Novel constructs for chimeric antigen receptors

Abstract

The present disclosure relates to immune cells comprising chimeric antigen receptors (CARs) and methods of using immune cells comprising CARs.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application Nos. 63 / 034,873 (filed June 4, 2020), 63 / 044,934 (filed June 26, 2020), and 63 / 082,584 (filed September 24, 2020), the contents of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Immunotherapies are being explored for many diseases and disorders, including cancer, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), systemic amyloidosis, prion diseases, heart disease, atherosclerosis, and fibrosis, but there is still a need to address the functional limitations encountered so far.

[0003] Therefore, there is a need to develop new therapeutic modalities that are optimized to target specific antigens. Summary of the Invention

[0004] In one aspect, the present disclosure provides modified immune cells comprising a chimeric antigen receptor (CAR) comprising (a) an extracellular domain, (b) a transmembrane domain, and (c) an intracellular domain, the immune cells comprising macrophages, monocytes, or dendritic cells. While various CARs have been developed for the treatment of multiple indications in multiple cell types, the present disclosure encompasses the recognition that maximizing CAR and / or effector activity in monocytes, macrophages, and / or dendritic cells requires the use of customized CAR components. Accordingly, the present disclosure provides several examples of CARs, as well as supporting agents, moieties, and adjunctive compositions, that are particularly useful in treating any of a variety of conditions, including, for example, various cancers, diseases or disorders of the central nervous system (e.g., Alzheimer's disease, Parkinson's disease, and / or ALS), cardiovascular disease, fibrosis, etc.

[0005] In another aspect, the disclosure provides a method of modifying macrophages, monocytes, or dendritic cells, the method comprising delivering to macrophages, monocytes, or dendritic cells a viral vector comprising one or more nucleic acid sequences encoding (a) an extracellular domain, (b) a transmembrane domain, and (c) an intracellular domain, wherein the viral vector is packaged with at least one Vpx protein, and the modified macrophages, monocytes, or dendritic cells comprise a chimeric engineered receptor (CAR) comprising (a)-(c), and the modified macrophages, monocytes, or dendritic cells exhibit increased expression of the CAR compared to modified macrophages, monocytes, or dendritic cells comprising the CAR delivered with a viral vector encoding a CAR that was not packaged with at least one Vpx protein.

[0006] In some embodiments, the viral vector is or comprises a lentiviral vector. In some embodiments, the viral vector is delivered at a multiplicity of infection (MOI) of about 1 to about 50. In some embodiments, the viral vector is delivered at an MOI of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.

[0007] In some embodiments, the modified macrophages, monocytes, or dendritic cells exhibit about a 20%, about 30%, about 40%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more increase in expression of the CAR compared to modified macrophages, monocytes, or dendritic cells comprising a CAR delivered with a viral vector encoding a CAR that was not packaged with at least one Vpx protein.

[0008] In some embodiments, the modified macrophages, monocytes, or dendritic cells exhibit CAR expression over a longer period of time than unmodified macrophages, monocytes, or dendritic cells. In some embodiments, the longer period of CAR expression is at least 5 days, at least 10 days, at least 15 days, at least 20 days, at least 25 days, at least 30 days, or more. In some embodiments, the modified macrophages, monocytes, or dendritic cells exhibit increased effector activity compared to unmodified macrophages, monocytes, or dendritic cells. In some embodiments, the modified macrophages, monocytes, or dendritic cells exhibit increased tumor killing capacity or phagocytosis compared to unmodified macrophages, monocytes, or dendritic cells.

[0009] In some embodiments, the modified macrophages, monocytes, or dendritic cells do not exhibit increased polarization toward an M1 phenotype compared to macrophages, monocytes, or dendritic cells comprising a CAR delivered with a viral vector encoding the CAR that was not packaged with at least one Vpx protein (e.g., an adeno-associated viral vector (e.g., an Ad5 vector, e.g., Ad5f35)). In some embodiments, the modified macrophages, monocytes, or dendritic cells do not exhibit increased expression of one or more markers of the M1 phenotype compared to macrophages, monocytes, or dendritic cells comprising a CAR delivered with a viral vector encoding the CAR that was not packaged with at least one Vpx protein (e.g., an adeno-associated viral vector (e.g., an Ad5 vector, e.g., Ad5f35)). In some embodiments, the one or more markers of the M1 phenotype include or are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of CD86, CD80, MHC II, IL-1R, TLR2, TLR4, iNOS, SOCS3, CD83, PD-L1, CD69, MHC I, CD64, CD32, CD16, IL1R, an IFIT family member, or an ISG family member.

[0010] In some embodiments, the modified macrophages, monocytes, or dendritic cells exhibit increased production of one or more inflammatory cytokines compared to unmodified macrophages, monocytes, or dendritic cells. In some embodiments, the one or more inflammatory cytokines include or are one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve of TNFα, IL-6, IL-1a, IL-1b, IL-12, IL-18, IL-8, IL-2, IL-23, IFNα, IFNβ, IFNγ, IL-2, IL-8, IL33, CCL3, CXCL12, CCL22, CCL4, CXCL10, or CCL2.

[0011] In some embodiments, the modified macrophages, monocytes, or dendritic cells exhibit reduced SIRPα activity compared to unmodified macrophages, monocytes, or dendritic cells. In some embodiments, the modified macrophages, monocytes, or dendritic cells comprise deletion of SIRPα using one or more endonucleases. In some embodiments, the one or more endonucleases comprise or are one or more of a CRISPR / Cas system, zinc finger nuclease (ZFN), transcription activator-like effector-based nuclease (TALEN), or meganuclease. In some embodiments, the CRISPR / Cas system comprises Cas9, Cas12a, or C2c2. In some embodiments, the modified macrophages, monocytes, or dendritic cells have been treated with one or more anti-SIRPα antibodies. In some embodiments, the modified macrophages, monocytes, or dendritic cells comprise one or more anti-SIRPα antibodies. In some embodiments, the modified macrophages, monocytes, or dendritic cells comprise one or more siRNAs that downregulate SIRPα.

[0012] In some embodiments, the extracellular domain of the CAR comprises one or more antigen binding domains and an FcR extracellular domain, and / or the transmembrane domain of the CAR comprises an FcR transmembrane domain, and / or the intracellular domain of the CAR comprises an FcR intracellular domain. In some embodiments, the CAR comprises, from N-terminal to C-terminal, one or more extracellular binding domains, an FcR extracellular domain, an FcR transmembrane domain, and an FcR intracellular domain. In some embodiments, one or more of the FcR extracellular domain, FcR transmembrane domain, and FcR intracellular domain are or comprise human FcR domains. In some embodiments, the FcR extracellular domain, FcR transmembrane domain, and FcR intracellular domain together comprise a full-length FcR. In some embodiments, the FcR extracellular domain comprises a CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c, CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, or FcαRI (CD89) domain. In some embodiments, the FcR transmembrane domain comprises a CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c, CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, or FcαRI (CD89) domain. In some embodiments, the FcR cell intracellular domain comprises a CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c, CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, or FcαRI (CD89) domain.

[0013] In some embodiments, the extracellular domain of the CAR comprises one or more antigen binding domains and a Toll-like receptor (TLR) extracellular domain, and / or the transmembrane domain of the CAR comprises a TLR transmembrane domain, and / or the intracellular domain of the CAR comprises a TLR intracellular domain. In some embodiments, the CAR comprises, from N-terminal to C-terminal, one or more extracellular binding domains, a TLR extracellular domain, a TLR transmembrane domain, and a TLR intracellular domain. In some embodiments, one or more of the TLR extracellular domain, the TLR transmembrane domain, and the TLR intracellular domain are or comprise human TLR domains. In some embodiments, the TLR extracellular domain, the TLR transmembrane domain, and the TLR intracellular domain together comprise a full-length TLR. In some embodiments, the TLR extracellular domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain. In some embodiments, the TLR transmembrane domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain. In some embodiments, the TLR intracellular domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.

[0014] In some embodiments, the chimeric antigen receptor further comprises one or more of: (d) one or more extracellular leader domains, (e) one or more extracellular hinge domains, and (f) one or more intracellular costimulatory domains. In some embodiments, the one or more extracellular leader domains comprise a CD8 extracellular leader domain.

[0015] In some embodiments, the one or more extracellular antigen-binding domains comprise an scFv, a centyrin, or a darpin.

[0016] In some embodiments, the one or more extracellular hinge domains comprise a CD28 extracellular hinge domain, a CD8a extracellular hinge domain, or an IgG4 extracellular hinge domain.

[0017] In some embodiments, the transmembrane domain comprises a CD28, CD8a, CD64, CD32a, CD32c, CD16a, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, or TLR9 transmembrane domain.

[0018] In some embodiments, the intracellular domain comprises one or more intracellular signaling domains, such as CD3 zeta, FcRγ, CD64, CD32a, CD32c, CD16a, 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, VEG FR, CD40, CD19, CD20, 41BB, CD28, OX40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, V / I / LxYxxL / V, SIRPa, CD45, S iglec-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, Dectin-2, IL1R, IL2R, IL3R, IL4R, IL5R, IL6R, IL7R, IL8R, IL9R, IL10R, IL11R, IL12R, IL13R, IL14R, IL15R, IL17R, IFNaR, IFNgR, TNFR, CSF1R, CSF2R, Dap10, CD36, Dectin-1, ICOSL, and / or Syk intracellular signaling domains. In some embodiments, the one or more intracellular signaling domains comprise at least one intracellular costimulatory domain.

[0019] In another aspect, the present disclosure provides a pharmaceutical composition comprising the immune cells of any aspect or embodiment described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0020] In another aspect, the present disclosure provides a nucleic acid construct (e.g., a viral vector) comprising one or more nucleic acid sequences encoding (a) an extracellular domain, (b) a transmembrane domain, and (c) an intracellular domain, wherein the nucleic acid construct encodes a chimeric antigen receptor (CAR) comprising (a)-(c). In some embodiments, the nucleic acid construct further comprises one or more nucleic acid sequences encoding (e) one or more extracellular leader domains, (f) one or more extracellular hinge domains, (g) one or more intracellular costimulatory domains, (h) a truncation peptide, and (i) one or more peptide agents. In some embodiments, the truncation peptide is or comprises a P2A, F2A, E2A, or T2A peptide.

[0021] In another aspect, the present disclosure provides a pharmaceutical composition comprising the nucleic acid construct of any aspect or embodiment described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0022] In another aspect, the present disclosure provides a pharmaceutical composition comprising a macrophage, monocyte, or dendritic cell modified by the method of any aspect or embodiment described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0023] In another aspect, the present disclosure provides a method of treating a disease or disorder in a subject, the method comprising delivering to the subject a therapeutically effective amount of a pharmaceutical composition of any aspect or embodiment described herein.

[0024] In another aspect, the present disclosure provides a method of modifying an immune cell, the method comprising delivering to the immune cell a nucleic acid construct comprising one or more nucleic acid sequences encoding (a) an extracellular domain, (b) a transmembrane domain, and (c) an intracellular domain, wherein the modified immune cell comprises a chimeric engineered receptor comprising (a)-(c), and wherein the modified immune cell comprises a macrophage, monocyte, or dendritic cell.

[0025] In some embodiments, the modified macrophages, monocytes, or dendritic cells exhibit reduced SIRPα activity compared to unmodified macrophages, monocytes, or dendritic cells. In some embodiments, the modified macrophages, monocytes, or dendritic cells comprise or express at least one CAR comprising one, two, three, or four of an anti-HER2 scFv, a CD8 hinge, a CD8 transmembrane domain, or a CD3 zeta intracellular signaling domain. In some embodiments, the at least one CAR comprises a CD8a signal peptide. In some embodiments, delivering a nucleic acid construct encoding (a)-(c) to the modified macrophages, monocytes, or dendritic cells comprises transducing with a viral vector. In some embodiments, the viral vector comprises or is an adenoviral vector, an adeno-associated viral vector, or a retroviral vector (e.g., a lentiviral vector or a gammaretroviral vector). In some embodiments, the adeno-associated viral vector comprises or is an Ad5 vector (e.g., an Ad5f35 viral vector).

[0026] In some embodiments, the modified macrophages, monocytes, or dendritic cells comprise deletion of SIRPα using one or more endonucleases. In some embodiments, the one or more endonucleases comprise or are one or more of a CRISPR / Cas system, a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or a meganuclease. In some embodiments, the CRISPR / Cas system comprises one or more of Cas9, Cas12a, or C2c2. In some embodiments, the modified macrophages, monocytes, or dendritic cells have been treated with one or more anti-SIRPα antibodies. In some embodiments, the modified macrophages, monocytes, or dendritic cells comprise one or more anti-SIRPα antibodies. In some embodiments, the modified macrophages, monocytes, or dendritic cells comprise one or more siRNAs that downregulate SIRPα.

[0027] The drawings are for purposes of illustration only and are not intended to be limiting. [Brief explanation of the drawings]

[0028] [Figure 1A] Graphs showing HER2 expression from macrophages transfected with constructs described herein (see Figures 8-44). Percentage of viable cells and CAR:HER2 expression are shown in dot plots for selected samples. [Figure 1B] Graphs showing HER2 expression from macrophages transfected with constructs described herein (see Figures 8-44). Percentage of viable cells and CAR:HER2 expression are shown in dot plots for all samples (Figure 1B).

[0029] [Figure 2A] Graphs showing HER2 expression from monocytes transfected with constructs described herein (see Figures 8-44). Percentage of viable cells and CAR:HER2 expression in selected samples are shown in dot plots. [Figure 2B-1] Graphs showing HER2 expression from monocytes transfected with constructs described herein (see Figures 8-44). Percentage of viable cells and CAR:HER2 expression in all samples are shown in dot plots. [Figure 2B-2] Graphs showing HER2 expression from monocytes transfected with constructs described herein (see Figures 8-44). Percentage of viable cells and CAR:HER2 expression in all samples are shown in dot plots.

