, MOI, of 40) or retrovirus (SinVec, approximately 400pl each) encoding constructs having a first expression cassette encoding an anti-GPC3 CAR and a second expression cassette encoding IL15, with the two expression cassettes in a head-to-head bidirectional orientation. Constructs varied in the intracellular domains of the CAR, having 4- IBB and CD3-zeta signaling domains (41BBz), CD28 and CD3-zeta signaling domains (CD28z), 0X40 and CD3-zeta signaling domains (OX40z) or a KIR3DS1 signaling domain (KIR3DS1), and transductions using either a lentivirus or a retrovirus system were compared for each construct. As a control, transductions were also performed with retroviruses and lentiviruses encoding each of the same CARs, but without the IL15 expression cassette (“CAR-only). After transduction, NK cells were rested in the same plate for 3 days before transfer to a 24-well non-adherent cell-optimized plate. NK cells were expanded to a total of 5 ml with a first cytokine spike-in on day 7 following transduction and a second cytokine spike-in on day 15 (each spike-in included 500 lU/ml IL12 for the CAR+IL15 transductions and the CAR-only transductions, and lOng/ml IL 15 for the CAR only constructs).

On days five and seven following transduction, CAR expression was assessed by flow cytometry for each construct. Day seven CAR expression from cells transduced with lentivirus encoding a bidirectional CAR + IL 15 bidirectional construct and cells transduced with a lentivirus encoding the CAR-only is shown in FIG. 2. Day seven CAR expression from cells transduced with retrovirus encoding a bidirectional CAR + IL 15 bidirectional construct and cells transduced with a retrovirus encoding the CAR-only is shown in FIG. 3. Day fifteen CAR expression from cells transduced with lentivirus encoding a bidirectional CAR + IL15 bidirectional construct and cells transduced with a lentivirus encoding the CAR-only is shown in FIG. 4. Day fifteen CAR expression from cells transduced with retrovirus encoding a bidirectional CAR + IL 15 bidirectional construct and cells transduced with a retrovirus encoding the CAR-only is shown in FIG. 5.

On day seven following transduction, a payload assay was conducted to assess IL15 levels for each construct. 200,000 cells per well were plated in 200pl media (NK MACs complete media with IL2) in a 96-well plate. NK cells were incubated for 48 hours, and then IL15 levels were assessed by immunoassay. IL15 expression is shown in FIG. 6.

Co-culture killing assays were then performed. 25,000 target cells (a Huh7 mKate cell line or a HepG2 mKate cell line) per well were plated in a 96-well plate. Effector cells (the NK cells expressing each construct) were added to the plate at effector to target (E to T) cell ratios of 1: 1 or 0.5: 1, and the cells were cultured with NK MACs complete media without cytokines in a total volume of 200pl. Two to three days following co-culture, real-time, fluorescence-based assays to measure mKate levels were performed to assess target cell killing. Killing by lentivirus-transduced NK cells expressing each construct is shown in FIG. 7, and killing by retrovirus-transduced NK cells expressing each construct is shown in FIG. 8.

Results

CAR expression from NK cells transduced with each construct was assessed. As shown in FIG. 2, at day seven transduced NK cells had measurable CAR expression for each construct, with at least 10% of cells in each transduced population positive for CAR expression. As shown in FIG. 3, at day fifteen lentivirus-transduced NK cells had measurable CAR expression for each construct (top panel), with at least 20% of cells in each transduced population positive for CAR expression. Additionally, as shown in FIG. 3, retrovirus-transduced NK cells expressing the 28z CAR + IL 15 bidirectional construct had measurable CAR expression, with at least 42% of cells in the transduced population positive for CAR expression.

IL15 expression by NK cells transduced with each construct was also assessed. Assay of IL15 expression by non-transduced cells and Ox40z CAR-only cells was performed as a negative control. As shown in FIG. 6, retrovirus-transduced NK cells expressing bidirectional CAR + IL15 had statistically significant increase in IL15 production over reciprocal lentivirus- transduced NK cells.

Killing by NK cells transduced with each construct was then assessed. As shown in FIG. 7, lentivirus-transduced NK cells expressing the CAR + IL 15 bidirectional construct had statistically significant increased killing over lentivirus-transduced NK cells expressing the CAR alone (without the IL15 expression cassette). As shown in FIG. 8, retrovirus-transduced NK cells expressing the CAR + IL 15 bidirectional construct had statistically significant increased killing over retrovirus-transduced NK cells expressing the CAR alone (without the IL 15 expression cassette).

