Novel anti-mesothelin chimeric antigen receptors and modified immune cells

Abstract

The present disclosure relates to modified immune cells comprising an anti-mesothelin chimeric antigen receptor, and methods of using and making immune cells comprising an anti-mesothelin chimeric antigen receptor. In one aspect, the disclosure provides a modified immune cell comprising a chimeric antigen receptor (CAR), the CAR comprising: (a) an extracellular domain, (b) a transmembrane domain, and (c) one or more intracellular domains, wherein the extracellular domain is or comprises an anti-mesothelin antigen binding domain comprising an amino acid sequence at least 80% identical to a sequence selected from Table 3, and the modified immune cell is or comprises a macrophage, monocyte, dendritic cell, or stem cell.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application Nos. 63 / 412,622, filed October 3, 2023, and 63 / 459,903, filed April 17, 2023, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Mesothelin is a tumor antigen highly expressed in many human cancers, including malignant mesothelioma and adenocarcinomas of the pancreas, ovary, and lung. It is an attractive target for cancer immunotherapy because its normal expression in humans is restricted to mesothelial cells. Therefore, there is a need to develop new therapeutic modalities optimized to target mesothelin. Summary of the Invention [Means for solving the problem]

[0003] The present disclosure encompasses, inter alia, compositions comprising modified immune cells (e.g., stem cells, macrophages, monocytes, and / or dendritic cells) comprising an anti-mesothelin chimeric antigen receptor (CAR), as well as methods of making and using such compositions.

[0004] In one aspect, the disclosure provides a modified immune cell comprising a chimeric antigen receptor (CAR), the CAR comprising (a) an extracellular domain, (b) a transmembrane domain, and (c) one or more intracellular domains, wherein the extracellular domain is or comprises an anti-mesothelin antigen binding domain comprising an amino acid sequence at least 80% identical to a sequence selected from Table 3, and the modified immune cell is or comprises a macrophage, monocyte, dendritic cell, or stem cell.

[0005] In some embodiments, the extracellular domain is or comprises an scFv, VHH antibody, centrin, darpin, or nanobody. In some embodiments, the transmembrane domain is or comprises a CD8, CD8a, CD28, CD40, MyD88 CD64, CD32a, CD32c, CD16a, CD3 zeta, ICOS, Dectin-1, DNGR1, SLAMF7, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, or TLR9 transmembrane domain. In some embodiments, one or more intracellular domains are selected from the group consisting of CD3ζ, FcRγ, MyD88, CD40, CD64, CD32a, CD32c, CD16a, CD89, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphA1, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROI, ROI S1, RYK, TIE2, TRK, VEGFR, CD19, CD20, 41BB, CD28, GCSFR (CD114), RAGE, CD30, CD160, DR3, Fn14, HVEM, CD160, NGFR, RANK, TNFR2, TROY, XEDAR, TRIF, OX40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, V / I / LxYxxL / V, SI RPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPb, CD22, PI R-B, LILRB1, 41BBL (TNFSF9), CD27, OX40L, CD32b, CD11b, ITGAM, SLAMF7, CD206, CD163, CD209, Dectin-2, IL1R, The intracellular domains include 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, a portion of any of the foregoing domains, or a combination thereof.In some embodiments, the one or more intracellular domains comprise a CD3ζ intracellular domain or an FcRγ intracellular domain.

[0006] In some embodiments, the CAR further comprises an extracellular leader domain. In some embodiments, the extracellular leader domain comprises a CD8a extracellular leader domain. In some embodiments, the CAR further comprises an extracellular hinge domain. In some embodiments, the extracellular hinge domain comprises a CD8 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, or an IgG4 extracellular hinge domain.

[0007] In some embodiments, the CAR comprises, from N-terminus to C-terminus, a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD8 extracellular hinge domain, a CD8 transmembrane domain, and a CD3ζ intracellular domain; a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and a CD3ζ intracellular domain; a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and an FcRγ intracellular domain; a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and an FcRγ intracellular domain; domain, anti-mesothelin antigen-binding domain, CD8 extracellular hinge domain, CD8 transmembrane domain, CD3ζ intracellular domain, P2A cleavage peptide, and CD40 ligand (CD40L); CD8a leader domain, anti-mesothelin antigen-binding domain, CD28 extracellular hinge domain, CD28 transmembrane domain, CD3ζ intracellular domain, P2A cleavage peptide, and CD40 ligand (CD40L); CD8a leader domain, anti-mesothelin antigen-binding domain, CD28 extracellular hinge domain, CD28 transmembrane domain, MyD88 cell CD8a leader domain, anti-mesothelin antigen-binding domain, CD28 extracellular hinge domain, CD28 transmembrane domain, MyD88 intracellular domain, CD40 intracellular domain, and CD3ζ intracellular domain; CD8a leader domain, anti-mesothelin antigen-binding domain, CD28 extracellular hinge domain, CD28 transmembrane domain, cleaved MyD88 intracellular domain, CD40 intracellular domain, and CD3ζ intracellular domain; CD8a leader domain, anti-mesothelin antigen-binding domain, CD 28 extracellular hinge domain, CD28 transmembrane domain, FcRγ intracellular domain, P2A cleavage peptide, and CD40 ligand (CD40L); CD8a leader domain, anti-mesothelin antigen binding domain, CD28 extracellular hinge domain, CD28 transmembrane domain, MyD88 intracellular domain, and FcRγ intracellular domain; CD8a leader domain, anti-mesothelin antigen binding domain, CD28 extracellular hinge domain, CD28 transmembrane domain, MyD88 intracellular domain, CD40 intracellular domain, and FcRγ intracellular domain;or a CD8a leader domain, an anti-mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a truncated MyD88 intracellular domain, a CD40 intracellular domain, and an FcRγ intracellular domain;

[0008] In some embodiments, the CAR has or comprises (a) an amino acid sequence selected from Table 2, (b) an amino acid sequence that differs from a sequence selected from Table 2 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 2.

[0009] In another aspect, the present disclosure provides pharmaceutical compositions comprising the modified immune cells described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0010] In another aspect, the disclosure provides a nucleic acid construct comprising one or more nucleic acid sequences encoding (a) an extracellular binding domain, (b) a transmembrane domain, and (c) one or more intracellular domains, wherein the extracellular domain is or comprises an anti-mesothelin antigen binding domain comprising an amino acid sequence at least 80% identical to a sequence selected from Table 5, and the nucleic acid construct encodes a chimeric antigen receptor (CAR) comprising (a)-(c). In some embodiments, the nucleic acid construct further comprises one or more nucleic acid sequences encoding (d) one or more extracellular leader domains, (e) one or more extracellular hinge domains, (f) one or more truncation peptides, or a combination thereof. In some embodiments, the truncation peptides are or comprise P2A, F2A, E2A, or T2A peptides.

[0011] In some embodiments, the nucleic acid construct comprises, from N-terminus to C-terminus, a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD8 extracellular hinge domain, a CD8 transmembrane domain, and a CD3ζ intracellular domain; a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and a CD3ζ intracellular domain; a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and an FcRγ intracellular domain; a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and an FcRγ intracellular domain; CD8a leader domain, anti-mesothelin antigen-binding domain, CD8 extracellular hinge domain, CD8 transmembrane domain, CD3ζ intracellular domain, P2A cleavage peptide, and CD40 ligand (CD40L); CD8a leader domain, anti-mesothelin antigen-binding domain, CD28 extracellular hinge domain, CD28 transmembrane domain, CD3ζ intracellular domain, P2A cleavage peptide, and CD40 ligand (CD40L); CD8a leader domain, anti-mesothelin antigen-binding domain, CD28 extracellular hinge domain, CD28 transmembrane domain, MyD88 CD8a leader domain, anti-mesothelin antigen-binding domain, CD28 extracellular hinge domain, CD28 transmembrane domain, MyD88 intracellular domain, CD40 intracellular domain, and CD3ζ intracellular domain; CD8a leader domain, anti-mesothelin antigen-binding domain, CD28 extracellular hinge domain, CD28 transmembrane domain, cleaved MyD88 intracellular domain, CD40 intracellular domain, and CD3ζ intracellular domain; CD8a leader domain, anti-mesothelin antigen-binding domain, C D28 extracellular hinge domain, CD28 transmembrane domain, FcRγ intracellular domain, P2A cleavage peptide, and CD40 ligand (CD40L); CD8a leader domain, anti-mesothelin antigen binding domain, CD28 extracellular hinge domain, CD28 transmembrane domain, MyD88 intracellular domain, and FcRγ intracellular domain; CD8a leader domain, anti-mesothelin antigen binding domain, CD28 extracellular hinge domain, CD28 transmembrane domain, MyD88 intracellular domain, CD40 intracellular domain, and FcRγ intracellular domain;or encoding a CD8a leader domain, an anti-mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a truncated MyD88 intracellular domain, a CD40 intracellular domain, and an FcRγ intracellular domain;

[0012] In some embodiments, the nucleic acid construct has or comprises (a) a nucleotide sequence selected from Table 4, (b) a nucleotide sequence that differs from a sequence selected from Table 4 by no more than five substitutions, additions, or deletions, or (c) a nucleotide sequence that is at least 80% identical to a sequence selected from Table 4.

[0013] In some embodiments, the nucleic acid construct of the present disclosure further comprises one or more introns, wherein the one or more introns comprise one or more inhibitory nucleic acids, and the one or more inhibitory nucleic acids encode one or more inhibitory RNAs. In some embodiments, the one or more inhibitory RNAs are or comprise one or more shRNAs. In some embodiments, the one or more shRNAs comprise a guide strand. In some embodiments, the guide strand comprises a nucleic acid sequence that is reverse-complementary to a target gene transcript that comprises the target nucleic acid sequence. In some embodiments, 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, PPARα, PPARγ, PTGS2, Siglec-10, SIRPα, SLAMF3, SLAMF4, SLC15A3, STAT3, STAT6, TNFRSF1B, TOX, TREM2, YTHDF2, or ZFP36. In some embodiments, the target gene transcript encodes a human antiphagocytic receptor selected from the group consisting of SIRPα, LILRB1, Siglec-10, PD1, SLAMF3, SLAMF4, CLEC1A, and CD32b. In some embodiments, the target gene transcript encodes human SIRPα.

[0014] In another aspect, the present disclosure provides pharmaceutical compositions comprising the nucleic acid constructs described herein. In some embodiments, the pharmaceutical compositions comprise a pharmaceutically acceptable carrier.

[0015] In another aspect, the present disclosure provides a method of treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein, wherein at least one sign or symptom of the disease or disorder is improved in the subject after administration. In some embodiments, the administering step is or includes intraarterial, subcutaneous, intravenous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, or intraperitoneal delivery.

[0016] In another aspect, the disclosure provides a method of modifying an immune cell, comprising delivering a nucleic acid construct described herein to an immune cell, thereby generating a modified immune cell, wherein the modified immune cell is or comprises a macrophage, monocyte, dendritic cell, or stem cell.

[0017] In some embodiments, the nucleic acid construct comprises DNA or messenger RNA (mRNA). In some embodiments, the nucleic acid construct comprises a modification selected from modified nucleotides, modifications to the 5' untranslated region (UTR), modifications to the 3' UTR, a cap structure, a poly(A) tail, or a combination thereof. In some embodiments, the cap structure comprises AGCap1, m6AGCap1, or an anti-reverse cap analog (ARCA). In some embodiments, the modified nucleotide comprises pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine / pseudouridine (5meC PsU), N1-methyl-pseudouridine (N1mPsU), or a combination thereof.

[0018] In some embodiments, the nucleic acid construct is a purified nucleic acid construct. In some embodiments, the purified nucleic acid construct is produced by a method including silica membrane purification, high performance liquid chromatography (HPLC), Dynabeads, LiCl precipitation, phenol-chloroform extraction, resin-based purification, polyA isolation, RNeasy, or a combination thereof. In some embodiments, the nucleic acid construct is codon-optimized. In some embodiments, the nucleic acid construct is codon-optimized for expression in stem cells, monocytes, macrophages, or dendritic cells.

[0019] In some embodiments, delivery comprises electroporation or transfection with the nucleic acid construct. In some embodiments, the nucleic acid construct is encapsulated within a delivery vehicle. In some embodiments, the delivery vehicle is or comprises a liposome, lipid nanoparticle, polymer, adeno-associated virus (AAV) vector, adenoviral vector, retroviral vector, or a combination thereof. In some embodiments, the liposome or lipid nanoparticle comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, one or more PEG-modified lipids, or a combination thereof. In some embodiments, the retroviral vector comprises a lentiviral vector or a gamma retroviral vector. In some embodiments, the lentiviral vector is packaged with a Vpx protein. In some embodiments, the adenoviral vector comprises an Ad2 vector or an Ad5 vector. In some embodiments, the Ad5 vector comprises an Ad5f35 adenoviral vector.

[0020] In some embodiments, the methods of the present disclosure further comprise delivering an additional payload to the immune cells. In some embodiments, the additional payload is or comprises a pathogen recognition receptor agonist, polyinosinic:polycytidylic acid (poly I:C), TLR7 / 8 agonist, CpG oligodeoxynucleotide, NOD-like receptor (NLR) agonist, RIG-I-like receptor (RLR) agonist, C-type lectin receptor (CLR) agonist, cytoplasmic DNA sensing, cyclic GMP-AMP synthase stimulator of interferon genes (cGAS-STING) agonist, interferon-inducible protein 16 (IFI16) agonist, DEAD-box helicase 41 (DDX41) agonist, LRR-binding FLII-interacting protein 1 (LRRFIP1) agonist, absent in melanoma 2 (AIM2) agonist, aryl hydrocarbon receptor (AhR) ligand, or a combination thereof. In some embodiments, the nucleic acid construct and additional payload are encapsulated within a delivery vehicle.

[0021] The drawings are for purposes of illustration only and not limitation. [Brief explanation of the drawings]

[0022] [Figure 1A] Figures 1A, 1B, and 1C show graphs of exemplary macrophage viability (Figure 1A) and CAR expression (Figure 1B and Figure 1C) after electroporation with mRNA encoding 20 different anti-mesothelin binders (M1-M20) used in a CD8 framework chimeric antigen receptor (CAR). [Figure 1B] Figures 1A, 1B, and 1C show graphs of exemplary macrophage viability (Figure 1A) and CAR expression (Figure 1B and Figure 1C) after electroporation with mRNA encoding 20 different anti-mesothelin binders (M1-M20) used in a CD8 framework chimeric antigen receptor (CAR). [Figure 1C]Figures 1A, 1B, and 1C show graphs of exemplary macrophage viability (Figure 1A) and CAR expression (Figure 1B and Figure 1C) after electroporation with mRNA encoding 20 different anti-mesothelin binders (M1-M20) used in a CD8 framework chimeric antigen receptor (CAR).

[0023] [Figure 2] FIG. 1 shows a graph of exemplary target cell killing mediated by macrophages after electroporation with mRNA encoding four different anti-mesothelin binders (M11, M14, M15, and M17) used in a CD8 framework CAR.

[0024] [Figure 3] FIG. 1 shows a graph of exemplary TNFα cytokine release mediated by macrophages after electroporation with mRNA encoding four different anti-mesothelin binders (M11, M14, M15, and M17) used in a CD8 framework CAR.

[0025] [Figure 4] FIG. 1 shows a graph of exemplary anti-mesothelin-mediated phagocytosis by macrophages after electroporation with mRNA encoding four different anti-mesothelin binders (M11, M14, M15, and M17) used in a CD8 framework CAR.

[0026] [Figure 5] 1 shows an exemplary graph of macrophage viability after transduction with Ad5f35 vectors containing CTX_269 (anti-mesothelin CAR) at various exemplary MOIs.

[0027] [Figure 6] 1 shows a graph of exemplary anti-mesothelin CAR expression after transduction of macrophages with Ad5f35 vectors containing CTX_269 (anti-mesothelin CAR) at various exemplary MOIs.

[0028] [Figure 7A] Figures 7A, 7B, and 7C show graphs of exemplary expression of exemplary M1-associated markers (CD80, CD86, and HLA-DR) after transduction of macrophages with various exemplary MOIs of Ad5f35 vectors containing CTX_269 (anti-mesothelin CAR). [Figure 7B] Figures 7A, 7B, and 7C show graphs of exemplary expression of exemplary M1-associated markers (CD80, CD86, and HLA-DR) after transduction of macrophages with various exemplary MOIs of Ad5f35 vectors containing CTX_269 (anti-mesothelin CAR). [Figure 7C] Figures 7A, 7B, and 7C show graphs of exemplary expression of exemplary M1-associated markers (CD80, CD86, and HLA-DR) after transduction of macrophages with various exemplary MOIs of Ad5f35 vectors containing CTX_269 (anti-mesothelin CAR).

[0029] [Figure 8] 1 shows a graph of exemplary macrophage expression of exemplary M2-associated markers (CD163 and CD206) after transduction of macrophages with Ad5f35 vectors containing CTX_269 (anti-mesothelin CAR) at various exemplary MOIs.

[0030] [Figure 9] FIG. 1 shows a graph of an exemplary M2-associated marker (CD163) expression after transduction of macrophages with Ad5f35 vector containing CTX_269 (anti-mesothelin CAR).

[0031] [Figure 10] FIG. 1 shows a graph of an exemplary cytokine M1-associated marker (CD86) expression after transduction of macrophages with Ad5f35 vector containing CTX_269 (anti-mesothelin CAR).

[0032] [Figure 11]FIG. 1 shows a graph of exemplary M2-associated marker (CD163 and CD206) expression after transduction of macrophages with an Ad5f35 vector containing CTX_269 (anti-mesothelin CAR) and exposure of the macrophages to mesothelin.

[0033] [Figure 12] FIG. 1 shows an exemplary graph of anti-mesothelin-mediated phagocytosis of A549 lung adenocarcinoma cells by macrophages after transduction of macrophages with an Ad5f35 vector containing CTX_269 (anti-mesothelin CAR).

[0034] [Figure 13] 1 shows a graph of exemplary antimesothelin-mediated phagocytosis of MES-OV ovarian cystadenocarcinoma cells by macrophages after transduction of macrophages with an Ad5f35 vector containing CTX_269 (anti-mesothelin CAR).

[0035] [Figure 14] FIG. 1 shows a graph of exemplary anti-mesothelin-mediated killing of mesothelin-expressing A549 lung adenocarcinoma cells by macrophages after transduction of macrophages with an Ad5f35 vector containing CTX_269 (anti-mesothelin CAR).

[0036] [Figure 15] FIG. 1 shows a graph of exemplary anti-mesothelin-mediated killing of mesothelin-expressing ovarian cystadenocarcinoma cells by macrophages after transduction of macrophages with an Ad5f35 vector containing CTX_269 (anti-mesothelin CAR).

[0037] [Figure 16] FIG. 1 shows graphs of exemplary cytokine (TNFα and IL-1β) release after transduction of macrophages with Ad5f35 vectors containing CTX_269 (anti-mesothelin CAR) and exposure of macrophages to mesothelin.

[0038] [Figure 17]Graph of exemplary cytokine (TNFα) release after transduction of macrophages with Ad5f35 vector containing CTX_269 (anti-mesothelin CAR) and exposure of macrophages to target cells (A549 lung adenocarcinoma cells or MES-OV ovarian cystadenocarcinoma cells) expressing mesothelin.

[0039] [Figure 18] FIG. 1 shows an exemplary experimental timeline for treatment of an in vivo mouse tumor model with macrophages transduced with an Ad5f35 vector containing CTX_269 (anti-mesothelin CAR).

[0040] [Figure 19] 1 shows an exemplary graph of tumor burden in mice treated with macrophages transduced with Ad5f35 vector containing CTX_269 (anti-mesothelin CAR).

[0041] [Figure 20] 1 shows a graph of exemplary anti-mesothelin CAR expression after transduction of macrophages with a lentiviral vector containing a CD28-based anti-mesothelin CAR or a lentiviral vector containing a CD8-based anti-mesothelin CAR.

[0042] [Figure 21] 1 shows a graph of exemplary target cell killing mediated by macrophages after transduction of macrophages with a lentiviral vector containing a CD28-based anti-mesothelin CAR or a lentiviral vector containing a CD8-based anti-mesothelin CAR.

[0043] [Figure 22] FIG. 1 shows a graph of exemplary cytokine (TNFα) release after transduction of macrophages with a lentiviral vector containing a CD28-based anti-mesothelin CAR or a lentiviral vector containing a CD8-based anti-mesothelin CAR and exposure of the transduced macrophages to mesothelin.

[0044] [Figure 23] Graphs of exemplary M2-associated marker (CD163 and CD206) expression after transduction of macrophages with Ad5f35 vectors containing either CTX_269 (anti-mesothelin CAR containing a CD8-framework) or CTX_293 (anti-mesothelin CAR containing a CD28-framework) and exposure of the transduced macrophages to IL-10 are shown.

[0045] [Figure 24] Graphs of exemplary M1-associated marker (CD80 and CD86) expression after transduction of macrophages with Ad5f35 vectors containing either CTX_269 (anti-mesothelin CAR containing a CD8-framework) or CTX_293 (anti-mesothelin CAR containing a CD28-framework) and exposure of the transduced macrophages to IL-10 are shown.

[0046] [Figure 25] FIG. 1 shows a graph of exemplary antimesothelin-mediated killing of mesothelin-expressing A549 lung adenocarcinoma cells by macrophages after transduction of macrophages with Ad5f35 vectors containing either CTX_269 (anti-mesothelin CAR containing a CD8-framework) or CTX_293 (anti-mesothelin CAR containing a CD28-framework).

[0047] [Figure 26A] Figures 26A, 26B, 26C, 26D, 26E, and 26F show graphs of exemplary phenotypic markers for monocytes transduced with either CTX_269 (CD8-based anti-mesothelin CAR) or CTX_001 (anti-HER2 CAR), monocytes transduced with CTX_269 or CTX_001 and then differentiated into macrophages, and macrophages transduced with CTX_269 or CTX_001 after differentiation from monocytes. [Figure 26B]Figures 26A, 26B, 26C, 26D, 26E, and 26F show graphs of exemplary phenotypic markers for monocytes transduced with either CTX_269 (CD8-based anti-mesothelin CAR) or CTX_001 (anti-HER2 CAR), monocytes transduced with CTX_269 or CTX_001 and then differentiated into macrophages, and macrophages transduced with CTX_269 or CTX_001 after differentiation from monocytes. [Figure 26C] Figures 26A, 26B, 26C, 26D, 26E, and 26F show graphs of exemplary phenotypic markers for monocytes transduced with either CTX_269 (CD8-based anti-mesothelin CAR) or CTX_001 (anti-HER2 CAR), monocytes transduced with CTX_269 or CTX_001 and then differentiated into macrophages, and macrophages transduced with CTX_269 or CTX_001 after differentiation from monocytes. [Figure 26D] Figures 26A, 26B, 26C, 26D, 26E, and 26F show graphs of exemplary phenotypic markers for monocytes transduced with either CTX_269 (CD8-based anti-mesothelin CAR) or CTX_001 (anti-HER2 CAR), monocytes transduced with CTX_269 or CTX_001 and then differentiated into macrophages, and macrophages transduced with CTX_269 or CTX_001 after differentiation from monocytes. [Figure 26E] Figures 26A, 26B, 26C, 26D, 26E, and 26F show graphs of exemplary phenotypic markers for monocytes transduced with either CTX_269 (CD8-based anti-mesothelin CAR) or CTX_001 (anti-HER2 CAR), monocytes transduced with CTX_269 or CTX_001 and then differentiated into macrophages, and macrophages transduced with CTX_269 or CTX_001 after differentiation from monocytes. [Figure 26F]Figures 26A, 26B, 26C, 26D, 26E, and 26F show graphs of exemplary phenotypic markers for monocytes transduced with either CTX_269 (CD8-based anti-mesothelin CAR) or CTX_001 (anti-HER2 CAR), monocytes transduced with CTX_269 or CTX_001 and then differentiated into macrophages, and macrophages transduced with CTX_269 or CTX_001 after differentiation from monocytes.

[0048] [Figure 27] Graphs showing exemplary anti-mesothelin-mediated killing of mesothelin-expressing ovarian cystadenocarcinoma cells (left graph) or mesothelin-expressing A549 lung adenocarcinoma cells (right graph) by monocytes transduced with either CTX_269 (CD8-based anti-mesothelin CAR) or CTX_001 (anti-HER2 CAR), monocytes transduced with CTX_269 or CTX_001 and then differentiated into macrophages, and macrophages transduced with CTX_269 or CTX_001 after differentiation from monocytes.

[0049] [Figure 28] FIG. 1 shows a schematic diagram of an exemplary anti-mesothelin CAR construct comprising M15 scFv.

[0050] [Figure 29] FIG. 1 shows a schematic diagram of an exemplary anti-mesothelin CAR construct comprising M17 scFv.

[0051] [Figure 30] 1 shows a graph of exemplary anti-mesothelin CAR expression at days 2 and 14 after transduction of macrophages with an Ad5f35 vector containing CTX_269 (anti-mesothelin CAR).

[0052] [Figure 31] 1 shows an exemplary graph of anti-mesothelin-mediated phagocytosis of K562 cells by macrophages after transduction of macrophages with an Ad5f35 vector containing CTX_269 (anti-mesothelin CAR).