[0030] [Figure 3A] Graphs showing CAR expression, CAR+ percentage, survival percentage, and tumor-killing capacity of CAR macrophages. [Figure 3B]Graphs showing CAR expression, CAR+ percentage, survival percentage, and tumor-killing capacity of CAR macrophages. [Figure 3C] Graphs showing CAR expression, CAR+ percentage, survival percentage, and tumor-killing capacity of CAR macrophages. [Figure 3D] Graphs showing CAR expression, CAR+ percentage, survival percentage, and tumor-killing capacity of CAR macrophages.

[0031] [Figure 4A] Graphs showing chimeric FcR (CFR) and Toll-like receptor (TAR) expression, percentage of CFR+ and TAR+, and survival percentage of CFR and TAR macrophages. [Figure 4B] Graphs showing chimeric FcR (CFR) and Toll-like receptor (TAR) expression, percentage of CFR+ and TAR+, and survival percentage of CFR and TAR macrophages. [Figure 4C] Graphs showing chimeric FcR (CFR) and Toll-like receptor (TAR) expression, percentage of CFR+ and TAR+, and survival percentage of CFR and TAR macrophages.

[0032] [Figure 5A] 1 is a graph showing tumor killing capacity after incubation of macrophages and CAR macrophages with CD40 ligand (CD40L). [Figure 5B] Graph showing induction of M1 and M2 marker expression after incubation of macrophages and CAR macrophages with CD40 ligand (CD40L).

[0033] [Figure 6A] 1 is a graph showing tumor killing capacity after incubation of macrophages and CAR macrophages with 4-1BB. [Figure 6B]Graph showing induction of M1 and M2 marker expression after incubation of macrophages and CAR macrophages with 4-1BB.

[0034] [Figure 7A] 1 is a graph showing the killing capacity of macrophages and CAR macrophages after incubation with 4-1BB ligand (4-1BBL). [Figure 7B] Graph showing induction of M1 and M2 marker expression after incubation of macrophages and CAR macrophages with 4-1BB ligand (4-1BBL).

[0035] [Figure 8] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 9] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 10] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 11] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 12] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 13] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 14] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 15] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 16]FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 17] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 18] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 19] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 20] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 21] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 22] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 23] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 24] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 25] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 26] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 27] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 28] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 29]FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 30] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 31] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 32] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 33] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 34] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 35] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 36] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 37] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 38] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 39] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 40] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 41] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 42]FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 43] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 44] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 45] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 46] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 47] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 48] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 49] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 50] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 51] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 52] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 53] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 54] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 55]FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 56] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 57] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 58] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 59] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 60] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 61] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 62] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 63] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 64] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 65] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 66] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 67] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 68]FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 69] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 70] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 71] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 72] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 73] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 74] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 75] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 76] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 77] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 78] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 79] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 80] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 81]FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 82] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 83] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 84] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 85] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 86] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 87] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 88] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 89] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 90] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 91] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 92] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 93] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 94]FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 95] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 96] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 97] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 98] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 99] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 100] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 101] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 102] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 103] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 104] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 105] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 106] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 107]FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 108] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 109] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 110] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 111] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 112] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 113] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 114] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 115] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 116] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above. [Figure 117] FIG. 1 is a schematic diagram of exemplary chimeric antigen receptor (CAR) constructs, including those shown in the figures above.

[0036] [Figure 118] Graph showing the survival percentage of CAR macrophages expressing CAR029, CAR061, CAR062, CAR063, CAR064, CAR071, CAR072, CAR074, CAR074, CAR077, CAR078, CAR079, CAR080, and CAR081.

[0037] [Figure 119] Graph showing the percentage of CAR expression in macrophages transfected with CAR029, CAR061, CAR062, CAR063, CAR064, CAR071, CAR072, CAR074, CAR074, CAR077, CAR078, CAR079, CAR080, and CAR081.

[0038] [Figure 120] Graph showing the intensity (MFI) of CAR expression in macrophages transfected with CAR029, CAR061, CAR062, CAR063, CAR064, CAR071, CAR072, CAR074, CAR074, CAR077, CAR078, CAR079, CAR080, and CAR081.

[0039] [Figure 121A] 1 shows an exemplary graph illustrating the viability of CAR macrophages after treatment with cytokines. [Figure 121B] 1 shows an exemplary graph illustrating the mean fluorescence intensity of the indicated surface markers after treatment with cytokines. [Figure 121C] 1 shows an exemplary graph illustrating the viability of CAR macrophages after treatment with cytokines. [Figure 121D] 1 shows an exemplary graph illustrating the mean fluorescence intensity of the indicated surface markers after treatment with cytokines. [Figure 121E] 1 shows an exemplary graph illustrating the mean fluorescence intensity of the indicated surface markers after treatment with cytokines. [Figure 121F] 1 shows an exemplary graph illustrating the mean fluorescence intensity of the indicated surface markers after treatment with cytokines.

[0040] [Figure 122]1 shows an exemplary graph illustrating the persistence of CAR expression in macrophages treated with interferon cytokines.

[0041] [Figure 123A] 1 shows exemplary graphs illustrating the viability, CAR expression, and mean fluorescence intensity of CAR macrophages after transfection with CAR mRNA and treatment with various IFN-β concentrations. [Figure 123B] 1 shows an exemplary graph illustrating the induction of M1 markers after transfection with CAR mRNA and treatment with various IFN-β concentrations.

[0042] [Figure 124A] 1 shows an exemplary graph illustrating CAR macrophage M2 and M1 marker mean fluorescence intensity 2 days after electroporation with CAR mRNA and treatment with IFN-β. [Figure 124B] 1 shows an exemplary graph illustrating CAR macrophage M2 and M1 marker mean fluorescence intensity 7 days after electroporation with CAR mRNA and treatment with IFN-β.

[0043] [Figure 125A] 1 shows an exemplary graph illustrating the anti-tumor function of CAR macrophages. Results are shown for macrophages transfected with CAR mRNA with or without priming with various concentrations of IFN-β. [Figure 125B] 1 shows an exemplary graph illustrating the anti-tumor function of CAR macrophages. Results from macrophages transfected with CAR mRNA containing various modifications and treated with IFN-β are shown. [Figure 125C] 1 shows an exemplary graph illustrating the anti-tumor function of CAR macrophages. Results are shown from macrophages transfected with CAR mRNA and treated with interferon cytokines.

[0044] [Figure 126]1 shows an exemplary graph illustrating the effect of treating CAR macrophages with interferon on cytokine secretion.

[0045] [Figure 127A] 1 shows exemplary graphs illustrating the effect of interferon treatment on the persistence of CAR mRNA in macrophages and the duration that CAR macrophages function. 1 shows the results of a study of viability and CAR expression in CAR macrophages treated with interferon cytokines. [Figure 127B] 1 shows exemplary graphs illustrating the effect of interferon treatment on the persistence of CAR mRNA in macrophages and the duration that CAR macrophages function. Tumor growth results are shown for cancer cells cultured with CAR mRNA electroporated macrophages and treated with interferon cytokines. [Figure 127C] 1 shows exemplary graphs illustrating the effect of interferon treatment on the persistence of CAR mRNA in macrophages and the duration that CAR macrophages function. Tumor growth results are shown for cancer cells cultured with CAR mRNA electroporated macrophages and treated with interferon cytokines.

[0046] [Figure 128A] 1 shows exemplary graphs illustrating the effect of interferon treatment on macrophage viability, CAR expression, M1 marker expression, and CAR macrophage functionality. Shown are the viability, CAR expression, and M1 marker expression of macrophages transfected with CAR mRNA and treated with interferon. [Figure 128B]1 shows exemplary graphs illustrating the effect of interferon treatment on macrophage viability, CAR expression, M1 marker expression, and CAR macrophage functionality. Tumor killing results are shown in cancer cells cultured with CAR mRNA electroporated macrophages and treated with interferon cytokines. [Figure 128C] 1 shows exemplary graphs illustrating the effect of interferon treatment on macrophage viability, CAR expression, M1 marker expression, and CAR macrophage functionality. Tumor killing results are shown in cancer cells cultured with CAR mRNA electroporated macrophages and treated with interferon cytokines.

[0047] [Figure 129] 1 shows an exemplary graph illustrating the effect of IFN-γ on transfected macrophages.

[0048] [Figure 130A] 1 shows exemplary graphs illustrating the effect of RNaseL inhibitors on CAR macrophages. 1 shows mCherry expression in transfected macrophages after treatment with IFN-γ and the RNaseL inhibitor sunitinib. [Figure 130B] 1 shows exemplary graphs illustrating the effect of RNaseL inhibitors on CAR macrophages. 2 shows tumor growth curves in cancer cells cultured with CAR macrophages treated with sunitinib. [Figure 130C] 1 shows exemplary graphs illustrating the effect of RNaseL inhibitors on CAR macrophages. 2 shows the tumor-killing activity of CAR macrophages treated with sunitinib.

[0049] [Figure 131]1 shows exemplary graphs illustrating viability and mCherry expression in macrophages co-transfected with mRNA encoding mCherry and mRNA encoding the RNaseL inhibitor ABCE1.

[0050] [Figure 132] FIG. 1 is a schematic diagram of an exemplary CAR construct comprising a CD28 hinge domain.

[0051] [Figure 133A] Graphs showing CAR+ percentage, CAR expression, survival percentage, and tumor-killing capacity of CAR macrophages. [Figure 133B] Graphs showing CAR+ percentage, CAR expression, survival percentage, and tumor-killing capacity of CAR macrophages. [Figure 133C] Graphs showing CAR+ percentage, CAR expression, survival percentage, and tumor-killing capacity of CAR macrophages. [Figure 133D] Graphs showing CAR+ percentage, CAR expression, survival percentage, and tumor-killing capacity of CAR macrophages. [Figure 133E] Graphs showing CAR+ percentage, CAR expression, survival percentage, and tumor-killing capacity of CAR macrophages.

[0052] [Figure 134] Graph showing secretion of TNFα and IL6 by CAR macrophages.

[0053] [Figure 135] FIG. 1 is a schematic diagram of an exemplary CAR construct.

[0054] [Figure 136]Graph showing expression of a CAR construct (CTX_001, containing an anti-HER2 scFv, a CD8 hinge, a CD8 transmembrane domain, and a CD3 zeta signaling domain; FIG. 8) in macrophages transduced with or without VPX lentivirus at MOIs of 0 to 50. Untransduced (UTD) macrophages served as a control.

[0055] [Figure 137]

[0023] Figure 1 is a series of graphs showing the viability (% Survival) and CAR expression (% CAR) of CTX_001 over 30 days in macrophages transduced with VPX lentivirus at an MOI of 1, 5, and 10. UTD macrophages were used as a control.

[0056] [Figure 138]

[0023] Figure 1 is a series of graphs showing surface protein expression of CTX_001 with alpha-trastuzumab-AF647 in macrophages transduced with VPX lentivirus at an MOI of 1, 5, and 10, at days 4, 7, 15, 22, and 29 post-transduction. UTD macrophages were used as a control.

[0057] [Figure 139]

[0023] Figure 1 is a series of graphs showing mean fluorescence intensity (MFI) of CTX_001 viability (survival) and CAR expression (CAR+) over 30 days in macrophages transduced with VPX lentivirus at an MOI of 1, 5, and 10. UTD macrophages were used as a control.

[0058] [Figure 140]This is a series of graphs showing the killing function of CTX_001 in macrophages transduced with VPX lentivirus at MOI:1, MOI:5, and MOI:10 after 4 and 7 days. Different effector macrophage:target tumor cell ratios (E:T) were 4:1, 2:1, 1:1, and 1:2. Integrated intensity indicates tumor burden. Results are graphed 72 hours after co-culture of tumor cells and macrophages. UTD macrophages and AU565 HER2+ breast cancer cells without macrophages served as controls.

[0059] [Figure 141A]

[0023] Figure 10 is a series of graphs showing surface protein expression of CTX_001 and CTX_003 (CARs comprising an anti-HER2 scFv, a CD8 hinge, and a CD8 transmembrane domain; Figure 10) in macrophages transduced with VPX lentivirus at an MOI of 2 and an MOI of 5, as assessed with alpha-trastuzumab-AF647. UTD macrophages were used as a control. [Figure 141B]

[0023] Figure 10 is a series of graphs showing the viability (% survival) and CAR expression (% CAR) of CTX_001 and CTX_003 (CARs comprising an anti-HER2 scFv, CD8 hinge, and CD8 transmembrane domain; Figure 10) in macrophages transduced with VPX lentivirus at an MOI of 2 and an MOI of 5. UTD macrophages were used as a control.

[0060] [Figure 142]

[0033] Figure 1 is a series of graphs of MFI showing viability (survival) and CAR expression (CAR+) (assessed using alpha-trastuzumab-AF647) of CTX_001 and CTX_003 in macrophages transduced with VPX lentivirus at an MOI of 2 and an MOI of 5. UTD macrophages were used as a control.

[0061] [Figure 143]

[0033] Figure 1 is a series of graphs of MFI of M2 macrophage markers (CD163 and CD206) and M1 macrophage markers (CD80 and CD86) in CTX_001 and CTX_003 macrophages transduced with VPX lentivirus at an MOI of 2 and an MOI of 5. UTD macrophages were used as a control.

[0062] [Figure 144]

[0023] Figure 1 is a series of graphs showing M1 polarization of VPX-lentivirus-transduced CTX_001 macrophages compared to Ad5f35-transduced macrophages, as assessed by CD80 and CD86. UTD macrophages were used as a control.

[0063] [Figure 145]

[0023] Figure 1 is a series of graphs showing the killing function of CTX_001 macrophages transduced with VPX lentivirus or Ad5f35 against SKOV3 ovarian cancer cells at 7 days and 21 days post-transduction. The E:T ratio was 4:1. UTD macrophages and SKOV3 cells without macrophages were used as controls.

