Compositions and methods relating to intronic shrna chimeric antigen receptor constructs

Abstract

The present disclosure pertains to nucleic acid constructs comprising a promoter, a gene of interest, a 3' untranslated region (UTR), and one or more introns, wherein the one or more introns comprise one or more inhibitory nucleic acids, wherein the one or more inhibitory nucleic acids encode one or more inhibitory RNAs, and, wherein the gene of interest encodes a chimeric antigen receptor (CAR) and methods of using and making immune cells comprising said nucleic acid constructs. The present disclosure also pertains to modified immune cells comprising anti-HER2 chimeric antigen receptors and methods of using and making monocytes comprising anti-HER2 chimeric antigen receptors.

Description

COMPOSITIONS AND METHODS RELATING TO INTRONIC SHRNA CHIMERICANTIGEN RECEPTOR CONSTRUCTSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 464,755, filed May 8, 2023, U.S. Provisional Patent Application No.63 / 587,387, filed October 2, 2023, U.S. Provisional Patent Application No. 63 / 594,644, filed October 31, 2023, and U.S. Provisional Patent Application No. 63 / 642,495, filed May 3, 2024, the contents of which are incorporated herein by reference in their entirety.BACKGROUND

[0002] Macrophages are naturally equipped with both pro- and anti-inflammatory capabilities. Knocking down anti-inflammatory, anti-phagocytic, or other regulatory molecules can enhance anti-tumor functions of chimeric antigen receptor (CAR) macrophages. SIRPa knockout via electroporation of CAR macrophages to deliver CRISPR / Cas components enhances CAR macrophage function. However, electroporating CAR macrophages to deliver CRISPR / Cas components has several drawbacks as a gene editing strategy, including cost, recovery of cells, manufacturing complexity, manual cell handling, electroporation, risks of permanent off-target genomic edits, a heterogeneous cell product (because electroporation and delivery of CAR are independent), and a limit to the number of genes that can be targeted simultaneously). Accordingly, a need exists for a single-step gene editing process for delivering CARs and knocking down anti-inflammatory, anti-phagocytic, or other regulatory molecules in immune cells. Additionally, while its expression in normal tissues is low, HER2 is highly expressed in a wide range of solid tumors and plays an active role in both malignant transformation and tumor aggressiveness by promoting cancer cell proliferation, invasion and metastasis. Therefore, a need exists for the development of new therapeutic modalities optimized to target HER2.SUMMARY OF THE INVENTION

[0003] The present disclosure encompasses, among other things, nucleic acid constructs comprising one or more nucleic acid sequences comprising a promoter, a gene of interest, a 3’ untranslated region (UTR), and one or more introns, wherein the one or more introns comprise one or more inhibitory nucleic acids and wherein the gene of interest encodes a chimeric antigen receptor (CAR) and methods of producing the same. The present disclosure also encompasses, among other things, compositions comprising modified immune cells (e.g., stem cells, macrophages, monocytes, and / or dendritic cells) comprising chimeric antigen receptors (CARs) and / or inhibitory nucleic acids and methods of producing the same. The present disclosure also provides, among other things, methods of designing inhibitory nucleic acids of the present disclosure.

[0004] In one aspect, the present disclosure provides nucleic acid constructs comprising a promoter, a gene of interest, a 3’ untranslated region (UTR), and one or more introns, wherein the one or more introns comprise one or more inhibitory nucleic acids, wherein the one or more inhibitory nucleic acids encode one or more inhibitory RNAs, and, wherein the gene of interest encodes a chimeric antigen receptor (CAR).

[0005] In some embodiments, one or more inhibitory RNAs are selected from the group consisting of antisense RNA (asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), guide RNA (gRNA), long noncoding RNA (IncRNA), microRNA (miRNA), piwi- interacting RNA (piRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), transacting siRNA (tasiRNA), and repeat associated siRNA (rasiRNA).

[0006] In some embodiments, one or more inhibitory RNAs are or comprise one or more shRNA. In some embodiments, one or more shRNA comprise an miRNA scaffold. In some embodiments, an miRNA scaffold comprises an miRNA-155 5’ scaffold, an miRNA-155 3’ scaffold, an miRNA-30 5’ scaffold, an miRNA-30 3’ scaffold, an miRNA- 16 5’ scaffold, an miRNA-16 3’ scaffold, an miRNA-125 5’ scaffold, an miRNA-125 3’ scaffold, an miRNA-223 5’ scaffold, or an miRNA-223 3’ scaffold.

[0007] In some embodiments, one or more shRNA comprise a guide strand. In some embodiments, a guide strand is about 19-24 bases in length. In some embodiments, a guide strand has a G / C content of about 36%-50%. In some embodiments, a guide strand comprises anucleic acid sequence that is reverse complementary to a target gene transcript comprising a target nucleic acid sequence.

[0008] In some embodiments, one or more shRNA comprise a passenger strand. In some embodiments, a passenger strand is 1-2 bases shorter than a guide strand. In some embodiments, a passenger strand is not fully complementary to a guide strand.

[0009] In some embodiments, one or more shRNA comprise a loop.

[0010] In some embodiments, one or more shRNA comprise: (a) a nucleic acid sequence selected from Table la, Table lb, Table 1c, Table Id or Table le; (b) a nucleic acid sequence that differs from a sequence selected from Table la, Table lb, Table 1c, Table Id or Table le by no more than 5 substitutions, additions, or deletions; or (c) a nucleic acid sequence that is at least 80% identical to a sequence selected from Table la, Table lb, Table 1c, Table Id or Table le.

[0011] In some embodiments, one or more inhibitory RNAs arc or comprise one or more miRNA. In some embodiments, one or more miRNA are selected from Table 2.

[0012] In some embodiments, a target gene transcript is mammalian. In some embodiments, a target gene transcript is human. In some embodiments, contacting a target gene transcript with one or more inhibitory RNAs reduces translation of the target gene transcript.

[0013] In some embodiments, a target gene transcript is expressed in an immune cell. In some embodiments, an immune cell is a stem cell, macrophage, monocyte, or dendritic cell. In some embodiments, an immune cell is an induced pluripotent stem cell (iPSC)-derived monocyte, macrophage, or dendritic cell. In some embodiments, a target gene transcript is expressed in a macrophage. In some embodiments, reduced translation of a target gene transcript is associated with an Ml phenotype. In some embodiments, reduced translation of a target gene transcript is associated with an M2 phenotype.

[0014] In some embodiments, a target nucleic acid sequence comprises: (a) a nucleic acid sequence selected from Table 3; (b) a nucleic acid sequence that differs from a sequence selected from Table 3 by no more than 5 substitutions, additions, or deletions; or (c) a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 3.

[0015] In some embodiments, a target gene transcript encodes human ATG7, C / EBP- alpha, C / EBP-beta, CD32b, CD36, CLEC1A, FATS, GOLM1, HAVCR2, ITGAD, KLF4,KLF6, LILRB1 , LILRB2, LILRB4, MAF, MafB, PD1 , PD-LI, PIK3CG, PIK3CG, PPARa, PPARy, PTGS2, SIGLEC10, SIRPa, SLAMF3, SLAMF4, SLC15A3, STAT3, STAT6, TNFRSF1B, TOX, TREM2, YTHDF2, or ZFP36. In some embodiments, a target gene transcript encodes a human anti-phagocytic receptor selected from the group consisting of: SIRPa, LILRB1, SIGLEC10, PD1, SLAMF3, SLAMF4, CLEC1A, and CD32b. In some embodiments, a target gene transcript encodes human SIRPa. In some embodiments, a target gene transcript encodes human PD-LI. In some embodiments, a target gene transcript encodes human PD1. In some embodiments, a target gene transcript encodes human LILRB1. In some embodiments, a target gene transcript encodes human SIGLEC10. In some embodiments, a target gene transcript encodes human SLAMF3. In some embodiments, a target gene transcript encodes human SLAMF4. In some embodiments, a target gene transcript encodes human CLEC1A. In some embodiments, a target gene transcript encodes human CD32b.

[0016] In some embodiments, one or more introns comprise one, two, or three introns. In some embodiments, one or more introns are located downstream of the promoter. In some embodiments, one or more introns are located within a gene of interest. In some embodiments, one or more introns are located within the 3’ UTR. In some embodiments, one or more inhibitory nucleic acids comprise one inhibitory nucleic acid. In some embodiments, one or more inhibitory nucleic acids comprise at least two, three, four or five inhibitory nucleic acids. In some embodiments, at least two, three, four, or five inhibitory nucleic acids are in tandem. In some embodiments, at least two, three, four, or five inhibitory nucleic acids comprise identical sequences. In some embodiments, at least two, three, four, or five inhibitory nucleic acids comprise at least two different sequences. In some embodiments, at least two, three, four, or five inhibitory nucleic acids target at least two, three, four, or five different anti-phagocytic receptors. In some embodiments, at least two, three, four, or five inhibitory nucleic acids encode inhibitory RNAs comprising nucleic acid sequences that are reverse complementary to the same target gene transcript. In some embodiments, at least two, three, four, or five inhibitory nucleic acids encode inhibitory RNAs comprising nucleic acid sequences that are reverse complementary to different target gene transcripts. In some embodiments, at least two, three, four, or five inhibitory nucleic acids encode inhibitory RNAs comprising identical miRNA scaffolds. In some embodiments, at least two, three, four, or five inhibitory nucleic acids encode inhibitory RNAs comprising at least two different miRNA scaffolds. In some embodiments, at least two different miRNA scaffoldsare selected from the group consisting of an miRNA- 155 5’ scaffold, an miRNA-155 3’ scaffold, an miRNA-30 5’ scaffold, and an miRNA-30 3’ scaffold.

[0017] In some embodiments, an intron comprises three inhibitory nucleic acids, wherein the three inhibitory nucleic acids encode three inhibitory RNAs, wherein the three inhibitory RNAs comprise the following miRNA scaffolds in order from 5’ to 3’: (a) miRNA 30, miRNA 30, miRNA 30, (b) miRNA 30, miRNA 155, miRNA 30, (c) miRNA 155, miRNA 30, miRNA 155, or (d) miRNA 155, miRNA 155, miRNA 155.

[0018] In some embodiments, expression of a CAR is increased relative to a reference nucleic acid construct that lacks one or more introns.

[0019] In some embodiments, a CAR comprises: (a) one or more extracellular domains; (b) a transmembrane domain; and (c) one or more intracellular domains.

[0020] In some embodiments, one or more extracellular domains are or comprise an scFv, VHH antibody, centyrin, darpin, or nanobody.

[0021] In some embodiments, a transmembrane domain is or comprises a CD8a, CD28, CD40, MyD88, CD64, CD32a, CD32c, CD16a, CD3zeta, DAP10, DAP12, Dectin-1, DNGR1, ICOS, NKG2D, RAGE, SLAMF7, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, or TLR9 transmembrane domain.

[0022] In some embodiments, one or more intracellular domains comprise one or more of a CD3^, FcRy, MyD88, CD40, CD64, CD32a, CD32c, CD16a, CD89, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD19, CD20, 41BB, CD28, GCSFR (GDI 14), RAGE, CD30, CD160, DR3, Fnl4, HVEM, CD160, NGFR, RANK, TNFR2, TROY, XEDAR, TRIF, 0X40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, V / RLxYxxL / V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPb, CD22, PIR-B, LILRB1, 41BBL (TNFSF9), CD27, OX40L, CD32b, CDl lb, ITGAM, SLAMF7, CD206, CD163, CD209, Dectin-2, IL1R, IL2R, IL3R, IL4R, IL5R, IL6R, IL7R, IL8R, IL9R, IL10R, IL11R, IL12R, IL13R, IL14R, IL15R, IL17R, IFNaR, IFNgR, TNFR, CSF1R, CSF2R, DAP10, CD36, Dectin-1, ICOSL, Syk, TRAM, ZAP-70, or NKG2D intracellular domain, a portion of any of the foregoing, or combinationsthereof. In some embodiments, one or more intracellular domains further comprise one or more of a CD3< , FcRy, MyD88, CD40, CD64, CD32a, CD32c, CD16a, CD89, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, R0R1, ROS1, RYK, TIE2, TRK, VEGFR, CD19, CD20, 41BB, CD28, GCSFR (CD114), RAGE, CD30, CD160, DR3, Fnl4, HVEM, CD160, NGFR, RANK, TNFR2, TROY, XEDAR, TRIF, 0X40, GITR, TREM-1, TREM-2, DAP 12, MR, ICOS, V / I / LxYxxL / V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPb, CD22, PIR-B, LILRB1, 41BBL (TNFSF9), CD27, OX40L, CD32b, CD1 lb, ITGAM, SLAMF7, CD206, CD163, CD209, Dectin-2, IL1R, IL2R, IL3R, IL4R, IL5R, IL6R, IL7R, IL8R, IL9R, IL10R, IL11R, IL12R, IL13R, IL14R, IL15R, IL17R, IFNaR, IFNgR, TNFR, CSF1R, CSF2R, DAP10, CD36, Dectin-1, ICOSL, Syk, TRAM, ZAP-70, or NKG2D intracellular domain, a portion of any of the foregoing, or combinations thereof.

[0023] In some embodiments, a CAR further comprises an extracellular leader domain. In some embodiments, an extracellular leader domain is or comprises a CD8a extracellular leader domain.

[0024] In some embodiments, a CAR further comprises an extracellular hinge domain. In some embodiments, an extracellular hinge domain is or comprises: a DAP10 extracellular hinge domain, a DAP12 extracellular hinge domain, a CD8a extracellular hinge domain, a CD28 extracellular hinge domain, a DNGR-1 extracellular hinge domain, a Dectin-1 extracellular hinge domain, and / or an IgG4 extracellular hinge domain.

[0025] In some embodiments, a CAR comprises, from N-terminus to C-terminus: a CD8a leader domain, an anti-HER2 antigen binding domain, a CD8 extracellular hinge domain, a CD8 transmembrane domain, and a CD3y intracellular domain.

[0026] In some embodiments, a CAR further comprises a cleavage peptide. In some embodiments, a cleavage peptide is or comprises a P2A, F2A, E2A or T2A peptide.

[0027] In some embodiments, a CAR comprises: (a) an amino acid sequence selected from Table 5; (b) an amino acid sequence that differs from a sequence selected from Table 5 by no more than five substitutions, additions, or deletions; or (c) an amino acid sequence that is at least 80% identical to a sequence selected from Table 5. In some embodiments, a nucleic acidconstruct comprises: (a) a nucleic acid sequence selected from Table 4; (b) a nucleic acid sequence that differs from a sequence selected from Table 4 by no more than five substitutions, additions, or deletions; or (c) a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 4.

[0028] In some embodiments, a nucleic acid construct comprises a modification selected from: a modified nucleotide, an alteration to the 5’ UTR, an alteration to the 3’ UTR, or combinations thereof. In some embodiments, a modified nucleotide comprises pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine / pseudouridine (5meC PsU), Nl-methyl- pseudouridine (NlmPsU), or combinations thereof.

[0029] In some embodiments, a nucleic acid construct is a purified nucleic acid construct.

[0030] In some embodiments, at least a portion of the nucleic acid construct is codon- optimized. In some embodiments, at least a portion of the nucleic acid construct is codon- optimized for expression in a stem cell, monocyte, macrophage, or dendritic cell.

[0031] In another aspect, the present disclosure provides modified immune cells comprising a nucleic acid construct of the present disclosure. In some embodiments, a cell is or comprises a stem cell, macrophage, monocyte, or dendritic cell. In some embodiments, a cell is an induced pluripotent stem cell (iPSC)-derived monocyte, macrophage, or dendritic cell.

[0032] In another aspect, the present disclosure provides pharmaceutical compositions comprising a nucleic acid construct of the present disclosure or a modified immune cell of the present disclosure. In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0033] In another aspect, the present disclosure provides methods of treating a disease or disorder in a subject, the methods comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition of the present disclosure, wherein at least one sign or symptom of the disease or disorder is improved in the subject. In some embodiments, a step of administering is or comprises transarterial, subcutaneous, intravenous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, or intraperitoneal delivery.

[0034] In another aspect, the present disclosure provides methods of modifying an immune cell, the methods comprising: delivering to the immune cell a nucleic acid construct ofthe present disclosure, thereby producing a modified immune cell, wherein the modified immune cell is or comprises a macrophage, monocyte, dendritic cell, or stem cell.

[0035] In some embodiments, a nucleic acid construct comprises DNA or RNA.

[0036] In some embodiments, delivering comprises electroporation or transfection with the nucleic acid construct. In some embodiments, a nucleic acid construct is within a delivery vehicle. In some embodiments, a delivery vehicle is or comprises a liposome, a lipid nanoparticle, a polymer, an adeno-associated viral (AAV) vector, an adenoviral vector, a retroviral vector, a transposon, a retrotransposon, a site-directed insertion vector or combinations thereof. In some embodiments, a retroviral vector comprises a lentiviral vector or a gammaretroviral vector. In some embodiments, a lentiviral vector is packaged with a Vpx protein. In some embodiments, an adenoviral vector comprises an Ad2 vector or an Ad5 vector. In some embodiments, an Ad5 vector comprises an Ad5f35 adenoviral vector. In some embodiments, a retrotransposon comprises sleeping beauty or piggyBac. In some embodiments, a site-directed insertion vector comprises a CRISPR / Cas system.

[0037] In some embodiments, a method of the present disclosure further comprises delivering to an immune cell an additional payload. In some embodiments, an additional payload is or comprises a pathogen recognition receptor agonist, polyinosinic:polycytidylic acid (poly I:C), a TLR7 / 8 agonist, a CpG oligodeoxynucleotide, a NOD-like receptor (NLR) agonist, a RIG-I-like receptor (RLR) agonist, a C-type lectins receptor (CLR) agonist, a cytosolic DNA sensing, the cyclic GMP-AMP synthase- stimulator of interferon genes (cGAS-STING) agonist, an interferon-inducible protein 16 (IFI16) agonist, a DEAD-box helicase 41 (DDX41) agonist, an LRR binding FLII interacting protein 1 (LRRFIP1) agonist, an absent in melanoma 2 (AIM2) agonist, an aryl hydrocarbon receptor (AhR) ligand, or combinations thereof. In some embodiments, a nucleic acid construct and an additional payload are encapsulated within a delivery vehicle.

[0038] In another aspect, the present disclosure provides methods of designing an shRNA comprising a guide strand, a passenger strand, and a loop, the methods comprising the steps of: (a) selecting a target region of a target gene transcript, (b) designing the guide strand, wherein the guide strand is a reverse complement of the target region, and wherein the guide strand comprises, from 5’ to 3’, an adenine or uracil at position 1, an adenine, guanine, or cytosine atposition 10, and a G / C content between 36% and 45% that is increases from 5’ to 3’; and (c) designing the passenger strand, wherein the passenger strand comprises the sequence of the target region, from 5’ to 3’, with the following variations: (i) deletion of a nucleotide at position 7 relative to the loop, (ii) deletion of a nucleotide at position 11 relative to the loop, (iii) mutating an adenine to guanine and / or mutating a cytosine to uracil near the deletions at positions 7 and 11, wherein the mutations create two partial G-U base pairs between the passenger strand and the guide strand; and wherein the passenger strand is 2 base pairs shorter than the guide strand.

[0039] In some embodiments, a guide strand comprises a higher G / C content of the 10 bases at the 3’ end of the guide strand relative to the G / C content of the 10 bases at the 5’ end of the guide strand. In some embodiments, a target region of a target gene transcript is 21 -22 base pairs in length.

[0040] In another aspect, the present disclosure provides effector cells comprising a nucleic acid construct of the present disclosure, wherein one or more inhibitory RNAs are secreted from the cell. In some embodiments, one or more inhibitory RNAs are packaged within an extracellular vesicle. In some embodiments, one or more inhibitory RNAs are packaged within an exosome. In some embodiments, secreted inhibitory RNA affect the phenotype of bystander cells. In some embodiments, bystander cells are bystander macrophages, bystander monocytes, bystander dendritic cells, or bystander stem cells. In some embodiments, bystander macrophages are skewed to an M 1 phenotype. In some embodiments, bystander macrophages are skewed to an M2 phenotype. In some embodiments, bystander cells are skewed to an antitumor phenotype. In some embodiments, bystander cells are skewed to an anti-inflammatory phenotype.BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The drawings are for illustration purposes only, not for limitation.

[0042] Figure 1 shows exemplary CAR constructs with or without an intron (e.g., an intron comprising shRNA).

[0043] Figure 2 shows exemplary effects on CAR expression and gene knockdown from a CAR construct comprising an intron (e.g., an intron comprising shRNA).

[0044] Figure 3 shows exemplary experimental timeline for plasmid electroporation (EP) and analysis by flow cytometry (FCM).

[0045] Figure 4 shows an exemplary CAR construct comprising an inhibitory nucleic acid, here a primary microRNA sequence (pri-miRNA) located downstream of a promoter (e.g., EFla) and CAR gene of interest (GOI).

[0046] Figure 5 shows exemplary SIRPa knockdown in cells with and without CAR expression using various shRNA species and control groups.

[0047] Figure 6A and Figure 6B show exemplary CAR constructs comprising an inhibitory nucleic acid, here a primary microRNA contained within an intron contained within the CAR GOI (Figure 6A) or a primary microRNA contained within an intron contained downstream of the CAR GOI (Figure 6B).

[0048] Figure 7 shows exemplary SIRPa knockdown and CAR expression in cells transduced with various intronic shRNA constructs, where the intron is positioned in different locations within the construct.

[0049] Figure 8 shows exemplary CAR expression in cells transduced with constructs with and without intronic shRNA using lentivirus vector.

[0050] Figure 9 shows exemplary shRNA species (584, 1457, and 4074) in tandem within a sequence.

[0051] Figure 10 shows exemplary SIRPa knockdown and CAR expression in cells transduced with various intronic shRNA constructs, where the intron is positioned in different locations within the construct and the shRNA tandems utilize various scaffolding configurations.

[0052] Figure 11 shows representative CAR expression of an exemplary intronic shRNA construct (Tandem 4) and a CRISPR control.

[0053] Figure 12 shows exemplary CAR constructs, including a standard construct lacking an intron (CTX_001), a construct comprising an intron with 3 shRNA species in tandem wherein the intron is located within the CAR-coding region (CTX_13O8), and a construct comprising an intron with 3 shRNA species in tandem wherein the intron is located downstream of the CAR-coding region (CTX_1309).

[0054] Figure 13 shows exemplary experimental timeline for adenovirus (AdV) transduction or CRISPR electroporation (EP), and later analysis by flow cytometry (FCM).

[0055] Figure 14 shows exemplary CAR expression in cells transduced with AdV encoding various intronic shRNA constructs at different multiplicity of infection (MOI) over three time points, where the intron is positioned in different locations within the construct.

[0056] Figure 15 shows exemplary SIRPa knockdown in cells transduced AdV encoding with various intronic shRNA constructs at different MOI over three time points, where the intron is positioned in different locations within the construct.

[0057] Figure 16 shows exemplary viability in cells transduced with AdV encoding various intronic shRNA constructs at different MOI over three time points, where the intron is positioned in different locations within the construct.

[0058] Figure 17 shows representative flow cytometry gating of CAR and SIRPa expression from day 3 in cells transduced with AdV encoding various intronic shRNA constructs.

[0059] Figure 18 shows exemplary experimental timeline for adenovirus (AdV) transduction or CRISPR EP, and later analysis of M1 / M2 phenotype, tumor cell killing, and phagocytosis.

[0060] Figure 19 shows exemplary CAR expression in primary human macrophages transduced with standard CAR AdV, transduced with standard CAR AdV and electroporated with CRISPR / Cas9, or transduced with intronic shRNA CAR AdV.

[0061] Figure 20 shows exemplary SIRPa knockdown in primary human macrophages transduced with standard CAR AdV, transduced with standard CAR AdV and electroporated with CR1SPR / Cas9, or transduced with intronic shRNA CAR AdV.

[0062] Figure 21 shows exemplary phenotypic markers as measured by flow cytometry in primary human macrophages transduced with standard CAR AdV, transduced with standard CAR AdV and electroporated with CRISPR / Cas9, or transduced with intronic shRNA CAR AdV. M2 marker (CD 163, CD206) expression and Ml marker (CD80, CD86) expression were assessed.

[0063] Figure 22 shows exemplary Ml polarization scores in primary human macrophages transduced with standard CAR AdV, transduced with standard CAR AdV and electroporated with CRISPR / Cas9, or transduced with intronic shRNA CAR AdV.

[0064] Figure 23 shows exemplary cell viability in primary human macrophages transduced with standard CAR AdV, transduced with standard CAR AdV and electroporated with CRISPR / Cas9, or transduced with intronic shRNA CAR AdV.

[0065] Figure 24 shows exemplary phagocytosis of tumor SKOV3 cells in primary human macrophages transduced with standard CAR AdV, transduced with standard CAR AdV and electroporated with CRISPR / Cas9, or transduced with intronic shRNA CAR AdV.

[0066] Figure 25 shows exemplary tumor SKOV3 cell killing in primary human macrophages transduced with standard CAR AdV, transduced with standard CAR AdV and electroporated with CRISPR / Cas9, or transduced with intronic shRNA CAR AdV.

[0067] Figure 26 shows exemplary CAR expression in primary human macrophages transduced with standard CAR AdV, transduced with standard CAR AdV and electroporated with CR1SPR / Cas9, or transduced with intronic shRNA CAR AdV at different MOI.

[0068] Figure 27 shows exemplary SIRPa knockdown in primary human macrophages transduced with standard CAR AdV, transduced with standard CAR AdV and electroporated with CRISPR / Cas9, or transduced with intronic shRNA CAR AdV at different MOI.

[0069] Figure 28 shows exemplary phagocytosis of tumor SKOV3 cells in primary human macrophages transduced with standard CAR AdV, transduced with standard CAR AdV and electroporated with CRISPR / Cas9, or transduced with intronic shRNA CAR AdV at different MOI.

[0070] Figure 29 shows exemplary tumor SK.OV3 cell killing in primary human macrophages transduced with standard CAR AdV, transduced with standard CAR AdV and electroporated with CRISPR / Cas9, or transduced with intronic shRNA CAR AdV at MOI of 10.

[0071] Figure 30 shows exemplary CAR constructs with or without an intron (e.g., an intron comprising shRNA) and the effects on CAR expression and SIRPa knockdown.