[0053] [Figure 32] FIG. 1 shows a graph of an exemplary cytokine (TNFα) release after transduction of macrophages with an Ad5f35 vector containing CTX_269 (anti-mesothelin CAR) and exposure of the macrophages to target cells (K562 cells) expressing mesothelin.

[0054] [Figure 33] Representative tissue sections of mouse lung immunohistochemistry (IHC) stained for human mesothelin in mice treated with macrophages transduced with Ad5f35 vectors containing CTX_269 (anti-mesothelin CAR). Scale bar 1 mm.

[0055] [Figure 34] 1 shows exemplary tumor nodule quantification from mouse lungs treated with macrophages transduced with Ad5f35 vector containing CTX_269 (anti-mesothelin CAR).

[0056] [Figure 35A] FIG. 1 shows an exemplary graph of anti-mesothelin CAR expression and cell viability after transduction of monocytes with Ad5f35 virus containing CTX_964 (anti-mesothelin CAR) or CTX_1461 (anti-mesothelin CAR plus intronic shRNA against SIRPα). [Figure 35B] FIG. 1 shows an exemplary graph of anti-mesothelin CAR expression and cell viability after transduction of monocytes with Ad5f35 virus containing CTX_964 (anti-mesothelin CAR) or CTX_1461 (anti-mesothelin CAR plus intronic shRNA against SIRPα).

[0057] [Figure 36A] 1 shows an exemplary graph of SIRPα expression after transduction of monocytes with Ad5f35 virus containing CTX_964 (anti-mesothelin CAR) or CTX_1461 (anti-mesothelin CAR + intronic shRNA against SIRPα). [Figure 36B]1 shows an exemplary graph of SIRPα expression after transduction of monocytes with Ad5f35 virus containing CTX_964 (anti-mesothelin CAR) or CTX_1461 (anti-mesothelin CAR + intronic shRNA against SIRPα).

[0058] [Figure 37] FIG. 1 shows a graph of exemplary cytokine (TNFα) release after transduction of monocytes with Ad5f35 virus containing CTX_964 (anti-mesothelin CAR) or CTX_1461 (anti-mesothelin CAR + intronic shRNA against SIRPα) and exposure of the cells to either recombinant human mesothelin or recombinant mesothelin + recombinant human CD47.

[0059] [Figure 38A] FIG. 1 shows a graph of exemplary anti-mesothelin-mediated killing of mesothelin-expressing target cells by monocytes and macrophages after transduction of the cells with Ad5f35 vectors containing either CTX_964 (anti-mesothelin CAR) or CTX_1461 (anti-mesothelin CAR + intronic shRNA against SIRPα). [Figure 38B] FIG. 1 shows a graph of exemplary anti-mesothelin-mediated killing of mesothelin-expressing target cells by monocytes and macrophages after transduction of the cells with Ad5f35 vectors containing either CTX_964 (anti-mesothelin CAR) or CTX_1461 (anti-mesothelin CAR + intronic shRNA against SIRPα).

[0060] [Figure 39] Graph showing exemplary inhibition of tumor growth by CAR-monocyte-derived CAR-macrophages after transduction of cells with Ad5f35 vectors containing either CTX_964 (anti-mesothelin CAR) or CTX_1461 (anti-mesothelin CAR + intronic shRNA against SIRPα). DETAILED DESCRIPTION OF THE INVENTION

[0061] definition In order that the present invention may be more readily understood, certain terms are first defined below. Further definitions for these and other terms are set forth throughout the specification. Publications and other reference materials referred to herein are incorporated by reference to describe the background of the invention and to provide further detail regarding its practice.

[0062] 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 element or more than one element.

[0063] Approximately or About: As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value similar to a stated reference value. In certain embodiments, unless otherwise stated or otherwise clear from the context, the term "approximately" or "about" refers to a range of values ​​that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% in either direction (above or below) of the stated reference value (except where such number exceeds 100% of the possible values).

[0064] Activated: As used herein, the term "activated" refers to the state of a cell, e.g., a monocyte, macrophage, or dendritic cell, that has been sufficiently stimulated to induce detectable cell proliferation or to exert its effector function. Activation may also be associated with induced cytokine production, phagocytosis, cell signaling, target cell killing, and / or antigen processing and presentation.

[0065] Activated Monocyte / Macrophage / Dendritic Cell: As used herein, the term "activated monocyte / macrophage / dendritic cell" refers, inter alia, to a monocyte / macrophage / dendritic cell that is undergoing cell division or exerting an effector function. The term "activated monocyte / macrophage / dendritic cell" refers, inter alia, to a cell that is performing an effector function or exhibiting some activity not found in the resting state, including phagocytosis, cytokine secretion, proliferation, changes in gene expression, changes in metabolism, and other functions.

[0066] Drug: As used herein, the term "drug" (or "biological agent" or "therapeutic agent") refers to a molecule that can be expressed, released, secreted, or delivered to a target by the modified cells described herein. Drugs include, but are not limited to, nucleic acids, antibiotics, anti-inflammatory agents, antibodies or fragments thereof, antibody agents or fragments thereof, growth factors, cytokines, enzymes, proteins (e.g., RNAse inhibitors), peptides, fusion proteins, synthetic molecules, organic molecules (e.g., small molecules), carbohydrates, lipids, hormones, microsomes, derivatives or variants thereof, and any combination thereof. Drugs can bind to any cellular moiety, such as a receptor, antigenic determinant, or other binding site present on the target or target cell. Drugs can diffuse or be transported into the cell and act within the cell.

[0067] Antibody: As used herein, the term "antibody" refers to a polypeptide containing sufficient canonical immunoglobulin sequence elements to confer specific binding to a particular target antigen. As is known in the art, intact antibodies, as produced in nature, are tetrameric agents of approximately 150 kD, composed of two identical heavy chain polypeptides (approximately 50 kD each) and two identical light chain polypeptides (approximately 25 kD each) that associate with each other into what is commonly referred to as a "Y-shaped" structure. Each heavy chain is composed of at least four domains, each approximately 110 amino acids long: an amino-terminal variable (VH) domain (located at the tip of the Y structure) followed by three constant domains: CH1, CH2, and a carboxy-terminal CH3 domain (located at the base tip of the Y). A short region known as the "switch" connects the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect two heavy chain polypeptides to each other in intact antibodies. Each light chain is composed of two domains: an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, which are separated from each other by another "switch." An intact antibody tetramer is composed of two heavy-light chain dimers, in which the heavy and light chains are linked to each other by one disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to each other, thereby connecting the dimers to form a tetramer. Naturally produced antibodies are typically glycosylated in the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" formed from two β-sheets (e.g., a three-, four-, or five-stranded sheet) packed together in a compressed antiparallel β-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 a natural antibody folds, the FR regions form beta sheets, providing a structural framework for the domain, and the CDR loop regions of both the heavy and light chains join in three-dimensional space to create a single hypervariable antigen-binding site located at the tip of a Y-structure. The Fc region of a naturally occurring antibody binds to components of the complement system and also to receptors on effector cells, e.g., effector cells that mediate cytotoxicity. The affinity and / or other binding properties of the Fc region for the Fc receptor can be modulated via glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized in accordance with the present invention (e.g., as components of chimeric switch receptors or CARs) comprise a glycosylated Fc domain, e.g., an Fc domain with modified or engineered glycosylation. In some embodiments, any polypeptide, or complex of polypeptides, comprising a sufficient immunoglobulin domain sequence as found in a natural antibody can be referred to and / or used as an "antibody," regardless of whether such polypeptide is produced naturally (e.g., by an animal in response to an antigen) or produced by recombinant genetic engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, the antibody is polyclonal. In some embodiments, the antibody is monoclonal. In some embodiments, the antibody has constant region sequences characteristic of murine, rabbit, primate, or human antibodies. In some embodiments, the antibody sequence elements are humanized, primatized, chimeric, etc., as known in the art. Furthermore, the term "antibody," as used herein, can refer, in appropriate embodiments (unless otherwise stated or clear from the context), to any of the constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations.For example, in some embodiments, antibodies utilized in accordance with the present invention include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific 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 Fv, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), camelid antibodies, masked antibodies (e.g., Probodies®), Small Modular ImmunoPharmaceuticals ("SMIPs™"), single-chain diabodies or tandem diabodies (TandAb®), VHH, Anticalins®, Nanobodies® minibodies, BiTE®, Anrikin repeat proteins or DARPINs®, Avimers®, DART, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, and KALBITOR®. In some embodiments, the antibody may lack covalent modifications (e.g., glycan attachment) that it would have if produced naturally. In some embodiments, antibodies can contain covalent modifications (e.g., the attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant groups (e.g., polyethylene glycol, etc.).

[0068] 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 contains sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences characteristic of murine, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more antibody sequence elements that are humanized, primatized, chimeric, etc., as known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations.For example, in some embodiments, antibody agents utilized in accordance with the present invention include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific 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 Fv, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), camelid antibodies, masked antibodies (e.g., Probodies®), Small Modular ImmunoPharmaceuticals ("SMIPs™"), single-chain or tandem diabodies (TandAb®), VHH, Anticalins®, Nanobodies® minibodies, BiTE®, Anrikin repeat proteins or DARPINs®, Avimers®, DART, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, and KALBITOR®. In some embodiments, the antibody agent may lack covalent modifications (e.g., glycan attachment) that it would have if produced in nature. In some embodiments, an antibody agent can include a covalent modification (e.g., the attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., polyethylene glycol, etc.). In many embodiments, an antibody agent is or includes a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs).In some embodiments, an antibody agent is or comprises a polypeptide that includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) whose amino acid sequence is substantially identical to that found in a reference antibody. In some embodiments, the included CDR is substantially identical to the reference CDR in that it is either identical in sequence or contains one to five amino acid substitutions compared to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that it exhibits 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, the included CDR is substantially identical to the reference CDR in that it exhibits 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid within the included CDRs is deleted, added, or substituted when compared to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to that of the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that 1-5 amino acids within the included CDRs are deleted, added, or substituted when compared to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to that of the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid within the included CDRs is substituted when compared to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to that of the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that 1-5 amino acids within the included CDRs are deleted, added, or substituted when compared to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to that of 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 that is homologous to an immunoglobulin binding domain or that is largely homologous to an immunoglobulin binding domain. In some embodiments, an antibody agent is not a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain and / or does not include 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 can be or include a molecule or composition that does not include immunoglobulin structural elements (e.g., a receptor or other naturally occurring molecule that includes at least one antigen binding domain).

[0069] Antibody fragment: As used herein, the term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the 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.

[0070] 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.

[0071] 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.

[0072] Synthetic antibody: As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, where the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0073] Antigen: As used herein, the term "antigen" or "Ag" refers to a molecule capable of eliciting an immune response. This immune response may include either antibody production, activation of specific immunologically competent cells, or both. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen," as the term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the 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 two or more genes, and that these nucleotide sequences may be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that antigens can be synthetically produced or derived from biological samples. Such biological samples include, but are not limited to, tissue samples, tumor samples, cells, or body fluids.

[0074] Anti-tumor effect: As used herein, the term "anti-tumor effect" refers to a biological effect that may be manifested by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" may also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the invention in preventing the development of an initial tumor.

[0075] Autologous: As used herein, the term "autologous" refers to any material derived from the same individual that is later reintroduced into the individual.

[0076] 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.

[0077] Xenogeneic: As used herein, the term "xenogeneic" refers to any material (e.g., a population of cells) derived from an animal of a different species.

[0078] Cancer: As used herein, the term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream or lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colon cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like. In certain embodiments, the cancer is medullary thyroid cancer.

[0079] Conservative sequence modification: As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change 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 antibodies for various embodiments by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those 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 in the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family, and the altered antibodies can be tested for their ability to bind to antigen using the functional assays described herein.

[0080] Costimulatory Ligand: As used herein, the term "costimulatory ligand" refers to a molecule on an antigen-presenting cell (e.g., an APC, a dendritic cell, a B cell, etc.) that specifically binds to a cognate costimulatory molecule on a monocyte / macrophage / dendritic cell, thereby providing a signal that mediates a monocyte / macrophage / dendritic cell response, including, but not limited to, proliferation, activation, differentiation, etc. Costimulatory ligands can include, but are 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, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands also include, inter alia, antibodies that specifically bind to costimulatory molecules present on monocytes / macrophages / dendritic cells, including, but not limited to, ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0081] Cytotoxic: As used herein, the term "cytotoxic" or "cytotoxicity" refers to killing or damaging cells. In one embodiment, metabolically enhanced cells have improved cytotoxicity, e.g., increased cytolytic activity of macrophages.

[0082] Effective amount: As used herein, "effective amount" and "therapeutically effective amount" are used interchangeably and refer to an amount of a compound, formulation, material, or composition described herein that is effective in achieving a particular biological result or provides a manufacturing, therapeutic, or prophylactic benefit. Such results include, but are not limited to, anti-tumor activity as determined by any means suitable in the art.

[0083] Effector function: As used herein, "effector function" or "effector activity" refers to a specific activity carried out by an immune cell in response to immune cell stimulation. For example, an effector function of a macrophage is to engulf and digest cellular debris, foreign bodies, microorganisms, cancer cells, and other unhealthy cells through phagocytosis.

[0084] Encode: As used herein, "encode" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined nucleotide (i.e., rRNA, tRNA, and mRNA) sequence or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0085] Endogenous: As used herein, "endogenous" refers to any substance that originates or is produced within a particular organism, cell, tissue, or system.

[0086] Exogenous: As used herein, the term "exogenous" refers to any substance that is introduced from or produced outside a particular organism, cell, tissue, or system.

[0087] Expansion: As used herein, the term "expansion" refers to an increase in number, as in increasing the number of cells, e.g., monocytes, macrophages, and / or dendritic cells. In one embodiment, ex vivo expanded monocytes, macrophages, or dendritic cells are increased in number compared to the number initially present in the culture. In another embodiment, ex vivo expanded monocytes, macrophages, or dendritic cells are increased in number compared to other cell types in the culture. In some embodiments, expansion can occur in vivo. As used herein, the term "ex vivo" refers to cells removed from a living organism (e.g., a human) and grown outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0088] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product from the nucleic acid sequence. In some embodiments, the gene product can be a transcript. In some embodiments, the gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence includes one or more of the following: (1) generation of an RNA template from the DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end formation), (3) translation of the RNA into a polypeptide or protein, and / or (4) post-translational modification of the polypeptide or protein.

[0089] Expression Vector: As used herein, the term "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting 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 vectors 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).

[0090] Fragment: As used herein, the term "fragment" or "portion" refers to a structure that comprises a distinct portion of a whole, but lacks one or more portions found in the whole structure. In some embodiments, the fragment consists of such a distinct portion. In some embodiments, the fragment consists of or comprises a characteristic structural element or portion 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 acid) found throughout the nucleotide sequence. 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 nucleotide whole. The whole substance or entity may, in some embodiments, be referred to as the "parent" of the whole.

[0091] Homology: As used herein, the term "homology" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymer 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, polymer 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., contain residues with related chemical properties at corresponding positions). As will be understood by those skilled in the art, various algorithms are available that allow for comparison of sequences to determine the degree of homology, including, for example, allowing gaps of a specified length in one sequence relative to another sequence when considering which residues in different sequences "correspond" to each other. Calculating the percent homology between two nucleic acid sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second nucleic acid sequences for optimal alignment, and non-corresponding sequences can be ignored for comparison purposes). In certain embodiments, the length of the aligned sequence 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 reference sequence. Next, nucleotides at corresponding nucleotide positions are compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position; when a position in the first sequence is occupied by a nucleotide that is similar to the corresponding position in the second sequence, the molecules are similar at that position.The percent homology between 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 that need to be introduced for optimal alignment of the two sequences.

[0092] Identity: As used herein, the term "identity" refers to the subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, for example, between two polypeptide molecules. If two amino acid sequences have the same residue at the same position, for example, if each position in two polypeptide molecules is occupied by arginine, they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position 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; for example, if half of the positions in the two sequences (e.g., 5 positions in a 10-amino acid-long polymer) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are matching or identical, the two amino acid sequences are 90% identical.

[0093] Substantial identity: As used herein, the term "substantial identity" refers to a comparison between amino acid or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues at corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercially available computer programs, such as BLASTN for nucleotide sequences, BLASTP for amino acid sequences, gapped BLAST, and PSI-BLAST. In some embodiments, two sequences are considered 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 the relevant stretch of residues. In some embodiments, the relevant stretch is the entire sequence. In some embodiments the relevant extension 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 that is at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the reference CDR.

[0094] Immune cell: As used herein, the term "immune cell" refers to a cell that is involved in an immune response, for example, promoting 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. The source of immune cells (e.g., macrophages, monocytes, or dendritic cells) can be obtained from a subject.

[0095] 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 an antigen molecule as foreign, induce the formation of antibodies, and / or activate lymphocytes to eliminate the antigen.

[0096] Immunoglobulin: As used herein, the terms "immunoglobulin" or "Ig" refer to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. Five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody present in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response of most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses and is important for defense against bacteria and viruses. IgD is an immunoglobulin with no known antibody function but may function as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by triggering the release of mediators from mast cells and basophils upon exposure to allergens.

[0097] Isolated: As used herein, the term "isolated" refers to something that has been modified or removed from its natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-native environment, such as a host cell.

[0098] Lentivirus: As used herein, the term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells and deliver significant amounts of genetic information into the DNA of host cells, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Lentivirus-derived vectors provide a means to achieve significant levels of gene transfer in vivo.

[0099] Modified: As used herein, the term "modified" refers to an altered state or structure of a molecule or cell of the invention. Molecules can be modified in many ways, including chemically, structurally, and functionally. Cells can be modified by the introduction of nucleic acids.

[0100] Modulate: As used herein, the term "modulate" refers to mediating a detectable increase or decrease in the level and / or change in the nature of a response in a subject compared to the level and / or nature of the response in the subject in the absence of the treatment or compound, and / or compared to the level and / or nature of the response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a natural signal or response in a subject, preferably a human, thereby mediating a beneficial therapeutic response.

[0101] Nucleic Acid: As used herein, the term "nucleic acid" refers to a polymer of at least three nucleotides. In some embodiments, nucleic acids include DNA. In some embodiments, nucleic acids include RNA. In some embodiments, nucleic acids are single-stranded. In some embodiments, nucleic acids are double-stranded. In some embodiments, nucleic acids include both single-stranded and double-stranded portions. In some embodiments, nucleic acids include a backbone that includes one or more phosphodiester bonds. In some embodiments, nucleic acids include a backbone that includes both phosphodiester and non-phosphodiester bonds. For example, in some embodiments, nucleic acids can include a backbone that includes one or more phosphorothioate or 5'-N-phosphoramidite bonds and / or one or more peptide bonds, e.g., "peptide nucleic acids." In some embodiments, nucleic acids include one or more or all naturally occurring residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, nucleic acids include one or more or all non-naturally occurring residues. In some embodiments, the non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 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, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, the non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to that of the natural residue. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as an RNA or a polypeptide.In some embodiments, the nucleic acid has a nucleotide sequence that includes one or more introns. In some embodiments, the nucleic acid can be prepared by isolation from a natural source, enzymatic synthesis (e.g., polymerization based on a complementary template in vivo or in vitro, replication in a recombinant cell or system, or chemical synthesis. In some embodiments, the nucleic acid has at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 8 0, 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 in length.

[0102] Operably linked: As used herein, the term "operably linked" refers to a 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 to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous, in the same reading frame, and, where necessary, to join two protein-coding regions.

[0103] 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 the tumor antigen in cells from a diseased area, such as a solid tumor, within a particular tissue or organ of a patient, compared to the level of expression in normal cells from that tissue or organ. Patients with solid tumors or hematologic malignancies characterized by overexpression of tumor antigens can be determined by standard assays known in the art.

[0104] Polynucleotide: As used herein, the term "polynucleotide" refers to a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Therefore, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides and can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cellular genomes using conventional cloning techniques and PCR™, etc., as well as by synthetic means.

[0105] Polypeptide: As used herein, the term "polypeptide" refers to any polymeric chain of residues (e.g., amino acids) typically linked by peptide bonds. In some embodiments, a polypeptide has a naturally occurring amino acid sequence. In some embodiments, a polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, a polypeptide has an engineered amino acid sequence, in that it is artificially designed and / or created. In some embodiments, a polypeptide can comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide can comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide can comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide can comprise only D-amino acids. In some embodiments, a polypeptide can comprise only L-amino acids. In some embodiments, a polypeptide can include one or more pendant groups or other modifications, e.g., modification of or attachment to one or more amino acid side chains, at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, and the like (including combinations thereof). In some embodiments, a polypeptide may be cyclic and / or include a cyclic moiety. In some embodiments, a polypeptide is not cyclic and / or does not include a cyclic moiety. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or include a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to the name of a reference polypeptide, activity, or structure, and in such cases, it is used herein to refer to polypeptides that share a related activity or structure and therefore can be considered members of the same class or family of polypeptides.For each such class, exemplary polypeptides within the class are provided herein, and / or those of skill in the art will be aware of, whose amino acid sequences and / or functions are known. In some embodiments, such exemplary polypeptides are reference polypeptides of a class or family of polypeptides. In some embodiments, members of a polypeptide class or family exhibit significant sequence homology or identity with the reference polypeptide of the class (and in some embodiments, with all polypeptides within the class), share common sequence motifs (e.g., characteristic sequence elements), and / or share a common activity (in some embodiments, at a similar level or within a specified range) with the reference polypeptide of the class (and in some embodiments, with all polypeptides within the class). For example, in some embodiments, a member polypeptide exhibits an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and / or contains at least one region (e.g., a conserved region, which in some embodiments may be or may include a distinctive sequence element) that exhibits very high sequence identity, often greater than 90%, or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region typically encompasses at least 3-4, and often up to 20 or more, amino acids, and in some embodiments, the 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, hi some embodiments, a useful polypeptide may comprise or consist of multiple fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to each other than that found in the polypeptide of interest (e.g., a fragment directly linked to the parent may be spatially separated in the polypeptide of interest, or vice versa, and / or the fragments may be present in a different order in the polypeptide of interest than in the parent), and thus the polypeptide of interest is a derivative of that parent polypeptide.

[0106] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a string of at least two amino acids linked together by peptide bonds). A protein may contain moieties other than amino acids (e.g., it may be a glycoprotein, proteoglycan, etc.) and / or may be otherwise processed or modified. Those of skill in the art will understand that a "protein" may be an entire polypeptide chain (with or without a signal sequence) produced by a cell, or a characteristic portion thereof. Those of skill in the art will understand that a protein may in some cases comprise two or more polypeptide chains linked, for example, by one or more disulfide bonds or associated by other means. Polypeptides can contain L-amino acids, D-amino acids, or both, and can contain various amino acid modifications or analogs known to those of skill in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, a protein can comprise natural amino acids, unnatural 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, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0107] Signal Transduction Pathway: As used herein, the term "signal transduction pathway" refers to the biochemical relationships between various signaling molecules that play a role in transmitting a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and complexes of molecules that can receive a signal and transmit the signal across the plasma membrane of a cell.

[0108] Single-chain antibody: As used herein, the term "single-chain antibody" refers to an antibody formed by recombinant DNA technology in which immunoglobulin heavy and light chain fragments are linked to the Fv region via an engineered amino acid stretch. Various methods for producing single-chain antibodies are known, including those described in U.S. Patent 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.

[0109] Specific binding: As used herein, the term "specific binding," with respect to an antigen-binding domain such as an antibody agent, refers to an antigen-binding domain or antibody agent that recognizes a specific antigen but does not substantially recognize or bind to 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 antigens from more than one species. However, such cross-species reactivity does not, in itself, alter the classification of the 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, in itself, alter the classification of the antigen-binding domain or antibody agent as specific. In some cases, the terms "specific binding" or "specifically bind" can be used in reference to the interaction of an antigen-binding domain or antibody agent, protein, or peptide with a second chemical species and may mean that the interaction is dependent on 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 the entire protein. If an antigen-binding domain or antibody agent is specific for epitope "A," then in a reaction involving labeled "A" and an antigen-binding domain or antibody agent, the presence of molecules containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0110] Stimulation: As used herein, the term "stimulation" refers to a primary response induced by a stimulatory molecule (e.g., an FcR complex, a TLR complex, or a TCR / CD3 complex) binding to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signaling via the Fc receptor mechanism, via a chimeric switch receptor, or via a synthetic CAR. Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-beta and / or rearrangement of cytoskeletal structure. As used herein, the term "stimulatory molecule" refers to a molecule on a monocyte, macrophage, or dendritic cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell. In some embodiments, the stimulatory molecule comprises an FcR extracellular domain comprising a CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c, CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, FcαRI (CD89), or CD40 domain. In some embodiments, the 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., aAPC, macrophage, dendritic cell, B cell, etc.) or tumor cell, specifically binds to 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, etc. Stimulatory ligands are well known in the art and include, among others, Toll-like receptor (TLR) ligands, anti-toll-like receptor antibodies, agonists, and antibodies against monocyte / macrophage receptors. In addition, cytokines such as interferon-gamma are potent stimulators of macrophages.

