Novel anti-mesothelin chimeric antigen receptor and modified immune cell

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.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority and the benefit of U.S. Provisional Patent Application No. 63 / 412,622, filed on October 3, 2023, and U.S. Provisional Patent Application No. 63 / 459,903, filed on April 17, 2023, the contents of which are incorporated herein by reference in their entirety. Background of the Invention

[0003] Mesothelin is a tumor antigen that is highly expressed in many human cancers, including malignant mesothelioma, pancreatic cancer, ovarian adenocarcinoma, and lung adenocarcinoma. It is a target for cancer immunotherapy because its normal expression in the human body is limited to mesothelial cells. Therefore, there is a need to develop novel therapeutic modalities optimized to target mesothelin. Summary of the Invention

[0004] The present disclosure particularly encompasses compositions comprising modified immune cells (such as, stem cells, macrophages, monocytes, and / or dendritic cells) containing an anti - mesothelin chimeric antigen receptor (CAR), as well as methods for producing and using such compositions.

[0005] In one aspect, the present disclosure provides modified immune cells comprising a chimeric antigen receptor (CAR), wherein the CAR comprises: (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 that is at least 80% identical to a sequence selected from Table 3; and wherein the modified immune cells are or comprise macrophages, monocytes, dendritic cells, or stem cells.

[0006] In some embodiments, the extracellular domain is or comprises an scFv, a VHH antibody, a centyrin, a designed ankyrin repeat protein (darpin), or a nanobody. In some embodiments, the transmembrane domain is or comprises a CD8, CD8a, CD28, CD40, MyD88 CD64, CD32a, CD32c, CD16a, CD3ζ, ICOS, dendritic cell-associated C-type lectin-1 (Dectin-1), DNGR1, SLAMF7, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, or TLR9 transmembrane domain. In some embodiments, one or more intracellular domains comprise: 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, 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, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPb, CD22, PIR-B, LILRB1, 41BBL (TNFSF9), CD27, OX40L, CD32b, CD11b, ITGAM, SLAMF7, CD206, CD163, CD209, dendritic cell-associated C-type lectin-2 (Dectin-2), IL1R, IL2R, IL3R, IL4R, IL5R, IL6R, IL7R, IL8R, IL9R, IL10R, IL11R, IL12R, IL13R, IL14R, IL15R, IL17R, IFNaR, IFNgR, TNFR, CSF1R, CSF2R, Dap10, CD36, dendritic cell-associated C-type lectin-1, ICOSL, or Syk intracellular domain, or a portion of any of the foregoing, or a combination thereof.In some embodiments, one or more intracellular domains include a CD3ζ intracellular domain or an FcRγ intracellular domain.

[0007] In some embodiments, the CAR further includes an extracellular leader domain. In some embodiments, the extracellular leader domain includes a CD8a extracellular leader domain. In some embodiments, the CAR further includes an extracellular hinge domain. In some embodiments, the extracellular hinge domain includes: a CD8 extracellular hinge domain, a CD8a extracellular hinge domain, a CD28 extracellular hinge domain, a DNGR-1 extracellular hinge domain, a dendritic cell-associated C-type lectin-1 extracellular hinge domain, or an IgG4 extracellular hinge domain.

[0008] In some embodiments, the CAR comprises, from the N-terminus to the C-terminus: a CD8a leader domain, a mesothelin antigen-binding domain, a CD8 extracellular hinge domain, a CD8 transmembrane domain, and a CD3ζ intracellular domain; a CD8a leader domain, a mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and a CD3ζ intracellular domain; a CD8a leader domain, a mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and an FcRγ intracellular domain; a CD8a leader domain, a 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); a CD8a leader domain, a 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); a CD8a leader domain, a mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a MyD88 intracellular domain, and a CD3ζ intracellular domain; a CD8a leader domain, a 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; a CD8a leader domain, a 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; a CD8a leader domain, a 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); a CD8a leader domain, a 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, a 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 a CD8a leader domain, a 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.

[0009] 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.

[0010] In another aspect, the present disclosure provides a pharmaceutical composition comprising the modified immune cells as described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0011] In another aspect, the present 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 that is at least 80% identical to a sequence selected from Table 5; and wherein the nucleic acid construct encodes a chimeric antigen receptor (CAR) comprising (a) to (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 cleavage peptides, or a combination thereof. In some embodiments, the cleavage peptide is or comprises a P2A, F2A, E2A or T2A peptide.

[0012] In some embodiments, the nucleic acid construct encodes, from the N-terminus to the C-terminus: a CD8a leader domain, a mesothelin antigen-binding domain, a CD8 extracellular hinge domain, a CD8 transmembrane domain, and a CD3ζ intracellular domain; a CD8a leader domain, a mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and a CD3ζ intracellular domain; a CD8a leader domain, a mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and an FcRγ intracellular domain; a CD8a leader domain, a 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); a CD8a leader domain, a 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); a CD8a leader domain, a mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a MyD88 intracellular domain, and a CD3ζ intracellular domain; a CD8a leader domain, a 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; a CD8a leader domain, a 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; a CD8a leader domain, a 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); a CD8a leader domain, a 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, a 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 a CD8a leader domain, a 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.

[0013] 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 5 substitutions, additions, or deletions; or (c) a nucleotide sequence that is at least 80% identical to a sequence selected from Table 4.

[0014] In some embodiments, the nucleic acid constructs of the present disclosure further comprise one or more introns, wherein the one or more introns comprise one or more inhibitory nucleic acids, and wherein the one or more inhibitory nucleic acids encode one or more inhibitory RNAs. 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 comprising a target nucleic acid sequence. In some embodiments, the target gene transcript encodes human ATG7, C / EBP-α, C / EBP-β, CD32b, CD36, CLEC1A, FATS, GOLM1, HAVCR2, ITGAD, KLF4, KLF6, LILRB1, LILRB2, LILRB4, MAF, MafB, PD1, PD-LI, PIK3CG, PIK3CG, PPARα, PPARγ, PTGS2, SIGLEC10, SIRPα, SLAMF3, SLAMF4, SLC15A3, STAT3, STAT6, TNFRSF1B, TOX, TREM2, YTHDF2 or ZFP36. In some embodiments, the target gene transcript encodes a human anti-phagocytic receptor selected from the group consisting of: SIRPα, LILRB1, SIGLEC10, PD1, SLAMF3, SLAMF4, CLEC1A and CD32b. In some embodiments, the target gene transcript encodes human SIRPα.

[0015] In another aspect, the present disclosure provides a pharmaceutical composition comprising a nucleic acid construct as described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0016] In another aspect, the present disclosure provides a method of treating a disease or disorder in a subject, the method comprising: administering to the subject a therapeutically effective amount of a pharmaceutical composition as described herein, wherein at least one sign or symptom of the disease or disorder in the subject is improved after administration. In some embodiments, the administering step is or comprises arterial, subcutaneous, intravenous, intradermal, intratumoral, intranodal, intramedullary, intramuscular or intraperitoneal delivery.

[0017] In another aspect, the present disclosure provides a method of modifying an immune cell, the method comprising: delivering a nucleic acid construct as described herein to the immune cell, thereby producing a modified immune cell, wherein the modified immune cell is or comprises a macrophage, a monocyte, a dendritic cell or a stem cell.

[0018] 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 the following: modified nucleotides, alterations to the 5' untranslated region (UTR), alterations to the 3' UTR, cap structure, poly(A) tail, or combinations thereof. In some embodiments, the cap structure comprises AGCap1, m6AGCap1, or anti-reverse cap analog (ARCA). In some embodiments, the modified nucleotides comprise pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine / pseudouridine (5meC PsU), N1-methyl-pseudouridine (N1mPsU), or combinations thereof.

[0019] 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 comprising silica membrane purification, high performance liquid chromatography (HPLC), Dynabeads, LiCl precipitation, phenol-chloroform extraction, resin-based purification, polyA isolation, RNeasy, or combinations 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.

[0020] 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 liposomes, lipid nanoparticles, polymers, adeno-associated virus (AAV) vectors, adenovirus vectors, retrovirus vectors, or combinations thereof. In some embodiments, the liposomes or lipid nanoparticles comprise one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, one or more PEG-modified lipids, or combinations thereof. In some embodiments, the retrovirus vector comprises a lentivirus vector or a gamma retrovirus vector. In some embodiments, the lentivirus vector is packaged with the Vpx protein. In some embodiments, the adenovirus vector comprises an Ad2 vector or an Ad5 vector. In some embodiments, the Ad5 vector comprises an Ad5f35 adenovirus vector.

[0021] In some embodiments, the methods of the present disclosure further include delivering an additional payload to immune cells. In some embodiments, the additional payload is or comprises a pathogen recognition receptor agonist, polyinosinic acid: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, cytosolic 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 the additional payload are encapsulated within a delivery vehicle. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings are for illustrative purposes only and are not intended to be limiting.

[0023] Figure 1A 、 Figure 1B and Figure 1C show graphs of exemplary macrophage viability ( Figure 1A ) and CAR expression ( Figure 1B and Figure 1C ) following electroporation with mRNA encoding 20 different mesothelin-binding agents (M1-M20) for a CD8-framework chimeric antigen receptor (CAR).

[0024] Figure 2 show graphs of exemplary macrophage-mediated target cell killing following electroporation with mRNA encoding 4 different mesothelin-binding agents (M11, M14, M15, and M17) for a CD8-framework CAR.

[0025] Figure 3 show graphs of exemplary TNFα cytokine release mediated by macrophages following electroporation with mRNA encoding 4 different mesothelin-binding agents (M11, M14, M15, and M17) for a CD8-framework CAR.

[0026] Figure 4 show graphs of exemplary mesothelin-mediated phagocytosis by macrophages following electroporation with mRNA encoding 4 different mesothelin-binding agents (M11, M14, M15, and M17) for a CD8-framework CAR.

[0027] Figure 5Shown is a graph of exemplary macrophage viability following transduction with Ad5f35 vectors containing CTX_269, an anti-mesothelin CAR, at different exemplary MOIs.

[0028] Figure 6 Shown is a graph of exemplary anti-mesothelin CAR expression following transduction of macrophages with Ad5f35 vectors containing CTX_269 (an anti-mesothelin CAR) at different exemplary MOIs.

[0029] Figure 7A , Figure 7B and Figure 7C A graph showing exemplary expression of exemplary M1-associated markers (CD80, CD86, and HLA-DR) following transduction of macrophages with Ad5f35 vectors containing CTX_269, an anti-mesothelin CAR, at different exemplary MOIs is shown.

[0030] Figure 8 Shown is a graph of exemplary macrophage expression of exemplary M2-associated markers (CD163 and CD206) after macrophages were transduced with Ad5f35 vectors containing CTX_269, an anti-mesothelin CAR, at different exemplary MOIs.

[0031] Figure 9 Shown is a graph of exemplary M2-associated marker (CD163) expression following transduction of macrophages with Ad5f35 vectors containing CTX_269, an anti-mesothelin CAR.

[0032] Figure 10 Shown is a graph of exemplary cytokine M1-associated marker (CD86) expression following transduction of macrophages with Ad5f35 vector containing CTX_269, an anti-mesothelin CAR.

[0033] Figure 11 Shown is a graph of exemplary M2-associated marker (CD163 and CD206) expression after macrophages were transduced with an Ad5f35 vector containing CTX_269, an anti-mesothelin CAR, and exposed to mesothelin.

[0034] Figure 12 Shown is a graph of exemplary anti-mesothelin-mediated phagocytosis of A549 lung adenocarcinoma cells by macrophages after transduction of macrophages with Ad5f35 vectors containing CTX_269, an anti-mesothelin CAR.

[0035] Figure 13Shows a graph of exemplary mesothelin-mediated phagocytosis of MES-OV ovarian cystadenocarcinoma cells by macrophages after transduction of the macrophages with an Ad5f35 vector containing CTX_269, an anti-mesothelin CAR.

[0036] Figure 14 Shows a graph of exemplary mesothelin-mediated killing of A549 lung adenocarcinoma cells expressing mesothelin by macrophages after transduction of the macrophages with an Ad5f35 vector containing CTX_269, an anti-mesothelin CAR.

[0037] Figure 15 Shows a graph of exemplary mesothelin-mediated killing of ovarian cystadenocarcinoma cells expressing mesothelin by macrophages after transduction of the macrophages with an Ad5f35 vector containing CTX_269, an anti-mesothelin CAR.

[0038] Figure 16 Shows a graph of the release of exemplary cytokines (TNFα and IL-1β) after transduction of macrophages with an Ad5f35 vector containing CTX_269, an anti-mesothelin CAR and exposure of the macrophages to mesothelin.

[0039] Figure 17 Shows a graph of the release of an exemplary cytokine (TNFα) after transduction of macrophages with an Ad5f35 vector containing CTX_269, an anti-mesothelin CAR and exposure of the macrophages to target cells (A549 lung adenocarcinoma cells or MES-OV ovarian cystadenocarcinoma cells) expressing mesothelin.

[0040] Figure 18 Shows an exemplary experimental timeline for treating a murine tumor model in vivo with macrophages transduced with an Ad5f35 vector containing CTX_269, an anti-mesothelin CAR.

[0041] Figure 19 Shows an exemplary graph of the tumor burden of mice treated with macrophages transduced with an Ad5f35 vector containing CTX_269, an anti-mesothelin CAR.

[0042] Figure 20 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 with a lentiviral vector containing a CD8-based anti-mesothelin CAR.

[0043] Figure 21 Shows a graph of macrophage-mediated exemplary target cell killing after transduction of macrophages with a lentiviral vector containing a CD28-based anti-mesothelin CAR or with a lentiviral vector containing a CD8-based anti-mesothelin CAR.

[0044] Figure 22 Shown is a graph of exemplary cytokine (TNFα) release following transduction of macrophages with a lentiviral vector comprising a CD28-based anti-mesothelin CAR or with a lentiviral vector comprising a CD8-based anti-mesothelin CAR and exposure of the transduced macrophages to mesothelin.

[0045] Figure 23 A graph showing the expression of exemplary M2-associated markers (CD163 and CD206) after macrophages were transduced with Ad5f35 vectors containing CTX_269 (an anti-mesothelin CAR containing a CD8-framework) or CTX_293 (an anti-mesothelin CAR containing a CD28-framework) and the transduced macrophages were exposed to IL-10.

[0046] Figure 24 Shown is a graph of the expression of exemplary M1-associated markers (CD80 and CD86) after macrophages were transduced with Ad5f35 vectors containing CTX_269 (an anti-mesothelin CAR containing a CD8-framework) or CTX_293 (an anti-mesothelin CAR containing a CD28-framework) and the transduced macrophages were exposed to IL-10.

[0047] Figure 25 Shown is a graph of exemplary anti-mesothelin-mediated killing of mesothelin-expressing A549 lung adenocarcinoma cells by macrophages after macrophages were transduced with Ad5f35 vectors containing CTX_269 (an anti-mesothelin CAR containing a CD8-framework) or CTX_293 (an anti-mesothelin CAR containing a CD28-framework).

[0048] Figure 26A , Figure 26B , Figure 26C , Figure 26D , Figure 26E and Figure 26F Shown are graphs of exemplary phenotypic markers of monocytes transduced with CTX_269 (a CD8-based anti-mesothelin CAR) or CTX_001 (an 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 27Graph showing exemplary mesothelin-mediated killing of mesothelin-expressing ovarian cystadenocarcinoma cells (left panel) or mesothelin-expressing A549 lung adenocarcinoma cells (right panel) by monocytes transduced with CTX_269, a CD8-based anti-mesothelin CAR, or CTX_001, an 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.

[0050] Figure 28 Schematic diagram showing an exemplary anti-mesothelin CAR construct containing M15 scFv.

[0051] Figure 29 Schematic diagram showing an exemplary anti-mesothelin CAR construct containing M17 scFv.

[0052] Figure 30 Graph showing exemplary anti-mesothelin CAR expression on day 2 and day 14 after transduction of macrophages with an Ad5f35 vector containing CTX_269, an anti-mesothelin CAR.

[0053] Figure 31 Graph showing exemplary mesothelin-mediated phagocytosis of K562 cells by macrophages after transduction of macrophages with an Ad5f35 vector containing CTX_269, an anti-mesothelin CAR.

[0054] Figure 32 Graph showing exemplary cytokine (TNFα) release after transduction of macrophages with an Ad5f35 vector containing CTX_269, an anti-mesothelin CAR, and exposure of the macrophages to mesothelin-expressing target cells (K562 cells).

[0055] Figure 33 Representative tissue section of murine lung immunohistochemistry (IHC) for human mesothelin staining in mice treated with macrophages transduced with an Ad5f35 vector containing CTX_269, an anti-mesothelin CAR. Scale bar, 1 mm.

[0056] Figure 34 Graph showing exemplary quantification of tumor nodules in murine lung treated with macrophages transduced with an Ad5f35 vector containing CTX_269, an anti-mesothelin CAR.

[0057] Figure 35A and Figure 35BShows a graph of exemplary anti-mesothelin CAR expression and cell viability after transduction of monocytes with Ad5f35 virus containing CTX_964 (an anti-mesothelin CAR) or CTX_1461 (an anti-mesothelin CAR + intronic shRNA against SIRPα).

[0058] Figure 36A and Figure 36B Shows a graph of exemplary SIRPα expression after transduction of monocytes with Ad5f35 virus containing CTX_964 (an anti-mesothelin CAR) or CTX_1461 (an anti-mesothelin CAR + intronic shRNA against SIRPα).

[0059] Figure 37 Shows a graph of exemplary cytokine (TNFα) release after transduction of monocytes with Ad5f35 virus containing CTX_964 (an anti-mesothelin CAR) or CTX_1461 (an anti-mesothelin CAR + intronic shRNA against SIRPα) and exposing the cells to recombinant human mesothelin or recombinant mesothelin + recombinant human CD47.

[0060] Figure 38A and Figure 38B Shows a graph of exemplary anti-mesothelin-mediated killing of mesothelin-expressing target cells by monocytes and macrophages after transduction of cells with an Ad5f35 vector containing CTX_964 (an anti-mesothelin CAR) or CTX_1461 (an anti-mesothelin CAR + intronic shRNA against SIRPα).

[0061] Figure 39 Shows a graph of exemplary inhibition of tumor growth by CAR-monocyte-derived CAR macrophages after transduction of cells with an Ad5f35 vector containing CTX_964 (an anti-mesothelin CAR) or CTX_1461 (an anti-mesothelin CAR + intronic shRNA against SIRPα).

[0062] Definitions

[0063] To facilitate understanding of the present invention, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification. Publications and other reference materials cited herein to describe the background of the present invention and to provide additional details regarding its practice are hereby incorporated by reference.

[0064] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. For example, "an element" refers to one element or more than one element.

[0065] About or approximately: As used herein, when applied to one or more values of interest, the terms “about” or “approximately” refer to a value similar to the stated reference value. In certain embodiments, the terms “about” or “approximately” refer to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) of the stated reference value, unless otherwise specified or clear from the context (except in cases where such numbers would exceed 100% of a possible value).

[0066] 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 has been stimulated to perform its effector function. Activation can also be associated with induced cytokine production, phagocytosis, cell signaling, target cell killing, and / or antigen processing and presentation.

[0067] Activated monocyte / macrophage / dendritic cell: As used herein, the term “activated monocyte / macrophage / dendritic cell” particularly refers to a monocyte / macrophage / dendritic cell that is undergoing cell division or performing its effector function. The term “activated monocyte / macrophage / dendritic cell” particularly refers to a cell that performs an effector function or exhibits any activity not seen in the resting state (including phagocytosis, cytokine secretion, proliferation, changes in gene expression, metabolic changes, and other functions).

[0068] Agent: As used herein, the term “agent” (or “biological agent” or “therapeutic agent”) refers to a molecule that can be expressed, released, secreted or delivered to a target by a modified cell as described herein. Agents 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 (such as RNase inhibitors), peptides, fusion proteins, synthetic molecules, organic molecules (such as small molecules), carbohydrates, lipids, hormones, microparticles, derivatives or variants thereof, and any combination thereof. An agent can bind to any cellular moiety, such as a receptor, epitope or other binding site present on a target or target cell. An agent can diffuse or be transported into a cell, where it can act intracellularly.

[0069] Antibody: As used herein, the term “antibody” refers to a polypeptide that includes the typical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As is known in the art, a complete antibody as it occurs in nature is a tetrameric agent of approximately 150 kD that contains two identical heavy chain polypeptides (each approximately 50 kD) and two identical light chain polypeptides (each approximately 25 kD), which associate with one another to form a structure commonly referred to as a “Y-shaped” structure. Each heavy chain contains at least four domains (each domain being approximately 110 amino acids in length)—an amino-terminal variable (VH) domain (at the apex of the Y structure), followed by three constant domains: CH1, CH2, and the carboxyl-terminal CH3 (at the base of the stem of the Y). A short region called the “switch” joins the heavy chain variable region and the constant region. The “hinge” joins the CH2 and CH3 domains to the remainder of the antibody. Two disulfide bonds in this hinge region link the two heavy chain polypeptides in the complete antibody to one another. Each light chain contains two domains—an amino-terminal variable (VL) domain, followed by a carboxyl-terminal constant (CL) domain, which are separated from one another by another “switch”. The complete antibody tetramer contains two heavy chain-light chain dimers, where the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds link the heavy chain hinge regions to one another such that the dimers are linked to one another and form the tetramer. Naturally occurring antibodies are also typically glycosylated on the CH2 domain. Each domain in a native antibody has a structure characterized by an “immunoglobulin fold” formed by two β-sheets (e.g., 3-strand, 4-strand, or 5-strand folds) that stack against one another in a compact, antiparallel β-barrel. Each variable domain contains three hypervariable loops called “complementary determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). When a native antibody folds, the FR regions form β-sheets that provide a structural framework for the domain, and the CDR loop regions from the heavy and light chains come together in three-dimensional space such that they create a single hypervariable antigen-binding site at the apex of the Y structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and also binds to receptors on effector cells (including, for example, effector cells that mediate cytotoxicity). The affinity of the Fc region for Fc receptors and / or other binding properties can be modulated by glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized according to the invention (e.g., as components of chimeric switch receptors or CARs) include glycosylated Fc domains, including Fc domains having modified or engineered glycosylation.In some embodiments, any polypeptide or polypeptide complex comprising sufficient immunoglobulin domain sequences as found in natural antibodies can be referred to as and / or used as an "antibody", whether such polypeptide is naturally produced (e.g., generated by an organism in response to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methods. 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. Additionally, as used herein, the term "antibody" in appropriate embodiments (unless otherwise stated or clear from the context) can refer to any construct or form known or developed in the art that utilizes the structural and functional features of an antibody in an alternative presentation. For example, in some embodiments, the antibodies utilized according to the present invention are forms selected from, but not limited to, the following: intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or collections thereof; single-chain Fv; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies, such as IgNAR or fragments thereof); cameloid antibodies; masking antibodies (e.g., ); Small Modular ImmunoPharmaceuticals ("SMIPs TM "); single-chain or tandem diabodies ( ); VHH; minibodies; ankyrin repeat proteins or DART; TCR-like antibodies; A Trans- microproteins; and In some embodiments, the antibody may lack covalent modifications (e.g., attachment of glycans) that it would have when naturally produced. In some embodiments, the antibody may contain covalent modifications (e.g., attachment of glycans, payloads [e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.] or other side groups [e.g., polyethylene glycol, etc.].