[0064] [Figure 146]

[0023] Figure 1 is a series of graphs showing the killing function of CTX_001 and CTX_003 macrophages transduced with VPX lentivirus at an MOI of 2 and an MOI of 5 against AU565 cells over a 72-hour period. Different E:T ratios were 4:1 and 1:2. UTD macrophages and AU565 cells without macrophages were used as controls.

[0065] [Figure 147]

[0023] Figure 1 is a series of graphs showing the killing function of CTX_001 and CTX_003 macrophages transduced with VPX lentivirus at an MOI of 2 and an MOI of 5 against SKOV3 ovarian cancer cells. The E:T ratio was 4:1. UTD macrophages and SKOV3 cells without macrophages were used as controls.

[0066] [Figure 148]

[0023] Figure 1 is a series of graphs showing TNFα and IL-6 production by CTX001 or CTX_003 macrophages transduced with VPX lentivirus at an MOI of 2 and an MOI of 5 and cultured with or without plate-bound HER2 for 24 hours. UTD macrophages were used as a control.

[0067] [Figure 149]

[0023] Figure 1 is a series of graphs showing viability (% alive) and CAR expression (% CAR+) of CTX_001 and CTX_003 over 21 days in macrophages transduced with VPX lentivirus at an MOI of 2 and an MOI of 5. UTD macrophages were used as a control.

[0068] [Figure 150]

[0033] Figure 1 is a series of graphs of MFI showing viability (survival) and CAR expression (CAR+) (assessed with alpha-trastuzumab-AF647) of CTX_001 and CTX_003 over 21 days in macrophages transduced with VPX lentivirus at an MOI of 2 and an MOI of 5. UTD macrophages were used as a control.

[0069] [Figure 151]

[0023] Figure 1 is a series of graphs showing the killing function of CTX_001 and CTX_003 macrophages transduced with VPX lentivirus at an MOI of 2 and an MOI of 5 against AU565 cells over a 72-hour period starting 21 days post-transduction at different E:T ratios of 4:1 and 1:2. UTD macrophages were used as a control.

[0070] [Figure 152]

[0023] Figure 1 is a series of graphs showing the killing function of CTX_001 and CTX_003 macrophages transduced with VPX lentivirus at an MOI of 2 and an MOI of 5 against SKOV3 cells over a 72-hour period, starting 21 days post-transduction. The E:T ratio was 4:1. UTD macrophages were used as a control.

[0071] [Figure 153A]

[0023] Figure 1 is a series of graphs showing viability following transduction of CD14+ monocytes with CTX_001 using VPX lentivirus at an MOI of 2, 5, or 10. Ad5f35-transduced CAR monocytes and CAR macrophages are shown for comparison. UTD macrophages were also used as a control for CAR expression histograms. [Figure 153B]

[0023] Figure 1 is a series of graphs showing the percentage of CAR expression after transduction of CD14+ monocytes with CTX_001 using VPX lentivirus at an MOI of 2, 5, or 10. Ad5f35-transduced CAR monocytes and CAR macrophages are shown for comparison. UTD macrophages were also used as a control for CAR expression histograms. [Figure 153C]

[0023] Figure 1 is a series of graphs showing the MFI of CAR expression after transduction of CD14+ monocytes with CTX_001 using VPX lentivirus at an MOI of 2, 5, or 10. Ad5f35-transduced CAR monocytes and CAR macrophages are shown for comparison. UTD macrophages were also used as a control for CAR expression histograms. [Figure 153D]

[0023] Figure 1 is a series of graphs showing CAR expression histograms after transduction of CD14+ monocytes with CTX_001 using VPX lentivirus at an MOI of 2, 5, or 10. Ad5f35-transduced CAR monocytes and CAR macrophages are shown for comparison. UTD macrophages were also used as a control for CAR expression histograms.

[0072] [Figure 154A]

[0023] Figure 1 is a series of graphs showing SIRPα knockout in CTX_001 macrophages from two donors as assessed by SIRPα-APC%POS. Ribonucleoprotein (RNP) concentrations were 165, 312, 625, and 1250 nM except for flow cytometry, and 312 and 1250 nM for flow cytometry. Non-treated (NT) CAR macrophages or Cas9 alone were used as controls. [Figure 154B]

[0023] Figure 1 is a series of graphs showing SIRPα knockout in CTX_001 macrophages from two donors as assessed by MFI. Ribonucleoprotein (RNP) concentrations were 165, 312, 625, and 1250 nM except for flow cytometry, and 312 and 1250 nM for flow cytometry. Naive (NT) CAR macrophages or Cas9 alone were used as controls. [Figure 154C]

[0023] Figure 1 is a series of graphs showing SIRPα knockout in CTX_001 macrophages from two donors as assessed by flow cytometry. Ribonucleoprotein (RNP) concentrations were 165, 312, 625, and 1250 nM except for flow cytometry, and 312 and 1250 nM for flow cytometry. Non-treated (NT) CAR macrophages or Cas9 alone were used as controls. [Figure 154D]

[0023] Figure 1 is a series of graphs showing SIRPα knockout in CTX_001 macrophages from two donors as assessed by cell viability. Ribonucleoprotein (RNP) concentrations were 165, 312, 625, and 1250 nM except for flow cytometry, and 312 and 1250 nM for flow cytometry. Non-treated (NT) CAR macrophages or Cas9 alone were used as controls.

[0073] [Figure 155A]

[0023] Figure 1 is a series of graphs showing SIRPα knockout in CTX_001 macrophages by Cas9 and gRNA targeting SIRPα using flow cytometry. Non-treated (NT) CAR macrophages, gRNA only, and UTD macrophages were used as controls. [Figure 155B]

[0023] Figure 1 is a series of graphs showing the expression of CTX_001 in SIRPα knockout macrophages using flow cytometry. Non-treated (NT) CAR macrophages, gRNA only, and UTD macrophages were used as controls. [Figure 155C] 1 is a graph showing phagocytosis of SKOV-3 ovarian cancer cells by SIRPα knockout CTX_001 macrophages over 12 hours. Non-treated (NT) CAR macrophages, gRNA only, and UTD macrophages were used as controls. [Figure 155D] Figure 1 shows total phagocytosis measured over 12 hours, quantified as area under the curve. Non-treated (NT) CAR macrophages, gRNA only, and UTD macrophages were used as controls.

[0074] [Figure 156A]

[0023] Figure 1 is a series of graphs showing SIRPα knockout in CTX_001 macrophages with Cas9 and gRNA targeting SIRPα using flow cytometry. NT CAR macrophages, gRNA only, and UTD macrophages were used as controls. [Figure 156B]

[0023] Figure 1 is a series of graphs showing the expression of CTX_001 in SIRPα knockout macrophages using flow cytometry. NT CAR macrophages, gRNA only, and UTD macrophages were used as controls. [Figure 156C]

[0023] Figure 1 is a series of graphs showing phagocytosis of HCC-1954 human breast cancer cells by SIRPα knockout CTX_001 macrophages over a 12 hour period. NT CAR macrophages, gRNA only, and UTD macrophages were used as controls. [Figure 156D]

[0023] Figure 1 is a series of graphs showing total phagocytosis measured over 12 hours, quantified as area under the curve. NT CAR macrophages, gRNA only, and UTD macrophages were used as controls.

[0075] [Figure 157A]

[0023] Figure 1 is a series of graphs showing SIRPα knockout in CTX_001 macrophages from different donor cells using Cas9 and gRNA targeting SIRPα using flow cytometry. NT CAR macrophages, gRNA only, and UTD macrophages were used as controls. [Figure 157B]

[0023] Figure 1 is a series of graphs showing the expression of CTX_001 in SIRPα knockout macrophages using flow cytometry. NT CAR macrophages, gRNA only, and UTD macrophages were used as controls. [Figure 157C] Figure 1 shows SIRPα knockout CTX_001 macrophages killing SKOV-3 cells expressing nuclight green fluorescent protein over 24 hours by normalized tumor burden. NT CAR macrophages, gRNA only, and UTD macrophages were used as controls. [Figure 157D] Figure 1 shows tumor killing kinetics based on the time required for 50% clearance of target tumor cells. NT CAR macrophages, gRNA only, and UTD macrophages were used as controls. [Figure 157E] Graph showing total phagocytosis over 12 hours. NT CAR macrophages, gRNA only, and UTD macrophages were used as controls.

[0076] definition In order that the present invention may be more readily understood, certain terms are first defined below. Further definitions of these and other terms are found throughout the specification. Publications and other reference materials referred to herein to describe the background of the invention and to provide further details regarding its practice are hereby incorporated by reference.

[0077] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0078] Approximately or about: As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value equivalent to a stated reference value. In certain embodiments, unless otherwise specified or clear from the context, the term "approximately" or "about" refers to a range of values ​​that falls 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 or less) of the stated reference value (except where such number exceeds 100% of possible values).

[0079] Activated: As used herein, the term "activated" refers to the state of a cell (e.g., a monocyte, macrophage, or dendritic cell) that has been sufficiently stimulated to induce detectable cell proliferation or to exert an effector function. Activation can also be associated with induced cytokine production, cytokine secretion, phagocytosis, cell signaling (e.g., changes in gene expression), target cell killing, metabolic changes, production of inflammatory mediators, proliferation, epigenetic reprogramming, macrophage phenotypic switching (e.g., M1 polarization), tumor-promoting or M2 macrophage suppression, tumor-promoting or M2 macrophage phenotypic switching, and / or antigen processing and presentation.

[0080] Activated Monocyte / Macrophage / Dendritic Cell: As used herein, the term "activated monocyte / macrophage / dendritic cell" refers, inter alia, to a monocyte / macrophage / dendritic cell that is undergoing cell division or exerting an effector function. The term "activated monocyte / macrophage / dendritic cell" refers, inter alia, to a cell that is performing an effector function or exerting any activity not found in a resting state, including phagocytosis, cytokine secretion, proliferation, changes in gene expression, metabolic changes, production of inflammatory mediators, proliferation, epigenetic reprogramming, phenotypic switching of macrophages (e.g., M1 polarization), tumor-promoting or M2 macrophage suppression, phenotypic switching of tumor-promoting or M2 macrophages, and other functions.

[0081] Drug: As used herein, the term "drug" (or "biological agent" or "therapeutic agent") refers to a molecule that can be expressed, released, secreted, or delivered to a target by the modified cells described herein. Drugs include, but are not limited to, nucleic acids, antibiotics, anti-inflammatory agents, antibodies or fragments thereof, antibody drugs or fragments thereof, growth factors, cytokines, enzymes, proteins, peptides, fusion proteins, synthetic molecules, organic molecules (e.g., small molecules), carbohydrates, lipids, hormones, microsomes, derivatives or variations thereof, formulations or compositions containing one or more of these, and any combination thereof. Drugs can bind to any cellular moiety present on the target or target cell, such as a receptor, antigenic determinant, or other binding site. Drugs can diffuse or transport into the cell and act within the cell.

[0082] Antibody: As used herein, the term "antibody" refers to a polypeptide containing sufficient canonical immunoglobulin sequence elements to confer specific binding to a particular target antigen. As is known in the art, intact antibodies produced in nature are approximately 150 kD tetrameric agents comprising two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (25 kD each), which associate with each other to form what is commonly referred to as a "Y" structure. Each heavy chain contains at least four domains (each about 110 amino acids long): an amino-terminal variable (VH) domain (located at the tip of the Y structure) followed by three constant domains: CH1, CH2, and the carboxy-terminal CH3 (located at the base of the stem of the Y). A short region known as the "switch" connects the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region interconnect the two heavy chain polypeptides in an intact antibody. Each light chain contains two domains: an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, separated from each other by another "switch." An intact antibody tetramer contains two heavy-light chain dimers, with the heavy and light chains linked to each other by one disulfide bond and two other disulfide bonds connecting the heavy chain hinge regions to form a tetramer. Naturally produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold," which is formed by two beta sheets (e.g., three-, four-, or five-stranded sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as "complementarity-determining regions" (CDR1, CDR2, and CDR3) and four "framework" regions (FR1, FR2, FR3, and FR4) that are somewhat invariant.When a natural antibody folds, the FR regions form beta sheets that provide a structural framework for the domains, and the CDR loop regions from both the heavy and light chains come together in three-dimensional space to create a single hypervariable antigen-binding site located at the tip of a Y-structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and also to receptors on effector cells (including, e.g., effector cells that mediate cytotoxicity). The affinity and / or other binding properties of the Fc region for Fc receptors can be modulated by glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized in accordance with the present invention (e.g., as components of CARs) comprise a glycosylated Fc domain (including an Fc domain with modified or engineered glycosylation). In some embodiments, any polypeptide or polypeptide complex that comprises a sufficient immunoglobulin domain sequence as found in a natural antibody can be referred to and / or used as an "antibody," regardless of whether such polypeptide is produced naturally (e.g., generated by an organism that responds to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, the antibody is polyclonal. In some embodiments, the antibody is monoclonal. In some embodiments, the antibody has constant region sequences characteristic of murine, rabbit, primate, or human antibodies. In some embodiments, the antibody sequence elements are humanized, primatized, chimeric, etc., as known in the art. Furthermore, as used herein, the term "antibody" can, in appropriate embodiments (unless otherwise stated or clear from the context), refer to any of the constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations.For example, in some embodiments, antibodies utilized in accordance with the present invention include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies; bi- or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fv; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular The antibody may take a format selected from ImmunoPharmaceuticals ("SMIP™"); single-chain or tandem diabodies (TandAb®); VHH; Anticalins®; Nanobodies® minibodies; BiTE®; ankyrin repeat proteins or DARPIN®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®, Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®. In some embodiments, the antibody may lack covalent modifications (e.g., glycan attachments) that it would have if produced naturally. In some embodiments, the antibody may include covalent modifications (e.g., glycan attachments), a payload (e.g., detectable moiety, therapeutic moiety, catalytic moiety, etc.), or other pendant groups (e.g., poly-ethylene glycol, etc.).