[0111] Subject: As used herein, the term "subject" refers to an organism, e.g., 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, the human subject is an adult, an adolescent, or a pediatric subject. In some embodiments, the subject is afflicted with a disease, disorder, or condition, e.g., a disease, disorder, or condition that can be treated as provided herein, e.g., a cancer or tumor listed herein. In some embodiments, the subject is predisposed to a disease, disorder, or condition; in some embodiments, a predisposed subject is predisposed to and / or exhibits an increased risk (compared to the average risk observed in a reference subject or a reference population) of developing the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms of a disease, disorder, or condition. In some embodiments, the subject does not exhibit a particular symptom (e.g., clinical symptoms of a disease) or characteristic of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual for whom and / or who is receiving a diagnosis and / or therapy.

[0112] Substantially purified: As used herein, the term "substantially purified," e.g., when applied to cells, refers to cells that are essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they are normally associated in their naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous cell population. In other instances, the term simply refers to cells that have been separated from cells with which they are naturally associated in their native state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0113] Target: As used herein, the term "target" refers to a cell, tissue, organ, or site in the body that is the subject of the provided methods, systems, and / or compositions, e.g., a cell, tissue, organ, or site in the body that is in need of treatment or that is preferentially bound by, for example, an antibody (or fragment thereof) or chimeric switch receptor, i.e., a CAR.

[0114] Target site: As used herein, the term "target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0115] T cell receptor: As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins involved in the activation of T cells in response to antigen presentation. TCRs are responsible for recognizing antigens bound to major histocompatibility complex molecules. TCRs are composed of a heterodimer of alpha (α) and beta (β) chains, although in some cells, TCRs are composed of gamma and delta (γ / δ) chains. TCRs can exist in alpha / beta and gamma / delta forms, which are structurally similar but have different anatomical locations and functions. Each chain is composed of two extracellular domains: a variable domain and a constant domain. In some embodiments, TCRs can be modified on any cell containing a TCR, including helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and γδ T cells.

[0116] Therapeutic: As used herein, the term "therapeutic" refers to treatment and / or prophylaxis. A therapeutic effect is achieved by suppressing, ameliorating, or eradicating a disease state.

[0117] Transfected: As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. This cell includes the primary subject cell and its progeny.

[0118] Treat: As used herein, the terms "treat," "treatment," or "treating" refer to the partial or complete alleviation, amelioration, delay in onset, inhibition, prevention, mitigation, 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 can be administered to a subject who does not exhibit signs or characteristics 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 characteristics of a disease, disorder, and / or condition, e.g., for the purpose of reducing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment can be administered to a subject who exhibits established, severe, and / or late signs of a disease, disorder, or condition. In some embodiments, treatment can include 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.

[0119] Tumor: As used herein, the term "tumor" refers to an abnormal growth of cells or tissues. In some embodiments, a tumor may comprise precancerous (e.g., benign), malignant, premetastatic, metastatic, and / or non-metastatic cells. In some embodiments, a tumor is associated with or is a sign of cancer. In some embodiments, a tumor may be a dispersed tumor or a liquid tumor. In some embodiments, a tumor may be a solid tumor.

[0120] Vector: As used herein, the term "vector" refers to a composition of matter that contains an isolated nucleic acid and can be used to introduce the isolated nucleic acid into 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 autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0121] Throughout this disclosure, various aspects of the present invention may 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 present invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0122] Detailed Description The present disclosure encompasses, inter alia, compositions comprising modified immune cells (e.g., stem cells, macrophages, monocytes, and / or dendritic cells) comprising the novel chimeric antigen receptors (CARs) described herein, as well as methods of using and producing such compositions. The present disclosure also encompasses, inter alia, compositions comprising modified immune cells (e.g., stem cells, macrophages, monocytes, and / or dendritic cells) comprising novel nucleic acid constructs encoding the CARs described herein, as well as methods of using and producing such compositions. In some embodiments, a CAR of the present disclosure comprises an anti-mesothelin antigen-binding domain described herein. In some embodiments, a CAR of the present disclosure comprises one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain.

[0123] In some embodiments, modified immune cells described herein that comprise or express a CAR described herein exhibit increased tumor killing, for example, compared to modified immune cells of the same type that comprise a similar CAR (e.g., a CAR that includes a different anti-mesothelin antigen-binding domain and / or does not include (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but includes the other components of a comparative CAR). In some embodiments, modified immune cells described herein that comprise or express a CAR described herein do not exhibit killing of tumor cells that do not express the target antigen (e.g., mesothelin), for example, compared to modified immune cells of the same type that comprise a similar CAR (e.g., a CAR that includes a different anti-mesothelin antigen-binding domain and / or does not include (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but includes the other components of a comparative CAR). In some embodiments, tumor killing includes or is one or more of phagocytosis, lysis, apoptosis, or production of tumor-killing cytokines (e.g., TNFα). In some embodiments, the modified immune cells described herein that comprise or express a CAR described herein exhibit increased tumor killing over a particular period of time. In some embodiments, the modified immune cells described herein that comprise or express a CAR described herein exhibit increased tumor killing for at least one week. In some embodiments, the modified immune cells described herein that comprise or express a CAR described herein exhibit increased tumor killing for at least two weeks.

[0124] In some embodiments, modified immune cells described herein that comprise or express a CAR described herein exhibit increased viability, for example, compared to modified immune cells of the same type that comprise a similar CAR (e.g., a CAR that includes a different anti-mesothelin antigen binding domain and / or does not include (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but includes the other components of a comparative CAR). In some embodiments, modified immune cells described herein that comprise or express a CAR described herein exhibit increased CAR expression, for example, compared to modified immune cells of the same type that comprise a similar CAR (e.g., a CAR that includes a different anti-mesothelin antigen binding domain and / or does not include (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but includes the other components of a comparative CAR).

[0125] In some embodiments, modified immune cells described herein that comprise or express a CAR described herein exhibit increased expression of M1 markers (e.g., one or both of CD80 or CD86) compared to, for example, modified immune cells of the same type that comprise a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen binding domain and / or does not include (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but includes the other components of a comparative CAR). In some embodiments, modified immune cells described herein that comprise or express a CAR described herein exhibit decreased expression of M2 markers (e.g., one or both of CD163 or CD206) compared to, for example, modified immune cells of the same type that comprise a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen binding domain and / or does not include (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but includes the other components of a comparative CAR).

[0126] In some embodiments, a CAR described herein comprises: (a) an extracellular domain comprising an anti-mesothelin binding domain described herein, (b) a transmembrane domain (e.g., a CD28 transmembrane domain or a CD8 transmembrane domain), and (c) one or more intracellular domains. In some embodiments, the one or more intracellular domains comprise a CD3 zeta (CD3ζ) intracellular domain. In some embodiments, the one or more intracellular domains comprise an FcRγ intracellular domain. In some embodiments, the CAR further comprises one or more extracellular hinge domains. In some embodiments, the one or more extracellular hinge domains comprise a CD28 extracellular hinge domain or a CD8a extracellular hinge domain. In some embodiments, a CAR described herein comprises, from N-terminus to C-terminus, a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD8 extracellular hinge domain, a CD8 transmembrane domain, and a CD3ζ intracellular domain. In some embodiments, a CAR described herein comprises, from N-terminus to C-terminus, a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and a CD3ζ intracellular domain. In some embodiments, a CAR described herein comprises, from N-terminus to C-terminus, a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and an FcRγ intracellular domain. In some embodiments, a CAR described herein comprises, from N-terminus to C-terminus, a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD8 extracellular hinge domain, a CD8 transmembrane domain, a CD3ζ intracellular domain, a P2A cleavage peptide, and a CD40 ligand (CD40L). In some embodiments, a CAR described herein comprises, from N-terminus to C-terminus, a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a CD3ζ intracellular domain, a P2A cleavage peptide, and a CD40 ligand (CD40L). In some embodiments, a CAR described herein comprises, from N-terminus to C-terminus, a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a MyD88 intracellular domain, and a CD3ζ intracellular domain. In some embodiments, a CAR described herein comprises, from N-terminus to C-terminus, a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a MyD88 intracellular domain, a CD40 intracellular domain, and a CD3ζ intracellular domain. In some embodiments, a CAR described herein comprises, from N-terminus to C-terminus, a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a truncated MyD88 intracellular domain, a CD40 intracellular domain, and a CD3ζ intracellular domain.In some embodiments, a CAR described herein comprises, from N-terminus to C-terminus, a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, an FcRγ intracellular domain, a P2A cleavage peptide, and a CD40 ligand (CD40L). In some embodiments, a CAR described herein comprises, from N-terminus to C-terminus, a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a MyD88 intracellular domain, and an FcRγ intracellular domain. In some embodiments, a CAR described herein comprises, from N-terminus to C-terminus, a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a MyD88 intracellular domain, a CD40 intracellular domain, and an FcRγ intracellular domain. In some embodiments, a CAR described herein comprises, from N-terminus to C-terminus, a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a truncated MyD88 intracellular domain, a CD40 intracellular domain, and an FcRγ intracellular domain.

[0127] immune cells The present disclosure provides, inter alia, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising at least one chimeric antigen receptor (CAR) described herein. In some embodiments, the population of immune cells described herein comprises stem cells, monocytes, macrophages, dendritic cells, and / or their precursors. In some embodiments, the population of immune cells comprises a substantially purified population or cell line of stem cells, monocytes, macrophages, or dendritic cells.

[0128] In some embodiments, immune cells are activated, e.g., the immune cells exhibit increased cytokine production, chemokine production, phagocytosis, cell signaling, target cell killing, and / or antigen presentation, e.g., compared to inactive cells. In some embodiments, activated immune cells exhibit altered gene expression, e.g., induction of pro-inflammatory gene expression, e.g., compared to inactive cells. In some embodiments, activated immune cells exhibit altered gene expression, e.g., induction of anti-inflammatory gene expression, e.g., compared to inactive cells. In certain embodiments, activated immune cells undergo cell division. In some embodiments, the target effector activity of immune cells is enhanced by inhibition of CD47 and / or SIRPα activity. CD47 and / or SIRPα activity can be inhibited by treating immune cells with anti-CD47 or anti-SIRPα antibodies, or by any method known to those of skill in the art.

[0129] In some embodiments, immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) are obtained (e.g., isolated) from a subject. The cells can be obtained from many 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, the cells can be obtained from a unit of blood collected from a subject using any number of separation techniques known to those of skill in the art, such as Ficoll separation. In some embodiments, cells from the subject's circulating blood are obtained by apheresis or leukapheresis. Cells collected by apheresis can be washed to remove the plasma fraction and resuspended in various buffers (e.g., phosphate-buffered saline (PBS)) or culture media. In some embodiments, enrichment of immune cells (e.g., monocytes) involves plastic adherence. In some embodiments, differentiation of the enriched immune cells (e.g., monocytes) involves stimulation with GM-CSF. In some embodiments, a composition comprising blood cells (e.g., monocytes, lymphocytes, platelets, plasma, and / or red blood cells), such as a leukopheresis composition (e.g., leukopheresis) is used for enrichment. In some embodiments, the leukopheresis composition (e.g., leukopheresis) 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) involves CD14-positive selection using microbeads (e.g., MACS® MicroBeads on a CliniMACS Prodigy device). In some embodiments, immune cell precursors (e.g., precursors of macrophages, monocytes, or dendritic cells, including, but not limited to, induced pluripotent stem cells, or iPSCs) are used in the compositions and methods described herein. Immune cell precursors can be differentiated into immune cells in vivo or ex vivo. Non-limiting examples of immune progenitor cells include hematopoietic stem cells, common myeloid progenitor cells, myeloblasts, monoblasts, promonocytes, or intermediates thereof. For example, induced pluripotent stem cells can be used to generate monocytes, macrophages, and / or dendritic cells.Induced pluripotent stem cells (iPSCs) can be derived from normal human tissues such as peripheral blood, fibroblasts, skin, keratinocytes, or renal epithelial cells. Autologous, allogeneic, or universal donor iPSCs can differentiate into myeloid lineages (e.g., monocytes, macrophages, dendritic cells, or their precursors).

[0130] Immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein can be isolated from peripheral blood, e.g., by lysing red blood cells and depleting lymphocytes and red blood cells, e.g., by centrifugation through a PERCOLL™ gradient. Alternatively, immune cells can be isolated from umbilical cord tissue. Specific subpopulations 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, CD34, CD3, CD4, CD8, CD56, CD66b, CD19, or CD20. In some embodiments, enrichment of immune cell populations, e.g., by negative selection, can be achieved using a combination of antibodies directed against surface markers unique to the negatively selected cells. As non-limiting examples, cell selection can also include negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells.

[0131] During the 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, the concentration and surface (e.g., particles such as beads) of the immune cells can be varied. To ensure maximum contact area between the cells and the beads, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together.

[0132] In some embodiments, prior to administration, the modified immune cells described herein (e.g., stem cells, macrophages, monocytes, or dendritic cells) (e.g., comprising at least one CAR described herein) are treated with a pro-inflammatory agent. In some embodiments, treatment with a pro-inflammatory agent increases the anti-tumor activity of the modified immune cells described herein. In some embodiments, treatment with at least one pro-inflammatory agent promotes an M1 phenotype (e.g., a switch from M2 to M1 phenotype) in the modified immune cells described herein. In some embodiments, the at least one pro-inflammatory agent includes or is a CD40 agonist (e.g., CD40L). In some embodiments, the at least one pro-inflammatory agent includes or is a 41BB-ligand agonist (e.g., 4-1BB). In some embodiments, the at least one pro-inflammatory agent includes or is a CD40 agonist (e.g., CD40L) and a 41BB ligand agonist (e.g., 4-1BB).

[0133] In some embodiments, the modified immune cells described herein (e.g., stem cells, macrophages, monocytes, or dendritic cells) (e.g., comprising at least one CAR described herein) have been treated with one or more pro-inflammatory agents. In some embodiments, the modified immune cells described herein exhibit increased anti-tumor activity compared to unmodified cells of the same type. In some embodiments, the one or more pro-inflammatory agents include or are a CD40 agonist (e.g., CD40L). In some embodiments, the one or more pro-inflammatory agents include or are a 41BB-ligand agonist (e.g., 4-1BB). In some embodiments, the one or more pro-inflammatory agents include or are a CD40 agonist (e.g., CD40L) and a 41BB ligand agonist (e.g., 4-1BB). The present disclosure provides methods of treating a disease or disorder in a subject, the method comprising delivering to the subject a therapeutically effective amount of a pharmaceutical composition comprising modified macrophages, monocytes, or dendritic cells described herein.

[0134] The present disclosure also provides methods of modifying immune cells described herein (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR described herein, the method comprising treating the immune cells described herein with one or more pro-inflammatory agents, thereby generating modified immune cells described herein that exhibit increased anti-tumor activity compared to the same type of immune cell comprising a CAR or a similar CAR that has not been treated with the one or more pro-inflammatory agents. In some embodiments, the one or more pro-inflammatory agents include or are a CD40 agonist (e.g., CD40L). In some embodiments, the one or more pro-inflammatory agents include or are a 41BB-ligand agonist (e.g., 4-1BB). In some embodiments, the one or more pro-inflammatory agents include or are a CD40 agonist (e.g., CD40L) and a 41BB ligand agonist (e.g., 4-1BB). The present disclosure provides methods of treating a disease or disorder in a subject, the method comprising delivering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an immune cell described herein that has been modified by a method described herein.

[0135] In some embodiments, the modified immune cells described herein (e.g., stem cells, macrophages, monocytes, or dendritic cells) (e.g., comprising a CAR described herein) are administered to a subject in combination with a pro-inflammatory agent. In some embodiments, the modified immune cells described herein (e.g., stem cells, macrophages, monocytes, or dendritic cells) (e.g., comprising a CAR described herein) are administered to a subject substantially simultaneously with, before, or after, the pro-inflammatory agent. In some embodiments, the pro-inflammatory agent is administered as a nucleic acid (e.g., in a construct packaged with the CAR and a truncated peptide such as a P2A, F2A, E2A, and / or T2A peptide). In some embodiments, administration with a pro-inflammatory agent increases the anti-tumor activity of the modified immune cells described herein. In some embodiments, administration with a pro-inflammatory agent promotes an M1 phenotype (e.g., a switch from M2 to M1 phenotype) in the immune cells described herein. In some embodiments, the pro-inflammatory agent includes or is a CD40 agonist (e.g., CD40L). In some embodiments, the pro-inflammatory agent comprises or is a 41BB-ligand agonist (eg, 4-1BB). macrophages Macrophages are immune cells specialized for the detection, phagocytosis, and destruction of target cells, such as pathogens or tumor cells. Macrophages are potent effectors of the innate immune system and can perform at least three distinct antitumor functions: 1) phagocytosis of dead or dying cells, microorganisms, cancer cells, cell debris, or other foreign bodies, 2) cytotoxicity against tumor cells, and 3) presentation of tumor antigens to orchestrate adaptive antitumor immune responses.

[0136] Macrophages are abundant in the tumor microenvironment of many cancers and can adopt many phenotypes, collectively referred to as tumor-associated macrophages (TAMs). The immunosuppressive nature of the tumor microenvironment typically results in more M2-like TAMs, which further contributes to the general suppression of antitumor immune responses. However, recent studies have confirmed that TAMs can be "reprogrammed" through pro-inflammatory signals and that a switch from an M2 phenotype to a more M1 phenotype is associated with a productive antitumor immune response. Engineering macrophages that induce endogenous TAMs to switch to M1 cells and are unable to subvert to M2 would significantly improve antitumor immunotherapy, representing a significant advance in the field.

[0137] In some embodiments, the macrophages comprise or are undifferentiated or M0 macrophages. In certain embodiments, the macrophages comprise or express one, two, three, four, five, or six of CD14, CD16, CD64, CD68, CD71, or CCR5. Upon exposure to various stimuli, M0 macrophages can be induced to polarize into several different populations that can be identified by macrophage phenotypic markers, cytokine production, and / or chemokine secretion.

[0138] In some embodiments, the macrophages comprise or are polarized macrophages. Under classical conditions of activation, M0 macrophages can be exposed to pro-inflammatory signals, such as LPS, IFNγ, or GM-CSF, and polarize into pro-inflammatory (i.e., M1) macrophages. Generally, pro-inflammatory (M1) macrophages are associated with pro-inflammatory immune responses, such as Th1 and Th17 T cell responses. Upon exposure to other stimuli, macrophages can polarize into various "alternatively activated" or anti-inflammatory (i.e., M2) macrophage populations.

[0139] In some embodiments, the macrophages comprise or are pro-inflammatory (M1) macrophages. In some embodiments, the macrophages express one or more markers of pro-inflammatory (M1) macrophages (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, six, seven, eight, nine, ten, eleven, twelve, thirteen, thirteen, fourteen, fifteen, six, seven ...

[0140] In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit relatively higher levels of one or more inflammatory cytokines (e.g., IL-1, TNF, IL-12, IL-18, IL-23, IFNα, IFNβ, IFNγ, IL-2, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-18, IL-23, IFNα, IFNβ, IFNγ, IL-2, IL-6, IL-11), compared to macrophages comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen binding domain and / or does not comprise (i) a CD8 or CD28 extracellular hinge domain and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises other components of a comparative CAR). or 12 of IL-8, or IL-33) or a chemokine (e.g., one or both of a CC or CXC chemokine) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 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 CC chemokines, e.g., one of a CX3C chemokine, e.g., one or both of a C chemokine). In some embodiments, macrophages comprising or expressing at least one CAR described herein stimulate an immune response and / or inflammation compared to macrophages comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or that does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises other components of the comparative CAR).

[0141] In some embodiments, the macrophages comprise or are anti-inflammatory (M2) macrophages (e.g., M2a, M2b, M2c, and M2d macrophages). M2a macrophages can be induced by IL-4, IL-13, and / or fungal infection. M2b macrophages can be induced by IL-1R ligands, immune complexes, and / or LPS. M2c macrophages can be induced by IL-10 and / or TGFβ. M2d macrophages can be induced by IL-6 and / or adenosine. In some embodiments, macrophages comprising or expressing at least one CAR described herein reduce an immune response in a subject compared to macrophages comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of the CD8 or CD28 transmembrane domains, but comprises other components of the comparative CAR). In some embodiments, the macrophages express one or more markers of anti-inflammatory (M2) macrophages (e.g., one, two, or three of CD206, CD163, or CD209). In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased secretion of one or more anti-inflammatory cytokines (e.g., one or both of IL-10 or TGFβ), e.g., compared to macrophages comprising a similar CAR (e.g., a CAR described herein).

[0142] In some embodiments, the macrophages comprise at least one upregulated pro-inflammatory (M1) marker and / or at least one downregulated anti-inflammatory (M2) marker compared to control macrophages that do not include at least one CAR provided herein and / or the same macrophages prior to delivery of at least one CAR described herein. In some embodiments, at least one pro-inflammatory (M1) marker (e.g., HLA DR, CD86, CD80, PD-L1, CD83, CD69, MHC I, CD64, CD32, CD16, IL1R, IFIT family members, and / or ISG family members) is upregulated in the macrophages. In some embodiments, at least one anti-inflammatory (M2) marker (e.g., CD206, CD163, and / or CD209) is downregulated in the macrophages.

[0143] In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased phagocytosis, for example, compared to macrophages comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or that does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises the other components of a comparative CAR). In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased cytotoxicity against tumor cells, for example, compared to macrophages comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or that does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises the other components of a comparative CAR). In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased tumor antigen presentation (e.g., presentation after phagocytosis) and / or increased antigen processing, for example, compared to macrophages comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises other components of a comparative CAR). In some embodiments, macrophages comprising or expressing at least one CAR exhibit increased tumor killing (e.g., by phagocytosis, lysis, apoptosis, or production of tumor-killing cytokines (e.g., TNFα)), for example, compared to macrophages comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises other components of a comparative CAR).

[0144] In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased effector function (e.g., phagocytosis, targeted cell cytotoxicity, antigen presentation, or cytokine secretion) (e.g., increased cellular cytotoxicity ... For example, the cells exhibit either or both increased expression of one or more genes typically associated with an effector function (e.g., phagocytosis, targeted cell cytotoxicity, antigen presentation, or cytokine secretion) (e.g., CD163, CD206, TGFβ, IL-10, and / or IL4), or decreased expression of one or more genes typically associated with a reduced effector function (e.g., phagocytosis, targeted cell cytotoxicity, antigen presentation, or cytokine secretion) (e.g., CD80, CD86, MHC-I, MHC-II, CD40, 41BBL, TNF, IFN-α, IFN-β, IFN-γ, IL2, IL12, IL6, IL8, IL1b, and / or CXCL12). In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased ROS production, for example, compared to macrophages comprising a similar CAR (e.g., a CAR that includes a different anti-mesothelin antigen-binding domain and / or does not include one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but includes other components of the comparative CAR). In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased ROS production, for example, compared to macrophages comprising a similar CAR (e.g., a CAR that includes a different anti-mesothelin antigen-binding domain and / or does not include one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but includes other components of the comparative CAR).Macrophage maturation signaling, dendritic cell maturation signaling, CD3-zeta signaling, FcRγ signaling, CD64 signaling, CD32a signaling, CD32c signaling, CD16a 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, EphA1 signaling, INSR signaling, cMET signaling, MUSK signaling, PDGFR signaling, PTK7 signaling, RET signaling, ROR1 signaling, ROS1 signaling, RYK signaling, TIE2 signaling, TRK signaling, VEGFR signaling, CD40 signaling, CD19 signaling, CD20 signaling, 41BB signaling, CD28 signaling, OX40 signaling, GITR signaling, TREM-1 signaling, TREM-2 signaling, DAP12 signaling, MR signaling, ICOS signaling, MyD88 signaling, V / I / LxYxxL / V signaling, SIRPα signaling, CD45 signaling, Siglec-10 signaling, PD1 signaling, SHP-1 signaling, SHP-2 signaling, KIR-2DL signaling, KIR-3DL signaling, NKG2A signaling, CD170 signaling, CD33 signaling, BTLA signaling, CD32b signaling, SIRPβ signaling, CD22 signaling, PIR-B signaling, and / or LILRB1 signaling). In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit induction of cell survival mechanisms, for example, compared to macrophages comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen binding domain and / or that does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises other components of the comparative CAR).Macrophages comprising or expressing at least one CAR described herein exhibit induction of cell death mechanisms, for example, compared to macrophages comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen binding domain and / or that does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of the CD8 or CD28 transmembrane domain, but comprises other components of the comparative CAR). In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit one, two, three, four, or five of the following, for example, increased resistance to phagocytic checkpoints, increased expression of chemokine receptors to assist trafficking, increased expression of chemokines to recruit other immune cells, increased expression of ECM-degrading enzymes (e.g., MMPs that degrade tumor ECM and / or exhibit anti-fibrotic activity), and / or increased proliferation, compared to macrophages comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or does not comprise (i) a CD8 or CD28 extracellular hinge domain and / or one or both of (ii) a CD8 or CD28 transmembrane domain, but comprises other components of a comparative CAR). In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit one, two, three, or four of the following: improved duration of CAR expression, improved stability of the CAR on the cell surface, increased levels of CAR expression, and / or decreased background activity of the CAR, as compared to macrophages comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen binding domain and / or does not comprise (i) a CD8 or CD28 extracellular hinge domain, and / or one or both of (ii) a CD8 or CD28 transmembrane domain, but comprises other components of the comparative CAR).