[0070] Antibody agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, the antibody agent may comprise one or more constant region sequences having the characteristics of murine, rabbit, primate, or human antibodies. In some embodiments, the antibody agent may include one or more 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 of the constructs or forms known or developed in the art that utilize antibody structural and functional characteristics in alternative presentations. For example, in some embodiments, the antibody agents utilized in accordance with the present invention are forms selected from, but not limited to, the following: intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or collections thereof; single-chain Fv; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies, such as IgNAR or fragments thereof); cameloid antibodies; masking antibodies (e.g., ); Small Modular ImmunoPharmaceuticals ("SMIPsTM"); single-chain or tandem diabodies ( ); VHH; minibodies; ankyrin repeat proteins or DART; TCR-like antibodies; Trans- microproteins; F and In some embodiments, the antibody agent may lack covalent modifications (e.g., attachment of glycans) that would be present when produced naturally. In some embodiments, the antibody agent may contain covalent modifications (such as attachment of glycans, payloads [such as detectable moieties, therapeutic moieties, catalytic moieties, etc.] or other side groups [such as polyethylene glycol, etc.]). In many embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence comprises one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs); in some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence comprises at least one CDR (such as at least one heavy-chain CDR and / or at least one light-chain CDR) that is substantially identical to a CDR found in a reference antibody. In some embodiments, the included CDR is substantially identical to the reference CDR because it is identical in sequence or contains 1-5 amino acid substitutions compared to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR because it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR because it exhibits at least 96%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR because at least one amino acid within the included CDR has been deleted, added or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR because 1-5 amino acids within the included CDR have been deleted, added or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR because at least one amino acid within the included CDR has been substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR because 1-5 amino acids within the included CDR have been deleted, added or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence comprises structural elements recognized by those skilled in the art as immunoglobulin variable domains. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain.In some embodiments, the antibody agent is not and / or does not comprise a polypeptide whose amino acid sequence includes structural elements that are recognized by those skilled in the art as immunoglobulin variable domains. In some embodiments, the antibody agent can be or comprise 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).

[0071] Antibody fragment: As used herein, the term "antibody fragment" refers to a portion of a full-length antibody and refers to the antigen-determining variable regions of a full-length 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 their human and humanized forms.

[0072] Antibody heavy chain: As used herein, the term "antibody heavy chain" refers to the larger of the two types of polypeptide chains that are present in all antibody molecules in their naturally occurring conformation.

[0073] Antibody light chain: As used herein, the term "antibody light chain" refers to the smaller of the two types of polypeptide chains that are present in all antibody molecules in their naturally occurring conformation.

[0074] Synthetic antibody: As used herein, the term "synthetic antibody" refers to an antibody generated using recombinant DNA techniques, such as an antibody expressed by a phage as described herein. The term should also be construed to mean an antibody generated by synthesizing a DNA molecule encoding the antibody (the DNA molecule expressing the antibody protein) or specifying the amino acid sequence of the antibody, wherein the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0075] Antigen: As used herein, the term "antigen" or "Ag" refers to a molecule that is capable of eliciting an immune response. Such an immune response can involve antibody production, activation of specific immunocompetent cells, or both. Those skilled in the art will understand that any macromolecule, including substantially all proteins or peptides, can be used as an antigen. In addition, an antigen can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA that contains a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen" as used herein. In addition, those skilled in the art will understand that an antigen does not need to be encoded solely by the full-length nucleotide sequence of a gene. It is obvious that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in various combinations to elicit the desired immune response. In addition, those skilled in the art will understand that an antigen does not need to be encoded by a "gene" at all. It is obvious that an antigen can be synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to, a tissue sample, a tumor sample, a cell, or a biological fluid.

[0076] Antitumor effect: As used herein, the term "antitumor effect" refers to a biological effect that can be manifested as a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancerous disorder. The "antitumor effect" can also be manifested as the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to prevent the occurrence of tumors in the first place.

[0077] Autologous: As used herein, the term "autologous" refers to any material derived from an individual that is subsequently reintroduced into the same individual.

[0078] Allogeneic: As used herein, the term "allogeneic" refers to any material (e.g., a cell population) derived from different animals of the same species.

[0079] Xenogeneic: As used herein, the term "xenogeneic" refers to any material (e.g., a cell population) derived from animals of different species.

[0080] 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 through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. In certain embodiments, the cancer is medullary thyroid cancer.

[0081] Conservative sequence modifications: As used herein, the term "conservative sequence modification" refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody in a manner compatible with the various embodiments by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are amino acid substitutions 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 having the following: 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Accordingly, one or more amino acid residues within the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family, and the ability of the altered antibody to bind antigen can be tested using the functional assays described herein.

[0082] Co-stimulatory ligands: As used herein, the term "co-stimulatory ligand" refers to a molecule on an antigen-presenting cell (e.g., APC, dendritic cell, B cell, etc.) that specifically binds to a cognate co-stimulatory molecule on a monocyte / macrophage / dendritic cell, thereby providing a signal that mediates a response by the monocyte / macrophage / dendritic cell, including but not limited to proliferation, activation, differentiation, etc. Co-stimulatory ligands can include but are not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds to the Toll ligand receptor, and a ligand that specifically binds to B7-H3. Co-stimulatory ligands also particularly encompass antibodies that specifically bind to co-stimulatory molecules present on a monocyte / macrophage / dendritic cell such as but not limited 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 a ligand that specifically binds to CD83.

[0083] Cytotoxicity: As used herein, the term "cytotoxic" or "cytotoxicity" refers to killing or destroying cells. In one embodiment, the cytotoxicity of metabolically enhanced cells is improved, such as an increase in the lytic activity of macrophages.

[0084] Effective amount: As used herein, "effective amount" and "therapeutically effective amount" are used interchangeably and refer to the amount of a compound, formulation, material, or composition that is effective to achieve a particular biological result or provide a manufacturing, therapeutic, or prophylactic benefit as described herein. Such results can include, but are not limited to, anti-tumor activity as determined by any suitable means in the art.

[0085] Effector function: As used herein, "effector function" or "effector activity" refers to a specific activity performed by an immune cell in response to stimulation of the immune cell. For example, the effector function of macrophages to engulf and digest cell debris, foreign substances, microorganisms, cancer cells, and other unhealthy cells by phagocytosis.

[0086] Encoding: As used herein, "encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) to serve as a template in biological processes for the synthesis of other polymers and macromolecules having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the resulting biological properties. Thus, if transcription and translation of the mRNA corresponding to a gene produce a protein in a cell or other biological system, the gene encodes that protein. Both the coding strand, which has the same nucleotide sequence as the mRNA sequence and is typically provided in the sequence listing, and the non-coding strand, which serves as the transcription template for the gene or cDNA, can be said to encode the protein or other product of the gene or cDNA.

[0087] Endogenous: As used herein, "endogenous" refers to any material that is derived from or produced within a particular organism, cell, tissue, or system.

[0088] Exogenous: As used herein, the term "exogenous" refers to any material that is introduced from or produced outside of a particular organism, cell, tissue, or system.

[0089] Amplification: As used herein, the term "amplification" refers to an increase in quantity, such as an increase in the number of cells (e.g., monocytes, macrophages, and / or dendritic cells). In one embodiment, the number of in vitro amplified monocytes, macrophages, or dendritic cells is increased relative to the number initially present in the culture. In another embodiment, the in vitro amplified monocytes, macrophages, or dendritic cells are increased in number relative to other cell types in the culture. In some embodiments, amplification can occur in vivo. The term "in vitro" as used herein refers to cells that have been removed from a living organism (e.g., a human) and propagated outside the body (e.g., in a culture dish, test tube, or bioreactor).

[0090] Expression: As used herein, the "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, the expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of 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.

[0091] Expression vector: As used herein, the term "expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. The 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 those expression 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).

[0092] Fragment: As used herein, the term "fragment" or "portion" refers to a structure that includes discrete portions of a whole but lacks one or more portions found in the whole structure. In some embodiments, the fragment consists of such discrete portions. In some embodiments, the fragment consists of or comprises characteristic structural elements or portions 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 monomer units (e.g., nucleic acids) found in the whole nucleotide. In some embodiments, a nucleotide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomer units (e.g., residues) found in the whole. In some embodiments, the whole material or entity may be referred to as the "parent" of the whole.

[0093] Homology: As used herein, the term "homology" refers to the overall relatedness between polymeric molecules (e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules). In some embodiments, polymeric molecules are considered to be "homologous" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical. In some embodiments, polymeric molecules are considered to be "homologous" to each other 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, a variety of algorithms can be utilized to compare sequences to determine the degree of their homology, including allowing for gaps of a specified length in one sequence relative to another when considering which residues in different sequences "correspond" to each other. For example, the calculation of the percentage of homology between two nucleic acid sequences can be carried out by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second nucleic acid sequences to achieve optimal alignment, and non-corresponding sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or substantially 100% of the length of the reference sequence. The nucleotides at the corresponding nucleotide positions are then compared. When the position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at this position; when the position in the first sequence is occupied by a nucleotide similar to the corresponding position in the second sequence, the molecules are similar at this position. Taking into account the number of gaps and the length of each gap (which are introduced to achieve optimal alignment of the two sequences), the percentage of homology between the two sequences is a function of the number of identical and similar positions shared by these sequences.

[0094] Identity: As used herein, the term "identity" refers to the subunit sequence identity between two polymer molecules, particularly between two amino acid molecules such as between two polypeptide molecules. Two amino acid sequences are identical at a position when they have the same residue at that position; for example, if the position in each of 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 typically expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half of the positions in two sequences (e.g., five positions in a polymer ten amino acids in length) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., nine out of ten) match or are identical, the two amino acid sequences are 90% identical.

[0095] 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 one of ordinary skill in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues in corresponding positions. As is well known in the art, any of a variety of algorithms (including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gap BLAST, and PSI-BLAST for amino acid sequences) can be used to compare amino acid or nucleic acid sequences. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over a relevant segment of residues. In some embodiments, the relevant segment is the complete sequence. In some embodiments, the relevant segment 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" generally means 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 a reference CDR.

[0096] Immune cells: As used herein, the term "immune cell" refers to a cell that participates in (e.g., promotes) 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 the immune cells (e.g., macrophages, monocytes, or dendritic cells) can be obtained from a subject.

[0097] Immune response: As used herein, the term "immune response" refers to the cellular and / or systemic response to an antigen that occurs when lymphocytes recognize an antigen molecule as foreign and induce the formation of antibodies and / or activate lymphocytes to remove the antigen.

[0098] Immunoglobulin: As used herein, the term "immunoglobulin" or "Ig" refers to a class of proteins that act as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is a primary antibody present in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and urogenital 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 effective immunoglobulin in terms of agglutination, complement fixation, and other antibody responses and is important in defense against bacteria and viruses. IgD is an immunoglobulin that does not have a known antibody function but can be used as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity reactions by causing the release of mediators from mast cells and basophils upon exposure to an allergen.

[0099] Isolated: As used herein, the term "isolated" refers to a substance that has been changed or removed from its natural state. For example, a nucleic acid or peptide that occurs naturally in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the substances with which it coexists in its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-natural environment such as, for example, a host cell.

[0100] Lentivirus: As used herein, the term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are able to infect non-dividing cells, which is unique among retroviruses; they can deliver large amounts of genetic information into the DNA of host cells and are thus one of the most effective methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0101] 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 introducing nucleic acids.

[0102] Modulate: As used herein, the term "modulate" refers to mediating a detectable increase or decrease in the level of response and / or an alteration in the nature of the response in a subject as compared to the level of response and / or nature of the response in a subject that has not received treatment or a compound and / or as compared to the level of response and / or nature of the response in a subject that is otherwise identical but untreated. The term encompasses disrupting and / or affecting a natural signal or response such that a beneficial therapeutic response is mediated in a subject, preferably a human.

[0103] Nucleic acid: As used herein, the term "nucleic acid" refers to a polymer of at least three nucleotides. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises RNA. In some embodiments, the nucleic acid is single-stranded. In some embodiments, the nucleic acid is double-stranded. In some embodiments, the nucleic acid contains both single-stranded and double-stranded portions. In some embodiments, the nucleic acid contains a backbone comprising one or more phosphodiester linkages. In some embodiments, the nucleic acid contains a backbone comprising phosphodiester linkages and non-phosphodiester linkages. For example, in some embodiments, the nucleic acid may contain a backbone comprising one or more phosphorothioate or 5'-N-phosphoramidite linkages and / or one or more peptide bonds, such as, for example, in "peptide nucleic acid". In some embodiments, the nucleic acid contains one or more or all natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, the nucleic acid contains one or more or all non-natural residues. In some embodiments, the non-natural residues include nucleoside analogs (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 residues contain one or more modified sugars compared to those sugars in natural residues (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose). In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product such as RNA or a polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence comprising one or more introns. In some embodiments, the nucleic acid can be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, such as in vivo or in vitro), replication in a recombinant cell or system, or chemical synthesis.In some embodiments, the nucleic acid has a length of at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues.

[0104] Operably linked: As used herein, the term "operably linked" refers to, for example, a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence such that the latter is expressed. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the two are in a functional relationship. 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 and, where necessary to join two protein-coding regions, are in the same reading frame.

[0105] Overexpressed tumor antigen: As used herein, the term "overexpressed" tumor antigen or "overexpression" of a tumor antigen refers to an abnormal level of expression of a tumor antigen in cells from a diseased area such as a solid tumor within a tissue or organ, relative to the level of expression in normal cells from the same tissue or organ of a patient. Patients having a solid tumor or hematological malignancy characterized by overexpression of a tumor antigen can be identified by standard assays known in the art.

[0106] Polynucleotide: As used herein, the term "polynucleotide" refers to a chain of nucleotides. In addition, a nucleic acid is a polymer of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. Those of ordinary skill in the art have the general knowledge that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including but not limited to recombinant means (i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR TM etc.) as well as by synthetic means.

[0107] Polypeptide: As used herein, the term "polypeptide" refers to any polymeric chain of residues (e.g., amino acids) that are typically linked by peptide bonds. In some embodiments, the polypeptide has an amino acid sequence that occurs in nature. In some embodiments, the polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, the polypeptide has an engineered amino acid sequence as it is designed and / or produced through the action of man. In some embodiments, the polypeptide may comprise natural amino acids, unnatural amino acids, or both, or consist of these amino acids. In some embodiments, the polypeptide may comprise only natural amino acids or only unnatural amino acids, or consist of only these amino acids. In some embodiments, the polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, the polypeptide may comprise only D-amino acids. In some embodiments, the polypeptide may comprise only L-amino acids. In some embodiments, the polypeptide may comprise one or more side groups or other modifications, such as modifications at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or attached to one or more amino acid side chains, or any combination thereof. In some embodiments, such side groups or modifications are optionally selected from the group consisting of acetylation, amidation, lipidation, methylation, polyethylene glycolylation, etc., including combinations thereof. In some embodiments, the polypeptide may be cyclic and / or may comprise a cyclic moiety. In some embodiments, the polypeptide is not cyclic and / or does not comprise any cyclic moiety. In some embodiments, the polypeptide is linear. In some embodiments, the 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; in such cases, it is used herein to refer to a polypeptide that shares the relevant activity or structure and is thus considered a member of the same class or family of polypeptides. For each such class, the present specification provides and / or those skilled in the art will know exemplary polypeptides within the class, whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are the reference polypeptides of the polypeptide class or family. In some embodiments, members of a polypeptide class or family exhibit significant sequence homology or identity with the reference polypeptide of the class (in some embodiments, with all polypeptides within the class), share a common sequence motif (e.g., a characteristic sequence element), and / or share a common activity (in some embodiments, at a comparable level or within a specified range).For example, in some embodiments, a member polypeptide shows an overall sequence homology or identity degree of at least about 30-40% and typically greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more with a reference polypeptide, and / or includes at least one region showing a very high sequence identity (usually greater than 90% or even 95%, 96%, 97%, 98% or 99%) (e.g., a conserved region that can be or include a characteristic sequence element in some embodiments). Such conserved regions typically span at least 3-4 and usually at most 20 or more amino acids; in some embodiments, the conserved region spans at least a 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, useful polypeptides can comprise or consist of fragments of a parent polypeptide. In some embodiments, useful polypeptides can comprise or consist of multiple fragments, each fragment being found in the same parent polypeptide in a different spatial arrangement relative to each other than that found in the target polypeptide (e.g., fragments that are directly linked in the parent can be spatially separated in the target polypeptide and vice versa, and / or the fragments can be present in the target polypeptide in a different order than in the parent), such that the target polypeptide is a derivative of its parent polypeptide.

[0108] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a string of at least two amino acids linked to each other by peptide bonds). A protein can include moieties other than amino acids (e.g., it can be a glycoprotein, a proteoglycan, etc.) and / or can be otherwise processed or modified. One of ordinary skill in the art will understand that a "protein" can be a complete polypeptide chain produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. One of ordinary skill in the art will appreciate that a protein can sometimes include, for example, more than one polypeptide chain linked by one or more disulfide bonds or associated by other means. A polypeptide can contain L-amino acids, D-amino acids, or both, and can contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, a protein can comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, a protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0109] Signal transduction pathway: As used herein, the term "signal transduction pathway" refers to the biochemical relationships among multiple signal transduction molecules that function in the transfer of a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and molecular complexes capable of receiving a signal and transmitting the signal across the plasma membrane of the cell.

[0110] 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 joined to the Fv region via engineered amino acid segments. Various methods for generating single-chain antibodies are known, including those described in the following references: 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 334:544-545; Skerra et al. (1988) Science 242:1038-1041.

[0111] 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 substantially does not recognize or bind other molecules in a sample. For example, an antigen-binding domain or antibody agent that specifically binds to an antigen from one species may also bind to an antigen from one or more species. However, such cross-species reactivity by itself does not change the classification of the antigen-binding domain or antibody reagent 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 by itself does not change the classification of the antigen-binding domain or antibody agent as specific. In some cases, the terms "specifically bind" or "specific binding" may be used in reference to the interaction of an antigen-binding domain or antibody agent, protein, or peptide with a second chemical species, meaning that the interaction depends on the presence of a specific 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 a general protein. If an antigen-binding domain or antibody agent is specific for epitope "A", then in a reaction containing labeled "A" and the antigen-binding domain or antibody agent, the presence of molecules containing epitope A (or free unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0112] Stimulation: As used herein, the term "stimulation" refers to a primary response caused by a stimulating molecule (e.g., an FcR complex, a TLR complex, or a TCR / CD3 complex) binding to its cognate ligand, for example, thereby mediating signal transduction events (such as, but not limited to, signal transduction via the Fc receptor mechanism, via a chimeric switch receptor, or via a synthetic CAR). Stimulation can mediate certain molecular expression changes (such as downregulation of TGF-β) and / or reorganization of the cytoskeletal structure, etc. As used herein, the term "stimulating molecule" refers to a molecule of a monocyte, macrophage, or dendritic cell that specifically binds to a cognate stimulating ligand present on an antigen-presenting cell. In some embodiments, the stimulating molecule comprises an FcR extracellular domain that comprises a CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c, CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, FcαRI (CD89), or CD40 domain. In some embodiments, the stimulating molecule comprises a TLR extracellular domain that comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain. As used herein, the term "stimulating ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., an aAPC, macrophage, dendritic cell, B cell, etc.) or a tumor cell, can specifically bind to a cognate binding partner (referred to herein as a "stimulating molecule") on a monocyte, macrophage, or dendritic cell, thereby mediating a response of the immune cell (including, but not limited to, activation, initiation of an immune response, proliferation, etc.). Stimulating ligands are well known in the art and particularly encompass Toll-like receptor (TLR) ligands, anti-Toll-like receptor antibodies, agonists, and antibodies to monocyte / macrophage receptors. Additionally, cytokines such as interferon-γ are effective macrophage stimulants.

[0113] Subject: As used herein, the term "subject" refers to an organism, such as a mammal (e.g., a human, non-human mammal, non-human primate, primate, laboratory animal, mouse, rat, hamster, gerbil, cat, or dog). In some embodiments, the human subject is an adult, adolescent, or pediatric subject. In some embodiments, the subject has a disease, disorder, or condition, such as a disease, disorder, or condition that can be treated as provided herein, such as cancer or tumor listed herein. In some embodiments, the subject is susceptible to a disease, disorder, or condition; in some embodiments, the susceptible subject tends to and / or shows an increased risk of developing a disease, disorder, or condition (compared to the average risk observed in a reference subject or population). 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 specific symptom (e.g., the clinical manifestation of a disease) or feature of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or features of a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom a diagnosis and / or therapy has been administered and / or has been administered.