Example 2: Expression of IL12 from Bidirectional Constructs Encoding a Regulatable IL12 and a Synthetic Transcription Factor (SynTF)

IL12 expression was assessed from NK cells transduced to express bidirectional constructs including a first expression cassette encoding a regulatable IL12 and a second expression cassette encoding a synthetic transcription factor. The regulatable IL12 is operably linked to a synthetic transcription factor-responsive promoter, which includes a ZF-10-1 binding site and a minimal promoter sequence (YBTATA). The synthetic transcription factor includes a DNA binding domain (an array of six zinc finger motifs known as ZF-10-1) and a transcriptional activation domain (Vpr). Between the DNA biding domain and the transcriptional activation domain is a protease domain (NS3) and cognate cleavage site for the protease. In the absence of an inhibitor of the protease, the protease induces cleavage at the cleavage site, resulting in a nonfunctional synthetic transcription factor. In the presence of the protease inhibitor, the synthetic transcription factor is not cleaved and is thus capable of modulating expression of the IL12. Constructs tested included IL12 expression cassettes having mRNA destabilization elements in the 3’ untranslated region. A cartoon diagram of the bidirectional orientation of the constructs is shown in FIG. 9.

Materials and Methods

Bidirectional constructs including two expression cassettes, a first expression cassette encoding a regulatable IL 12 and a second expression cassette encoding a small molecule- regulatable synthetic transcription factor, were produced. A first construct lacks an mRNA destabilization element (“WT”), and four constructs each include a different mRNA destabilization element added to the 5’ non-coding region. The four destabilization elements used were: 1) an AU-rich motif (“AU” or “1XAU”); 2) a stem-loop destabilization element (“SLDE” or “1XSLDE”); 3) a tandem AU motif and SLDE motif (“AuSLDE” or “IX AuSLDE”); and 4) two repeated AuSLDE motifs (2X AuSLDE). The destabilization elements were added to attempt to reduce leakiness of IL12 expression in the absence of the small molecule regulator of the synthetic promoter (e.g., grazoprevir).

Primary, donor-derived NK cells were expanded for ten days and grown in IL21 and IL15, with K562 feeder cells, and then were transduced with a multiplicity of infection (MOI) of 40 (as determined by infection units titer) in a retronectin-coated 24 well plate, following Bx795 pre-treatment. Transduction was performed with spinoculation, at 800g for 2 hours at 32°C.

On day three following transduction, NK cells were counted and seeded at le6 cells/mL with no drug or 0.1 uM grazoprevir (GRZ) for 24 hours.

On day four following transduction (with 24 hours of drug treatment), supernatant was harvested and analyzed for IL12 levels by immunoassay. IL12 concentrations for each cell type and condition are shown in FIG. 10.

Results

As shown in FIG. 10, NK cells transduced with each construct demonstrated increased IL12 expression following treatment with grazoprevir, as compared to the absence of drug. NK cells transduced with the IL12 lacking a destabilization element (“WT”) had greater than 19-fold induction of IL12 expression following treatment with grazoprevir. However, NK cells transduced with constructs that included destabilization tags demonstrated about a 457-fold, 58- fold, 50-fold, and 89-fold induction of IL12 upon treatment with grazoprevir for 2X AuSLDE, IX AuSLDE, IX AU, and IX SLDE, respectively. Additionally, each of the destabilization tags decreased the baseline IL12 expression in the absence of grazoprevir. Furthermore, the construct encoding an IL12 with a 2X AuSLDE destabilization element resulted in a non-detectable level of IL12 expression in the absence of grazoprevir.

Example 3: Expression and Function of anti-GPC3 CAR + IL15 Bidirectional Constructs

Protein expression, cellular activation, and killing activity of cells transduced with anti- GPC3 CAR + cleavable release IL 15 bidirectional constructs were assessed. The expression cassette encoding the cleavable release IL15 includes a chimeric polypeptide including the IL15 and a transmembrane domain. Between the IL15 and the transmembrane domain is a protease cleavage domain that is cleavable by a protease endogenous to NK cells. A cartoon diagram of the bidirectional construct encoding a cleavable release IL 15 is shown in FIG. 11.

Briefly, primary, donor-derived NK cells were transduced with viral vectors encoding constructs having a first expression cassette encoding an anti-GPC3 CAR and a second expression cassette encoding a cleavable release IL15 expression cassette, with the two expression cassettes in a head-to-head bidirectional orientation.

Culture Supernatant'. Spinoculation of NK cells was performed (day 0). A partial culture media exchange was performed on days 1, 2, and 6. Cell culture supernatant was harvested on day 8.

Flow cytometry: On day 10 following transduction, CAR and mbIL15 expression was assessed by flow cytometry for each construct. NK cells were stained with an IL- 15 primary antibody and PE-secondary, and rhGPC3-FITC and Sytox blue (viability stain). Cells were run on Cytoflex and analyzed using Flowjo for CAR/mbIL15 expression.