[0083] Antibody drug: As used herein, the term "antibody drug" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody drugs include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody drug can include one or more constant region sequences characteristic of murine, rabbit, primate, or human antibodies. In some embodiments, an antibody drug can include one or more sequence elements that are humanized, primatized, chimeric, etc., as known in the art. In many embodiments, the term "antibody drug" is used to refer to one or more constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations.For example, in some embodiments, antibody agents utilized in accordance with the present invention include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies; bi- or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fv; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular The antibody drug may take a format selected from ImmunoPharmaceuticals ("SMIP™"); single-chain or tandem diabodies (TandAb®); VHH; Anticalins®; Nanobodies® minibodies; BiTE®; ankyrin repeat proteins or DARPIN®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®, Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®. In some embodiments, the antibody drug may lack covalent modifications (e.g., glycan attachments) that it would have if produced naturally. In some embodiments, the antibody drug may include covalent modifications (e.g., glycan attachments), payloads (e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.), or other pendant groups (e.g., poly-ethylene glycol, etc.). In many embodiments, an antibody agent is or comprises a polypeptide having an amino acid sequence that includes one or more structural elements recognized by those skilled in the art as complementarity determining regions (CDRs).In some embodiments, an antibody agent is or comprises a polypeptide having an amino acid sequence comprising at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to a CDR found in a reference antibody. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they are sequence identical or comprise one to five amino acid substitutions relative to the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they exhibit 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 CDRs are substantially identical to the reference CDRs in that they exhibit at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid within the included CDRs has been deleted, added, or substituted relative to the reference CDRs, but the included CDRs otherwise have the same amino acid sequence as the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that one to five amino acids within the included CDRs have been deleted, added, or substituted relative to the reference CDRs, but the included CDRs otherwise have the same amino acid sequence as the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid within the included CDRs has been substituted relative to the reference CDRs, but the included CDRs otherwise have the same amino acid sequence as the reference CDRs. In some embodiments, the included CDR is substantially identical to the reference CDR in that 1 to 5 amino acids within the included CDR have been deleted, added, or substituted relative to the reference CDR, but the included CDR otherwise has the same amino acid sequence as the reference CDR.In some embodiments, an antibody drug is or comprises a polypeptide having an amino acid sequence comprising structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody drug is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. In some embodiments, an antibody drug is not and / or does not comprise a polypeptide having an amino acid sequence comprising structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody drug is or comprises a molecule or composition that does not comprise immunoglobulin structural elements (e.g., a receptor or other naturally occurring molecule comprising at least one antigen-binding domain).

[0084] Antibody fragment: As used herein, the term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments and humanized versions thereof.

[0085] Antibody heavy chain: As used herein, the term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their native conformation.

[0086] Antibody light chain: As used herein, the term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their native conformation.

[0087] Synthetic antibody: As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA technology (e.g., an antibody expressed by a bacteriophage described herein). The term should also be construed to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0088] Antigen: As used herein, the term "antigen" or "Ag" refers to a molecule capable of eliciting an immune response. This immune response may involve either or both antibody production and / or activation of specific immunocompetent cells. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences may be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be synthetically produced or derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0089] Anti-tumor effect: As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested by a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to prevent the development of tumors in the first place.

[0090] Autologous: As used herein, the term "autologous" refers to any material that originates from an individual and is later reintroduced into the same individual.

[0091] Allogeneic: As used herein, the term "allogeneic" refers to a graft (eg, a population of cells) derived from a different animal of the same species.

[0092] Xenogeneic: As used herein, the term "xenogeneic" refers to a graft (eg, a population of cells) derived from an animal of a different species.

[0093] Cancer: As used herein, the term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colon cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. In certain embodiments, the cancer is medullary thyroid cancer.

[0094] Conservative sequence modifications: As used herein, the term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing that amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into antibodies suitable for various embodiments by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Such families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), polar uncharged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), beta-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Thus, one or more amino acid residues in the CDR regions of an antibody can be substituted with other amino acid residues from the same side chain family, and the altered antibodies can be tested for their ability to bind antigen using the functional assays described herein.

[0095] Costimulatory Ligand: As used herein, the term "costimulatory ligand" refers to a molecule on an antigen-presenting cell (e.g., an APC, a dendritic cell, a B cell, etc.) that specifically binds to a cognate costimulatory molecule on a monocyte / macrophage / dendritic cell, thereby providing a signal that mediates a monocyte / macrophage / dendritic cell response (including, but not limited to, proliferation, activation, differentiation, etc.). Costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to the Toll ligand receptor, and ligands that specifically bind to B7-H3. Costimulatory ligands also include, inter alia, antibodies that specifically bind to costimulatory molecules present on monocytes / macrophages / dendritic cells, such as, but not limited to, ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0096] Cytotoxicity: As used herein, the term "cytotoxic" or "cytotoxicity" refers to killing or damaging cells. In one embodiment, the cytotoxicity of metabolically enhanced cells is improved, e.g., the cytolytic activity of macrophages is increased.

[0097] Effective amount: As used herein, "effective amount" and "therapeutically effective amount" are interchangeable and refer to an amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result or provide a therapeutic or prophylactic benefit. Such result can include, but is not limited to, anti-tumor activity as quantified by any means suitable in the art.

[0098] Effector function: As used herein, "effector function" or "effector activity" refers to a specific activity performed by an immune cell in response to an immune cell stimulus. For example, the effector function of a macrophage is to engulf and digest cellular debris, foreign substances, microorganisms, cancer cells, and other unhealthy cells through phagocytosis.

[0099] Encode: As used herein, "encode" refers to the inherent property of a particular nucleotide sequence within a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template 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 in biological processes, and the biological properties that result therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene results in the protein in a cell or other biological system. Both the coding strand (the nucleotide sequence which is identical to the base sequence of the mRNA and is usually shown in a sequence listing) and the non-coding strand (which is used as a template for transcription of the gene or cDNA) can be said to encode the protein or other product of that gene or cDNA.

[0100] Endogenous: As used herein, "endogenous" refers to a substance that is produced from or within a particular organism, cell, tissue, or system.

[0101] Exogenous: As used herein, the term "exogenous" refers to any material introduced or produced from outside a particular organism, cell, tissue, or system.

[0102] Expand: As used herein, the term "expand" refers to an increase in number, as in increasing the number of monocytes / macrophages. In one embodiment, ex vivo expanded monocytes, macrophages, or dendritic cells are increased in number relative to the number originally present in the culture. In another embodiment, ex vivo expanded monocytes, macrophages, or dendritic cells are increased in number relative to other cell types in the culture. As used herein, the term "ex vivo" refers to cells removed from an organism (e.g., a human) and grown outside of the organism (e.g., in a culture dish, test tube, or bioreactor).

[0103] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product from the nucleic acid sequence. In some embodiments, the gene product can be a transcript. In some embodiments, the gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence includes one or more of the following: (1) production of an RNA template from the DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end formation); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0104] Expression Vector: As used herein, the term "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements necessary for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) that incorporate the recombinant polynucleotide, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses (e.g., Ad5 vectors, e.g., Ad5f35)).

[0105] Homology: As used herein, the term "homology" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules), and / or between polypeptide molecules. In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., contain residues with related chemical properties at corresponding positions). As will be understood by those skilled in the art, various algorithms are available that allow for sequence comparison to determine the degree of homology, including those that allow gaps of a specified length in one sequence relative to another sequence when considering which residues in different sequences "correspond" to each other. Calculating the percent homology between two nucleic acid sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second nucleic acid sequences for optimal alignment, and non-corresponding sequences can be ignored for comparison purposes). In certain embodiments, the length of the aligned sequences 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. Next, the nucleotides at corresponding nucleotide positions are compared. If a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules at that position are identical; if a position in the first sequence is occupied by a nucleotide that is similar to the corresponding position in the second sequence, then the molecules at that position are similar.The percent homology between the two sequences is a function of the number of identical and similar positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.

[0106] Identity: As used herein, the term "identity" refers to the identity of the subunit sequences between two polymer molecules, particularly between two amino acid molecules, for example, between two polypeptide molecules. If two amino acid sequences have the same residue at the same position, for example, if each position in two polypeptide molecules is occupied by arginine, these sequences are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of positions that match or are identical; for example, if half of the two amino acid sequences (e.g., 5 positions in a 10-amino acid-long polymer) match, the two amino acid sequences are 50% identical, and if 90% of the positions (e.g., 9 out of 10) match or are identical, the two amino acid sequences are 90% identical.

[0107] Substantial identity: As used herein, the term "substantial identity" refers to a comparison between amino acid or nucleic acid sequences. As one of ordinary skill in the art will appreciate, two sequences are generally considered to be "substantially identical" if they contain identical residues at corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms. Such algorithms include those available in commercially available computer programs, such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. In some embodiments, two sequences are considered 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 the corresponding residues are identical over the relevant stretch of residues. In some embodiments, the relevant stretch is the complete sequence. In some embodiments, the stretch in question 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. When reference is made to "substantial identity" in the context of CDRs, it typically refers to a CDR having an amino acid sequence that is at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the reference CDR.

[0108] Immunoglobulin: As used herein, the term "immunoglobulin" or "Ig" refers to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. The five members of this protein class are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody present in bodily secretions (e.g., saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and urinary tracts). IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response of most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses and is important in defense against bacteria and viruses. IgD is an immunoglobulin with no known antibody function but can function as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by inducing the release of mediators from mast cells and basophils upon exposure to allergens.

[0109] Immune response: As used herein, the term "immune response" refers to a cellular response to an antigen, which occurs when lymphocytes recognize the antigen molecule as foreign and induce the formation of antibodies and / or activate lymphocytes to eliminate the antigen.

[0110] Isolated: As used herein, the term "isolated" refers to something that has been changed or removed from its natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-native environment, such as a host cell.

[0111] Lentivirus: As used herein, the term "lentivirus" refers to a genus in the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells and deliver significant amounts of genetic information into the DNA of host cells, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Lentivirus-derived vectors provide a means to achieve significant levels of gene transfer in vivo.

[0112] Modified: As used herein, the term "modified" refers to an altered state or structure of a molecule or cell of the invention. Molecules can be modified in many ways, including chemically, structurally, and functionally. Cells can be modified by the introduction of a nucleic acid.

[0113] Modulate: As used herein, the term "modulate" refers to mediating a detectable increase or decrease in the level of a response in a subject compared to the level of the response in the subject in the absence of a treatment or compound and / or compared to the level of the response in an otherwise identical, but untreated, subject. The term encompasses perturbing and / or affecting a native signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0114] Operably linked: As used herein, the term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed into a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it 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, in the same reading frame.

[0115] Overexpressed tumor antigen: As used herein, the term "overexpressed" tumor antigen or "overexpression" of a tumor antigen refers to an abnormal level of expression of the tumor antigen in cells from a diseased area, such as a solid tumor, within a particular tissue or organ of a patient, compared to the expression level in normal cells from that tissue or organ. Patients with solid tumors or hematologic malignancies characterized by overexpression of tumor antigens can be determined by standard assays known in the art.

[0116] Polynucleotide: As used herein, the term "polynucleotide" refers to a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Therefore, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide, which can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cellular genomes using conventional cloning techniques and PCR™, as well as synthetic means.

[0117] Polypeptide: As used herein, the term "polypeptide" refers to any polymeric chain of residues (e.g., amino acids), typically joined by peptide bonds. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced by human action. In some embodiments, a polypeptide can comprise or consist of natural amino acids, unnatural amino acids, or both. In some embodiments, a polypeptide can comprise or consist of only natural amino acids or only unnatural amino acids. In some embodiments, a polypeptide can comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide can comprise one or more pendant groups or other modifications (e.g., modification of or attachment to one or more amino acid side chains) at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications can be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, and the like (including combinations thereof). In some embodiments, the polypeptide can be cyclic and / or include a cyclic moiety. In some embodiments, the polypeptide is not cyclic and / or does not include any cyclic moieties. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide can be or include a stapled polypeptide.In some embodiments, the term "polypeptide" can be added to the name of a reference polypeptide, activity, or structure; in such cases, the term is used herein to refer to polypeptides that share a related activity or structure and therefore can be considered members of the same class or family of polypeptides. For each such class, the specification provides exemplary polypeptides within the class whose amino acid sequence and / or function are known, and / or those of skill in the art would recognize. In some embodiments, such exemplary polypeptides are the reference polypeptides in the polypeptide class or family. In some embodiments, members of a polypeptide class or family exhibit significant sequence homology or identity to reference polypeptides of the class (in some embodiments, all polypeptides in the class), share common sequence motifs (e.g., characteristic sequence elements), and / or share a common activity (in some embodiments, activity at a similar level or within a specified range) with reference polypeptides of the class (in some embodiments, all polypeptides in the class). For example, in some embodiments, a member polypeptide exhibits a degree of overall sequence homology with a reference polypeptide of at least about 30-40%, often about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, and / or contains at least one region (e.g., a conserved region which, in some embodiments, is or may contain a distinctive sequence element) that exhibits very high sequence identity, often greater than 90%, or even greater than 95%, 96%, 97%, 98%, or 99%. Such a conserved region typically encompasses at least 3-4, and often up to 20 or more, amino acids, and in some embodiments, a conserved region encompasses 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, a useful polypeptide can comprise or consist of a fragment of a parent polypeptide.In some embodiments, a useful polypeptide may comprise or consist of multiple fragments, each of which is found in a different spatial arrangement from each other within the same parent polypeptide than is found within the polypeptide of interest, such that the polypeptide of interest is a derivative of that parent polypeptide (e.g., fragments that are directly linked within the parent may be spatially separated within the polypeptide of interest, or vice versa, and / or fragments may be present in a different order within the polypeptide of interest than in the parent).