[0114] Substantially purified: As used herein, the term "substantially purified" when applied to cells, for example, refers to cells that are substantially 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 natural state. In some cases, a substantially purified cell population refers to a homogeneous cell population. In other cases, the term only refers to cells that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0115] Target: As used herein, the term "target" refers to a cell, tissue, organ, or site in the body that is the object of the provided methods, systems, and / or compositions, such as a cell, tissue, organ, or site in the body that needs treatment or is preferentially bound by, for example, an antibody (or fragment thereof), a chimeric switch receptor, or a CAR.

[0116] Target site: As used herein, the term "target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient to effect binding.

[0117] T cell receptor: As used herein, the term "T cell receptor" or "TCR" refers to a membrane protein complex that participates in activating T cells in response to the presentation of an antigen. The TCR is responsible for recognizing an antigen that binds to a major histocompatibility complex molecule. The TCR comprises a heterodimer of alpha (α) and beta (β) chains, although in some cells, the TCR comprises gamma and delta (γ / δ) chains. The TCR can exist in α / β and γ / δ forms, which are structurally similar but have different anatomical locations and functions. Each chain contains two extracellular domains, a variable and a constant domain. In some embodiments, the TCR can be modified on any cell that contains the TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and γδ T cells.

[0118] Therapeutic: As used herein, the term "therapeutic" refers to treatment and / or prevention. A therapeutic effect is obtained by inhibiting, alleviating, or eradicating a disease state.

[0119] Transfected: As used herein, the term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0120] Treat: As used herein, the term "treat (treat, treatment, treating)" refers to the partial or complete alleviation, amelioration, delay in the onset, inhibition, prevention, mitigation, and / or reduction in the incidence and / or severity of one or more symptoms or characteristics of a disease, disorder, and / or affliction. In some embodiments, treatment can be administered to a subject who does not exhibit signs or characteristics of a disease, disorder, and / or affliction (e.g., it can be prophylactic). In some embodiments, treatment can be administered to a subject who exhibits only early or mild signs or characteristics of a disease, disorder, and / or affliction, for example, for the purpose of reducing the risk of developing a pathology associated with the disease, disorder, and / or affliction. In some embodiments, treatment can be administered to a subject who exhibits established, severe, and / or advanced signs of a disease, disorder, or affliction. In some embodiments, treatment can include administering to or contacting an immune cell (e.g., a monocyte, macrophage, or dendritic cell) with a modulator of a pathway activated by in vitro transcribed mRNA.

[0121] Tumor: As used herein, the term "tumor" refers to an abnormal growth of cells or tissues. In some embodiments, a tumor can comprise pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic cells. In some embodiments, a tumor is associated with or is a manifestation of cancer. In some embodiments, a tumor can be a diffuse tumor or a liquid tumor. In some embodiments, a tumor can be a solid tumor.

[0122] Vector: As used herein, the term "vector" refers to a composition of matter that contains a nucleic acid isolated and can be used to deliver the isolated nucleic acid into a cell interior. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphoteric compounds, plasmids, and viruses. Thus, the term "vector" includes self-replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

[0123] Throughout this disclosure, various aspects of the 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 imposing a rigid limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as the individual values within that range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges (such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc.) and the individual values within that range (e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6). This applies regardless of the width of the range. Detailed Description

[0124] This disclosure particularly encompasses compositions comprising modified immune cells (e.g., stem cells, macrophages, monocytes, and / or dendritic cells) comprising the novel chimeric antigen receptors (CARs) described herein, and methods of producing and using such compositions. This disclosure particularly also encompasses 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, and methods of using and producing such compositions. In some embodiments, the CARs of this disclosure comprise an anti-mesothelin antigen-binding domain as described herein. In some embodiments, the CARs of this disclosure comprise one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain.

[0125] In some embodiments, the modified immune cells described herein that comprise or express a CAR as described herein exhibit increased tumor killing as compared to the same type of modified immune cells that comprise a similar CAR (e.g., that comprises different anti-mesothelin antigen-binding domains and / or that does not comprise one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but that has the other components of the comparator CAR). In some embodiments, the modified immune cells described herein that comprise or express a CAR as described herein do not exhibit killing of tumor cells that do not express the target antigen (e.g., mesothelin) as compared to the same type of modified immune cells that comprise a similar CAR (e.g., that comprises different anti-mesothelin antigen-binding domains and / or that does not comprise one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but that has the other components of the comparator CAR). In some embodiments, tumor killing comprises or is one or both 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 as described herein exhibit increased tumor killing over a particular length of time. In some embodiments, the modified immune cells described herein that comprise or express a CAR as 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 as described herein exhibit increased tumor killing for at least two weeks.

[0126] In some embodiments, the modified immune cells described herein that comprise or express a CAR as described herein exhibit increased viability as compared to the same type of modified immune cells that comprise a similar CAR (e.g., that comprises different anti-mesothelin antigen-binding domains and / or that does not comprise one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but that has the other components of the comparator CAR). In some embodiments, the modified immune cells described herein that comprise or express a CAR as described herein exhibit increased CAR expression as compared to the same type of modified immune cells that comprise a similar CAR (e.g., that comprises different anti-mesothelin antigen-binding domains and / or that does not comprise one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but that has the other components of the comparator CAR).

[0127] In some embodiments, the modified immune cells described herein that comprise or express a CAR as described herein exhibit increased expression of M1 markers (such as one or both of CD80 or CD86), as compared to the same type of modified immune cells that comprise a similar CAR (such as 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 has the other components of the comparator CAR). In some embodiments, the modified immune cells described herein that comprise or express a CAR as described herein exhibit decreased expression of M2 markers (such as one or both of CD163 or CD206), as compared to the same type of modified immune cells that comprise a similar CAR (such as 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 has the other components of the comparator CAR).

[0128] In some embodiments, the CARs described herein include: (a) an extracellular domain that includes an anti-mesothelin binding domain as described herein; (b) a transmembrane domain (such as 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 include a CD3 zeta (CD3ζ) intracellular domain. In some embodiments, the one or more intracellular domains include an FcRγ intracellular domain. In some embodiments, the CAR further includes one or more extracellular hinge domains. In some embodiments, the one or more extracellular hinge domains include a CD28 extracellular hinge domain or a CD8a extracellular hinge domain.

[0129] In some embodiments, the CARs described herein comprise, from the N-terminus to the C-terminus: a CD8a leader domain, a mesothelin antigen-binding domain, a CD8 extracellular hinge domain, a CD8 transmembrane domain, and a CD3ζ intracellular domain. In some embodiments, the CARs described herein comprise, from the N-terminus to the C-terminus: a CD8a leader domain, a mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and a CD3ζ intracellular domain. In some embodiments, the CARs described herein comprise, from the N-terminus to the C-terminus: a CD8a leader domain, a mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and an FcRγ intracellular domain. In some embodiments, the CARs described herein comprise, from the N-terminus to the C-terminus: a CD8a leader domain, a 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, the CARs described herein comprise, from the N-terminus to the C-terminus: a CD8a leader domain, a 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, the CARs described herein comprise, from the N-terminus to the C-terminus: a CD8a leader domain, a 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, the CARs described herein comprise, from the N-terminus to the C-terminus: a CD8a leader domain, a 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, the CARs described herein comprise, from the N-terminus to the C-terminus: a CD8a leader domain, a 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, the CARs described herein comprise, from the N-terminus to the C-terminus: a CD8a leader domain, a 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, the CARs described herein comprise, from the N-terminus to the C-terminus: a CD8a leader domain, a 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, the CARs described herein include, from the N-terminus to the C-terminus: a CD8a leader domain, a 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, the CARs described herein include, from the N-terminus to the C-terminus: a CD8a leader domain, a 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.

[0130] immune cell

[0131] The present disclosure particularly provides modified immune cells (such as, stem cells, macrophages, monocytes, or dendritic cells) comprising at least one chimeric antigen receptor (CAR) described herein. In some embodiments, the immune cell populations described herein include stem cells, monocytes, macrophages, dendritic cells, and / or their precursors. In some embodiments, the immune cell population includes a substantially purified population or cell line of stem cells, monocytes, macrophages, or dendritic cells.

[0132] In some embodiments, the immune cells are activated, for example, the immune cells exhibit increased cytokine production, chemokine production, phagocytosis, cell signaling, target cell killing, and / or antigen presentation as compared to inactive cells. In some embodiments, the activated immune cells exhibit altered gene expression, such as induction of pro-inflammatory gene expression, as compared to inactive cells. In some embodiments, the activated immune cells exhibit altered gene expression, such as induction of anti-inflammatory gene expression, as compared to inactive cells. In certain embodiments, the activated immune cells are undergoing cell division. In some embodiments, the targeted effector activity of immune cells is enhanced by inhibiting CD47 and / or SIRPα activity. CD47 and / or SIRPα activity can be inhibited by treating the immune cells with anti-CD47 or anti-SIRPα antibodies or by any method known to those skilled in the art.

[0133] In some embodiments, immune cells (such as stem cells, macrophages, monocytes, or dendritic cells) are obtained (e.g., isolated) from a subject. The cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, tumor, and / or induced pluripotent stem cells, such as embryonic stem cells (ESC). In certain embodiments, any number of isolation techniques known to those of skill in the art, such as Ficoll separation, can be used to obtain cells from a unit of blood collected from a subject. In some embodiments, cells from the circulating blood of a subject are obtained by apheresis or leukapheresis. The cells collected by apheresis can be washed to remove the plasma fraction and resuspended in a variety of buffers (e.g., phosphate buffered saline (PBS) or media). In some embodiments, enrichment of immune cells (such as monocytes) includes plastic adherence. In some embodiments, after enrichment, differentiation of immune cells (such as monocytes) includes stimulation with GM-CSF. In some embodiments, a composition comprising blood cells (such as monocytes, lymphocytes, platelets, plasma, and / or red blood cells), such as a leukapheresis composition (e.g., leukopak), is used for enrichment. In some embodiments, the leukapheresis composition (e.g., leukopak) contains a sample from a healthy human donor. In certain embodiments, immune cells (such as monocytes) are mobilized with GM-CSF after apheresis. In certain embodiments, selection of immune cells (such as monocytes) includes using beads (such as the beads) on a CliniMACS Prodigy device for CD14 positive selection. In some embodiments, immune cell precursors (such as precursors of macrophages, monocytes, or dendritic cells, including but not limited to induced pluripotent stem cells or iPSC) are used in the compositions and methods described herein. Immune cell precursors can differentiate into immune cells in vivo or ex vivo. Non-limiting examples of precursor immune cells include hematopoietic stem cells, common myeloid progenitors, myeloblasts, monoblasts, promonocytes, or intermediates thereof. For example, induced pluripotent stem cells can be used to generate monocytes, macrophages, and / or dendritic cells. Induced pluripotent stem cells (iPSC) can be derived from normal human tissues, such as peripheral blood, fibroblasts, skin, keratinocytes, or renal epithelial cells. Autologous, allogeneic, or universal donor iPSC can differentiate into the myeloid lineage (such as monocytes, macrophages, dendritic cells, or precursors thereof).

[0134] Lysis of red blood cells and depletion of lymphocytes and red blood cells can be performed, for example, via PERCOLL TMThe immune cells described herein (e.g., stem cells, macrophages, monocytes, or dendritic cells) are isolated from peripheral blood by gradient centrifugation. Alternatively, immune cells can be isolated from umbilical cord tissue. Specific immune cell subsets can be further isolated by positive or negative selection techniques. In some embodiments, cells expressing certain antigens can be depleted from the immune cells, including but not limited to CD34, CD3, CD4, CD8, CD56, CD66b, CD19, or CD20. In some embodiments, enrichment of the immune cell population (e.g., by negative selection) can be achieved using a combination of antibodies against cell-specific surface markers for negative selection. By way of non-limiting example, cell selection can also include negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies against cell surface markers present on the cells for negative selection.

[0135] During the isolation of the desired immune cell population (e.g., stem cells, macrophages, monocytes, or dendritic cells) by positive or negative selection as described herein, the immune cell concentration and surface (e.g., particles such as beads) can be altered. It may be desirable to significantly reduce the volume in which the beads and cells are mixed together to ensure maximum contact area between the cells and the beads.

[0136] 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 as 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 transition from an M2 to an M1 phenotype) in the modified immune cells described herein. In some embodiments, at least one pro-inflammatory agent comprises or is a CD40 agonist (e.g., CD40L). In some embodiments, at least one pro-inflammatory agent comprises or is a 41BB-ligand agonist (e.g., 4-1BB). In some embodiments, at least one pro-inflammatory agent comprises or is a CD40 agonist (e.g., CD40L) and a 41BB-ligand agonist (e.g., 4-1BB).

[0137] In some embodiments, the modified immune cells (e.g., stem cells, macrophages, monocytes or dendritic cells) described herein (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 relative to unmodified cells of the same type. In some embodiments, one or more pro-inflammatory agents comprise or are a CD40 agonist (e.g., CD40L). In some embodiments, one or more pro-inflammatory agents comprise or are a 41BB-ligand agonist (e.g., 4-1BB). In some embodiments, one or more pro-inflammatory agents comprise 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, comprising: delivering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified macrophages, monocytes or dendritic cells described herein.

[0138] The present disclosure also provides methods of modifying immune cells (e.g., stem cells, macrophages, monocytes or dendritic cells) described herein that comprise a CAR described herein, wherein the method comprises treating the immune cells described herein with one or more pro-inflammatory agents, thereby producing the modified immune cells described herein, which exhibit increased anti-tumor activity relative to the same type of immune cells that comprise a CAR or a similar CAR that has not been treated with one or more pro-inflammatory agents. In some embodiments, one or more pro-inflammatory agents comprise or are a CD40 agonist (e.g., CD40L). In some embodiments, one or more pro-inflammatory agents comprise or are a 41BB-ligand agonist (e.g., 4-1BB). In some embodiments, one or more pro-inflammatory agents comprise 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, comprising: delivering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the immune cells described herein modified by the methods described herein.

[0139] 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 a pro-inflammatory agent. In some embodiments, the pro-inflammatory agent is administered as a nucleic acid (e.g., in a construct packaged with a CAR and a cleavage 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 in the immune cells described herein (e.g., a transition from an M2 to an M1 phenotype). In some embodiments, the pro-inflammatory agent comprises or is a CD40 agonist (e.g., CD40L). In some embodiments, the pro-inflammatory agent comprises or is a 41BB-ligand agonist (e.g., 4-1BB).

[0140] Macrophages

[0141] Macrophages are immune cells specialized for detecting, engulfing, and destroying target cells such as pathogens or tumor cells. Macrophages are potent effectors of the innate immune system and can have at least three different anti-tumor functions: 1) phagocytosis of dead and dying cells, microorganisms, cancer cells, cell debris, or other foreign substances; 2) cytotoxicity against tumor cells; and 3) presentation of tumor antigens to coordinate an adaptive anti-tumor immune response.

[0142] Macrophages are abundant in the tumor microenvironment of many cancers and can adopt multiple 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 leads to a general suppression of the anti-tumor immune response. However, recent studies have found that TAMs can be "reprogrammed" via pro-inflammatory signals, and the transition from an M2 phenotype to a more M1 phenotype is associated with an effective anti-tumor immune response. Inducing endogenous TAMs to transform into M1-type cells and engineering macrophages that cannot be converted into M2 will greatly enhance anti-tumor immunotherapy and represent a significant advance in the field.

[0143] In some embodiments, the macrophages include 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. Exposure to various stimuli can induce polarization of M0 macrophages into several distinct populations, which can be identified by macrophage phenotypic markers, cytokine production, and / or chemokine secretion.

[0144] In some embodiments, the macrophages include or are polarized macrophages. Under classical activation conditions, M0 macrophages can be exposed to pro-inflammatory signals such as LPS, IFNγ, and 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. Exposure to other stimuli can polarize macrophages into distinct groups of "alternatively activated" or anti-inflammatory (i.e., M2) macrophages.

[0145] In some embodiments, the macrophages include or are pro-inflammatory (M1) macrophages. In some embodiments, the macrophages express one or more markers of pro-inflammatory (M1) macrophages (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of CD86, CD80, MHCII, IL-1R, TLR2, TLR4, iNOS, SOCS3, CD83, PD-L1, CD69, MHCI, CD64, CD32, CD16, IL1R, IFIT family members, or ISG family members).

[0146] In some embodiments, macrophages that comprise or express at least one CAR described herein secrete relatively high levels of one or more inflammatory cytokines (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of IL-1, TNF, IL-12, IL-18, IL-23, IFNα, IFNβ, IFNγ, IL-2, IL-6, IL-8, or IL33) or chemokines (e.g., one or two of CC or CXC chemokines) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the CXC chemokines; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 of the CC chemokines; e.g., one of the CX3C chemokines, e.g., one or two of the C chemokines), as compared to macrophages that comprise 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 contains other components of the comparator CAR). In some embodiments, macrophages that comprise or express at least one CAR described herein stimulate an immune response and / or inflammation, as compared to macrophages that comprise 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 contains other components of the comparator CAR).

[0147] In some embodiments, the macrophages include 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, relative to macrophages comprising a similar CAR (e.g., including different anti-mesothelin antigen-binding domains and / or not including one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparative CAR), macrophages comprising or expressing at least one CAR described herein reduce the immune response of a subject. 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, relative to macrophages comprising a similar CAR (e.g., the CARs described herein), 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).

[0148] In some embodiments, compared to control macrophages that do not comprise at least one CAR as provided herein and / or the same macrophages prior to delivery of at least one CAR described herein, the macrophages comprise at least one upregulated pro-inflammatory (M1) marker and / or at least one downregulated anti-inflammatory (M2) marker. 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.

[0149] In some embodiments, macrophages that comprise or express at least one CAR described herein exhibit increased phagocytosis, as compared to macrophages that comprise a similar CAR (e.g., that includes different anti-mesothelin antigen-binding domains and / or that does not include one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but that has other components of the comparator CAR). In some embodiments, macrophages that comprise or express at least one CAR described herein exhibit increased cytotoxicity against tumor cells, as compared to macrophages that comprise a similar CAR (e.g., that includes different anti-mesothelin antigen-binding domains and / or that does not include one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but that has other components of the comparator CAR). In some embodiments, macrophages that comprise or express at least one CAR described herein exhibit increased tumor antigen presentation (e.g., presentation after phagocytosis), as compared to macrophages that comprise a similar CAR (e.g., that includes different anti-mesothelin antigen-binding domains and / or that does not include one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but that has other components of the comparator CAR). In some embodiments, macrophages that comprise or express at least one CAR exhibit increased tumor killing (e.g., by phagocytosis, lysis, apoptosis, or production of tumor-killing cytokines (such as TNFα)), as compared to macrophages that comprise a similar CAR (e.g., that includes different anti-mesothelin antigen-binding domains and / or that does not include one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but that has other components of the comparator CAR).

[0150] In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit one or both of the following, as compared to macrophages comprising a similar CAR (e.g., including a different anti-mesothelin antigen-binding domain and / or lacking one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator CAR): increased expression of one or more genes typically associated with increased effector function (e.g., phagocytosis, targeted 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) or decreased expression of one or more genes typically associated with decreased effector function (e.g., phagocytosis, targeted cytotoxicity, antigen presentation, or cytokine secretion) (e.g., CD163, CD206, TGFβ, IL-10, and / or IL4). In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased ROS production, as compared to macrophages comprising a similar CAR (e.g., including a different anti-mesothelin antigen-binding domain and / or lacking one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator CAR).In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit (e.g., interferon signaling pathway, TH1 pathway, PTEN signaling, PI3K signaling, MTOR signaling, TLR signaling, CD40 signaling, 41BB signaling, 41BBL signaling, macrophage maturation signaling, dendritic cell maturation signaling, CD3-ζ 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) metabolic reprogramming as compared to macrophages comprising a similar CAR (e.g., including different anti-mesothelin antigen-binding domains and / or not including one or both of (i) a CD8 or CD28 extracellular hinge domain and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparative CAR).In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit induction of a cell survival mechanism, as compared to macrophages comprising a similar CAR (e.g., including a different anti-mesothelin antigen-binding domain and / or lacking one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator CAR). In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit induction of a cell death mechanism, as compared to macrophages comprising a similar CAR (e.g., including a different anti-mesothelin antigen-binding domain and / or lacking one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator CAR). In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit an increase in one, two, three, four, or five of increased resistance to phagocytic checkpoints, increased expression of chemokine receptors that aid trafficking, increased expression of chemokines that recruit other immune cells, increased expression of ECM-degrading enzymes (e.g., MMPs that degrade tumor ECM and / or exhibit antifibrotic activity), or increased proliferation, as compared to macrophages comprising a similar CAR (e.g., including a different anti-mesothelin antigen-binding domain and / or lacking one or both of (i) a CD8 or CD28 extracellular hinge domain and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator CAR). In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit one, two, three, or four of improved CAR expression duration, improved stability of the CAR on the cell surface, increased CAR expression levels, and / or reduced CAR background activity, as compared to macrophages comprising a similar CAR (e.g., including a different anti-mesothelin antigen-binding domain and / or lacking one or both of (i) a CD8 or CD28 extracellular hinge domain and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator CAR).

[0151] Monocytes

[0152] Monocytes are multipotent cells that circulate in the blood, bone marrow, and spleen and generally do not proliferate under steady-state conditions. The size of monocytes can vary significantly within a diameter range of about 10 - 30 μm. The nucleus-to-cytoplasm ratio of monocytes can range from about 2:1 to about 1:1. Typically, monocytes contain chemokine receptors and pathogen recognition receptors that mediate migration from the blood to tissues, such as during infection. Monocytes can produce inflammatory cytokines, uptake cells and / or toxic molecules, and differentiate into dendritic cells or macrophages.