Payload assay: On day 7 or 8 following transduction, a payload assay was conducted to assess IL15 levels for each construct. 200,000 cells per well were plated in 200 pl media (NK MACs complete media with IL2 only) in a 96-well plate, run in duplicates. Cells were incubated for 48 hours, and then cleaved IL15 levels were assessed by Luminex immunoassay.

Serial killing assay: Co-culture killing assays were performed. About 25,000 target cells (a Huh7 mKate cell line or a HepG2 mKate cell line) per well were plated in a 96-well plate. Effector cells (the NK cells expressing each construct) were added to the plate at effector to target (E to T) cell ratios of 1: 1 in triplicates, and the cells were cultured with NK MAC complete media (no cytokines) in a total volume of 200 pl. Real-time, fluorescence-based assays were used to measure mKate to assess target cell killing in a serial-killing assay performed at 37° C; initial killing was at day 9 post-transduction, serial one was at day 11 posttransduction, and serial 2 was at day 14 post transduction.

Over 150 IL15 cleavable release (crIL15) constructs were designed, and 33 constructs were selected for experimental testing, (see Table 7A). Each construct was tested in two viral backbones (e.g., SB06250 and SB06256, as shown in Table 7A). A summary of expression and killing activity of cells expressing a subset of bicistronic constructs is shown in Table 7B. Full- length sequences of a subset of constructs are shown in Table 7C. A summary of bicistronic constructs tested and their functional activities is provided in FIG. 12.

Table 7A.

Atty. Ref: STB-050WO

Client Ref: SNTI-0050-WQ

Table 7B. a Normalized to Target cells alone

* crIL-15 control did not function as expected

* crIL-15 control did not killed as expected

Table 7C

NK cells comprising CARs comprising 0X40 transmembrane (TM) and co-stimulatory (co-stim) domains, SB06251, SB06257, and SB06254, were assessed for expression of constructs as described above. Results as determined by flow cytometry are shown in FIG. 13A and FIG. 13B. Secreted IL-15 was measured as described above; results are summarized in FIG. 14A and FIG. 14B. To assess killing of the target cell population, cell growth was determined as described above (FIG. 15A and FIG. 15B).

Serial killing by the NK cells comprising SB06257 was also assessed. Target cells were added at Days 0, 2, and 5, and Huh7 target cell count was calculated using an Incucyte. Results are shown in FIG. 16.

NK cells comprising CARs comprising CD28 co-stimulatory (co-stim) domains, SB06252, SB06258, and SB06255, were assessed for expression of constructs as described above. Results as determined by flow cytometry FACS are shown in FIG. 17A and FIG. 17B. Secreted IL- 15 was measured as described above; results are summarized in FIG. 18A and FIG. 18B. To assess killing of the target cell population, cell growth was determined as described above (FIG. 19A and FIG. 19B).

Serial killing by the NK cells comprising SB06252 and SB06258 was also assessed. Target cells were added at Days 0, 2, and 5, and Huh7 target cell count was calculated using an Incucyte. Results are shown in FIG. 20.

Screening for bicistronic constructs

0.5e6 NK donor 7B cells were expanded in the presence of fresh irradiated mbIL21/IL15 K562 feeder cells on retronectin coated non-TC 24-well plates. Spinoculation was performed at 800g at 32°C for 2 hr. For viral transduction, 300 p l of virus added, for a total transduction volume of 500 pl.

Cells were cultured in the same plate for the entire expansion period, in 2 ml final volume. Three partial media exchanges were performed as described above before assessing expression and using the cells in functional assays. Results of expression and cytotoxicity against target cells are shown in Table 8. As shown, SB06261, SB6294, and SB6298 showed good CAR and IL- 15 expression levels as determined by flow and good cytotoxicity in serial killing assay (n=2). Flow cytometry expression data is shown in FIG. 21A and FIG. 21B, IL- 15 levels are shown in FIG. 22A and FIG. 22B, and cell growth of the target cell population (as a measure of cell killing by the NK cells) is shown in FIG. 23A and FIG. 23B.

Due to its high CAR and IL- 15 expression and performance in functional assays, SB06294, a retroviral vector with crIL15 2A 0X40 CAR design, was selected for further study.

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Table 8.

Analysis ofTACE-OPT constructs

Bicistronic TACE-OPT constructs comprising a TACE10 cleavage site, were analyzed for CAR and IL- 15 expression, CNA assay, and payload assay for secreted cytokines, as described above. A TACE10 cleavage site was modified to increase cleavage kinetics, resulting in “TACE-OPT,” which results in higher cytokine secretion levels as compared to the parent TACE10. Tricistronic constructs were analyzed for CAR and IL- 15 expression, and IL- 12 induction.

Briefly, 0.5e6 NK donor 7B cells were expanded in the presence of fresh irradiated mbIL21/IL15 K562 feeder cells on retronectin coated non-TC 24-well plates. Spinoculation was performed at 800g at 32°C for 2 hr. For viral transduction, 300 pl of virus was added, for a total transduction volume of 500 pl.