[0145] Monocytes Monocytes are multipotent cells that circulate in the blood, bone marrow, and spleen and generally do not proliferate in a steady state. Monocytes vary greatly in size, ranging from approximately 10 to 30 μm in diameter. The nucleus-to-cytoplasm ratio of monocytes can range from approximately 2:1 to approximately 1:1. Monocytes typically contain chemokine receptors and pathogen recognition receptors that mediate migration from the blood to tissues, such as during infection. Monocytes produce proinflammatory cytokines, internalize cells and / or toxic molecules, and can differentiate into dendritic cells or macrophages.

[0146] In some embodiments, monocytes comprise or express one or more phenotypic markers. Examples of phenotypic markers of human monocytic cells include CD9, CD11b, CD11c, CDw12, CD13, CD15, CDw17, 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, CD116, CD119, CDw121b, CDw123, CD127, CDw128, CDw131, CD147, CD155, CD156a, CD157, CD162 Examples of phenotypic markers for mouse monocytic cells include, but are not limited to, CD163, CD164, CD168, CD171, CD172a, CD180, CD206, CD131a1, CD2132, CDw210, CD226, CD281, CD282, CD284, and CD286. Examples of phenotypic markers for mouse monocytic cells include, but are not limited to, CD11a, CD11b, CD16, CD18, CD29, CD31, CD32, CD44, CD45, CD49d, CD115, CD116, Cdw131, CD281, CD282, CD284, CD286, F4 / 80, and CD49b. In certain embodiments, monocytes comprise one, two, or three of CD11b, CD14, or CD16. In certain embodiments, the monocytes comprise CD14+ CD16- monocytes, CD14+ CD16+ monocytes, or CD14- CD16+ monocytes.

[0147] In some embodiments, monocytes are differentiated into macrophages. In some embodiments, monocytes are differentiated into dendritic cells (DCs). Monocytes can be differentiated into macrophages or DCs by any technique known in the art. For example, differentiation of monocytes into macrophages is 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.

[0148] In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit 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), compared to, for example, monocytes comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or that does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but includes the other components of the comparative CAR). In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased phagocytosis, compared to, for example, monocytes comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or that does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but includes the other components of the comparative CAR). In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit improved survival, e.g., compared to monocytes comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or that does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises the other components of a comparative CAR). In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit enhanced differentiation into macrophages (e.g., M1 or M2 macrophages), e.g., compared to monocytes comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or that does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises the other components of a comparative CAR).In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit enhanced differentiation into DCs (e.g., resident or migratory DCs and / or lymphoid and non-lymphoid tissues), compared to, for example, monocytes comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or that does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises the other components of a comparative CAR). In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased cytotoxicity against tumor cells, compared to, for example, monocytes comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or that does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises the other components of a comparative CAR). In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased tumor antigen presentation (e.g., presentation after phagocytosis) and / or increased antigen processing, e.g., compared to monocytes comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or that does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises the other components of a comparative CAR). In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased tumor killing (e.g., by phagocytosis, lysis, apoptosis, or production of tumor-killing cytokines (e.g., TNFα)), e.g., compared to monocytes comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or that does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises the other components of a comparative CAR).

[0149] In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit one or both of increased expression of one or more genes typically associated with increased effector function (e.g., phagocytosis, targeted cell cytotoxicity, antigen presentation, or cytokine secretion), or decreased expression of one or more genes typically associated with decreased effector function (e.g., phagocytosis, targeted cell cytotoxicity, antigen presentation, or cytokine secretion), compared to, e.g., monocytes comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises other components of a comparison CAR). In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased ROS generation, compared to, e.g., monocytes that do not comprise a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit metabolic reprogramming, e.g., compared to monocytes comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or that does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises the other components of the comparative CAR). In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit induction of cell survival mechanisms, e.g., compared to monocytes comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or that does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises the other components of the comparative CAR).In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit induction of cell death mechanisms, e.g., compared to monocytes comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or that does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises other components of a comparative CAR). In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit one, two, three, four, or five of the following, e.g., increased resistance to phagocytic checkpoints, increased expression of chemokine receptors to assist 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, compared to monocytes that do not comprise a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit one, two, three, or four of the following, e.g., improved duration of CAR expression, improved stability of the CAR on the cell surface, increased levels of CAR expression, and / or decreased background activity of the CAR, compared to monocytes that do not comprise a CAR described herein.

[0150] dendritic cells Dendritic cells (DCs) are specialized bone marrow-derived antigen-presenting cells that are involved in initiating immune responses and maintaining tolerance of the immune system to self-antigens. Dendritic cells can be found in both lymphoid and non-lymphoid organs and are generally thought to originate from either the lymphoid or myeloid lineages.

[0151] In some embodiments, DCs comprise or express one or more phenotypic markers. Exemplary phenotypic markers of DCs include, but are not limited to, CD11c, CD83, CD1a, CD1c, CD141, CD207, CLEC9a, CD123, CD85, CD180, CD187, CD205, CD281, CD282, CD284, CD286, and in part, CD206, CD207, CD208, and CD209.

[0152] Immature DCs may be characterized by a high capacity for antigen capture but a relatively low capacity for T cell stimulation. Inflammatory mediators promote DC maturation. When DCs reach a mature stage, their properties change dramatically compared to immature DCs, including a decreased capacity for antigen capture and / or an increased capacity for T cell stimulation. In some embodiments, the DCs comprise or are immature DCs. In other embodiments, the DCs comprise or are mature DCs.

[0153] Without wishing to be bound by theory, it is believed that modifying DC cells to contain or express at least one CAR described herein may enable mature DCs to simultaneously exhibit increased antigen capture capacity and T cell stimulation, as compared to DCs containing a similar CAR (e.g., a CAR that contains a different anti-mesothelin antigen-binding domain and / or does not contain (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of the CD8 or CD28 transmembrane domain, but contains other components of the comparative CAR). In some embodiments, DCs containing or expressing at least one CAR described herein mediate tumor antigen presentation, e.g., increased tumor antigen presentation, as compared to DCs containing a similar CAR (e.g., a CAR that contains a different anti-mesothelin antigen-binding domain and / or does not contain (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of the CD8 or CD28 transmembrane domain, but contains other components of the comparative CAR). In some embodiments, DCs comprising or expressing at least one CAR described herein mediate tumor T cell stimulation, e.g., increased T cell stimulation, compared to DCs comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises other components of the comparative CAR).

[0154] In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit 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), compared to, for example, DCs comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises the other components of a comparative CAR). In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit increased phagocytosis, for example, compared to DCs comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises the other components of a comparative CAR). In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit increased tumor antigen presentation (e.g., presentation after phagocytosis), increased antigen processing, increased antigen cross-presentation, increased T cell priming, and / or T cell stimulation, for example, compared to DCs comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or does not comprise (i) a CD8 or CD28 extracellular hinge domain, and / or one or both of (ii) a CD8 or CD28 transmembrane domain, but comprises other components of a comparative CAR).

[0155] In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit one or both of increased expression of preferred genes or decreased expression of undesired genes, e.g., compared to DCs comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises the other components of a comparative CAR). In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit increased ROS generation, e.g., compared to DCs comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises the other components of a comparative CAR). In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit metabolic reprogramming, e.g., compared to DCs comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises the other components of a comparative CAR). In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit induction of cell survival mechanisms, e.g., compared to DCs comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but comprises the other components of a comparative CAR).

[0156] In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit induction of cell death mechanisms, e.g., compared to DCs comprising a similar CAR (e.g., a CAR that comprises a different anti-mesothelin antigen-binding domain and / or that does not comprise (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of the CD8 or CD28 transmembrane domains, but comprises other components of a comparative CAR). In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit one, two, three, four, or five of the following, e.g., increased resistance to phagocytic checkpoints, increased expression of chemokine receptors to assist 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, compared to DCs that do not comprise a CAR described herein. In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit one, two, three, or four of the following, e.g., improved duration of CAR expression, improved stability of the CAR on the cell surface, increased levels of CAR expression, and / or decreased background activity of the CAR, compared to DCs that do not comprise a CAR described herein.

[0157] Chimeric antigen receptor (CAR) As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial cell surface receptor engineered to be expressed on immune effector cells and specifically target the cells and / or bind to an antigen. CARs can be used as a therapy, e.g., using 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 the blood, tumor, or ascites) and modified to express a receptor specific for a particular form of antigen. In some embodiments, such modified immune cells are then reintroduced into the same or a different subject as a therapeutic agent. In some embodiments, the CAR is specifically expressed against an antigen, e.g., a tumor-associated antigen. In some embodiments, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain.

[0158] In some embodiments, modified immune cells, e.g., modified stem cells, macrophages, monocytes, or dendritic cells, are generated by expressing a CAR therein. In some embodiments, the immune cells comprise a CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain, and the immune cells comprise stem cells, macrophages, monocytes, or dendritic cells.

[0159] In some embodiments, the CAR may further comprise one or more extracellular leader domains, one or more extracellular hinge domains, and one or more intracellular costimulatory domains.

[0160] In some embodiments, the CAR comprises a spacer domain or hinge (i.e., an extracellular hinge domain) between the extracellular domain and the transmembrane domain. In some embodiments, the CAR comprises a spacer domain or hinge (i.e., an intracellular hinge domain) between the intracellular domain and the transmembrane domain. As used herein, the term "spacer domain" or "hinge" refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to either the extracellular or intracellular domain of a polypeptide chain. In some embodiments, the spacer domain or hinge may comprise up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids. In some embodiments, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, may form the link between the transmembrane and intracellular domains of the CAR. Examples of linkers include a glycine-serine doublet.

[0161] In some embodiments, an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR may comprise one or more regulatory systems, including, but not limited to, a safety switch (e.g., an on switch and an off switch, a suicide switch), and / or a logic gate (e.g., an AND gate (e.g., two or more CARs, each lacking one or more signaling domains such that activation of both / all CARs is required for activation or function of the complete immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell)), an OR gate (e.g., two or more CARs, each having an intracellular domain such as CD3ζ and a costimulatory domain), and / or a NOT gate (e.g., two or more CARs, one of which contains an inhibitory domain that antagonizes the function of the other CAR(s)).

[0162] The present disclosure also provides an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a nucleic acid (e.g., an isolated nucleic acid) 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 an intracellular domain, and the cell is a stem cell, macrophage, monocyte, or dendritic cell that expresses a CAR.

[0163] In some embodiments, the CAR comprises an extracellular domain operably linked to another domain of the CAR, such as a transmembrane domain or an intracellular domain, for expression in an immune cell. In some embodiments, the nucleic acid encoding the extracellular domain is operably linked to the nucleic acid encoding the transmembrane domain, which is operably linked to the nucleic acid encoding the intracellular domain.

[0164] In some embodiments, the effector activity of an immune cell comprising a CAR is directed against a target cell that contains an antigen that specifically binds to the antigen-binding domain of the CAR. In some embodiments, the targeted effector activity directed against the target cell is or includes phagocytosis, targeted cell cytotoxicity, antigen presentation, or cytokine secretion.

[0165] 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, a macrophage, a monocyte, or a dendritic cell). In some embodiments, a CAR described herein improves the effector activity of an immune cell described herein (e.g., a stem cell, a macrophage, a monocyte, or a dendritic cell), e.g., by enhancing inhibition of CD47 and / or SIRPα activity. In some embodiments, a CAR described herein binds to CD47 and acts as a dominant-negative receptor, inhibiting SIRPα activity (e.g., a CD47 sink). In some embodiments, a CAR described herein that binds to SIRPα comprises, e.g., an activating receptor (e.g., comprises a CD3z intracellular domain). In some embodiments, a CAR described herein inhibits at least one interaction between CD47 and SIRPα. In some embodiments, a CAR is or comprises a phagocytosis logic gate.

[0166] In some embodiments, an immune cell described herein (e.g., comprising at least one CAR described herein) comprises or expresses at least one variant or fragment of SIRPα (e.g., dominant-negative SIRPα or a high-affinity engineered variant of SIRPα (e.g., CV1)), 5F9 scFv, B6H12 scFv (e.g., humanized B6H12 scFv), PD1 (e.g., dominant-negative PD1 or HAC-I), anti-PD1 scFv (e.g., E27 or durvalumab), Siglec-10, Siglec-9, Siglec-11, and / or SHP-1. In some embodiments, the variant or fragment comprises a mutant intracellular domain. In some embodiments, the variant or fragment does not comprise or express at least one intracellular domain (e.g., the immune cell comprises or expresses an anti-CD47 scFv, a CD8 hinge domain, and a CD8 transmembrane). In some embodiments, an immune cell described herein (e.g., comprising or expressing at least one CAR described herein) comprises a dominant negative receptor, e.g., that blocks an inhibitory checkpoint.

[0167] In some embodiments, the payload comprising a CAR described herein further comprises at least one second CAR comprising a truncated peptide (e.g., a P2A, F2A, E2A, and / or T2A peptide) and / or at least one inhibitory domain of anti-phagocytic signaling. In some embodiments, the at least one second CAR comprises SIRPα (e.g., a high-affinity engineered variant of SIRPα (e.g., CV1)), 5F9 scFv, B6H12 scFv (e.g., humanized B6H12 scFv), or a CD47-binding extracellular domain or a fragment thereof. In some embodiments, the at least one second CAR comprises a SIRPα transmembrane domain or a fragment thereof. In certain embodiments, the second CAR further comprises a hinge domain (e.g., a CD8 hinge domain). In certain embodiments, at least one second CAR comprises (i) a leader sequence (e.g., a CD8 leader), ii) an extracellular domain (e.g., a SIRPα, CV1, 5F9 scFv, or B6H12 scFv (e.g., a humanized B6H12 scFv) extracellular domain), and ii) a transmembrane domain (e.g., a SIRPα transmembrane domain). In some embodiments, a payload comprising a CAR described herein further comprises a truncated peptide (e.g., a P2A peptide) and at least one marker protein (e.g., CD20 or a fragment thereof, CD19 or a fragment thereof, NGFR or a fragment thereof, a synthetic peptide, and / or a fluorescent protein).

[0168] In some embodiments, an immune cell described herein (e.g., comprising or expressing at least one CAR described herein) comprises or expresses one or more phosphatase dead domains (e.g., phosphatase dead Shp1, phosphatase dead 72-5ptase (INPP5E), phosphatase dead Shp2, and / or phosphatase dead SHIP-1 domains) and / or constitutively active kinase domains (e.g., constitutively active LYN domains). In some embodiments, a payload comprising a CAR described herein further comprises a truncated peptide (e.g., a P2A, F2A, E2A, and / or T2A peptide) and one or more phosphatase-dead domains (e.g., phosphatase-dead Shp1, 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).

[0169] In some embodiments, a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence at least 80% identical to a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence at least 85% identical to a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence at least 90% identical to a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence at least 95% identical to a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence at least 96% identical to a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence at least 97% identical to a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence at least 98% identical to a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence at least 99% identical to a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence identical to a sequence selected from Table 2.

[0170] In some embodiments, a CAR of the present disclosure binds mesothelin and comprises an amino acid sequence that differs by no more than 50 substitutions, additions, or deletions from a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure binds mesothelin and comprises an amino acid sequence that differs by no more than 40 substitutions, additions, or deletions from a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure binds mesothelin and comprises an amino acid sequence that differs by no more than 30 substitutions, additions, or deletions from a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure binds mesothelin and comprises an amino acid sequence that differs by no more than 20 substitutions, additions, or deletions from a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure binds mesothelin and comprises an amino acid sequence that differs by no more than 10 substitutions, additions, or deletions from a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure binds mesothelin and comprises an amino acid sequence that differs by no more than 5 substitutions, additions, or deletions from a sequence selected from Table 2. In some embodiments, a CAR of the disclosure binds mesothelin and comprises an amino acid sequence that differs by no more than four substitutions, additions, or deletions from a sequence selected from Table 2. In some embodiments, a CAR of the disclosure binds mesothelin and comprises an amino acid sequence that differs by no more than three substitutions, additions, or deletions from a sequence selected from Table 2. In some embodiments, a CAR of the disclosure binds mesothelin and comprises an amino acid sequence that differs by no more than two substitutions, additions, or deletions from a sequence selected from Table 2. In some embodiments, a CAR of the disclosure binds mesothelin and comprises an amino acid sequence that differs by no more than one substitution, addition, or deletion from a sequence selected from Table 2. In some embodiments, a CAR of the disclosure binds mesothelin and comprises an amino acid sequence that does not have any substitutions, additions, or deletions compared to a sequence selected from Table 2.

[0171] CAR extracellular domain The present disclosure provides chimeric antigen receptors (CARs) comprising one or more extracellular domains. In some embodiments, the CAR extracellular domain comprises an Fc receptor (FcR) extracellular domain. In some embodiments, the CAR extracellular domain comprises a toll-like receptor (TLR) extracellular domain. In some embodiments, the CAR extracellular domain comprises a leader domain. In some embodiments, the CAR extracellular domain comprises an antigen-binding domain. In some embodiments, the CAR extracellular domain comprises a hinge domain. In some embodiments, the CAR extracellular domain comprises 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, the CAR extracellular domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the CAR extracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0172] FcR extracellular domain In some embodiments, the FcR extracellular domain comprises a full-length FcR extracellular domain. In some embodiments, the FcR extracellular domain comprises a portion of a full-length FcR extracellular domain. In some embodiments, the FcR extracellular domain (or portion thereof) is or comprises a human FcR extracellular domain. In some embodiments, the FcR extracellular domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the FcR extracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the FcR extracellular domain comprises an FcRγ, CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c, CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, or FcαRI (CD89) domain.

[0173] TLR extracellular domains In some embodiments, the TLR ectodomain comprises a full-length TLR ectodomain. In some embodiments, the TLR ectodomain comprises a portion of a full-length TLR ectodomain. In some embodiments, the TLR ectodomain (or portion thereof) is or comprises a human TLR ectodomain. In some embodiments, the TLR ectodomain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the TLR ectodomain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the TLR ectodomain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.

[0174] Leader Domain In some embodiments, the CAR comprises one or more extracellular leader domains. In some embodiments, the nucleic acid encoding the 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, the extracellular leader domain is or comprises a human extracellular leader domain. In some embodiments, the extracellular leader domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the extracellular leader domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the extracellular leader domain comprises a CD8 extracellular leader domain. In some embodiments, the extracellular leader domain comprises a leader domain derived from a stimulatory or costimulatory domain (e.g., a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphA1, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD19, CD20, 41BB, CD28, OX40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88 domain).

[0175] antigen-binding domain In some embodiments, the CAR comprises an antigen-binding domain that binds to, for example, an antigen on a target cell. In some embodiments, the CAR comprises an antigen-binding domain that binds to an antigen associated with a cancer cell. In some embodiments, the CAR antigen-binding domain recognizes an antigen that acts as a cell surface marker on target cells associated with a particular disease state.

[0176] In some embodiments, the CAR antigen-binding domain binds to a tumor antigen, such as an antigen specific to a tumor or cancer of interest. In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes. In some embodiments, the tumor antigen comprises mesothelin.

[0177] In some embodiments, the CAR antigen binding domain binds mesothelin and comprises an amino acid sequence at least 80% identical to a sequence selected from Table 3. In some embodiments, the CAR antigen binding domain binds mesothelin and comprises an amino acid sequence at least 85% identical to a sequence selected from Table 3. In some embodiments, the CAR antigen binding domain binds mesothelin and comprises an amino acid sequence at least 90% identical to a sequence selected from Table 3. In some embodiments, the CAR antigen binding domain binds mesothelin and comprises an amino acid sequence at least 95% identical to a sequence selected from Table 3. In some embodiments, the CAR antigen binding domain binds mesothelin and comprises an amino acid sequence at least 96% identical to a sequence selected from Table 3. In some embodiments, the CAR antigen binding domain binds mesothelin and comprises an amino acid sequence at least 97% identical to a sequence selected from Table 3. In some embodiments, the CAR antigen binding domain binds mesothelin and comprises an amino acid sequence at least 98% identical to a sequence selected from Table 3. In some embodiments, the CAR antigen binding domain binds mesothelin and comprises an amino acid sequence at least 99% identical to a sequence selected from Table 3. In some embodiments, the CAR antigen-binding domain binds to mesothelin and comprises an amino acid sequence identical to a sequence selected from Table 3.

[0178] In some embodiments, the CAR antigen binding domain binds mesothelin and comprises an amino acid sequence that differs by no more than 10 substitutions, additions, or deletions from a sequence selected from Table 3. In some embodiments, the CAR antigen binding domain binds mesothelin and comprises an amino acid sequence that differs by no more than 9 substitutions, additions, or deletions from a sequence selected from Table 3. In some embodiments, the CAR antigen binding domain binds mesothelin and comprises an amino acid sequence that differs by no more than 8 substitutions, additions, or deletions from a sequence selected from Table 3. In some embodiments, the CAR antigen binding domain binds mesothelin and comprises an amino acid sequence that differs by no more than 7 substitutions, additions, or deletions from a sequence selected from Table 3. In some embodiments, the CAR antigen binding domain binds mesothelin and comprises an amino acid sequence that differs by no more than 6 substitutions, additions, or deletions from a sequence selected from Table 3. In some embodiments, the CAR antigen binding domain binds mesothelin and comprises an amino acid sequence that differs by no more than 5 substitutions, additions, or deletions from a sequence selected from Table 3. In some embodiments, the CAR antigen binding domain binds mesothelin and comprises an amino acid sequence that differs by no more than four substitutions, additions, or deletions from a sequence selected from Table 3. In some embodiments, the CAR antigen binding domain binds mesothelin and comprises an amino acid sequence that differs by no more than three substitutions, additions, or deletions from a sequence selected from Table 3. In some embodiments, the CAR antigen binding domain binds mesothelin and comprises an amino acid sequence that differs by no more than two substitutions, additions, or deletions from a sequence selected from Table 3. In some embodiments, the CAR antigen binding domain binds mesothelin and comprises an amino acid sequence that differs by no more than one substitution, addition, or deletion from a sequence selected from Table 3. In some embodiments, the CAR antigen binding domain binds mesothelin and comprises an amino acid sequence that does not have any substitutions, additions, or deletions compared to a sequence selected from Table 3.

[0179] In some embodiments, the CAR antigen-binding domain comprises any domain that binds to an antigen. In some embodiments, the 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, such as an scFv. In some embodiments, the CAR antigen-binding domain is or comprises an aptamer, a darpin, a centilin, a naturally occurring or synthetic receptor, an affibody, a nanobody, or other engineered protein recognition molecule. In some embodiments, the CAR antigen-binding domain is or comprises a mammalian antibody or a fragment thereof. In some embodiments, the 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, the antigen-binding domain of the CAR comprises a human antibody, a humanized antibody, or a fragment thereof (e.g., an scFv). In some embodiments, the CAR antigen-binding domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the CAR antigen-binding domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0180] In some embodiments, the CAR comprises one or more antigen-binding domains. In some embodiments, the CAR comprises two or more antigen-binding domains. In some embodiments, the CAR is a bispecific CAR. In some embodiments, the immune cells comprise two or more different CARs comprising one or more antigen-binding domains. In some embodiments, immune cells comprising bispecific CARs and / or two or more different CARs comprising one or more antigen-binding domains can reduce off-target and / or on-target extratissue effects by requiring the presence of two antigens. In some embodiments, the immune cells comprise bispecific CARs and / or two or more different CARs comprising one or more antigen-binding domains, where the CARs alone are insufficient to mediate activation of the modified cell, but together are synergistic and provide distinct signals that stimulate activation of the modified cell. In some embodiments, such a configuration may be referred to as an "AND" logic gate.

[0181] In some embodiments, immune cells containing bispecific CARs and / or two or more different CARs containing one or more antigen-binding domains can reduce off-target and / or on-target extratissue effects by requiring the presence of one antigen and the absence of a second, normal protein antigen before cellular activity is stimulated. In some embodiments, such a configuration may be referred to as a "NOT" logic gate. In contrast to AND gates, NOT-gate CAR-modified cells are activated by binding to a single antigen. However, binding of a second receptor to the second antigen functions to neutralize the activation signal perpetuated through the CAR. Typically, such inhibitory receptors target antigens that are abundantly expressed in normal tissues but absent in tumor tissues.

[0182] Hinge domain In some embodiments, the CAR comprises one or more extracellular hinge domains. In some embodiments, the CAR extracellular hinge domain is or comprises a human extracellular hinge domain. In some embodiments, the CAR extracellular hinge domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the CAR extracellular hinge domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the one or more CAR extracellular hinge domains comprise a CD8a extracellular hinge domain, a DNGR-1 extracellular hinge domain, a Dectin-1 extracellular hinge domain, or an IgG4 or CD28 extracellular hinge domain. In some embodiments, the CAR extracellular hinge domain optimizes the physicochemical parameters of the CAR, such as, for example, optimal size (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 to tumor antigens.

[0183] CAR transmembrane domain In some embodiments, the CAR comprises a transmembrane domain, for example, connecting the extracellular domain to the intracellular domain. In some embodiments, the CAR transmembrane domain is naturally associated with one or more other domain(s) of the CAR. In some embodiments, to minimize interaction with other members of the receptor complex, the CAR transmembrane domain can be modified to avoid binding of other surface membrane proteins to the transmembrane domain. In some embodiments, the CAR transmembrane domain can be derived from either a naturally occurring source or a synthetic source. In some embodiments, the CAR transmembrane domain is derived from a naturally occurring membrane-associated or transmembrane protein. In some embodiments, the CAR transmembrane domain is or comprises a human transmembrane domain. In some embodiments, the CAR transmembrane domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the CAR transmembrane domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the CAR transmembrane domain is selected from the group consisting of CD8, CD8a, CD64, CD32a, CD32c, CD16a, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphA1, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD19, CD20, 41BB, CD28, OX4 0, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, CD3 zeta, Dectin-1, DNGR1, SLAMF7, FcRγ, V / I / LxYxxL / V, SIRPα, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPβ, CD22, PIR-B, LILRB1, CD36, or Syk transmembrane domain.