[0072] Figure 31 shows exemplary structures involved in the design of intronic shRNA constructs (c.g., as described herein).

[0073] Figure 32A and Figure 32B show graphs of exemplary anti-mesothelin CAR expression and cell viability after transduction of monocytes with Ad5f35 virus comprising CTX_964 (an anti-mesothelin CAR) or CTX_1461 (an anti-mesothelin CAR + intronic shRNA against SIRPa).

[0074] Figure 33A and Figure 33B show graphs of exemplary SIRPa expression after transduction of monocytes with Ad5f35 virus comprising CTX_964 (an anti-mesothelin CAR) or CTX_1461 (an anti-mesothelin CAR + intronic shRNA against SIRPa).

[0075] Figure 34 shows graphs of exemplary cytokine (TNFa) release after transduction of monocytes with Ad5f35 virus comprising CTX_964 (an anti-mesothelin CAR) or CTX_1461 (an anti-mesothelin CAR + intronic shRNA against SIRPa) and exposure of the cells to either recombinant human mesothelin or recombinant mesothelin + recombinant human CD47.

[0076] Figure 35A and Figure 35B show graphs of exemplary anti-mesothelin mediated killing of mesothelin-expressing target cells by monocytes and macropahges after transduction of the cells with Ad5f35 vector comprising either CTX_964 (an anti-mesothelin CAR) or CTX_1461 (an anti-mesothelin CAR + intronic shRNA against SIRPa).

[0077] Figure 36 shows a graph of exemplary suppression of tumor growth by CAR- monocyte-derived CAR macrophages after transduction of the cells with Ad5f35 vector comprising either CTX_964 (an anti-mesothelin CAR) or CTX_1461 (an anti-mesothelin CAR + intronic shRNA against SIRPa).

[0078] Figure 37A, Figure 37B, Figure 37C, and Figure 37D show graphs of exemplary CAR expression, SIRPa knockdown, phagocytosis, and tumor cell killing for macrophages transduced with a standard construct lacking an intron (CTX_001), a construct comprising an intron with 3 shRNA species in tandem wherein the intron is located downstream of the CAR-coding region (CTX_1309), or a CAR construct and then subjected to CRISPR / Cas9 electroporation.

[0079] Figure 38A and Figure 38B show graphs exemplary body weight, body score, and suppression of tumor growth by CAR macrophages after transduction of the cells withAd5f35 vector comprising either CTX_001 (an anti-HER2 CAR) or CTX_1309 (an anti-HER2 CAR + intronic shRNA against SIRPa).

[0080] Figure 39A and Figure 39B show graphs of exemplary target cell killing mediated by anti-HER2 CAR monocyte (CAR-Mono)-derived macrophages relative to anti- HER2 CAR macrophages (CAR-Mac).

[0081] Figure 40 shows graphs of exemplary TNFa, IL-6, and IL-ip cytokine secretion from anti-HER2 CAR monocyte (CAR-Mono)-derived macrophages relative to anti-HER2 CAR macrophages (CAR-Mac).

[0082] Figure 41 shows a graph of exemplary persistence of intravenously administered anti-HER2 CAR monocytes (CAR-Mono) relative to anti-HER2 CAR macrophages (CAR-Mac) in mice.

[0083] Figure 42 shows a graph of exemplary trafficking of intravenously administered anti-HER2 CAR monocytes (CAR-Mono) relative to anti-HER2 CAR macrophages (CAR-Mac) to tumors in mice.

[0084] Figure 43 shows a graph of exemplary production yields of anti-HER2 CAR monocytes (CAR-Mono) relative to anti-HER2 CAR macrophages (CAR-Mac).DEFINITIONS

[0085] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.

[0086] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one clement or more than one clement.

[0087] Approximately or about: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated referencevalue. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0088] Activation: As used herein, the term “activation” refers to the state of a cell, for example a monocyte, macrophage, or dendritic cell that has been sufficiently stimulated to induce detectable cellular proliferation or has been stimulated to exert its effector function. Activation can also be associated with induced cytokine production, phagocytosis, cell signaling, target cell killing, and / or antigen processing and presentation.

[0089] Activated monocytes / macrophages / dendritic cells'. As used herein, the term “activated monocytes / macrophages / dendritic cells” refers to, among other things, monocyte / macrophage / dendritic cells that are undergoing cell division or exerting effector function. The term “activated monocytes / macrophages / dendritic cells” refers to, among others thing, cells that are performing an effector function or exerting any activity not seen in the resting state, including phagocytosis, cytokine secretion, proliferation, gene expression changes, metabolic changes, and other functions.

[0090] Agent'. As used herein, the term “agent” (or “biological agent” or “therapeutic agent”), refers to a molecule that may be expressed, released, secreted or delivered to a target by a modified cell described herein. An agent includes, but is not limited to, a nucleic acid, an antibiotic, an anti-inflammatory agent, an antibody or fragments thereof, an antibody agent or fragments thereof, a growth factor, a cytokine, an enzyme, a protein (e.g., an RNAse inhibitor), a peptide, a fusion protein, a synthetic molecule, an organic molecule (e.g., a small molecule), a carbohydrate, a lipid, a hormone, a microsome, a derivative or a variation thereof, and any combinations thereof. An agent may bind any cell moiety, such as a receptor, an antigenic determinant, or other binding site present on a target or target cell. An agent may diffuse or be transported into a cell, where it may act intracellularly.

[0091] Antibody. As used herein, the term “antibody” refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As is known in the art, intact antibodies as produced in nature areapproximately 150 kD tetrameric agents comprising two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a “Y-shaped” structure. Each heavy chain comprises at least four domains (each about 110 amino acids long) - an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CHI, CH2, and the carboxy-terminal CH3 (located at the base of the Y’s stem). A short region, known as the “switch”, connects the heavy chain variable and constant regions. The “hinge” connects CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to one another in an intact antibody. Each light chain comprises two domains - an amino-terminal variable (VL) domain, followed by a carboxy- terminal constant (CL) domain, separated from one another by another “switch”. Intact antibody tetramers comprise two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and a tetramer is formed. Naturally-produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold” formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as “complementarity determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). When natural antibodies fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three- dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure. The Fc region of naturally-occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. Affinity and / or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, antibodies produced and / or utilized in accordance with the present invention (e.g., as a component of a CAR) include glycosylated Fc domains, including Fc domains with modified or engineered glycosylation. In some embodiments, any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in natural antibodies can be referred toand / or used as an “antibody”, whether such polypeptide is naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology. In some embodiments, an antibody is polyclonal. In some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc, as is known in the art. Moreover, the term “antibody”, as used herein, can refer in appropriate embodiments (unless otherwise stated or clear from context) to any of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, in some embodiments, an antibody utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments. Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™”); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®;Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KAEBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc.].

[0092] Antibody agent'. As used herein, the term “antibody agent” refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. Exemplary antibody agents include, but are not limited to monoclonal antibodies or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more sequenceelements are humanized, primatized, chimeric, etc., as is known in the art. In many embodiments, the term “antibody agent” is used to refer to one or more of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, in some embodiments, an antibody agent utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPsTM”); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody agent may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody agent may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc.]. In many embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a referenceCDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain. In some embodiments, an antibody agent is not and / or does not comprise a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent may be or comprise a molecule or composition which does not include immunoglobulin structural elements (e.g., a receptor or other naturally occurring molecule which includes at least one antigen binding domain).

[0093] Antibody fragment'. As used herein, the term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments and human and humanized versions thereof.

[0094] Antibody heavy chain: As used herein, the term “antibody heavy chain” refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0095] Antibody light chain: As used herein, the term “antibody light chain” refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0096] Synthetic antibody: As used herein, the term “synthetic antibody” refers to an antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

[0097] Antigen: As used herein, the term “antigen” or “Ag” refers to a molecule that is capable of provoking an immune response. This immune response may involve either antibody production, the activation of specific immunologically-competent cells, or both. A skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA that comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0098] Anti-tumor effect: As used herein, the term “anti-tumor effect” refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An“anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of a tumor in the first place.

[0099] Autologous: As used herein, the term “autologous” refers to any material derived from an individual to which it is later to be re-introduced into the same individual.

[0100] Allogeneic: As used herein, the term “allogeneic” refers to any material (e.g., a population of cells) derived from a different animal of the same species.

[0101] Xenogenic: As used herein, the term “xenogeneic” refers to any material (e.g., a population of cells) derived from an animal of a different species.

[0102] Cancer: As used herein, the term “cancer” refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like. In certain embodiments, the cancer is medullary thyroid carcinoma.

[0103] Conservative sequence modifications: As used herein, the term “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions.Modifications can be introduced into an antibody compatible with various embodiments by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which an amino acid residue is replaced with an amino acid residue having a similar’ side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody can be replaced withother amino acid residues from the same side chain family and the altered antibody can be tested for the ability to bind antigens using the functional assays described herein.

[0104] Co-stimulatory ligand: As used herein, the term “co-stimulatory ligand” refers to a molecule on an antigen presenting cell (e.g., an APC, dendritic cell, B cell, and the like) that specifically binds a cognate co-stimulatory molecule on a monocyte / macrophage / dendritic cell, thereby providing a signal which mediates a monocyte / macrophage / dendritic cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A co- stimulatory ligand can include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3. A co-stimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a monocyte / macrophage / dendritic cell, such as, but not limited to, CD27, CD28, 4- IBB, 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function- associated antigen-1 (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.

[0105] Cytotoxic: As used herein, the term “cytotoxic” or “cytotoxicity” refers to killing or damaging cells. In one embodiment, cytotoxicity of the metabolically enhanced cells is improved, e.g. increased cytolytic activity of macrophages.

[0106] Effective amount: As used herein, “effective amount” and “therapeutically effective amount” are interchangeable, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a manufacturing, therapeutic or prophylactic benefit. Such results may include, but are not limited to, anti-tumor activity as determined by any means suitable in the art.

[0107] Effector function: As used herein, “effector function” or “effector activity” refers to a specific activity carried out by an immune cell in response to stimulation of the immune cell. For example, an effector function of macrophages to engulf and digest cellular debris, foreign substances, microbes, cancer cells and other unhealthy cells by phagocytosis.

[0108] Encoding: As used herein, “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve astemplates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.c., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0109] Endogenous: As used herein “endogenous” refers to any material from or produced inside a particular organism, cell, tissue or system.

[0110] Exogenous: As used herein, the term “exogenous” refers to any material introduced from or produced outside a particular organism, cell, tissue or system.

[0111] Expand: As used herein, the term “expand” refers to increasing in number, as in an increase in the number of cells, for example, monocytes, macrophages, and / or dendritic cells. In one embodiment, monocytes, macrophages, or dendritic cells that are expanded ex vivo increase in number relative to the number originally present in a culture. In another embodiment, monocytes, macrophages, or dendritic cells that are expanded ex vivo increase in number relative to other cell types in a culture. In some embodiments, expansion may occur in vivo. The term "ex vivo," as used herein, refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0112] Expression: As used herein, the term “expression” of a nucleic acid sequence refers to generation of any gene product from a nucleic acid sequence. In some embodiments, a gene product can be a transcript. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and / or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0113] Expression vector: As used herein, the term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cisacting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0114] Fragment: As used herein, the terms “fragment” or “portion” refers to a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole structure. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole. In some embodiments, a nucleotide fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more monomeric units (e.g., nucleic acids) as found in the whole nucleotide. In some embodiments, a nucleotide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the monomeric units (e.g., residues) found in the whole nucleotide. The whole material or entity may in some embodiments be referred to as the “parent” of the whole.

[0115] Homology: As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). As will be understood by those skilled in the ail, a variety of algorithms are available that permit comparison of sequences in order to determine theirdegree of homology, including by permitting gaps of designated length in one sequence relative to another when considering which residues “correspond” to one another in different sequences. Calculation of the percent homology between two nucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-corresponding sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules arc identical at that position; when a position in the first sequence is occupied by a similar nucleotide as the corresponding position in the second sequence, then the molecules are similar at that position. The percent homology between the two sequences is a function of the number of identical and similar positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.

[0116] Identity: As used herein, the term “identity” refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an Arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.

[0117] Substantial identity: As used herein, the term “substantial identity” refers to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those ofordinary skill in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues in corresponding positions. As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over a relevant stretch of residues. In some embodiments, the relevant stretch is a complete sequence. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues. In the context of a CDR, reference to “substantial identity” typically refers to a CDR having an amino acid sequence at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to that of a reference CDR.

[0118] Immune cell'. As used herein, the term “immune cell,” refers to a cell that is involved in an immune response, e.g., promotion of an immune response. Examples of immune cells include, but are not limited to, macrophages, monocytes, dendritic cells, neutrophils, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, T-lymphocytes, or B-lymphocytes. A source of immune cells (e.g., macrophages, monocytes, or dendritic cells) can be obtained from a subject.

[0119] Immune response: As used herein the term “immune response” refers to a cellular and / or systemic response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and / or activate lymphocytes to remove the antigen.

[0120] Immunoglobulin: As used herein, the term “immunoglobulin” or “Ig,” refers to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as a BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulatingantibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is an immunoglobulin that has no known antibody function, but may serve as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergen.

[0121] Isolated: As used herein, the term “isolated” refers to something altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0122] Modified: As used herein, the term “modified” refers to a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

[0123] Modulating: As used herein the term “modulating,” refers to mediating a detectable increase or decrease in the level of a response and / or a change in the nature of a response in a subject compared with the level and / or nature of a response in the subject in the absence of a treatment or compound, and / or compared with the level and / or nature of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0124] Nucleic acid: As used herein, the term “nucleic acid” refers to a polymer of at least three nucleotides. In some embodiments, a nucleic acid comprises DNA. In some embodiments, a nucleic acid comprises RNA. In some embodiments, a nucleic acid is single stranded. In some embodiments, a nucleic acid is double stranded. In some embodiments, a nucleic acid comprises both single and double stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioatc or 5'-N-phosphoramiditc linkages and / or one or more peptide bonds, e.g., as in a “peptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxy thymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises one or more, or all, non-natural residues. In some embodiments, a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5 -fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2- aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)- methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residue comprises one or more modified sugars (e.g., 2'- fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long.

[0125] Operably linked: As used herein, the term “operably linked” refers to functional linkage between, for example, a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the codingsequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0126] Overexpressed tumor antigen: As used herein, the term “overexpressed” tumor antigen or “overexpression” of a tumor antigen refers to an abnormal level of expression of a tumor antigen in a cell from a disease area like a solid tumor within a specific tissue or organ of the patient relative to the level of expression in a normal cell from that tissue or organ. Patients having solid tumors or a hematological malignancy characterized by overexpression of the tumor antigen can be determined by standard assays known in the art.

[0127] Polynucleotide: As used herein, the term “polynucleotide” refers to a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0128] Polypeptide: As used herein, the term “polypeptide” refers to any polymeric chain of residues (e.g., amino acids) that are typically linked by peptide bonds. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only nonnatural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L- amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at thepolypeptide’s C-terminus, or any combination thereof. Tn some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and / or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a useful polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide ofinterest or vice versa, and / or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.

[0129] Protein: As used herein, the term “protein” refers to a polypeptide (i.e. , a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and / or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and / or characteristic portions thereof.

[0130] Signal transduction pathway: As used herein, the term “signal transduction pathway” refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. The phrase “cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the plasma membrane of a cell.

[0131] Single chain antibodies: As used herein, the term “single chain antibodies” refers to antibodies formed by recombinant DNA techniques in which immunoglobulin heavy and light chain fragments are linked to the Fv region via an engineered span of amino acids. Various methods of generating single chain antibodies are known, including those described in U.S. Pat. No. 4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci.USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041.

[0132] Specifically binds: As used herein, the term “specifically binds,” with respect to an antigen binding domain, such as an antibody agent, refers to an antigen binding domain or antibody agent which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antigen binding domain or antibody agent that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antigen binding domain or antibody agent as specific. In another example, an antigen binding domain or antibody agent that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antigen binding domain or antibody agent as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antigen binding domain or antibody agent, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antigen binding domain or antibody agent recognizes and binds to a specific protein structure rather than to proteins generally. If an antigen binding domain or antibody agent is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antigen binding domain or antibody agent, will reduce the amount of labeled A bound to the antibody.

[0133] Stimulation: As used herein, the term “stimulation,” refers to a primary response induced by binding of a stimulatory molecule (e.g., an FcR complex, a TLR complex, or a TCR / CD3 complex), for example, with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via Fc receptor machinery, or via a synthetic CAR. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-beta, and / or reorganization of cytoskeletal structures, and the like. As used herein, the term “stimulatory molecule,” refers to a molecule of a monocyte, macrophage, or dendritic cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell. In some embodiments, a stimulatory molecule comprises an FcR extracellulardomain comprising a CD64 (FcyRI), CD32a (FcyRIIa), CD32b (FcyRIIb), CD32c, CD16a (FcyRIIIa), CD16b (FcyRIIIb), FceRI, FceRII, FcaRI (CD89) or CD40 domain. In some embodiments, a stimulatory molecule comprises a TLR extracellular domain comprising a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain. As used herein, the term “stimulatory ligand,” refers to a ligand that when present on an antigen presenting cell (e.g., an aAPC, a macrophage, a dendritic cell, a B-cell, and the like) or tumor cell can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a monocyte, macrophage, or dendritic cell thereby mediating a response by the immune cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, inter alia, Toll-like receptor (TLR) ligand, an anti-toll-like receptor antibody, an agonist, and an antibody for a monocyte / macrophage receptor. In addition, cytokines, such as interferon-gamma, are potent stimulants of macrophages.

[0134] Subject: As used herein, the term “subject” refers to an organism, for example, a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, or a dog). In some embodiments a human subject is an adult, adolescent, or pediatric subject. In some embodiments, a subject is suffering from a disease, disorder or condition, e.g., a disease, disorder, or condition that can be treated as provided herein, e.g., a cancer or a tumor listed herein. In some embodiments, a subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and / or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing the disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms of a disease, disorder, or condition. In some embodiments, a subject does not display a particular symptom (e.g., clinical manifestation of disease) or characteristic of a disease, disorder, or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0135] Substantially purified: As used herein, the term “substantially purified”, for example as applied to a cell, refers to a cell that is essentially free of other cell types. Asubstantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0136] Target: As used herein, the term “target” refers to a cell, tissue, organ, or site within the body that is the subject of provided methods, systems, and / or compositions, for example, a cell, tissue, organ or site within a body that is in need of treatment or is preferentially bound by, for example, an antibody (or fragment thereof), or a CAR.

[0137] Target site: As used herein, the term “target site” or “target sequence” refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.

[0138] T cell receptor: As used herein, the term “T cell receptor” or “TCR” refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen. A TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. A TCR comprises a heterodimer of an alpha (a) and beta (p) chain, although in some cells the TCR comprises gamma and delta (y / 8) chains. TCRs may exist in alpha / beta and gamma / delta forms, which arc structurally similar but have distinct anatomical locations and functions. Each chain comprises two extracellular domains, a variable and constant domain. In some embodiments, a TCR may be modified on any cell comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta 1’ cell.

[0139] Therapeutic: As used herein, the term “therapeutic” refers to a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0140] Transfected: As used herein, the term “transfected” or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has beentransfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0141] Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to partial or complete alleviation, amelioration, delay of onset of, inhibition, prevention, relief, and / or reduction in incidence and / or severity of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who does not exhibit signs or features of a disease, disorder, and / or condition (e.g., may be prophylactic). In some embodiments, treatment may be administered to a subject who exhibits only early or mild signs or features of the disease, disorder, and / or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who exhibits established, severe, and / or late-stage signs of the disease, disorder, or condition. In some embodiments, treating may comprise administering to an immune cell (e.g., a monocyte, macrophage, or dendritic cell) or contacting an immune cell with a modulator of a pathway activated by in vitro transcribed mRNA.

[0142] Tumor: As used herein, the term “tumor” refers to an abnormal growth of cells or tissue. In some embodiments, a tumor may comprise cells that are precancerous e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. In some embodiments, a tumor is associated with, or is a manifestation of, a cancer. In some embodiments, a tumor may be a disperse tumor or a liquid tumor. In some embodiments, a tumor may be a solid tumor.

[0143] Vector: As used herein, the term “vector” refers to a composition of matter that comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno- associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0144] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.DETAILED DESCRIPTION

[0145] The present disclosure encompasses, among other things, nucleic acid constructs comprising a promoter, a gene of interest, a 3’ untranslated region (UTR), and one or more introns, wherein the one or more introns comprise one or more inhibitory nucleic acids, wherein the one or more inhibitory nucleic acids encode one or more inhibitory RNAs, and, wherein the gene of interest encodes a protein of the present disclosure (e.g., a chimeric antigen receptor (CAR)) and methods of using and making immune cells comprising said nucleic acid constructs. The present disclosure also encompasses, among other things, compositions comprising modified immune cells (e.g., stem cells, macrophages, monocytes, and / or dendritic cells) comprising nucleic acid constructs as described herein and methods of using and producing the same.

[0146] Compositions and methods of the present disclosure allow for a one-step production of modified immune cells by delivering nucleic acid constructs comprising CARs to immune cells, wherein the nucleic acid constructs comprise one or more introns and the one or more introns comprise one or more inhibitory nucleic acids that encode one or more inhibitory RNAs.

[0147] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a nucleic acid construct comprising one or more introns comprising one or more inhibitory nucleic acids and encoding a chimeric antigen receptor (CAR) do not exhibit reduced viability relative to immune cells of the same type without a CAR. In some embodiments, modified immune cells (e.g., stem cells, macrophages,monocytes, or dendritic cells) comprising a nucleic acid construct comprising one or more introns comprising one or more inhibitory nucleic acids and encoding a CAR do not exhibit reduced viability relative to immune cells of the same type comprising a nucleic acid construct encoding a CAR, but without an intron as described herein. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a nucleic acid construct comprising one or more introns comprising one or more inhibitory nucleic acids and encoding a CAR do not exhibit reduced viability relative to immune cells of the same type comprising a nucleic acid construct encoding a CAR, but without an inhibitory nucleic acid as described herein.

[0148] In some embodiments, a modified immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) comprising a nucleic acid construct comprising one or more introns comprising one or more inhibitory nucleic acids and encoding a CAR expresses the CAR on the surface of the modified immune cell. In some embodiments, a modified immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) comprising a nucleic acid construct comprising one or more introns comprising one or more inhibitory nucleic acids and encoding a CAR expresses the CAR on the surface of the modified immune cell and expresses one or more inhibitory RNAs encoded by the one or more inhibitory nucleic acids. In some embodiments, a modified immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) comprising a nucleic acid construct comprising one or more introns comprising one or more inhibitory nucleic acids and encoding a CAR expresses the CAR on the surface of the modified immune cell and exhibits decreased expression of one or more target gene transcripts targeted by the one or more inhibitory nucleic acids.

[0149] In some embodiments, a modified immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) comprising a nucleic acid construct comprising one or more introns comprising one or more inhibitory nucleic acids and encoding a CAR exhibits increased pro- inflammatory (Ml) polarization relative to an immune cell of the same type that does not comprise a nucleic acid construct encoding a CAR. In some embodiments, a modified immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) comprising a nucleic acid construct comprising one or more introns comprising one or more inhibitory nucleic acids and encoding a CAR exhibits increased pro-inflammatory (Ml) polarization relative to an immune cell of thesame type comprising a nucleic acid construct encoding a CAR, but without an intron as described herein. In some embodiments, a modified immune cell (c.g., stem cell, macrophage, monocyte, or dendritic cell) comprising a nucleic acid construct comprising one or more introns comprising one or more inhibitory nucleic acids and encoding a CAR exhibits increased pro- inflammatory (Ml) polarization relative to an immune cell of the same type comprising a nucleic acid construct encoding a CAR, but without an inhibitory nucleic acid as described herein.

[0150] In some embodiments, a modified immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) comprising a nucleic acid construct comprising one or more introns comprising one or more inhibitory nucleic acids and encoding a CAR exhibits decreased antiinflammatory (M2) polarization relative to an immune cell of the same type that does not comprise a nucleic acid construct encoding a CAR. In some embodiments, a modified immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) comprising a nucleic acid construct comprising one or more introns comprising one or more inhibitory nucleic acids and encoding a CAR exhibits decreased anti-inflammatory (M2) polarization relative to an immune cell of the same type comprising a nucleic acid construct encoding a CAR, but without an intron as described herein. In some embodiments, a modified immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) comprising a nucleic acid construct comprising one or more introns comprising one or more inhibitory nucleic acids and encoding a CAR exhibits decreased antiinflammatory (M2) polarization relative to an immune cell of the same type comprising a nucleic acid construct encoding a CAR, but without an inhibitory nucleic acid as described herein.

[0151] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a nucleic acid construct comprising one or more introns comprising one or more inhibitory nucleic acids and encoding a CAR exhibit enhanced killing function relative to immune cells of the same type that do not comprise a nucleic acid construct encoding a CAR. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a nucleic acid construct comprising one or more introns comprising one or more inhibitory nucleic acids and encoding a CAR exhibit enhanced killing function relative to immune cells of the same type comprising a nucleic acid construct encoding a CAR, but without an intron as described herein. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprisinga nucleic acid construct comprising one or more introns comprising one or more inhibitory nucleic acids and encoding a CAR exhibit enhanced killing function relative to immune cells of the same type comprising a nucleic acid construct encoding a CAR, but without an inhibitory nucleic acid as described herein.