Bicistronic constructs SB6691 (comprising 41BB co- stimulatory domain), SB6692 (comprising 0X40 co- stimulatory domain), and SB6693 (comprising CD28 co-stimulatory domain) were assessed by flow cytometry for expression of CAR and IL- 15 (FIG. 24A). Copy number of each construct per cell is shown in Table 9. IL-15 secretion was quantified as described above at 48 hours and 24 weeks post-tranduction (FIG. 24B). While the TACE-OPT constructs tested have similar expression levels and cytokine secretion, SB06692 (comprising an 0X40 co-stimulatory domain) has the highest CAR expression.

Table 9.

SB06258, SB06257, SB06294 and SB06692 demonstrated high CAR expression, high crIL-15 expression (both membrane-bound and secreted), and high serial killing function in vitro. Example 4: Expression of IL12 from Bidirectional Constructs Encoding a Regulatable, Cleavable-Release IL12 and a Synthetic Transcription Factor

IL12 expression was assessed for NK cells transduced with bidirectional constructs encoding regulatable, cleavable release IL 12 and a synthetic transcription factor, with transductions performed as described in Example 3 above. The regulatable, cleavable IL 12 is operably linked to a synthetic transcription factor-responsive promoter, which includes a ZF-10- 1 binding site and a minimal promoter sequence. The synthetic transcription factor includes a DNA binding domain and a transcriptional activation domain. Between the DNA binding domain and the transcriptional activation domain is a protease domain that is regulatable by a protease inhibitor and cognate cleavage site for the protease. In the absence of an inhibitor of the protease, the protease induces cleavage at the cleavage site, resulting in a non-functional synthetic transcription factor. In the presence of the protease inhibitor, the synthetic transcription factor is not cleaved and is thus capable of modulating expression of the cleavable IL12. The expression cassette encoding the cleavable release IL12 includes a chimeric polypeptide including the IL 12 and a transmembrane domain. Between the IL 12 and the transmembrane domain is a protease cleavage domain that is cleavable by a protease endogenous to NK cells. A cartoon diagram of the bidirectional constructs encoding cleavable release 12 is shown in FIG. 25. Parameters of the constructs tested herein are summarized in Table 10. Designs tested include: cleavable-release IL12 (crIL12) regulated constructs (32 constructs tested), soluble IL12 (sIL12) regulated and/or WPRE and polyA + different destabilizing domains (32 constructs tested), destabilizing domain and/or WPRE and polyA (26 constructs tested). Initial studies demonstrated toxicity generally due to leaky expression of IL- 12, resulting in poor NK cell viability and expansion following transduction (data not shown). A screen was designed to discover constructs that could overcome or reduce IL- 12 associated toxicity by modifying the parameters in Table 10. A summary of screening criteria for is shown in Table 11A. Suitable candidates SB05058 and SB05042 (both gammaretroviral vectors) and SB04599 (lentiviral vector) were identified. A summary of these candidates is provided in Table 11B. Table 10.

Table 11A. Table 11B.

Assessment of gammaretroviral vectors and lentiviral vectors was performed. A grazoprevir (GRZ) dose response assay measuring IL12 secretion demonstrated that both gammaretroviral constructs showed higher sensitivity to GRZ as compared to the lentiviral construct (FIG. 26 and Table 12A). Table 12A.

Construct expression and cellular viability were determined 10-days following transduction of NK cells. Results are shown in Table 12B and demonstrate an above 10-fold cellular expansion in mid-scale plates, above 85% viability, and greater than 2 copies/cell. Gammaretroviral vectors displayed higher transduction efficiency of NK cells than lentiviral vectors, particularly for the bidirectional vectors tested.

Table 12B.

Additionally, IL12 induction was assessed in vivo. Briefly, mice were injected intravenously with transduced NK cells at a dose of 15e6 cells in a 200pL volume. Blood was collected 24 hours after injection and assayed for IL12 expression levels. SB05042 and SB05058 showed the highest IL12 fold-induction. No induction was observed in 10 mg/kg dose groups (data not shown). The percentage of %hNKs in mouse blood was determined to be less than 2% for all constructs. Results are summarized in Table 12C. IL12 levels are shown in FIG. 27A and fold change is shown in FIG. 27B. Table 12C

The gammaretroviral vectors (SB05042 and SB05058) demonstrated superior IL12 induction in vitro compared to the lentiviral vector (SB04599), while maintaining good viability and cell growth post-transduction. Importantly, both gammaretroviral vectors tested showed IL 12 induction in NK cells in vivo.

Full-length sequences of constructs described in this Example are shown in Table 13.

Table 13.