[0118] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a string of at least two amino acids linked together by peptide bonds). Proteins may include moieties other than amino acids (e.g., glycoproteins, proteoglycans, etc.) and / or may be subject to other processing or modification. Those of skill in the art will understand that a "protein" may refer to an entire polypeptide chain (with or without a signal sequence) produced by a cell, or a characteristic portion thereof. Those of skill in the art will understand that a protein may optionally include multiple polypeptide chains (e.g., linked by one or more disulfide bonds or associated by other means). Polypeptides can include L-amino acids, D-amino acids, or both, and can include 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, proteins can include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids in length. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0119] Signal transduction pathway: As used herein, the term "signal transduction pathway" refers to the biochemical relationships between various signaling molecules that play a role in transmitting a signal from one part of a cell to another. The phrase "cell surface receptor" includes molecules and complexes of molecules that are capable of receiving a signal and transmitting the signal across the plasma membrane of a cell.

[0120] Single-chain antibody: As used herein, the term "single-chain antibody" refers to an antibody formed by recombinant DNA techniques in which immunoglobulin heavy and light chain fragments are linked to the Fv region via an engineered span of amino acids. Various methods for producing single-chain antibodies are known, including those described in U.S. Patent No. 4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041.

[0121] Specific binding: As used herein, the term "specific binding" with respect to an antigen-binding domain (e.g., an antibody drug) refers to an antigen-binding domain or antibody drug that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antigen-binding domain or antibody drug that specifically binds to an antigen of one species may also bind to that antigen from one or more species. However, such cross-species reactivity does not itself change the classification of the specificity of the antigen-binding domain or antibody drug. In another example, an antigen-binding domain or antibody drug that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not itself change the classification of the specificity of the antigen-binding domain or antibody drug. In some cases, the term "specific binding" or "specifically binding" can be used in reference to the interaction of an antigen-binding domain or antibody drug, protein, or peptide with a second chemical species, meaning that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species, for example, meaning that the antigen-binding domain or antibody drug recognizes and binds to a specific protein structure rather than proteins in general. If an antigen-binding domain or antibody drug is specific for epitope "A," the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antigen-binding domain or antibody drug will reduce the amount of labeled A that binds to the antibody.

[0122] Stimulation: As used herein, the term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., an FcR complex, a TLR complex, or a TCR / CD3 complex) to a cognate ligand, thereby mediating a signal transduction event (e.g., signal transduction via the Fc receptor mechanism or a synthetic CAR, without limitation). Stimulation can mediate changes in the expression of certain molecules (e.g., downregulation of TGF-beta and / or rearrangement of cytoskeletal structure, etc.). As used herein, the term "stimulatory molecule" refers to a molecule of a monocyte, macrophage, or dendritic cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell. In some embodiments, the stimulatory molecule comprises an FcR extracellular domain containing a CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c, CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, or FcαRI (CD89) domain. In some embodiments, the stimulatory molecule comprises a TLR extracellular domain, including a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain. As used herein, the term "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., aAPC, macrophage, dendritic cell, B cell, etc.) or tumor cell, can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule") on a monocyte, macrophage, or dendritic cell, thereby mediating a response by the immune cell (including, but not limited to, activation, initiation of an immune response, proliferation, etc.). Stimulatory ligands are well known in the art and include, among others, Toll-like receptor (TLR) ligands, anti-Toll-like receptor antibodies, agonists, and antibodies against monocyte / macrophage receptors. Additionally, cytokines (e.g., interferon-gamma) are potent stimulators of macrophages.

[0123] Subject: As used herein, the term "subject" refers to an organism, e.g., a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, a dog). In some embodiments, a human subject is an adult, adolescent, or pediatric subject. In some embodiments, the subject is suffering from a disease, disorder, or condition (e.g., a disease, disorder, or condition that can be treated as provided herein, e.g., a cancer or tumor listed herein). In some embodiments, the subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and / or exhibits an increased risk of developing the 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 the disease, disorder, or condition. In some embodiments, the subject does not exhibit certain symptoms (e.g., clinical manifestations of the disease) or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual for whom diagnosis and / or treatment is and / or has been managed.

[0124] Substantially purified: As used herein, the term "substantially purified," e.g., as applied to cells, refers to cells that are essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they are normally associated in their naturally occurring state. In some cases, a population of substantially purified cells refers to a homogenous population of cells. In other cases, the term simply refers to cells that have been separated from the cells with which they are naturally associated. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0125] Target: As used herein, the term "target" refers to a cell, tissue, organ, or site in the body that requires treatment or to which, for example, an antibody (or fragment thereof) or a CAR preferentially binds.

[0126] 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 for binding to occur.

[0127] T cell receptor: As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins involved in the activation of T cells in response to antigen presentation. TCRs are responsible for recognizing antigens bound to major histocompatibility complex molecules. TCRs comprise a heterodimer of an alpha (α) chain and a beta (β) chain, although in some cells, TCRs comprise gamma and delta (γ / δ) chains. TCRs can exist in alpha / beta and gamma / delta forms, which are structurally similar but differ in anatomical location and function. Each chain comprises two extracellular domains, a variable domain and a constant domain. In some embodiments, TCRs can be modified on any cell containing a TCR (including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma delta T cells).

[0128] Therapeutic: As used herein, the term "therapeutic" refers to treatment and / or prophylaxis. A therapeutic benefit is achieved by suppressing, ameliorating, or eradicating the disease state.

[0129] Transfection: As used herein, the terms "transfection" or "transduction" or "transformation" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0130] Treat: As used herein, the terms "treat," "treatment," or "treating" refer to the partial or complete alleviation, amelioration, delay in onset, suppression, prevention, reduction, and / or reduction in the incidence and / or severity of one or more symptoms or characteristics of a disease, disorder, and / or condition. In some embodiments, treatment can be administered to a subject who does not exhibit signs or characteristics of a disease, disorder, and / or condition (e.g., may be prophylactic). In some embodiments, treatment can be administered to a subject who only exhibits signs or characteristics of an early or mild disease, disorder, and / or condition, e.g., for the purpose of reducing the risk of developing pathologies associated with the disease, disorder, and / or condition. In some embodiments, treatment can be administered to a subject who exhibits signs of an established, severe, and / or late-stage disease, disorder, or condition.

[0131] Tumor: As used herein, the term "tumor" refers to an abnormal growth of cells or tissue. In some embodiments, a tumor can comprise cells that are pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. In some embodiments, a tumor is associated with or is a manifestation of cancer. In some embodiments, a tumor can be a sporadic tumor or a liquid tumor. In some embodiments, a tumor can be a solid tumor.

[0132] Vector: As used herein, the term "vector" refers to a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides bound to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes a self-replicating plasmid or virus. The term should also be interpreted to include non-plasmid and non-viral compounds (e.g., polylysine compounds, liposomes, etc.) that facilitate the transfer of nucleic acid into a cell. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0133] 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 an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed each and every numerical value within that range, as well as all possible subranges. For example, a description of a range such as "1 to 6" should be considered to have specifically disclosed each and every numerical value within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6, as well as subranges such as "1 to 3," "1 to 4," "1 to 5," "2 to 4," "2 to 6," and "3 to 6." This applies regardless of the breadth of the range. DETAILED DESCRIPTION OF THE INVENTION

[0134] immune cells The present disclosure provides, inter alia, modified immune cells (e.g., macrophages, monocytes, or dendritic cells) comprising at least one chimeric antigen receptor (CAR) described herein. Thus, in some embodiments, the immune cells comprising at least one CAR comprise (a) an extracellular domain (e.g., an extracellular domain described herein), (b) a transmembrane domain (e.g., a transmembrane domain described herein), and (c) an intracellular domain (e.g., an intracellular domain described herein).

[0135] As used herein, the term "immune cell" refers to a cell that is involved in an immune response (e.g., promoting an immune response). Examples of immune cells include, but are not limited to, macrophages, monocytes, dendritic cells, neutrophils, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, T-lymphocytes, or B-lymphocytes. A source of immune cells (e.g., macrophages, monocytes, or dendritic cells) can be obtained from a subject. Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof.

[0136] In some embodiments, the populations of immune cells described herein comprise monocytes, macrophages, dendritic cells, and / or their precursors, hi some embodiments, the populations of immune cells comprise purified populations or cell lines of monocytes, macrophages, or dendritic cells.

[0137] In some embodiments, immune cells are activated, e.g., immune cells exhibit increased cytokine production, chemokine production, phagocytosis, cell signaling, target cell killing, and / or antigen presentation, e.g., compared to inactive cells. In some embodiments, activated immune cells exhibit altered gene expression, e.g., induction of pro-inflammatory gene expression (e.g., one, two, three, four, five, six, or seven of TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, or IL-1), e.g., compared to inactive cells. In certain embodiments, activated immune cells have undergone cell division. In some embodiments, target effector activity of immune cells is enhanced by inhibition of CD47 and / or SIRPα activity. CD47 and / or SIRPα activity can be inhibited by treating immune cells with anti-CD47 or anti-SIRPα antibodies, or by any method known to one of skill in the art.

[0138] In some embodiments, immune cells (e.g., macrophages, monocytes, or dendritic cells) are obtained (e.g., isolated) from a subject. The immune cells can be autologous or sourced from allogeneic or universal donors. Cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, tumors, and / or induced pluripotent stem cells (e.g., embryonic stem cells (ESCs)). In certain embodiments, cells can be obtained from a unit of blood collected from a subject using any number of separation techniques known to those of skill in the art (e.g., Ficoll separation). In some embodiments, cells from a subject's circulating blood are obtained by apheresis or leukapheresis. Cells collected by apheresis can be washed to remove the plasma fraction and resuspended in various buffers (e.g., phosphate-buffered saline (PBS)) or culture media. In some embodiments, enrichment of immune cells (e.g., monocytes) involves plastic adherence. In some embodiments, differentiation of immune cells (e.g., monocytes) following enrichment involves stimulation with GM-CSF. In some embodiments, a composition comprising blood cells (e.g., monocytes, lymphocytes, platelets, plasma, and / or red blood cells), e.g., a leukopheresis composition (e.g., a leukopak), is used for enrichment. In some embodiments, the leukopheresis composition (e.g., a leukopak) comprises a sample from a healthy human donor. In certain embodiments, apheresis of immune cells (e.g., monocytes) is followed by mobilization with GM-CSF. In certain embodiments, selection of immune cells (e.g., monocytes) involves CD14-positive selection using microbeads (e.g., MACS® MicroBeads on a CliniMACS Prodigy device). In some embodiments, immune cell precursors (e.g., precursors of macrophages, monocytes, or dendritic cells) are used in the compositions and methods described herein. Immune cell precursors can be differentiated into immune cells in vivo or ex vivo. Non-limiting examples of 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 (iPSCs) can be derived from normal human tissues (e.g., peripheral blood, fibroblasts, skin, keratinocytes, or renal epithelial cells). Autologous, allogeneic, or universal donor iPSCs can be differentiated toward myeloid lineages (e.g., monocytes, macrophages, dendritic cells, or their precursors).

[0139] Immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein can be isolated from peripheral blood by lysing red blood cells and depleting lymphocytes and red blood cells, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, immune cells may be isolated from umbilical cord tissue. Specific subpopulations of immune cells can be further isolated by positive or negative selection techniques. In some embodiments, immune cells can be depleted from cells expressing certain antigens (including, but not limited to, CD34, CD3, CD4, CD8, CD56, CD66b, CD19, or CD20). In some embodiments, enrichment of immune cell populations, e.g., by negative selection, can be achieved using a combination of antibodies directed against surface markers unique to the negatively selected cells. As non-limiting examples, cell selection can also include negative magnetic immunoadhesion or flow cytometry, which uses a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells.

[0140] During the isolation of desired populations of immune cells (e.g., macrophages, monocytes, or dendritic cells) by positive or negative selection as described herein, the concentration and surface (e.g., particles such as beads) of the immune cells can be varied. To ensure maximum contact area between the beads and cells, it may be desirable to significantly reduce the volume in which the beads and cells are mixed.

[0141] In some embodiments, prior to administration, immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein (e.g., comprising a CAR described herein) are treated with a pro-inflammatory agent. In some embodiments, treatment with a pro-inflammatory agent increases the anti-tumor activity of immune cells described herein. In some embodiments, treatment with at least one pro-inflammatory agent promotes an M1 phenotype (e.g., a switch from M2 to M1 phenotype) in immune cells described herein. In some embodiments, the at least one pro-inflammatory agent includes or is a CD40 agonist (e.g., CD40L). In some embodiments, the at least one pro-inflammatory agent includes or is a 41BB-ligand agonist (e.g., 4-1BB). In some embodiments, the at least one pro-inflammatory agent includes or is a CD40 agonist (e.g., CD40L) and a 41BB ligand agonist (e.g., 4-1BB).

[0142] In some embodiments, the modified macrophage, monocyte, or dendritic cell comprises a CAR described herein (e.g., a CAR comprising one or more extracellular domains, one or more transmembrane domains, and one or more intracellular domains). In some embodiments, the modified macrophage, monocyte, or dendritic cell has been treated with one or more pro-inflammatory agents. In some embodiments, the modified macrophage, monocyte, or dendritic cell exhibits increased anti-tumor activity compared to an unmodified cell of the same type. In some embodiments, the one or more pro-inflammatory agents include or are a CD40 agonist (e.g., CD40L). In some embodiments, the one or more pro-inflammatory agents include or are a 41BB-ligand agonist (e.g., 4-1BB). In some embodiments, the one or more pro-inflammatory agents include or are a CD40 agonist (e.g., CD40L) and a 41BB ligand agonist (e.g., 4-1BB). The present disclosure provides a method of treating a disease or disorder in a subject, the method comprising delivering to the subject a therapeutically effective amount of a pharmaceutical composition comprising modified macrophages, monocytes, or dendritic cells as described herein.