[0152] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a nucleic acid construct comprising one or more introns comprising one or more inhibitory nucleic acids and encoding a CAR exhibit increased secretion of cytokines (e.g., TNFa, IL-8, IL-6) relative to immune cells of the same type that do not comprise a nucleic acid construct encoding a CAR. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a nucleic acid construct comprising one or more introns comprising one or more inhibitory nucleic acids and encoding a CAR exhibit increased secretion of cytokines (e.g., TNFa, IL-8, IL-6) relative to immune cells of the same type comprising a nucleic acid construct encoding a CAR, but without an intron as described herein. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a nucleic acid construct comprising one or more introns comprising one or more inhibitory nucleic acids and encoding a CAR exhibit increased secretion of cytokines (e.g., TNFa, IL-8, IL-6) relative to immune cells of the same type comprising a nucleic acid construct encoding a CAR, but without an inhibitory nucleic acid as described herein.Inhibitory Nucleic Acids

[0153] Inhibitory nucleic acids of the present disclosure comprise any nucleic acids that can bind a target messenger RNA (mRNA). In some embodiments, an inhibitory nucleic acid of the present disclosure comprises RNA. In some embodiments, an inhibitory nucleic acid of the present disclosure comprises DNA. In some embodiments, an inhibitory nucleic acid of the present disclosure is a DNA / RNA hybrid. In some embodiments, an inhibitory nucleic acid of the present disclosure is an inhibitory RNA. In some embodiments, an inhibitory RNA silences expression of a target gene via RNA interference. In some embodiments, an inhibitory RNA is selected from the group consisting of antisense RNA (asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), guide RNA (gRNA), long noncoding RNA (IncRNA),microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), trans-acting siRNA (tasiRNA), and repeat associated siRNA (rasiRNA). In some embodiments, an inhibitory RNA is an shRNA.

[0154] In some embodiments, an shRNA of the present disclosure comprises a stem-loop structure. In some embodiments, an shRNA of the present disclosure comprises a stem-loop structure flanked by at least 10 bases on either side of the stem. In some embodiments, a stem is double-stranded RNA structure. In some embodiments, a stem is about 30-40 bases in length on each side. In some embodiments, a stem is about 30-39 bases in length on each side. In some embodiments, a stem is about 30-38 bases in length on each side. In some embodiments, a stem is about 30-37 bases in length on each side. In some embodiments, a stem is about 30-36 bases in length on each side. In some embodiments, a stem is about 30-35 bases in length on each side. In some embodiments, a stem is about 30-34 bases in length on each side. In some embodiments, a stem is about 30-33 bases in length on each side. In some embodiments, a stem is about 30-32 bases in length on each side. In some embodiments, a stem is about 30-31 bases in length on each side. In some embodiments, a stem is about 31-40 bases in length on each side. In some embodiments, a stem is about 32-40 bases in length on each side. In some embodiments, a stem is about 33-40 bases in length on each side. In some embodiments, a stem is about 34-40 bases in length on each side. In some embodiments, a stem is about 35-40 bases in length on each side. In some embodiments, a stem is about 36-40 bases in length on each side. In some embodiments, a stem is about 37-40 bases in length on each side. In some embodiments, a stem is about 38-40 bases in length on each side. In some embodiments, a stem is about 39-40 bases in length on each side. In some embodiments, a stem is 35 bases in length on each side. In some embodiments, an shRNA of the present disclosure comprises a scaffold. In some embodiments, an shRNA of the present disclosure comprises a 5’ scaffold. In some embodiments, an shRNA of the present disclosure comprises a 3’ scaffold. In some embodiments, an shRNA of the present disclosure comprises a guide strand. In some embodiments, an shRNA of the present disclosure comprises a passenger strand. In some embodiments, an shRNA of the present disclosure comprises a loop connecting the guide strand and the passenger strand. In some embodiments, a loop is about 8-20 bases in length. In some embodiments, a loop is about 8-19 bases in length. In some embodiments, a loop is about 8-18 bases in length. In some embodiments, a loop is about 8-17 bases in length. In someembodiments, a loop is about 8-16 bases in length. In some embodiments, a loop is about 8-15 bases in length. In some embodiments, a loop is about 8-14 bases in length. In some embodiments, a loop is about 8-13 bases in length. In some embodiments, a loop is about 8-12 bases in length. In some embodiments, a loop is about 8-11 bases in length. In some embodiments, a loop is about 8-10 bases in length. In some embodiments, a loop is about 8-9 bases in length. In some embodiments, a loop is about 9-20 bases in length. In some embodiments, a loop is about 10-20 bases in length. In some embodiments, a loop is about 11- 20 bases in length. In some embodiments, a loop is about 12-20 bases in length. In some embodiments, a loop is about 13-20 bases in length. In some embodiments, a loop is about 14- 20 bases in length. In some embodiments, a loop is about 15-20 bases in length. In some embodiments, a loop is about 16-20 bases in length. In some embodiments, a loop is about 17- 20 bases in length. In some embodiments, a loop is about 18-20 bases in length. In some embodiments, a loop is about 19-20 bases in length. In some embodiments, the sections of the stem closest to the loop comprises a guide strand and a passenger strand. In some embodiments, the sections of the stem furthest from the loop comprises a 5’ scaffold and a 3’ scaffold. In some embodiments, an shRNA of the present disclosure comprises, from 5’ to 3’, a 5’ scaffold, a guide strand, a loop, a passenger strand, and a 3’ scaffold. In some embodiments, an shRNA of the present disclosure comprises, from 5’ to 3’, a 5’ scaffold, a passenger strand, a loop, a guide strand, and a 3’ scaffold.

[0155] In some embodiments, a scaffold of the present disclosure comprises an miRNA scaffold. In some embodiments, an miRNA scaffold comprises an miRNA- 155 5’ scaffold, an miRNA- 155 3’ scaffold, an miRNA-30 5’ scaffold, an miRNA-30 3’ scaffold, an miRNA- 16 5’ scaffold, an miRNA-16 3’ scaffold, an miRNA-125 5’ scaffold, an miRNA-125 3’ scaffold, an miRNA-223 5’ scaffold, or an miRNA-223 3’ scaffold.

[0156] In some embodiments, a guide strand of the present disclosure is about 19-24 bases in length. In some embodiments, a guide strand of the present disclosure is 19-24 bases in length. In some embodiments, a guide strand is 18 bases in length. In some embodiments, a guide strand is 19 bases in length. In some embodiments, a guide strand is 20 bases in length. In some embodiments, a guide strand is 21 bases in length. In some embodiments, a guide strand is 22 bases in length. In some embodiments, a guide strand is 23 bases in length. In someembodiments, a guide strand is 24 bases in length. In some embodiments, a guide strand is 25 bases in length.

[0157] In some embodiments, a passenger strand of the present disclosure is about 17-24 bases in length. In some embodiments, a passenger strand of the present disclosure is about 17- 22 bases in length. In some embodiments, a passenger strand of the present disclosure is 17-22 bases in length. In some embodiments, a passenger strand is 17 bases in length. In some embodiments, a passenger strand is 18 bases in length. In some embodiments, a passenger strand is 19 bases in length. In some embodiments, a passenger strand is 20 bases in length. In some embodiments, a passenger strand is 21 bases in length. In some embodiments, a passenger strand is 22 bases in length. In some embodiments, a passenger strand is 23 bases in length. In some embodiments, a passenger strand is 22 bases in length. In some embodiments, a passenger strand is 24 bases in length. In some embodiments, a passenger strand is 1-2 bases shorter than a corresponding guide strand. In some embodiments, a passenger strand is 1 base shorter than a corresponding guide strand. In some embodiments, a passenger strand is 2 bases shorter than a corresponding guide strand. In some embodiments, a passenger strand is the same length as a corresponding guide strand.

[0158] In some embodiments, a guide strand of the present disclosure has a G / C content of about 36%-50%. In some embodiments, a guide strand of the present disclosure has a G / C content of 36%-50%. In some embodiments, a guide strand has a G / C content of 35%. In some embodiments, a guide strand has a G / C content of 36%. In some embodiments, a guide strand has a G / C content of 37%. In some embodiments, a guide strand has a G / C content of 38%. In some embodiments, a guide strand has a G / C content of 39%. In some embodiments, a guide strand has a G / C content of 40%. In some embodiments, a guide strand has a G / C content of 41%. In some embodiments, a guide strand has a G / C content of 42%. In some embodiments, a guide strand has a G / C content of 43%. In some embodiments, a guide strand has a G / C content of 44%. In some embodiments, a guide strand has a G / C content of 45%. In some embodiments, a guide strand has a G / C content of 46%. In some embodiments, a guide strand has a G / C content of 47%. In some embodiments, a guide strand has a G / C content of 48%. In some embodiments, a guide strand has a G / C content of 49%. In some embodiments, a guide strand has a G / C content of 50%. In some embodiments, a guide strand has a G / C content of 51%.

[0159] In some embodiments, a passenger strand of the present disclosure has a G / C content of about 36%-50%. In some embodiments, a passenger strand of the present disclosure has a G / C content of 36%-50%. In some embodiments, a passenger strand has a G / C content of 35%. In some embodiments, a passenger strand has a G / C content of 36%. In some embodiments, a passenger strand has a G / C content of 37%. In some embodiments, a passenger strand has a G / C content of 38%. In some embodiments, a passenger strand has a G / C content of 39%. In some embodiments, a passenger strand has a G / C content of 40%. In some embodiments, a passenger strand has a G / C content of 41%. In some embodiments, a passenger strand has a G / C content of 42%. In some embodiments, a passenger strand has a G / C content of 43%. In some embodiments, a passenger strand has a G / C content of 44%. In some embodiments, a passenger strand has a G / C content of 45%. In some embodiments, a passenger strand has a G / C content of 46%. In some embodiments, a passenger strand has a G / C content of 47%. In some embodiments, a passenger strand has a G / C content of 48%. In some embodiments, a passenger strand has a G / C content of 49%. In some embodiments, a passenger strand has a G / C content of 50%. In some embodiments, a passenger strand has a G / C content of 51%.

[0160] In some embodiments, a stemloop formed by a 5' scaffold, a guide strand, a passenger strand, and a 3' scaffold is recognized by Drosha. In some embodiments, Drosha cleaves 10-15 bases above the base of a stemloop. In some embodiments, a Drosha-cleaved stemloop will be recognized by Dicer. In some embodiments, Dicer cleaves 20-24 bases from the 5' and 3' ends of the stemloop.

[0161] In some embodiments, a guide strand of the present disclosure comprises a nucleic acid sequence that is reverse complementary to a target gene transcript comprising a target nucleic acid sequence. In some embodiments, a passenger strand of the present disclosure comprises a nucleic acid sequence that is not fully complementary to a corresponding guide strand. In some embodiments, a passenger strand comprises a nucleic acid sequence that is not fully complementary to a corresponding guide strand by one nucleotide. In some embodiments, a passenger strand of the present disclosure comprises a nucleic acid sequence that is not fully complementary to a corresponding guide strand by two nucleotides. In some embodiments, a passenger strand of the present disclosure comprises a nucleic acid sequence that is not fullycomplementary to a corresponding guide strand by three nucleotides. Tn some embodiments, a passenger strand of the present disclosure comprises a nucleic acid sequence that is not fully complementary to a corresponding guide strand by four nucleotides.

[0162] In some embodiments, an shRNA of the present disclosure comprises: (a) a nucleic acid sequence selected from Table la, Table lb, Table 1c, Table Id, or Table le; (b) a nucleic acid sequence that differs from a sequence selected from Table la, Table lb, Table 1c, Table Id, or Table le by no more than 5 substitutions, additions, or deletions; or (c) a nucleic acid sequence that is at least 80% identical to a sequence selected from Table la, Table lb, Table 1c, Table Id, or Table le. In some embodiments, an shRNA of the present disclosure comprises a nucleic acid sequence selected from Table la, Table lb, Table 1c, Table Id, or Table le. In some embodiments, an shRNA of the present disclosure comprises a nucleic acid sequence that differs from a sequence selected from Table la, Table lb, Table 1c, Table Id, or Table le by no more than 5 substitutions, additions, or deletions. In some embodiments, an shRNA of the present disclosure comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from Table la, Table lb, Table 1c, Table Id, or Table le.

[0163] In some embodiments, an inhibitory RNA of the present disclosure is or comprises an miRNA. In some embodiments, an miRNA of the present disclosure comprises: (a) a nucleic acid sequence selected from Table 2; (b) a nucleic acid sequence that differs from a sequence selected from Table 2 by no more than 5 substitutions, additions, or deletions; or (c) a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 2. In some embodiments, an miRNA of the present disclosure comprises a nucleic acid sequence selected from Table 2. In some embodiments, an miRNA of the present disclosure comprises a nucleic acid sequence that differs from a sequence selected from Table 2 by no more than 5 substitutions, additions, or deletions. In some embodiments, an miRNA of the present disclosure comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 2.Targets of Inhibitory Nucleic Acids

[0164] In some embodiments, inhibitory nucleic acids of the present disclosure regulate gene expression in modified immune cells of the present disclosure via RNA interference(RNAi). In some embodiments, inhibitory nucleic acids of the present disclosure target mRNA comprising a complementary sequence. In some embodiments, inhibitory nucleic acids of the present disclosure induce degradation of target mRNA. In some embodiments, inhibitory nucleic acids of the present disclosure repress translation of target mRNA.

[0165] In some embodiments, a target gene transcript is mammalian. In some embodiments, a target gene transcript is human. In some embodiments, contacting a target gene transcript with one or more inhibitory nucleic acids (e.g., RNAs) reduces translation of the target gene transcript. In some embodiments, a target gene transcript is expressed in an immune cell. In some embodiments, an immune cell is a stem cell, macrophage, monocyte, or dendritic cell. In some embodiments, a target gene transcript is expressed in a macrophage. In some embodiments, a target gene transcript is expressed in a monocyte. In some embodiments, reduced translation of a target gene transcript is associated with an Ml phenotype. In some embodiments, reduced translation of a target gene transcript is associated with an M2 phenotype. In some embodiments, increased expression of an inhibitory nucleic acid (e.g., RNA) of the present disclosure is associated with an MI phenotype. In some embodiments, increased expression of an inhibitory nucleic acid (e.g., RNA) of the present disclosure is associated with an M2 phenotype.

[0166] In some embodiments, a target gene transcript of the present disclosure comprises: (a) a target nucleic acid sequence selected from Table 3; (b) a target nucleic acid sequence that differs from a sequence selected from Table 3 by no more than 5 substitutions, additions, or deletions; or (c) a target nucleic acid sequence that is at least 80% identical to a sequence selected from Table 3. In some embodiments, a target gene transcript of the present disclosure comprises a target nucleic acid sequence selected from Table 3. In some embodiments, a target gene transcript of the present disclosure comprises a target nucleic acid sequence that differs from a sequence selected from Table 3 by no more than 5 substitutions, additions, or deletions. In some embodiments, a target gene transcript of the present disclosure comprises a target nucleic acid sequence that is at least 80% identical to a sequence selected from Table 3.

[0167] In some embodiments, a target gene transcript of the present disclosure encodes human ATG7, C / EBP-alpha, C / EBP-beta, CD36, CLEC1A, FATS, GOLM1, HAVCR2, ITGAD,KLF4, KLF6, LILRB 1 , LILRB2, LILRB4, MAF, MafB, PD-L1 , PIK3CG, PIK3CG, PPARa, PPARy, PTGS2, SIGLEC7, SIGLEC10, SIRPa, SLC15A3, STAT3, STAT6, TNFRSF1B, TOX, TREM2, YTHDF2, or ZFP36. In some embodiments, a target gene transcript of the present disclosure encodes human SIRPa. In some embodiments, a target gene transcript of the present disclosure encodes human PD-1 or PD-L1.

[0168] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises one or more inhibitory nucleic acids. In some embodiments, one or more inhibitory nucleic acids comprise one inhibitory nucleic acid. In some embodiments, one or more inhibitory nucleic acids comprise at least two, three, four or five inhibitory nucleic acids. In some embodiments, one or more inhibitory nucleic acids comprise two inhibitory nucleic acids. In some embodiments, one or more inhibitory nucleic acids comprise three inhibitory nucleic acids. In some embodiments, one or more inhibitory nucleic acids comprise four inhibitory nucleic acids. In some embodiments, one or more inhibitory nucleic acids comprise five inhibitory nucleic acids.

[0169] In some embodiments, at least two, three, four, or five inhibitory nucleic acids are in tandem. In some embodiments, at least two, three, four, or five inhibitory nucleic acids comprise identical sequences. In some embodiments, at least two, three, four, or five inhibitory nucleic acids comprise at least two different sequences. In some embodiments, at least three, four, or five inhibitory nucleic acids comprise at least three different sequences. In some embodiments, at least four or five inhibitory nucleic acids comprise at least four different sequences. In some embodiments, at least five inhibitory nucleic acids comprise at least five different sequences. In some embodiments, at least two, three, four, or five inhibitory nucleic acids encode inhibitory RNAs comprising nucleic acid sequences that are reverse complementary to the same target gene transcript. In some embodiments, at least two, three, four, or five inhibitory nucleic acids encode inhibitory RNAs comprising nucleic acid sequences that are reverse complementary to different target gene transcripts.

[0170] In some embodiments, at least two, three, four, or five inhibitory nucleic acids encode inhibitory RNAs comprising identical miRNA scaffolds. In some embodiments, at least two, three, four, or five inhibitory nucleic acids encode inhibitory RNAs comprising at least two different miRNA scaffolds. In some embodiments, at least three, four, or five inhibitory nucleicacids encode inhibitory RNAs comprising at least three different miRNA scaffolds. In some embodiments, at least four or five inhibitory nucleic acids encode inhibitory RNAs comprising at least four different miRNA scaffolds. In some embodiments, at least five inhibitory nucleic acids encode inhibitory RNAs comprising at least five different miRNA scaffolds. In some embodiments, at least two different miRNA scaffolds are selected from the group consisting of an miRNA-155 5’ scaffold, an miRNA-155 3’ scaffold, an miRNA-30 5’ scaffold, and an miRNA-303’ scaffold.Methods of Designing Inhibitory Nucleic Acids

[0171] The present disclosure also provides methods of designing inhibitory nucleic acids of the present disclosure. In some embodiments, the present disclosure provides methods of designing inhibitory RNA of the present disclosure. In some embodiments, the present disclosure provides methods of designing shRNA of the present disclosure. In some embodiments, methods of designing an shRNA comprising a guide strand, a passenger strand, and a loop, comprise the steps of: (a) selecting a target region of a target gene transcript, (b) designing the guide strand, and (c) designing the passenger strand. In some embodiments, a guide strand is a reverse complement of a target region. In some embodiments, a guide strand comprises, from 5’ to 3’, an adenine or uracil at position 1, an adenine, guanine, or cytosine at position 10, and a G / C content between 36% and 45% that is increases from 5’ to 3’. In some embodiments, a guide strand comprises a higher G / C content of the 10 bases at the 3’ end of the guide strand relative to the G / C content of the 10 bases at the 5’ end of the guide strand. In some embodiments, a passenger strand comprises the sequence of a target region, from 5’ to 3’, with the following variations: (i) deletion of a nucleotide at position 7 relative to the loop, (ii) deletion of a nucleotide at position 11 relative to the loop, and (iii) mutation of an adenine to guanine and / or mutation of a cytosine to uracil near the deletions at positions 7 and 11, wherein the mutations create two partial G-U base pairs between the passenger strand and the guide strand. In some embodiments, a passenger strand is 2 base pairs shorter than a guide strand. When nucleotide positions are counted relative to a loop, position 1 is nearest the loop and position counts increase moving away from the loop.

[0172] In some embodiments, methods of designing an shRNA comprising a guide strand, a passenger strand, and a loop, comprise the steps of: (a) selecting a target region of a target gene transcript, (b) designing the guide strand, wherein the guide strand is a reverse complement of the target region, and wherein the guide strand comprises, from 5’ to 3’, an adenine or uracil at position 1, an adenine, guanine, or cytosine at position 10, and a G / C content between 36% and 45% that is increases from 5’ to 3’; and (c) designing the passenger strand, wherein the passenger strand comprises the sequence of the target region, from 5’ to 3’, with the following variations: (i) deletion of a nucleotide at position 7 relative to the loop, (ii) deletion of a nucleotide at position 11 relative to the loop, (iii) mutating an adenine to guanine and / or mutating a cytosine to uracil near the deletions at positions 7 and 11, wherein the mutations create two partial G-U base pairs between the passenger strand and the guide strand; and wherein the passenger strand is 2 base pairs shorter than the guide strand.

[0173] In some embodiments, methods of designing an shRNA comprise a target region of a target gene transcript that is 21-22 base pairs in length.Nucleic Acid Constructs

[0174] In some embodiments, the present disclosure provides nucleic acid molecules comprising a promoter, a gene of interest, a 3’ untranslated region (UTR), and one or more introns. In some embodiments, one or more introns of the present disclosure comprise one or more inhibitory nucleic acids of the present disclosure. In some embodiments, one or more inhibitory nucleic acids of the present disclosure encode one or more inhibitory RNAs of the present disclosure. In some embodiments, a gene of interest encodes at least one chimeric antigen receptor (CAR) described herein or a fragment thereof. A modified immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure can comprise a nucleic acid molecule (e.g., an exogenous nucleic acid molecule) comprising one or more introns of the present disclosure and encoding at least one protein (e.g., a CAR of the present disclosure) described herein. In some embodiments, the present disclosure provides nucleic acid molecules wherein expression of a CAR is increased relative to a reference nucleic acid molecule that lacks one or more introns.

[0175] In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence at least 80% identical to a sequence selected from Table 4. In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence at least 85% identical to a sequence selected from Table 4. In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence at least 90% identical to a sequence selected from Table 4. In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence at least 95% identical to a sequence selected from Table 4. In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence at least 96% identical to a sequence selected from Table 4. In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence at least 97% identical to a sequence selected from Table 4. In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence at least 98% identical to a sequence selected from Table 4. In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence at least 99% identical to a sequence selected from Table 4. In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence identical to a sequence selected from Table 4.Introns

[0176] In some embodiments, one or more introns of the present disclosure comprise one, two, or three introns. In some embodiments, one or more introns of the present disclosure are located downstream of a promoter. In some embodiments, one or more introns of the present disclosure are located within a gene of interest. In some embodiments, one or more introns of the present disclosure are located within a 3’ UTR. In some embodiments, one or more introns of the present disclosure are located within a gene of interest and within a 3’ UTR. In some embodiments, one or more introns of the present disclosure comprise three inhibitory nucleic acids. In some embodiments, three inhibitory nucleic acids encode three inhibitory RNAs. In some embodiments, three inhibitory RNAs comprise the following miRNA scaffolds in order from 5’ to 3’: (a) miRNA 30, miRNA 30, miRNA 30, (b) miRNA 30, miRNA 155, miRNA 30, (c) miRNA 155, miRNA 30, miRNA 155, or (d) miRNA 155, miRNA 155, miRNA 155.

[0177] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that arc degenerate versions of each other and that encode the same amino acid sequence. The term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0178] The term “operably linked” or “transcriptional control” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.

[0179] Nucleic acid molecules encoding at least one protein (e.g., a CAR of the present disclosure) described herein or a fragment thereof can be a DNA molecule, an RNA molecule, or a combination thereof. In some embodiments, a nucleic acid molecule comprises or is a messenger RNA (mRNA) transcript encoding at least one protein (e.g., a CAR of the present disclosure) described herein or a fragment thereof. In some embodiments, a nucleic acid molecule comprises or is a DNA construct encoding at least one protein (e.g., a CAR of the present disclosure) described herein or a fragment thereof.

[0180] In some embodiments, all or a fragment of a protein (e.g., a CAR of the present disclosure) described herein is encoded by a codon optimized nucleic acid molecule, e.g., forexpression in a cell (e.g., a mammalian cell). A variety of codon optimization methods are known in the art, e.g., as disclosed in US Patent Nos. 5,786,464 and 6,114,148, each of which is hereby incorporated by reference in its entirety.

[0181] Expression of nucleic acids as described herein may be achieved by operably linking a nucleic acid encoding a protein (e.g., a CAR of the present disclosure) or fragment thereof to a promoter in an expression vector. Exemplary promoters (e.g., constitutive promoters) include, but are not limited to, an elongation factor- 1 a promoter (EF-1 a) promoter, immediate early cytomegalovirus (CMV) promoter, ubiquitin C promoter, phosphoglycerokinase (PGK) promoter, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Moloney murine leukemia virus (MoMuLV) promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, or a creatine kinase promoter. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter. A vector can also comprise additional promoter elements, e.g., enhancers, to regulate the frequency of transcriptional initiation.

[0182] In some embodiments, a vector comprising a nucleic acid molecule encoding a protein (e.g., a CAR of the present disclosure) or fragment thereof comprises or is a viral vector. Viral vector technology is well known in the art and is described (e.g., in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor- Press, NY). Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno- associated viral vectors, or retroviral vectors (e.g., a lentiviral vector or a gammaretroviral vector). In some embodiments, a vector comprises a lentiviral vector (e.g., as described in US Patent No. 9,149,519 or International Publication No. WO 2017 / 044487, each of which is hereby incorporated by reference in its entirety).

[0183] In some embodiments, a viral vector comprises an adenoviral vector. Adenoviruses are a large family of viruses containing double stranded DNA. They replicate within the nucleus of a host cell, using the host’s cell machinery to synthesize viral RNA, DNA and proteins. Adenoviruses are known in the art to affect both replicating and non-replicating cells, to accommodate large transgenes, and to code for proteins without integrating into the hostcell genome. In some embodiments, an adenoviral vector comprises an Ad2 vector or an Ad5 vector (c.g., Ad5f35 adenoviral vector, c.g., a helper-dependent Ad5F35 adenoviral vector).

[0184] In some embodiments, a viral vector is an adeno-associated virus (AAV) vector. AAV systems are generally well known in the art (see, e.g., Kelleher and Vos, Biotechniques, 17(6):1110-17 (1994); Cotten et al., P.N.A.S. U.S.A., 89(13):6094-98 (1992); Curiel, Nat Immun, 13(2-3): 141-64 (1994); Muzyczka, Curr Top Microbiol Immunol, 158:97-129 (1992); and Asokan A, et al., Mol. Ther., 20(4):699-708 (2012)). Methods for generating and using recombinant AAV (rAAV) vectors are described, for example, in U.S. Pat. Nos. 5,139,941 and 4,797,368.

[0185] Several AAV serotypes have been characterized, including AAV1, AAV2, AAV3 (e.g., AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, as well as variants thereof. Generally, any AAV serotype may be used to deliver a protein (e.g., a CAR of the present disclosure) or fragment thereof described herein. In some embodiments, an AAV serotype has a tropism for a particular tissue.

[0186] In some embodiments, CRISPR / Cas9 system has recently been shown to facilitate high levels of precise genome editing using adeno associated viral (AAV) vectors to serve as donor template DNA during homologous recombination (HR).