Example 5: Screening of GPC3 CAR / IL15 Expression Constructs

Assessment of the expression and function of the GPC3 CAR/IL15 expression constructs in NK cells was performed. 2e6 NK cells were plated into a 6-well non-TC treated, retronectin coated plate. A single viral transduction via spinoculation (MOI = 15) was performed on plated NK cells. The NK cells were transduced using lentivirus or retrovirus containing the expression construct. Expression of the CAR and membrane IL15 were assessed as seen in FIG. 28A. NK cells transduced with constructs SB06257, SB06258, SB06294, and SB06692 exhibited expression of greater than 65% of cells in the gated population. In addition, FIG. 28A shows the measured copy numbers of YP7 and IL 15 of each transduced NK cell population.

In addition to CAR expression being assessed, secreted IL-15 was also measured using the same expression constructs. To measure the levels of secreted IL- 15, 200,000 transduced NK cells were suspended in 200 [1L of MACS media in the presence of IL2. Secreted IL- 15 was measured 48 hours after transduction. The concentrations of secreted IL- 15 were measured for each construct and the results are shown in FIG. 28B.

Serial killing by NK cells transduced with the constructs was also assessed. Target cells were added at Days 0, 2, and 5, and target cell killing was measured over the course of the study. Results for serial NK cell killing of HepG2 target cells are shown in FIG. 28C and FIG. 29A. FIG. 29B shows results of serial NK cell killing of HuH-7 target cells.

Table 14 shows the exemplary constructs and their components used in this study.

Table 14

Example 6: Measuring GPC3 CAR / IL15 Expression and Function in Expanded NK cells In this study, the expression and function of GPC3 CAR/IL15 were measured for NK cells that were expanded using the G-Rex (Gas rapid expansion) system.

7-day-old donor-derived 7B NK cells (mbIL21/IL15 K562 feeders) were transduced and expanded in two different G-Rex experimental methods. Experiment 1 transduced 7-day donor 7B NK cells (mbIL21/IL15 K562 feeders) in G-Rex 6M culture containers for 11 days and harvested 11 days after transduction. Experiment 2 transduced 7-day donor 7B NK cells (mbIL21/IL15 K562 feeders) in G-Rex IL culture containers for 7 days and harvested 10 days after transduction. FIG. 30A demonstrated the effects of the different expansion conditions have on the expression of different proteins of interest in the engineered NK cells. FIG. 30B shows the serial killing assay measurements from the NK Cells derived from the different experiments. Table 15 shows a summary of the study performed in Example 6. The top number corresponds to results obtained from NK cells expanded using the method of Experiment 1. The bottom number corresponds to results obtained from NK cells expended using the method of Experiment 2.

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Table 15

Example 7: Assessment of GPC3 CAR / IL15 Bicistronic Constructs in a Xenograft Tumor Model

The in vivo function of selected engineered NK cells was assessed using a HepG2 xenotransplantation tumor model. Two studies were conducted: a double NK dose and a triple NK dose.

Double NK Dose In vivo Xenograft Tumor Model

The tumor was implanted in NSG mice at day 0. Mice were randomized at day 9. NK cells were injected twice over the course of the study on days 10 and 17. Table 16 summarizes the study set-up.

Table 16: Summary of double NK dosing in vivo xenograft tumor model

For this survival study, Jackson Labs NSG mice were also injected with 50,000 IU rhIL2 per mouse twice per week. Bioluminescence imaging (BLI), body weight, and overall health measurements were conducted twice a week. Upon euthanizing mice, tumor were collected, weighed, and formalin fixed paraffin embedded (FFPE) for histology. IP fluid and cells were collected from the IP space and the % NK cells were assessed by flow cytometry. FIG. 31 summarizes the results the fold change in normalized mean BLI measurement in the HepG2 xenotransplantation tumor model. SB06258 showed the lowest normalized mean BLI compared to other treatment groups and was found to be statistically significant compared to the no virus (NV) group. FIG. 32A shows a survival curve of animals and FIG. 32B shows a summary of the median survival of each of the treatment groups. Each of the different CAR constructs tested were found to be statistically significant compared to un-engineered NK cells.

FIG. 33 shows a time course of the mice treated with different CAR-NK cells as measured and observed through bioluminescence imaging (BLI). The animals shown here were imaged 3 days, 10 days, 34 days, 48 days, and 69 days after treatment. In FIG. 34, BLI measurements were normalized to day 10 (first dose). Triple Dosing - In Vivo HepG2 Xenograft Tumor Model

The in vivo function of selected engineered NK cells was assessed using a HepG2 xenotransplantation tumor model. The tumor was implanted in NSG mice at day 0 in another in vivo experiments. Mice were randomized at day 9 and day 20. 30e6 NK cells were injected (IP) three times over the course of the study on days 10, 15, and 22. Table 17 summarizes the study set-up. On day 21, half of the mice were euthanized. The other half were euthanized on day 50 of the study. Upon euthanizing mice, tumor were collected, weighed, and formalin fixed paraffin embedded (FFPE) for histology.