[0153] In some embodiments, monocytes comprise or express one or more phenotypic markers. Exemplary phenotypic markers of human monocytes include, but are not limited to, 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, CDwl2lb, CDw123, CD127, CDw128, CDw131, CD147, CD155, CD156a, CD157, CD162, CD163, CD164, CD168, CD171, CD172a, CD180, CD206, CD131a1, CD213 2, CDw210, CD226, CD281, CD282, CD284, and CD286. Exemplary phenotypic markers of mouse monocytes 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, monocytes include CD14+CD16− monocytes, CD14+CD16+ monocytes, or CD14−CD16+ monocytes.

[0154] In some embodiments, monocytes differentiate into macrophages. In some embodiments, monocytes differentiate into dendritic cells (DCs). Monocytes can be differentiated into macrophages or DCs by any technique known in the art. For example, monocytes can be induced to differentiate into macrophages by macrophage colony-stimulating factor (M-CSF). Monocytes can be induced to differentiate into DCs by a combination of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4.

[0155] In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit an increase in the secretion of one or more cytokines (such as one, two, three, four, five, six, or seven of TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, or IL-1), as compared to monocytes comprising a similar CAR (such as 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 has other components of the comparator CAR). In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased phagocytosis, as compared to monocytes comprising a similar CAR (such as 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 has other components of the comparator CAR). In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit enhanced survival, as compared to monocytes comprising a similar CAR (such as 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 has other components of the comparator CAR). In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit enhanced differentiation into macrophages (such as M1 or M2 macrophages), as compared to monocytes comprising a similar CAR (such as 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 has other components of the comparator CAR). In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit enhanced differentiation into DCs (such as resident or migratory DCs and / or in lymphoid and non-lymphoid tissues), as compared to monocytes comprising a similar CAR (such as 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 has other components of the comparator CAR). In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased cytotoxicity against tumor cells, as compared to monocytes comprising a similar CAR (such as 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 has other components of the comparator CAR).In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased tumor antigen presentation (e.g., presentation after phagocytosis), as compared to monocytes comprising a similar CAR (e.g., including different anti-mesothelin antigen-binding domains and / or lacking one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator 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 such as TNFα), as compared to monocytes comprising a similar CAR (e.g., including different anti-mesothelin antigen-binding domains and / or lacking one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator CAR).

[0156] In some embodiments, such as relative to monocytes comprising a similar CAR (e.g., including different anti-mesothelin antigen-binding domains and / or not including one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator CAR), monocytes comprising or expressing at least one CAR described herein exhibit one or both of the following: increased expression of one or more genes normally associated with increased effector function (e.g., phagocytosis, targeted cytotoxicity, antigen presentation, or cytokine secretion) or decreased expression of one or more genes normally associated with decreased effector function (e.g., phagocytosis, targeted cytotoxicity, antigen presentation, or cytokine secretion). In some embodiments, such as relative to monocytes not having a CAR described herein, monocytes comprising or expressing at least one CAR described herein exhibit increased ROS production. In some embodiments, such as relative to monocytes comprising a similar CAR (e.g., including different anti-mesothelin antigen-binding domains and / or not including one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator CAR), monocytes comprising or expressing at least one CAR described herein exhibit metabolic reprogramming. In some embodiments, such as relative to monocytes comprising a similar CAR (e.g., including different anti-mesothelin antigen-binding domains and / or not including one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator CAR), monocytes comprising or expressing at least one CAR described herein exhibit induction of cell survival mechanisms. In some embodiments, such as relative to monocytes comprising a similar CAR (e.g., including different anti-mesothelin antigen-binding domains and / or not including one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator CAR), monocytes comprising or expressing at least one CAR described herein exhibit induction of cell death mechanisms. In some embodiments, such as relative to monocytes not having a CAR described herein, monocytes comprising or expressing at least one CAR described herein exhibit one, two, three, four, or five of increased resistance to phagocytic checkpoints, increased expression of chemokine receptors that aid trafficking, increased expression of chemokines that recruit other immune cells, increased expression of ECM-degrading enzymes (e.g., MMPs that degrade tumor ECM and / or exhibit antifibrotic activity), and / or increased proliferation.In some embodiments, monocytes comprising or expressing at least one CAR as described herein exhibit one, two, three, or four of an improved duration of CAR expression, an improved stability of the CAR on the cell surface, an increased CAR expression level, and / or a reduced CAR background activity, as compared to monocytes that do not have a CAR as described herein.

[0157] Dendritic cell

[0158] Dendritic cells (DCs) are specialized antigen-presenting cells of bone marrow origin that are involved in initiating immune responses and maintaining immune system tolerance to self-antigens. Dendritic cells can be found in lymphoid and non-lymphoid organs and are generally thought to be of lymphoid or myeloid lineage.

[0159] 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 portions of CD206, CD207, CD208, and CD209.

[0160] Immature DCs can be characterized by high antigen capture ability but relatively low T cell-stimulating ability. Inflammatory mediators promote DC maturation. Once DCs reach the mature stage, there are significant changes in characteristics as compared to immature DCs, such as a decrease in antigen capture ability and / or an increase in the ability to stimulate T cells. In some embodiments, the DCs include or are immature DCs. In other embodiments, the DCs include or are mature DCs.

[0161] Without wishing to be bound by theory, it is believed that modifying DC cells to incorporate or express at least one CAR as described herein enables mature DCs to exhibit both antigen capture ability and increased T cell stimulation, such as relative to DCs comprising a similar CAR (e.g., including a different anti-mesothelin antigen-binding domain and / or lacking one or both of (i) the CD8 or CD28 extracellular hinge domain, and (ii) the CD8 or CD28 transmembrane domain, but having other components of the comparator CAR). In some embodiments, such as relative to DCs comprising a similar CAR (e.g., including a different anti-mesothelin antigen-binding domain and / or lacking one or both of (i) the CD8 or CD28 extracellular hinge domain, and (ii) the CD8 or CD28 transmembrane domain, but having other components of the comparator CAR), DCs comprising or expressing at least one CAR as described herein mediate tumor antigen presentation, such as increased tumor antigen presentation. In some embodiments, such as relative to DCs comprising a similar CAR (e.g., including a different anti-mesothelin antigen-binding domain and / or lacking one or both of (i) the CD8 or CD28 extracellular hinge domain, and (ii) the CD8 or CD28 transmembrane domain, but having other components of the comparator CAR), DCs comprising or expressing at least one CAR as described herein mediate tumor T cell stimulation, such as increased T cell stimulation.

[0162] In some embodiments, DCs that comprise or express 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), as compared to DCs that comprise a similar CAR (e.g., that includes a different anti-mesothelin antigen-binding domain and / or that does not include one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but that has other components of the comparator CAR). In some embodiments, DCs that comprise or express at least one CAR described herein exhibit increased phagocytosis, as compared to DCs that comprise a similar CAR (e.g., that includes a different anti-mesothelin antigen-binding domain and / or that does not include one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but that has other components of the comparator CAR). In some embodiments, DCs that comprise or express 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 stimulation of T cells, as compared to DCs that comprise a similar CAR (e.g., that includes a different anti-mesothelin antigen-binding domain and / or that does not include one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but that has other components of the comparator CAR).

[0163] In some embodiments, DCs that contain or express at least one CAR described herein exhibit either an increase in the expression of favorable genes or a decrease in the expression of unfavorable genes, or both, as compared to DCs that contain a similar CAR (e.g., including different anti-mesothelin antigen-binding domains and / or lacking one or both of (i) a CD8 or CD28 extracellular hinge domain and (ii) a CD8 or CD28 transmembrane domain, but having the other components of the comparator CAR). In some embodiments, DCs that contain or express at least one CAR described herein exhibit an increase in ROS production, as compared to DCs that contain a similar CAR (e.g., including different anti-mesothelin antigen-binding domains and / or lacking one or both of (i) a CD8 or CD28 extracellular hinge domain and (ii) a CD8 or CD28 transmembrane domain, but having the other components of the comparator CAR). In some embodiments, DCs that contain or express at least one CAR described herein exhibit metabolic reprogramming, as compared to DCs that contain a similar CAR (e.g., including different anti-mesothelin antigen-binding domains and / or lacking one or both of (i) a CD8 or CD28 extracellular hinge domain and (ii) a CD8 or CD28 transmembrane domain, but having the other components of the comparator CAR). In some embodiments, DCs that contain or express at least one CAR described herein exhibit induction of cell survival mechanisms, as compared to DCs that contain a similar CAR (e.g., including different anti-mesothelin antigen-binding domains and / or lacking one or both of (i) a CD8 or CD28 extracellular hinge domain and (ii) a CD8 or CD28 transmembrane domain, but having the other components of the comparator CAR).

[0164] In some embodiments, DCs comprising or expressing at least one CAR as described herein exhibit induction of cell death mechanisms, e.g., relative to DCs comprising a similar CAR (e.g., including different anti-mesothelin antigen-binding domains and / or not including one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator CAR). In some embodiments, DCs comprising or expressing at least one CAR as described herein exhibit an increase in one, two, three, four, or five of resistance to phagocytic checkpoints, increased expression of chemokine receptors that aid trafficking, increased expression of chemokines that recruit other immune cells, increased expression of ECM-degrading enzymes (e.g., MMPs that degrade tumor ECM and / or exhibit antifibrotic activity), and / or increased proliferation, e.g., relative to DCs not having a CAR as described herein. In some embodiments, DCs comprising or expressing at least one CAR as described herein exhibit one, two, three, or four of improved duration of CAR expression, improved stability of the CAR on the cell surface, increased CAR expression levels, and / or reduced CAR background activity, e.g., relative to DCs not having a CAR as described herein.

[0165] Chimeric antigen receptor (CAR)

[0166] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial cell surface receptor that is engineered to be expressed on an immune effector cell and that specifically targets a cell and / or binds an antigen. CARs can be used, for example, in therapies 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 blood, tumor, or ascites) and modified such that they express a receptor specific for a particular form of an antigen. In some embodiments, such modified immune cells are then re-introduced into the same or a different subject as a therapeutic agent. In some embodiments, CARs have been expressed that are specific for an antigen, e.g., a tumor-associated antigen. In some embodiments, a CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain.

[0167] 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, an immune cell comprises a CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the immune cell comprises a stem cell, a macrophage, a monocyte, or a dendritic cell.

[0168] In some embodiments, the CAR may further include one or more of the following: one or more extracellular leader domains, one or more extracellular hinge domains, and one or more intracellular co-stimulatory domains.

[0169] In some embodiments, the CAR includes a spacer domain or hinge (i.e., an extracellular hinge domain) between the extracellular domain and the transmembrane domain. In some embodiments, the CAR includes 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 the transmembrane domain to the extracellular domain or intracellular domain in a polypeptide chain. In some embodiments, the spacer domain or hinge may comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. In some embodiments, a short oligopeptide or polypeptide linker (preferably having a length between 2 and 10 amino acids) may form a linkage between the transmembrane domain and the intracellular domain of the CAR. Examples of linkers include glycine-serine doublets.

[0170] In some embodiments, an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR may include one or more control systems, including but not limited to: safety switches (e.g., on switches and off switches, suicide switches), and / or logic gates (e.g., AND gates (e.g., two or more CARs, each of which lacks one or more signaling domains such that full activation or function of the immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) requires activation of both / all CARs), OR gates (e.g., two or more CARs, each having an intracellular domain such as CD3ζ and a co-stimulatory domain), and / or NOT gates (e.g., two or more CARs, one of which includes an inhibitory domain that antagonizes the function of one or more of the other CARs)).

[0171] 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 sequence 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 wherein the cell is a stem cell, macrophage, monocyte, or dendritic cell that expresses the CAR.

[0172] 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, a nucleic acid encoding the extracellular domain is operably linked to a nucleic acid encoding the transmembrane domain, and the nucleic acid encoding the transmembrane domain is operably linked to a nucleic acid encoding the intracellular domain.

[0173] In some embodiments, the effector activity of an immune cell comprising a CAR is directed against a target cell comprising an antigen that specifically binds the antigen-binding domain of the CAR. In some embodiments, the targeted effector activity against the target cell is or comprises phagocytosis, targeted cytotoxicity, antigen presentation, or cytokine secretion.

[0174] In some embodiments, the CARs described herein comprise at least one domain (such as an extracellular domain, a transmembrane domain, and / or an intracellular domain) that inhibits anti-phagocytic signaling in immune cells (such as stem cells, macrophages, monocytes, or dendritic cells) described herein. In some embodiments, the CARs described herein improve the effector activity of immune cells (such as stem cells, macrophages, monocytes, or dendritic cells) described herein by enhancing inhibition of CD47 and / or SIRPα activity. In some embodiments, the CARs described herein bind to, e.g., CD47 and act as a dominant negative receptor, thereby inhibiting SIRPα activity (such as CD47 deposition). In some embodiments, the CARs described herein that bind SIRPα comprise, for example, an activating receptor (such as comprising a CD3z intracellular domain). In some embodiments, the CARs described herein inhibit at least one interaction of CD47 and SIRPα. In some embodiments, the CAR is or comprises a phagocytic logic gate.

[0175] In some embodiments, the immune cells described herein (e.g., comprising at least one CAR described herein) comprise or express at least one variant or fragment of the following: 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 includes a mutated intracellular domain. In some embodiments, the variant or fragment does not include or express at least one intracellular domain (e.g., the immune cell comprises or expresses anti-CD47 scFv, CD8 hinge domain, and CD8 transmembrane). In some embodiments, the immune cells described herein (e.g., comprising or expressing at least one CAR described herein) comprise a dominant-negative receptor, such as blocking an inhibitory checkpoint.

[0176] In some embodiments, the payload comprising the CAR described herein further comprises a cleavage peptide (e.g., P2A, F2A, E2A, and / or T2A peptide) and / or at least one second CAR comprising at least one inhibitory domain that includes at least one anti-phagocytosis signal transduction. In some embodiments, 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 fragment thereof. In some embodiments, at least one second CAR comprises a SIRPα transmembrane domain or fragment thereof. In certain embodiments, the second CAR further comprises a hinge domain (e.g., CD8 hinge domain). In certain embodiments, at least one second CAR comprises: (i) a leader sequence (e.g., CD8 leader sequence); ii) an extracellular domain (e.g., SIRPα, CV1, 5F9 scFv, or B6H12 scFv (e.g., humanized B6H12 scFv) extracellular domain); and ii) a transmembrane domain (e.g., SIRPα transmembrane domain). In some embodiments, the payload comprising the CAR described herein further comprises a cleavage peptide (e.g., P2A peptide) and at least one marker protein (e.g., CD20 or fragment thereof, CD19 or fragment thereof, NGFR or fragment thereof, synthetic peptide, and / or fluorescent protein).

[0177] In some embodiments, the immune cells described herein (e.g., comprising or expressing at least one CAR described herein) comprise or express one or more phosphatase death domains (e.g., phosphatase death Shp1, phosphatase death 72-5 phosphatase (INPP5E), phosphatase death Shp2, and / or phosphatase death SHIP-1 domain) and / or constitutively active kinase domains (e.g., constitutively active LYN domain). In some embodiments, the payload comprising the CAR described herein further comprises a cleavage peptide (e.g., P2A, F2A, E2A, and / or T2A peptide) and one or more phosphatase death domains (e.g., phosphatase death Shp1, phosphatase death 72-5 phosphatase (INPP5E), phosphatase death Shp2, and / or phosphatase death SHIP-1 domain) and / or constitutively active kinase domains (e.g., constitutively active LYN domain).

[0178] In some embodiments, the CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence that is at least 80% identical to a sequence selected from Table 2. In some embodiments, the CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence that is at least 85% identical to a sequence selected from Table 2. In some embodiments, the CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence that is at least 90% identical to a sequence selected from Table 2. In some embodiments, the CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence that is at least 95% identical to a sequence selected from Table 2. In some embodiments, the CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence that is at least 96% identical to a sequence selected from Table 2. In some embodiments, the CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence that is at least 97% identical to a sequence selected from Table 2. In some embodiments, the CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence that is at least 98% identical to a sequence selected from Table 2. In some embodiments, the CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence that is at least 99% identical to a sequence selected from Table 2. In some embodiments, the CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence identical to a sequence selected from Table 2.

[0179] In some embodiments, the CARs of the present disclosure bind to mesothelin and comprise an amino acid sequence that differs from a sequence selected from Table 2 by no more than 50 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and comprise an amino acid sequence that differs from a sequence selected from Table 2 by no more than 40 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and comprise an amino acid sequence that differs from a sequence selected from Table 2 by no more than 30 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and comprise an amino acid sequence that differs from a sequence selected from Table 2 by no more than 20 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and comprise an amino acid sequence that differs from a sequence selected from Table 2 by no more than 10 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and comprise an amino acid sequence that differs from a sequence selected from Table 2 by no more than 5 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and comprise an amino acid sequence that differs from a sequence selected from Table 2 by no more than 4 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and comprise an amino acid sequence that differs from a sequence selected from Table 2 by no more than 3 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and comprise an amino acid sequence that differs from a sequence selected from Table 2 by no more than 2 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and comprise an amino acid sequence that differs from a sequence selected from Table 2 by no more than 1 substitution, addition, or deletion. In some embodiments, the CARs of the present disclosure bind to mesothelin and comprise an amino acid sequence that has no substitutions, additions, or deletions relative to a sequence selected from Table 2.

[0180] CAR extracellular domain

[0181] 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 may be a domain endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, the CAR extracellular domain may be a domain not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).

[0182] FcR extracellular domain

[0183] 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 the 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 may be a domain endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, the FcR extracellular domain may be a domain not endogenous to a particular immune cell type (e.g., a modified immune cell as 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.

[0184] TLR extracellular domain

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

[0186] Leader domain

[0187] 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 immune cells. 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 endogenous to a particular immune cell type (e.g., a modified immune cell as 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 as 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 from a stimulatory or co-stimulatory domain (e.g., 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).

[0188] Antigen-binding domain

[0189] In some embodiments, the CAR comprises an antigen-binding domain that binds to an antigen, such as an antigen on a target cell. In some embodiments, the CAR comprises an antigen-binding domain that binds to an antigen associated with cancer. In some embodiments, the CAR antigen-binding domain recognizes an antigen that serves as a cell surface marker associated with a particular disease state on a target cell.

[0190] In some embodiments, the CAR antigen-binding domain binds to a tumor antigen, such as an antigen specific to a target tumor or cancer. In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes. In some embodiments, the tumor antigen comprises mesothelin.

[0191] In some embodiments, the CAR antigen-binding domain binds to mesothelin and comprises an amino acid sequence that is at least 80% 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 that is at least 85% 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 that is at least 90% 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 that is at least 95% 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 that is at least 96% 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 that is at least 97% 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 that is at least 98% 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 that is 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 that is identical to a sequence selected from Table 3.

[0192] In some embodiments, the CAR antigen-binding domain binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 3 by no more than 10 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 3 by no more than 9 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 3 by no more than 8 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 3 by no more than 7 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 3 by no more than 6 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 3 by no more than 5 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 3 by no more than 4 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 3 by no more than 3 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 3 by no more than 2 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 3 by no more than 1 substitution, addition, or deletion. In some embodiments, the CAR antigen-binding domain binds to mesothelin and comprises an amino acid sequence that has no substitutions, additions, or deletions relative to a sequence selected from Table 3.

[0193] 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 designed ankyrin repeat protein, a centyrin, 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 wholly or partially derived 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 may be a domain endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, the CAR antigen-binding domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).

[0194] 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 cell comprises two or more different CARs that each comprise one or more antigen-binding domains. In some embodiments, an immune cell comprising a bispecific CAR and / or comprising two or more different CARs that each comprise one or more antigen-binding domains can reduce off-target and / or on-target off-tissue effects by requiring the presence of two antigens. In some embodiments, the immune cell comprises a bispecific CAR and / or comprises two or more different CARs that each comprise one or more antigen-binding domains, wherein the CARs provide different signals that are insufficient, when separated, to mediate activation of the modified cell, but that co-stimulate activation of the modified cell together. In some embodiments, such a construct may be referred to as an "AND" logic gate.

[0195] In some embodiments, immune cells comprising a bispecific CAR and / or comprising two or more different CARs each comprising one or more antigen-binding domains can reduce off-target and / or on-target off-tissue effects by requiring the presence of one antigen but the absence of a second normal protein antigen prior to activation of the cell's activity. In some embodiments, such constructs may be referred to as "NOT" logic gates. In contrast to an AND gate, cells modified with a NOT-gated CAR are activated by binding to a single antigen. However, the binding of a second receptor to a second antigen serves to override the activation signal that persists through the CAR. Typically, such an inhibitory receptor will be targeted to an antigen that is highly expressed in normal tissue but absent in tumor tissue.

[0196] Hinge domain

[0197] In some embodiments, the CAR comprises one or more extracellular hinge domains. In some embodiments, the CAR extracellular domain is or comprises a human extracellular hinge domain. In some embodiments, the CAR extracellular hinge domain can be a domain endogenous to a particular immune cell type (e.g., a modified immune cell as 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 as provided herein). In some embodiments, one or more CAR extracellular hinge domains comprise a CD8a extracellular hinge domain, a DNGR-1 extracellular hinge domain, a dendritic cell-associated C-type lectin-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 optimal size relative to the tumor antigen (e.g., allowing exclusion of inhibitory molecules), optimal flexibility, optimal protein folding, optimal protein stability, optimal binding, optimal homodimerization, and / or lack of homodimerization.

[0198] CAR transmembrane domain

[0199] In some embodiments, the CAR includes, for example, a transmembrane domain that links an extracellular domain to an intracellular domain. In some embodiments, the CAR transmembrane domain naturally associates with one or more other domains of the CAR. In some embodiments, the CAR transmembrane domain can be modified to avoid binding to the transmembrane domains of other surface membrane proteins in order to minimize interactions with other members of the receptor complex. In some embodiments, the CAR transmembrane domain can be derived from a naturally occurring source or a synthetic source. In some embodiments, the CAR transmembrane domain is derived from a naturally occurring membrane-bound protein or transmembrane protein. In some embodiments, the CAR transmembrane domain is or includes a human transmembrane domain. In some embodiments, the CAR transmembrane domain can be a domain endogenous to a particular immune cell type (e.g., the modified immune cells 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., the modified immune cells provided herein). In some embodiments, the CAR transmembrane domain includes 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, OX40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, CD3-ζ, dendritic cell-associated C-type lectin-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.