[0187] In some embodiments, a vector comprises a gammaretroviral vector (e.g., as described in Tobias Maetzig et al., “Gammaretroviral Vectors: Biology, Technology and Application” Viruses. 2011 Jun; 3(6): 677-713, which is hereby incorporated by reference in its entirety). Exemplary gammaretroviral vectors include Murine Leukemia Virus (MLV), Spleen- Focus Forming Virus (SFFV), and Myeloproliferative Sarcoma Virus (MPSV), and vectors derived therefrom.

[0188] In some embodiments, a vector comprises two or more nucleic acid sequences encoding proteins, e.g., at least one CAR described herein, and a second CAR, e.g., a different CAR described herein. In some embodiments, two or more nucleic acid sequences encoding a CAR and a second CAR are encoded by a single nucleic molecule, e.g., in same frame and as a single polypeptide chain. In some embodiments, two or more CARs are separated by one or more cleavage peptide sites (e.g., an auto-cleavage site or a substrate for an intracellular protease). In certain embodiments, a cleavage peptide comprises a porcine teschovirus-1 (P2A)peptide, Thosea asigna vims (T2A) peptide, equine rhinitis A virus (E2A) peptide, foot-and- mouth disease virus (F2A) peptide, or a variant thereof.

[0189] In some embodiments, a vector comprises at least one nucleic acid sequence encoding a protein, e.g., at least one CAR described herein, and at least one nucleic acid encoding at least one gene co-expressed with a second protein, e.g., a cytokine described herein (e.g., TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, and / or IL-1) or a stimulatory ligand described herein (e.g., CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, 1COS-L, ICAM, CD30L, CD40, CD40L, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor, and / or a B7-H3 ligand).

[0190] In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 8. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that is at least 85% identical to a sequence selected from Table 8. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that is at least 90% identical to a sequence selected from Table 8. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that is at least 95% identical to a sequence selected from Table 8. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that is at least 96% identical to a sequence selected from Table 8. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that is at least 97% identical to a sequence selected from Table 8. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that is at least 98% identical to a sequence selected from Table 8. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that is at least 99% identical to a sequence selected from Table 8. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that is identical to a sequence selected from Table 8.

[0191] In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 8 by nomore than 100 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 8 by no more than 75 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 8 by no more than 50 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 8 by no more than 40 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 8 by no more than 30 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 8 by no more than 20 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 8 by no more than 10 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 8 by no more than 9 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 8 by no more than 8 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 8 by no more than 7 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 8 by no more than 6 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 8 by no more than 5 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 8 by no more than 4 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 8 by no more than 3 substitutions, additions, ordeletions. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 8 by no more than 2 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 8 by no more than 1 substitution, addition, or deletion. In some embodiments, a CAR of the present disclosure binds to HER2 and is encoded by a nucleic acid sequence that does not have any substitutions, additions, or deletions relative to a sequence selected from Table 8.

[0192] In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 9. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that is at least 85% identical to a sequence selected from Table 9. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that is at least 90% identical to a sequence selected from Table 9. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that is at least 95% identical to a sequence selected from Table 9. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that is at least 96% identical to a sequence selected from Table 9. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that is at least 97% identical to a sequence selected from Table 9. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that is at least 98% identical to a sequence selected from Table 9. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that is at least 99% identical to a sequence selected from Table 9. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that is identical to a sequence selected from Table 9.

[0193] In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 9 by no more than 50 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that differs from asequence selected from Table 9 by no more than 40 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 9 by no more than 30 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 9 by no more than 20 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 9 by no more than 10 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 9 by no more than 9 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 9 by no more than 8 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 9 by no more than 7 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 9 by no more than 6 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 9 by no more than 5 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 9 by no more than 4 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 9 by no more than 3 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 9 by no more than 2 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 9 by no more than 1 substitution, addition, or deletion. In some embodiments, a CAR antigen binding domain binds to HER2 and is encodedby a nucleic acid sequence that does not have any substitutions, additions, or deletions relative to a sequence selected from Table 9.Chimeric Antigen Receptors ( CAR )

[0194] The term “chimeric antigen receptor” or “CAR,” as used herein, refers to an artificial cell surface receptor that is engineered to be expressed on an immune effector cell and specifically targets a cell and / or binds an antigen. CARs may be used, for example, as a therapy with adoptive cell transfer. For example, in some embodiments, immune cells (e.g., stem cells, macrophages, monocytes, and / or dendritic cells) are removed from a patient (e.g., from blood, tumor or ascites fluid) and modified so that they express a receptor specific to a particular form of antigen. In some embodiments, such modified immune cells are then reintroduced to the same or a different subject as a therapeutic. In some embodiments, CARs have been expressed with specificity to an antigen, for example, a tumor associated antigen. In some embodiments, a CAR comprises an extracellular domain, a transmembrane domain and one or more intracellular moieties.

[0195] In some embodiments, a modified immune cell, for example, a modified stem cell, macrophage, monocyte, or dendritic cell, is generated by expressing a CAR therein. In some embodiments, an immune cell comprises a CAR comprising an extracellular domain, a transmembrane domain, and one or more intracellular moieties, wherein the immune cell comprises a stem cell, macrophage, monocyte, or dendritic cell.

[0196] In some embodiments, a CAR may further comprise one or more of: one or more extracellular leader domains, one or more extracellular hinge domains and one or more intracellular co-stimulatory domains.

[0197] In some embodiments, a CAR comprises a spacer domain or hinge between an extracellular domain and a transmembrane domain. In some embodiments, a CAR comprises a spacer domain or hinge between one or more intracellular moieties and a transmembrane domain. As used herein, the term “spacer domain” or “hinge” refers to any oligo- or polypeptide that functions to link a transmembrane domain to either an extracellular domain or to one or more intracellular moieties in a polypeptide chain. In some embodiments, a spacer domain orhinge may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. In some embodiments, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length, may form a linkage between a transmembrane domain and one or more intracellular moieties of a CAR. An example of a linker includes a glycine-serine doublet.

[0198] In some embodiments, an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR may comprise one or more control systems including, but not limited to: a safety switch (e.g., an on switch, and off switch, a suicide switch), a logic gate, for example an AND gate (e.g., two or more CARs, each of which lacks one or more signaling domains such that activation of both / all CARs is required for full immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) activation or function), an OR gate (e.g., two or more CARs, each with an intracellular domain such as CD3C and a co-stimulatory domain), and / or a NOT gate (e.g., two or more CARs, one of which includes an inhibitory domain that antagonizes the function of the other CAR[s]).

[0199] The present disclosure also provides immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a nucleic acid (e.g., a nucleic acid construct) encoding a CAR, wherein the nucleic acid comprises a nucleic acid sequence encoding an extracellular domain, a nucleic acid sequence encoding a transmembrane domain and a nucleic acid sequence encoding one or more intracellular moieties, wherein the nucleic acid also comprises a promoter, a 3’ UTR, and one or more introns, wherein the one or more introns comprise one or more inhibitory nucleic acids, and wherein the one or more inhibitory nucleic acids encode one or more inhibitory RNAs, and wherein the cell is a stem cell, macrophage, monocyte or dendritic cell that expresses the CAR and the one or more inhibitory RNAs.

[0200] In some embodiments, a CAR comprises an extracellular domain that is operably linked to another domain of the CAR, such as a transmembrane domain or one or more intracellular moieties, for expression in an immune cell. In some embodiments, a nucleic acid encoding an extracellular domain is operably linked to a nucleic acid encoding a transmembrane domain and the nucleic acid encoding the transmembrane domain is operably linked to a nucleic acid encoding one or more intracellular moieties.

[0201] In some embodiments, an effector activity of an immune cell comprising a CAR is directed against a target cell comprising an antigen that specifically binds an antigen binding domain of the CAR. In some embodiments, a targeted effector activity directed against a target cell is or comprises phagocytosis, targeted cellular cytotoxicity, antigen presentation, or cytokine secretion.

[0202] In some embodiments, a CAR described herein comprises at least one domain (e.g., an extracellular domain, a transmembrane domain, and / or an intracellular domain) that inhibits anti-phagocytic signaling in an immune cell described herein (e.g., a stem cell, macrophage, monocyte, or dendritic cell). In some embodiments, a CAR described herein improves effector activity of an immune cell described herein (e.g., a stem cell, macrophage, monocyte, or dendritic cell), e.g., by enhancing inhibition of CD47 and / or SIRPa activity. In some embodiments, a CAR described herein binds CD47, e.g., and serves as a dominant negative receptor, inhibiting SIRPa activity (e.g., a CD47 sink). In some embodiments, a CAR described herein that binds SIRPa, e.g., comprises an activating receptor (e.g., comprises a CD3z intracellular domain). In some embodiments, a CAR described herein inhibits at least one interaction of CD47 and SIRPa. In some embodiments, a CAR is or comprises a phagocytic logic gate.

[0203] In some embodiments, an immune cell described herein (e.g., comprising or expressing a CAR as described herein) comprises or expresses one or more inhibitory RNAs that target SIRPa transcripts. In some embodiments, an immune cell described herein (e.g., comprising or expressing a CAR as described herein) comprises or expresses at least one variant or fragment of: SIRPa (e.g., a dominant negative SIRPa or a high-affinity engineered variant of SIRPa (e.g., CV1)), 5F9 scFv, B6H12 scFv (e.g., a humanized B6H12 scFv), PD1 (e.g., a dominant negative PDI or HAC-I), anti-PDl scFv (e.g., E27 or durvalumab), Siglec-10, Siglec- 9, Siglec-11, and / or SHP-1. In some embodiments, a variant or fragment comprises a mutated intracellular domain. In some embodiments, an immune cell described herein (e.g., comprising or expressing a CAR as described herein) comprises a dominant negative receptor, e.g., blocking an inhibitory checkpoint.

[0204] In some embodiments, a CAR described herein further comprises a cleavage peptide (e.g., a P2A, F2A, E2A and / or T2A peptide) and at least one of a second CARcomprising at least one inhibitory domain of anti-phagocytic signaling. In some embodiments, at least one second CAR comprises a SIRPa (c.g., a high-affinity engineered variant of SIRPa (c.g., CV1)), 5F9 scFv, B6H12 scFv (e.g., a humanized B6H12 scFv), or a CD47 binding extracellular domain or a fragment thereof. In some embodiments, at least one second CAR comprises a SIRPa transmembrane domain or a fragment thereof. In certain embodiments, a second CAR further comprises a hinge domain (e.g., a CD8a hinge domain). In certain embodiments, at least one second CAR comprises: (i) a leader sequence (e.g., a CD8a leader); ii) an extracellular domain (e.g., a SIRPa, CV1, 5F9 scFv, or B6H12 scFv (e.g., a humanized B6H12 scFv) extracellular domain); and ii) a transmembrane domain (e.g., a SIRPa transmembrane domain). In some embodiments, a CAR described herein further comprises a cleavage peptide (e.g., a P2A peptide) and at least one marker protein (e.g., CD 20 or a fragment thereof, CD 19 or a fragment thereof, NGFR or a fragment thereof, a synthetic peptide, and / or a fluorescent protein).

[0205] In some embodiments, an immune cell described herein (e.g., comprising or expressing a CAR as described herein) comprises or expresses one or more phosphatase dead domains (e.g. a phosphatase dead Shpl, phosphatase dead 72-5ptase (INPP5E), phosphatase dead Shp2, and / or phosphatase dead SHIP-1 domain) and / or a constitutively active kinase domain (e.g., a constitutively active LYN domain). In some embodiments, a CAR described herein further comprises a cleavage peptide (e.g., a P2A, F2A, E2A and / or T2A peptide) and one or more phosphatase dead domains (e.g. a phosphatase dead Shpl, phosphatase dead 72-5ptase (INPP5E), phosphatase dead Shp2, and / or phosphatase dead SHIP-1 domain) and / or a constitutively active kinase domain (e.g., a constitutively active LYN domain).

[0206] In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 80% identical to a sequence selected from Table 4. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 85% identical to a sequence selected from Table 4. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 90% identical to a sequence selected from Table 4. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 95% identical to a sequence selected from Table 4. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 96% identical to a sequence selected from Table 4. In some embodiments, a CAR of the present disclosure is encoded by anucleic acid sequence at least 97% identical to a sequence selected from Table 4. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 98% identical to a sequence selected from Table 4. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 99% identical to a sequence selected from Table 4. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence identical to a sequence selected from Table 4.

[0207] In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 80% identical to a sequence selected from Table 8. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 85% identical to a sequence selected from Table 8. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 90% identical to a sequence selected from Table 8. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 95% identical to a sequence selected from Table 8. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 96% identical to a sequence selected from Table 8. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 97% identical to a sequence selected from Table 8. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 98% identical to a sequence selected from Table 8. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 99% identical to a sequence selected from Table 8. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence identical to a sequence selected from Table 8.

[0208] In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 80% identical to a sequence selected from Table 5. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 85% identical to a sequence selected from Table 5. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 90% identical to a sequence selected from Table 5. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 95% identical to a sequence selected from Table 5. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 96% identical to a sequence selected from Table 5. In some embodiments, a CAR of the present disclosure comprises an amino acidsequence at least 97% identical to a sequence selected from Table 5. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 98% identical to a sequence selected from Table 5. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 99% identical to a sequence selected from Table 5. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence identical to a sequence selected from Table 5.

[0209] In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 80% identical to a sequence selected from Table 6. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 85% identical to a sequence selected from Table 6. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 90% identical to a sequence selected from Table 6. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 95% identical to a sequence selected from Table 6. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 96% identical to a sequence selected from Table 6. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 97% identical to a sequence selected from Table 6. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 98% identical to a sequence selected from Table 6. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 99% identical to a sequence selected from Table 6. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence identical to a sequence selected from Table 6.CAR Extracellular Domains

[0210] The present disclosure provides chimeric antigen receptors (CAR) comprising one or more extracellular domains. In some embodiments, a CAR extracellular domain comprises an Fc receptor (FcR) extracellular domain. In some embodiments, a CAR extracellular domain comprises a toll-like receptor (TLR) extracellular domain. In some embodiments, a CAR extracellular domain comprises a leader domain. In some embodiments, a CAR extracellular domain comprises an antigen binding domain. In some embodiments, a CAR extracellular domain comprises a hinge domain. In some embodiments, a CAR extracellular domaincomprises one or more of an FcR extracellular domain, a TLR extracellular domain, a leader domain, an antigen binding domain and a hinge domain. In some embodiments, a CAR extracellular domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a CAR extracellular domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).FcR Extracellular Domains

[0211] In some embodiments, an FcR extracellular domain comprises a full-length FcR extracellular domain. In some embodiments, an FcR extracellular domain comprises a portion of a full-length FcR extracellular domain. In some embodiments, an FcR extracellular domain (or portion thereof) is or comprises a human FcR extracellular domain. In some embodiments, an FcR extracellular domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an FcR extracellular domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an FcR extracellular’ domain comprises a CD64 (FcyRl), CD32a (FcyRIla), CD32b (FcyRllb), CD32c, CD16a (FcyRIlIa), CD16b (FcyRIIIb), FcsRI, FcsRII, or FcaRI (CD89) domain.TLR Extracellular Domains

[0212] In some embodiments, a TLR extracellular domain comprises a full-length TLR extracellular domain. In some embodiments, a TLR extracellular domain comprises a portion of a full-length TLR extracellular domain. In some embodiments, a TLR extracellular domain (or portion thereof) is or comprises a human TLR extracellular domain. In some embodiments, a TLR extracellular domain may be a domain that is endogenous to a particular’ immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a TLR extracellular domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a TLR extracellular domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.Leader Domains

[0213] In some embodiments, a CAR comprises one or more extracellular leader domains. In some embodiments, a nucleic acid encoding a CAR comprises a nucleic acid sequence encoding an extracellular leader domain, but the extracellular leader domain is cleaved from the CAR before the CAR is expressed in an immune cell. In some embodiments, an extracellular leader domain is or comprises a human extracellular leader domain. In some embodiments, an extracellular leader domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an extracellular’ leader domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an extracellular leader domain comprises a CD8a extracellular leader domain. In some embodiments, an extracellular leader domain comprises a leader domain from a stimulatory or co-stimulatory domain (e.g., a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD19, CD20, 41BB, CD28, 0X40, GITR, TREM-1, TREM-2, DAP10, DAP12, MR, ICOS, MyD88 domain). In some embodiments, an extracellular leader domain comprises a DAP 10 leader domain. In some embodiments, an extracellular leader domain comprises a DAP 12 leader domain.Antigen Binding Domains

[0214] In some embodiments, a CAR comprises at least one antigen binding domain that binds to an antigen, for example, on a target cell. In some embodiments, a CAR comprises an antigen binding domain that binds to an antigen associated with viral infection, bacterial infection, parasitic infection, autoimmune disease, and / or cancer cells. In some embodiments, a CAR antigen binding domain recognizes an antigen that acts as a cell surface marker on a target cell associated with a particular disease state.

[0215] In some embodiments, a CAR antigen binding domain binds to a tumor antigen, such as an antigen that is specific for a tumor or cancer of interest. In some embodiments a tumor antigen comprises one or more antigenic cancer epitopes. In some embodiments, a tumor antigen comprises CD19; CD123; CD22; CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2);ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l -l)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Scr / Thr)); pro state- specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (R0R1); Fms-Like Tyrosine Kinase 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPC AM); B7H3 (CD276); KIT (CD117); Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha (IL-l lRa); prostate stem cell antigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascular' endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stagespecific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2 (Her2 / neu); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100 (gplOO); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2 (EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2- 3)bDGalp(l-4)bDGlcp(l-l)Cer); transglutaminase 5 (TGS5); high molecular weight-melanoma- associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61);CD97; CD 179a; anaplastic lymphoma kinase (ALK); Poly sialic acid; placenta- specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer / testis antigen 1 (NY-ESO-1); Cancer / testis antigen 2 (LAGE-la); Melanoma- associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6, located on chromosome 12p(ETV6-AML); sperm protein 17 (SPA 17); X Antigen Family, Member 1 A (XAGE1 ); angiopoictin-binding cell surface receptor 2 (Tic 2); melanoma cancer testis antigen- 1 (MAD- CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase; prostate carcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML- ZAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V (NA 17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin Bl; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1 (CYP1B1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Glycation Endproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like modulecontaining mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); or immunoglobulin lambda-like polypeptide 1 (IGLL1). In certain embodiments, a tumor antigen comprises ERBB2 (Her2 / neu). In certain embodiments, a tumor antigen comprises PSMA. In certain embodiments, a tumor antigen comprises Mesothelin.

[0216] In some embodiments, an antigen binding domain binds to a misfolded protein antigen or a protein of a protein aggregate, such as a protein that is specific for a disease / disorder of interest. In some embodiments, the disease / disorder is a neurodegenerative disease / disorder, an inflammatory disease / disorder, a cardiovascular disease / disorder, a fibrotic disease / disorder,or amyloidosis (e.g., mediated by protein aggregates of immunoglobulin light chains or of transthyretin). In some embodiments, the ncurodcgcncrativc discasc / disordcr is selected from the group consisting of tauopathy, asynucleopathy, presenile dementia, senile dementia, Alzheimer's disease (mediated by protein aggregates ofbeta- amyloid), Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), Pick' s disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, Parkinson's disease with dementia, dementia with Lewy bodies, Down syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS), Hallervorden-Spatz syndrome, polyglutamine disease, trinucleotide repeat disease, Familial British dementia, Fatal Familial Insomnia, Gerstmann-Straussler-Scheinker Syndrome, Hereditary cerebral hemorrhage with amyloidosis (Icelandic) (HCHW A-I), Sporadic Fatal Insomnia (sFI), Variably Protease- Sensitive Prionopathy (VPSPr), Familial Danish dementia, and prion disease (such as Creutzfeldt- Jakob disease, CJD and Variant Creutzfeldt- Jakob Disease (vCJD)).

[0217] In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence that is at least 80% identical to a sequence selected from Table 7. In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence that is at least 85% identical to a sequence selected from Table 7. In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence that is at least 90% identical to a sequence selected from Table 7. In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence that is at least 95% identical to a sequence selected from Table 7. In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence that is at least 96% identical to a sequence selected from Table 7. In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence that is at least 97% identical to a sequence selected from Table 7. In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence that is at least 98% identical to a sequence selected from Table 7. In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence that is at least 99% identical to a sequence selected from Table 7. In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence identical to a sequence selected from Table 7.

[0218] In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence that differs from a sequence selected from Table 7 by no more than 10 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence that differs from a sequence selected from Table 7 by no more than 9 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence that differs from a sequence selected from Table 7 by no more than 8 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence that differs from a sequence selected from Table 7 by no more than 7 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence that differs from a sequence selected from Table 7 by no more than 6 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence that differs from a sequence selected from Table 7 by no more than 5 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence that differs from a sequence selected from Table 7 by no more than 4 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence that differs from a sequence selected from Table 7 by no more than 3 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence that differs from a sequence selected from Table 7 by no more than 2 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence that differs from a sequence selected from Table 7 by no more than 1 substitution, addition, or deletion. In some embodiments, a CAR antigen binding domain binds to HER2 and comprises an amino acid sequence that does not have any substitutions, additions, or deletions relative to a sequence selected from Table 7.

[0219] In some embodiments, an antigen binding domain comprises any domain that binds to an antigen. In some embodiments, a CAR antigen binding domain is or comprises a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, or any fragment thereof, for example an scFv. In some embodiments, a CAR antigen binding domain is or comprises an aptamer, a darpin, acentyrin, a naturally occurring or synthetic receptor, an affibody, or other engineered protein recognition molecule. In some embodiments, a CAR antigen binding domain is or comprises a mammalian antibody or a fragment thereof. In some embodiments, a CAR antigen binding domain is derived, in whole or in part, from the same species in which the CAR will ultimately be used. For example, for use in humans, an antigen binding domain of a CAR comprises a human antibody, a humanized antibody, or a fragment thereof (e.g. a scFv). In some embodiments, a CAR antigen binding domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a CAR antigen binding domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).

[0220] In some embodiments, a CAR comprises one or more antigen binding domains. In some embodiments, a CAR comprises two or more antigen binding domains. In some embodiments, a CAR is a bispecific CAR. In some embodiments, an immune cell comprises two or more different CARs comprising one or more antigen binding domains. In some embodiments, an immune cell comprising a bispecific CAR and / or comprising two or more different CARs comprising one or more antigen binding domains can reduce off-target and / or on-target off-tissue effects by requiring that two antigens are present. In some embodiments, an immune cell comprises a bispecific CAR and / or comprises two or more different CARs comprising one or more antigen binding domains, wherein the CARs provide distinct signals that in isolation arc insufficient to mediate activation of the modified cell, but arc synergistic together, stimulating activation of the modified cell. In some embodiments, such a construct may be referred to as an ‘AND’ logic gate.

[0221] In some embodiments, an immune cell comprising a bispecific CAR and / or comprising two or more different CARs comprising one or more antigen binding domains can reduce off-target and / or on-target off-tissue effects by requiring that one antigen is present and a second, normal protein antigen is absent before the cell’s activity is stimulated. In some embodiments, such a construct may be referred to as a ‘NOT’ logic gate. In contrast to AND gates, NOT gated CAR-modified cells are activated by binding to a single antigen. However, the binding of a second receptor to the second antigen functions to override the activating signalbeing perpetuated through the CAR. Typically, such an inhibitory receptor would be targeted against an antigen that is abundantly expressed in a normal tissue but is absent in tumor tissue.Hinge Domains

[0222] In some embodiments, a CAR comprises one or more extracellular hinge domains. In some embodiments, a CAR extracellular hinge domain is or comprises a human extracellular hinge domain. In some embodiments, a CAR extracellular hinge domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a CAR extracellular hinge domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, one or more CAR extracellular hinge domains comprise a CD8a extracellular hinge domain, a CD28 extracellular hinge domain, a DAP 10 extracellular hinge domain, a DAP12 extracellular hinge domain, a DNGR-1 extracellular hinge domain, a Dectin- 1 extracellular hinge domain, or an IgG4 extracellular hinge domain. In some embodiments, one or more CAR extracellular hinge domains comprise a CD8a extracellular hinge domain, an IgG4 extracellular’ hinge domain, or a CD28 extracellular hinge domain. In some embodiments, a CAR extracellular hinge domain optimizes the physicochemical parameters of a CAR, e.g., optimal size relative to tumor antigen (e.g., allowing for exclusion of inhibitory molecules), optimal flexibility, optimal protein folding, optimal protein stability, optimal binding, optimal homodimerization, and / or lack of homodimerization.CAR Transmembrane Domains

[0223] In some embodiments, a CAR comprises a transmembrane domain, for example, that connects an extracellular domain to one or more intracellular’ moieties. In some embodiments, a CAR transmembrane domain is naturally associated with one or more other domain(s) of a CAR. In some embodiments, a CAR transmembrane domain can be modified to avoid binding to transmembrane domains of other surface membrane proteins, in order to minimize interactions with other members of a receptor complex. In some embodiments, a CAR transmembrane domain may be derived either from a naturally-occurring or from a synthetic source. In some embodiments a CAR transmembrane domain is derived from a naturally- occurring membrane -bound or transmembrane protein. In some embodiments, a CARtransmembrane domain is or comprises a human transmembrane domain. In some embodiments, a CAR transmembrane domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a CAR transmembrane domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a CAR transmembrane domain comprises a CD8a, CD64, CD32a, CD32c, CD16a, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD19, CD20, 41BB, CD28, 0X40, GITR, TREM-1, TREM-2, DAP10, DAP12, MR, ICOS, MyD88, CD3- zeta, FcRy, V / I / LxYxxL / V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRP , CD22, PIR-B, LILRB1, CD36, or Syk transmembrane domain. In some embodiments, a CAR transmembrane domain comprises a CD28 transmembrane domain (e.g., in some embodiments, with a CD28 hinge domain).FcR Transmembrane Domains

[0224] In some embodiments, an FcR transmembrane domain comprises a full-length FcR transmembrane domain. In some embodiments, an FcR transmembrane domain comprises a portion of a full-length FcR transmembrane domain. In some embodiments, an FcR transmembrane domain is or comprises a human FcR transmembrane domain, or portion thereof. In some embodiments, an FcR transmembrane domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an FcR transmembrane domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an FcR transmembrane domain comprises a CD64 (FcyRI), CD32a (FcyRIIa), CD32b (FcyRIIb), CD32c, CD 16a (FcyRIIIa), CD 16b (FcyRIIIb), FcsRI, FcsRII, or FcaRI (CD89) domain.TLR Transmembrane Domains

[0225] In some embodiments, a TLR transmembrane domain comprises a full-length TLR transmembrane domain. In some embodiments, a TLR transmembrane domain comprises a portion of a full-length TLR transmembrane domain. In some embodiments, a TLR transmembrane domain is or comprises a human TLR transmembrane domain, or portion thereof.In some embodiments, a TLR transmembrane domain may be a domain that is endogenous to a particular immune cell type (c.g., a modified immune cell as provided herein). In some embodiments, a TLR transmembrane domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a TLR transmembrane domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.CAR Intracellular Domains

[0226] In some embodiments, a CAR comprises one or more intracellular domains. In some embodiments, an intracellular domain is or comprises a human intracellular domain, or portion thereof. In some embodiments, an intracellular domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an intracellular domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an intracellular domain and / or other cytoplasmic domain of a CAR is responsible for activation of the cell in which the CAR is expressed (e.g., an immune cell). In some embodiments, an intracellular domain of a CAR is responsible for signal activation and / or transduction in an immune cell comprising said CAR.