Table 17: Study Design of HepG2 xenograft model

For this survival study, Jackson Labs NSG mice were also injected with 50,000 IU rhIL2 per mouse twice per week. Bioluminescence imaging (BLI), body weight, and overall health measurements were conducted twice a week. IP fluid and cells were collected from the IP space and the % NK cells were assessed by flow cytometry. FIG. 35A shows a representative BLI image at day 23 of the study. FIG. 35B summarizes the results the fold change in normalized mean BLI measurement in the HepG2 xenograft tumor model.

The fold change of BLI measurements were assessed at different stages of the experiments to assess the effect of a single or double dose of the engineered NK cells had an effect. FIG. 36A shows the fold change of BLI measurements on day 13, in which the mice had undergone one dose of the engineered NK cells. FIG. 36B shows the fold change of BLI measurements on day 20, in which the mice had undergone two doses of the engineered NK cells. Comparison of the results from the two in vivo experiments are presented in FIG. 37 A. and FIG. 37B. In FIG. 37A, the different CAR constructs were tested in a xenograft model, plotting fold change of BLI over the course of the study. As seen in FIG. 37A and FIG. 37B, the two in vivo experiments exhibit differences in antitumor function of SB06257 and SB06258. GPC3 CAR- crIL-15 NK cell therapy shows statically significant in vivo anti-tumor efficacy compared to unengineered NK cells in an IP HCC (HepG2+luciferase) xenotransplantation model. All 3 groups treated with GPC3 CAR-crIL-15 engineered NK cells show significant increased survival over untreated (PBS) and unengineered NK cell-treated groups.

In vivo Xenograft model - Intratumoral Injection ofNK cells

Another experimental approach was used to demonstrate NK-mediated anti-tumor killing for an HepG2 (HCC) subcutaneous xenograft tumor model. In this survival study, mice were injected three times with 3e6 NK cells on days 20, 25, and 32. FIG. 38A demonstrates tumor growth in mice in the absence or presence of injected engineered NK cells. GPC3 CAR- crIL-15 NK cell therapy shows significant in vivo anti-tumor efficacy compared to unengineered NK cells injected intratumorally (IT) within a subcutaneous HCC (HepG2+luciferase) xenotransplantation model. NK cells transduced with SB05605 show significantly increased survival over untreated (PBS) and unengineered NK cell-treated groups. Table 18 provides the constructs used for intratumoral injection of NK cells. SB05009 and SB06205 contain IL15 and the GPC3 CAR that are separate, and their expression is driven by separate promoters (SV40 promoter = GPC3 CAR, hPGK promoter = IL15). In addition, these constructs are oriented such that the reading frames are oriented in opposing directions.

Table 18

Example 8: Assessment of Grazoprevir induction of IL12 in natural killer cells

For this study, the induction of IL12 was measured in the presence and absence of grazoprevir, an inhibitor of the HCV NS3 protease. The construct used in this study has been previously described in Example 2. Two regulatable IL- 12 constructs demonstrated controlled crIL-12 expression by GRZ in a dose-response manner and show low donor-to-donor variability The tested construct candidates resulted in low IL- 12 basal levels in the absence of GRZ (less than 100 pg/ml) and greater than 100-fold induction of IL- 12 by 0.1 M of GRZ (p=<0.0001). FIGs. 39A-39B show two different time points (24 hours and 72 hours, respectively) after addition of GRZ to NK cells expressing the SB05042 and SB05058 constructs.

To assess whether the grazoprevir can be used to transition the circuit in an on to off or off to on state in a mouse model, the following study was designed. On day 0, NK cells were injected (IV) in the presence of grazoprevir or vehicle. On days 1, 9, and 10, another dose of grazoprevir or vehicle was injected. Mice were bled on days 2, 9, and 11 to assess expression of IL- 12. FIG. 40 shows the results of the study. On day 2, IL12 expression increased in the presence of 20, 50, and 100 mg/kg GRZ as compared to the control. On day 9, where GRZ administration has not occurred for 8 days, expression of IL12 is decreased as compared to sampling on day 2. On day 11, expression has increased once again in relation to the control.

Example 9: Assessment of Co-transduction of GPC3 CAR / IL15 and Regulated IL12 constructs

Function and expression of GPC3 CAR, IL15 and IL12 were assessed in NK cells that were co-transduced with GPC/IL15 constructs and the regulated IL 12 construct.