[0184] FcR transmembrane domain In some embodiments, the FcR transmembrane domain comprises a full-length FcR transmembrane domain. In some embodiments, the FcR transmembrane domain comprises a portion of a full-length FcR transmembrane domain. In some embodiments, the FcR transmembrane domain is or comprises a human FcR transmembrane domain or a portion thereof. In some embodiments, the FcR transmembrane domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the FcR transmembrane domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the FcR transmembrane domain comprises an FcRγ, CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c, CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, or FcαRI (CD89) domain.

[0185] TLR transmembrane domains In some embodiments, the TLR transmembrane domain comprises a full-length TLR transmembrane domain. In some embodiments, the TLR transmembrane domain comprises a portion of a full-length TLR transmembrane domain. In some embodiments, the TLR transmembrane domain is or comprises a human TLR transmembrane domain or a portion thereof. In some embodiments, the TLR transmembrane domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the TLR transmembrane domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the TLR transmembrane domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.

[0186] CAR intracellular domain In some embodiments, a CAR comprises one or more intracellular domains. In some embodiments, the CAR intracellular domain is or comprises a human intracellular domain or a portion thereof. In some embodiments, the CAR intracellular domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the CAR intracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the CAR intracellular domain and / or other cytoplasmic domains of a CAR are involved in activation of a cell in which the CAR is expressed (e.g., a modified immune cell provided herein). In some embodiments, the CAR intracellular domain of a CAR is involved in signal activation and / or transduction in an immune cell comprising the CAR.

[0187] In some embodiments, the CAR intracellular domain of the CAR comprises at least one domain involved in signal activation and / or transduction. In some embodiments, the CAR intracellular domain is or comprises at least one of a costimulatory molecule and a signaling domain. In some embodiments, the CAR intracellular domain of the CAR comprises dual signaling domains. In some embodiments, the CAR intracellular domain of the CAR comprises three or more signaling domains.

[0188] In some embodiments, the CAR intracellular domain comprises the cytoplasmic portion of a surface receptor. In some embodiments, the CAR intracellular domain comprises a costimulatory molecule. In some embodiments, the CAR intracellular domain comprises a molecule that acts to initiate signal transduction in immune cells.

[0189] In some embodiments, the intracellular domain of the CAR comprises any portion of one or more costimulatory molecules, including at least one signaling domain from CD3ζ, Fc epsilon RI gamma chain, any derivative or variant thereof, any synthetic sequence thereof having the same functional capability, and any combination thereof.

[0190] FcR intracellular domain In some embodiments, the FcR intracellular domain comprises a full-length FcR intracellular domain. In some embodiments, the FcR intracellular domain comprises a portion of a full-length FcR intracellular domain. In some embodiments, the FcR intracellular domain is or comprises a human FcR intracellular domain or a portion thereof. In some embodiments, the FcR intracellular domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the FcR intracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the FcR intracellular domain comprises an FcRγ, CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c, CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, or FcαRI (CD89) domain.

[0191] TLR intracellular domain In some embodiments, the TLR intracellular domain comprises a full-length TLR intracellular domain. In some embodiments, the TLR intracellular domain comprises a portion of a full-length TLR intracellular domain. In some embodiments, the TLR intracellular domain is or comprises a human TLR intracellular domain or a portion thereof. In some embodiments, the TLR intracellular domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the TLR intracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the TLR intracellular domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.

[0192] Signaling domains In some embodiments, the CAR comprises one or more intracellular signaling domains. In some embodiments, the CAR intracellular signaling domain is or comprises a human intracellular signaling domain or a portion thereof. In some embodiments, the CAR signaling domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the CAR signaling domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0193] In some embodiments, one or more CAR intracellular signaling domains are selected from the group consisting of CD3 zeta (CD3ζ), FcRγ, CD64, CD32a, CD32c, CD16a, CD40, CD89, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphA1, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD19, CD20, 41BB, CD28, OX40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, V / I / LxYxxL / V, SIRPα, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRP β, CD22, PIR-B, LILRB1, Syk, 41BB ligand (41BBL; TNFSF9), CD27, OX40L, CD32b, CD11b, ITGAM, SLAMF7, CD206, CD163, CD209, GCSFR (CD114), RAGE, CD30, CD160, DR3, Fn14, HVEM, CD160, NGFR, RANK, TNFR2, TROY, XEDAR, TRIF, Dectin-2, or or one or more cytokine receptor signaling domains (e.g., an 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, or ICOSL intracellular signaling domain).

[0194] In some embodiments, the intracellular domain of the CAR comprises dual signaling domains, e.g., 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, CD14, CD36, LOX-1, CD11b, in any combination with any of the signaling domains listed in the paragraph above.

[0195] Costimulatory domain As used herein, "costimulatory molecule" or "costimulatory domain" refers to a molecule in an immune cell that is used to enhance or attenuate the initial stimulus. For example, pathogen-associated pattern recognition receptors such as TLRs or the CD47 / SIRPα axis are molecules on immune cells that enhance or attenuate the initial stimulus, respectively.In some embodiments, the CAR costimulatory domain is selected from the group consisting of TCR, CD3 zeta (CD3ζ), CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc epsilon R1b), CD79a, CD79b, Fc gamma RIIa, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, Ligands that specifically bind to CD7, LIGHT, NKG2C, B7-H3, CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RAN KL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D ), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, other costimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a costimulatory molecule having the same functional capability, and any combination thereof.

[0196] In some embodiments, the CAR costimulatory domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the CAR costimulatory domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0197] As used herein, a "co-stimulatory signal" refers to a signal that, in combination with a primary signal, such as activation of a CAR on an immune cell, results in activation of the immune cell.

[0198] Cleaved peptide As used herein, a cleavage peptide refers to a peptide that can induce cleavage of a recombinant protein within a cell. In some embodiments, the cleavage peptide is a 2A peptide. In some embodiments, the cleavage peptide is or comprises a P2A, F2A, E2A, or T2A peptide. In some embodiments, the nucleic acids described herein comprise one or more nucleic acid sequences encoding one or more cleavage peptides. In some embodiments, the nucleic acid comprising the nucleic acid sequence encoding the 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 the one or more peptide agents by the cleavage peptides. In some embodiments, a first promoter is operably linked to one or more 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, the nucleic acid sequence comprising a CAR and optionally one or more peptide agents further comprises an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA, facilitating initiation of translation.

[0199] CAR peptide agents As used herein, a CAR peptide agent refers to a peptide that is co-expressed with a CAR in an immune cell. In some embodiments, the CAR peptide agent is co-expressed with a CAR to ensure stoichiometric balance and optimal signaling of the CAR. In some embodiments, a CAR peptide agent forms a homodimer with the same peptide agent. In some embodiments, a CAR peptide agent forms a heterodimer with a different peptide agent. In some embodiments, a nucleic acid 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.

[0200] In some embodiments, a CAR peptide agent comprises any peptide, protein, receptor, secreted antibody, or fragment thereof (e.g., 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 IL-1, IL-2, IL-6, IL-8, TNF-α, IFNa, IFNb, IFN-γ, GMCSF, or MCSF), CD40-L, dominant negative SIRPα, dominant negative PD1, dominant negative CD45, dominant negative Siglec-10, or dominant negative LILRB.

[0201] Fc receptors (FcRs) In some embodiments, the 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, the CAR comprises, from N-terminus to C-terminus, one or more extracellular binding 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 are or comprise human FcR domains. In some embodiments, the FcR extracellular domain, the FcR transmembrane domain, and the FcR intracellular domain together comprise a full-length FcR. In some embodiments, the FcR extracellular domain, the FcR transmembrane domain, and the FcR intracellular domain together comprise a portion of a full-length FcR. In some embodiments, the FcR extracellular domain comprises a portion of a full-length FcR extracellular domain. In some embodiments, the FcR transmembrane domain comprises a portion of a full-length FcR transmembrane domain. In some embodiments, the FcR intracellular domain comprises a portion of a full-length FcR intracellular domain.

[0202] Toll-like receptors (TLRs) In some embodiments, the 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, the CAR comprises, from N-terminus to C-terminus, one or more extracellular binding 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 are or comprise human TLR domains. In some embodiments, the TLR extracellular domain, the TLR transmembrane domain, and the TLR intracellular domain together comprise a full-length TLR. In some embodiments, the TLR extracellular domain, the TLR transmembrane domain, and the TLR intracellular domain together comprise a portion of a full-length TLR. In some embodiments, the TLR extracellular domain comprises a portion of a full-length TLR extracellular domain. In some embodiments, the TLR transmembrane domain comprises a portion of a full-length TLR transmembrane domain. In some embodiments, the TLR intracellular domain comprises a portion of a full-length TLR intracellular domain.

[0203] Methods for modifying immune cells The present disclosure provides, inter alia, methods of modifying immune cells (e.g., stem cells, monocytes, macrophages, or dendritic cells), the methods comprising delivering to the immune cells a nucleic acid construct comprising one or more nucleic acids encoding one or more CARs described herein. The method may comprise delivering to the immune cells (e.g., stem cells, monocytes, macrophages, or dendritic cells) a nucleic acid construct comprising one or more nucleic acids encoding at least one extracellular domain described herein, at least one transmembrane domain described herein, and at least one intracellular domain described herein.

[0204] In some embodiments, the disclosure provides methods of generating modified immune cells (e.g., stem cells, monocytes, macrophages, or dendritic cells) in a subject, the method comprising administering to the subject a composition described herein comprising: (a) one or more nucleic acid molecules, at least a portion of which encodes a CAR and / or CAR peptide agent; and (b) a delivery vehicle. Thus, in some embodiments, after administration of the composition, the one or more nucleic acid molecules are translated in the immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) to generate a modified immune cell comprising a CAR and / or CAR peptide agent. In some embodiments, the modified immune cell comprising a CAR and / or CAR peptide agent has target effector activity.

[0205] Delivery method Nucleic acid constructs comprising one or more nucleic acid sequences encoding at least one CAR described herein can be introduced into immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) by physical, chemical, or biological methods. In some embodiments, the present disclosure provides methods for modifying immune cells, comprising producing the modified immune cells (e.g., stem cells, monocytes, macrophages, or dendritic cells) ex vivo. In some embodiments, the present disclosure provides methods for modifying immune cells, comprising producing the modified immune cells (e.g., stem cells, monocytes, macrophages, or dendritic cells) within a subject (i.e., in vivo).

[0206] Physical methods for introducing the nucleic acid constructs described herein into immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) can include electroporation, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, or a combination thereof. Nucleic acid constructs can be introduced into immune cells using commercially available methods, such as 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)). Nucleic acid constructs can also be introduced into immune cells using biolistic delivery systems such as mRNA transfection, cationic liposome-mediated transfection, lipofection, polymer encapsulation, peptide-mediated transfection, or "gene guns" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001), which is incorporated herein by reference in its entirety).

[0207] Biological methods for introducing the nucleic acid constructs described herein into immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) include the use of DNA and RNA vectors. In one embodiment, the vector comprises a plasmid vector, a viral vector, a transposon, a retrotransposon (e.g., piggybac, Sleeping Beauty), a site-specific insertion vector (e.g., CRISPR, zinc finger nuclease, TALEN), a suicide expression vector, or another vector known in the art. Viral vectors, particularly retroviral vectors, have become widely used to insert genes into mammalian cells (e.g., human cells). Viral vectors can also be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses (e.g., Ad5f35), or adeno-associated viruses (see, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362, which are incorporated by reference in their entireties). Retroviral vectors, such as lentiviruses, are suitable tools for achieving long-term gene transfer, allowing for long-term and stable integration of the transgene and its propagation in daughter cells. In some embodiments, the lentiviral vector is packaged with a VPX protein (e.g., as described in International Publication No. WO2017 / 044487, incorporated herein by reference in its entirety). In some embodiments, the VPX comprises a virion-associated protein (e.g., an accessory protein for viral replication). In some embodiments, the VPX protein is encoded by human immunodeficiency virus type 2 (HIV-2). In some embodiments, the VPX protein is encoded by simian immunodeficiency virus (SIV). In some embodiments, immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein are transfected with a lentiviral vector packaged with a VPX protein. In some embodiments, the VPX inhibits at least one antiviral factor in the immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein.In some embodiments, lentiviral vectors packaged with a VPX protein exhibit increased transfection efficiency of immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein, for example, compared to lentiviral vectors not packaged with a VPX protein. In some embodiments, immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein are electroporated and / or transfected with at least one VPX mRNA prior to transfection with a viral vector (e.g., an adenoviral vector, e.g., an Ad2 vector or an Ad5 vector (e.g., an Ad5f35 adenoviral vector, e.g., a helper-dependent Ad5F35 adenoviral vector)).

[0208] Chemical means for introducing the nucleic acid constructs described herein into immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) include colloidal dispersion systems, polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems (e.g., oil-in-water emulsions, micelles, mixed micelles, nanoparticles, liposomes, and lipofectamine-nucleic acid complexes).

[0209] An exemplary system for delivering the nucleic acid constructs described herein is a lipid-based system. The nucleic acid constructs described herein may be encapsulated in the aqueous interior of liposomes, dispersed within lipid bilayers, attached to liposomes via linking molecules, attached to lipid nanoparticles (LNPs) via linking molecules, entrapped in liposomes, entrapped in LNPs, complexed with liposomes, complexed with LNPs, dispersed in a solution or suspension containing lipids, mixed with lipids, complexed with micelles, or otherwise associated with lipids. The lipids used in the methods described herein may be naturally occurring lipids or synthetic lipids. Lipids may 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 approximately -20°C. In some embodiments, the lipid-based system can include one or more lipids that facilitate targeting of the composition to a desired cell type(s) (e.g., stem cells, monocytes, macrophages, or dendritic cells). In some embodiments, the delivery vehicle allows the composition to be preferentially taken up (e.g., endocytosed, phagocytosed) by immune cells (e.g., stem cells, monocytes, macrophages, or dendritic cells) compared to a composition that does not include the delivery vehicle.

[0210] targeting part In some embodiments, the delivery vehicle may include one or more targeting moieties. In some embodiments, the targeting moiety may facilitate passive targeting of the composition to a desired target. In some embodiments, the targeting moiety may facilitate active targeting of the composition to a desired target.

[0211] In some embodiments, the targeting moiety may be or include one or more of an antibody (e.g., monoclonal, polyclonal, synthetic, human, humanized, non-human antibody) or any fragment thereof, e.g., an scFv, an aptamer, a darpin, a centrin, a naturally occurring or synthetic receptor, an affibody, or other engineered protein recognition molecule, for binding to one or more of CD14, CD11b, CD163, CD206, CD33, CD209. In some embodiments, the targeting moiety may be or include a small molecule.

[0212] In some embodiments, the targeting moiety may be or include a combination of specific lipids or hydrophobic entities, for example, present on or forming the outer surface of a liposome or lipid nanoparticle (e.g., for targeting to a specific cell type(s)).

[0213] nucleic acid molecule In some embodiments of the present disclosure, the one or more nucleic acid molecules are or comprise DNA. In some embodiments of the present disclosure, the one or more nucleic acid molecules are or comprise messenger RNA (mRNA). In some embodiments, mRNA according to the present disclosure can be synthesized as unmodified or modified mRNA. Typically, mRNA is modified to increase stability. Modification of mRNA can include, for example, modifications of nucleotides of the RNA. Thus, modified mRNA according to the present disclosure can include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, modifying the mRNA includes including modified nucleotides, modifications to the 5' or 3' untranslated region (UTR), a cap structure, and / or a poly(A) tail in the mRNA.

[0214] In some embodiments, the mRNA of the present disclosure (e.g., mRNA encoding one or more CARs described herein) can include RNA backbone modifications. Typically, backbone modifications are modifications in which the backbone phosphate of the nucleotides contained in the RNA is chemically modified. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methyl phosphonate, methyl phosphoramidate, phosphoramidate, phosphorothioate (e.g., cytidine 5'-O-(1-thiophosphate)), boranophosphate, positively charged guanidinium group, etc., which include replacing phosphodiester bonds with other anionic, cationic, or neutral groups.

[0215] In some embodiments, an mRNA of the present disclosure (e.g., an mRNA encoding one or more CARs described herein) can include a sugar modification. Exemplary sugar modifications are chemical modifications of the sugars of the nucleotides involved, such as 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyl oligoribonucleotides, 2'-deoxy Sugar modifications include, but are not limited to, sugar modifications selected from the group consisting of 2'-C-alkyl oligoribonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligoribonucleotides and their isomers (2'-aracytidine 5'-triphosphate, 2'-arauidine 5'-triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0216] In some embodiments, an mRNA of the disclosure (e.g., an mRNA encoding one or more CARs described herein) comprises modified nucleotides including pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine / pseudouridine (5meC PsU), N1-methyl-pseudouridine (N1mPsU), or combinations thereof.

[0217] In some embodiments, an mRNA of the present disclosure (e.g., an mRNA encoding one or more CARs described herein) can include a modification of the base of a nucleotide (base modification). A modified nucleotide that includes a base modification is also referred to as a base-modified nucleotide.

[0218] Typically, mRNA synthesis involves the addition of a "cap" to the N-terminal (5') end and a "tail" to 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 the "tail" serves to protect the mRNA from exonuclease degradation.

[0219] Thus, in some embodiments, an mRNA of the disclosure (e.g., an mRNA encoding one or more CARs described herein) comprises a 5' cap structure. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate via a guanylyltransferase to generate a 5' triphosphate linkage; and the 7-nitrogen of guanine is 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, the cap comprises a Cap0 structure. The Cap0 structure lacks a 2'-O-methyl residue on the ribose attached to bases 1 and 2. In some embodiments, the cap comprises an AGCap1 structure. The AGCap1 structure has a 2'-O-methyl residue at base 2. In some embodiments, the cap comprises a Cap2 structure. The Cap2 structure has 2'-O-methyl residues attached to both bases 2 and 3. In some embodiments, the cap structure comprises AGCap1, m6AGCap1, or an anti-reverse cap analog (ARCA). In some embodiments, modified mRNAs of the present disclosure comprise m6AGCap1 and modified nucleotides including pseudouridine (PsU).

[0220] In some embodiments, an mRNA of the disclosure (e.g., an mRNA encoding one or more CARs described herein) comprises a 3' poly(A) tail structure. The poly(A) tail at the 3' end of the mRNA typically comprises about 10-400 adenosine nucleotides (SEQ ID NO: 73) (e.g., about 100-400 adenosine nucleotides, about 10-200 adenosine nucleotides, about 10-150 adenosine nucleotides, about 10-100 adenosine nucleotides, about 20-70 adenosine nucleotides, or about 20-60 adenosine nucleotides). In some embodiments, the mRNA comprises a 3' poly(C) tail structure. A suitable poly(C) tail at the 3' end of an mRNA typically contains about 10-200 cytosine nucleotides (SEQ ID NO: 74) (e.g., about 10-150 cytosine nucleotides, about 10-100 cytosine nucleotides, about 20-70 cytosine nucleotides, about 20-60 cytosine nucleotides, or about 10-40 cytosine nucleotides). The poly(C) tail may be in addition to or alternative to a poly(A) tail.

[0221] In some embodiments, an mRNA of the disclosure (e.g., an mRNA encoding one or more CARs described herein) comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region comprises one or more elements that affect mRNA stability or translation, e.g., an iron-responsive element. In some embodiments, the 5' untranslated region can be between about 50 and 500 nucleotides in length.

[0222] In some embodiments, the 3' untranslated region includes one or more of a polyadenylation signal, a binding site for a protein that affects the stability of the mRNA's location within a cell, or one or more binding sites for an miRNA. In some embodiments, the 3' untranslated region can be 50 to 500 or more nucleotides in length.

[0223] Administration of additional payloads In some embodiments, the methods of the disclosure include one or more steps of treating immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) during the process of modifying the immune cells. In some embodiments, the methods of the disclosure include one or more steps of administering to the subject an additional payload to modulate the immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) during the process of modifying the immune cells (e.g., with a payload comprising a CAR). In some embodiments, the composition may include one or more additional payloads. In some embodiments, the composition may include one or more additional payloads in the same delivery vehicle as the one or more nucleic acid molecules. In some embodiments, the composition may include one or more additional payloads in a delivery vehicle different from that used for the one or more nucleic acid molecules.

[0224] In some embodiments, the methods of the present disclosure include treating immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with a modulator of a pathway activated by in vitro transcribed mRNA. In some embodiments, the additional payload can be or include 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, 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 leads to inflammation associated with activation of type 1 interferon (IFN), tumor necrosis factor (TNF), interleukin-6 (IL-6), IL-12, and a cascade of transcriptional programs. Collectively, these create a pro-inflammatory microenvironment poised to induce specific immune responses. Furthermore, downstream effects such as slowing down translation via eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, promoting RNA degradation via ribonuclease L (RNase L), and overexpression and inhibition of self-amplifying mRNA replication are relevant to the pharmacokinetics and pharmacodynamics of IVT mRNA.

[0225] In some embodiments, the modulator of a pathway activated by in vitro transcribed mRNA comprises an RNase inhibitor. In some embodiments, the modulator of a pathway activated by in vitro transcribed mRNA comprises an RNase L, RNase T2, or RNase 1 inhibitor. In some embodiments, the modulator of a pathway activated by in vitro transcribed mRNA comprises an RNase L inhibitor. In some embodiments, the RNase L inhibitor comprises sunitinib. In some embodiments, the RNase L inhibitor comprises ABCE1.

[0226] In some embodiments, treating immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with an RNaseL inhibitor increases mRNA stability in the modified immune cells compared to mRNA stability in modified immune cells of the same type that have not been treated with the RNaseL inhibitor. In some embodiments, treating immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with an RNaseL inhibitor increases CAR expression in the modified immune cells compared to CAR expression in modified immune cells of the same type that have not been treated with the RNaseL inhibitor. In some embodiments, treating immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with an RNaseL inhibitor increases effector activity in the modified immune cells compared to effector activity in modified immune cells of the same type that have not been treated with the RNaseL inhibitor.

[0227] In some embodiments, administering an RNaseL inhibitor to a subject increases mRNA stability in modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) compared to mRNA stability in modified immune cells of the same type in a subject to which the RNaseL inhibitor was not administered. In some embodiments, administering an RNaseL inhibitor to a subject increases CAR expression in the modified immune cells compared to CAR expression in modified immune cells of the same type in a subject to which the RNaseL inhibitor was not administered. In some embodiments, administering an RNaseL inhibitor to a subject increases effector activity in modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) compared to effector activity in modified immune cells of the same type in a subject to which the RNaseL inhibitor was not administered.

[0228] In some embodiments of the present disclosure, the step of treating immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) occurs before the step of delivering mRNA to the immune cells. In some embodiments of the present disclosure, the step of administering an additional payload to the subject occurs before the step of administering a composition comprising mRNA to the subject.

[0229] In some embodiments, the methods of the disclosure include culturing immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with a cytokine or an immunostimulatory recombinant protein. In some embodiments, the methods of the disclosure include administering to a subject a cytokine or immunostimulatory recombinant protein. In some embodiments, the cytokine is selected from the group consisting of IFN-α, IFN-β, IFN-γ, TNFα, IL-6, STNGL, LPS, CD40 agonist, 4-1BB ligand, recombinant 4-1BB, CD19 agonist, TLR agonist (e.g., TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, ​​TLR-7, TLR-8, or TLR-9), TGF-β (e.g., TGF-β1, TGF-β2, or TGF-β3), glucocorticoids, and the like. Id, immune complex, interleukin-1 alpha (IL-1α), IL-1β, 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-β, CD1 54, lymphotoxin beta (LT-β), A proliferation-inducing ligand (APRIL), CD70, CD153, 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), and TNF-related weak apoptosis-inducing factor (TNF-IL). WEAK), TNF-related activation-inducing 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), nucleotide-binding oligomerization domain-containing protein (NOD) ligand (e.g., NOD1, NOD2, or NOD1 / 2 agonist),RIG-I-like receptor (RLR) ligands (e.g., 5'ppp-dsRNA, 3p-hpRNA, Poly(I:C), or Poly(dA:dT)), C-type lectin receptor (CLR) ligands (e.g., curdlan, β-glucan, HKCA, laminarin, pustulan, scleroglucan, dispersible WGP, soluble WGP, zymosan, zymosan-degraded, furfurman, b-GlcCer, GlcC14C18, HKMT, TDB, TDB-HS15, or TDM), cyclic dinucleotide sensor ligands (e.g., C-Gas agonists or stimulator of interferon genes (STING) ligands), inflammasome inducers (e.g., alum, ATP, CPPD crystals, hemozoin, MSU crystals, nanoSiO2, nigericin, or TDB), aryl hydrocarbon (AhR) ligands (e.g., FICZ, indirubin, ITE, or L-kynurenine), alpha protein kinase 1 (ALPK1) ligands, multi-PRR ligands, NFKB / NFAT activators (e.g., concanavalin A, ionomycin, PHA-P, or PMA), or combinations thereof. In some embodiments, the cytokine comprises IFN-β.