[0227] In some embodiments, an intracellular domain of a CAR includes at least one domain responsible for signal activation and / or transduction. In some embodiments, an intracellular domain is or comprises at least one of a co- stimulatory molecule and a signaling domain. In some embodiments, an intracellular domain of a CAR comprises dual signaling domains. In some embodiments, an intracellular domain of a CAR comprises more than two signaling domains.

[0228] In some embodiments, an intracellular domain comprises a cytoplasmic portion of a surface receptor. In some embodiments, an intracellular domain comprises a co-stimulatory molecule. In some embodiments, an intracellular domain comprises a molecule that acts to initiate signal transduction in an immune cell.

[0229] In some embodiments, an intracellular domain of a CAR includes any portion of one or more co-stimulatory molecules, such as at least one signaling domain from CD3, Fc epsilon RI gamma chain, any derivative or variant thereof, any synthetic sequence thereof that has the same functional capability, and any combination thereof.FcR Intracellular Domains

[0230] In some embodiments, an FcR intracellular domain comprises a full-length FcR intracellular domain. In some embodiments, an FcR intracellular’ domain comprises a portion of a full-length FcR intracellular domain. In some embodiments, an FcR intracellular- domain is or comprises a human FcR intracellular domain, or portion thereof. In some embodiments, an FcR intracellular- domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an FcR intracellular domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an FcR intracellular domain comprises a CD64 (FcyRI), CD32a (FcyRIIa), CD32b (FcyRIIb), CD32c, CD16a (FcyRIIIa), CD16b (FcyRIIIb), FceRI, FceRII, or FcaRI (CD89) domain.TLR Intracellular Domains

[0231] In some embodiments, a TLR intracellular’ domain comprises a full-length TLR intracellular domain. In some embodiments, a TLR intracellular domain comprises a portion of a full-length TLR intracellular domain. In some embodiments, a TLR intracellular domain is or comprises a human TLR intracellular’ domain, or portion thereof. In some embodiments, a TLR intracellular domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a TLR intracellular domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a TLR intracellular domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.Signaling Domains

[0232] In some embodiments, a CAR comprises one or more intracellular’ signaling domains. In some embodiments, an intracellular signaling domain is or comprises a human intracellular signaling domain, or portion thereof. In some embodiments, a signaling domainmay be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a signaling domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).

[0233] In some embodiments, one or more intracellular signaling domains may be or comprise a CD3-zeta, FcR y, CD64, CD32a, CD32c, CD16a, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, T1E2, TRK, VEGFR, CD40, CD 19, CD20, 4 IBB, CD28, 0X40, GITR, TREM-1, TREM-2, DAP10, DAP12, MR, ICOS, MyD88, V / I / LxYxxL / V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPp, CD22, PIR-B, LILRBl ,Syk, 41 BB ligand (41 BBL; TNFSF9), CD27, OX40L, CD32b, CDllb, ITGAM, SLAMF7, CD206, CD163, CD209, Dectin-2, or one or more cytokine receptor signaling domains (e.g., an IL1R, an IL2R, an IL3R, an IL4R, an IL5R, an IL6R, an IL7R, an IL8R, an IL9R, an IL10R, an IL11R, an IL12R, an IL13R, an IL14R, an IL15R, an IL17R, an IFNaR, an IFNgR, an TNFR, an CSF1R, an CSF2R, DaplO, CD36, Dectin-1, or ICOSL intracellular signaling domain).

[0234] In some embodiments, an intracellular domain of a CAR may be or comprise dual signaling domains, such as 41BB, CD28, ICOS, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, CD116 receptor beta chain, CSF1-R, LRP1 / CD91, SR-A1, SR-A2, MARCO, SR-CL1 , SR-CL2, SR-C, SR-E, CR1 , CR3, CR4, dectin 1 , DEC-205, DC- SIGN, CD 14, CD36, LOX-1, CD1 lb, together with any of the signaling domains listed in the above paragraph in any combination.Co-stimulatory Domains

[0235] As used herein, a “co-stimulatory molecule” or “co- stimulatory domain” refers to a molecule in an immune cell that is used to heighten or dampen an initial stimulus. For example, pathogen-associated pattern recognition receptors, such as TLR or the CD47 / SIRPa axis, are molecules on immune cells that, respectively, heighten or dampen an initial stimulus. In some embodiments, a CAR co-stimulatory domain comprises TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD2,CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1 , GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CDl lb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp3O, NKp46, NKG2D, other costimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a co-stimulatory molecule that has the same functional capability, and any combinations thereof.

[0236] In some embodiments, a co-stimulatory domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a co-stimulatory domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).

[0237] As used herein, a “co-stimulatory signal” refers to a signal, which in combination with a primary signal, such as activation of a CAR on an immune cell, leads to activation of the immune cell.Cleavage Peptides

[0238] As used herein, a cleavage peptide refers to a peptide that can induce the cleaving of a recombinant protein in a cell. In some embodiments, a cleavage peptide is a 2A peptide. In some embodiments, a cleavage peptide is or comprises a P2A, F2A, E2A or T2A peptide. In some embodiments, a nucleic acid as described herein comprises one or more nucleic acid sequences encoding one or more cleavage peptides. In some embodiments, a nucleic acid comprising a nucleic acid sequence encoding a cleavage peptide also comprises one or more nucleic acid sequences encoding one or more intracellular domains and one or more nucleic acid sequences comprising one or more peptide agents, wherein translation of the nucleic acid results in a protein comprising one or more intracellular domains separated from one or more peptide agents by a cleavage peptide. In some embodiments, a first promoter is operably linked to one ormore nucleic acids encoding a CAR and a second promoter is operably linked to one or more nucleic acids encoding a peptide agent. In some embodiments, a nucleic acid sequence comprising a CAR, and optionally one or more peptide agents, further comprises an internal ribosome entry site (IRES) sequence. An IRES sequence may be any viral, chromosomal or artificially designed sequence that initiates cap-independent ribosome binding to mRNA facilitates the initiation of translation.CAR Peptide Agents

[0239] As used herein, a CAR peptide agent refers to a peptide co-expressed with a CAR in an immune cell. In some embodiments, a CAR peptide agent is co-cxprcsscd with a CAR to ensure stoichiometric balance and optimal signaling of a CAR. In some embodiments, a CAR peptide agent forms a homodimer with an identical peptide agent. In some embodiments, a CAR peptide agent forms a heterodimer with a different peptide agent. In some embodiments, a nucleic acid as described herein comprises one or more nucleic acid sequences encoding one or more CAR peptide agents. In some embodiments, a CAR peptide agent is or comprises an FcR gamma chain.

[0240] In some embodiments, a CAR peptide agent comprises any peptide, protein, receptor, secreted antibody or a fragment thereof (e.g., an scFv, Fab, Fab', F(ab')2, Fc, or nanobody). In some embodiments, a CAR peptide agent comprises one or more cytokines (e.g., one or more of IE-1, IE-2, IE-6, IL-8, TNF-a, IFN-a, IFN-P, IFN-y, GMCSF, or MCSF), CD40- L, dominant negative SIRPa, dominant negative PD1, dominant negative CD45, dominant negative SIGEEC 10, or dominant negative EIERB.Fc Receptors (FcR)

[0241] In some embodiments, a CAR comprises one or more antigen binding domains and an FcR extracellular domain, and / or the transmembrane domain of the CAR comprises an FcR transmembrane domain, and / or the intracellular domain of the CAR comprises an FcR intracellular domain. In some embodiments, a CAR comprises, from N-terminus to C-terminus, one or more extracellular domains, an FcR extracellular domain, an FcR transmembrane domain,and an FcR intracellular domain. In some embodiments, one or more of the FcR extracellular domain, the FcR transmembrane domain and the FcR intracellular domain is or comprises a human FcR domain. In some embodiments, an FcR extracellular domain, an FcR transmembrane domain and an FcR intracellular domain together comprise a full-length FcR. In some embodiments, an FcR extracellular domain, an FcR transmembrane domain and an FcR intracellular domain together comprise a portion of a full-length FcR. In some embodiments, an FcR extracellular domain comprises a portion of a full-length FcR extracellular domain. In some embodiments, an FcR transmembrane domain comprises a portion of a full-length FcR transmembrane domain. In some embodiments, an FcR intracellular domain comprises a portion of a full-length FcR intracellular domain.Toll-Like Antigen Receptors (TLR)In some embodiments, a CAR comprises one or more antigen binding domains and a toll-like receptor (TLR) extracellular domain and / or the transmembrane domain of the CAR comprises a TLR transmembrane domain and / or the intracellular domain of the CAR comprises a TLR intracellular domain. In some embodiments, a CAR comprises, from N-terminus to C-terminus, one or more extracellular domains, a TLR extracellular domain, a TLR transmembrane domain, and a TLR intracellular domain. In some embodiments, one or more of the TLR extracellular domain, the TLR transmembrane domain and the TLR intracellular domain is or comprises a human TLR domain. In some embodiments, a TLR extracellular domain, a TLR transmembrane domain and a TLR intracellular domain together comprise a full-length TLR. In some embodiments, a TLR extracellular domain, a TLR transmembrane domain and a TLR intracellular domain together comprise portion of a full-length TLR. In some embodiments, a TLR extracellular domain comprises a portion of a full-length TLR extracellular domain. In some embodiments, a TLR transmembrane domain comprises a portion of a full-length TLR transmembrane domain. In some embodiments, a TLR intracellular domain comprises a portion of a full-length TLR intracellular domain.Immune Cells

[0242] The present disclosure provides, among other things, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a chimeric antigen receptor (CAR) as described herein.

[0243] In some embodiments, a population of immune cells as described herein comprises stem cells, monocytes, macrophages, dendritic cells, and / or precursors thereof. In some embodiments, a population of immune cells comprises a substantially purified population of stem cells, monocytes, macrophages, or dendritic cells, or a cell line.

[0244] In some embodiments, an immune cell is activated, e.g., an immune cell exhibits increased cytokine production, chemokine production, phagocytosis, cell signaling, target cell killing, and / or antigen presentation in response to a particular stimuli, e.g., relative to an inactive cell. In some embodiments, an activated immune cell exhibits changes in gene expression, e.g., an induction of pro-inflammatory gene expression, e.g., relative to an inactive cell. In some embodiments, an activated immune cell exhibits changes in gene expression, e.g., an induction of anti-inflammatory gene expression, e.g., relative to an inactive cell. In certain embodiments, activated immune cells are undergoing cell division. In some embodiments, targeted effector activity of an immune cell is enhanced by inhibition of CD47 and / or SIRPa activity. CD47 and / or SIRPa activity may be inhibited by treating an immune cell with an anti-CD47 or anti- SIRPa antibody or by any method known to those skilled in the art.

[0245] In some embodiments, immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) are obtained (e.g., isolated) from a subject. Immune cells may be autologous or sourced from allogeneic or universal donors. Cells can be obtained from a number of sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, tumors, and / or induced pluripotent stem cells, such as embryonic stem cells (ESCs). In certain embodiments, cells can be obtained from a unit of blood collected from a subject using any number of separation techniques known to a skilled artisan, such as Ficoll separation. In some embodiments, cells from circulating blood of a subject are obtained by apheresis or leukapheresis. Cells collected by apheresis may be washed to remove a plasma fraction and resuspended in a variety of buffers (e.g., phosphate buffered saline (PBS)) or culture media). In some embodiments, enrichment of immune cells (e.g. monocytes) comprises plastic adherence. In some embodiments, following enrichment, differentiation of immune cells (e.g.monocytes) comprises stimulation with GM-CSF. In some embodiments, a composition comprising blood cells (c.g., monocytes, lymphocytes, platelets, plasma, and / or red blood cells), such as a leukapheresis composition (e.g., a leukopak) is used for enrichment. In some embodiments, a leukapheresis composition (e.g., a leukopak) comprises a sample from a healthy human donor. In certain embodiments, apheresis of immune cells (e.g. monocytes) is followed by mobilization with GM-CSF. In certain embodiments, selection of immune cells (e.g., monocytes) comprises CD14 positive selection using microbeads (e.g., MACS® MicroBeads on a CliniMACS Prodigy device). In some embodiments, an immune cell precursor (e.g., precursors to macrophages, monocytes, or dendritic cells including, but not limited to induced pluripotent stem cells, or iPSCs) is used in compositions and methods described herein. Immune cell precursors may be differentiated in vivo or ex vivo into immune cells. Non-limiting examples of precursor immune cells include hematopoietic stem cells, common myeloid progenitors, myeloblasts, monoblasts, promonocytes, or intermediates thereof. For example, induced pluripotent stem cells may be used to generate monocytes, macrophages, and / or dendritic cells. Induced pluripotent stem cells (iPSCs) may be derived from normal human tissue, such as peripheral blood, fibroblasts, skin, keratinocytes, or renal epithelial cells. Autologous, allogeneic, or universal donor iPSCs could be differentiated toward a myeloid lineage (e.g., a monocyte, macrophage, dendritic cell, or precursor thereof).

[0246] Immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) as described herein can be isolated from peripheral blood, for example, by lysing red blood cells and depleting lymphocytes and red blood cells, such as by centrifugation through a PERCOLL™ gradient. Alternatively, immune cells can be isolated from umbilical cord tissue. A specific subpopulation of immune cells can be further isolated by positive or negative selection techniques. In some embodiments, immune cells can be depleted of cells expressing certain antigens, including, but not limited to, CD14, CD16, CD34, CD3, CD4, CD8a, CD56, CD66b, CD19, or CD20. In some embodiments, immune cells can be depleted of macrophages but not monocytes. In some embodiments, enrichment of an immune cell population, for example, by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. By way of non-limiting example, cell selection can also comprise negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on negatively selected cells.

[0247] During isolation of a desired population of immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) as described herein by positive or negative selection, immune cell concentration and surface (e.g., particles, such as beads) can be varied. It may be desirable to significantly decrease volume in which beads and cells are mixed together to ensure maximum contact area of cells and beads.

[0248] In some embodiments, prior to administration, immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) as described herein (e.g., comprising a CAR as described herein) are treated with a pro-inflammatory agent. In some embodiments, treatment with a pro-inflammatory agent promotes an anti-inflammatory (i.e., M2) phenotype in immune cells as described herein. In some embodiments, a pro-inflammatory agent comprises or is a pro- inflammatory cytokine. In some embodiments, a pro-inflammatory agent comprises or is IFN-a, IFN-P, IFN- , IFN-y, TNF-a, IL-6, IL-lb, IL-2, IL-8, IL-12, IL-15, IL-18, IL-17, IL-la, IL-3, IL-21, IL-33, IL-23, IL-37, or IL-36. In some embodiments, a pro-inflammatory agent comprises or is an IFN-y agonist (e.g., IFN-y).

[0249] In some embodiments, prior to administration, immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) as described herein (e.g., comprising a CAR as described herein) are treated with an anti-inflammatory agent. In some embodiments, treatment with an anti-inflammatory agent promotes a pro-inflammatory (i.e., Ml) phenotype in immune cells as described herein. In some embodiments, an anti-inflammatory agent comprises or is an anti-inflammatory cytokine. In some embodiments, an anti-inflammatory agent comprises or is IL-10, TGF- , IL-4, IL-13, IL-11, or IL-35. In some embodiments, an anti-inflammatory agent comprises or is an IL-10 agonist (e.g., IL-10).

[0250] In some embodiments, immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) as described herein (e.g., comprising a CAR as described herein) are administered to a subject in combination with a pro-inflammatory agent. In some embodiments, immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) as described herein (e.g., comprising a CAR as described herein) are administered to a subject substantially simultaneously, before, or after a pro-inflammatory agent. In some embodiments, administration with a pro-inflammatory agent promotes an anti-inflammatory (i.e., M2) phenotype in immunecells as described herein. In some embodiments, a pro-inflammatory agent comprises or is an IFN-y agonist (e.g., IFN-y).

[0251] In some embodiments, immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) as described herein (e.g., comprising a CAR as described herein) are administered to a subject in combination with an anti-inflammatory agent. In some embodiments, immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) as described herein (e.g., comprising a CAR as described herein) are administered to a subject substantially simultaneously, before, or after an anti-inflammatory agent. In some embodiments, administration with an anti-inflammatory agent increases anti-tumor activity of immune cells as described herein. In some embodiments, administration with an anti-inflammatory agent promotes a pro-inflammatory (i.e., Ml) phenotype in immune cells as described herein. In some embodiments, an anti-inflammatory agent comprises or is an IL-10 agonist (e.g., IL-10).

[0252] In some embodiments, immune cells are modified in a subject (e.g., via in vivo delivery mechanisms). In some embodiments, the present disclosure provides modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in a subject, wherein the subject is administered a composition as described herein comprising: (a) one or more nucleic acid molecules, wherein at least a portion of the one or more nucleic acid molecules encodes a CAR and / or CAR peptide agent, and (b) a delivery vehicle. Accordingly, in some embodiments, following administration of the composition the one or more nucleic acid molecules are translated in an immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) to produce a modified immune cell comprising the CAR and / or CAR peptide agent. In some embodiments, the modified immune cell comprising the CAR and / or CAR peptide agent possesses targeted effector activity.Macrophages

[0253] Macrophages are immune cells specialized for detection, phagocytosis, and destruction of target cells, such as pathogens or tumor cells. Macrophages are potent effectors of the innate immune system and are capable of at least three distinct anti-tumor functions: 1) phagocytosis of dead and dying cells, microorganisms, cancer cells, cellular debris, or otherforeign substances; 2) cytotoxicity against tumor cells; and 3) presentation of tumor antigens to orchestrate an adaptive anti-tumor immune response.

[0254] Accumulating evidence suggests that macrophages are abundant in the tumor microenvironment of numerous cancers and can adopt a number of phenotypes that are collectively referred to as tumor-associated macrophages (TAMs). The immunosuppressive nature of the tumor microenvironment typically results in more M2-like TAMs, which further contribute to the general suppression of anti-tumor immune responses. Recent studies, however, have identified that TAMs are able to be “reprogrammed” via pro-inflammatory signals, and that the switch from a M2 phenotype to a more Ml phenotype is associated with productive antitumor immune responses. Inducing endogenous TAMs to switch to Ml -type cells and engineering macrophages that cannot be subverted into M2 would greatly enhance anti-tumor immunotherapy and represent a significant advance in the field.

[0255] In some embodiments, a macrophage comprises or is an undifferentiated or MO macrophage. In certain embodiments, a macrophage comprises or expresses one, two, three, four, five, or six of CD14, CD16, CD64, CD68, CD71, or CCR5. Exposure to various stimuli can induce MO macrophages to polarize into several distinct populations, which may be identified by macrophage phenotype markers, cytokine production, and / or chemokine secretion.

[0256] In some embodiments, a macrophage comprises or is a polarized macrophage. Under classical conditions of activation, MO macrophages can be exposed to pro-inflammatory signals, such as LPS, IFN-y, and GM-CSF, and polarize into pro-inflammatory (i.e., Ml) macrophages. Generally, pro-inflammatory (Ml) macrophages are associated with pro- inflammatory immune responses, such as Thl and Thl7 T cell responses. Exposure to other stimuli can polarize macrophages into a diverse group of “alternatively activated” or antiinflammatory (i.e., M2) macrophages.

[0257] In some embodiments, a macrophage comprises or is a pro-inflammatory (Ml) macrophage. In some embodiments, a macrophage expresses one or more markers of pro- inflammatory (Ml) macrophages (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of CD86, CD80, MHC II, IL-1R, TLR2, TLR4, iNOS, SOCS3, CD83, PD-L1, CD69, MHC I, CD64, CD32, CD16, IL1R, a IFIT family member, or an ISG family member).

[0258] In some embodiments, a macrophage comprising or expressing a CAR as described herein secretes relatively high levels of one or more inflammatory cytokines (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of IL-1, TNF, IL-12, IL-18, IL-23, IFN-a, IFN-P, IFN-y, IL-2, IL- 6, IL-8, or IL33) or chemokines (e.g., one or both of CC or CXC chemokines) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the CXC chemokines; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 of the CC chemokines; e.g., one of the CX3C chemokines, e.g., one or both of the C chemokines), e.g., relative to a macrophage without a CAR as described herein. In some embodiments, a macrophage comprising or expressing a CAR as described herein stimulates an immune response and / or inflammation, e.g., relative to a macrophage without a CAR as described herein.

[0259] In some embodiments, a macrophage comprises or is an anti-inflammatory (M2) macrophage (e.g., an M2a, M2b, M2c, and M2d macrophage). An M2a macrophage can be induced by IL-4, IL-13, and / or fungal infection. An M2b macrophage can be induced by IL-1R ligands, an immune complex, and / or LPS. An M2c macrophage can be induced by IL- 10 and / or TGFp. An M2d macrophage can be induced by IL-6 and / or adenosine. In some embodiments, a macrophage comprising or expressing a CAR as described herein decreases an immune response in a subject, e.g., relative to a macrophage without a CAR as described herein. In some embodiments, a macrophage expresses one or more markers of anti-inflammatory (M2) macrophages (e.g., one, two, or three of CD206, CD163, or CD209). In some embodiments, a macrophage comprising or expressing a CAR as described herein exhibits increased secretion of one or more anti-inflammatory cytokines (e.g., one or both of IL- 10 or TGFP), e.g., relative to a macrophage without a CAR as described herein.

[0260] In some embodiments, a macrophage comprises at least one upregulated pro- inflammatory (Ml) marker and / or at least one downregulated anti-inflammatory (M2) marker as compared to a control macrophage that does not comprise a CAR as described herein and / or the same macrophage before delivery of a CAR as described herein. In some embodiments, at least one pro-inflammatory (Ml) marker (e.g., HLA DR, CD86, CD80, PD-L1, CD83, CD69, MHC I, CD64, CD32, CD16, IL1R, an IFIT family member, and / or an ISG family member) is upregulated in a macrophage. In some embodiments, at least one anti-inflammatory (M2) marker (e.g., CD206, CD 163, and / or CD209) is downregulated in a macrophage.

[0261] In some embodiments, a macrophage comprising or expressing a CAR as described herein exhibits increased phagocytosis, e.g., relative to a macrophage without a CAR as described herein. In some embodiments, a macrophage comprising or expressing a CAR as described herein exhibits increased cytotoxicity against a tumor cell, e.g., relative to a macrophage without a CAR as described herein. In some embodiments, a macrophage comprising or expressing a CAR as described herein exhibits increased tumor antigen presentation (e.g., post-phagocytosis presentation) and / or increased antigen processing, e.g., relative to a macrophage without a CAR as described herein. In some embodiments, a macrophage comprising or expressing at a CAR as described herein exhibits increased tumor killing (e.g., by phagocytosis, lysis, apoptosis, or production of tumor killing cytokines (e.g., TNFa)), e.g., relative to a macrophage without a CAR as described herein.

[0262] In some embodiments, a macrophage comprising or expressing a CAR as described herein exhibits one or both of increased expression of one or more genes typically associated with increased effector function (e.g., phagocytosis, targeted cellular cytotoxicity, antigen presentation, or cytokine secretion) (e.g., CD80, CD86, MHC-I, MHC-II, CD40, 41 BBL, TNF, IFN-a, IFN-0, IFN-y, IL2, IL12, IL6, IL8, ILlb, and / or CXCL12) or decreased expression of one or more genes typically associated with decreased effector function (e.g., phagocytosis, targeted cellular cytotoxicity, antigen presentation, or cytokine secretion) (e.g., CD163, CD206, TGFp, IL- 10, and / or IL4), e.g., relative to a macrophage without a CAR as described herein. In some embodiments, a macrophage comprising or expressing a CAR as described herein exhibits increased production of ROS, e.g., relative to a macrophage without a CAR as described herein. In some embodiments, a macrophage comprising or expressing a CAR as described herein exhibits metabolic reprogramming (e.g., of an interferon signaling pathway, TH1 pathway, PTEN signaling, PI3K signaling, MTOR signaling, TLR signaling, CD40 signaling, 41BB signaling, 41BBL signaling, macrophage maturation signaling, dendritic cell maturation signaling, CD3-zeta signaling, FcR y signaling, CD64 signaling, CD32a signaling, CD32c signaling, CD 16a signaling, TLR1 signaling, TLR2 signaling, TLR3 signaling, TLR4 signaling, TLR5 signaling, TLR6 signaling, TLR7 signaling, TLR8 signaling, TLR9 signaling, ALK signaling, AXL signaling, DDR2 signaling, EGFR signaling, Eph Al signaling, INSR signaling, cMET signaling, MUSK signaling, PDGFR signaling, PTK7 signaling, RET signaling, ROR1 signaling, ROS 1 signaling, RYK signaling, TIE2 signaling, TRK signaling, VEGFR signaling,CD40 signaling, CD 19 signaling, CD20 signaling, 41 BB signaling, CD28 signaling, 0X40 signaling, GITR signaling, TREM-1 signaling, TREM-2 signaling, DAP 12 signaling, MR signaling, ICOS signaling, MyD88 signaling, V / I / LxYxxL / V signaling, SIRPa signaling, CD45 signaling, Siglec-10 signaling, PD1 signaling, SHP-1 signaling, SHP-2 signaling, KIR-2DL signaling, KIR-3DL signaling, NKG2A signaling, CD 170 signaling, CD33 signaling, BTLA signaling, CD32b signaling, SIRPP signaling, CD22 signaling, PIR-B signaling, and / or LILRB 1 signaling), e.g., relative to a macrophage without a CAR as described herein. In some embodiments, a macrophage comprising or expressing a CAR as described herein exhibits induction of cell survival mechanisms, e.g., relative to a macrophage without a CAR as described herein. In some embodiments, a macrophage comprising or expressing a CAR as described herein exhibits induction of cell death mechanisms, e.g., relative to a macrophage without a CAR as described herein. In some embodiments, a macrophage comprising or expressing a CAR as described herein exhibits one, two, three, four, or five of: increased resistance to phagocytic checkpoints, increased expression of chemokine receptors to aid in trafficking, increased expression of chemokines to recruit other immune cells, increased expression of ECM degrading enzymes (e.g., MMPs to degrade tumor ECM and / or exhibit anti fibrotic activity), and / or increased proliferation, e.g., relative to a macrophage without a CAR as described herein. In some embodiments, a macrophage comprising or expressing a CAR as described herein exhibits one, two, three, or four of improved duration of CAR expression, improved stability of the CAR on the cell surface, increased level of CAR expression, and / or decreased background activity of the CAR, e.g., relative to a macrophage without a CAR as described herein.