Expression of GPC3 CAR / IL15

Three construct combinations were tested: 1) SB05042 + SB0257, 2) SB05042 + SB06258, and 3) SB05042 and SB06294. NK cells co-transduced with SB05042 + SB06257 or SB05042 + SB06258 expressed GPC3 CAR and IL15 populations and similar copies per cell. NK cells co-transduced with SB06294 exhibited a higher double positive (GPC+/IL15+) population with a slight decrease in CAR only population and with similar copies per cell (FIG. 41)

Expression of secreted IL12 and IL15

Expression of secreted IL12 and IL15 were measured in NK cells in the presence or absence of grazoprevir was tested. 200,000 transduced NK cells were suspended in 200 pL of NK MACS media supplemented with IL-2. Grazoprevir was added to “+” conditions at a molar concentration of 0.1 pM. NK cells were incubated for 48 hours at 37C prior to measurement of the supernatant for IL15 (FIG. 42A) and IL12 (FIG. 42B) concentration. IL15 expression increased slightly in the presence of grazoprevir, with the co-transduced NK cells showing statistically significant IL 15 expression in the presence of GRZ. NK cells co-transduced with SB05042 +SB06257 expressed 2201 pg/mL IL12 in the presence of grazoprevir, as compared to 12 pg/mL in the absence of grazoprevir (1100-fold induction). SB05042 +SB06258 cotransduction exhibited 1003-fold induction in the presence of grazoprevir. SB05042 +SB06294 co transduction exhibited 736-fold induction. The three co-transduction combinations were statistically significant compared to NK cells transduced with SB05042 alone. Assessing IL12 expression, NK cells transduced with SB05042 alone showed induction of IL12 in the presence of grazoprevir, showing an 390-fold increase in expression.

Cytokine Secretion during Serial Killing (Huh7)

Serial killing of target cells were carried out as previously described using NK cells singly transduced or co-transduced with GPC3 CAR/IL15 (SB06257, SB06258, SB06294) and /or IL12 constructs (SB05042).

Co-transduced samples maintained low amounts of IL12 induction into the 3rd round in the presence of GRZ. Overall cytokine secretion decreases overtime in both IL 12 and IL 15 (FIG. 43). In the presence of grazoprevir, SB05042 and SB05042 + SB06257 transductions showed significant induction of IL12 expression in the first round of killing. In the second round, the three co-transductions with the different GPC3 CAR expressing constructs (SB06257, SB06258, SB06294) and SB05042 showed statistically significant induction of IL12. In the third round, only SB05042 + SB06257 and SB05042 + SB06294 showed significant IL12 induction.

Serial Killing Assays with Co-transduced NK cells

The cell killing effect of NK cells that were co-transduced with GPC3 CAR/IL15 (SB06257, SB06258, SB06294) and /or IL12 constructs (SB05042) were assessed using a serial killing assay. NK cells co-transduced with SB05042 + SB06258 (FIG. 44A), SB05042 + SB06257 (FIG. 44B) and SB05042 + SB06294 (FIG. 44C) were used in a serial killing assay in which GRZ was added at the first and third rounds of cell killing. When co-cultured with HepG2 we see a greater difference between +/- GRZ (induced IL 12 or not) as compared to huh7. FIG. 44D shows a combination of the data shown in FIGs. 44A-44C.

Example 10: Selection of GPC3 CAR / IL15 clones

Selection of clones were performed by transducing NK cells that have stably integrated the expression construct. A lower MOI was used (MOI=3) was used for clonal selection of SB06258. A control transient transduction (MOI = 15) was also performed used in SB06258 and SB07273 (identical to SB06258 but contains a kanamycin resistance marker instead of an ampicillin resistance marker). 8 days after transduction, the cells were assessed. The copies per cell was lower in the primary cell bank (PCB) clones as compared to the transient transduction using SB06258 (FIG. 45A). CAR expression was relatively constant across the different PCB clones (FIG. 45B), as well as the IL15+ population (FIG. 45C). Secreted IL15 of PCB clones was measured to be greater than 30 pg/mL (FIG. 45D).

Flow cytometry was also used to assess the expression of the GPC3 CAR and IL15 in the PCM clones. As a control, SB07473 was used to transduced NK cells at an MOI=15. PCB clones were transduced at an MOI of 3.0. For all PCR clones, GPC3 CAR expression was greater than 20% (FIG. 46A).

For select clones, SB05042 was also co-transduced to assess the expression of the GPC3 CAR, membrane bound IL15 and membrane bound IL12 9 days after transduction. Clone 3 (MOI=3.0) and clone 4 (MOI=3.0) was co-transduced with SB05042 (MOI = 0.05). During cotransduction, there was similar expression of the GPC3 CAR and membrane bound IL12 (FIG. 46B). Table 19 shows a summary of the expression levels of the PCB clones transduced with SB06258.