[0200] FcR transmembrane domain

[0201] 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 transmembrane domain or a portion thereof. In some embodiments, the FcR transmembrane domain can be a domain endogenous to a particular immune cell type (e.g., a modified immune cell as 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 as 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.

[0202] TLR transmembrane domain

[0203] 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 transmembrane domain or a portion thereof. In some embodiments, the TLR transmembrane domain can be a domain endogenous to a particular immune cell type (e.g., a modified immune cell as 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 as provided herein). In some embodiments, the TLR transmembrane domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.

[0204] CAR intracellular domain

[0205] In some embodiments, the 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 endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, the CAR intracellular domain can be a domain not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, the CAR intracellular domain and / or other cytoplasmic domains of the CAR are responsible for activating the cell in which the CAR is expressed (e.g., a modified immune cell as provided herein). In some embodiments, the CAR intracellular domain of the CAR is responsible for signal activation and / or transduction in immune cells comprising the CAR.

[0206] In some embodiments, the CAR intracellular domain of the CAR comprises at least one domain responsible for 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 a dual signaling domain. In some embodiments, the CAR intracellular domain of the CAR comprises more than two signaling domains.

[0207] 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 initiates signal transduction in immune cells.

[0208] In some embodiments, the intracellular domain of the CAR comprises any portion of one or more costimulatory molecules, such as at least one signaling domain from CD3ζ, the FcεRIγ chain, any derivative or variant thereof, any synthetic sequence having the same functional capacity, and any combination thereof.

[0209] FcR intracellular domain

[0210] 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 the 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 an endogenous domain of a particular immune cell type (e.g., a modified immune cell as 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 as 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.

[0211] TLR intracellular domain

[0212] 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 the 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 an endogenous domain of a particular immune cell type (e.g., a modified immune cell as 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 as provided herein). In some embodiments, the TLR intracellular domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.

[0213] Signal transduction domain

[0214] In some embodiments, the CAR comprises one or more intracellular signal transduction domains. In some embodiments, the CAR intracellular signal transduction domain is or comprises a human intracellular signal transduction domain or a portion thereof. In some embodiments, the CAR signal transduction domain can be an endogenous domain of a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, the CAR signal transduction domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).

[0215] In some embodiments, one or more intracellular signaling domains of the CAR cells include 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, SIRPβ, 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, dendritic cell-associated C-type lectin-2, or one or more cytokine receptor signaling domains (e.g., IL1R, IL2R, IL3R, IL4R, IL5R, IL6R, IL7R, IL8R, IL9R, IL10R, IL11R, IL12R, IL13R, IL14R, IL15R, IL17R, IFNaR, IFNgR, TNFR, CSF1R, CSF2R, Dap10, CD36, dendritic cell-associated C-type lectin-1 or ICOS ligand intracellular signaling domain).

[0216] In some embodiments, the intracellular domain of the CAR includes dual signaling domains such as 41BB, CD28, ICOS, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, the beta chain of the CD116 receptor, CSF1-R, LRP1 / CD91, SR-A1, SR-A2, MARCO, SR-CL1, SR-CL2, SR-C, SR-E, CR1, CR3, CR4, dendritic cell-associated C-type lectin-1, DEC-205, DC-SIGN, CD14, CD36, LOX-1, CD11b, and any combination of the signaling domains listed in the foregoing paragraphs.

[0217] Co-stimulatory domain

[0218] As used herein, "costimulatory molecule" or "costimulatory domain" refers to a molecule in an immune cell that enhances or attenuates an 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 an initial stimulus, respectively. In some embodiments, CAR costimulatory domains include TCR, CD3 zeta (CD3ζ), CD3γ, CD3δ, CD3ε, CD86, common FcRγ, FcRβ (FcεR1b), CD79a, CD79b, FcγRIIa, 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, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD 19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA 1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGA D, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF 8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, other costimulatory molecules described herein, any derivatives, variants or fragments thereof, any synthetic sequences of costimulatory molecules having the same functional capacity, and any combinations thereof.

[0219] In some embodiments, the CAR co-stimulatory domain can be a domain endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, the CAR co-stimulatory domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).

[0220] 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), causes activation of the immune cell.

[0221] Cleavage peptide

[0222] As used herein, a cleavage peptide refers to a peptide that can induce cleavage of a recombinant protein in a cell. In some embodiments, the cleavage peptide is a 2A peptide. In some embodiments, the cleavage peptide is or includes 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, a nucleic acid comprising a nucleic acid sequence encoding a cleavage peptide further 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 produces a protein comprising one or more intracellular domains separated from the one or more peptide agents by the cleavage peptide. 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 engineered sequence that initiates cap-independent ribosome binding to the mRNA, facilitating initiation of translation.

[0223] CAR peptide agent

[0224] 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 the CAR to ensure stoichiometric balance and optimal CAR signaling. In some embodiments, the CAR peptide agent forms a homodimer with the same peptide agent. In some embodiments, the CAR peptide agent forms a heterodimer with a different peptide agent. In some embodiments, the nucleic acids described herein comprise one or more nucleic acid sequences encoding one or more CAR peptide agents. In some embodiments, the CAR peptide agent is or includes the FcRγ chain.

[0225] In some embodiments, the CAR peptide agent includes any peptide, protein, receptor, secreted antibody, or fragment thereof (e.g., scFv, Fab, Fab', F(ab')2, Fc, or nanobody). In some embodiments, the CAR peptide agent includes one or more cytokines (e.g., one or more of IL-1, IL-2, IL-6, IL-8, TNF-α, IFNα, IFNβ, IFN-γ, GMCSF, or MCSF), CD40-L, dominant negative SIRPα, dominant negative PD1, dominant negative CD45, dominant negative SIGLEC 10, or dominant negative LILRB.

[0226] Fc receptor (FcR)

[0227] In some embodiments, the CAR includes one or more antigen-binding domains and the extracellular domain of FcR, and / or the transmembrane domain of the CAR includes the transmembrane domain of FcR, and / or the intracellular domain of the CAR includes the intracellular domain of FcR. In some embodiments, the CAR includes, from the N-terminus to the C-terminus, one or more extracellular binding domains, the extracellular domain of FcR, the transmembrane domain of FcR, and the intracellular domain of FcR. In some embodiments, one or more of the extracellular domain of FcR, the transmembrane domain of FcR, and the intracellular domain of FcR are or include human FcR domains. In some embodiments, the extracellular domain of FcR, the transmembrane domain of FcR, and the intracellular domain of FcR together constitute a full-length FcR. In some embodiments, the extracellular domain of FcR, the transmembrane domain of FcR, and the intracellular domain of FcR together constitute a part of the full-length FcR. In some embodiments, the extracellular domain of FcR includes a part of the full-length extracellular domain of FcR. In some embodiments, the transmembrane domain of FcR includes a part of the full-length transmembrane domain of FcR. In some embodiments, the intracellular domain of FcR includes a part of the full-length intracellular domain of FcR.

[0228] Toll-like antigen receptor (TLR)

[0229] 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 the N-terminus to the 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 constitute a full-length TLR. In some embodiments, the TLR extracellular domain, the TLR transmembrane domain, and the TLR intracellular domain together constitute 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.

[0230] Method for modifying immune cells

[0231] The present disclosure particularly provides a method for modifying immune cells (such as, stem cells, monocytes, macrophages or dendritic cells), the method 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 (such as, 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.

[0232] In some embodiments, the present disclosure provides a method for generating modified immune cells (such as, stem cells, monocytes, macrophages or dendritic cells) in a subject, which comprises administering to the subject a composition as described herein, the composition comprising: (a) one or more nucleic acid molecules, wherein at least a portion of the one or more nucleic acid molecules encodes a CAR and / or a CAR peptide agent, and (b) a delivery vehicle. Thus, in some embodiments, after administering the composition, the one or more nucleic acid molecules are translated in immune cells (such as, stem cells, macrophages, monocytes or dendritic cells) to generate modified immune cells comprising a CAR and / or a CAR peptide agent. In some embodiments, the modified immune cells comprising a CAR and / or a CAR peptide agent have targeted effector activity.

[0233] Delivery methods

[0234] A nucleic acid construct comprising one or more nucleic acid sequences encoding at least one CAR described herein can be introduced into immune cells (e.g., stem cells, monocytes, macrophages or dendritic cells) by physical, chemical or biological methods. In some embodiments, the present disclosure provides methods for modifying immune cells, which include generating 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, which include generating modified immune cells (e.g., stem cells, monocytes, macrophages or dendritic cells) in a subject (i.e., in vivo).

[0235] 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 combinations thereof. Commercially available methods can be used to introduce nucleic acid constructs into immune cells, including electroporation (Amaxa Nucleofector - (Amaxa Biosystems, Cologne, Germany), ECM 830 BTX (Harvard Instruments, Boston, Mass.), Gene Pulser (BioRad, Denver, Colo.) or (Eppendort, Hamburg, Germany)). Nucleic acid constructs can also be introduced into immune cells using mRNA transfection, such as cationic lipid-mediated transfection, lipofection, polymer encapsulation, peptide-mediated transfection, or a biolistic particle delivery system (such as a "gene gun") (see, e.g., Nishikawa et al., Hum Gene Ther., 12(8):861 - 70 (2001), which is hereby incorporated by reference in its entirety).

[0236] 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 includes a plasmid vector, a viral vector, a transposon, a retrotransposon (e.g., piggyBac, Sleeping Beauty), a site-specific integration vector (e.g., CRISPR, zinc finger nuclease, TALEN), a suicide expression vector, or another vector known in the art. Viral vectors, and particularly retroviral vectors, have been 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. Patent Nos. 5,350,674 and 5,585,362, which are hereby incorporated by reference in their entirety). Retroviral vectors such as lentiviruses are suitable tools for achieving long-term gene transfer, which allows for the long-term, 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. WO 2017 / 044487, which is hereby incorporated by reference in its entirety). In some embodiments, VPX comprises a virion-associated protein (e.g., an accessory protein for viral replication). In some embodiments, 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, the immune cells described herein (e.g., stem cells, macrophages, monocytes, or dendritic cells) are transfected with a lentiviral vector packaged with a VPX protein. In some embodiments, VPX inhibits at least one antiviral factor of the immune cells described herein (e.g., stem cells, macrophages, monocytes, or dendritic cells). In some embodiments, the lentiviral vector packaged with a VPX protein exhibits an increased transfection efficiency of the immune cells described herein (e.g., stem cells, macrophages, monocytes, or dendritic cells) as compared to a lentiviral vector not packaged with a VPX protein. In some embodiments, the immune cells described herein (e.g., stem cells, macrophages, monocytes, or dendritic cells) are electroporated or transfected with at least one VPX mRNA, or both, prior to transfection with a viral vector (e.g., an adenoviral vector, such as an Ad2 vector or an Ad5 vector (e.g., an Ad5f35 adenoviral vector, such as an Ad5F35 helper virus-dependent adenoviral vector)).

[0237] 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, macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (e.g., oil-in-water emulsions, micelles, mixed micelles, nanoparticles, liposomes, and Lipofectamine-nucleic acid complexes).

[0238] An exemplary system for delivering the nucleic acid constructs described herein is a lipid-based system. The nucleic acid constructs described herein can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer, attached to liposomes via linking molecules, attached to lipid nanoparticles (LNPs) via linking molecules, entrapped within liposomes, entrapped within LNPs, complexed with liposomes, complexed with LNPs, dispersed in a lipid-containing solution or suspension, mixed with lipids, complexed with micelles, or otherwise associated with lipids. The lipids used in the methods described herein can be naturally occurring or synthetic lipids. Lipids can also be obtained from commercial sources. For example, dimyristoyl 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 dimyristoyl phosphatidylglycerol can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20 °C. In some embodiments, the lipid-based system can include one or more lipids that facilitate targeting of the composition to one or more desired cell types, such as 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, such as stem cells, monocytes, macrophages, or dendritic cells, relative to a composition that does not include the delivery vehicle.

[0239] Targeting moiety

[0240] In some embodiments, the delivery vehicle can comprise one or more targeting moieties. In some embodiments, the targeting moiety can facilitate passive targeting of the composition to a desired target. In some embodiments, the targeting moiety can facilitate active targeting of the composition to a desired target.

[0241] In some embodiments, the targeting moiety can be or include one or more antibodies (e.g., monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies) or any fragment thereof, such as scFv, aptamers, engineered ankyrin repeat proteins, centyrins, naturally occurring or synthetic receptors, affibodies, or other engineered protein recognition molecules, e.g., to bind to one or more of CD14, CD11b, CD163, CD206, CD33, CD209. In some embodiments, the targeting moiety can be or include small molecules.

[0242] In some embodiments, the targeting moiety can be or include a specific lipid or combination of hydrophobic entities, such as those present on or forming the outer surface of a liposome or lipid nanoparticle (e.g., for targeting a specific cell type or types).

[0243] Nucleic acid molecule

[0244] In some embodiments of the present disclosure, one or more nucleic acid molecules are or include DNA. In some embodiments of the present disclosure, one or more nucleic acid molecules are or include messenger RNA (mRNA). In some embodiments, the mRNA according to the present disclosure can be synthesized as unmodified or modified mRNA. Generally, the mRNA is modified to enhance stability. Modifications of mRNA can include, for example, modifications of the nucleotides of the RNA. Thus, the modified mRNA according to the present disclosure can include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, the step of modifying the mRNA includes causing the mRNA to include modified nucleotides, alterations of the 5' or 3' untranslated regions (UTRs), a cap structure, and / or a poly(A) tail.

[0245] In some embodiments, the mRNA of the present disclosure (e.g., the mRNA encoding one or more CARs described herein) can contain RNA backbone modifications. Generally, a backbone modification is a modification in which the phosphate ester of the backbone of the nucleotides contained in the RNA is chemically modified. Exemplary backbone modifications generally include, but are not limited to, modifications selected from the group consisting of methylphosphonate, methylaminophosphate, aminophosphate, phosphorothioate (e.g., cytidine 5'-O-(1-thiophosphate)), boranophosphate, a positively charged guanidinium group, etc., which includes replacing the phosphodiester bond with other anionic, cationic, or neutral groups.

[0246] In some embodiments, the mRNA of the present disclosure (e.g., mRNA encoding one or more CARs as described herein) may contain sugar modifications. Typical sugar modifications are chemical modifications of the sugars of the nucleotides it contains, including but not limited to sugar modifications selected from the group consisting of: 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamino-oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyl oligoribonucleotides, 2'-deoxy-2'-C-alkyl oligoribonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligoribonucleotides and their isomers (2'-arabinosylcytidine 5'-triphosphate, 2'-arabinosyluridine 5'-triphosphate) or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0247] In some embodiments, the mRNA of the present disclosure (e.g., mRNA encoding one or more CARs as described herein) includes modified nucleotides containing pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine / pseudouridine (5meC PsU), N1-methylpseudouridine (N1mPsU) or combinations thereof.

[0248] In some embodiments, the mRNA of the present disclosure (e.g., mRNA encoding one or more CARs as described herein) may contain modifications of the bases of the nucleotides (base modifications). Modified nucleotides containing base modifications are also referred to as base-modified nucleotides.

[0249] Typically, mRNA synthesis includes adding a "cap" at the N-terminus (5') end and a "tail" at the C-terminus (3') end. The presence of the cap is important for providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" is used to protect the mRNA from exonuclease degradation.

[0250] Thus, in some embodiments, the mRNA of the present disclosure (e.g., mRNA encoding one or more CARs as described herein) includes a 5' cap structure. The 5' cap is typically added as follows: First, an RNA terminal phosphatase removes a terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; then guanosine triphosphate (GTP) is added to the terminal phosphate via guanylyltransferase, generating a 5' triphosphate linkage; then 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 includes a Cap0 structure. The Cap0 structure lacks 2'-O-methyl residues attached to the riboses of bases 1 and 2. In some embodiments, the cap includes an AGCap1 structure. The AGCap1 structure has a 2'-O-methyl residue at base 2. In some embodiments, the cap includes a Cap2 structure. The Cap2 structure has 2'-O-methyl residues attached to bases 2 and 3. In some embodiments, the cap structure includes AGCap1, m6AGCap1, or an anti-reverse cap analog (ARCA). In some embodiments, the modified mRNA of the present disclosure contains m6AGCap1 and modified nucleotides containing pseudouridine (PsU).

[0251] In some embodiments, the mRNA of the present disclosure (e.g., mRNA encoding one or more CARs as described herein) includes a 3' poly(A) tail structure. The poly(A) tail on the 3' end of the mRNA typically includes from about 10 to 400 adenosine nucleotides (SEQ ID NO:73) (e.g., from about 100 to 400 adenosine nucleotides, from about 10 to 200 adenosine nucleotides, from about 10 to 150 adenosine nucleotides, from about 10 to 100 adenosine nucleotides, from about 20 to 70 adenosine nucleotides, or from about 20 to 60 adenosine nucleotides). In some embodiments, the mRNA includes a 3' poly(C) tail structure. A suitable poly(C) tail on the 3' end of the mRNA typically includes from about 10 to 200 cytosine nucleotides (SEQ ID NO:74) (e.g., from about 10 to 150 cytosine nucleotides, from about 10 to 100 cytosine nucleotides, from about 20 to 70 cytosine nucleotides, from about 20 to 60 cytosine nucleotides, or from about 10 to 40 cytosine nucleotides). The poly(C) tail can be added to the poly(A) tail, or the poly(C) tail can replace the poly(A) tail.

[0252] In some embodiments, the mRNA of the present disclosure (e.g., mRNA encoding one or more CARs described herein) includes 5' and / or 3' untranslated regions. In some embodiments, the 5' untranslated region includes one or more elements that affect the stability or translation of the mRNA, such as an iron-responsive element. In some embodiments, the length of the 5' untranslated region can be between about 50 and 500 nucleotides.

[0253] 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 mRNA localization in a cell, or a binding site for one or more miRNAs. In some embodiments, the length of the 3' untranslated region can be between 50 and 500 nucleotides or longer.

[0254] Administration of additional payloads

[0255] In some embodiments, the methods of the present disclosure include one or more steps of treating an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) during the process of modifying the immune cell. In some embodiments, the methods of the present disclosure include one or more steps of administering an additional payload to a subject during the process of modifying an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) (e.g., with a payload comprising a CAR) to modulate the immune cell. In some embodiments, the composition can comprise one or more additional payloads. In some embodiments, the composition can comprise one or more additional payloads in the same delivery vehicle as one or more nucleic acid molecules. In some embodiments, the composition can comprise one or more additional payloads in a delivery vehicle different from the delivery vehicle used with one or more nucleic acid molecules.

[0256] In some embodiments, the methods of the present disclosure include the step of treating immune cells (such as stem cells, macrophages, monocytes, or dendritic cells) with a regulator of a pathway activated by in vitro transcribed mRNA. In some embodiments, the additional payload can be or include a regulator of a pathway activated by in vitro transcribed mRNA. In vitro transcribed (IVT) mRNA is recognized by various endosomal innate immune receptors (Toll-like receptor 3 (TLR3), TLR7, and TLR8) and cytoplasmic innate immune receptors (RNA-activated protein kinase (PKR), retinoic acid-inducible gene I protein (RIG-I), melanoma differentiation-associated protein 5 (MDA5), and 2'-5'-oligoadenylate synthetase (OAS)). Signaling through these different pathways generates inflammation associated with the activation of type I interferon (IFN), tumor necrosis factor (TNF), interleukin-6 (IL-6), IL-12, and a transcriptional program cascade. Collectively, these create a pro-inflammatory microenvironment primed to induce a specific immune response. In addition, downstream effects (such as the translational slowdown caused by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, enhanced RNA degradation caused by ribonuclease L (RNase L), and overexpression and inhibition of self-amplifying mRNA replication) are all related to the pharmacokinetics and pharmacodynamics of IVT mRNA.

[0257] In some embodiments, the regulator of a pathway activated by in vitro transcribed mRNA includes an RNase inhibitor. In some embodiments, the regulator of a pathway activated by in vitro transcribed mRNA includes an RNase L, RNase T2, or RNase 1 inhibitor. In some embodiments, the regulator of a pathway activated by in vitro transcribed mRNA includes an RNase L inhibitor. In some embodiments, the RNase L inhibitor includes sunitinib. In some embodiments, the RNase L inhibitor includes ABCE1.

[0258] In some embodiments, treatment of immune cells (such as stem cells, macrophages, monocytes, or dendritic cells) with an RNase L inhibitor increases mRNA stability in the modified immune cells relative to the mRNA stability in the same type of modified immune cells not treated with an RNase L inhibitor. In some embodiments, treatment of immune cells (such as stem cells, macrophages, monocytes, or dendritic cells) with an RNase L inhibitor increases CAR expression in the modified immune cells relative to the CAR expression in the same type of modified immune cells not treated with an RNase L inhibitor. In some embodiments, treatment of immune cells (such as stem cells, macrophages, monocytes, or dendritic cells) with an RNase L inhibitor increases effector activity in the modified immune cells relative to the effector activity in the same type of modified immune cells not treated with an RNase L inhibitor.

[0259] In some embodiments, relative to the mRNA stability in the same type of modified immune cells in a subject not administered an RNase L inhibitor, administration of an RNase L inhibitor to a subject increases the mRNA stability in modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells). In some embodiments, relative to the CAR expression in the same type of modified immune cells in a subject not administered an RNase L inhibitor, administration of an RNase L inhibitor to a subject increases the CAR expression in modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells). In some embodiments, relative to the effector activity in the same type of modified immune cells in a subject not administered an RNase L inhibitor, administration of an RNase L inhibitor to a subject increases the effector activity in modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells).

[0260] 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 a subject occurs before the step of administering a composition comprising mRNA to the subject.