[0263] In some embodiments, a macrophage comprising or expressing a CAR as described herein decreases one or more signs and / or symptoms of an infection (e.g., of an infectious agent) in a subject, e.g., relative to a macrophage without a CAR as described herein. In some embodiments, an infectious agent comprises or is a virus, a protozoa (e.g., trypanosome, malaria, or toxoplasma), a bacteria (e.g., mycobacterium, salmonella, or listeria), a fungi (e.g., Candida), or a combination thereof. In some embodiments, a virus comprises hepatitis virus (e.g., hepatitis A, hepatitis B, hepatitis C, or hepatitis E), retrovirus, human immunodeficiency virus (e.g., HIV1 or HIV2), T cell leukemia virus, a Lymphotropic vims (e.g., HTLV1 or HTLV2), herpes simplex vims (e.g., herpes simplex vims type 1 or type 2), Epstein-Barr virus, cytomegalovims, varicella-zoster virus, poliovirus, measles vims, Rubella virus, Japaneseencephalitis virus, mumps vims, influenza vims, adenovirus, enterovims, rhinovirus, coronavims (c.g., severe acute respiratory syndrome (SARS) virus, Middle East respiratory syndrome (MERS) vims, or severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)), Ebola vims, West Nile virus, or a variant or combination thereof.

[0264] In some embodiments, a macrophage comprising or expressing a CAR as described herein decreases formation and / or degrades existing aggregates via phagocytosis of at least one protein aggregate in a subject (e.g., a subject having a neurodegenerative disease, an inflammatory disease, a cardiovascular disease, a fibrotic disease, amyloidosis, or a combination thereof), e.g., relative to a macrophage without a CAR as described herein. In some embodiments, a neurodegenerative disease is selected from the group consisting of tauopathy, a- synucleopathy, presenile dementia, senile dementia, Alzheimer's disease, progressive supranuclear palsy (PSP), Pick's disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, dementia with Lewy bodies, Down's syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS), Hallervorden-Spatz syndrome, polyglutamine disease, trinucleotide repeat disease, and prion disease. In some embodiments, an inflammatory disease is selected from the group consisting of systemic lupus erythematosus, vasculitis, rheumatoid arthritis, periodontitis, ulcerative colitis, sinusitis, asthma, tuberculosis, Crohn’s disease, chronic infection, hereditary periodic fever, a malignancy, systemic vasculitides, cystic fibrosis, bronchiectasis, epidermolysis bullosa, cyclic neutropenia, an immunodeficiency, Muckle-Wells (MWS) disease, and Familiar Mediterranean Fever (FMF). In some embodiments, amyloidosis is selected from the group consisting of Primary Amyloidosis (AL), Secondary Amyloidosis (AA), Familial Amyloidosis (ATTR) , Beta-2 Microglobulin Amyloidosis, Localized Amyloidosis, Heavy Chain Amyloidosis (AH), Light Chain Amyloidosis (AL), Primary Systemic Amyloidosis, ApoAI Amyloidosis, ApoAII Amyloidosis, ApoAIV Amyloidosis, Apolipoprotein C2 Amyloidosis, Apolipoprotein C3 Amyloidosis, Corneal lactoferrin amyloidosis, Transthyretin-Related Amyloidosis, Dialysis amyloidosis, Fibrinogen amyloidosis, Lect2 amyloidosis (ALECT2), and Lysozyme amyloidosis. In some embodiments, a cardio vascular disease is selected from the group consisting of atherosclerosis, coronary artery disease, peripheral artery disease, hypertensive heart disease, metabolic syndrome, hypertension, cerebrovascular disease, and heart failure. In some embodiments, a fibrotic disease is selected from the group consisting of pulmonary fibrosis, idiopathicpulmonary fibrosis, cirrhosis, cystic fibrosis, scleroderma, cardiac fibrosis, radiation-induced lung injury, stcatohcpatitis, glomerulosclerosis, interstitial lung disease, liver fibrosis, mediastinal fibrosis, retroperitoneal cavity fibrosis, bone marrow fibrosis, and skin fibrosis.Monocytes

[0265] Monocytes are multipotent cells that circulate in the blood, bone marrow, and spleen, and generally do not proliferate when in a steady state. Monocytes can vary in size significantly in the range of about 10-30 pm in diameter. A ratio of nucleus to cytoplasm for a monocyte can range from about 2:1 to about 1 :1. Typically, monocytes comprise chemokine receptors and pathogen recognition receptors that mediate migration from blood to tissues, such as during an infection. Monocytes can produce inflammatory cytokines, take up cells and / or toxic molecules, and differentiate into dendritic cells or macrophages.

[0266] In some embodiments, a monocyte comprises or expresses one or more phenotypic markers. Exemplarily phenotypic markers for human monocyte cells include, but are not limited to, CD9, CDllb, CDl lc, CDwl2, CD13, CD15, CDwl7, CD31, CD32, CD33, CD35, CD36, CD38, CD43, CD49b, CD49e, CD49f, CD63, CD64, CD65s, CD68, CD84, CD85, CD86, CD87, CD89, CD91, CDw92, CD93, CD98, CD101, CD102, CD111, CD112, CD115, CD 116, CD 1 19, CDwl21b, CDwl23, CD 127, CDwl28, CDwl31 , CD 147, CD 155, CD 156a, CD157, CD162 CD163, CD164, CD168, CD171, CD172a, CD180, CD206, CD131al, CD213 2, CDw210, CD226, CD281, CD282, CD284, and CD286. Exemplarily phenotypic markers for mouse monocyte cells include, but are not limited to, CD1 la, CD1 lb, CD16, CD18, CD29, CD31, CD32, CD44, CD45, CD49d, CD115, CD116, Cdwl31, CD281, CD282, CD284, CD286, F4 / 80, and CD49b. In certain embodiments, monocytes comprise one, two, or three of CD1 lb, CD 14, or CD 16. In certain embodiments, monocytes comprise CD 14+ CD 16- monocytes, CD14+ CD16+ monocytes, or CD14- CD16+ monocytes. In some embodiments, a population of monocytes comprises CD14+ CD16- monocytes, CD14+ CD16+ monocytes, and CD14- CD16+ monocytes. In some embodiments, a population of monocytes comprises CD 14+ CD 16- monocytes and CD 14+ CD 16+ monocytes. In some embodiments, a population of monocytes comprises CD14+ CD16- monocytes and CD14- CD16+ monocytes. In some embodiments, a population of monocytes comprises CD 14+ CD 16+ monocytes and CD 14- CD 16+ monocytes.In some embodiments, a population of monocytes comprises CD14+ CD16- monocytes. In some embodiments, a population of monocytes comprises CD14+ CD16+ monocytes. In some embodiments, a population of monocytes comprises CD14- CD16+ monocytes. In some embodiments, a population of monocytes comprises classical monocytes, intermediate monocytes, and / or non-classical monocytes.

[0267] In some embodiments, a monocyte differentiates into a macrophage. In some embodiments, a monocyte differentiates into a dendritic cell (DC). Monocytes can be differentiated into macrophages or DCs by any technique known in the art. For example, differentiation of monocytes into macrophages can be induced by macrophage colony stimulating factor (M-CSF). Differentiation of monocytes into DCs can be induced by granulocyte-macrophage colony stimulating factor (GM-CSF) in combination with IL-4.

[0268] In some embodiments, a monocyte comprising or expressing a CAR as described herein exhibits increased secretion of one or more cytokines (e.g., one, two, three, four, five, six, or seven of TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, or IL-1), e.g., relative to a monocyte without a CAR as described herein. In some embodiments, a monocyte comprising or expressing a CAR as described herein exhibits increased phagocytosis, e.g., relative to a monocyte without a CAR as described herein. In some embodiments, a monocyte comprising or expressing a CAR as described herein exhibits enhanced survival, e.g., relative to a monocyte without a CAR as described herein. In some embodiments, a monocyte comprising or a CAR as described herein exhibits enhanced differentiation into macrophages (e.g., Ml or M2 macrophages), e.g., relative to a monocyte without a CAR as described herein. In some embodiments, a monocyte comprising or expressing a CAR as described herein as described herein exhibits enhanced differentiation into DCs (e.g., resident or migrating DCs and / or in lymphoid and non-lymphoid tissue), e.g., relative to a monocyte without a CAR as described herein. In some embodiments, a monocyte comprising or expressing a CAR as described herein exhibits increased cytotoxicity against a tumor cell, e.g., relative to a monocyte without a CAR as described herein. In some embodiments, a monocyte comprising or expressing a CAR as described herein exhibits increased tumor antigen presentation (e.g., post-phagocytosis presentation) and / or increased antigen processing, e.g., relative to a monocyte without a CAR as described herein. In some embodiments, a monocyte comprising or expressing a CAR asdescribed herein exhibits increased tumor killing (e.g., by phagocytosis, lysis, apoptosis, or production of tumor killing cytokines (e.g., TNFa), e.g., relative to a monocyte without a CAR as described herein.

[0269] In some embodiments, a monocyte comprising or expressing a CAR as described herein exhibits one or both of increased expression of one or more genes typically associated with increased effector function (e.g., phagocytosis, targeted cellular cytotoxicity, antigen presentation, or cytokine secretion) or decreased expression of one or more genes typically associated with decreased effector function (e.g., phagocytosis, targeted cellular cytotoxicity, antigen presentation, or cytokine secretion), e.g., relative to a monocyte without a CAR as described herein. In some embodiments, a monocyte comprising or expressing a CAR as described herein exhibits increased production of ROS, e.g., relative to a monocyte without a CAR as described herein. In some embodiments, a monocyte comprising or expressing a CAR as described herein exhibits metabolic reprogramming, e.g., relative to a monocyte without a CAR as described herein. In some embodiments, a monocyte comprising or expressing a CAR as described herein exhibits induction of cell survival mechanisms, e.g., relative to a monocyte without a CAR as described herein. In some embodiments, a monocyte comprising or expressing a CAR as described herein exhibits induction of cell death mechanisms, e.g., relative to a monocyte without a CAR as described herein. In some embodiments, a monocyte comprising or expressing a CAR as described herein exhibits one, two, three, four, or five of increased resistance to phagocytic checkpoints, increased expression of chemokine receptors to aid in trafficking, increased expression of chemokines to recruit other immune cells, increased expression of ECM degrading enzymes (e.g., MMPs to degrade tumor ECM and / or exhibit anti fibrotic activity), or increased proliferation, e.g., relative to a monocyte without a CAR as described herein. In some embodiments, a monocyte comprising or expressing a CAR as described herein exhibits one, two, three, or four of improved duration of CAR expression, improved stability of the CAR on the cell surface, increased level of CAR expression, and / or decreased background activity of the CAR, e.g., relative to a monocyte without a CAR as described herein.Dendritic Cells

[0270] Dendritic cells (DCs) are bone marrow-derived, specialized antigen presenting cells that arc involved in initiating immune responses and maintaining tolerance of the immune system to self-antigens. Dendritic cells may be found in both lymphoid and non-lymphoid organs and are generally thought to arise from lymphoid or myeloid lineages.

[0271] In some embodiments, a DC comprises or expresses one or more phenotypic markers. Exemplarily phenotypic markers for DCs include, but are not limited to, CD11c, CD83, CDla, CDlc, CD141, CD207, CLEC9a, CD123, CD85, CD180, CD187, CD205, CD281, CD282, CD284, CD286 and partially CD206, CD207, CD208 and CD209.

[0272] Immature DCs can be characterized by a high capacity for antigen capture, but relatively low T cell stimulatory capability. Inflammatory mediators promote DC maturation. Once DCs reach the mature stage, there is a dramatic change in properties relative to immature DCs, such as a decrease in antigen capture ability and / or an increased ability to stimulate T cells. In some embodiments, a DC comprises or is an immature DC. In other embodiments, a DC comprises or is a mature DC.

[0273] Without wishing to be bound by theory, it is believed that modification of a DC cell to comprise or express a CAR as described herein as described herein can allow mature DCs to simultaneously exhibit increased antigen capture ability and T cell stimulation, e.g., relative to a DC without a CAR as described herein. In some embodiments, a DC comprising or expressing a CAR as described herein mediates tumor antigen presentation, e.g., increased tumor antigen presentation relative to a DC without a CAR as described herein. In some embodiments, a DC comprising or expressing a CAR as described herein mediates tumor T cell stimulation, e.g., increased T cell stimulation relative to a DC without a CAR as described herein.

[0274] In some embodiments, a DC comprising or expressing a CAR as described herein exhibits increased secretion of one or more cytokines (e.g., one, two, three, four, five, six, or seven of TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, or IL-1), e.g., relative to a DC without a CAR as described herein. In some embodiments, a DC comprising or expressing a CAR as described herein exhibits increased phagocytosis, e.g., relative to a DC without a CAR as described herein. In some embodiments, a DC comprising or expressing a CAR as described herein exhibits increased tumor antigen presentation (e.g., post-phagocytosis presentation),increased antigen processing, increased antigen cross presentation, increased T cell priming, and / or stimulation of T cells, c.g., relative to a DC without a CAR as described herein.

[0275] In some embodiments, a DC comprising or expressing a CAR as described herein exhibits one or both of increased expression of favorable genes or decreased expression of unfavorable genes, e.g., relative to a DC without a CAR as described herein. In some embodiments, a DC comprising or expressing a CAR as described herein exhibits increased production of ROS, e.g., relative to a DC without a CAR as described herein. In some embodiments, a DC comprising or expressing a CAR as described herein exhibits metabolic reprogramming, e.g., relative to a DC without a CAR as described herein. In some embodiments, a DC comprising or expressing a CAR as described herein exhibits induction of cell survival mechanisms, e.g., relative to a DC without a CAR as described herein.

[0276] In some embodiments, a DC comprising or expressing a CAR as described herein exhibits induction of cell death mechanisms, e.g., relative to a DC without a CAR as described herein. In some embodiments, a DC comprising or expressing a CAR as described herein exhibits one, two, three, four, or five of increased resistance to phagocytic checkpoints, increased expression of chemokine receptors to aid in trafficking, increased expression of chemokines to recruit other immune cells, increased expression of ECM degrading enzymes (e.g., MMPs to degrade tumor ECM and / or exhibit anti fibrotic activity), or increased proliferation, e.g., relative to a DC without a CAR as described herein. In some embodiments, a DC comprising or expressing a CAR as described herein exhibits one, two, three, or four of improved duration of CAR expression, improved stability of the CAR on the cell surface, increased level of CAR expression, and / or decreased background activity of the CAR, e.g., relative to a DC without a CAR as described herein.Methods of Immune Cell Modification

[0277] The present disclosure provides, among other things, methods for modifying an immune cell (e.g., a stem cell, monocyte, macrophage, or dendritic cell) comprising delivering to the immune cell a nucleic acid construct into an immune cell, wherein the nucleic acid construct comprises one or more nucleic acids encoding a CAR as described herein or a fragment thereofand one or more introns comprising one or more inhibitory nucleic acids of the present disclosure.

[0278] In some embodiments, the present disclosure provides methods of producing a modified immune cell (e.g., a stem cell, monocyte, macrophage, or dendritic cell) in a subject comprising administering to a subject a composition as described herein comprising: (a) one or more nucleic acid molecules, wherein at least a portion of one or more nucleic acid molecules encodes a CAR and / or CAR peptide agent, and (b) a delivery vehicle. Accordingly, in some embodiments, following administration of the composition one or more nucleic acid molecules are translated in an immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) to produce a modified immune cell comprising the CAR and / or CAR peptide agent. In some embodiments, the modified immune cell comprising the CAR and / or CAR peptide agent possesses targeted effector activity.Delivery Methods

[0279] A nucleic acid construct comprising one or more nucleic acid sequences encoding at least one CAR described herein, can be introduced into an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) by physical, chemical, or biological methods. In some embodiments, the present disclosure provides methods for modifying an immune cell comprising producing a modified immune cell (e.g., a stem cell, monocyte, macrophage, or dendritic cell) ex vivo. In some embodiments, the present disclosure provides methods for modifying an immune cell comprising producing a modified immune cell (e.g., a stem cell, monocyte, macrophage, or dendritic cell) in a subject (i.e., in vivo).

[0280] Physical methods for introducing a nucleic acid construct as described herein into an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) can comprise electroporation, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, or a combination thereof. A nucleic acid construct can be introduced into immune cells using commercially available methods, including electroporation (Amaxa Nucleofector-II® (Amaxa Biosystems, Cologne, Germany), ECM 830 BTX (Harvard Instruments, Boston, Mass.) Gene Pulser II® (BioRad, Denver, Colo.), or Multiporator® (Eppendort, Hamburg Germany)). A nucleic acid construct can also be introduced into immunecells using mRNA transfection, e.g., cationic liposome-mediated transfection, lipofection, polymer encapsulation, pcptidc-mcdiatcd transfection, or biolistic particle delivery systems, such as “gene guns” (See, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001), which is hereby incorporated by reference in its entirety).

[0281] Biological methods for introducing a nucleic acid construct as described herein into an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) include use of DNA and RNA vectors. In one embodiment, a vector comprises a plasmid vector, a viral vector, a transposon, a retrotransposon (e.g., piggyBac, sleeping beauty), a site directed insertion vector (e.g., CRISPR, Zn finger nucleases, TALEN), suicide expression vector, or another vector known in the art. Viral vectors, and especially retroviral vectors, have become widely used for inserting genes into mammalian cells (e.g., human cells). Viral vectors can also be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses (e.g. Ad5f35), or adeno-associated viruses (See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362, which are hereby incorporated by reference in their entirety). Retroviral vectors, such as lentivirus, are suitable tools to achieve long-term gene transfer that allow for long-term, stable integration of a transgene and its propagation in daughter cells. In some embodiments, a lentiviral vector is packaged with a Vpx protein (e.g., as described in International Publication No. WO 2017 / 044487, which is hereby incorporated by reference in its entirety). In some embodiments, Vpx comprises a virion- associated protein (e.g., an accessory protein for viral replication). In some embodiments, a Vpx protein is encoded by human immunodeficiency virus type 2 (HIV-2). In some embodiments, a Vpx protein is encoded by simian immunodeficiency virus (SIV). In some embodiments, an immune cell as described herein (e.g., a stem cell, macrophage, monocyte, or dendritic cell) is transfected with a lentiviral vector packaged with a Vpx protein. In some embodiments, Vpx inhibits at least one antiviral factor of an immune cell as described herein (e.g., a stem cell, macrophage, monocyte, or dendritic cell). In some embodiments, a lentiviral vector packaged with a Vpx protein exhibits increased transfection efficiency of an immune cell as described herein (e.g., a stem cell, macrophage, monocyte, or dendritic cell), e.g., relative to a lentiviral vector not packaged with a Vpx protein. In some embodiments, an immune cell as described herein (e.g., a stem cell, macrophage, monocyte, or dendritic cell) is one or both of electroporated or transfected with at least one VPX mRNA prior to transfection with a viralvector (e.g., an adenoviral vector, e.g., an Ad2 vector or an Ad5 vector (e.g., Ad5f35 adenoviral vector, e.g., a helper-dependent Ad5F35 adenoviral vector)).

[0282] Chemical means for introducing a nucleic acid construct as described herein into an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) include colloidal dispersion systems, macromolecule complexes, nanocapsules, microspheres, beads, and lipid- based systems (e.g., oil-in-water emulsions, micelles, mixed micelles, nanoparticles, liposomes, and lipofectamine-nucleic acid complexes).

[0283] An exemplary system for delivery of a nucleic acid construct as described herein is a lipid-based system. A nucleic acid construct as described herein may be encapsulated in an aqueous interior of a liposome, interspersed within a lipid bilayer, attached to a liposome via a linking molecule, attached to a lipid nanoparticle (LNP) via a linking molecule, entrapped in a liposome, entrapped in an LNP, complexed with a liposome, complexed with an LNP, dispersed in a solution or suspension comprising a lipid, mixed with a lipid, complexed with a micelle, or otherwise associated with a lipid. Lipids for use in methods described herein may be naturally occurring or synthetic lipids. Lipids can also be obtained from commercial sources. For example, dimyristyl phosphatidylcholine can be obtained from Sigma (St. Louis, MO); dicetyl phosphate can be obtained from K & K Laboratories (Plainview, NY); cholesterol can be obtained from Calbiochem-Behring; and dimyristyl phosphatidylglycerol can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. In some embodiments, a lipid-based system may comprise one or more lipids that facilitate targeting of the composition to a desired cell type or cell types (e.g., stem cells, monocytes, macrophages, or dendritic cells). In some embodiments, a delivery vehicle allows a composition to be preferentially taken up (e.g. endocytosed, phagocytosed) by an immune cell (e.g., stem cell, monocyte, macrophage, or dendritic cell) relative to a composition that does not comprise the delivery vehicle.Targeting Moieties

[0284] In some embodiments, a delivery vehicle may comprise one or more targeting moieties. In some embodiments, a targeting moiety may facilitate passive targeting of acomposition to a desired target. In some embodiments, a targeting moiety may facilitate active targeting of a composition to a desired target.

[0285] In some embodiments, a targeting moiety may be or comprise one of more of an antibody (e.g., a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody) or any fragment thereof, for example an scFv, an aptamer, a darpin, a centyrin, a naturally occurring or synthetic receptor, an affibody, or other engineered protein recognition molecule, for example, to bind to one or more of CD 14, CD1 lb, CD163, CD206, CD33, CD209. In some embodiments, a targeting moiety may be or comprise a small molecule.

[0286] In some embodiments, a targeting moiety may be or comprise a particular lipid or combination of hydrophobic entities, for example, present in or forming an exterior surface of a liposome or lipid nanoparticle (e.g., for targeting to a particular cell type or cell types).Nucleic Acid Molecules

[0287] In some embodiments of the present disclosure, one or more nucleic acid molecules are or comprise DNA. In some embodiments of the present disclosure, one or more nucleic acid molecules are or comprise messenger RNA (mRNA). In some embodiments, mRNA according to the present disclosure may be synthesized as unmodified or modified mRNA. Typically, mRNAs are modified to enhance stability. Modifications of mRNA can include, for example, modifications of the nucleotides of the RNA. A modified mRNA according to the present disclosure can thus include, for example, backbone modifications, sugar modifications or base modifications. In some embodiments, a step of modifying an mRNA comprises causing the mRNA to include a modified nucleotide, an alteration to the 5’ or 3’ untranslated region (UTR), a cap structure, and / or a poly(A) tail. In some embodiments, provided nucleic acid molecules may be delivered to a population of immune cells (e.g., in a subject) via an in vivo delivery method (e.g., via a liposome or lipid nanoparticle).

[0288] In some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding CARs) may contain RNA backbone modifications. Typically, a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNAare modified chemically. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of mcthylphosphonatcs, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g. cytidine 5'-O-(l- thiophosphate)), boranophosphates, positively charged guanidinium groups etc., which comprises replacing the phosphodiester linkage by other anionic, cationic or neutral groups.

[0289] In some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding CARs) may contain sugar modifications. A typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 2'-deoxy-2'-fluoro-oligoribonucleotide (2'- fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'- deamine-oligoribonucleotide (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'- deoxyuridine 5 '-triphosphate), 2'-O-alkyloligoribonucleotide, 2'-deoxy-2'-C- alkyloligoribonucleotide (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyloligoribonucleotide, and isomers thereof (2'-aracytidine 5'-triphosphate, 2'-arauridine 5'-triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'- deoxyuridine 5'-triphosphate).

[0290] In some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding CARs) comprise modified nucleotide comprising pseudouridine (PsU), 5-methoxy uridine (5moU), 5-methylcytidine / pseudouridine (5meC PsU), Nl -methyl-pseudouridine (NlmPsU), or combinations thereof.

[0291] In some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding CARs) may contain modifications of the bases of the nucleotides (base modifications). A modified nucleotide which contains a base modification is also called a base-modified nucleotide.

[0292] Typically, mRNA synthesis includes the addition of a “cap” on the N-terminal (5’) end, and a “tail” on the C-terminal (3’) end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation.

[0293] Thus, in some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding CARs) include a 5’ cap structure. A 5’ cap is typically added as follows: first, an RNAterminal phosphatase removes one of the terminal phosphate groups from the 5’ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5’ triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G. In some embodiments, a cap comprises a CapO structure. A capO structures lack a 2'-O-methyl residue of the ribose attached to bases 1 and 2. In some embodiments, a cap comprises an AGCapl structure. An AGCapl structures has a 2'-O-methyl residue at base 2. In some embodiments, a cap comprises a Cap2 structure. Cap2 structures have a 2'-O-methyl residue attached to both bases 2 and 3. In some embodiments, a cap structure comprises AGCapl, m6AGCapl, or Anti-Reverse Cap Analog (ARCA). In some embodiments, a modified mRNA of the present disclosure comprises an m6AGCapl and modified nucleotides comprising pseudouridine (PsU).