[0143] The present disclosure also provides methods for modifying macrophages, monocytes, or dendritic cells comprising a CAR described herein (e.g., a CAR comprising one or more extracellular domains, one or more transmembrane domains, and one or more intracellular domains), the methods comprising treating the macrophages, monocytes, or dendritic cells with one or more pro-inflammatory agents, thereby producing modified macrophages, monocytes, or dendritic cells that exhibit increased anti-tumor activity compared to unmodified cells of the same type. In some embodiments, the one or more pro-inflammatory agents include or are a CD40 agonist (e.g., CD40L). In some embodiments, the one or more pro-inflammatory agents include or are a 41BB-ligand agonist (e.g., 4-1BB). In some embodiments, the one or more pro-inflammatory agents include or are a CD40 agonist (e.g., CD40L) and a 41BB ligand agonist (e.g., 4-1BB). The present disclosure includes a method of treating a disease or disorder in a subject, the method comprising delivering to the subject a therapeutically effective amount of a pharmaceutical composition comprising modified macrophages, monocytes, or dendritic cells by the methods described herein.

[0144] In some embodiments, the immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein (e.g., those comprising a CAR described herein) are administered to a subject in combination with a pro-inflammatory agent. In some embodiments, the immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein (e.g., those comprising a CAR described herein) are administered to a subject substantially simultaneously with, before, or after the pro-inflammatory agent. In some embodiments, administration with a pro-inflammatory agent increases the anti-tumor activity of the immune cells described herein. In some embodiments, administration with a pro-inflammatory agent promotes an M1 phenotype (e.g., a switch from M2 to M1 phenotype) in the immune cells described herein. In some embodiments, the pro-inflammatory agent includes or is a CD40 agonist (e.g., CD40L). In some embodiments, the pro-inflammatory agent includes or is a 41BB-ligand agonist (e.g., 4-1BB).

[0145] macrophages Macrophages are immune cells specialized for the detection, phagocytosis, and destruction of target cells (e.g., pathogens or tumor cells). Macrophages are potent effectors of the innate immune system and are capable of at least three distinct antitumor functions: phagocytosis of dead and dying cells, microorganisms, cancer cells, cellular debris, or other foreign substances; cytotoxicity against tumor cells; and presentation of tumor antigens to orchestrate adaptive antitumor immune responses.

[0146] Accumulating evidence suggests that macrophages are abundant within the tumor microenvironment of many cancers and can adopt many phenotypes, collectively referred to as tumor-associated macrophages (TAMs). The immunosuppressive nature of the tumor microenvironment typically results in a greater number of M2-like TAMs, which further contribute to the overall suppression of antitumor immune responses. However, recent studies have revealed that TAMs can be "reprogrammed" by proinflammatory signals, and that a switch from the M2 phenotype to a more M1 phenotype is linked to a productive antitumor immune response. Engineering macrophages that induce endogenous TAMs to switch to the M1 type and cannot be compromised into M2 would greatly enhance antitumor immunotherapy and represent a significant advance in this field.

[0147] In some embodiments, the macrophages comprise or are undifferentiated or M0 macrophages. In certain embodiments, the macrophages comprise or express one, two, three, four, five, or six of CD14, CD16, CD64, CD68, CD71, or CCR5. Upon exposure to various stimuli, M0 macrophages can be induced to polarize into several different populations, which can be distinguished by macrophage phenotypic markers, cytokine production, and / or chemokine secretion.

[0148] In some embodiments, the macrophages comprise or are polarized macrophages. Under classical activation conditions, M0 macrophages can be exposed to pro-inflammatory signals (e.g., LPS, IFNγ, and GM-CSF) and polarized into M1 macrophages. Generally, M1 macrophages are associated with pro-inflammatory immune responses (Th1 and Th17 T cell responses). Exposure to other stimuli can polarize macrophages into various "alternatively activated" populations or M2 macrophages.

[0149] In some embodiments, the macrophages comprise or are M1 macrophages. In some embodiments, the macrophages express one or more markers of M1 macrophages (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, or eighteen of CD86, CD80, MHC II, IL-1R, TLR2, TLR4, iNOS, SOCS3, CD83, PD-L1, CD69, MHC I, CD64, CD32, CD16, IL1R, an IFIT family member, or an ISG family member).

[0150] In some embodiments, macrophages comprising or expressing at least one CAR described herein produce relatively higher levels of one or more inflammatory cytokines (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve 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., , CC or CXC chemokines) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the CXC chemokines; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 of the CC chemokines; e.g., one of the CX3C chemokines, e.g., one or both of the C chemokines). In some embodiments, macrophages comprising or expressing at least one CAR described herein stimulate an immune response and / or inflammation, e.g., compared to macrophages without a CAR described herein. In some embodiments, the macrophages comprise or are M2 macrophages (e.g., M2a, M2b, M2c, and M2d macrophages). M2a macrophages can be induced by IL-4, IL-13, and / or fungal infection. M2b macrophages can be induced by IL-1R ligands, immune complexes, and / or LPS. M2c macrophages can be induced by IL-10 and / or TGFβ. M2d macrophages can be induced by IL-6 and / or adenosine. In some embodiments, macrophages comprising or expressing at least one CAR described herein reduce the immune response in a subject, for example, compared to macrophages without a CAR described herein.In some embodiments, the macrophages express one or more markers of M2 macrophages (e.g., one, two, or three of CD206, CD163, or CD209). In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased secretion of one or more anti-inflammatory cytokines (e.g., one or both of IL-10 or TGFβ), e.g., compared to macrophages without a CAR described herein.

[0151] In some embodiments, macrophages comprise at least one upregulated M1 marker and / or at least one downregulated M2 marker. In some embodiments, at least one 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 macrophages. In some embodiments, at least one M2 marker (e.g., CD206, CD163, and / or CD209) is downregulated in macrophages.

[0152] In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased phagocytosis, e.g., compared to macrophages without a CAR described herein. In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased cytotoxicity against tumor cells, e.g., compared to macrophages without a CAR described herein. In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased tumor antigen presentation (e.g., presentation after phagocytosis) and / or increased antigen processing, e.g., compared to macrophages without a CAR described herein. In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased tumor killing (e.g., by phagocytosis, lysis, apoptosis, or production of tumor-killing cytokines (e.g., TNFα)), e.g., compared to macrophages without a CAR described herein.

[0153] In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit one or both of increased expression of favorable genes (e.g., CD80, CD86, MHC-I, MHC-II, CD40, 41BBL, TNF, IFNa, IFNb, IFNg, IL2, IL12, IL6, IL8, IL1b, and / or CXCL12) or decreased expression of unfavorable genes (e.g., CD163, CD206, TGFb, IL10, and / or IL4), e.g., compared to macrophages without a CAR described herein. In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased production of ROS, e.g., compared to macrophages without a CAR described herein.In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit reduced metabolic reprogramming (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 zeta signaling, FcR signaling), compared to macrophages without a CAR described herein. γ 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 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, SIRPa 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, SIRPb signaling, CD22 signaling, PIR-B signaling, and / or LILRB1 signaling (metabolic reprogramming).In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit an induction of cell survival mechanisms, e.g., compared to macrophages without a CAR described herein. In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit an induction of cell death mechanisms, e.g., compared to macrophages without a CAR described herein. In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit one, two, three, four, or five of the following, e.g., increased resistance to phagocytic checkpoints, increased expression of chemokine receptors that assist in 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 anti-fibrotic activity), or increased proliferation, e.g., compared to macrophages without a CAR described herein. In some embodiments, macrophages comprising or expressing at least one CAR 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, or reduced background activity of the CAR, e.g., compared to macrophages without a CAR described herein.

[0154] In some embodiments, macrophages comprising or expressing at least one CAR described herein reduce infection (e.g., infection with an infectious pathogen) in a subject, e.g., compared to macrophages without a CAR described herein. In some embodiments, the infectious pathogen comprises or is a virus, a protozoan (e.g., Trypanosoma, Malaria, or Toxoplasma), a bacterium (e.g., Mycobacterium, Salmonella, or Listeria), a fungus (e.g., Candida), or a combination thereof. In some embodiments, the virus comprises a hepatitis virus (e.g., hepatitis A, hepatitis B, hepatitis C, or hepatitis E), a retrovirus, a human immunodeficiency virus (e.g., HIV1 or HIV2), a T-cell leukemia virus, a lymphotropic virus (e.g., HTLV1 or HTLV2), a herpes simplex virus (e.g., herpes simplex virus type 1 or 2), Epstein-Barr virus, cytomegalovirus, varicella-zoster virus, poliovirus, measles virus, rubella virus, Japanese encephalitis virus, mumps virus, influenza virus, adenovirus, enterovirus, rhinovirus, a coronavirus (e.g., severe acute respiratory syndrome (SARS) virus, Middle East respiratory syndrome (MERS) virus, or coronavirus disease 2019 (COVID-19)), Ebola virus, West Nile virus, or a variant or combination thereof.

[0155] In some embodiments, macrophages comprising or expressing at least one CAR described herein reduce the formation of and / or degrade existing aggregates by phagocytosis of at least one protein aggregate in a subject (e.g., a subject with a neurodegenerative disease, an inflammatory disease, a cardiovascular disease, a fibrotic disease, an amyloidosis, or a combination thereof), e.g., compared to macrophages without a CAR described herein. In some embodiments, the neurodegenerative disease is selected from the group consisting of tauopathies, a-synucleinopathies, presenile dementia, senile dementia, Alzheimer's disease, progressive supranuclear palsy (PSP), Pick's disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, dementia with Lewy bodies, Down's syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS), Hallervorden-Spatz syndrome, polyglutamine diseases, trinucleotide repeat diseases, and prion diseases. In some embodiments, the inflammatory disease is selected from the group consisting of systemic lupus erythematosus, vasculitis, rheumatoid arthritis, periodontal disease, ulcerative colitis, sinusitis, asthma, tuberculosis, Crohn's disease, chronic infections, hereditary periodic fevers, malignancies, systemic vasculitis, cystic fibrosis, bronchiectasis, epidermolysis bullosa, cyclic neutropenia, immunodeficiency, Muckle-Wells (MWS) disease, and familial Mediterranean fever (FMF). In some embodiments, the amyloidosis is selected from the group consisting of primary amyloidosis (AL), secondary amyloidosis (AA), familial amyloidosis (ATTR), beta2 microglobulin amyloidosis, localized amyloidosis, heavy chain amyloidosis (AH), light chain amyloidosis (AL), primary systemic amyloidosis, ApoA1 amyloidosis, ApoA2 amyloidosis, ApoA4 amyloidosis, apolipoprotein C2 amyloidosis, apolipoprotein C3 amyloidosis, corneal lactoferrin amyloidosis, transthyretin-related amyloidosis, dialysis amyloidosis, fibrinogen amyloidosis, Lect2 amyloidosis (ALECT2), and lysozyme amyloidosis.In some embodiments, the cardiovascular disease is selected from the group consisting of atherosclerosis, coronary artery disease, peripheral artery disease, hypertensive heart disease, metabolic syndrome, hypertension, cerebrovascular disease, and heart failure. In some embodiments, the fibrotic disease is selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis, liver cirrhosis, cystic fibrosis, scleroderma, cardiac fibrosis, radiation-induced lung injury, steatohepatitis, glomerulosclerosis, interstitial lung disease, liver fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, bone marrow fibrosis, and skin fibrosis.

[0156] Monocytes Monocytes are multipotent cells that circulate in the blood, bone marrow, and spleen and generally do not proliferate in a steady state. Monocyte size can vary widely, ranging from approximately 10 to 30 μm in diameter. The nucleus:cytoplasm ratio of monocytes can range from approximately 2:1 to approximately 1:1. Generally, monocytes contain chemokine receptors and pathogen recognition receptors, which mediate migration from the blood to tissues, such as during infection. Monocytes produce proinflammatory cytokines, internalize cells and / or toxic molecules, and can differentiate into dendritic cells or macrophages.

[0157] In some embodiments, monocytes comprise or express one or more phenotypic markers. Exemplary phenotypic markers for 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, CD w92, 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 comprise CD14+CD16- monocytes, CD14+CD16+ monocytes, or CD14-CD16+ monocytes.

[0158] In some embodiments, monocytes are differentiated into macrophages. In some embodiments, monocytes are differentiated into dendritic cells (DCs). Monocytes can be differentiated into macrophages or DCs by any technique known in the art. For example, differentiation of monocytes into macrophages can be induced by macrophage colony-stimulating factor (M-CSF). Differentiation of monocytes into DCs can be induced by a combination of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4.

[0159] In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased secretion of one or more cytokines (e.g., one, two, three, four, five, six, or seven of TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, or IL-1), e.g., compared to monocytes without a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased phagocytosis, e.g., compared to monocytes without a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit enhanced survival, e.g., compared to monocytes without a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit enhanced differentiation into macrophages (e.g., M1 or M2 macrophages), e.g., compared to monocytes without a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit enhanced differentiation into DCs (e.g., resident or migratory DCs and / or those in lymphoid and non-lymphoid tissues), e.g., compared to monocytes without a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased cytotoxicity against tumor cells, e.g., compared to monocytes without a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased tumor antigen presentation (e.g., presentation after phagocytosis) and / or increased antigen processing, e.g., compared to monocytes without a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased tumor killing (e.g., by phagocytosis, lysis, apoptosis, or production of tumor-killing cytokines (e.g., TNFα)), e.g., compared to monocytes without a CAR described herein.