[0261] In some embodiments, the methods of the present disclosure include culturing immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with cytokines or immunostimulatory recombinant proteins. In some embodiments, the methods of the present disclosure include administering cytokines or immunostimulatory recombinant proteins to a subject. In some embodiments, the cytokines include IFN-α, IFN-β, IFN-γ, TNFα, IL-6, STNGL, LPS, CD40 agonists, 4-1BB ligand, recombinant 4-1BB, CD19 agonists, TLR agonists (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, immune complexes, interleukin-1α (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, CD154, lymphotoxin β (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), TNF-related weak inducer of apoptosis (TWEAK), TNF-related activation-induced cytokine (TRANCE), erythropoietin (Epo), thyroid peroxidase precursor (Tpo), FMS-related tyrosine kinase 3 ligand (FLT-3L), stem cell factor (SCF), macrophage colony-stimulating factor (M-CSF), merozoite surface protein (MSP), nucleotide-binding oligomerization domain-containing protein (NOD) ligands (e.g., NOD1, NOD2, or NOD1 / 2 agonists), 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, usnic acid, scleroglucan, dispersible WGP, soluble WGP, zymosan, depleted zymosan, 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, malaria pigment, MSU crystals, nano-SiO2, nigericin or TDB), aryl hydrocarbon (AhR) ligands (e.g., FICZ, indirubin, ITE or L-kynurenine), α-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.,

[0262] 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 an immune-stimulating recombinant protein to a subject occurs after the step of administering a composition comprising mRNA to the subject.

[0263] 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 relative to the same type of modified immune cells cultured without 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 as described herein) expression in the modified immune cells relative to the same type of modified immune cells cultured without 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 as described herein) expression relative to the same type of modified immune cells cultured without 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 relative to the same type of modified immune cells cultured without 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 pro-inflammatory (M1) polarization of the modified immune cells relative to the same type of modified immune cells cultured without cytokines or immunostimulatory recombinant proteins.

[0264] In some embodiments, administering a cytokine or an 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, as compared to the same type of modified immune cells in a subject not administered the cytokine or immunostimulatory recombinant protein. In some embodiments, administering a cytokine or an immunostimulatory recombinant protein to a subject increases the protein (e.g., at least one CAR as described herein) expression of modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in the subject, as compared to the same type of modified immune cells in a subject not administered the cytokine or immunostimulatory recombinant protein. In some embodiments, administering a cytokine or an immunostimulatory recombinant protein to a subject increases the longevity of protein (e.g., at least one CAR as described herein) expression in modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in the subject, as compared to the same type of modified immune cells in a subject not administered the cytokine or immunostimulatory recombinant protein. In some embodiments, administering a cytokine or an 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, as compared to the same type of modified immune cells in a subject not administered the cytokine or immunostimulatory recombinant protein. In some embodiments, administering a cytokine or an 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, as compared to the same type of modified immune cells in a subject not administered the cytokine or immunostimulatory recombinant protein.

[0265] Methods of altering the inflammatory phenotype of a cell population

[0266] In some embodiments, the methods of the present disclosure include altering the inflammatory phenotype of a cell population. In some embodiments, methods of altering the inflammatory phenotype of a cell population include contacting the cell population with modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) as described herein. In some embodiments, the cell population includes macrophages, monocytes, dendritic cells, T cells, NK cells, or combinations thereof. In some embodiments, the modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) as described herein secrete one or more inhibitory RNAs that alter the inflammatory phenotype of the cell population (e.g., bystander cells). In some embodiments, the one or more inhibitory RNAs are packaged within an extracellular vehicle. In some embodiments, the one or more inhibitory RNAs are packaged within an exosome.

[0267] In some embodiments, the inflammatory phenotype of a cell population is changed from anti-inflammatory to non-activated. In some embodiments, the inflammatory phenotype of a cell population is changed from pro-inflammatory to non-activated. In some embodiments, the inflammatory phenotype of a cell population is changed from anti-inflammatory to pro-inflammatory. In some embodiments, the inflammatory phenotype of a cell population is changed from pro-inflammatory to anti-inflammatory.

[0268] Modified immune cells

[0269] 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 as described herein. In some embodiments, the modified immune cells comprise one or more nucleic acids encoding at least one CAR as described herein. In some embodiments, at least one CAR as 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 target gene, a 3' untranslated region (UTR), and one or more introns, wherein one or more of the introns comprise one or more inhibitory nucleic acids, wherein one or more of the inhibitory nucleic acids encode one or more inhibitory RNAs, and wherein the target gene 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.

[0270] 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 as described herein, wherein the CAR comprises an extracellular domain as described herein that binds to a tumor antigen (such as an antigen specific to a target tumor or cancer). In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes. In some embodiments, the tumor antigen comprises mesothelin.

[0271] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a modified mRNA encoding at least one CAR provided herein exhibits increased viability relative to the same type of modified immune cell comprising a similar CAR (e.g., including a different anti-mesothelin antigen-binding domain and / or lacking one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator CAR). In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a modified mRNA encoding at least one CAR provided herein exhibits increased expression of the mRNA encoding at least one CAR provided herein relative to the same type of modified immune cell comprising an unmodified mRNA encoding a similar CAR (e.g., including a different anti-mesothelin antigen-binding domain and / or lacking one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator CAR). In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising at least one CAR provided herein exhibits increased CAR expression relative to the same type of modified immune cell comprising an unmodified mRNA encoding a similar CAR (e.g., including a different anti-mesothelin antigen-binding domain and / or lacking one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator CAR). In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a modified mRNA encoding at least one CAR provided herein exhibits increased longevity of the mRNA encoding at least one CAR relative to the same type of modified immune cell comprising an unmodified mRNA encoding a similar CAR (e.g., including a different anti-mesothelin antigen-binding domain and / or lacking one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator CAR). In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a modified mRNA encoding a CAR provided herein exhibits increased longevity of the CAR relative to the same type of modified immune cell comprising a similar CAR (e.g., including a different anti-mesothelin antigen-binding domain and / or lacking one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator CAR).In some embodiments, modified immune cells (such as stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as provided herein exhibit increased effector activity relative to the same type of modified immune cells comprising an unmodified mRNA encoding a similar CAR (e.g., including a different anti-mesothelin antigen-binding domain and / or not including one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator CAR). In some embodiments, modified immune cells (such as stem cells, macrophages, monocytes, or dendritic cells) comprising a modified mRNA encoding a CAR as provided herein exhibit increased pro-inflammatory (M1) polarization relative to the same type of modified immune cells comprising an unmodified mRNA encoding a similar CAR (e.g., including a different anti-mesothelin antigen-binding domain and / or not including one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparator CAR).

[0272] In some embodiments, modified immune cells (such as stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as provided herein maintain a pro-inflammatory phenotype over time. In some embodiments, modified immune cells (such as stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as 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 modifying the immune cells with a nucleic acid encoding the CAR.

[0273] In some embodiments, modified immune cells (such as stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as provided herein maintain an anti-inflammatory phenotype over time. In some embodiments, modified immune cells (such as stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as 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 modifying the immune cells with a nucleic acid encoding the CAR.

[0274] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as provided herein maintain a pro-inflammatory phenotype and / or otherwise resist subversion when attacked by anti-inflammatory cytokines. In some embodiments, the sensitivity of the modified immune cells to environmental cytokines is measured by generating a dose-response curve of pro-inflammatory markers by treating the modified immune cells comprising a CAR as provided herein. In some embodiments, the sensitivity of the modified immune cells to environmental cytokines is measured by generating a dose-response curve of pro-inflammatory markers by treating the modified immune cells comprising a CAR as provided herein.

[0275] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as provided herein maintain an anti-inflammatory phenotype and / or otherwise resist subversion when attacked by pro-inflammatory cytokines. In some embodiments, the sensitivity of the modified immune cells to environmental cytokines is measured by generating a dose-response curve of anti-inflammatory markers by treating the modified immune cells comprising a CAR as provided herein with increasing concentrations of pro-inflammatory cytokines. In some embodiments, the sensitivity of the modified immune cells to environmental cytokines is measured by generating a dose-response curve of anti-inflammatory markers by treating the modified immune cells comprising a CAR as provided herein with increasing concentrations of anti-inflammatory cytokines.

[0276] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as provided herein have minimal effect on neighboring cells. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as provided herein have a significant effect on neighboring cells. In some embodiments, the effect of modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as 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 immune cells and the unmodified immune cells can be co-cultured in a culture dish where the modified immune cells and the unmodified immune cells are in contact with each other. In some embodiments, the modified immune cells and the unmodified immune cells can be co-cultured in a culture dish where the modified immune cells and the unmodified immune cells are separated by a transwell assay membrane.

[0277] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as provided herein have minimal cytotoxic effects on neighboring cells. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as provided herein have significant cytotoxic effects on neighboring cells (e.g., cancer cells). In some embodiments, modifying immune cells to comprise a CAR as provided herein is non-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 cytotoxic effects.

[0278] In some embodiments, relative to unmodified immune cells or modified cells comprising a similar CAR (e.g., including a different anti-mesothelin antigen-binding domain and / or not including one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but having other components of the comparison CAR), expression of the CARs provided herein in modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) increases at least one targeted effector function of the modified immune cells (e.g., phagocytosis, targeted cellular cytotoxicity, antigen presentation, or cytokine secretion).

[0279] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as provided herein comprise one or more control systems, including but not limited to: safety switches (e.g., on switches, off switches, suicide switches), transcriptional control (e.g., cell-specific promoters, cell-state-specific promoters, promoters downstream of CAR activation, promoters downstream of endogenous signaling pathways, or drug-induced transcription), post-transcriptional control of CAR mRNA (e.g., RNA-based inhibition with endogenous or recombinant miRNAs), or post-translational control of CAR structure or stability (e.g., a CAR whose intracellular domain associates conditionally with the intact structure by drug / light-induced association (to permit signaling) or dissociation (to inhibit signaling), or a CAR whose stability is regulated by a drug to effect induced stabilization (to permit signaling) or degradation (to inhibit signaling)). These control systems can be combined to create logic gates, such as AND gates (e.g., a CAR having an inducible promoter for the CAR and a cytoplasmic domain that associates in a drug-dependent manner, thus requiring CAR activation and the presence of a small molecule), OR gates (e.g., a CAR controlled by a promoter that is transcriptionally active upon CAR activation or addition of a small molecule), and / or NOT gates (e.g., a CAR whose mRNA is degraded by an endogenous miRNA expressed in the native immune cell signaling state (such as a miRNA upregulated by a specific cytokine signaling pathway, thus expressing the CAR only in the absence of such a cytokine)).

[0280] In some embodiments, the modified immune cells of the disclosure (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprise one or more inhibitory RNAs selected from the group consisting of: antisense RNA (asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), guide RNA (gRNA), long 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).

[0281] In some embodiments, the modified immune cells of the present disclosure (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprise one or more shRNAs. In some embodiments, the modified immune cells of the present disclosure (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprise one or more shRNAs that include miRNA scaffolds. In some embodiments, the miRNA scaffolds include miRNA-155 5' scaffold, miRNA-155 3' scaffold, miRNA-30 5' scaffold, miRNA-30 3' scaffold, miRNA-16 5' scaffold, miRNA-16 3' scaffold, miRNA-125 5' scaffold, miRNA-125 3' scaffold, miRNA-223 5' scaffold, or miRNA-223 3' scaffold.

[0282] In some embodiments, the modified immune cells of the present disclosure (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprise one or more shRNAs that include guide strands. In some embodiments, the guide strands are about 19-24 bases in length. In some embodiments, the guide strands have a G / C content of about 36%-50%. In some embodiments, the guide strands comprise a nucleic acid sequence that is reverse complementary to a target gene transcript comprising a target nucleic acid sequence.

[0283] In some embodiments, the modified immune cells of the present disclosure (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprise one or more shRNAs that include passenger strands. In some embodiments, the passenger strands are 1-2 bases shorter than the corresponding guide strands. In some embodiments, the passenger strands are not fully complementary to the guide strands.

[0284] In some embodiments, the modified immune cells of the present disclosure (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprise one or more shRNAs that include loops.

[0285] In some embodiments, the modified immune cells of the present disclosure (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprise target gene transcripts encoding human ATG7, C / EBP-α, C / EBP-β, CD36, CLEC1A, FATS, GOLM1, HAVCR2, ITGAD, KLF4, KLF6, LILRB2, LILRB4, MAF, MafB, PD-LI, PIK3CG, PIK3CG, PPARα, PPARγ, PTGS2, SIGLEC10, SIRPα, SLC15A3, STAT3, STAT6, TNFRSF1B, TOX, TREM2, YTHDF2, or ZFP36. In some embodiments, the modified immune cells of the present disclosure (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprise a target gene transcript encoding human SIRPα. In some embodiments, the modified immune cells of the present disclosure (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprise a target gene transcript encoding human PD-L1.

[0286] In some embodiments, the modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as described herein maintain a pro-inflammatory phenotype over time. In some embodiments, the modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as 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 modifying the immune cells with the nucleic acid encoding the CAR.

[0287] In some embodiments, the modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as described herein maintain an anti-inflammatory phenotype over time. In some embodiments, the modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as 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 modifying the immune cells with the nucleic acid encoding the CAR.

[0288] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as described herein maintain a pro-inflammatory phenotype and / or otherwise resist subversion when attacked by anti-inflammatory cytokines. In some embodiments, the sensitivity of the modified immune cells to environmental cytokines is measured by generating a dose-response curve of pro-inflammatory markers by treating the modified immune cells comprising a CAR as described herein with increasing concentrations of anti-inflammatory cytokines. In some embodiments, the sensitivity of the modified immune cells to environmental cytokines is measured by generating a dose-response curve of pro-inflammatory markers by treating the modified immune cells comprising a CAR as described herein with increasing concentrations of pro-inflammatory cytokines.

[0289] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as described herein maintain an anti-inflammatory phenotype and / or otherwise resist subversion when attacked by pro-inflammatory cytokines. In some embodiments, the sensitivity of the modified immune cells to environmental cytokines is measured by generating a dose-response curve of anti-inflammatory markers by treating the modified immune cells comprising a CAR as described herein with increasing concentrations of pro-inflammatory cytokines. In some embodiments, the sensitivity of the modified immune cells to environmental cytokines is measured by generating a dose-response curve of anti-inflammatory markers by treating the modified immune cells comprising a CAR as described herein with increasing concentrations of anti-inflammatory cytokines.

[0290] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as described herein have minimal effect on neighboring cells. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as described herein have a significant effect on neighboring cells. In some embodiments, the effect of modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as described herein on unmodified cells (e.g., immune cells not comprising a CAR as described 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 immune cells and unmodified immune cells can be co-cultured in a culture dish in which the modified immune cells and unmodified immune cells are in contact with each other. In some embodiments, the modified immune cells and unmodified immune cells can be co-cultured in a culture dish in which the modified immune cells and unmodified immune cells are separated by a transwell assay membrane.

[0291] In some embodiments, modified immune cells (such as, stem cells, macrophages, monocytes or dendritic cells) comprising a CAR as described herein have minimal cytotoxic effects on neighboring cells. In some embodiments, modified immune cells (such as, stem cells, macrophages, monocytes or dendritic cells) comprising a CAR as described herein have significant cytotoxic effects on neighboring cells (such as cancer cells). In some embodiments, modifying immune cells to comprise a CAR as described herein is non-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 cytotoxic effects.

[0292] In some embodiments, modified immune cells (such as, stem cells, macrophages, monocytes or dendritic cells) comprising a CAR as described herein can comprise one or more control systems, including but not limited to: safety switches (such as, on-switch, off-switch, suicide switch), transcriptional control (such as, cell-specific promoter, cell-state specific promoter, promoter downstream of CAR activation, promoter downstream of an endogenous signaling pathway or drug-induced transcription), post-transcriptional control of CAR mRNA (such as, RNA-based inhibition with endogenous or recombinant miRNAs), or post-translational control of CAR structure or stability (such as, a CAR whose intracellular domain associates conditionally with the intact structure by drug / light-induced association (to allow signal transduction) or dissociation (to inhibit signal transduction), or whose stability is regulated by a drug to achieve induced stabilization (to allow signal transduction) or degradation (to inhibit signal transduction)). These control systems can be combined to create logic gates, such as AND gates (such as, a CAR having a CAR-inducible promoter and a cytoplasmic domain that associates in a drug-dependent manner, thus requiring CAR activation and the presence of a small molecule), OR gates (such as, a CAR controlled by a promoter that is transcriptionally active upon CAR activation or addition of a small molecule) and / or NOT gates (such as, a CAR whose mRNA is degraded by an endogenous miRNA expressed in the native immune cell signaling state (such as an miRNA upregulated by a specific cytokine signaling pathway, thus expressing the CAR only in the absence of such a cytokine)).

[0293] In some embodiments, effector cells comprising a nucleic acid construct as described herein (i.e., the modified immune cells of the present disclosure (such as, stem cells, macrophages, monocytes or dendritic cells)) secrete one or more inhibitory RNAs of the present disclosure. In some embodiments, one or more inhibitory RNAs are packaged within extracellular vesicles. In some embodiments, one or more inhibitory RNAs are packaged within exosomes. In some embodiments, the secreted inhibitory RNAs affect 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 towards an anti-tumor phenotype. In some embodiments, the bystander cells are biased towards an anti-inflammatory phenotype. In some embodiments, the bystander macrophages are biased towards an M1 phenotype. In some embodiments, the bystander macrophages are biased towards an M2 phenotype.

[0294] Assay

[0295] A variety of assays can be performed to confirm the presence of a nucleic acid construct as described herein and / or the presence of a protein (such as, a CAR) in immune cells (such as, stem cells, macrophages, monocytes or dendritic cells). For example, such assays include molecular biology assays well known to those skilled in the art, such as DNA and RNA blotting, RT-PCR and PCR; and biochemical assays, such as detecting the presence or absence of a specific peptide 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 assays, mass spectrometry (MS), RNA-Seq and functional assays.

[0296] A variety of assays can be performed to determine various characteristics of the modified immune cells (such as, stem cells, macrophages, monocytes or dendritic cells), such as but not limited to immune cell viability, nucleic acid expression, nucleic acid lifespan, protein (such as, a CAR) expression, protein (such as, a CAR) lifespan, 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 assays of target tumor cells.

[0297] Nucleic acid construct

[0298] The present disclosure particularly provides nucleic acid molecules encoding at least one CAR or a fragment thereof as described herein. Immune cells (such as, stem cells, macrophages, monocytes or dendritic cells) can comprise a nucleic acid molecule (such as, an exogenous nucleic acid molecule) encoding at least one polypeptide as described herein (such as, one or more CARs of the present disclosure).

[0299] Unless otherwise specified, the phrase "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate to each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns to the extent that the nucleotide sequence encoding the protein may contain one or more introns in some forms. The term "encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) to serve as a template in a biological process for synthesizing other polymers and macromolecules having a defined nucleotide sequence (such as rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties that result therefrom. Thus, if transcription and translation of an mRNA corresponding to a gene produce a protein in a cell or other biological system, the gene, cDNA, or RNA encodes that protein. Both the coding strand, which is identical to the mRNA sequence and is typically provided in the sequence listing, and the non-coding strand that serves as the transcription template for a gene or cDNA can be said to encode the protein or other product of that gene or cDNA.

[0300] The term "operably linked" or "transcriptionally controlled" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence such that the heterologous nucleic acid sequence is expressed. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first and second nucleic acid sequences are in a functional relationship. 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 contiguous with each other and, as in the case where two protein-coding regions need to be joined, in the same reading frame.

[0301] The nucleic acid molecule encoding at least one protein (such as 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 (such as 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 (such as a CAR of the present disclosure) or a fragment thereof described herein.

[0302] In some embodiments, all or a fragment of the protein (such as at least one CAR of the present disclosure) described herein is encoded by a codon-optimized nucleic acid molecule, such as for expression in a cell (such as a mammalian cell). A variety of codon optimization methods are known in the art, such as those disclosed in U.S. Patent Nos. 5,786,464 and 6,114,148, each of which is hereby incorporated by reference in its entirety.

[0303] 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) promoter, the immediate early cytomegalovirus (CMV) promoter, the ubiquitin C promoter, the phosphoglycerate kinase (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 metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter. The vector can also include additional promoter elements such as enhancers to regulate the frequency of transcriptional initiation.

[0304] In some embodiments, a vector comprising a 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 technologies are well known in the art and are described (e.g., in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Volumes 1-4, Cold Spring Harbor Press, NY). Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, or retroviral vectors (e.g., lentiviral vectors or γ-retroviral vectors). In some embodiments, the vector comprises a lentiviral vector (e.g., as described in U.S. Patent No. 9,149,519 or International Publication No. WO 2017 / 044487, each of which is hereby incorporated by reference in its entirety).

[0305] In some embodiments, the viral vector comprises an adenoviral vector. Adenoviruses are a large family of viruses containing double-stranded DNA. They replicate in the nucleus of host cells by 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., the Ad5f35 adenoviral vector, such as the helper virus-dependent Ad5F35 adenoviral vector).

[0306] In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. The AAV system is generally well-known in the art (see, e.g., Kelleher and Vos, Biotechniques, 17(6):1110-17 (1994); Cotten et al., P.N.A.S. U.S.A., 89(13):6094-98 (1992); Curiel, Nat Immun, 13(2-3):141-64 (1994); Muzyczka, Curr Top Microbiol Immunol, 158:97-129 (1992); and Asokan A et al., Mol. Ther., 20(4):699-708 (2012)). Methods for generating and using recombinant AAV (rAAV) vectors are described, for example, in U.S. Patent Nos. 5,139,941 and 4,797,368.

[0307] 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 the proteins described herein (e.g., at least one CAR of the present disclosure) or fragments thereof. In some embodiments, the AAV serotype has tropism for a particular tissue.

[0308] In some embodiments, the CRISPR / Cas9 system has recently been shown to facilitate high levels of precise genome editing using an adeno-associated virus (AAV) vector as a donor template DNA during homologous recombination (HR).

[0309] In some embodiments, the vector includes a gamma-retroviral vector (e.g., as described in Tobias Maetzig et al., “Gammaretroviral Vectors: Biology, Technology and Application” Viruses. June 2011; 3(6):677–713, which is hereby incorporated by reference in its entirety). Exemplary gamma-retroviral vectors include murine leukemia virus (MLV), spleen focus-forming virus (SFFV), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom.