[0294] In some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding CARs) include a 3’ poly(A) tail structure. A poly(A) tail on the 3' terminus of mRNA typically includes about 10 to 400 adenosine nucleotides (SEQ ID NO: 273) (e.g., about 100 to 400 adenosine nucleotides, about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, mRNAs include a 3’ poly(C) tail structure. A suitable poly(C) tail on the 3' terminus of mRNA typically include about 10 to 200 cytosine nucleotides (SEQ ID NO: 274) (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). A poly(C) tail may be added to a poly(A) tail or may be a substitute for the poly(A) tail.

[0295] In some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding CARs) include a 5’ and / or 3’ untranslated region. In some embodiments, a 5’ untranslated region includes one or more elements that affect an mRNA’s stability or translation, for example, an iron responsive element. In some embodiments, a 5’ untranslated region may be between about 50 and 500 nucleotides in length.

[0296] In some embodiments, a 3’ untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA’s stability of location in acell, or one or more binding sites for miRNAs. In some embodiments, a 3’ untranslated region may be between 50 and 500 nucleotides in length or longer.Administration of Additional Payloads

[0297] In some embodiments, methods of the present disclosure comprise one or more steps of treating an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) during the process of modifying the immune cell. In some embodiments, methods of the present disclosure comprise one or more steps of administering to a subject an additional payload for modulating an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) during the process of modifying the immune cell. In some embodiments, a composition may comprise one or more additional payloads. In some embodiments, a composition may comprise one or more additional pay loads in the same delivery vehicle as one or more nucleic acid molecules. In some embodiments, a composition may comprise one or more additional payloads in a different delivery vehicle than the one used with one or more nucleic acid molecules.

[0298] In some embodiments, methods of the present disclosure comprise a step of treating an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) with a modulator of a pathway activated by in vitro transcribed mRNA. In some embodiments, an additional payload may be or comprise a modulator of a pathway activated by in vitro transcribed mRNA. In vitro transcribed (IVT) mRNA is recognized by various endosomal innate immune receptors (Toll-like receptor 3 (TLR3), TLR7 and TLR8) and cytoplasmic innate immune receptors (protein kinase RNA-activated (PKR), retinoic acid-inducible gene I protein (RIG-I), melanoma differentiation- associated protein 5 (MDA5) and 2'-5 '-oligoadenylate synthase (OAS)). Signaling through these different pathways results in inflammation associated with type 1 interferon (IFN), tumor necrosis factor (TNF), interleukin-6 (IL-6), IL- 12 and the activation of cascades of transcriptional programs. Overall, these create a pro-inflammatory microenvironment poised for inducing specific immune responses. Moreover, downstream effects such as slow-down of translation by eukaryotic translation initiation factor 2a (eIF2a) phosphorylation, enhanced RNA degradation by ribonuclease L (RNaseL), and overexpression and inhibition of replication of self-amplifying mRNA are of relevance for the pharmacokinetics and pharmacodynamics of IVT mRNA.

[0299] In some embodiments, a modulator of a pathway activated by in vitro transcribed mRNA comprises an RNase inhibitor. In some embodiments, a modulator of a pathway activated by in vitro transcribed mRNA comprises an RNaseL, RNase T2 or RNasel inhibitor. In some embodiments, a modulator of a pathway activated by in vitro transcribed mRNA comprises an RNaseL inhibitor. In some embodiments, an RNaseL inhibitor comprises sunitinib. In some embodiments, an RNaseL inhibitor comprises ABCE1.

[0300] In some embodiments, treating an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) with an RNaseL inhibitor increases mRNA stability in a modified immune cell relative to mRNA stability in a modified immune cell of the same type that was not treated with an RNaseL inhibitor. In some embodiments, treating an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) with an RNaseL inhibitor increases CAR expression in a modified immune cell relative to CAR expression in a modified immune cell of the same type that was not treated with an RNaseL inhibitor. In some embodiments, treating an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) with an RNaseL inhibitor increases effector activity in a modified immune cell relative to effector activity in a modified immune cell of the same type that was not treated with an RNaseL inhibitor.

[0301] In some embodiments, administering to a subject an RNaseL inhibitor increases mRNA stability in a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) relative to mRNA stability in a modified immune cell of the same type in a subject that that was not administered an RNaseL inhibitor. In some embodiments, administering to a subject an RNaseL inhibitor increases CAR expression in a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) relative to CAR expression in a modified immune cell of the same type in a subject that was not administered an RNaseL inhibitor. In some embodiments, administering to a subject an RNaseL inhibitor increases effector activity in a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) relative to effector activity in a modified immune cell of the same type in a subject that was not administered an RNaseL inhibitor.

[0302] In some embodiments of the present disclosure, a step of treating an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) occurs before a step of delivering an mRNA to the immune cell. In some embodiments of the present disclosure, a step ofadministering an additional payload to a subject occurs before a step of administering a composition comprising an mRNA to the subject.

[0303] In some embodiments, methods of the present disclosure comprise a step of culturing an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) with a cytokine or immune stimulating recombinant protein. In some embodiments, methods of the present disclosure comprise a step of administering to a subject a cytokine or immune stimulating recombinant protein. In some embodiments, a cytokine comprises IFN-a, 1FN-P, IFN-y, TNFa, IL-6, STNGL, LPS, a CD40 agonist, a 4-1BB ligand, recombinant 4-1BB, a CD19 agonist, a TLR agonist (e.g., TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8 or TLR-9), TGF-0 (e.g., TGF-pi , TGF- p2, or TGF-P3), a glucocorticoid, an immune complex, interleukin-1 alpha (IL-la), IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, granulocyte-macrophage colony- stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), Leukemia inhibitory factor (LIF), oncostatin M (OSM), TNF-p, CD154, lymphotoxin beta (LT-P), an A proliferation-inducing ligand (APRIL), CD70, CDI53, glucocorticoid-induced TNF receptor ligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L (CD252), TALL-1 (Tumor necrosis factor ligand superfamily member 13B - TNFSF13B), TNF-related apoptosis-inducing ligand (TRAIL), TNF-related weak inducer of apoptosis (TWEAK), TNF-related activation-induced cytokine (TRANCE), erythropoietin (Epo), thyroid peroxidase precursor (Tpo), FMS-related tyrosine kinase 3 ligand (FLT-3L), stem cell factor (SCF), macrophage colony-stimulating factor (M-CSF), merozoite surface protein (MSP), a Nucleotide-binding oligomerization domaincontaining protein (NOD) ligand (e.g., NODI, NOD2, or NOD 1 / 2 agonists), a RIG-I-like receptor (RLR) ligand (e.g., 5'ppp-dsRNA, 3p-hpRNA, Poly(EC), or Poly(dA:dT)), a C-type lectin receptor (CLR) ligand (e.g., curdlan, P-glucan, HKCA, laminarin, pustulan, scleroglucan, WGP dispersible, WGP soluble, zymosan, zymosan depleted, furfurman, b-GlcCer, GlcC14C18, HKMT, TDB, TDB-HS15, or TDM), a cyclic dinucleotide sensor ligand (e.g., C-Gas agonist or stimulator of interferon gene (STING) ligand), an inflammasome inducer (e.g., alum, ATP, CPPD crystals, hemozoin, MSU crystals, Nano-SiO2, Nigericin, or TDB), an aryl hydrocarbon (AhR) ligand (e.g., FICZ, indirubin, ITE, or L-kynurenine), an alpha-protein kinase 1 (ALPK 1) ligand, a multi-PRR ligand, an NFKB / NFAT activator (e.g., concavalin A, ionomycin, PHA-P, or PMA) or combinations thereof. In some embodiments, a cytokine comprises IFN- .

[0304] In some embodiments of the present disclosure, a step of culturing an immune cell (c.g., a stem cell, macrophage, monocyte, or dendritic cell) occurs after a step of delivering an mRNA to the immune cell. In some embodiments of the present disclosure, a step of administering to a subject a cytokine or immune stimulating recombinant protein occurs after a step of administering a composition comprising an mRNA to the subject.

[0305] In some embodiments, culturing a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) with a cytokine or immune stimulating recombinant protein increases the viability of the modified immune cell relative to a modified immune cell of the same type that was not cultured with the cytokine or immune stimulating recombinant protein. In some embodiments, culturing a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) with a cytokine or immune stimulating recombinant protein increases protein (e.g., CAR) expression in the modified immune cell relative to a modified immune cell of the same type that was not cultured with the cytokine or immune stimulating recombinant protein. In some embodiments, culturing a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) with a cytokine or immune stimulating recombinant protein increases longevity of protein (e.g., CAR) expression relative to a modified immune cell of the same type that was not cultured with the cytokine or immune stimulating recombinant protein. In some embodiments, culturing a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) with a cytokine or immune stimulating recombinant protein increases effector activity of the modified immune cell relative to a modified immune cell of the same type that was not cultured with the cytokine or immune stimulating recombinant protein. In some embodiments, culturing a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) with a cytokine or immune stimulating recombinant protein increases pro-inflammatory (Ml) polarization of the modified immune cell relative to a modified immune cell of the same type that was not cultured with the cytokine or immune stimulating recombinant protein.

[0306] In some embodiments, administering to a subject a cytokine or immune stimulating recombinant protein increases the viability of a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulatingrecombinant protein. In some embodiments, administering to a subject a cytokine or immune stimulating recombinant protein increases protein (c.g., CAR) expression of a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulating recombinant protein. In some embodiments, administering to a subject a cytokine or immune stimulating recombinant protein increases longevity of protein (e.g., CAR) expression in a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulating recombinant protein. In some embodiments, administering to a subject a cytokine or immune stimulating recombinant protein increases effector activity of a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulating recombinant protein. In some embodiments, administering to a subject a cytokine or immune stimulating recombinant protein increases pro- inflammatory (Ml) polarization of a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulating recombinant protein.Methods of Altering the Inflammatory Phenotype of a Population of Cells

[0307] In some embodiments, methods of the present disclosure comprise altering the inflammatory phenotype of a population of cells. In some embodiments, methods of altering the inflammatory phenotype of a population of cells comprises contacting the population of cells with a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) as described herein. In some embodiments, a population of cells comprises macrophages, monocytes, dendritic cells, T cells, NK cells, or combinations thereof. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) as described herein secretes one or more inhibitory RNAs, which alters the inflammatory phenotype of a population of cells (e.g., bystander cells). In some embodiments, one or more inhibitory RNAs are packaged within an extracellular vehicle. In some embodiments, one or more inhibitory RNAs are packaged within an exosome.

[0308] In some embodiments, the inflammatory phenotype of the population of cells is altered from anti-inflammatory to non-activatcd. In some embodiments, the inflammatory phenotype of the population of cells is altered from pro-inflammatory to non-activated. In some embodiments, the inflammatory phenotype of the population of cells is altered from antiinflammatory to pro-inflammatory. In some embodiments, the inflammatory phenotype of the population of cells is altered from pro-inflammatory to anti-inflammatory.Modified Immune Cells

[0309] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure is made by methods of the present disclosure. In some embodiments, a modified immune cell of the present disclosure comprises one or more nucleic acids constructs of the present disclosure. In some embodiments, a modified immune cell of the present disclosure comprises one or more nucleic acids constructs comprising a promoter, a gene of interest, a 3’ untranslated region (UTR), and one or more introns, wherein the one or more introns comprise one or more inhibitory nucleic acids, wherein the one or more inhibitory nucleic acids encode one or more inhibitory RNAs, and, wherein the gene of interest encodes a chimeric antigen receptor (CAR). In some embodiments, a modified immune cell of the present disclosure comprises a CAR and one or more inhibitory RNAs.

[0310] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more inhibitory RNAs selected from the group consisting of antisense RNA (asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), guide RNA (gRNA), long noncoding RNA (IncRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), trans-acting siRNA (tasiRNA), and repeat associated siRNA (rasiRNA).

[0311] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more shRNA. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more shRNA comprising an miRNA scaffold. In some embodiments, an miRNA scaffold comprises an miRNA-155 5’ scaffold, an miRNA-155 3’ scaffold, an miRNA-30 5’ scaffold, an miRNA-30 3’ scaffold, an miRNA-16 5’ scaffold, anmiRNA-163’ scaffold, an miRNA-125 5’ scaffold, an miRNA-125 3’ scaffold, an miRNA-223 5’ scaffold, or an miRNA-223 3’ scaffold.

[0312] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more shRNA comprising a guide strand. In some embodiments, a guide strand is about 19-24 bases in length. In some embodiments, a guide strand has a G / C content of about 36%-50%. In some embodiments, a guide strand comprises a nucleic acid sequence that is reverse complementary to a target gene transcript comprising a target nucleic acid sequence.

[0313] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more shRNA comprising a passenger strand. In some embodiments, a passenger strand is 1-2 bases shorter than a corresponding guide strand. In some embodiments, a passenger strand is not fully complementary to the guide strand.

[0314] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more shRNA comprising a loop.

[0315] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more shRNA comprising (a) a nucleic acid sequence selected from Table la, Table lb, Table 1c, Table Id or Table le; a nucleic acid sequence that differs from a sequence selected from Table la, Table lb, Table 1c, Table Id or Table le by no more than five substitutions, additions, or deletions; or (c) a nucleic acid sequence that is at least 80% identical to a sequence selected from Table la, Table lb, Table 1c, Table Id or Table le.

[0316] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more shRNA comprising a nucleic acid sequence at least 80% identical to a sequence selected from Table la, Table lb, Table 1c, Table Id or Table le. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more shRNA comprising a nucleic acid sequence at least 85% identical to a sequence selected from Table la, Table lb, Table 1c, Table Id or Table le. In some embodiments, a modifiedimmune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more shRNA comprising a nucleic acid sequence at least 90% identical to a sequence selected from Table la, Table lb, Table 1c, Table Id or Table le. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more shRNA comprising a nucleic acid sequence at least 95% identical to a sequence selected from Table la, Table lb, Table 1c, Table Id or Table le. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more shRNA comprising a nucleic acid sequence at least 96% identical to a sequence selected from Table la, Table lb, Table 1c, Table Id or Table le. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more shRNA comprising a nucleic acid sequence at least 97% identical to a sequence selected from Table la, Table lb, Table 1c, Table Id or Table le. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more shRNA comprising a nucleic acid sequence at least 98% identical to a sequence selected from Table la, Table lb, Table 1c, Table Id or Table le. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more shRNA comprising a nucleic acid sequence at least 99% identical to a sequence selected from Table la, Table lb, Table 1c, Table Id or Table le. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more shRNA comprising a nucleic acid sequence identical to a sequence selected from Table la, Table lb, Table 1c, Table Id or Table le.

[0317] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more miRNA of the present disclosure.

[0318] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more miRNA comprising (a) a nucleic acid sequence selected from Table 2; a nucleic acid sequence that differs from asequence selected from Table 2 by no more than five substitutions, additions, or deletions; or (c) a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 2.

[0319] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more miRNA comprising a nucleic acid sequence at least 80% identical to a sequence selected from Table 2. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more miRNA comprising a nucleic acid sequence at least 85% identical to a sequence selected from Table 2. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more miRNA comprising a nucleic acid sequence at least 90% identical to a sequence selected from Table 2. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more miRNA comprising a nucleic acid sequence at least 95% identical to a sequence selected from Table 2. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more miRNA comprising a nucleic acid sequence at least 96% identical to a sequence selected from Table 2. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more miRNA comprising a nucleic acid sequence at least 97% identical to a sequence selected from Table 2. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more miRNA comprising a nucleic acid sequence at least 98% identical to a sequence selected from Table 2. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more miRNA comprising a nucleic acid sequence at least 99% identical to a sequence selected from Table 2. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more miRNA comprising a nucleic acid sequence identical to a sequence selected from Table 2.

[0320] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more target gene transcripts. In some embodiments, one or more target gene transcripts are mammalian. In someembodiments, one or more target gene transcripts are human. In some embodiments, contacting a target gene transcript with one or more inhibitory RNAs of the present disclosure reduces translation of the target gene transcript. In some embodiments, reduced translation of the target gene transcript in a modified cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure is associated with an Ml phenotype. In some embodiments, reduced translation of the target gene transcript in a modified cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure is associated with an M2 phenotype. In some embodiments, reduced translation of the target gene transcript in a modified cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure is associated with a pro-inflammatory phenotype. In some embodiments, reduced translation of the target gene transcript in a modified cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure is associated with an anti-inflammatory phenotype.

[0321] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more target gene transcripts comprising (a) a nucleic acid sequence selected from Table 3; a nucleic acid sequence that differs from a sequence selected from Table 3 by no more than five substitutions, additions, or deletions; or (c) a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 3.

[0322] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more target gene transcripts comprising a nucleic acid sequence at least 80% identical to a sequence selected from Table 3. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more target gene transcripts comprising a nucleic acid sequence at least 85% identical to a sequence selected from Table 3. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more target gene transcripts comprising a nucleic acid sequence at least 90% identical to a sequence selected from Table 3. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more target gene transcripts comprising a nucleic acid sequence at least 95% identical to a sequence selected fromTable 3. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more target gene transcripts comprising a nucleic acid sequence at least 96% identical to a sequence selected from Table 3. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more target gene transcripts comprising a nucleic acid sequence at least 97% identical to a sequence selected from Table 3. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more target gene transcripts comprising a nucleic acid sequence at least 98% identical to a sequence selected from Table 3. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more target gene transcripts comprising a nucleic acid sequence at least 99% identical to a sequence selected from Table 3. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises one or more target gene transcripts comprising a nucleic acid sequence identical to a sequence selected from Table 3.

[0323] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a target gene transcript encoding human ATG7, C / EBP-alpha, C / EBP-beta, CD36, CLEC1A, FATS, GOLM1, HAVCR2, ITGAD, KLF4, KLF6, LILRB2, LILRB4, MAF, MafB, PD-LI, PIK3CG, PIK3CG, PPARa, PPARy, PTGS2, SIGLEC10, SIRPa, SLC15A3, STAT3, STAT6, TNFRSF1B, TOX, TREM2, YTHDF2, or ZFP36. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a target gene transcript encoding human SIRPa. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a target gene transcript encoding human PD-LI.

[0324] In some embodiments, a modified immune cell of the present disclosure comprises a CAR comprising one or more extracellular domains, a transmembrane domain and one or more intracellular domains. In some embodiments, one or more extracellular domains are or comprise an scFv, VHH antibody, centyrin, darpin, or nanobody.

[0325] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR comprising a transmembrane domain comprising a CD8a, CD28, CD40, MyD88 CD64, CD32a, CD32c, CD 16a, CD3zeta, ICOS, DAP 10, DAP 12, Dectin- 1, DNGR1, SLAMF7, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, or TLR9 transmembrane domain.

[0326] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR comprising one or more intracellular domains comprising one or more of a CD3(^, FcRy, MyD88, CD40, CD64, CD32a, CD32c, CD16a, CD89, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphA 1 , INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1 , ROS1 , RYK, TIE2, TRK, VEGFR, CD19, CD20, 41BB, CD28, GCSFR (CD114), RAGE, CD30, CD160, DR3, Fnl4, HVEM, CD 160, NGFR, RANK, TNFR2, TROY, XEDAR, TRIF, 0X40, GITR, TREM-1, TREM-2, DAP 12, MR, ICOS, V / I / LxYxxL / V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD 170, CD33, BTLA, CD32b, SIRPb, CD22, PIR-B, LILRB1, 41BBL (TNFSF9), CD27, OX40L, CD32b, CD1 lb, ITGAM, SLAMF7, CD206, CD163, CD209, Dectin-2, IL1R, IL2R, IL3R, IL4R, IL5R, IL6R, IL7R, IL8R, IL9R, IL10R, IL11R, IL12R, IL13R, IL14R, IL15R, IL17R, IFNaR, IFNgR, TNFR, CSF1R, CSF2R, DAP10, CD36, Dectin- 1, ICOSL, or Syk intracellular domain, a portion of any of the foregoing, or combinations thereof. In some embodiments, one or more intracellular domains further comprise one or more of a CD20, CD40, DAP10, DAP12, Dectin-1, FcRy, MyD88, RAGE, SLAMF7, or TLR2 intracellular domain, a portion of any of the foregoing, or combinations thereof.

[0327] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR further comprising an extracellular leader domain. In some embodiments, an extracellular leader domain is or comprises a CD8a extracellular leader domain.

[0328] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR further comprising an extracellular hinge domain. In some embodiments, an extracellular hinge domain is or comprises: a DAP10 extracellular hinge domain, a DAP12 extracellular hinge domain, a CD8aextracellular hinge domain, a CD28 extracellular hinge domain, a DNGR-1 extracellular hinge domain, a Dcctin-1 extracellular hinge domain, or an IgG4 extracellular hinge domain.

[0329] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR comprising (a) an amino acid sequence selected from Table 5; an amino acid sequence that differs from a sequence selected from Table 5 by no more than five substitutions, additions, or deletions; or (c) an amino acid sequence that is at least 80% identical to a sequence selected from Table 5.

[0330] In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR comprising (a) an amino acid sequence selected from Table 6; an amino acid sequence that differs from a sequence selected from Table 6 by no more than five substitutions, additions, or deletions; or (c) an amino acid sequence that is at least 80% identical to a sequence selected from Table 6.

[0331] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR comprising an amino acid sequence at least 80% identical to a sequence selected from Table 5. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR comprising an amino acid sequence at least 85% identical to a sequence selected from Table 5. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR comprising an amino acid sequence at least 90% identical to a sequence selected from Table 5. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR comprising an amino acid sequence at least 95% identical to a sequence selected from Table 5. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR comprising an amino acid sequence at least 96% identical to a sequence selected from Table 5. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR comprising an amino acid sequence at least 97% identical to a sequence selected from Table 5. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR comprising an amino acid sequence at least 98%identical to a sequence selected from Table 5. In some embodiments, a modified immune cell (c.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR comprising an amino acid sequence at least 99% identical to a sequence selected from Table 5. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR comprising an amino acid sequence identical to a sequence selected from Table 5.

[0332] In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR comprising an amino acid sequence at least 80% identical to a sequence selected from Table 6. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR comprising an amino acid sequence at least 85% identical to a sequence selected from Table 6. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR comprising an amino acid sequence at least 90% identical to a sequence selected from Table 6. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR comprising an amino acid sequence at least 95% identical to a sequence selected from Table 6. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR comprising an amino acid sequence at least 96% identical to a sequence selected from Table 6. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR comprising an amino acid sequence at least 97% identical to a sequence selected from Table 6. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR comprising an amino acid sequence at least 98% identical to a sequence selected from Table 6. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR comprising an amino acid sequence at least 99% identical to a sequence selected from Table 6. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR comprising an amino acid sequence identical to a sequence selected from Table 6.

[0333] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR comprising an amino acid sequence that differs from a sequence selected from Table 5 by no more than five substitutions, additions, or deletions. In some embodiments, a modified immune cell (e.g., aIllstem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR comprising an amino acid sequence that differs from a sequence selected from Table 5 by no more than four substitutions, additions, or deletions. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR comprising an amino acid sequence that differs from a sequence selected from Table 5 by no more than three substitutions, additions, or deletions. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR comprising an amino acid sequence that differs from a sequence selected from Table 5 by no more than two substitutions, additions, or deletions. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR comprising an amino acid sequence that differs from a sequence selected from Table 5 by no more than one substitution, addition, or deletion. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR comprising an amino acid sequence that differs from a sequence selected from Table 5 by zero substitutions, additions, or deletions.

[0334] In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR comprising an amino acid sequence that differs from a sequence selected from Table 6 by no more than five substitutions, additions, or deletions. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR comprising an amino acid sequence that differs from a sequence selected from Table 6 by no more than four substitutions, additions, or deletions. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR comprising an amino acid sequence that differs from a sequence selected from Table 6 by no more than three substitutions, additions, or deletions. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR comprising an amino acid sequence that differs from a sequence selected from Table 6 by no more than two substitutions, additions, or deletions. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR comprising an amino acid sequence that differs from a sequence selected from Table 6 by no more than one substitution, addition, or deletion. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises aCAR comprising an amino acid sequence that differs from a sequence selected from Table 6 by zero substitutions, additions, or deletions.

[0335] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR encoded by a nucleic acid sequence at least 80% identical to a sequence selected from Table 4. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR encoded by a nucleic acid sequence at least 85% identical to a sequence selected from Table 4. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR encoded by a nucleic acid sequence at least 90% identical to a sequence selected from Table 4. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR encoded by a nucleic acid sequence at least 95% identical to a sequence selected from Table 4. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR encoded by a nucleic acid sequence at least 96% identical to a sequence selected from Table 4. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR encoded by a nucleic acid sequence at least 97% identical to a sequence selected from Table 4. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR encoded by a nucleic acid sequence at least 98% identical to a sequence selected from Table 4. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR encoded by a nucleic acid sequence at least 99% identical to a sequence selected from Table 4. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR encoded by a nucleic acid sequence identical to a sequence selected from Table 4. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR encoded by a nucleic acid sequence that differs from a sequence selected from Table 4, but encodes an amino acid sequence identical to a sequence selected from Table 5.

[0336] In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR encoded by a nucleic acid sequence at least 80% identical to a sequence selected from Table 8. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR encoded by a nucleic acid sequence at least 85% identical to a sequence selected from Table 8. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR encoded by a nucleic acid sequence at least 90% identical to a sequence selected from Table 8. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR encoded by a nucleic acid sequence at least 95% identical to a sequence selected from Table 8. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR encoded by a nucleic acid sequence at least 96% identical to a sequence selected from Table 8. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR encoded by a nucleic acid sequence at least 97 % identical to a sequence selected from Table 8. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR encoded by a nucleic acid sequence at least 98% identical to a sequence selected from Table 8. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR encoded by a nucleic acid sequence at least 99% identical to a sequence selected from Table 8. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR encoded by a nucleic acid sequence identical to a sequence selected from Table 8. In some embodiments, a modified immune cell (e.g., a monocyte) of the present disclosure comprises a CAR encoded by a nucleic acid sequence that differs from a sequence selected from Table 8, but encodes an amino acid sequence identical to a sequence selected from Table 6.