[0230] In some embodiments of the present disclosure, the step of culturing immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) occurs after the step of delivering mRNA to the immune cells. In some embodiments of the present disclosure, the step of administering a cytokine or immunostimulatory recombinant protein to the subject occurs after the step of administering a composition comprising mRNA to the subject.

[0231] In some embodiments, culturing modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with cytokines or immunostimulatory recombinant proteins increases the viability of the modified immune cells compared to modified immune cells of the same type that have not been cultured with cytokines or immunostimulatory recombinant proteins. In some embodiments, culturing modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with cytokines or immunostimulatory recombinant proteins increases protein (e.g., at least one CAR described herein) expression in the modified immune cells compared to modified immune cells of the same type that have not been cultured with cytokines or immunostimulatory recombinant proteins. In some embodiments, culturing modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with cytokines or immunostimulatory recombinant proteins increases the longevity of protein (e.g., at least one CAR described herein) expression in the modified immune cells compared to modified immune cells of the same type that have not been cultured with cytokines or immunostimulatory recombinant proteins. In some embodiments, culturing modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with cytokines or immunostimulatory recombinant proteins increases the effector activity of the modified immune cells compared to modified immune cells of the same type that have not been cultured with cytokines or immunostimulatory recombinant proteins. In some embodiments, culturing modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with cytokines or immunostimulatory recombinant proteins increases pro-inflammatory (M1) polarization of the modified immune cells compared to modified immune cells of the same type that have not been cultured with cytokines or immunostimulatory recombinant proteins.

[0232] In some embodiments, administering a cytokine or immunostimulatory recombinant protein to a subject increases the viability of modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in the subject, compared to modified immune cells of the same type in a subject that has not been administered the cytokine or immunostimulatory recombinant protein. In some embodiments, administering a cytokine or immunostimulatory recombinant protein to a subject increases protein (e.g., at least one CAR described herein) expression in modified immune cells in the subject, compared to modified immune cells of the same type (e.g., stem cells, macrophages, monocytes, or dendritic cells) in a subject that has not been administered the cytokine or immunostimulatory recombinant protein. In some embodiments, administering a cytokine or immunostimulatory recombinant protein to a subject increases the longevity of protein (e.g., at least one CAR described herein) expression in modified immune cells in the subject, compared to modified immune cells of the same type (e.g., stem cells, macrophages, monocytes, or dendritic cells) in a subject that has not been administered the cytokine or immunostimulatory recombinant protein. In some embodiments, administration of a cytokine or immunostimulatory recombinant protein to a subject increases the effector activity of modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in the subject compared to the same type of modified immune cells in a subject that has not been administered the cytokine or immunostimulatory recombinant protein. In some embodiments, administration of a cytokine or immunostimulatory recombinant protein to a subject increases the pro-inflammatory (M1) polarization of modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in the subject compared to the same type of modified immune cells in a subject that has not been administered the cytokine or immunostimulatory recombinant protein.

[0233] Methods for altering the inflammatory phenotype of a population of cells In some embodiments, the methods of the present disclosure include altering the inflammatory phenotype of a population of cells. In some embodiments, the method of altering the inflammatory phenotype of a population of cells includes contacting the population of cells with modified immune cells described herein (e.g., stem cells, macrophages, monocytes, or dendritic cells). In some embodiments, the population of cells includes macrophages, monocytes, dendritic cells, T cells, NK cells, or a combination thereof. In some embodiments, the modified immune cells described herein (e.g., stem cells, macrophages, monocytes, or dendritic cells) secrete one or more inhibitory RNAs that alter the inflammatory phenotype of the population of cells (e.g., bystander cells). In some embodiments, the one or more inhibitory RNAs are packaged in an extracellular vehicle. In some embodiments, the one or more inhibitory RNAs are packaged in an exosome.

[0234] In some embodiments, the inflammatory phenotype of a population of cells is changed from an anti-inflammatory to a non-activated state. In some embodiments, the inflammatory phenotype of a population of cells is changed from a pro-inflammatory to a non-activated state. In some embodiments, the inflammatory phenotype of a population of cells is changed from an anti-inflammatory to a pro-inflammatory. In some embodiments, the inflammatory phenotype of a population of cells is changed from a pro-inflammatory to an anti-inflammatory.

[0235] modified immune cells In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) are produced by the methods of the present disclosure. In some embodiments, the modified immune cells comprise at least one CAR described herein. In some embodiments, the modified immune cells comprise one or more nucleic acids encoding at least one CAR described herein. In some embodiments, the at least one CAR described herein comprises at least one extracellular domain, at least one transmembrane domain, and at least one intracellular domain. In some embodiments, the modified immune cells of the present disclosure comprise one or more 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). In some embodiments, the modified immune cells of the present disclosure comprise a CAR and one or more inhibitory RNAs.

[0236] In some embodiments, the modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) of the present disclosure comprise at least one CAR described herein, which comprises an extracellular domain described herein that binds to a tumor antigen, such as an antigen specific to a tumor or cancer of interest. In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes. In some embodiments, the tumor antigen comprises mesothelin.

[0237] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a modified mRNA encoding at least one CAR provided herein exhibit increased survival compared to modified immune cells of the same type comprising a similar CAR (e.g., a CAR that includes a different anti-mesothelin antigen binding domain and / or does not include (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but includes the other components of a comparison CAR). In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a modified mRNA encoding at least one CAR described herein exhibit increased expression of mRNA encoding at least one CAR described herein compared to modified immune cells of the same type comprising an unmodified mRNA encoding a similar CAR (e.g., a CAR that includes a different anti-mesothelin antigen binding domain and / or does not include (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but includes the other components of a comparison CAR). In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising at least one CAR provided herein exhibit increased CAR expression compared to modified immune cells of the same type that contain an unmodified mRNA encoding a similar CAR (e.g., a CAR that includes a different anti-mesothelin antigen-binding domain and / or does not include (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of the CD8 or CD28 transmembrane domains, but includes other components of the comparative CAR).In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a modified mRNA encoding at least one CAR provided herein exhibit increased longevity of the mRNA encoding the at least one CAR compared to modified immune cells of the same type comprising an unmodified mRNA encoding a similar CAR (e.g., a CAR that includes a different anti-mesothelin antigen binding domain and / or does not include (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but includes the other components of a comparison CAR). In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a modified mRNA encoding a CAR provided herein exhibit increased longevity of the CAR compared to modified immune cells of the same type comprising a similar CAR (e.g., a CAR that includes a different anti-mesothelin antigen binding domain and / or does not include (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but includes the other components of a comparison CAR). In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR provided herein exhibit increased effector activity compared to modified immune cells of the same type comprising an unmodified mRNA encoding a similar CAR (e.g., a CAR that includes a different anti-mesothelin antigen-binding domain and / or that does not include (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but includes the other components of a comparative CAR). In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a modified mRNA encoding a CAR provided herein exhibit increased pro-inflammatory (M1) polarization compared to modified immune cells of the same type comprising an unmodified mRNA encoding a similar CAR (e.g., a CAR that includes a different anti-mesothelin antigen-binding domain and / or that does not include (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but includes the other components of a comparative CAR).

[0238] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR provided herein maintain a pro-inflammatory phenotype over time. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR provided herein maintain a pro-inflammatory phenotype for at least 4 hours, 2 days, 4 days, 7 days, 14 days, 28 days, and / or 40 days after the immune cells are modified with a nucleic acid encoding a CAR.

[0239] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR provided herein maintain an anti-inflammatory phenotype over time. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR provided herein maintain an anti-inflammatory phenotype for at least 4 hours, 2 days, 4 days, 7 days, 14 days, 28 days, and / or 40 days after the immune cells are modified with a nucleic acid encoding a CAR.

[0240] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR provided herein maintain a pro-inflammatory phenotype and / or otherwise resist destruction when exposed to anti-inflammatory cytokines. In some embodiments, the sensitivity of modified immune cells to environmental cytokines is measured by generating a dose-response curve of pro-inflammatory markers by treating modified immune cells comprising a CAR provided herein. In some embodiments, the sensitivity of modified immune cells to environmental cytokines is measured by generating a dose-response curve of pro-inflammatory markers by treating modified immune cells comprising a CAR provided herein.

[0241] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR provided herein maintain an anti-inflammatory phenotype and / or otherwise resist destruction when exposed to pro-inflammatory cytokines. In some embodiments, the sensitivity of modified immune cells to environmental cytokines is measured by generating a dose-response curve for anti-inflammatory markers by treating modified immune cells comprising a CAR provided herein with increasing concentrations of a pro-inflammatory cytokine. In some embodiments, the sensitivity of modified immune cells to environmental cytokines is measured by generating a dose-response curve for anti-inflammatory markers by treating modified immune cells comprising a CAR provided herein with increasing concentrations of an anti-inflammatory cytokine.

[0242] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR provided herein have minimal impact on surrounding cells. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR provided herein have a significant impact on surrounding cells. In some embodiments, the impact of modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR provided herein can be tested by co-culturing the modified immune cells with unmodified immune cells and analyzing the expression of pro-inflammatory and anti-inflammatory markers in the unmodified cells using flow cytometry. In some embodiments, the modified and unmodified immune cells can be co-cultured in a culture dish where the modified and unmodified immune cells are in contact with each other. In some embodiments, the modified and unmodified immune cells can be co-cultured in a culture dish where the modified and unmodified immune cells are separated by a transwell assay membrane.

[0243] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR provided herein confer a minimal cytotoxic effect on surrounding cells. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR provided herein confer a significant cytotoxic effect on surrounding cells (e.g., cancer cells). In some embodiments, the modification of immune cells to include a CAR provided herein is not cytotoxic to the modified immune cells. In some embodiments, RNAseq data from the modified immune cells is examined to determine whether there is upregulation of genes indicative of a cytotoxic effect.

[0244] In some embodiments, expression of a CAR provided herein in a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) increases at least one targeted effector function of the modified immune cell (e.g., phagocytosis, targeted cell cytotoxicity, antigen presentation, or cytokine secretion) compared to an unmodified immune cell or a modified cell containing a similar CAR (e.g., a CAR that contains a different anti-mesothelin antigen-binding domain and / or does not contain (i) a CD8 or CD28 extracellular hinge domain, and (ii) one or both of a CD8 or CD28 transmembrane domain, but contains other components of the comparative CAR).

[0245] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR provided herein comprise one or more regulatory systems, including, but not limited to, a safety switch (e.g., an on switch, an off switch, a suicide switch), transcriptional control (e.g., a cell-specific promoter, a cell state-specific promoter, a downstream promoter of CAR activation, a downstream promoter of an endogenous signaling pathway, or drug-inducible transcription), post-transcriptional control of CAR mRNA (e.g., RNA-based inhibition by endogenous or recombinant miRNA), or post-translational control of CAR structure or stability (e.g., a CAR whose intracellular domain conditionally associates with the complete structure by drug / light-induced association (allowing signaling) or dissociation (inhibiting signaling), or a CAR whose stability is regulated by a drug for inducible stabilization (allowing signaling) or degradation (inhibiting signaling)). These control systems can be combined to create logic gates, e.g., AND gates (e.g., CARs with CAR-inducible promoters and cytosolic domains that associate in a drug-dependent manner and thus require CAR activation and the presence of a small molecule), OR gates (e.g., CARs under the control of a promoter that is transcriptionally activated after either CAR activation or the addition of a small molecule), and / or NOT gates (e.g., CARs whose mRNA is degraded by an endogenous miRNA that is expressed in native immune cell signaling states (such as a miRNA that is upregulated by a specific cytokine signaling pathway and therefore the CAR is only expressed in the absence of this cytokine)).

[0246] In some embodiments, the modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) of the present disclosure comprise one or more inhibitory RNAs selected from the group consisting of antisense RNA (asRNA), cis-naturally occurring antisense transcripts (cis-NAT), CRISPR RNA (crRNA), guide RNA (gRNA), long non-coding RNA (lncRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), trans-acting siRNA (tasiRNA), and repeat-associated siRNA (rasiRNA).

[0247] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) of the present disclosure comprise one or more shRNAs. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) of the present disclosure comprise one or more shRNAs comprising an miRNA scaffold. In some embodiments, the miRNA scaffold comprises a miRNA-155 5' scaffold, a miRNA-155 3' scaffold, a miRNA-30 5' scaffold, a miRNA-30 3' scaffold, a miRNA-16 5' scaffold, a miRNA-16 3' scaffold, a miRNA-125 5' scaffold, a miRNA-125 3' scaffold, a miRNA-223 5' scaffold, or a miRNA-223 3' scaffold.

[0248] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) of the present disclosure comprise one or more shRNAs that comprise a guide strand. In some embodiments, the guide strand is about 19-24 bases in length. In some embodiments, the guide strand has a G / C content of about 36%-50%. In some embodiments, the guide strand comprises a nucleic acid sequence that is reverse complementary to a target gene transcript that comprises the target nucleic acid sequence.

[0249] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) of the present disclosure comprise one or more shRNAs that comprise a passenger strand. In some embodiments, the passenger strand is 1-2 bases shorter than the corresponding guide strand. In some embodiments, the passenger strand is not perfectly complementary to the guide strand.

[0250] In some embodiments, the modified immune cells (eg, stem cells, macrophages, monocytes, or dendritic cells) of the present disclosure comprise one or more shRNAs that include a loop.

[0251] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) of the disclosure comprise 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, PPARα, PPARγ, PTGS2, Siglec-10, SIRPα, SLC15A3, STAT3, STAT6, TNFRSF1B, TOX, TREM2, YTHDF2, or ZFP36. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) of the disclosure comprise a target gene transcript encoding human SIRPα. In some embodiments, the modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) of the disclosure comprise a target gene transcript encoding human PD-L1.

[0252] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR described herein maintain a pro-inflammatory phenotype over time. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR described herein maintain a pro-inflammatory phenotype for at least 4 hours, 2 days, 4 days, 7 days, 14 days, and / or 28 days after the immune cells are modified with a nucleic acid encoding a CAR.

[0253] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR described herein maintain an anti-inflammatory phenotype over time. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR described herein maintain an anti-inflammatory phenotype for at least 4 hours, 2 days, 4 days, 7 days, 14 days, and / or 28 days after the immune cells are modified with a nucleic acid encoding a CAR.

[0254] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR described herein maintain a pro-inflammatory phenotype and / or otherwise resist destruction when exposed to an anti-inflammatory cytokine. In some embodiments, the sensitivity of modified immune cells to environmental cytokines is measured by generating a dose-response curve of a pro-inflammatory marker by treating modified immune cells comprising a CAR described herein with increasing concentrations of an anti-inflammatory cytokine. In some embodiments, the sensitivity of modified immune cells to environmental cytokines is measured by generating a dose-response curve of a pro-inflammatory marker by treating modified immune cells comprising a CAR described herein with increasing concentrations of a pro-inflammatory cytokine.

[0255] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR described herein maintain an anti-inflammatory phenotype and / or otherwise resist destruction when exposed to pro-inflammatory cytokines. In some embodiments, the sensitivity of modified immune cells to environmental cytokines is measured by generating a dose-response curve for anti-inflammatory markers by treating modified immune cells comprising a CAR described herein with increasing concentrations of a pro-inflammatory cytokine. In some embodiments, the sensitivity of modified immune cells to environmental cytokines is measured by generating a dose-response curve for anti-inflammatory markers by treating modified immune cells comprising a CAR described herein with increasing concentrations of an anti-inflammatory cytokine.

[0256] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR described herein have minimal impact on surrounding cells. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR described herein have a significant impact on surrounding cells. In some embodiments, the impact of modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR described herein on unmodified cells (e.g., immune cells not comprising a CAR described herein) can be tested by co-culturing the modified immune cells with the unmodified immune cells and analyzing the expression of pro-inflammatory and anti-inflammatory markers in the unmodified cells using flow cytometry. In some embodiments, the modified and unmodified immune cells can be co-cultured in a culture dish where the modified and unmodified immune cells are in contact with each other. In some embodiments, the modified and unmodified immune cells can be co-cultured in a culture dish where the modified and unmodified immune cells are separated by a transwell assay membrane.

[0257] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR described herein confer a minimal cytotoxic effect on surrounding cells. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR described herein confer a significant cytotoxic effect on surrounding cells (e.g., cancer cells). In some embodiments, the modification of immune cells to include a CAR described herein is not cytotoxic to the modified immune cells. In some embodiments, RNAseq data from the modified immune cells is examined to determine whether there is upregulation of genes indicative of a cytotoxic effect.

[0258] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR described herein may comprise one or more regulatory systems, including, but not limited to, a safety switch (e.g., an on switch, an off switch, a suicide switch), transcriptional control (e.g., a cell-specific promoter, a cell state-specific promoter, a downstream promoter of CAR activation, a downstream promoter of an endogenous signaling pathway, or drug-inducible transcription), post-transcriptional control of CAR mRNA (e.g., RNA-based inhibition by endogenous or recombinant miRNA), or post-translational control of CAR structure or stability (e.g., a CAR whose intracellular domain conditionally associates with the complete structure by drug / light-induced association (allowing signaling) or dissociation (inhibiting signaling), or a CAR whose stability is regulated by a drug for inducible stabilization (allowing signaling) or degradation (inhibiting signaling)). These control systems can be combined to create logic gates, e.g., AND gates (e.g., CARs with CAR-inducible promoters and cytosolic domains that associate in a drug-dependent manner and thus require CAR activation and the presence of a small molecule), OR gates (e.g., CARs under the control of a promoter that is transcriptionally activated after either CAR activation or the addition of a small molecule), and / or NOT gates (e.g., CARs whose mRNA is degraded by an endogenous miRNA that is expressed in native immune cell signaling states (such as a miRNA that is upregulated by a specific cytokine signaling pathway and therefore the CAR is only expressed in the absence of this cytokine)).

[0259] In some embodiments, effector cells (i.e., modified immune cells of the present disclosure (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a nucleic acid construct described herein secrete one or more inhibitory RNAs of the present disclosure. In some embodiments, the one or more inhibitory RNAs are packaged within extracellular vesicles. In some embodiments, the one or more inhibitory RNAs are packaged within exosomes. In some embodiments, the secreted inhibitory RNA affects the phenotype of bystander cells. In some embodiments, the bystander cells are bystander macrophages, bystander monocytes, bystander dendritic cells, or bystander stem cells. In some embodiments, the bystander cells are biased toward an anti-tumor phenotype. In some embodiments, the bystander cells are biased toward an anti-inflammatory phenotype. In some embodiments, the bystander macrophages are biased toward an M1 phenotype. In some embodiments, the bystander macrophages are biased toward an M2 phenotype.

[0260] Assay Various assays can be performed to confirm the presence of a nucleic acid construct described herein and / or the presence of a protein (e.g., a CAR) in immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells). For example, such assays include molecular biological assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR, as well as biochemical assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blotting). Other assays of the present disclosure include, for example, fluorescence-activated cell sorting (FACS), immunofluorescence microscopy, MSD cytokine analysis, mass spectrometry (MS), RNA-Seq, and functional assays.

[0261] Various assays can be performed to determine various characteristics of the modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells), including, but not limited to, immune cell viability, nucleic acid expression, nucleic acid longevity, protein (e.g., CAR) expression, protein (e.g., CAR) longevity, effector activity, and pro-inflammatory (M1) polarization. For example, such assays include flow cytometry, quantitative PCR, and in vitro functional assays such as cytokine / chemokine secretion, phagocytosis, and specific lysis of target tumor cells.

[0262] nucleic acid construct The present disclosure provides, inter alia, a nucleic acid molecule encoding at least one CAR or fragment thereof described herein. An immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) can contain a nucleic acid molecule (e.g., an exogenous nucleic acid molecule) encoding at least one polypeptide described herein (e.g., one or more CARs of the present disclosure).

[0263] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" can also include introns to the extent that a nucleotide sequence encoding a protein, depending on its type, may contain intron(s). "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined nucleotide (e.g., rRNA, tRNA, and mRNA) sequence or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and usually provided in a sequence listing, and the non-coding strand used as a template for transcription of a gene or cDNA can be said to encode the protein or other product of that gene or cDNA.

[0264] The term "operably linked" or "transcriptional control" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, 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 adjacent to each other and, where necessary to join two protein-coding regions, in the same reading frame.

[0265] A nucleic acid molecule encoding at least one protein (e.g., a CAR of the present disclosure) or a fragment thereof described herein can be a DNA molecule, an RNA molecule, or a combination thereof. In some embodiments, the nucleic acid molecule comprises or is a messenger RNA (mRNA) transcript encoding at least one protein (e.g., a CAR of the present disclosure) or a fragment thereof described herein. In some embodiments, the nucleic acid molecule comprises or is a DNA construct encoding at least one protein (e.g., a CAR of the present disclosure) or a fragment thereof described herein.

[0266] In some embodiments, all or a fragment of a protein described herein (e.g., at least one CAR of the present disclosure) is encoded by a codon-optimized nucleic acid molecule, e.g., for expression in a cell (e.g., a mammalian cell). Various codon optimization methods are known in the art, e.g., as disclosed in U.S. Patent Nos. 5,786,464 and 6,114,148, each of which is incorporated herein by reference in its entirety.

[0267] Expression of the nucleic acids described herein can be achieved by operably linking a nucleic acid encoding a protein (e.g., at least one CAR of the present disclosure) or a fragment thereof to a promoter in an expression vector. Exemplary promoters (e.g., constitutive promoters) include, but are not limited to, the elongation factor-1α promoter (EF-1α), the immediate-early cytomegalovirus (CMV) promoter, the ubiquitin C promoter, the phosphoglycerokinase (PGK) promoter, the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the Moloney murine leukemia virus (MoMuLV) promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate-early promoter, the Rous sarcoma virus promoter, the actin promoter, the myosin promoter, the hemoglobin promoter, or the creatine kinase promoter. Examples of inducible promoters include, but are not limited to, the metallothionine promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter. The vector can also contain additional promoter elements, such as enhancers, to control the frequency of transcription initiation.

[0268] In some embodiments, the vector comprising the nucleic acid molecule encoding a protein (e.g., at least one CAR of the present disclosure) or a fragment thereof comprises or is a viral vector. Viral vector technology is well known and described in the art (e.g., 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., lentiviral vectors or gamma retroviral vectors). In some embodiments, the vector comprises a lentiviral vector (e.g., as described in U.S. Pat. No. 9,149,519 or International Publication No. WO2017 / 044487, each of which is incorporated herein by reference in its entirety).

[0269] In some embodiments, the viral vector comprises an adenoviral vector. Adenoviruses are a large family of viruses that contain double-stranded DNA. Adenoviruses replicate in the nucleus of host cells, using the host's cellular machinery to synthesize viral RNA, DNA, and proteins. Adenoviruses are known in the art to infect both replicating and non-replicating cells, accommodate large transgenes, and encode proteins without integrating into the host cell genome. In some embodiments, the adenoviral vector comprises an Ad2 vector or an Ad5 vector (e.g., an Ad5f35 adenoviral vector, e.g., a helper-dependent Ad5F35 adenoviral vector).

[0270] In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. AAV systems are generally well known in the art (see, for example, Kelleher and Vos, Biotechniques, 17(6):1110-17(1994); Cotten et al., PNASUSA, 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 producing and using recombinant AAV (rAAV) vectors are described, for example, in U.S. Patent Nos. 5,139,941 and 4,797,368.

[0271] 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 can be used to deliver a protein described herein (e.g., at least one CAR of the present disclosure) or a fragment thereof. In some embodiments, the AAV serotype has tropism for a specific tissue.

[0272] In some embodiments, the CRISPR / Cas9 system facilitates high-level, precise genome editing using adeno-associated virus (AAV) vectors, which have recently been shown to serve as donor template DNA during homologous recombination (HR).

[0273] In some embodiments, the 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 incorporated herein by reference in its entirety). Exemplary gammaretroviral vectors include murine leukemia virus (MLV), spleen-limited focus-forming virus (SFFV), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom.

[0274] In some embodiments, the vector comprises two or more nucleic acid sequences encoding a CAR, e.g., at least one CAR described herein, and a second CAR, e.g., a different CAR described herein. In some embodiments, the two or more nucleic acid sequences encoding the CAR and the second CAR are encoded by a single nucleic acid molecule, e.g., in the same frame as a single polypeptide chain. In some embodiments, the two or more CARs are separated by one or more cleavage peptide sites (e.g., autocleavage sites or substrates for intracellular proteases). In certain embodiments, the cleavage peptide comprises a porcine teschovirus I (P2A) peptide, a Thosea asigna virus (T2A) peptide, an equine rhinitis A virus (E2A) peptide, a foot-and-mouth disease virus (F2A) peptide, or a variant thereof.

[0275] In some embodiments, the vector comprises at least one nucleic acid sequence encoding a CAR (e.g., at least one CAR described herein) and at least one nucleic acid encoding at least one gene co-expressed with the CAR (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, ICOS-L, ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds to a Toll ligand receptor, and / or a B7-H3 ligand)).

[0276] In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that is at least 85% identical to a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that is at least 90% identical to a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that is at least 95% identical to a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that is at least 96% identical to a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that is at least 97% identical to a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that is at least 98% identical to a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that is at least 99% identical to a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that is identical to a sequence selected from Table 4.