[0160] In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit one or both of increased expression of favorable genes or decreased expression of unfavorable genes, e.g., compared to monocytes without a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased production of ROS, e.g., compared to monocytes without a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit metabolic reprogramming, e.g., compared to monocytes without a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit induction of cell survival mechanisms, e.g., compared to monocytes without a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit induction of cell death mechanisms, e.g., compared to monocytes without a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit one, two, three, four, or five of the following, e.g., compared to monocytes that do not comprise a CAR described herein: increased resistance to phagocytic checkpoints; increased expression of chemokine receptors that assist in 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 anti-fibrotic activity); or increased proliferation. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit one, two, three, or four of the following, e.g., improved duration of CAR expression, improved stability of the CAR on the cell surface, increased levels of CAR expression, or reduced background activity of the CAR, compared to monocytes without a CAR described herein.

[0161] dendritic cells Dendritic cells (DCs) are specialized antigen-presenting cells derived from the bone marrow that are involved in initiating immune responses and maintaining tolerance of the immune system to self-antigens. Dendritic cells can be found in lymphoid and non-lymphoid organs and are generally thought to arise from either the lymphoid or myeloid lineage.

[0162] In some embodiments, DCs comprise or express one or more phenotypic markers. Exemplary phenotypic markers of DCs include, but are not limited to, CD11c, CD83, CD1a, CD1c, CD141, CD207, CLEC9a, CD123, CD85, CD180, CD187, CD205, CD281, CD282, CD284, CD286, and in part, CD206, CD207, CD208, and CD209.

[0163] Immature DCs can be characterized by a high capacity for antigen capture but a relatively low capacity for T cell stimulation. Inflammatory mediators promote DC maturation. As DCs reach a mature stage, they undergo dramatic changes in properties compared to immature DCs (e.g., a decreased capacity for antigen capture and / or an increased capacity for T cell stimulation). In some embodiments, the DCs comprise or are immature DCs. In other embodiments, the DCs comprise or are mature DCs.

[0164] Without wishing to be bound by theory, it is believed that by modifying DC cells to contain or express at least one CAR described herein, the mature DCs can simultaneously exhibit increased antigen capture capacity and T cell stimulation, e.g., compared to DCs without a CAR described herein. In some embodiments, DCs containing or expressing at least one CAR described herein mediate tumor antigen presentation, e.g., increased tumor antigen presentation, compared to DCs without a CAR described herein. In some embodiments, DCs containing or expressing at least one CAR described herein mediate tumor T cell stimulation, e.g., increased T cell stimulation, compared to DCs without a CAR described herein.

[0165] In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit increased secretion of one or more cytokines (e.g., one, two, three, four, five, six, or seven of TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, or IL-1), e.g., compared to DCs without a CAR described herein. In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit increased phagocytosis, e.g., compared to DCs without a CAR described herein. In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit increased tumor antigen presentation (e.g., presentation after phagocytosis), increased antigen processing, increased antigen cross-presentation, increased T cell priming, and / or T cell stimulation, e.g., compared to DCs without a CAR described herein.

[0166] In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit one or both of increased expression of favorable genes or decreased expression of unfavorable genes, e.g., compared to DCs without a CAR described herein. In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit increased production of ROS, e.g., compared to DCs without a CAR described herein. In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit metabolic reprogramming, e.g., compared to DCs without a CAR described herein. In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit induction of cell survival mechanisms, e.g., compared to DCs without a CAR described herein. In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit induction of cell death mechanisms, e.g., compared to DCs without a CAR described herein. In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit one, two, three, four, or five of the following, e.g., compared to DCs that do not comprise a CAR described herein: increased resistance to phagocytic checkpoints; increased expression of chemokine receptors that assist in 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 anti-fibrotic activity); or increased proliferation. In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit one, two, three, or four of the following, e.g., compared to DCs without a CAR described herein: improved duration of CAR expression; improved stability of the CAR on the cell surface; increased levels of CAR expression; or decreased background activity of the CAR.

[0167] Chimeric antigen receptor (CAR) As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial cell surface receptor engineered to be expressed on immune effector cells and specifically target the cells and / or bind to an antigen. CARs can be used as therapeutics, e.g., using adoptive cell transfer. For example, in some embodiments, monocytes, macrophages, and / or dendritic cells are removed from a patient (e.g., from blood, tumor, or ascites) and modified to express a receptor specific for a particular antigen form. In some embodiments, the CAR is specifically expressed for an antigen, e.g., a tumor-associated antigen. In some embodiments, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain.

[0168] In some embodiments, modified immune cells (e.g., modified macrophages, monocytes, or dendritic cells) are generated by expressing a CAR therein. In some embodiments, the immune cells comprise a CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain, and the immune cells comprise macrophages, monocytes, or dendritic cells.

[0169] In some embodiments, the CAR can further comprise one or more extracellular leader domains, one or more extracellular hinge domains, and one or more intracellular costimulatory domains.

[0170] In some embodiments, a CAR comprises a spacer domain or hinge between the extracellular domain and the transmembrane domain. In some embodiments, a CAR comprises a spacer domain or hinge 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 within a polypeptide chain to connect a transmembrane domain to either the extracellular or intracellular domain. In some embodiments, a spacer domain or hinge can comprise up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids. In some embodiments, a short oligopeptide or polypeptide linker (preferably 2-10 amino acids in length) can form the link between the transmembrane domain and the intracellular domain of a CAR. An example of a linker is a glycine-serine doublet.

[0171] In some embodiments, an immune cell comprising a CAR can include, but is not limited to, a safety switch (e.g., an on switch and an off switch, a suicide switch), a logic gate, such as an AND gate (e.g., two or more CARs, each lacking one or more signaling domains such that activation of both / all CARs is required for intact immune cell (e.g., macrophage, monocyte, or dendritic cell) activation or function), an OR gate (e.g., two or more CARs, each having an intracellular domain such as CD3ζ and a costimulatory domain), and / or a NOT gate (e.g., two or more CARs, one of which includes an inhibitory domain that antagonizes the function of the other CAR(s)).

[0172] The present disclosure also provides an immune cell comprising a nucleic acid sequence (e.g., an isolated nucleic acid sequence) 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 the cell is a monocyte, macrophage, or dendritic cell that expresses the CAR.

[0173] In some embodiments, the CAR comprises an extracellular domain that is operably linked to another domain of the CAR (a transmembrane domain or an intracellular domain) for expression in an immune cell. In some embodiments, the nucleic acid encoding the extracellular domain is operably linked to the nucleic acid encoding the transmembrane domain, and the nucleic acid encoding the transmembrane domain is operably linked to the nucleic acid encoding the intracellular domain.

[0174] In some embodiments, the effector activity of an immune cell comprising a CAR is directed against a target cell that comprises an antigen that specifically binds to the antigen-binding domain of the CAR. In some embodiments, the target effector activity directed against the target cell is or includes phagocytosis, target cell cytotoxicity, antigen presentation, or cytokine secretion.

[0175] In some embodiments, a CAR described herein comprises at least one domain (e.g., an extracellular domain, a transmembrane domain, and / or an intracellular domain) that inhibits anti-phagocytic signaling in an immune cell described herein (e.g., a macrophage, a monocyte, or a dendritic cell). In some embodiments, a CAR described herein improves the effector activity of an immune cell described herein (e.g., a macrophage, a monocyte, or a dendritic cell) compared to the same type of cell without the CAR, e.g., by enhancing inhibition of CD47 and / or SIRPα activity. In some embodiments, a CAR described herein binds to CD47, e.g., functions as a dominant-negative receptor, and inhibits SIRPα activity (e.g., a CD47 sink). In some embodiments, a CAR described herein that binds to SIRPα comprises, e.g., an activating receptor (e.g., comprises a CD3z intracellular domain). In some embodiments, a CAR described herein inhibits at least one interaction of CD47 and SIRPα. In some embodiments, a CAR is or comprises a phagocytosis logic gate.

[0176] In some embodiments, an immune cell described herein (e.g., one comprising or expressing a CAR described herein) comprises or expresses a variant or fragment of at least one of the following: SIRPα (e.g., a 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., a dominant-negative PD1 or HAC-I), anti-PD1 scFv (e.g., E27 or durvalumab), Siglec (e.g., Siglec-10, Siglec-9, and / or Siglec-11), and / or SHP-1. In some embodiments, the variant or fragment comprises 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 includes or expresses an anti-CD47 scFv, a CD8 hinge domain, and a CD8 transmembrane domain). In some embodiments, the immune cells described herein (e.g., those that include or express a CAR described herein) include a dominant-negative receptor, e.g., that blocks an inhibitory checkpoint.

[0177] In some embodiments, the CARs described herein further comprise at least one second CAR comprising a truncated peptide (e.g., a P2A, F2A, E2A, and / or T2A peptide) and at least one inhibitory domain of anti-phagocytic signaling. In some embodiments, the at least one second CAR comprises SIRPα (e.g., a high-affinity engineered variant of SIRPα (e.g., CV1)), 5F9 scFv, B6H12 scFv (e.g., humanized B6H12 scFv), or a CD47-binding extracellular domain, or a fragment thereof. In some embodiments, the at least one second CAR comprises a SIRPα transmembrane domain or a fragment thereof. In certain embodiments, the second CAR further comprises a hinge domain (e.g., a CD8 hinge domain). In certain embodiments, at least one second CAR comprises (i) a leader sequence (e.g., a CD8 leader); ii) an extracellular domain (e.g., an extracellular domain of SIRPα, CV1, 5F9 scFv, or B6H12 scFv (e.g., humanized B6H12 scFv)); and ii) a transmembrane domain (e.g., the transmembrane domain of SIRPα). In some embodiments, the CARs described herein further comprise a truncated peptide (e.g., a P2A peptide) and at least one marker protein (e.g., CD20 or a fragment thereof, CD19 or a fragment thereof, NGFR or a fragment thereof, a synthetic peptide, and / or a fluorescent protein).

[0178] In some embodiments, an immune cell described herein (e.g., one comprising or expressing a CAR described herein) comprises or expresses one or more phosphatase death domains (e.g., phosphatase-dead Shp1, phosphatase-dead 72-5ptase (INPP5E), phosphatase-dead Shp2, and / or phosphatase-dead SHIP-1 domains), and / or a constitutively active kinase domain (e.g., a constitutively active LYN domain). In some embodiments, a CAR described herein further comprises a truncation peptide (e.g., a P2A, F2A, E2A, and / or T2A peptide), and one or more phosphatase death domains (e.g., phosphatase-dead Shp1, phosphatase-dead 72-5ptase (INPP5E), phosphatase-dead Shp2, and / or phosphatase-dead SHIP-1 domains), and / or a constitutively active kinase domain (e.g., a constitutively active LYN domain).

[0179] Extracellular domain The present disclosure provides a chimeric antigen receptor (CAR) comprising an extracellular domain. In some embodiments, the extracellular domain comprises an Fc receptor (FcR) extracellular domain. In some embodiments, the extracellular domain comprises a Toll-like receptor (TLR) extracellular domain. In some embodiments, the extracellular domain comprises a leader domain. In some embodiments, the extracellular domain comprises an antigen-binding domain. In some embodiments, the extracellular domain comprises a hinge domain. In some embodiments, the 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 extracellular domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the extracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0180] FcR extracellular domain In some embodiments, the FcR extracellular domain comprises a full-length FcR extracellular domain. In some embodiments, the FcR extracellular domain comprises a portion of a full-length FcR extracellular domain. In some embodiments, the FcR extracellular domain (or a portion thereof) is or comprises a human FcR extracellular domain. In some embodiments, the FcR extracellular domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the FcR extracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the FcR extracellular domain comprises a CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c, CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, or FcαRI (CD89) domain.

[0181] TLR extracellular domains In some embodiments, the TLR ectodomain comprises a full-length TLR ectodomain. In some embodiments, the TLR ectodomain comprises a portion of a full-length TLR ectodomain. In some embodiments, the TLR ectodomain (or portion thereof) is or comprises a human TLR ectodomain. In some embodiments, the TLR ectodomain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the TLR ectodomain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the TLR ectodomain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.

[0182] Leader Domain In some embodiments, the CAR comprises one or more extracellular leader domains. In some embodiments, the nucleic acid encoding the CAR comprises a nucleic acid sequence encoding an extracellular leader domain, but the extracellular leader domain is cleaved from the CAR before the CAR is expressed in an immune cell. In some embodiments, the extracellular leader domain is or comprises a human extracellular leader domain. In some embodiments, the extracellular leader domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the extracellular leader domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the extracellular leader domain comprises a CD8 extracellular leader domain. In some embodiments, the extracellular leader domain comprises a leader domain from a stimulatory or costimulatory domain (e.g., a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphA1, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD19, CD20, 41BB, CD28, OX40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, or MyD88 domain).

[0183] antigen-binding domain In some embodiments, the CAR comprises an antigen-binding domain that binds to an antigen (e.g., on a target cell). In some embodiments, the CAR comprises an antigen-binding domain that binds to an antigen associated with a viral infection, a bacterial infection, a parasitic infection, an autoimmune disease, and / or a cancer cell. In some embodiments, the antigen-binding domain recognizes an antigen that acts as a cell surface marker on a target cell associated with a particular disease state.