[0337] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a CAR comprising an extracellular domain that binds a tumor antigen, such as an antigen that is specific for a tumor or cancer of interest. In some embodiments a tumor antigen comprises one or more antigenic cancer epitopes. In some embodiments, a tumor antigen comprises CD 19; CD 123; CD22;CD30; CD171 ; CS-1 (also referred to as CD2 subset 1 , CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l -l)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Scr / Thr)); prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (R0R1); Fms-Like Tyrosine Kinase 3 (FLT3); Tumor- associated glycoprotein 72 (TAG72); CD38; CD44v6; Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha (IL- HRa); prostate stem cell antigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage- specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha; Receptor tyrosine -protein kinase ERBB2 (Her2 / neu); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100 (gplOO); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type- A receptor 2 (EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer); transglutaminase 5 (TGS5); high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta- specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51 E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer / testis antigen 1 (NY-ESO- 1); Cancer / testis antigen 2 (LAGE-la); Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17(SPA 17); X Antigen Family, Member 1 A (XAGE1 ); angiopoietin-binding cell surface receptor 2 (Tic 2); melanoma cancer testis antigcn-1 (MAD-CT-1); melanoma cancer testis antigcn-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase; prostate carcinoma tumor antigen- 1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N- Acetyl glucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin Bl; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP- 2); Cytochrome P450 1B1 (CYP1B1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Glycation Endproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucinlike hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); or immunoglobulin lambda-like polypeptide 1 (IGLL1). In certain embodiments, a tumor antigen comprises ERBB2 (Her2 / neu). In certain embodiments, a tumor antigen comprises PSMA. In certain embodiments, a tumor antigen comprises Mesothelin.

[0338] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as described herein maintains a pro-inflammatory phenotype over time. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as described herein maintains a pro-inflammatory phenotype at least 4 hours, 2 days, 4 days, 7 days, 14 days, and / or 28 days after an immune cell is modified with a nucleic acid encoding the CAR.

[0339] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as described herein maintains an antiinflammatory phenotype over time. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as described herein maintains an anti-inflammatory phenotype at least 4 hours, 2 days, 4 days, 7 days, 14 days, and / or 28 days after an immune cell is modified with a nucleic acid encoding the CAR.

[0340] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as described herein maintains a pro-inflammatory phenotype and / or otherwise resists subversion when challenged by anti-inflammatory cytokines. In some embodiments, the sensitivity of a modified immune cell to environmental cytokines is measured by generating a dose-response curve of pro-inflammatory markers by treating modified immune cells comprising a CAR as described herein with increasing concentrations of antiinflammatory cytokines. In some embodiments, the sensitivity of a modified immune cell to environmental cytokines is measured by generating a dose-response curve of pro-inflammatory markers by treating modified immune cells comprising a CAR as described herein with increasing concentrations of pro-inflammatory cytokines.

[0341] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as described herein maintains an antiinflammatory phenotype and / or otherwise resists subversion when challenged by pro- inflammatory cytokines. In some embodiments, the sensitivity of a modified immune cell to environmental cytokines is measured by generating a dose-response curve of anti-inflammatory markers by treating modified immune cells comprising a CAR as described herein with increasing concentrations of pro-inflammatory cytokines. In some embodiments, the sensitivity of a modified immune cell to environmental cytokines is measured by generating a doseresponse curve of anti-inflammatory markers by treating modified immune cells comprising a CAR as described herein with increasing concentrations of anti-inflammatory cytokines.

[0342] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as described herein has minimal effects onneighboring cells. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as described herein has significant effects on neighboring cells. In some embodiments, the effect of a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as described herein on an unmodified cell (e.g., an immune cell that doesn’t comprise a CAR as described herein) can be tested by co-culturing modified immune cells with unmodified immune cells and using flow cytometry to analyze the expression of pro-inflammatory and anti-inflammatory markers in the unmodified cells. In some embodiments, modified immune cells and unmodified immune cells can be co-cultured in a culture dish where the modified immune cells and unmodified immune cells contact each other. In some embodiments, modified immune cells and unmodified immune cells can be co-cultured in a culture dish where the modified immune cells and unmodified immune cells are separated by a transwell assay membrane.

[0343] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as described herein has minimal cytotoxic effects on neighboring cells. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as described herein has significant cytotoxic effects on neighboring cells (e.g., cancer cells). In some embodiments, modifying an immune cell to comprise a CAR as described herein is not cytotoxic to the modified immune cell. In some embodiments, RNAseq data from modified immune cells are examined to determine if upregulation of genes indicative of cytotoxic effects is present.

[0344] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as described herein may comprise one or more control systems including, but not limited to: a safety switch (e.g., an on switch, an off switch, a suicide switch), transcriptional control (e.g. cell-specific promoters, cell-state specific promoters, promoters downstream of CAR activation, promoters downstream of endogenous signaling pathways, or drug-inducible transcription), post-transcriptional control of CAR mRNA (e.g. RNA-based inhibition with endogenous or recombinant miRNA), or post-translational control of a CAR’s structure or stability (e.g. a CAR whose intracellular domain conditionally associates with the full structure by drug / light-inducible association (to allow signaling) or dissociation (to inhibit signaling), or whose stability is drug-regulated for inducible stabilization (to allowsignaling) or degradation (to inhibit signaling)). These control systems can be combined to create logic gates, for example an AND gate (c.g. a CAR with a CAR-inducible promoter and cytosolic domain that associates in a drug-dependent manner, thus requiring CAR activation and the presence of a small molecule), an OR gate (e.g. a CAR under control of a promoter that is transcriptionally active following CAR activation or small molecule addition), and / or a NOT gate (e.g. a CAR whose mRNA is degraded by endogenous miRNAs expressed in natural immune cell signaling states (such as miRNAs upregulated by a particular cytokine signaling pathway, thus only expressing CAR in the absence of this cytokine)).

[0345] In some embodiments, an effector cell (i.e., a modified immune cell of the present disclosure (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a nucleic acid construct as described herein secretes one or more inhibitory RNAs of the present disclosure. In some embodiments, one or more inhibitory RNAs are packaged within an extracellular vesicle. In some embodiments, one or more inhibitory RNAs are packaged within an exosome. In some embodiments, secreted inhibitory RNA affects the phenotype of bystander cells. In some embodiments, bystander cells are bystander macrophages, bystander monocytes, bystander dendritic cells, or bystander stem cells. In some embodiments, bystander cells are skewed to an anti-tumor phenotype. In some embodiments, bystander cells are skewed to an antiinflammatory phenotype. In some embodiments, bystander macrophages are skewed to an Ml phenotype. In some embo...

Claims

CLAIMS1. A nucleic acid construct comprising a promoter, a gene of interest, a 3’ untranslated region (UTR), and one or more introns, wherein the one or more introns comprise one or more inhibitory nucleic acids, wherein the one or more inhibitory nucleic acids encode one or more inhibitory RNAs, and, wherein the gene of interest encodes a chimeric antigen receptor (CAR).

2. The nucleic acid construct of claim 1, wherein the one or more inhibitory RNAs are selected from the group consisting of antisense RNA (asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), guide RNA (gRNA), long noncoding RNA (IncRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), trans-acting siRNA (tasiRNA), and repeat associated siRNA (rasiRNA).

3. The nucleic acid construct of claim 1 or 2, wherein the one or more inhibitory RNAs are or comprise one or more shRNA.

4. The nucleic acid construct of claim 3, wherein the one or more shRNA comprise an miRNA scaffold.

5. The nucleic acid construct of claim 4, wherein the miRNA scaffold comprises an miRNA- 155 5’ scaffold, an miRNA- 155 3’ scaffold, an miRNA-30 5’ scaffold, an miRNA-30 3’ scaffold, an miRNA- 16 5’ scaffold, an miRNA- 16 3’ scaffold, an miRNA- 125 5’ scaffold, an miRNA- 125 3’ scaffold, an miRNA-223 5’ scaffold, or an miRNA-223 3’ scaffold.

6. The nucleic acid construct of any one of claims 3-5, wherein the one or more shRNA comprise a guide strand.

7. The nucleic acid construct of claim 6, wherein the guide strand is about 19-24 bases in length.

8. The nucleic acid construct of claim 6 or 7, wherein the guide strand has a G / C content of about 36%-50%.

9. The nucleic acid construct of any one of claims 6-8, wherein the guide strand comprises a nucleic acid sequence that is reverse complementary to a target gene transcript comprising a target nucleic acid sequence.

10. The nucleic acid construct of any one of claims 6-9, wherein the one or more shRNA comprise a passenger strand.

11. The nucleic acid construct of claim 10, wherein the passenger strand is 1-2 bases shorter than the guide strand.

12. The nucleic acid construct of claim 10 or 11, wherein the passenger strand is not fully complementary to the guide strand.

13. The nucleic acid construct of any one of claims 3-12, wherein the one or more shRNA comprise a loop.

14. The nucleic acid construct of any one of claims 3-13, wherein the one or more shRNA comprise:(a) a nucleic acid sequence selected from Table la, Table lb, Table 1c, Table Id or Table le;(b) a nucleic acid sequence that differs from a sequence selected from Table la, Table lb, Table 1c, Table Id or Table le by no more than 5 substitutions, additions, or deletions; or(c) a nucleic acid sequence that is at least 80% identical to a sequence selected from Table la, Table lb, Table le, Table Id or Table le.

15. The nucleic acid construct of claim 1 or 2, wherein the one or more inhibitory RNAs are or comprise one or more miRNA.

16. The nucleic acid construct of claim 3, wherein the one or more miRNA arc selected from Table 2.

17. The nucleic acid construct of claim 11, wherein the target gene transcript is mammalian.

18. The nucleic acid construct of claim 11 or 17, wherein the target gene transcript is human.

19. The nucleic acid construct of any one of claims 11-18, wherein contacting the target gene transcript with the one or more inhibitory RNAs reduces translation of the target gene transcript.

20. The nucleic acid construct of claim 19, wherein the target gene transcript is expressed in an immune cell.

21. The nucleic acid construct of claim 20, wherein the immune cell is a stem cell, macrophage, monocyte, or dendritic cell.

22. The nucleic acid construct of claim 20, wherein the immune cell is an induced pluripotent stem cell (iPSC)-derived monocyte, macrophage, or dendritic cell.

23. The nucleic acid construct of claim 20, 21 or 22, wherein the target gene transcript is expressed in a macrophage.

24. The nucleic acid construct of claim 23, wherein reduced translation of the target gene transcript is associated with an Ml phenotype.

25. The nucleic acid construct of claim 23, wherein reduced translation of the target gene transcript is associated with an M2 phenotype.

26. The nucleic acid construct of any one of claims 11-25, wherein the target nucleic acid sequence comprises:(a) a nucleic acid sequence selected from Table 3;(b) a nucleic acid sequence that differs from a sequence selected from Table 3 by no more than 5 substitutions, additions, or deletions; or(c) a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 3.

27. The nucleic acid construct of any one of claims 11-26, wherein the target gene transcript encodes human ATG7, C / EBP-alpha, C / EBP-beta, CD32b, CD36, CLEC1A, FATS, GOLM1, HAVCR2, ITGAD, KLF4, KLF6, LILRB1, LILRB2, LILRB4, MAF, MafB, PD1, PD-LI, PIK3CG, PIK3CG, PPARa, PPARy, PTGS2, SIGLEC10, SIRPa, SLAMF3, SLAMF4, SLC15A3, STAT3, STAT6, TNFRSF1B, TOX, TREM2, YTHDF2, or ZFP36.

28. The nucleic acid construct of any one of claims 11-27, wherein the target gene transcript encodes a human anti-phagocytic receptor selected from the group consisting of: SIRPa, LILRB1, SIGLEC10, PD1, SLAMF3, SLAMF4, CLEC1A, and CD32b.

29. The nucleic acid construct of any one of claims 11-27, wherein the target gene transcript encodes human SIRPa.

30. The nucleic acid construct of any one of claims 11-27, wherein the target gene transcript encodes human PD-LI.

31. The nucleic acid construct of any one of claims 11-27, wherein the target gene transcript encodes human PD 1.

32. The nucleic acid construct of any one of claims 11-27, wherein the target gene transcript encodes human LILRB 1.

33. The nucleic acid construct of any one of claims 11-27, wherein the target gene transcript encodes human SIGLEC10.

34. The nucleic acid construct of any one of claims 1 1 -27, wherein the target gene transcript encodes human SLAMF3.

35. The nucleic acid construct of any one of claims 11-27, wherein the target gene transcript encodes human SLAMF4.

36. The nucleic acid construct of any one of claims 11-27, wherein the target gene transcript encodes human CLEC1A.

37. The nucleic acid construct of any one of claims 11-27, wherein the target gene transcript encodes human CD32b.

38. The nucleic acid construct of any one of the preceding claims, wherein the one or more introns comprise one, two, or three introns.

39. The nucleic acid construct of any one of the preceding claims, wherein the one or more introns are located downstream of the promoter.

40. The nucleic acid construct of claim 39, wherein the one or more introns are located within the gene of interest.

41. The nucleic acid construct of claim 39, wherein the one or more introns are located within the 3’ UTR.

42. The nucleic acid construct of any one of the preceding claims, wherein the one or more inhibitory nucleic acids comprise one inhibitory nucleic acid.

43. The nucleic acid construct of any of claims 1-41, wherein the one or more inhibitory nucleic acids comprise at least two, three, four or five inhibitory nucleic acids.

44. The nucleic acid construct of claim 43, wherein the at least two, three, four, or five inhibitory nucleic acids arc in tandem.

45. The nucleic acid construct of claim 43 or 44, wherein the at least two, three, four, or five inhibitory nucleic acids comprise identical sequences.

46. The nucleic acid construct of claim 43 or 44, wherein the at least two, three, four, or five inhibitory nucleic acids comprise at least two different sequences.

47. The nucleic acid construct of claim 43 or 44, wherein the at least two, three, four, or five inhibitory nucleic acids target at least two, three, four, or five different anti-phagocytic receptors.

48. The nucleic acid construct of claim 43 or 44, wherein the at least two, three, four, or five inhibitory nucleic acids encode inhibitory RNAs comprising nucleic acid sequences that are reverse complementary to the same target gene transcript.

49. The nucleic acid construct of claim 43 or 44, wherein the at least two, three, four, or five inhibitory nucleic acids encode inhibitory RNAs comprising nucleic acid sequences that are reverse complementary to different target gene transcripts.

50. The nucleic acid construct of claim 43 or 3444 wherein the at least two, three, four, or five inhibitory nucleic acids encode inhibitory RNAs comprising identical miRNA scaffolds.

51. The nucleic acid construct of claim 43 or 44, wherein the at least two, three, four, or five inhibitory nucleic acids encode inhibitory RNAs comprising at least two different miRNA scaffolds.

52. The nucleic acid construct of claim 51 , wherein the at least two different miRNA scaffolds are selected from the group consisting of an miRNA-155 5’ scaffold, an miRNA-155 3’ scaffold, an miRNA-30 5’ scaffold, and an miRNA-303’ scaffold.

53. The nucleic acid construct of claim 43 or 44, wherein the intron comprises three inhibitory nucleic acids, wherein the three inhibitory nucleic acids encode three inhibitory RNAs, wherein the three inhibitory RNAs comprise the following miRNA scaffolds in order from 5’ to 3’:(a) miRNA 30, miRNA 30, miRNA 30,(b) miRNA 30, miRNA 155, miRNA 30,(c) miRNA 155, miRNA 30, miRNA 155, or(d) miRNA 155, miRNA 155, miRNA 155.

54. The nucleic acid construct of any one of the preceding claims, wherein expression of the CAR is increased relative to a reference nucleic acid construct that lacks the one or more introns.

55. The nucleic acid construct of any one of the preceding claims, wherein the CAR comprises:(a) one or more extracellular domains;(b) a transmembrane domain; and(c) one or more intracellular domains.

56. The nucleic acid construct of claim 55, wherein the one or more extracellular domains are or comprise an scFv, VHH antibody, centyrin, darpin, or nanobody.

57. The nucleic acid construct of claim 55 or 56, wherein the transmembrane domain is or comprises a CD8a, CD28, CD40, MyD88, CD64, CD32a, CD32c, CD16a, CD3zeta, DAP10, DAP12, Dectin-1, DNGR1, ICOS, NKG2D, RAGE, SLAMF7, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, or TLR9 transmembrane domain.

58. The nucleic acid construct of any one of claims 55-57, wherein the one or more intracellular domains comprise one or more of a CD3(^, FcRy, MyD88, CD40, CD64, CD32a, CD32c, CD16a, CD89, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK,TIE2, TRK, VEGFR, CD19, CD20, 41BB, CD28, GCSFR (CD114), RAGE, CD30, CD160, DR3, Fnl4, HVEM, CD 160, NGFR, RANK, TNFR2, TROY, XEDAR, TRIF, 0X40, GITR, TREM-1, TREM-2, DAP 12, MR, ICOS, V / I / LxYxxL / V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD 170, CD33, BTLA, CD32b, SIRPb, CD22, PIR-B, LILRB1, 41BBL (TNFSF9), CD27, OX40L, CD32b, CDllb, ITGAM, SLAMF7, CD206, CD163, CD209, Dectin-2, IL1R, IL2R, IL3R, IL4R, IL5R, IL6R, IL7R, IL8R, IL9R, IL10R, IL11R, IL12R, IL13R, IL14R, IL15R, IL17R, IFNaR, IFNgR, TNFR, CSF1R, CSF2R, DAP10, CD36, Dectin- 1, ICOSL, Syk, TRAM, ZAP-70, or NKG2D intracellular domain, a portion of any of the foregoing, or combinations thereof.

59. The nucleic acid construct of any one of claims 55-58, wherein the one or more intracellular domains further comprise one or more of a CD3(^, FcRy, MyD88, CD40, CD64, CD32a, CD32c, CD16a, CD89, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD19, CD20, 41BB, CD28, GCSFR (CD114), RAGE, CD30, CD160, DR3, Fnl4, HVEM, CD160, NGFR, RANK, TNFR2, TROY, XEDAR, TRIF, 0X40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, V / I / LxYxxL / V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD 170, CD33, BTLA, CD32b, SIRPb, CD22, PIR-B, LILRB1, 41BBL (TNFSF9), CD27, OX40L, CD32b, CDl lb, ITGAM, SLAMF7, CD206, CD163, CD209, Dectin-2, IL1R, IL2R, IL3R, IL4R, IL5R, IL6R, IL7R, IL8R, IL9R, IL10R, IL11R, IL12R, IL13R, IL14R, IL15R, IL17R, IFNaR, IFNgR, TNFR, CSF1R, CSF2R, DAP10, CD36, Dectin-1, ICOSL, Syk, TRAM, ZAP-70, or NKG2D intracellular domain, a portion of any of the foregoing, or combinations thereof.

60. The nucleic acid construct of any one of claims 55-59, wherein the CAR further comprises an extracellular leader domain.

61. The nucleic acid construct of claim 60, wherein the extracellular leader domain is or comprises a CD8a extracellular leader domain.

62. The nucleic acid construct of any one of clams 55-61 , wherein the CAR further comprises an extracellular hinge domain.

63. The nucleic acid construct of claim 62, wherein the extracellular hinge domain is or comprises: a DAP10 extracellular hinge domain, a DAP12 extracellular hinge domain, a CD8a extracellular hinge domain, a CD28 extracellular hinge domain, a DNGR-1 extracellular hinge domain, a Dectin- 1 extracellular hinge domain, and / or an IgG4 extracellular hinge domain.

64. The nucleic acid construct of any one of claims 55-63, wherein the CAR further comprises a cleavage peptide.

65. The nucleic acid construct of claim 64, wherein the cleavage peptide is or comprises a P2A, F2A, E2A or T2A peptide.

66. The nucleic acid construct of any one of claims 55-65, wherein the CAR comprises:(a) an amino acid sequence selected from Table 5;(b) an amino acid sequence that differs from a sequence selected from Table 5 by no more than five substitutions, additions, or deletions; or(c) an amino acid sequence that is at least 80% identical to a sequence selected from Table 5.

67. The nucleic acid construct of any one of claims 55-65, wherein the nucleic acid construct comprises:(a) a nucleic acid sequence selected from Table 4;(b) a nucleic acid sequence that differs from a sequence selected from Table 4 by no more than five substitutions, additions, or deletions; or(c) a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 4.

68. The nucleic acid construct of any one of the preceding claims, wherein the nucleic acid construct comprises a modification selected from: a modified nucleotide, an alteration to the 5’ UTR, an alteration to the 3’ UTR, or combinations thereof.

69. The nucleic acid construct of claim 68, wherein the modified nucleotide comprises pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine / pseudouridine (5meC PsU), Nl-methyl-pseudouridine (NlmPsU), or combinations thereof.

70. The nucleic acid construct of any one of the preceding claims, wherein the nucleic acid construct is a purified nucleic acid construct.

71. The nucleic acid construct of any one of the preceding claims, wherein at least a portion of the nucleic acid construct is codon-optimized.

72. The nucleic acid construct of claim 71, wherein at least a portion of the nucleic acid construct is codon-optimized for expression in a stem cell, monocyte, macrophage, or dendritic cell.

73. A modified immune cell comprising the nucleic acid construct of any one of the preceding claims.

74. The modified immune cell of claim 73, wherein the cell is or comprises a stem cell, macrophage, monocyte, or dendritic cell.

75. The nucleic acid construct of claim 73, wherein the cell is an induced pluripotent stem cell (iPSC)-derived monocyte, macrophage, or dendritic cell.

76. A pharmaceutical composition comprising the nucleic acid construct of any of claims 1-60 or the modified immune cell of claim 73 or 74.

77. The pharmaceutical composition of claim 76, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

78. A method of treating a disease or disorder in a subject, the method comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 76 or 77, wherein at least one sign or symptom of the disease or disorder is improved in the subject.

79. The method of claim 78, wherein the step of administering is or comprises transarterial, subcutaneous, intravenous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, or intraperitoneal delivery.

80. A method of modifying an immune cell, the method comprising: delivering to the immune cell the nucleic acid construct of any one of claims 1-72, thereby producing a modified immune cell, wherein the modified immune cell is or comprises a macrophage, monocyte, dendritic cell, or stem cell.

81. The method of claim 80, wherein the nucleic acid construct comprises DNA or RNA.

82. The method of claim 80 or 81, wherein the delivering comprises electroporation or transfection with the nucleic acid construct.

83. The method of claim 80 or 81, wherein the nucleic acid construct is within a delivery vehicle.

84. The method of claim 83, wherein the delivery vehicle is or comprises a liposome, a lipid nanoparticle, a polymer, an adeno-associated viral (AAV) vector, an adenoviral vector, a retroviral vector, a transposon, a retrotransposon, a site-directed insertion vector or combinations thereof.

85. The method of claim 84, wherein the retroviral vector comprises a lentiviral vector or a gammaretroviral vector.

86. The method of claim 85, wherein the lentiviral vector is packaged with a Vpx protein.

87. The method of claim 84, wherein the adenoviral vector comprises an Ad2 vector or anAd5 vector.

88. The method of claim 87, wherein the Ad5 vector comprises an Ad5f35 adenoviral vector.

89. The method of claim 84, wherein the retrotransposon comprises sleeping beauty or piggyBac.

90. The method of claim 84, wherein the site-directed insertion vector comprises a CRISPR / Cas system.

91. The method of any of claims 80-90, the method further comprising delivering to the immune cell an additional payload.

92. The method of claim 91, wherein the additional payload is or comprises a pathogen recognition receptor agonist, polyinosinic:polycy tidy lie acid (poly I:C), a TLR7 / 8 agonist, a CpG oligodeoxynucleotide, a NOD-like receptor (NLR) agonist, a RIG-I-like receptor (RLR) agonist, a C-type lectins receptor (CLR) agonist, a cytosolic DNA sensing, the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) agonist, an interferon-inducible protein 16 (IFI16) agonist, a DEAD-box helicase 41 (DDX41) agonist, an LRR binding FLII interacting protein 1 (LRRFIP1) agonist, an absent in melanoma 2 (AIM2) agonist, an aryl hydrocarbon receptor (AhR) ligand, or combinations thereof.

93. The method of claim 91 or 92, wherein the nucleic acid construct and the additional payload are encapsulated within the delivery vehicle.

94. A method of designing an shRNA comprising a guide strand, a passenger strand, and a loop, the method comprising the steps of:(a) selecting a target region of a target gene transcript,(b) designing the guide strand, wherein the guide strand is a reverse complement of the target region, and wherein the guide strand comprises, from 5’ to 3’, an adenine or uracil at position 1, an adenine, guanine, or cytosine at position 10, and a G / C content between 36% and 45% that is increases from 5’ to 3’; and(c) designing the passenger strand, wherein the passenger strand comprises the sequence of the target region, from 5’ to 3’, with the following variations:(i) deletion of a nucleotide at position 7 relative to the loop,(ii) deletion of a nucleotide at position 11 relative to the loop,(iii) mutating an adenine to guanine and / or mutating a cytosine to uracil near the deletions at positions 7 and 11, wherein the mutations create two partial G-U base pairs between the passenger strand and the guide strand; and wherein the passenger strand is 2 base pairs shorter than the guide strand.

95. The method of claim 94, wherein the guide strand comprises a higher G / C content of the 10 bases at the 3’ end of the guide strand relative to the G / C content of the 10 bases at the 5’ end of the guide strand.

96. The method of claim 94, wherein the target region of the target gene transcript is 21-22 base pairs in length.

97. An effector cell comprising the nucleic acid construct of any one of claims 1-72 wherein the one or more inhibitory RNAs are secreted from the cell.

98. The effector cell of claim 97, wherein the one or more inhibitory RNAs are packaged within an extracellular vesicle.

99. The effector cell of claim 97, wherein the one or more inhibitory RNAs arc packaged within an exo some.

100. The effector cell of any one of claims 97-99, wherein the secreted inhibitory RNA affects the phenotype of bystander cells.

101. The effector cell of claim 100, wherein the bystander cells are bystander macrophages, bystander monocytes, bystander dendritic cells, or bystander stem cells.

102. The effector cell of claim 101, wherein the bystander macrophages are skewed to an Ml phenotype.

103. The effector cell of claim 101, wherein the bystander macrophages are skewed to an M2 phenotype.

104. The effector cell of any one of claims 100-102, wherein the bystander cells are skewed to an anti-tumor phenotype.

105. The effector cell of any one of claims, 100, 101, or 103, wherein the bystander cells are skewed to an anti-inflammatory phenotype.

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