[0277] In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 100 substitutions, additions, or deletions from a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 75 substitutions, additions, or deletions from a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 50 substitutions, additions, or deletions from a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 40 substitutions, additions, or deletions from a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 30 substitutions, additions, or deletions from a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 20 substitutions, additions, or deletions from a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 10 substitutions, additions, or deletions from a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 9 substitutions, additions, or deletions from a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 8 substitutions, additions, or deletions from a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 7 substitutions, additions, or deletions from a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 6 substitutions, additions, or deletions from a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 5 substitutions, additions, or deletions from a sequence selected from Table 4.In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than four substitutions, additions, or deletions from a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than three substitutions, additions, or deletions from a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than two substitutions, additions, or deletions from a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than one substitution, addition, or deletion from a sequence selected from Table 4. In some embodiments, a CAR of the disclosure binds mesothelin and is encoded by a nucleic acid sequence that does not have any substitutions, additions, or deletions compared to a sequence selected from Table 4.

[0278] In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that is at least 85% identical to a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that is at least 90% identical to a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that is at least 95% identical to a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that is at least 96% identical to a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that is at least 97% identical to a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that is at least 98% identical to a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that is at least 99% identical to a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that is at least 99% identical to a sequence selected from Table 5.

[0279] In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 50 substitutions, additions, or deletions from a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 40 substitutions, additions, or deletions from a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 30 substitutions, additions, or deletions from a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 20 substitutions, additions, or deletions from a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 10 substitutions, additions, or deletions from a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 9 substitutions, additions, or deletions from a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 8 substitutions, additions, or deletions from a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 7 substitutions, additions, or deletions from a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 6 substitutions, additions, or deletions from a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 5 substitutions, additions, or deletions from a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than 4 substitutions, additions, or deletions from a sequence selected from Table 5.In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than three substitutions, additions, or deletions from a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than two substitutions, additions, or deletions from a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that differs by no more than one substitution, addition, or deletion from a sequence selected from Table 5. In some embodiments, the CAR antigen binding domain binds mesothelin and is encoded by a nucleic acid sequence that does not have any substitutions, additions, or deletions compared to a sequence selected from Table 5.

[0280] In some embodiments, the present disclosure provides a nucleic acid molecule comprising a promoter, a gene of interest, a 3' untranslated region (UTR), and one or more introns. In some embodiments, the one or more introns of the present disclosure comprise one or more inhibitory nucleic acids of the present disclosure. In some embodiments, the one or more inhibitory nucleic acids of the present disclosure encode one or more inhibitory RNAs of the present disclosure. In some embodiments, the gene of interest encodes at least one chimeric antigen receptor (CAR) or fragment thereof described herein. Modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) of the present disclosure can comprise a nucleic acid molecule (e.g., an exogenous nucleic acid molecule) that comprises one or more introns of the present disclosure and encodes at least one protein described herein (e.g., a CAR of the present disclosure). In some embodiments, the present disclosure provides a nucleic acid molecule that increases expression of a CAR compared to a reference nucleic acid molecule lacking one or more introns.

[0281] Introns In some embodiments, the one or more introns of the present disclosure comprise one, two, or three introns. In some embodiments, the one or more introns of the present disclosure are located downstream of a promoter. In some embodiments, the one or more introns of the present disclosure are located within a gene of interest. In some embodiments, the one or more introns of the present disclosure are located within a 3' UTR. In some embodiments, the one or more introns of the present disclosure are located within a gene of interest and within a 3' UTR. In some embodiments, the one or more introns of the present disclosure comprise three inhibitory nucleic acids. In some embodiments, the three inhibitory nucleic acids encode three inhibitory RNAs. In some embodiments, the three inhibitory RNAs comprise, in 5' to 3' order, the following miRNA scaffolds: (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.

[0282] inhibitory nucleic acid Inhibitory nucleic acids of the present disclosure include any nucleic acid capable of binding to a target messenger RNA (mRNA). In some embodiments, inhibitory nucleic acids of the present disclosure include RNA. In some embodiments, inhibitory nucleic acids of the present disclosure include DNA. In some embodiments, inhibitory nucleic acids of the present disclosure are DNA / RNA hybrids. In some embodiments, inhibitory nucleic acids of the present disclosure are inhibitory RNA. In some embodiments, the inhibitory RNA silences expression of a target gene via RNA interference. In some embodiments, the inhibitory RNA is selected from the group consisting of antisense RNA (asRNA), cis-naturally occurring antisense transcripts (cis-NAT), CRISPR RNA (crRNA), guide RNA (gRNA), long non-coding RNA (lncRNA), 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, the inhibitory RNA is an shRNA.

[0283] In some embodiments, shRNAs of the present disclosure comprise a stem-loop structure. In some embodiments, shRNAs of the present disclosure comprise a stem-loop structure flanked on either side by at least 10 bases. In some embodiments, the stem is a double-stranded RNA structure. In some embodiments, the stem is about 30-40 bases long on each side. In some embodiments, the stem is about 30-39 bases long on each side. In some embodiments, the stem is about 30-38 bases long on each side. In some embodiments, the stem is about 30-37 bases long on each side. In some embodiments, the stem is about 30-36 bases long on each side. In some embodiments, the stem is about 30-35 bases long on each side. In some embodiments, the stem is about 30-34 bases long on each side. In some embodiments, the stem is about 30-33 bases long on each side. In some embodiments, the stem is about 30-32 bases long on each side. In some embodiments, the stem is about 30-31 bases long on each side. In some embodiments, the stem is about 31-40 bases long on each side. In some embodiments, the stem is about 32-40 bases long on each side. In some embodiments, the stem is about 33-40 bases long on each side. In some embodiments, the stem is about 34-40 bases long on each side. In some embodiments, the stem is about 35-40 bases long on each side. In some embodiments, the stem is about 36-40 bases long on each side. In some embodiments, the stem is about 37-40 bases long on each side. In some embodiments, the stem is about 38-40 bases long on each side. In some embodiments, the stem is about 39-40 bases long on each side. In some embodiments, the stem is 35 bases long on each side. In some embodiments, the shRNA of the present disclosure comprises a scaffold. In some embodiments, the shRNA of the present disclosure comprises a 5' scaffold. In some embodiments, the shRNA of the present disclosure comprises a 3' scaffold. In some embodiments, the shRNA of the present disclosure comprises a guide strand. In some embodiments, the shRNA of the present disclosure comprises a passenger strand.In some embodiments, the shRNA of the present disclosure comprises a loop connecting the guide strand and the passenger strand. In some embodiments, the loop is about 8 to 20 bases in length. In some embodiments, the loop is about 8 to 19 bases in length. In some embodiments, the loop is about 8 to 18 bases in length. In some embodiments, the loop is about 8 to 17 bases in length. In some embodiments, the loop is about 8 to 16 bases in length. In some embodiments, the loop is about 8 to 15 bases in length. In some embodiments, the loop is about 8 to 14 bases in length. In some embodiments, the loop is about 8 to 13 bases in length. In some embodiments, the loop is about 8 to 12 bases in length. In some embodiments, the loop is about 8 to 11 bases in length. In some embodiments, the loop is about 8 to 10 bases in length. In some embodiments, the loop is about 8 to 9 bases in length. In some embodiments, the loop is about 9 to 20 bases in length. In some embodiments, the loop is about 10 to 20 bases in length. In some embodiments, the loop is about 11-20 bases in length. In some embodiments, the loop is about 12-20 bases in length. In some embodiments, the loop is about 13-20 bases in length. In some embodiments, the loop is about 14-20 bases in length. In some embodiments, the loop is about 15-20 bases in length. In some embodiments, the loop is about 16-20 bases in length. In some embodiments, the loop is about 17-20 bases in length. In some embodiments, the loop is about 18-20 bases in length. In some embodiments, the loop is about 19-20 bases in length. In some embodiments, the section of the stem closest to the loop comprises a guide strand and a passenger strand. In some embodiments, the section of the stem furthest from the loop comprises a 5' scaffold and a 3' scaffold. In some embodiments, shRNAs of the present disclosure comprise, 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 disclosure comprises, from 5' to 3', a 5' scaffold, a passenger strand, a loop, a guide strand, and a 3' scaffold.

[0284] In some embodiments, the scaffold of the present disclosure comprises an miRNA scaffold. In some embodiments, the miRNA scaffold comprises a miRNA-155 5' scaffold, a miRNA-155 3' scaffold, a miRNA-30 5' scaffold, a miRNA-30 3' scaffold, a miRNA-16 5' scaffold, a miRNA-16 3' scaffold, a miRNA-125 5' scaffold, a miRNA-125 3' scaffold, a miRNA-223 5' scaffold, or a miRNA-223 3' scaffold.

[0285] In some embodiments, the guide strand of the present disclosure is about 19-24 bases in length. In some embodiments, the guide strand of the present disclosure is 19-24 bases in length. In some embodiments, the guide strand is 18 bases in length. In some embodiments, the guide strand is 19 bases in length. In some embodiments, the guide strand is 20 bases in length. In some embodiments, the guide strand is 21 bases in length. In some embodiments, the guide strand is 22 bases in length. In some embodiments, the guide strand is 23 bases in length. In some embodiments, the guide strand is 24 bases in length. In some embodiments, the guide strand is 25 bases in length.

[0286] In some embodiments, passenger strands of the present disclosure are about 17-24 bases in length. In some embodiments, passenger strands of the present disclosure are about 17-22 bases in length. In some embodiments, passenger strands of the present disclosure are 17-22 bases in length. In some embodiments, passenger strands are 17 bases in length. In some embodiments, passenger strands are 18 bases in length. In some embodiments, passenger strands are 19 bases in length. In some embodiments, passenger strands are 20 bases in length. In some embodiments, passenger strands are 21 bases in length. In some embodiments, passenger strands are 22 bases in length. In some embodiments, passenger strands are 23 bases in length. In some embodiments, passenger strands are 22 bases in length. In some embodiments, passenger strands are 24 bases in length. In some embodiments, passenger strands are 1-2 bases shorter than the corresponding guide strand. In some embodiments, passenger strands are 1 base shorter than the corresponding guide strand. In some embodiments, the passenger strand is two bases shorter than the corresponding guide strand. In some embodiments, the passenger strand is the same length as the corresponding guide strand.

[0287] In some embodiments, the guide strand of the present disclosure has a G / C content of about 36%-50%. In some embodiments, the guide strand of the present disclosure has a G / C content of 36%-50%. In some embodiments, the guide strand has a G / C content of 35%. In some embodiments, the guide strand has a G / C content of 36%. In some embodiments, the guide strand has a G / C content of 37%. In some embodiments, the guide strand has a G / C content of 38%. In some embodiments, the guide strand has a G / C content of 39%. In some embodiments, the guide strand has a G / C content of 40%. In some embodiments, the guide strand has a G / C content of 41%. In some embodiments, the guide strand has a G / C content of 42%. In some embodiments, the guide strand has a G / C content of 43%. In some embodiments, the guide strand has a G / C content of 44%. In some embodiments, the guide strand has a G / C content of 45%. In some embodiments, the guide strand has a G / C content of 46%. In some embodiments, the guide strand has a G / C content of 47%. In some embodiments, the guide strand has a G / C content of 48%. In some embodiments, the guide strand has a G / C content of 49%. In some embodiments, the guide strand has a G / C content of 50%. In some embodiments, the guide strand has a G / C content of 51%.

[0288] In some embodiments, the passenger strand of the present disclosure has a G / C content of about 36%-50%. In some embodiments, the passenger strand of the present disclosure has a G / C content of 36%-50%. In some embodiments, the passenger strand has a G / C content of 35%. In some embodiments, the passenger strand has a G / C content of 36%. In some embodiments, the passenger strand has a G / C content of 37%. In some embodiments, the passenger strand has a G / C content of 38%. In some embodiments, the passenger strand has a G / C content of 39%. In some embodiments, the passenger strand has a G / C content of 40%. In some embodiments, the passenger strand has a G / C content of 41%. In some embodiments, the passenger strand has a G / C content of 42%. In some embodiments, the passenger strand has a G / C content of 43%. In some embodiments, the passenger strand has a G / C content of 44%. In some embodiments, the passenger strand has a G / C content of 45%. In some embodiments, the passenger strand has a G / C content of 46%. In some embodiments, the passenger strand has a G / C content of 47%. In some embodiments, the passenger strand has a G / C content of 48%. In some embodiments, the passenger strand has a G / C content of 49%. In some embodiments, the passenger strand has a G / C content of 50%. In some embodiments, the passenger strand has a G / C content of 51%.

[0289] In some embodiments, the stem-loop formed by the 5' scaffold, guide strand, passenger strand, and 3' scaffold is recognized by Drosha. In some embodiments, Drosha cleaves 10-15 bases above the base of the stem-loop. In some embodiments, the Drosha-cleaved stem-loop will be recognized by Dicer. In some embodiments, Dicer cleaves 20-24 bases from the 5' and 3' ends of the stem-loop.

[0290] In some embodiments, the guide strand of the present disclosure comprises a nucleic acid sequence that is reverse-complementary to a target gene transcript that comprises a target nucleic acid sequence. In some embodiments, the passenger strand of the present disclosure comprises a nucleic acid sequence that is not fully complementary to the corresponding guide strand. In some embodiments, the passenger strand comprises a nucleic acid sequence that is not fully complementary to the corresponding guide strand by one nucleotide. In some embodiments, the passenger strand of the present disclosure comprises a nucleic acid sequence that is not fully complementary to the corresponding guide strand by two nucleotides. In some embodiments, the passenger strand of the present disclosure comprises a nucleic acid sequence that is not fully complementary to the corresponding guide strand by three nucleotides. In some embodiments, the passenger strand of the present disclosure comprises a nucleic acid sequence that is not fully complementary to the corresponding guide strand by four nucleotides.

[0291] In some embodiments, shRNAs of the disclosure comprise (a) a nucleic acid sequence selected from Table 6a, Table 6b, Table 6c, Table 6d, or Table 6e; (b) a nucleic acid sequence that differs from a sequence selected from Table 6a, Table 6b, Table 6c, Table 6d, or Table 6e 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 6a, Table 6b, Table 6c, Table 6d, or Table 6e. In some embodiments, shRNAs of the disclosure comprise a nucleic acid sequence selected from Table 6a, Table 6b, Table 6c, Table 6d, or Table 6e. In some embodiments, shRNAs of the disclosure comprise a nucleic acid sequence that differs from a sequence selected from Table 6a, Table 6b, Table 6c, Table 6d, or Table 6e by no more than five substitutions, additions, or deletions. In some embodiments, an shRNA of the disclosure comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 6a, Table 6b, Table 6c, Table 6d, or Table 6e.

[0292] 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 7; (b) a nucleic acid sequence that differs from a sequence selected from Table 7 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 7. In some embodiments, an miRNA of the present disclosure comprises a nucleic acid sequence selected from Table 7. In some embodiments, an miRNA of the present disclosure comprises a nucleic acid sequence that differs from a sequence selected from Table 7 by no more than five 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 7.

[0293] Inhibitory nucleic acid targets In some embodiments, the inhibitory nucleic acids of the present disclosure regulate gene expression in the modified immune cells of the present disclosure via RNA interference (RNAi). In some embodiments, the inhibitory nucleic acids of the present disclosure target mRNAs that contain complementary sequences. In some embodiments, the inhibitory nucleic acids of the present disclosure induce degradation of the target mRNA. In some embodiments, the inhibitory nucleic acids of the present disclosure suppress translation of the target mRNA.

[0294] In some embodiments, the target gene transcript is mammalian. In some embodiments, the target gene transcript is human. In some embodiments, contacting the target gene transcript with one or more inhibitory nucleic acids (e.g., RNA) decreases translation of the target gene transcript. In some embodiments, the target gene transcript is expressed in an immune cell. In some embodiments, the immune cell is a stem cell, macrophage, monocyte, or dendritic cell. In some embodiments, the target gene transcript is expressed in a macrophage. In some embodiments, the target gene transcript is expressed in a monocyte. In some embodiments, decreased translation of the target gene transcript is associated with an M1 phenotype. In some embodiments, decreased translation of the 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 M1 phenotype. In some embodiments, increased expression of an inhibitory nucleic acid (e.g., RNA) of the present disclosure is associated with an M2 phenotype.

[0295] In some embodiments, a target gene transcript of the disclosure comprises (a) a target nucleic acid sequence selected from Table 8; (b) a target nucleic acid sequence that differs from a sequence selected from Table 8 by no more than five substitutions, additions, or deletions; or (c) a target nucleic acid sequence that is at least 80% identical to a sequence selected from Table 8. In some embodiments, a target gene transcript of the disclosure comprises a target nucleic acid sequence selected from Table 8. In some embodiments, a target gene transcript of the disclosure comprises a target nucleic acid sequence that differs from a sequence selected from Table 8 by no more than five substitutions, additions, or deletions. In some embodiments, a target gene transcript of the disclosure comprises a target nucleic acid sequence that is at least 80% identical to a sequence selected from Table 8.

[0296] In some embodiments, the target gene transcript of the present disclosure encodes human ATG7, C / EBP-alpha, C / EBP-beta, CD36, CLEC1A, FATS, GOLM1, HAVCR2, ITGAD, KLF4, KLF6, LILRB1, LILRB2, LILRB4, MAF, MafB, PD-L1, PIK3CG, PIK3CG, PPARα, PPARγ, PTGS2, Siglec-7, Siglec-10, SIRPα, SLC15A3, STAT3, STAT6, TNFRSF1B, TOX, TREM2, YTHDF2, or ZFP36. In some embodiments, the target gene transcript of the present disclosure encodes human SIRPα. In some embodiments, the target gene transcript of the present disclosure encodes human PD-1 or PD-L1.

[0297] In some embodiments, the inhibitory nucleic acids of the present disclosure comprise one or more inhibitory nucleic acids. In some embodiments, the one or more inhibitory nucleic acids comprise one inhibitory nucleic acid. In some embodiments, the one or more inhibitory nucleic acids comprise at least two, three, four, or five inhibitory nucleic acids. In some embodiments, the one or more inhibitory nucleic acids comprise two inhibitory nucleic acids. In some embodiments, the one or more inhibitory nucleic acids comprise three inhibitory nucleic acids. In some embodiments, the one or more inhibitory nucleic acids comprise four inhibitory nucleic acids. In some embodiments, the one or more inhibitory nucleic acids comprise five inhibitory nucleic acids.

[0298] In some embodiments, at least two, three, four, or five inhibitory nucleic acids are present 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.

[0299] In some embodiments, at least two, three, four, or five inhibitory nucleic acids encode inhibitory RNAs comprising the same miRNA scaffold. 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 nucleic acids 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, the at least two different miRNA scaffolds are selected from the group consisting of miRNA-155 5' scaffold, miRNA-155 3' scaffold, miRNA-30 5' scaffold, and miRNA-30 3' scaffold.

[0300] Methods for designing inhibitory nucleic acids The present disclosure also provides methods for designing inhibitory nucleic acids of the present disclosure. In some embodiments, the present disclosure provides methods for designing inhibitory RNAs of the present disclosure. In some embodiments, the present disclosure provides methods for designing shRNAs of the present disclosure. In some embodiments, a method for designing an shRNA comprising a guide strand, a passenger strand, and a loop comprises 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, the guide strand is the reverse complement of the target region. In some embodiments, the guide strand comprises, from 5' to 3', an adenine or uracil at position 1 and an adenine, guanine, or cytosine at position 10, and a G / C content of between 36% and 45% increasing from 5' to 3'. In some embodiments, the guide strand comprises a higher G / C content of the 10 bases at the 3' end of the guide strand compared to the G / C content of the 10 bases at the 5' end of the guide strand. In some embodiments, the passenger strand comprises the sequence of the target region from 5' to 3' with the following modifications: (i) a deletion of the nucleotide at position 7 relative to the loop, (ii) a deletion of the nucleotide at position 11 relative to the loop, and (iii) an adenine to guanine and / or a cytosine to uracil mutation near the deletion at positions 7 and 11, where the mutations generate two partial GU base pairs between the passenger strand and the guide strand. In some embodiments, the passenger strand is two base pairs shorter than the guide strand. When nucleotide positions are counted relative to the loop, position 1 is closest to the loop, and positions further from the loop are counted increasing.

[0301] In some embodiments, a method for designing an shRNA comprising a guide strand, a passenger strand, and a loop includes the steps of: (a) selecting a target region of a target gene transcript; (b) designing a guide strand, wherein the guide strand is a reverse complement of the target region, and the guide strand comprises, from 5' to 3', an adenine or uracil at position 1 and an adenine, guanine, or cytosine at position 10, with a G / C content between 36% and 45% increasing from 5' to 3'; and (c) designing a passenger strand. and designing a passenger strand, wherein the passenger strand comprises, from 5' to 3', the sequence of the target region with the following modifications: (i) a deletion of the nucleotide at position 7 relative to the loop, (ii) a deletion of the nucleotide at position 11 relative to the loop, and (iii) an adenine to guanine and / or a cytosine to uracil mutation near the deletions at positions 7 and 11 (the mutations create two partial GU base pairs between the passenger strand and the guide strand, and the passenger strand is two base pairs shorter than the guide strand). In some embodiments, the method of designing an shRNA comprises a target region of a target gene transcript that is 21-22 base pairs in length.

[0302] Pharmaceutical Composition The present disclosure provides, inter alia, pharmaceutical compositions comprising modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising one or more CARs described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. The present disclosure also provides, inter alia, pharmaceutical compositions comprising nucleic acids encoding one or more CARs described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0303] Where a "therapeutically effective amount," "immunologically effective amount," "anti-immune response effective amount," or "immune response inhibiting effective amount" is indicated, the exact amount of the pharmaceutical compositions described herein can be determined by a physician, taking into account individual differences in the age, weight, immune response, and condition of the patient (subject).

[0304] The pharmaceutical compositions described herein may include buffers such as neutral buffered saline or phosphate buffered saline (PBS), carbohydrates such as glucose, mannose, sucrose, dextran, or mannitol, proteins, polypeptides, or amino acids (e.g., glycine), antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), preservatives such as serum and cryoprotectants. In some embodiments, the pharmaceutical compositions are substantially free of contaminants, e.g., free of detectable levels of contaminants (e.g., endotoxins).

[0305] The pharmaceutical compositions described herein can be administered in a manner appropriate for the disease, disorder, or condition to be treated or prevented. The amount and frequency of administration will be determined by factors such as the condition of the patient and the type and severity of the patient's disease, disorder, or condition, but the appropriate dosage can be determined by clinical trials.

[0306] The pharmaceutical compositions described herein may be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. Preferred compositions may be injectable or infusible solutions. The pharmaceutical compositions described herein may be formulated for intravenous, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intraarterial, or intraperitoneal administration.

[0307] In some embodiments, the pharmaceutical compositions described herein are formulated for parenteral (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular) administration. In some embodiments, the pharmaceutical compositions described herein are formulated for intravenous infusion or injection. In some embodiments, the pharmaceutical compositions described herein are formulated for intramuscular or subcutaneous injection. The pharmaceutical compositions described herein can be formulated for administration using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988, which is incorporated herein by reference in its entirety).

[0308] As used herein, the terms "parenteral administration" and "parenterally administered" refer to modes of administration other than enteral and topical administration, usually by injection or infusion, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intratumor, and intrasternal injection and infusion.

[0309] A pharmaceutical composition comprising the modified immune cells described herein may be administered in an amount of about 10 4 ~about 10 9 cells / kg body weight (e.g., approximately 10 5 ~about 10 6 In some embodiments, the dose of immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein is at least about 1 x 10 cells / kg body weight, including all integer values ​​within these ranges. 6 , about 1.1×10 6 , about 2×10 6 , about 3.6×10 6 , about 5×10 6 , about 1×10 7 , about 1.8×10 7 , about 2×10 7 , about 5×10 7 , about 1×10 8 , about 2×10 8 , about 5×10 8 , about 1×10 9, about 2×10 9 , or about 5 × 10 9 The pharmaceutical compositions described herein may also be administered multiple times at a particular dosage. The optimal dosage and treatment regime for a particular patient can be readily determined by one of skill in the art by monitoring the patient for symptoms of a disease, disorder, or condition and adjusting the treatment accordingly.

[0310] It may be desirable to administer a pharmaceutical composition described herein to a subject, followed by redrawing blood (or performing apheresis), activating the collected immune cells, and reinfusing the activated immune cells into the subject. This process can be performed multiple times, for example, every few weeks. Immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) can be activated from a blood draw of about 10 cc to about 400 cc. In some embodiments, immune cells (e.g., macrophages, monocytes, or dendritic cells) are activated from a blood draw of about 20 cc, about 30 cc, about 40 cc, about 50 cc, about 60 cc, about 70 cc, about 80 cc, about 90 cc, or about 100 cc. Without being bound by theory, methods involving multiple blood draws and reinfusions as described herein may be selected for certain immune cell populations.