[0310] In some embodiments, the vector comprises two or more nucleic acid sequences encoding a CAR (e.g., at least one CAR as described herein) and a second CAR (e.g., a different CAR as described herein). In some embodiments, the two or more nucleic acid sequences encoding the CAR and the second CAR are, for example, in the same frame and are encoded by a single nucleic acid molecule as a single polypeptide chain. In some embodiments, the two or more CARs are separated by one or more cleavage peptide sites (e.g., self-cleaving sites or substrates of intracellular proteases). In certain embodiments, the cleavage peptide comprises a porcine teschovirus-1 (P2A) peptide, a Thosea asigna virus (T2A) peptide, an equine rhinitis A virus (E2A) peptide, a foot-and-mouth disease virus (F2A) peptide, or variants thereof.

[0311] In some embodiments, the vector comprises at least one nucleic acid sequence encoding a CAR (e.g., at least one CAR as described herein) and at least one nucleic acid encoding at least one gene co-expressed with the CAR (e.g., a cytokine as described herein (e.g., TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, and / or IL-1) or a stimulatory ligand as 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 β receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds to a Toll ligand receptor, and / or a B7-H3 ligand)).

[0312] In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 4. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by a nucleic acid sequence that is at least 85% identical to a sequence selected from Table 4. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by a nucleic acid sequence that is at least 90% identical to a sequence selected from Table 4. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by a nucleic acid sequence that is at least 95% identical to a sequence selected from Table 4. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by a nucleic acid sequence that is at least 96% identical to a sequence selected from Table 4. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by a nucleic acid sequence that is at least 97% identical to a sequence selected from Table 4. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by a nucleic acid sequence that is at least 98% identical to a sequence selected from Table 4. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by a nucleic acid sequence that is at least 99% identical to a sequence selected from Table 4. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by a nucleic acid sequence that is identical to a sequence selected from Table 4.

[0313] In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by nucleic acid sequences that differ from the sequences selected from Table 4 by no more than 100 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by nucleic acid sequences that differ from the sequences selected from Table 4 by no more than 75 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by nucleic acid sequences that differ from the sequences selected from Table 4 by no more than 50 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by nucleic acid sequences that differ from the sequences selected from Table 4 by no more than 40 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by nucleic acid sequences that differ from the sequences selected from Table 4 by no more than 30 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by nucleic acid sequences that differ from the sequences selected from Table 4 by no more than 20 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by nucleic acid sequences that differ from the sequences selected from Table 4 by no more than 10 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by nucleic acid sequences that differ from the sequences selected from Table 4 by no more than 9 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by nucleic acid sequences that differ from the sequences selected from Table 4 by no more than 8 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by nucleic acid sequences that differ from the sequences selected from Table 4 by no more than 7 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by nucleic acid sequences that differ from the sequences selected from Table 4 by no more than 6 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by nucleic acid sequences that differ from the sequences selected from Table 4 by no more than 5 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by nucleic acid sequences that differ from the sequences selected from Table 4 by no more than 4 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by nucleic acid sequences that differ from the sequences selected from Table 4 by no more than 3 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by nucleic acid sequences that differ from the sequences selected from Table 4 by no more than 2 substitutions, additions, or deletions. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by nucleic acid sequences that differ from the sequences selected from Table 4 by no more than 1 substitution, addition, or deletion. In some embodiments, the CARs of the present disclosure bind to mesothelin and are encoded by nucleic acid sequences that have no substitutions, additions, or deletions relative to the sequences selected from Table 4.

[0314] In some embodiments, the CAR antigen-binding domain binds to 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to mesothelin and is encoded by a nucleic acid sequence that is identical to a sequence selected from Table 5.

[0315] In some embodiments, the CAR antigen-binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 50 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 40 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 30 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 20 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 10 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 9 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 8 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 7 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 6 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 5 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 4 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 3 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 2 substitutions, additions, or deletions. In some embodiments, the CAR antigen-binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 1 substitution, addition, or deletion. In some embodiments, the CAR antigen-binding domain binds to mesothelin and is encoded by a nucleic acid sequence that has no substitutions, additions, or deletions relative to a sequence selected from Table 5.

[0316] In some embodiments, the present disclosure provides nucleic acid molecules comprising a promoter, a target gene, a 3' untranslated region (UTR), and one or more introns. In some embodiments, one or more introns of the present disclosure comprise one or more inhibitory nucleic acids of the present disclosure. In some embodiments, one or more inhibitory nucleic acids of the present disclosure encode one or more inhibitory RNAs of the present disclosure. In some embodiments, the target gene encodes at least one chimeric antigen receptor (CAR) or a fragment thereof described herein. A modified immune cell of the present disclosure (e.g., a stem cell, macrophage, monocyte, or dendritic cell) 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 nucleic acid molecules in which the expression of the CAR is increased relative to a reference nucleic acid molecule lacking one or more introns.

[0317] Intron

[0318] In some embodiments, one or more introns of the present disclosure comprise one, two, or three introns. In some embodiments, one or more introns of the present disclosure are located downstream of the promoter. In some embodiments, one or more introns of the present disclosure are located within the target gene. In some embodiments, one or more introns of the present disclosure are located within the 3’UTR. In some embodiments, one or more introns of the present disclosure are located within the target gene and within the 3’UTR. In some embodiments, 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 the following miRNA scaffolds in order from 5' to 3': (a) miRNA 30, miRNA 30, miRNA 30, (b) miRNA 30, miRNA 155, miRNA 30, (c) miRNA 155, miRNA 30, miRNA 155, or (d) miRNA 155, miRNA 155, miRNA 155.

[0319] Inhibitory nucleic acid

[0320] The inhibitory nucleic acids of the present disclosure include any nucleic acid that can bind to a target messenger RNA (mRNA). In some embodiments, the inhibitory nucleic acids of the present disclosure include RNA. In some embodiments, the inhibitory nucleic acids of the present disclosure include DNA. In some embodiments, the inhibitory nucleic acids of the present disclosure are DNA / RNA hybrids. In some embodiments, the inhibitory nucleic acids of the present disclosure are inhibitory RNAs. In some embodiments, the inhibitory RNA silences the 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-natural antisense transcript (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 shRNA.

[0321] In some embodiments, the shRNAs of the present disclosure include a stem-loop structure. In some embodiments, the shRNAs of the present disclosure include a stem-loop structure flanked on either side of the stem by at least 10 bases. In some embodiments, the stem is a double-stranded RNA structure. In some embodiments, the length of the stem on each side is about 30-40 bases. In some embodiments, the length of the stem on each side is about 30-39 bases. In some embodiments, the length of the stem on each side is about 30-38 bases. In some embodiments, the length of the stem on each side is about 30-37 bases. In some embodiments, the length of the stem on each side is about 30-36 bases. In some embodiments, the length of the stem on each side is about 30-35 bases. In some embodiments, the length of the stem on each side is about 30-34 bases. In some embodiments, the length of the stem on each side is about 30-33 bases. In some embodiments, the length of the stem on each side is about 30-32 bases. In some embodiments, the length of the stem on each side is about 30-31 bases. In some embodiments, the length of the stem on each side is about 31-40 bases. In some embodiments, the length of the stem on each side is about 32-40 bases. In some embodiments, the length of the stem on each side is about 33-40 bases. In some embodiments, the length of the stem on each side is about 34-40 bases. In some embodiments, the length of the stem on each side is about 35-40 bases. In some embodiments, the length of the stem on each side is about 36-40 bases. In some embodiments, the length of the stem on each side is about 37-40 bases. In some embodiments, the length of the stem on each side is about 38-40 bases. In some embodiments, the length of the stem on each side is about 39-40 bases. In some embodiments, the length of the stem on each side is 35 bases. In some embodiments, the shRNAs of the present disclosure include a scaffold. In some embodiments, the shRNAs of the present disclosure include a 5' scaffold. In some embodiments, the shRNAs of the present disclosure include a 3' scaffold. In some embodiments, the shRNAs of the present disclosure include a guide strand. In some embodiments, the shRNAs of the present disclosure include a passenger strand. In some embodiments, the shRNAs of the present disclosure include a loop connecting the guide strand and the passenger strand. In some embodiments, the length of the loop is about 8-20 bases. In some embodiments, the length of the loop is about 8-19 bases. In some embodiments, the length of the loop is about 8-18 bases. In some embodiments, the length of the loop is about 8-17 bases. In some embodiments, the length of the loop is about 8-16 bases. In some embodiments, the length of the loop is about 8-15 bases. In some embodiments, the length of the loop is about 8-14 bases. In some embodiments, the length of the loop is about 8-13 bases.In some embodiments, the length of the loop is about 8 - 12 bases. In some embodiments, the length of the loop is about 8 - 11 bases. In some embodiments, the length of the loop is about 8 - 10 bases. In some embodiments, the length of the loop is about 8 - 9 bases. In some embodiments, the length of the loop is about 9 - 20 bases. In some embodiments, the length of the loop is about 10 - 20 bases. In some embodiments, the length of the loop is about 11 - 20 bases. In some embodiments, the length of the loop is about 12 - 20 bases. In some embodiments, the length of the loop is about 13 - 20 bases. In some embodiments, the length of the loop is about 14 - 20 bases. In some embodiments, the length of the loop is about 15 - 20 bases. In some embodiments, the length of the loop is about 16 - 20 bases. In some embodiments, the length of the loop is about 17 - 20 bases. In some embodiments, the length of the loop is about 18 - 20 bases. In some embodiments, the length of the loop is about 19 - 20 bases. In some embodiments, the portion of the stem closest to the loop comprises a guide strand and a messenger strand. In some embodiments, the portion of the stem farthest from the loop comprises a 5' scaffold and a 3' scaffold. In some embodiments, the shRNA of the present disclosure comprises, from 5' to 3', a 5' scaffold, a guide strand, a loop, a messenger strand, and a 3' scaffold. In some embodiments, the shRNA of the present disclosure comprises, from 5' to 3', a 5' scaffold, a messenger strand, a loop, a guide strand, and a 3' scaffold.

[0322] In some embodiments, the scaffolds of the present disclosure comprise miRNA scaffolds. In some embodiments, the miRNA scaffolds comprise miRNA-155 5' scaffolds, miRNA-155 3' scaffolds, miRNA-30 5' scaffolds, miRNA-30 3' scaffolds, miRNA-16 5' scaffolds, miRNA-16 3' scaffolds, miRNA-125 5' scaffolds, miRNA-125 3' scaffolds, miRNA-223 5' scaffolds, or miRNA-223 3' scaffolds.

[0323] In some embodiments, the length of the guide strand of the present disclosure is about 19 - 24 bases. In some embodiments, the length of the guide strand of the present disclosure is 19 - 24 bases. In some embodiments, the length of the guide strand is 18 bases. In some embodiments, the length of the guide strand is 19 bases. In some embodiments, the length of the guide strand is 20 bases. In some embodiments, the length of the guide strand is 21 bases. In some embodiments, the length of the guide strand is 22 bases. In some embodiments, the length of the guide strand is 23 bases. In some embodiments, the length of the guide strand is 24 bases. In some embodiments, the length of the guide strand is 25 bases.

[0324] In some embodiments, the length of the messenger strand of the present disclosure is about 17-24 bases. In some embodiments, the length of the messenger strand of the present disclosure is about 17-22 bases. In some embodiments, the length of the messenger strand of the present disclosure is 17-22 bases. In some embodiments, the length of the messenger strand is 17 bases. In some embodiments, the length of the messenger strand is 18 bases. In some embodiments, the length of the messenger strand is 19 bases. In some embodiments, the length of the messenger strand is 20 bases. In some embodiments, the length of the messenger strand is 21 bases. In some embodiments, the length of the messenger strand is 22 bases. In some embodiments, the length of the messenger strand is 23 bases. In some embodiments, the length of the messenger strand is 22 bases. In some embodiments, the length of the messenger strand is 24 bases. In some embodiments, the messenger strand is 1-2 bases shorter than the corresponding guide strand. In some embodiments, the messenger strand is 1 base shorter than the corresponding guide strand. In some embodiments, the messenger strand is 2 bases shorter than the corresponding guide strand. In some embodiments, the messenger strand has the same length as the corresponding guide strand.

[0325] 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%.

[0326] In some embodiments, the messenger strand of the present disclosure has a G / C content of about 36%-50%. In some embodiments, the messenger strand of the present disclosure has a G / C content of 36%-50%. In some embodiments, the messenger strand has a G / C content of 35%. In some embodiments, the messenger strand has a G / C content of 36%. In some embodiments, the messenger strand has a G / C content of 37%. In some embodiments, the messenger strand has a G / C content of 38%. In some embodiments, the messenger strand has a G / C content of 39%. In some embodiments, the messenger strand has a G / C content of 40%. In some embodiments, the messenger strand has a G / C content of 41%. In some embodiments, the messenger strand has a G / C content of 42%. In some embodiments, the messenger strand has a G / C content of 43%. In some embodiments, the messenger strand has a G / C content of 44%. In some embodiments, the messenger strand has a G / C content of 45%. In some embodiments, the messenger strand has a G / C content of 46%. In some embodiments, the messenger strand has a G / C content of 47%. In some embodiments, the messenger strand has a G / C content of 48%. In some embodiments, the messenger strand has a G / C content of 49%. In some embodiments, the messenger strand has a G / C content of 50%. In some embodiments, the messenger strand has a G / C content of 51%.

[0327] In some embodiments, the stem-loop formed by the 5' scaffold, the guide strand, the messenger strand, and the 3' scaffold is recognized by Drosha. In some embodiments, Drosha cleaves 10-15 bases above the stem-loop bases. In some embodiments, the stem-loop cleaved by Drosha will be recognized by Dicer. In some embodiments, Dicer cleaves 20-24 bases from the 5' and 3' ends of the stem-loop.

[0328] In some embodiments, the guide strand of the present disclosure comprises a nucleic acid sequence that is reverse complementary to a target gene transcript comprising a target nucleic acid sequence. In some embodiments, the messenger strand of the present disclosure comprises a nucleic acid sequence that is not fully complementary to the corresponding guide strand. In some embodiments, the messenger strand comprises a nucleic acid sequence that is not fully complementary to the corresponding guide strand by one nucleotide. In some embodiments, the messenger 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 messenger 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 messenger strand of the present disclosure comprises a nucleic acid sequence that is not fully complementary to the corresponding guide strand by four nucleotides.

[0329] In some embodiments, the shRNAs of the present disclosure include: (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 5 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, the shRNAs of the present disclosure include a nucleic acid sequence selected from Table 6a, Table 6b, Table 6c, Table 6d, or Table 6e. In some embodiments, the shRNAs of the present disclosure include 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 5 substitutions, additions, or deletions. In some embodiments, the shRNAs of the present disclosure include 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.

[0330] In some embodiments, the inhibitory RNAs of the present disclosure are or include miRNAs. In some embodiments, the miRNAs of the present disclosure include: (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 5 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, the miRNAs of the present disclosure include a nucleic acid sequence selected from Table 7. In some embodiments, the miRNAs of the present disclosure include a nucleic acid sequence that differs from a sequence selected from Table 7 by no more than 5 substitutions, additions, or deletions. In some embodiments, the miRNAs of the present disclosure include a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 7.

[0331] Targets of Inhibitory Nucleic Acids

[0332] 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 containing complementary sequences. In some embodiments, the inhibitory nucleic acids of the present disclosure induce the degradation of target mRNAs. In some embodiments, the inhibitory nucleic acids of the present disclosure inhibit the translation of target mRNAs.

[0333] 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) reduces the translation of the target gene transcript. In some embodiments, the target gene transcript is expressed in immune cells. In some embodiments, the immune cells are stem cells, macrophages, monocytes or dendritic cells. In some embodiments, the target gene transcript is expressed in macrophages. In some embodiments, the target gene transcript is expressed in monocytes. In some embodiments, the reduction in the translation of the target gene transcript is associated with the M1 phenotype. In some embodiments, the reduction in the translation of the target gene transcript is associated with the M2 phenotype. In some embodiments, the increased expression of the inhibitory nucleic acids (e.g., RNA) of the present disclosure is associated with the M1 phenotype. In some embodiments, the increased expression of the inhibitory nucleic acids (e.g., RNA) of the present disclosure is associated with the M2 phenotype.

[0334] In some embodiments, the target gene transcripts of the present disclosure include: (a) a target nucleic acid sequence selected from Table 8; (b) a target nucleic acid sequence that differs from the sequence selected from Table 8 by no more than 5 substitutions, additions or deletions; or (c) a target nucleic acid sequence that is at least 80% identical to the sequence selected from Table 8. In some embodiments, the target gene transcripts of the present disclosure include a target nucleic acid sequence selected from Table 8. In some embodiments, the target gene transcripts of the present disclosure include a target nucleic acid sequence that differs from the sequence selected from Table 8 by no more than 5 substitutions, additions or deletions. In some embodiments, the target gene transcripts of the present disclosure include a target nucleic acid sequence that is at least 80% identical to the sequence selected from Table 8.

[0335] In some embodiments, the target gene transcripts of the present disclosure encode human ATG7, C / EBP-α, C / EBP-β, CD36, CLEC1A, FATS, GOLM1, HAVCR2, ITGAD, KLF4, KLF6, LILRB1, LILRB2, LILRB4, MAF, MafB, PD-L1, PIK3CG, PIK3CG, PPARα, PPARγ, PTGS2, SIGLEC7, SIGLEC10, SIRPα, SLC15A3, STAT3, STAT6, TNFRSF1B, TOX, TREM2, YTHDF2 or ZFP36. In some embodiments, the target gene transcripts of the present disclosure encode human SIRPα. In some embodiments, the target gene transcripts of the present disclosure encode human PD-1 or PD-L1.

[0336] In some embodiments, the inhibitory nucleic acids of the present disclosure include one or more inhibitory nucleic acids. In some embodiments, the one or more inhibitory nucleic acids include one inhibitory nucleic acid. In some embodiments, the one or more inhibitory nucleic acids include at least two, three, four, or five inhibitory nucleic acids. In some embodiments, the one or more inhibitory nucleic acids of the present disclosure include two inhibitory nucleic acids. In some embodiments, the one or more inhibitory nucleic acids of the present disclosure include three inhibitory nucleic acids. In some embodiments, the one or more inhibitory nucleic acids of the present disclosure include four inhibitory nucleic acids. In some embodiments, the one or more inhibitory nucleic acids of the present disclosure include five inhibitory nucleic acids.

[0337] In some embodiments, at least two, three, four, or five inhibitory nucleic acids are in tandem. In some embodiments, at least two, three, four, or five inhibitory nucleic acids contain the same sequence. In some embodiments, at least two, three, four, or five inhibitory nucleic acids contain at least two different sequences. In some embodiments, at least three, four, or five inhibitory nucleic acids contain at least three different sequences. In some embodiments, at least four or five inhibitory nucleic acids contain at least four different sequences. In some embodiments, at least five inhibitory nucleic acids contain 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.

[0338] 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, 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.

[0339] Methods of designing inhibitory nucleic acids

[0340] The present disclosure also provides methods for designing the inhibitory nucleic acids of the present disclosure. In some embodiments, the present disclosure provides methods for designing the inhibitory RNAs of the present disclosure. In some embodiments, the present disclosure provides methods for designing the shRNAs of the present disclosure. In some embodiments, the 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', adenine or uracil at position 1, adenine, guanine, or cytosine at position 10, and a G / C content that increases from 5' to 3' by 36% to 45%. In some embodiments, the guide strand comprises a higher G / C content of 10 bases at the 3' end of the guide strand relative to the G / C content of 10 bases at the 5' end of the guide strand. In some embodiments, the passenger strand comprises, from 5' to 3', the sequence of the target region with the following alterations: (i) a deletion of a nucleotide at position 7 relative to the loop, (ii) a deletion of a nucleotide at position 11 relative to the loop, and (iii) a mutation of adenine to guanine and / or cytosine to uracil near the deletions at positions 7 and 11, wherein the mutations create two partial G-U base pairs between the passenger strand and the guide strand. In some embodiments, the passenger strand is 2 base pairs shorter than the guide strand. When calculating nucleotide positions relative to the loop, position 1 is closest to the loop, and the position count increases as it moves away from the loop.

[0341] In some embodiments, the 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, wherein the guide strand is the reverse complement of the target region, and wherein the guide strand comprises, from 5' to 3', adenine or uracil at position 1, adenine, guanine, or cytosine at position 10, and a G / C content that increases from 5' to 3' by 36% to 45%; and (c) designing the passenger strand, wherein the passenger strand comprises, from 5' to 3', the sequence of the target region with the following alterations: (i) a deletion of a nucleotide at position 7 relative to the loop, (ii) a deletion of a nucleotide at position 11 relative to the loop, (iii) a mutation of adenine to guanine and / or cytosine to uracil near the deletions at positions 7 and 11, wherein the mutations create two partial G-U base pairs between the passenger strand and the guide strand; and wherein the passenger strand is 2 base pairs shorter than the guide strand. In some embodiments, the method for designing an shRNA comprises a target region of a target gene transcript that is 21-22 base pairs in length.

[0342] Drug composition

[0343] The present disclosure particularly provides a pharmaceutical composition comprising a modified immune cell (such as a stem cell, macrophage, monocyte or dendritic cell) containing one or more CARs as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. The present disclosure particularly also provides a pharmaceutical composition comprising a nucleic acid encoding one or more CARs as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

[0344] When indicating a "therapeutically effective amount", "immunologically effective amount", "anti-immune response effective amount" or "immune response inhibitory effective amount", the precise amount of the pharmaceutical composition 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).

[0345] The pharmaceutical composition described herein may comprise a buffer, such as neutral buffered saline or phosphate buffered saline (PBS); carbohydrates, such as glucose, mannose, sucrose, dextran or mannitol; proteins, polypeptides or amino acids (such as glycine); antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); serum and preservatives, such as cryoprotectants. In some embodiments, the pharmaceutical composition is substantially free of contaminants, such as no detectable level of contaminants (such as endotoxin).