Table 19

Example 11: Kinetics of the crIL12 small molecule induction

In this example, induction of crIL12 was assessed in the presence of different small molecule inducers, such as grazoprevir or endoxifen (a tamoxifen metabolite).

Grazoprevir induction

Donor derived natural killer cells were transduced with SB05042 and assessed for expression kinetics of IL12. crIL12 expression is regulated by GRZ in a dose-response manner and shows low donor-to-donor variability (FIG. 47A).

GRZ-regulatable IL-12 constructs demonstrate low crIL12 basal levels in the absence of GRZ (~30pg/le6 cells in 24h), while induction with O.luM of GRZ lead to a significant increased crIL12 expression after only 4 hours (p= 0.0013) and ~300-fold (-10,400 pg/le6 cells) in 24h. p= 0.0025 (FIG. 47B).

Endoxifen induction

A second regulatable IL- 12 construct was designed to incorporate an endoxifen- responsive element (estrogen receptor variant, ERT2) instead of the HCV NS 3 protease (responsive to grazoprevir). Donor derived natural killer cells were transduced with a regulatable IL- 12 construct having 2 different ERT2 domains (ERT mutant 81 or ERT mutant 77) that regulate the expression of crIL12 (Table 26). crIL12 expression is regulated by TAM in a dose-response manner (FIG. 47C). Endoxifen-regulated IL12 constructs demonstrate low crIL12 basal levels in the absence of drug (<10 pg/le6 cells in 24 hours), while an increased crIL12 expression is observed in a drugdosage dependent manner, reaching a -100-fold in 24 hours at 1 nM (approximate physiologically relevant level in humans). crIL-12 levels continue to increase overtime post drug treatment (InM) when IL12 concentrations are measured 48 and 72 hours after exposure to endoxifen (FIG. 47D). Table 26: Sequences for Tamoxifen-responsive ACP

Example 12: Characterization of Cell Combinations

In this example, a comparative study was performed to assess the differences in cell activity between a population of co-transduced engineered NK cells and a mixed population engineered NK cells. For the population of co-transduced cells, cells were co-transduced with both GPC3/IL15 and crIL12 constructs. For the mixed population cells, cells were individually transduced with either the GPC3/IL15 construct or the crIL12 construct and mixed creating a combination of cell populations having the GPC3/IL15 transduced population and the crIL12 transduced population. Both engineered cell populations were individually co-cultured with cancer cells. Further, M2 M cells were provided at a certain time point to provide an immunosuppressive environment during data collection. Cell cultures were further drug-induced to allow for controlled release of IL- 12 after creating the M2 M induced immunosuppressive environment.

For the co-transduction assay: o Freshly isolated CD 14+ monocytes were differentiated to MO macrophages (MO) for 4 days in ImmunoCult SF-Macrophage media with M-CSF followed by 2-3 days of M2 polarization in the presence of IL- 10 and TGFp. o In parallel, Day 7 activated NK cells (mbIL21) were transduced with crIL-15 GPC3 (VI SB06258, MOI 5) and regulatable crIL-12 (V2 SB05042, MOI 0.05). o Day 17 post-transduction NK cells were cytokine starved for 18 hours in complete media. o Engineered NK cells were co-cultured with M2 M<I> for 24 hours (5 M2 M<I>: 1 NK or 2.5 M2 M : 1 NK) in the presence or absence of 0.1 pM grazoprevir. o Cell trace violet labeled K562 target cells (50K) were added into the co-culture. o 18 hours later, supernatants were collected for Luminex and cells were processed for flow cytometry.

For the mixed cell populations: o Freshly isolated CD 14+ monocytes were differentiated to MO macrophages (M ) for 4 days in ImmunoCult SF-Macrophage media with M-CSF followed by 2-3 days of M2 polarization in the presence of IE- 10 and TGFp. o In parallel, Day 7 activated NK cells (mbIL21) were transduced with crIL-15 GPC3 (VI SB06258, MOI 5) or regulatable crIL-12 (V2 SB05042, MOI 0.05). o Day 17 post-transduction NK cells were cytokine starved for 18 hours in complete media. o VI and V2 cells were mixed in 1 : 1 ratio (25k+25k) and co-cultured with M2 MO for 24 hours (5 M2 MO: 1 NK or 2.5 M2 MO: 1 NK) in the presence or absence of 0.1 pM grazoprevir. o Cell trace violet labeled K562 target cells (50K) were added into the co-culture. o 18 hours later, supernatants were collected for Luminex and cells were processed for flow cytometry

FIGs. 48A (Co-transduced population) and 48B (Mixed cell population) demonstrate similar cell characterization between the different methods to generate the cell product.

Table 20

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Table 21:

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Table 22: Full Length CAR and Component Amino Acid Sequences

Interpretations

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. In the claims, as well as in the specification above, all transitional phrases such as

“comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.