[0311] In some embodiments, the pharmaceutical compositions described herein are administered in combination with (e.g., before, simultaneously with, or after) a second therapy. For example, the second therapy can include antiviral therapy (e.g., cidofovir, interleukin-2, cytarabine (ARA-C), or natalizumab), chimeric antigen receptor-T cell (CAR-T) therapy, T cell receptor (TCR)-T cell therapy, chemotherapy, radiation, immunosuppressants (e.g., cyclosporine, azathioprine, methotrexate, mycophenolate, FK506 antibody, or glucocorticoids), antagonists (e.g., PD-1 antagonists, PD-L1 antagonists), or immunosuppressants (e.g., cyclosporine, azathioprine, methotrexate, mycophenolate, FK506 antibody, or glucocorticoids). The therapeutic agent includes, but is not limited to, one or more of an anti-CD52 antibody (e.g., alemtuzumab), an anti-CD3 antibody, cytoxin, fludarivine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, or irradiation.

[0312] In some embodiments, the pharmaceutical compositions described herein are administered in combination with (e.g., before, concurrently with, or after) bone marrow transplantation or lymphocyte depletion therapy using chemotherapeutic agents (e.g., fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or Rituxan). In certain embodiments, the subject undergoes standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives an infusion of a pharmaceutical composition comprising the immune cells described herein. The pharmaceutical compositions described herein can be administered before or after surgery.

[0313] The dosage of any of the aforementioned therapies administered to a subject varies depending on the disease, disorder, or condition being treated and on the specific subject. Scaling of dosages for human administration can be performed according to art-recognized practices. For example, the dosage of alemtuzumab is generally about 1 mg to about 100 mg for an adult, usually administered daily for a period of about 1 day to about 30 days, e.g., a daily dose of about 1 mg to about 10 mg per day (e.g., as described in U.S. Patent No. 6,120,766, the entire contents of which are incorporated herein by reference).

[0314] Treatment method The present disclosure provides, inter alia, methods of treating a disease or disorder (e.g., a disease or disorder described herein) in a subject, the method comprising delivering a pharmaceutical composition described herein. In some embodiments, a therapeutically effective amount of a pharmaceutical composition described herein is administered to a subject having a disease or disorder. The pharmaceutical compositions described herein may be for use in the manufacture of a medicament for treating a disease or disorder in a subject or for stimulating an immune response in a subject.

[0315] The subject treated with the methods described herein can be a mammal, e.g., a primate, e.g., a human (e.g., a patient having or at risk of having a disease or disorder described herein). In some embodiments, the modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) can be autologous, allogeneic, or xenogeneic with respect to the subject. The pharmaceutical compositions described herein can be administered to a subject alone or in combination with one or more therapeutic agents, procedures, or modalities according to the dosing regimens described herein.

[0316] The pharmaceutical compositions described herein can be used to treat or prevent tumor or cancer-related diseases.

[0317] Methods are provided for treating (e.g., reducing, inhibiting, or delaying the progression of) cancer or tumor in a subject using the pharmaceutical compositions described herein. The subject may have an adult or childhood form of cancer. The cancer may be in early, intermediate, or late stage, or may be metastatic. Cancers can include, but are not limited to, solid tumors, hematological cancers (e.g., leukemia, lymphoma, or myeloma, such as multiple myeloma), or metastatic lesions. Examples of solid tumors include malignant tumors of various organ systems, such as sarcomas and carcinomas, e.g., adenocarcinomas, including those affecting the lung, breast, ovary, lymphatic system, gastrointestinal (e.g., colon), anus, genital and genitourinary tract (e.g., kidney, urothelium, bladder cells, prostate), pharynx, CNS (e.g., brain, neural or glial cells), head and neck, skin (e.g., melanoma, e.g., cutaneous melanoma), pancreas, and bone (e.g., chordoma).

[0318] In some embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC) (e.g., non-small cell lung cancer (NSCLC) of squamous and / or non-squamous tissue, or NSCLC adenocarcinoma), or small cell lung cancer (SCLC)), skin cancer (e.g., Merkel cell carcinoma or melanoma (e.g., advanced melanoma)), ovarian cancer, mesothelioma, bladder cancer, soft tissue sarcoma (e.g., hemangiopericytoma (HPC)), bone cancer (osteosarcoma), kidney cancer (e.g., renal carcinoma (e.g., renal cell carcinoma)), or small cell lung cancer (SCLC)). cancer), liver cancer (e.g., hepatocellular carcinoma), cholangiocarcinoma, sarcoma, myelodysplastic syndrome (MDS), prostate cancer, breast cancer (e.g., breast cancer that does not express one, two, or all of the estrogen receptor, progesterone receptor, or Her2 / neu, e.g., triple-negative breast cancer), colorectal cancer (e.g., recurrent colorectal cancer or metastatic colorectal cancer, e.g., microsatellite unstable colorectal cancer, microsatellite stable colorectal cancer, mismatch repair deficient (mismatch repair deficient), repair proficient, or mismatch repair deficient colorectal cancer), nasopharyngeal cancer, duodenal cancer, endometrial cancer, pancreatic cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), anal cancer, gastroesophageal cancer, thyroid cancer (e.g., anaplastic thyroid carcinoma), cervical cancer (e.g., cervical squamous cell carcinoma), neuroendocrine tumor (NET) (e.g., atypical pulmonary carcinoid tumor)), lymphoproliferative disorder (e.g., post-transplant lymphoproliferative disorder), lymphoma (e.g., T-cell lymphoma, B-cell lymphoma, non-Hodgkin's lymphoma), myeloma (e.g., multiple myeloma), or leukemia (e.g., myeloid leukemia or lymphocytic leukemia).

[0319] In some embodiments, the cancer is a brain tumor, e.g., glioblastoma, gliosarcoma, or recurrent brain tumor. In some embodiments, the cancer is pancreatic cancer, e.g., advanced pancreatic cancer. In some embodiments, the cancer is skin cancer, e.g., melanoma (e.g., stage II-IV melanoma, HLA-A2-positive melanoma, unresectable melanoma, or metastatic melanoma), or Merkel cell carcinoma. In some embodiments, the cancer is kidney cancer, e.g., renal cell carcinoma (RCC) (e.g., metastatic renal cell carcinoma). In some embodiments, the cancer is breast cancer, e.g., metastatic breast cancer or stage IV breast cancer, e.g., triple-negative breast cancer (TNBC). In some embodiments, the cancer is a virus-associated cancer. In some embodiments, the cancer is anal canal cancer (e.g., squamous cell carcinoma of the anal canal). In some embodiments, the cancer is cervical cancer (e.g., squamous cell carcinoma of the cervix). In some embodiments, the cancer is gastric cancer (e.g., Epstein-Barr virus (EBV)-positive gastric cancer, or gastric or gastroesophageal junction cancer). In some embodiments, the cancer is head and neck cancer (e.g., HPV-positive and -negative squamous cell carcinoma of the head and neck (SCCHN)). In some embodiments, the cancer is nasopharyngeal carcinoma (NPC). In some embodiments, the cancer is colorectal cancer, e.g., recurrent colorectal cancer, metastatic colorectal cancer, e.g., microsatellite-unstable colorectal cancer, microsatellite-stable colorectal cancer, mismatch repair-proficient colorectal cancer, or mismatch repair-deficient colorectal cancer.

[0320] In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is a leukemia, such as acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic leukemia, or acute leukemia. In some embodiments, the cancer is a lymphoma, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma, lymphocytic lymphoma, or diffuse large B-cell lymphoma (DLBCL) (e.g., relapsed or refractory HL or DLBCL). In some embodiments, the cancer is a myeloma, such as multiple myeloma.

[0321] The pharmaceutical compositions described herein can be used to enhance or regulate immune responses in a subject. In one embodiment, the pharmaceutical compositions described herein enhance, stimulate, or increase immune responses in a subject (e.g., a subject with or at risk of a disease or disorder described herein). In certain embodiments, the subject is immunocompromised or at risk of being immunocompromised. For example, the subject is undergoing or has undergone chemotherapy and / or radiation therapy.

[0322] Administration of the pharmaceutical compositions described herein can be by any convenient method (e.g., injection, ingestion, infusion, inhalation, implantation, or implantation). In some embodiments, the pharmaceutical compositions described herein are administered by injection or infusion. The pharmaceutical compositions described herein can be administered to a patient intraarterially, subcutaneously, intravenously, intradermally, intratumorally, intranodally, intramuscularly, or intraperitoneally. In some embodiments, the pharmaceutical compositions described herein are administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or intramuscularly). In some embodiments, the pharmaceutical compositions described herein are administered by intravenous infusion or injection. In some embodiments, the pharmaceutical compositions described herein are administered by intramuscular or subcutaneous injection. The pharmaceutical compositions described herein can be injected directly into the subject at the site of inflammation, the site of local disease, a lymph node, an organ, a tumor, or the site of infection.

[0323] All publications, patent applications, patents, and other references mentioned herein, including GenBank accession numbers, are incorporated by reference in their entirety. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0324] The present disclosure is further illustrated by the following examples, which are provided for illustrative purposes only and should not be construed as limiting the scope or content of the present disclosure in any way. [Example]

[0325] The following examples are provided to illustrate to one of ordinary skill in the art how to make and use the methods and compositions described herein and are not intended to limit the scope of the disclosure.

[0326] As shown below, Table 1 includes exemplary CAR constructs described herein. Figures 28 and 29 show schematic diagrams of these exemplary CAR constructs. [Table 1]

[0327] Example 1: Screening for anti-mesothelin binders This example evaluates 20 different anti-mesothelin binders (M1-M20) used in a CD8 framework CAR. All 20 CARs were produced as 5-methoxyuridine (5moU) mRNA. CAR mRNA was electroporated into human primary macrophages using a MaxCyte Atx instrument. 24 hours after electroporation, CAR expression was assessed by biotinylated mesothelin followed by APC-streptavidin binding. SS1-based CAR adenovirus was used as a positive control for mesothelin staining, and CAR001 (anti-HER2 CAR) mRNA was used as a positive control for electroporation and mRNA quality.

[0328] As shown in Figures 1A, 1B, and 1C, electroporation of macrophages with mRNA encoding a CAR did not adversely affect cell viability. Furthermore, as shown in Figures 1B and 1C, of ​​the 20 different anti-mesothelin CARs tested, M14, M15, and M17 showed the best expression in macrophages.

[0329] Example 2: Killing and cytokine secretion of macrophages expressing anti-mesothelin binders This example evaluates four different anti-mesothelin binders (M11, M14, M15, and M17) used in a CD8 framework CAR. All four CARs were produced as 5-methoxyuridine (5moU) mRNA. CAR mRNA was electroporated into human primary macrophages using a MaxCyte ATx instrument. Twenty-four hours after electroporation, cytokine release was assessed by 24-hour co-incubation with K562 WT (mesothelin-negative) or K562 MESO (mesothelin-positive) cells at a 1:1 effector to target cell (E:T) ratio. Killing was also assessed 24 hours after electroporation by measuring the change in GFP fluorescence over 72 hours using an Incucyte S3 Live-Cell Analysis System. An SS1-based CAR adenovirus was used as a positive control for killing and cytokine release, and CAR001 (anti-HER2 CAR) mRNA was used as a non-mesothelin-targeting CAR control.

[0330] As shown in Figure 2, of the four anti-mesothelin binders tested (M11, M14, M15, and M17), only M15 and M17 mediated target cell killing. When macrophages expressing exemplary anti-mesothelin CARs were co-incubated with K562 cells, M15 and M17CAR macrophages mediated TNFα cytokine release from mesothelin-positive K562 target cells. These data are shown in Figure 3.

[0331] Example 3: Phagocytosis screening of macrophages expressing anti-mesothelin binders This example evaluates four different anti-mesothelin binders (M11, M14, M15, and M17) used with CD8 framework CARs. All four CARs were produced as 5-methoxyuridine (5moU) mRNA. CAR mRNA was electroporated into human primary macrophages using a MaxCyte ATx instrument. 24 hours after electroporation, phagocytosis was assessed by 4 hours of co-incubation with K562 WT (mesothelin-negative) or K562 MESO (mesothelin-positive) at a 1:1 E:T ratio. At the end of the study, cells were analyzed via FACS, and CD11β+ / GFP+ events were defined as phagocytosis. Adenovirus containing the CD8-framework SS1 anti-mesothelin scFv CAR (SS1 virus) was used as a positive control for phagocytosis, and CAR001 (anti-HER2 CAR) mRNA was used as a non-mesothelin-targeting CAR control.

[0332] As shown in Figure 4, macrophages expressing all four anti-mesothelin binders (M11, M14, M15, and M17) mediated phagocytosis of mesothelin-positive target cells.

[0333] Example 4: Phenotyping of CTX_269-transduced macrophages In this example, we evaluate the phenotype of macrophages transduced with CTX_269. Pre-frozen macrophages from three donors were thawed and transduced with Ad5f35 vectors containing CTX_269 at various MOIs. After 48 hours, cells were analyzed by FACS for viability, CAR expression, and M1 / M2 polarization.

[0334] As shown in Figure 5, the Ad5f35 vector containing CTX_269 did not signifi...

Claims

1. A modified immune cell comprising a chimeric antigen receptor (CAR), the CAR comprising: (a) the extracellular domain; (b) a transmembrane domain, and (c) comprises one or more intracellular domains; the extracellular domain is or comprises an anti-mesothelin antigen-binding domain comprising an amino acid sequence at least 80% identical to a sequence selected from Table 3; The modified immune cell is or comprises a macrophage, monocyte, dendritic cell, or stem cell.

2. The modified immune cell of claim 1 , wherein the extracellular domain is or comprises an scFv, a VHH antibody, a centrin, a darpin, or a nanobody.

3. 3. The modified immune cell of claim 1 or 2, wherein the transmembrane domain is or comprises a CD8, CD8a, CD28, CD40, MyD88 CD64, CD32a, CD32c, CD16a, CD3 zeta, ICOS, Dectin-1, DNGR1, SLAMF7, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, or TLR9 transmembrane domain.

4. The one or more intracellular domains may be CD3ζ, FcRγ, MyD88, CD40, CD64, CD32a, CD32c, CD16a, CD89, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphA1, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TR K, VEGFR, CD19, CD20, 41BB, CD28, GCSFR (CD114), RAGE, CD30, CD160, DR3, Fn14, HVEM, CD160, NGFR, RANK, TNFR2 , TROY, XEDAR, TRIF, OX40, GITR, TREM-1, TREM-2, 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, SIRPb, CD22, PIR-B, LILRB1, 41BBL (T NFSF9), CD27, OX40L, CD32b, CD11b, ITGAM, SLAMF7, CD206, CD163, CD209, Dectin-2, IL1R, IL2R, IL3R, IL4R, IL5R, 4. The modified immune cell of any one of claims 1 to 3, comprising an 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 said domains, or a combination thereof.

5. The modified immune cell of any one of claims 1 to 4, wherein the one or more intracellular domains comprise a CD3ζ intracellular domain or an FcRγ intracellular domain.

6. The modified immune cell of any one of claims 1 to 5, wherein the CAR further comprises an extracellular leader domain.

7. The modified immune cell of claim 6, wherein the extracellular leader domain comprises a CD8a extracellular leader domain.

8. The modified immune cell of any one of claims 1 to 7, wherein the CAR further comprises an extracellular hinge domain.

9. 9. The modified immune cell of claim 8, wherein the extracellular hinge domain comprises a CD8 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, or an IgG4 extracellular hinge domain.

10. The CAR comprises, from the N-terminus to the C-terminus: a CD8a leader domain, an anti-mesothelin antigen-binding domain, a CD8 extracellular hinge domain, a CD8 transmembrane domain, and a CD3ζ intracellular domain; a CD8a leader domain, an anti-mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and a CD3ζ intracellular domain; a CD8a leader domain, an anti-mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and an FcRγ intracellular domain; CD8a leader domain, anti-mesothelin antigen-binding domain, CD8 extracellular hinge domain, CD8 transmembrane domain, CD3ζ intracellular domain, P2A cleavage peptide, and CD40 ligand (CD40L), CD8a leader domain, anti-mesothelin antigen-binding domain, CD28 extracellular hinge domain, CD28 transmembrane domain, CD3ζ intracellular domain, P2A cleavage peptide, and CD40 ligand (CD40L), a CD8a leader domain, an anti-mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a MyD88 intracellular domain, and a CD3ζ intracellular domain; CD8a leader domain, anti-mesothelin antigen-binding domain, CD28 extracellular hinge domain, CD28 transmembrane domain, MyD88 intracellular domain, CD40 intracellular domain, and CD3ζ intracellular domain, a CD8a leader domain, an anti-mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a truncated MyD88 intracellular domain, a CD40 intracellular domain, and a CD3ζ intracellular domain; CD8a leader domain, anti-mesothelin antigen binding domain, CD28 extracellular hinge domain, CD28 transmembrane domain, FcRγ intracellular domain, P2A cleavage peptide, and CD40 ligand (CD40L); a CD8a leader domain, an anti-mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a MyD88 intracellular domain, and an FcRγ intracellular domain; a CD8a leader domain, an anti-mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a MyD88 intracellular domain, a CD40 intracellular domain, and an FcRγ intracellular domain, or CD8a leader domain, anti-mesothelin antigen binding domain, CD28 extracellular hinge domain, CD28 transmembrane domain, truncated MyD88 intracellular domain, CD40 intracellular domain, and FcRγ intracellular domain 10. The modified immune cell of claim 8 or 9, comprising:

11. The CAR is (a) an amino acid sequence selected from Table 2; (b) an amino acid sequence that differs from a sequence selected from Table 2 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 2 11. The modified immune cell of claim 10, having or comprising:

12. A pharmaceutical composition comprising a modified immune cell according to any one of the preceding claims.

13. 13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

14. (a) an extracellular binding domain; (b) a transmembrane domain, and (c) one or more intracellular domains; 1. A nucleic acid construct comprising one or more nucleic acid sequences encoding the extracellular domain is or comprises an anti-mesothelin antigen-binding domain comprising an amino acid sequence at least 80% identical to a sequence selected from Table 5; The nucleic acid construct, wherein the nucleic acid construct encodes a chimeric antigen receptor (CAR) comprising (a) to (c).

15. (d) one or more extracellular leader domains; (e) one or more extracellular hinge domains; (f) one or more truncated peptides; or a combination thereof, 15. The nucleic acid construct of claim 14, further comprising one or more nucleic acid sequences encoding:

16. 16. The nucleic acid construct of claim 15, wherein the truncated peptide is or comprises a P2A, F2A, E2A, or T2A peptide.

17. From the N-terminus to the C-terminus, a CD8a leader domain, an anti-mesothelin antigen-binding domain, a CD8 extracellular hinge domain, a CD8 transmembrane domain, and a CD3ζ intracellular domain; a CD8a leader domain, an anti-mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and a CD3ζ intracellular domain; a CD8a leader domain, an anti-mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and an FcRγ intracellular domain; CD8a leader domain, anti-mesothelin antigen-binding domain, CD8 extracellular hinge domain, CD8 transmembrane domain, CD3ζ intracellular domain, P2A cleavage peptide, and CD40 ligand (CD40L), CD8a leader domain, anti-mesothelin antigen-binding domain, CD28 extracellular hinge domain, CD28 transmembrane domain, CD3ζ intracellular domain, P2A cleavage peptide, and CD40 ligand (CD40L), a CD8a leader domain, an anti-mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a MyD88 intracellular domain, and a CD3ζ intracellular domain; CD8a leader domain, anti-mesothelin antigen-binding domain, CD28 extracellular hinge domain, CD28 transmembrane domain, MyD88 intracellular domain, CD40 intracellular domain, and CD3ζ intracellular domain, a CD8a leader domain, an anti-mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a truncated MyD88 intracellular domain, a CD40 intracellular domain, and a CD3ζ intracellular domain; CD8a leader domain, anti-mesothelin antigen binding domain, CD28 extracellular hinge domain, CD28 transmembrane domain, FcRγ intracellular domain, P2A cleavage peptide, and CD40 ligand (CD40L); a CD8a leader domain, an anti-mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a MyD88 intracellular domain, and an FcRγ intracellular domain; a CD8a leader domain, an anti-mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a MyD88 intracellular domain, a CD40 intracellular domain, and an FcRγ intracellular domain, or 17. The nucleic acid construct of claim 15 or 16, encoding a CD8a leader domain, an anti-mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a truncated MyD88 intracellular domain, a CD40 intracellular domain, and an FcRγ intracellular domain.

18. (a) a nucleotide sequence selected from Table 4; (b) a nucleotide sequence that differs from a sequence selected from Table 4 by no more than five substitutions, additions, or deletions; or (c) a nucleotide acid sequence that is at least 80% identical to a sequence selected from Table 4.

18. The nucleic acid construct of claim 17, having or comprising:

19. 15. The nucleic acid construct of claim 14, further comprising one or more introns, wherein the one or more introns comprise one or more inhibitory nucleic acids, and the one or more inhibitory nucleic acids encode one or more inhibitory RNAs.

20. 20. The nucleic acid construct of claim 19, wherein the one or more inhibitory RNAs are or comprise one or more shRNAs.

21. 21. The nucleic acid construct of claim 20, wherein the one or more shRNAs comprise a guide strand.

22. 22. The nucleic acid construct of claim 21, wherein the guide strand comprises a nucleic acid sequence that is reverse complementary to a target gene transcript that comprises a target nucleic acid sequence.

23. 23. The nucleic acid construct of claim 22, 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, PPARα, PPARγ, PTGS2, Siglec-10, SIRPα, SLAMF3, SLAMF4, SLC15A3, STAT3, STAT6, TNFRSF1B, TOX, TREM2, YTHDF2, or ZFP36.

24. 24. The nucleic acid construct of claim 22 or 23, wherein the target gene transcript encodes a human anti-phagocytic receptor selected from the group consisting of SIRPα, LILRB1, Siglec-10, PD1, SLAMF3, SLAMF4, CLEC1A, and CD32b.

25. The nucleic acid construct of any one of claims 22 to 24, wherein the target gene transcript encodes human SIRPα.

26. A pharmaceutical composition comprising the nucleic acid construct according to any one of claims 14 to 25.

27. 27. The pharmaceutical composition of claim 26, comprising a pharmaceutically acceptable carrier.

28. 1. A method of treating a disease or disorder in a subject, comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 12, 13, 26, or 27; The method, wherein at least one sign or symptom of the disease or disorder is improved in the subject after administration.

29. 29. The method of claim 28, wherein the administering step is or includes intraarterial, subcutaneous, intravenous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, or intraperitoneal delivery.

30. 1. A method for modifying immune cells, comprising: delivering to said immune cells a nucleic acid construct according to any one of claims 14 to 25, thereby generating modified immune cells; The method, wherein the modified immune cells are or comprise macrophages, monocytes, dendritic cells, or stem cells.

31. 31. The method of claim 30, wherein the nucleic acid construct comprises DNA or messenger RNA (mRNA).

32. 32. The method of claim 30 or 31, wherein the nucleic acid construct comprises a modification selected from modified nucleotides, a modification to the 5' untranslated region (UTR), a modification to the 3' UTR, a cap structure, a poly(A) tail, or a combination thereof.

33. 33. The method of claim 32, wherein the cap structure comprises AGCap1, m6AGCap1, or an anti-reverse cap analog (ARCA).

34. 34. The method of claim 32 or 33, wherein the modified nucleotide comprises pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine / pseudouridine (5meC PsU), N1-methyl-pseudouridine (N1mPsU), or a combination thereof.

35. The method of any one of claims 30 to 34, wherein the nucleic acid construct is a purified nucleic acid construct.

36. 36. The method of claim 35, wherein the purified nucleic acid construct is produced by a method comprising silica membrane purification, high performance liquid chromatography (HPLC), Dynabeads, LiCl precipitation, phenol-chloroform extraction, resin-based purification, polyA isolation, RNeasy, or a combination thereof.

37. The method of any one of claims 30 to 36, wherein the nucleic acid construct is codon optimized.

38. 38. The method of claim 37, wherein the nucleic acid construct is codon-optimized for expression in stem cells, monocytes, macrophages, or dendritic cells.

39. The method of any one of claims 30 to 38, wherein the delivery comprises electroporation or transfection with the nucleic acid construct.

40. The method of any one of claims 30 to 38, wherein the nucleic acid construct is encapsulated in a delivery vehicle.

41. 41. The method of claim 40, wherein the delivery vehicle is or comprises a liposome, a lipid nanoparticle, a polymer, an adeno-associated virus (AAV) vector, an adenoviral vector, a retroviral vector, or a combination thereof.

42. 42. The method of claim 41, wherein the liposome or lipid nanoparticle comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, one or more PEG-modified lipids, or a combination thereof.

43. 42. The method of claim 41, wherein the retroviral vector comprises a lentiviral vector or a gammaretroviral vector.

44. 44. The method of claim 43, wherein the lentiviral vector is packaged with a Vpx protein.

45. 42. The method of claim 41, wherein the adenoviral vector comprises an Ad2 vector or an Ad5 vector.

46. 46. ​​The method of claim 45, wherein the Ad5 vector comprises an Ad5f35 adenoviral vector.

47. 47. The method of any one of claims 30 to 46, further comprising delivering an additional payload to the immune cells.

48. The additional payload may be selected from the group consisting of 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 lectin receptor (CLR) agonist, a cytoplasmic DNA sensing agonist, a 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 (absent in melanoma ...cytoplasmic DNA sensing agonist, a cytoplasmic DNA sensing agonist, a 48. The method of claim 47, wherein the therapeutic agent is or comprises an anti-inflammatory drug (AIM2) agonist, an aryl hydrocarbon receptor (AhR) ligand, or a combination thereof.

49. 49. The method of claim 47 or 48, wherein the nucleic acid construct and the additional payload are encapsulated within the delivery vehicle.

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