[0346] The pharmaceutical composition described herein can be administered in a manner suitable 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 appropriate doses can be determined through clinical trials.

[0347] The pharmaceutical composition described herein can be in a variety of forms. These forms 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 can be injectable or infusible solutions. The pharmaceutical composition described herein can be formulated for intravenous, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intraarterial or intraperitoneal administration.

[0348] 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 by using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988, which is hereby incorporated by reference in its entirety).

[0349] As used herein, the terms "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration that are typically effected by injection or infusion, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intratumoral, and intrasternal injection and infusion.

[0350] A pharmaceutical composition comprising the modified immune cells described herein can be administered at a dose of about 10 4 to about 10 9 cells / kg body weight (e.g., about 10 5 to about 10 6 cells / kg body weight) (including all integer values within those ranges). In some embodiments, the dose of the immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein comprises at least about 1x10 6 、about 1.1x10 6 、about 2x10 6 、about 3.6x10 6 、about 5x10 6 、about 1x10 7 、about 1.8x10 7 、about 2x10 7 、about 5x10 7 、about 1x10 8 、about 2x10 8 、about 5x10 8 、about 1x10 9 、about 2x10 9 or about 5x10 9cells. The pharmaceutical compositions described herein can also be administered in multiple doses. Those skilled in the art can readily determine the optimal dosage and treatment regimen for a particular patient by monitoring the signs of the patient's disease, disorder or condition and adjusting the treatment accordingly.

[0351] It may be desirable to administer the pharmaceutical compositions described herein to a subject, then draw blood (or perform apheresis), activate the collected immune cells, and reinfuse the activated immune cells into the subject. This process can be performed multiple times, such as 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 including multiple blood draws and reinfusions described herein can be selected for certain immune cell populations.

[0352] In some embodiments, the pharmaceutical compositions described herein are administered in combination with a second therapy (e.g., before, simultaneously with or after the second therapy). For example, the second therapy can include, but is not limited to, antiviral therapies (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 mofetil, FK506 antibody or glucocorticoids), antagonists (e.g., one or more of a PD-1 antagonist, a PD-L1 antagonist, a CTLA4 antagonist, a CD47 antagonist, a SIRPα antagonist, a CD40 agonist, a CSF1 / CSF1R antagonist or a STING agonist) or immune ablative agents (e.g., anti-CD52 antibody (e.g., alemtuzumab), anti-CD3 antibody, cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228 or irradiation).

[0353] In some embodiments, the pharmaceutical compositions described herein are administered in combination with (e.g., before, concurrently with, or after) a bone marrow transplant or lymphodepleting therapy using chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide, or rituximab (Rituxan). In certain embodiments, the subject undergoes standard therapy with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives an infusion of the pharmaceutical composition comprising the immune cells described herein. The pharmaceutical compositions described herein can be administered before or after surgery.

[0354] The dosage of any of the foregoing therapies to be administered to a subject will vary with the disease, disorder, or condition being treated and will be based on the particular subject. Dosage scaling for human administration can be carried out according to practices accepted in the art. For example, for an adult, the dosage of alemtuzumab will generally be from about 1 mg to about 100 mg, typically administered daily for a period of about 1 day to about 30 days, such as a daily dosage of about 1 mg to about 10 mg (e.g., as described in U.S. Patent No. 6,120,766, which is hereby incorporated by reference in its entirety).

[0355] Methods of Treatment

[0356] The present disclosure particularly provides methods of treating a disease or disorder (e.g., a disease or disorder described herein) in a subject, which comprise delivering the pharmaceutical compositions described herein. In some embodiments, a therapeutically effective amount of the pharmaceutical compositions described herein is administered to a subject having a disease or disorder. The pharmaceutical compositions described herein can be used to manufacture a medicament for treating a disease or disorder in a subject or for stimulating an immune response in a subject.

[0357] The subject to be treated by the methods described herein can be a mammal, such as a primate, such as a human (e.g., a patient having a disease or disorder described herein 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 to the subject. The pharmaceutical compositions described herein can be administered to the subject alone or in combination with one or more therapeutic agents, therapeutic procedures, or modalities according to the dosage regimens described herein.

[0358] The pharmaceutical compositions described herein can be used to treat or prevent diseases associated with tumors or cancer.

[0359] Methods are provided for treating (e.g., reducing, inhibiting, or delaying one or more in its progression) cancer or tumor in a subject with the pharmaceutical compositions described herein. The subject can have cancer in adult or pediatric form. The cancer can be in an early, intermediate, or late stage, or can be metastatic cancer. The cancer can include, but is not limited to, solid tumors, blood cancers (such as leukemia, lymphoma, or myeloma, such as multiple myeloma), or metastatic lesions. Examples of solid tumors include malignancies such as sarcomas and carcinomas, such as adenocarcinomas of various organ systems, such as those affecting the lung, breast, ovary, lymph, gastrointestinal (such as colon), anal, genital, and urogenital tracts (such as kidney, urothelial, bladder cell, prostate), pharynx, CNS (such as brain, nerve, or glial cells), head and neck, skin (such as melanoma, such as cutaneous melanoma), pancreas, and bone (such as chordoma).

[0360] In some embodiments, the cancer is selected from lung cancer (such as non-small cell lung cancer (NSCLC) (such as NSCLC with squamous and / or non-squamous histology or NSCLC adenocarcinoma) or small cell lung cancer (SCLC)), skin cancer (such as Merkel cell carcinoma or melanoma (such as advanced melanoma)), ovarian cancer, mesothelioma, bladder cancer, soft tissue sarcoma (such as hemangiopericytoma (HPC)), bone cancer (osteosarcoma), kidney cancer (such as renal cancer (such as renal cell carcinoma)), liver cancer (such as hepatocellular carcinoma), cholangiocarcinoma, sarcoma, myelodysplastic syndrome (MDS), prostate cancer, breast cancer (such as breast cancer that does not express one, two, or all of estrogen receptor, progesterone receptor, or Her2 / neu, such as triple-negative breast cancer), colorectal cancer (such as recurrent colorectal cancer or metastatic colorectal cancer, such as microsatellite unstable colorectal cancer, microsatellite stable colorectal cancer, mismatch repair-proficient colorectal cancer, or mismatch repair-deficient colorectal cancer), nasopharyngeal cancer, duodenal cancer, endometrial cancer, pancreatic cancer, head and neck cancer (such as head and neck squamous cell carcinoma (HNSCC)), anal cancer, gastroesophageal cancer, thyroid cancer (such as undifferentiated thyroid cancer), cervical cancer (such as cervical squamous cell carcinoma), neuroendocrine tumor (NET) (such as atypical pulmonary carcinoid tumor), lymphoproliferative disorder (such as post-transplant lymphoproliferative disorder), lymphoma (such as T cell lymphoma, B cell lymphoma, or non-Hodgkin lymphoma), myeloma (such as multiple myeloma), or leukemia (such as myeloid leukemia or lymphoid leukemia).

[0361] In some embodiments, the cancer is a brain tumor, such as glioblastoma, gliosarcoma, or recurrent brain tumor. In some embodiments, the cancer is pancreatic cancer, such as advanced pancreatic cancer. In some embodiments, the cancer is skin cancer, such as 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, such as renal cell carcinoma (RCC) (e.g., metastatic renal cell carcinoma). In some embodiments, the cancer is breast cancer, such as metastatic breast cancer or stage IV breast cancer, such as triple-negative breast cancer (TNBC). In some embodiments, the cancer is a virus-associated cancer. In some embodiments, the cancer is anal cancer (e.g., anal squamous cell carcinoma). In some embodiments, the cancer is cervical cancer (e.g., cervical squamous cell carcinoma). 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 head and neck squamous cell carcinoma (SCCHN)). In some embodiments, the cancer is nasopharyngeal cancer (NPC). In some embodiments, the cancer is colorectal cancer, such as recurrent colorectal cancer or metastatic colorectal cancer, such as microsatellite unstable colorectal cancer, microsatellite stable colorectal cancer, mismatch repair-proficient colorectal cancer, or mismatch repair-deficient colorectal cancer).

[0362] In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is 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 lymphoma, such as Hodgkin lymphoma (HL), non-Hodgkin's lymphoma, lymphocytic lymphoma, or diffuse large B-cell lymphoma (DLBCL) (e.g., recurrent or refractory HL or DLBCL). In some embodiments, the cancer is myeloma, such as multiple myeloma.

[0363] The pharmaceutical compositions described herein can be used to enhance or modulate the immune response of a subject. In one embodiment, the pharmaceutical compositions described herein enhance, stimulate, or increase the immune response of a subject (e.g., a subject having or at risk of having 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 treatment and / or radiation therapy.

[0364] The administration of the pharmaceutical compositions described herein can be carried out in any convenient manner (e.g., injection, ingestion, infusion, inhalation, implantation, or transplantation). 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 via artery, subcutaneously, intravenously, intradermally, intratumorally, intra-articularly, intramedullary, 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 directly injected into the inflammatory site, local disease site, lymph node, organ, tumor, or infection site of a subject.

[0365] All publications, patent applications, patents, and other references (including GenBank accession numbers) mentioned herein are incorporated by reference in their entirety. Additionally, the materials, methods, and examples are illustrative only and 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.

[0366] The present disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only. They should not be construed as limiting the scope or content of the present disclosure in any way.

[0367] Examples

[0368] The following examples are provided to describe to the skilled artisan how to make and use the methods and compositions described herein and are not intended to limit the scope of the present disclosure.

[0369] As shown below, Table 1 includes the exemplary CAR constructs described herein. Figure 28 and 29 shows schematic diagrams of these exemplary CAR constructs.

[0370] Table 1. Exemplary CAR constructs described herein

[0371]

[0372] Example 1: Mesothelin-Binding Agent Screening

[0373] This example evaluated 20 different mesothelin binders (M1 - M20) for use in a CD8 - framework CAR. All 20 CARs were manufactured as 5 - methoxyuridine (5moU) mRNA. The CAR mRNA was electroporated into human primary macrophages using a MaxCyte Atx instrument. Twenty - four hours after electroporation, CAR expression was evaluated via the binding of biotinylated mesothelin followed by APC - streptavidin. A CAR adenovirus based on SS1 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.

[0374] As Figure 1A , 1B and shown in 1C, electroporating macrophages with mRNA encoding the CAR did not have a negative impact on cell viability. Additionally, as Figure 1B and Figure 1C illustrate, among the 20 different anti - mesothelin CARs tested, M14, M15, and M17 had the best expression in macrophages.

[0375] Example 2: Killing and cytokine secretion of macrophages expressing anti - mesothelin binders

[0376] This example evaluated four different mesothelin binders (M11, M14, M15, and M17) for use in a CD8 - framework CAR. All four CARs were manufactured as 5 - methoxyuridine (5moU) mRNA. The CAR mRNA was electroporated into human primary macrophages using a MaxCyte ATx instrument. Twenty - four hours after electroporation, cytokine release was evaluated by co - incubating for 24 hours with K562 WT (mesothelin - negative) or K562MESO (mesothelin - positive) cells at a 1:1 effector - to - target (E:T) ratio. Killing 24 hours after electroporation was also evaluated by measuring the change in GFP fluorescence over 72 hours on an Incucyte S3 live - cell analysis system. A CAR adenovirus based on SS1 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.

[0377] As Figure 2 shown, among the four anti - mesothelin binders (M11, M14, M15, and M17) tested, only M15 and M17 mediated target - cell killing. When macrophages expressing an exemplary anti - mesothelin CAR were co - incubated with K562 cells, M15 and M17 CAR macrophages mediated the release of the TNFα cytokine from mesothelin - positive K562 target cells. These data are shown in Figure 3 .

[0378] Example 3: Phagocytosis Screening of Macrophages Expressing Anti-Mesothelin Binding Agents

[0379] This example evaluated four different anti-mesothelin binding agents (M11, M14, M15, and M17) for use in a CD8 framework CAR. All four CARs were manufactured as 5-methoxyuridine (5moU) mRNA. The CAR mRNA was electroporated into human primary macrophages using a MaxCyte ATx instrument. Twenty-four hours after electroporation, phagocytosis was evaluated by co-incubating with K562 WT (mesothelin negative) or K562MESO (mesothelin positive) at a 1:1 E:T for 4 hours. At the end time point, cells were analyzed via FACS, and CD11β+ / GFP+ events were defined as phagocytosis. An adenovirus containing a 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-targeted CAR control.

[0380] As Figure 4 shown, macrophages expressing all four anti-mesothelin binding agents (M11, M14, M15, and M17) mediated phagocytosis of mesothelin-positive target cells.

[0381] Example 4: Phenotypic Analysis of Macrophages Transduced with CTX_269

[0382] This example evaluated the phenotype of macrophages transduced with CTX_269. Previously cryopreserved macrophages from three donors were thawed and transduced with an Ad5f35 vector containing CTX_269 at different exemplary MOIs. Forty-eight hours later, cell viability, CAR expression, and M1 / M2 polarization were analyzed via FACS.

[0383] As Figure 5 shown, the Ad5f35 vector containing CTX_269 had no significant effect on viability at the tested MOIs. Additionally, as Figure 6 shown, anti-mesothelin CAR (CTX_269) expression was robust and could be titrated based on the viral MOI. Figure 7A 、 7B and 7C show that infecting macrophages with an Ad5f35 vector containing CTX_269 induced upregulation of the exemplary M1-related markers CD80, CD86, and HLA-DR. M1 marker expression increased with increasing adenovirus MOI. In addition to the upregulation of M1-related markers, infecting macrophages with an Ad5f35 vector containing CTX_269 also induced downregulation of the exemplary M2-related markers CD163 and CD206 ([[]] Figure 8 shown). M2 marker expression decreased with increasing adenovirus MOI.

[0384] Example 5: Resistance of Macrophages Transduced with Anti-Mesothelin Binding Agents to M2 Cytokines

[0385] This example evaluated the resistance of CTX_269-transduced macrophages to M2 cytokines. Previously frozen macrophages from three donors were thawed and transduced with an Ad5f35 vector containing CTX_269 at 3000 MOI. An anti-HER2 CAR delivered via Ad5f35 (CT0508) at an MOI of 3000 was used as a positive control. After 48 hours, the Ad5f35 virus was removed from the macrophage cultures. Seventy-two hours after transduction, medium containing 10 ng / mL IL10 was added to the macrophages. Twenty-four hours after adding the cytokine (IL10), the macrophages were phenotypically analyzed for M1 / M2 marker expression.

[0386] As Figure 9 shown, when exposed to IL10, macrophages transduced with CTX_269 showed a reduced upregulation of CD163 relative to untransduced (UTD) controls, indicating less of an M2-like phenotype. Similarly, as Figure 10 shown, when exposed to IL10, macrophages transduced with CTX_269 showed a reduced downregulation of CD86 relative to untransduced (UTD) controls, indicating maintenance of the M1 phenotype.

[0387] Example 6: Changes in M1 / M2 Polarization of Macrophages Transduced with Anti-Mesothelin Binding Agents after Mesothelin Stimulation

[0388] This example evaluated changes in M1 / M2 polarization of macrophages transduced with CTX_269 after exposure to mesothelin. Previously frozen macrophages from two donors were thawed and transduced with an Ad5f35 vector containing CTX_269 at 3000 MOI. The macrophages were plated on plates containing an exemplary titration of mesothelin. After 24 hours, the macrophages were removed and analyzed for M1 / M2 polarization via flow cytometry.

[0389] As Figure 11 shown, exposure of macrophages expressing anti-mesothelin CAR to mesothelin reduced the expression of M2-associated markers CD163 and CD206 relative to untransduced (UTD) control cells.

[0390] Example 7: Phagocytosis Assay of Macrophages Transduced with Anti-Mesothelin Binding Agents

[0391] This example evaluated the phagocytic ability of macrophages tr...

Claims

1. A modified immune cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises: (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 that is at least 80% identical to a sequence selected from Table 3; and wherein the modified immune cell is or comprises a macrophage, a monocyte, a dendritic cell, or a stem cell.

2. The modified immune cell according to claim 1, wherein the extracellular domain is or comprises a scFv, a VHH antibody, a centyrin, a designed ankyrin repeat protein, or a nanobody.

3. The modified immune cell according to claim 1 or 2, wherein the transmembrane domain is or comprises a CD8, CD8a, CD28, CD40, MyD88 CD64, CD32a, CD32c, CD16a, CD3ζ, ICOS, dendritic cell-associated C-type lectin-1, DNGR1, SLAMF7, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, or TLR9 transmembrane domain.

4. The modified immune cell according to any one of claims 1-3, wherein the one or more intracellular domains comprise: 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, 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, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPb, CD22, PIR-B, LILRB1, 41BBL (TNFSF9), CD27, OX40L, CD32b, CD11b, ITGAM, SLAMF7, CD206, CD163, CD209, dendritic cell-associated C-type lectin-2, IL1R, IL2R, IL3R, IL4R, IL5R, IL6R, IL7R, IL8R, IL9R, IL10R, IL11R, IL12R, IL13R, IL14R, IL15R, IL17R, IFNaR, IFNgR, TNFR, CSF1R, CSF2R, Dap10, CD36, dendritic cell-associated C-type lectin-1, ICOSL or the intracellular domain of Syk, or a portion of any one of the foregoing substances, or a combination thereof.

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

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

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

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

9. The modified immune cell according to claim 8, wherein the extracellular hinge domain comprises: CD8 extracellular hinge domain, CD8a extracellular hinge domain, CD28 extracellular hinge domain, DNGR-1 extracellular hinge domain, dendritic cell-associated C-type lectin-1 extracellular hinge domain or IgG4 extracellular hinge domain.

10. The modified immune cell according to claim 8 or 9, wherein the CAR comprises, from the N-terminus to the C-terminus: a CD8a leader domain, a mesothelin antigen-binding domain, a CD8 extracellular hinge domain, a CD8 transmembrane domain, and a CD3ζ intracellular domain; a CD8a leader domain, a mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and a CD3ζ intracellular domain; a CD8a leader domain, a mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and an FcRγ intracellular domain; a CD8a leader domain, a 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); a CD8a leader domain, a 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); a CD8a leader domain, a mesothelin antigen-binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a MyD88 intracellular domain, and a CD3ζ intracellular domain; a CD8a leader domain, a 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; a CD8a leader domain, a 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; a CD8a leader domain, a 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); a CD8a leader domain, a 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, a 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 a CD8a leader domain, a 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.

11. The modified immune cell according to claim 10, wherein the CAR has or comprises: (a) an amino acid sequence selected from Table 2; (b) an amino acid sequence that differs from the 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.

12. A pharmaceutical composition comprising the modified immune cells as described in any one of the preceding claims.

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

14. 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 that is at least 80% identical to a sequence selected from Table 5; and wherein the nucleic acid construct encodes a chimeric antigen receptor (CAR) comprising (a) to (c).

15. The nucleic acid construct according to claim 14, which 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 cleavage peptides, or a combination thereof.

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

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

18. The nucleic acid construct according to claim 17, wherein the nucleic acid construct has or comprises: (a) A nucleotide sequence selected from Table 4; (b) A nucleotide sequence that differs from the 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 the sequence selected from Table 4.

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

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

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

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

23. The nucleic acid construct according to claim 22, wherein the target gene transcript encodes human ATG7, C / EBP-α, C / EBP-β, CD32b, CD36, CLEC1A, FATS, GOLM1, HAVCR2, ITGAD, KLF4, KLF6, LILRB1, LILRB2, LILRB4, MAF, MafB, PD1, PD-LI, PIK3CG, PIK3CG, PPARα, PPARγ, PTGS2, SIGLEC10, SIRPα, SLAMF3, SLAMF4, SLC15A3, STAT3, STAT6, TNFRSF1B, TOX, TREM2, YTHDF2, or ZFP36.

24. The nucleic acid construct according to claim 22 or 23, wherein the target gene transcript encodes a human anti-phagocytosis receptor selected from the group consisting of SIRPα, LILRB1, SIGLEC10, PD1, SLAMF3, SLAMF4, CLEC1A, and CD32b.

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

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

27. The pharmaceutical composition according to claim 26, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

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

29. The method according to claim 28, wherein the administering step is or comprises delivery by arterial, subcutaneous, intravenous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, or intraperitoneal routes.

30. A method of modifying an immune cell, the method comprising: delivering the nucleic acid construct according to any one of claims 14-25 to the immune cell, thereby generating a modified immune cell, wherein the modified immune cell is or comprises a macrophage, a monocyte, a dendritic cell, or a stem cell.

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

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

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

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

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

36. The method according to 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, poly(A) separation, RNeasy, or a combination thereof.

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

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

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

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

41. The method according to claim 40, wherein the delivery vehicle is or comprises liposomes, lipid nanoparticles, polymers, adeno-associated virus (AAV) vectors, adenovirus vectors, retrovirus vectors or combinations thereof.

42. The method according to claim 41, wherein the liposomes or lipid nanoparticles comprise one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, one or more PEG-modified lipids or combinations thereof.

43. The method according to claim 41, wherein the retrovirus vector comprises a lentivirus vector or a gamma-retrovirus vector.

44. The method according to claim 43, wherein the lentivirus vector is packaged with a Vpx protein.

45. The method according to claim 41, wherein the adenovirus vector comprises an Ad2 vector or an Ad5 vector.

46. The method according to claim 45, wherein the Ad5 vector comprises an Ad5f35 adenovirus vector.

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

48. The method according to claim 47, wherein the additional payload is or comprises a pathogen recognition receptor agonist, polyinosinic acid: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, cytosolic DNA sensing, a stimulator of interferon genes cyclic GMP-AMP synthase (cGAS-STING) agonist, an interferon-inducible protein 16 (IFI16) agonist, a DEAD-box helicase 41 (DDX41) agonist, a LRR-binding FLII-interacting protein 1 (LRRFIP1) agonist, a melanoma absent factor 2 (AIM2) agonist, an aryl hydrocarbon receptor (AhR) ligand or combinations thereof.

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

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