[0184] In some embodiments, the antigen binding domain binds to a tumor antigen (e.g., an antigen specific to a tumor or cancer of interest). In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes. In some embodiments, the tumor antigen is selected from the group consisting of CD19; CD123; CD22; CD30; CD171; CS-1 (CD2 subset 1, also referred to as CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule 1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor family member B-cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha 2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (Testisin or PR SS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX);Proteasome (macropain) subunit, beta type 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high-molecular-weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2) ; folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5 member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer-testis antigen 1 (MAD-CT-1); melanoma cancer-testis antigen 2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase;Prostate cancer tumor antigen 1 (PCTA-1 or galectin-8), T cell type 1-recognized melanoma antigen (MelanA or MART1); Rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS; Brother of the Regulator of Imprinted Sites), T cell-recognized squamous cell carcinoma antigen 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); kinase anchor protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of the IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2). CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican 3 (GPC3); Fc receptor-like 5 (FCRL5);Alternatively, the tumor antigen comprises immunoglobulin lambda-like polypeptide 1 (IGLL1). In certain embodiments, the tumor antigen comprises ERBB2 (Her2 / neu). In certain embodiments, the tumor antigen comprises PSMA. In certain embodiments, the tumor antigen comprises mesothelin. In some embodiments, the antigen-binding domain binds to a misfolded protein antigen or a protein in a protein aggregate (e.g., a protein specific to a disease / disorder of interest). In some embodiments, the disease / disorder is a neurodegenerative disease / disorder, an inflammatory disease / disorder, a cardiovascular disease / disorder, a fibrotic disease / disorder, or an amyloidosis (e.g., mediated by protein aggregates of immunoglobulin light chains or transthyretin). In some embodiments, the neurodegenerative disease / disorder is a tauopathy, a-synucleinopathy, presenile dementia, senile dementia, Alzheimer's disease (mediated by beta-amyloid protein aggregates), Parkinson's disease linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), Pick's disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, Parkinson's disease with dementia, dementia with Lewy bodies, Down syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS), Hallervorden-Spatz syndrome, polyglutamine disease, trinucleotide repeat disease, familial British dementia, fatal familial insomnia, Gerstmann-Straussler-Scheinker syndrome, Hereditary Cerebral Hemorrhage with Amyloidosis (Iceland) (HCHW) AI), sporadic fatal insomnia (sFI), variably protease-sensitive prion disease (VPSPr), familial Danish dementia, and prion diseases (such as Creutzfeldt-Jakob disease, CJD, and variant Creutzfeldt-Jakob disease (vCJD));

[0185] In some embodiments, the antigen-binding domain comprises any domain that binds to an antigen. In some embodiments, the 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 (e.g., scFv). In some embodiments, the antigen-binding domain is or comprises an aptamer, a darpin, a centyrin, a natural or synthetic receptor, an affibody, or other engineered protein recognition molecule. In some embodiments, the antigen-binding domain is or comprises a mammalian antibody or a fragment thereof. In some embodiments, the antigen-binding domain is derived, in whole or in part, from the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen-binding domain of the CAR comprises a human antibody, a humanized antibody, or a fragment thereof (e.g., scFv). In some embodiments, the antigen-binding domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the antigen-binding domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0186] In some embodiments, the CAR comprises one or more antigen-binding domains. In some embodiments, the CAR comprises two or more antigen-binding domains. In some embodiments, the CAR is a bispecific CAR. In some embodiments, the immune cells comprise two or more different CARs comprising one or more antigen-binding domains. In some embodiments, immune cells comprising bispecific CARs and / or two or more different CARs comprising one or more antigen-binding domains can reduce off-target and / or on-target extratissue effects by requiring the presence of two antigens. In some embodiments, the immune cells comprise bispecific CARs and / or two or more different CARs comprising one or more antigen-binding domains, where the CARs provide different signals that, alone, are insufficient to mediate activation of the modified cells, but together are synergistic and stimulate activation of the modified cells. In some embodiments, such a construct may be referred to as an "AND" logic gate.

[0187] In some embodiments, immune cells containing bispecific CARs and / or two or more different CARs containing one or more antigen-binding domains can reduce off-target and / or on-target extra-tissue effects by requiring the presence of one antigen and the absence of a second, normal protein antigen before cellular activity is stimulated. In some embodiments, such constructs may be referred to as "NOT" logic gates. In contrast to AND gates, NOT-gate CAR-modified cells are activated by binding to one antigen. However, when a second receptor binds to the second antigen, it functions to neutralize the sustained activation signal via the CAR. Typically, such inhibitory receptors would target antigens that are abundantly expressed in normal tissues but absent in tumor tissues.

[0188] Hinge domain In some embodiments, the CAR comprises one or more extracellular hinge domains. In some embodiments, the extracellular hinge domain is or comprises a human extracellular hinge domain. In some embodiments, the extracellular hinge domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the extracellular hinge domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the one or more extracellular hinge domains comprise a CD8a extracellular hinge domain, or an IgG4 or CD28 extracellular hinge domain. In some embodiments, the one or more extracellular hinge domains comprise a CD28 extracellular hinge domain. In some embodiments, the extracellular hinge domain optimizes the physicochemical parameters of the CAR, for example, optimal size (e.g., a size that allows for the exclusion of inhibitory molecules), optimal flexibility, optimal protein folding, optimal protein stability, optimal binding, optimal homodimerization, and / or lack of homodimerization to tumor antigens.

[0189] Transmembrane domain In some embodiments, the CAR comprises a transmembrane domain, e.g., connecting the extracellular domain to the intracellular domain. In some embodiments, the transmembrane domain is naturally associated with one or more other domain(s) of the CAR. In some embodiments, the transmembrane domain can be modified to avoid binding to the transmembrane domains of other surface membrane proteins to minimize interaction with other members of the receptor complex. In some embodiments, the transmembrane domain can be derived from either natural or synthetic sources. In some embodiments, the transmembrane domain is derived from a naturally occurring membrane-associated or transmembrane protein. In some embodiments, the transmembrane domain is or comprises a human transmembrane domain. In some embodiments, the transmembrane domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the transmembrane domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the transmembrane domain is selected from the group consisting of CD28, 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, 41B B, CD28, OX40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, CD3 zeta, FcRγ, 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, CD36, or Syk transmembrane domain.

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

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

[0192] Intracellular domain In some embodiments, the CAR comprises one or more intracellular domains. In some embodiments, the intracellular domain is or comprises a human intracellular domain or a portion thereof. In some embodiments, the intracellular domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the intracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the intracellular domain and / or other cytoplasmic domain of the CAR is responsible for activation of the cell (e.g., immune cell) in which the CAR is expressed. In some embodiments, the intracellular domain of the CAR is responsible for activation and / or transmission of a signal within the immune cell comprising the CAR.

[0193] In some embodiments, the intracellular domain of the CAR comprises at least one domain responsible for signal activation and / or transmission. In some embodiments, the intracellular domain is or comprises at least one of a costimulatory molecule and a signaling domain. In some embodiments, the intracellular domain of the CAR comprises dual signaling domains. In some embodiments, the intracellular domain of the CAR comprises more than two signaling domains.

[0194] In some embodiments, the intracellular domain comprises the cytoplasmic portion of a surface receptor. In some embodiments, the intracellular domain comprises a costimulatory molecule. In some embodiments, the intracellular domain comprises a molecule that acts to initiate signaling within an immune cell.

[0195] In some embodiments, the intracellular domain of the CAR comprises any portion of one or more costimulatory molecules (e.g., at least one signaling domain from CD3, Fc epsilon RI gamma chain, any derivative or variant thereof, any synthetic sequence thereof having the same functional capability, and any combination thereof).

[0196] FcR intracellular domain In some embodiments, the FcR intracellular domain comprises a full-length FcR intracellular domain. In some embodiments, the FcR intracellular domain comprises a portion of a full-length FcR intracellular domain. In some embodiments, the FcR intracellular domain is or comprises a human FcR intracellular domain or a portion thereof. In some embodiments, the FcR intracellular domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the FcR intracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the FcR intracellular domain comprises a CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c (FcgammaRIIc), CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, or FcαRI (CD89) domain.

[0197] TLR intracellular domain In some embodiments, the TLR intracellular domain comprises a full-length TLR intracellular domain. In some embodiments, the TLR intracellular domain comprises a portion of a full-length TLR intracellular domain. In some embodiments, the TLR intracellular domain is or comprises a human TLR intracellular domain or a portion thereof. In some embodiments, the TLR intracellular domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the TLR intracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the TLR intracellular domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.

[0198] Signaling domains In some embodiments, the CAR comprises one or more intracellular signaling domains. In some embodiments, the intracellular signaling domain is or comprises a human intracellular signaling domain or a portion thereof. In some embodiments, the signaling domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the signaling domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0199] In some embodiments, one or more intracellular signaling domains are selected from the group consisting of CD3 zeta, FcRγ, CD64, CD32a, CD32c, CD16a, 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, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NK G2A, CD170, CD33, BTLA, CD32b, SIRPb, CD22, PIR-B, LILRB1, Syk, 41BBL ligand (41BBL; TNFSF9), CD27, OX40L, CD32b, CD11b, ITGAM, SLAMF7, CD206, CD163, CD209, Dectin-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, Dectin-1, or ICOSL intracellular signaling domains).

[0200] In some embodiments, the intracellular domain of the CAR comprises dual signaling domains, e.g., 41BB, CD28, ICOS, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, CD116 receptor beta chain, CSF1-R, LRP1 / CD91, SR-A1, SR-A2, MARCO, SR-CL1, SR-CL2, SR-C, SR-E, CR1, CR3, CR4, dectin 1, DEC-205, DC-SIGN, CD14, CD36, LOX-1, CD11b, in any combination, with the signaling domains listed in the paragraph above.

[0201] Costimulatory domain As used herein, "costimulatory molecule" or "costimulatory domain" refers to a molecule in an immune cell that is used to enhance or attenuate initial stimulation. For example, pathogen-associated pattern recognition receptors, such as TLRs or the CD47 / SRPα axis, are molecules on immune cells that enhance or attenuate initial stimulation, respectively. In some embodiments, the costimulatory domain is selected from the group consisting of TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc epsilon R1b), 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, L IGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / R ANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D) , CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, other costimulatory molecules described herein, derivatives, variants, or fragments thereof, any synthetic sequence of a costimulatory molecule having the same functional capability, and any combination thereof.

[0202] In some embodiments, the costimulatory domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the costimulatory domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0203] As used herein, a "co-stimulatory signal" refers to a signal that, in combination with a primary signal (e.g., activation of a CAR on an immune cell), results in activation of an immune cell.

[0204] Cleaved peptide As used herein, a cleavage peptide refers to a peptide capable of inducing cleavage of a recombinant protein within a cell. In some embodiments, the cleavage peptide is a 2A peptide. In some embodiments, the cleavage peptide is or 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, the nucleic acid comprising the nucleic acid sequence encoding the cleavage peptide also comprises one or more nucleic acid sequences encoding one or more intracellular domains and one or more nucleic acid sequences comprising one or more peptide drugs, such that translation of the nucleic acid results in a protein comprising one or more intracellular domains separated from the one or more peptide drugs by the cleavage peptides. In some embodiments, a first promoter is operably linked to one or more nucleic acids encoding a CAR, and a second promoter is operably linked to one or more nucleic acids encoding a peptide drug. In some embodiments, the nucleic acid sequence comprising a CAR and, optionally, one or more peptide drugs, further comprises an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA, facilitating initiation of translation.

[0205] Peptide drugs As used herein, a peptide drug refers to a peptide that is co-expressed with a CAR in an immune cell. In some embodiments, the peptide drug is co-expressed with a CAR to ensure stoichiometric balance and optimal signaling of the CAR. In certain embodiments, a peptide drug forms a homodimer with the same peptide drug. In some embodiments, a peptide drug forms a heterodimer with a different peptide drug. In some embodiments, the nucleic acids described herein comprise one or more nucleic acid sequences encoding one or more peptide drugs. In some embodiments, the peptide drug is or comprises an FcR gamma chain. In some embodiments, the peptide drug comprises any peptide, protein, receptor, secreted antibody, or fragment thereof (e.g., scFv, Fab, Fab', F(ab')2, Fc, or nanobody). In some embodiments, the peptide agent comprises one or more cytokines (e.g., one or more of IL-1, IL-2, IL-6, IL-8, TNF-α, IFNa, IFNb, IFN-γ, GMCSF, or MCSF), CD40-L, dominant negative SIRPa, dominant negative PD1, dominant negative CD45, dominant negative SIGLEC 10, or dominant negative LILRB.

[0206] Fc receptor (FcR) In some embodiments, the CAR comprises one or more antigen-binding domains and an FcR extracellular domain, and / or the transmembrane domain of the CAR comprises an FcR transmembrane domain, and / or the intracellular domain of the CAR comprises an FcR intracellular domain. In some embodiments, the CAR comprises, from N-terminal to C-terminal, one or more extracellular binding domains, an FcR extracellular domain, an FcR transmembrane domain, and an FcR intracellular domain. In some embodiments, one or more of the FcR extracellular domain, the FcR transmembrane domain, and the FcR intracellular domain are or comprise human FcR domains. In some embodiments, the FcR extracellular domain, the FcR transmembrane domain, and the FcR intracellular domain together comprise a full-length FcR. In some embodiments, the FcR extracellular domain, the FcR transmembrane domain, and the FcR intracellular domain together comprise a portion of a full-length FcR. In some embodiments, the FcR extracellular domain comprises a portion of a full-length FcR extracellular domain. In some embodiments, the FcR transmembrane domain comprises a portion of a full-length FcR transmembrane domain. In some embodiments, the FcR intracellular domain comprises a portion of a full-length FcR intracellular domain.

[0207] Toll-like receptors (TLRs) In some embodiments, the CAR comprises one or more antigen binding domains and a Toll-like receptor (TLR) extracellular domain, and / or the transmembrane domain of the CAR comprises a TLR transmembrane domain, and / or the intracellular domain of the CAR comprises a TLR intracellular domain. In some embodiments, the CAR comprises, from N-terminal to C-terminal, one or more extracellular binding domains, a TLR extracellular domain, a TLR transmembrane domain, and a TLR intracellular domain. In some embodiments, one or more of the TLR extracellular domain, the TLR transmembrane domain, and the TLR intracellular domain are or comprise human TLR domains. In some embodiments, the TLR extracellular domain, the TLR transmembrane domain, and the TLR intracellular domain together comprise a full-length TLR. In some embodiments, the TLR extracellular domain, the TLR transmembrane domain, and the TLR intracellular domain together comprise a portion of a full-length TLR. In some embodiments, the TLR extracellular domain comprises a portion of a full-length TLR extracellular domain. In some embodiments, the TLR transmembrane domain comprises a portion of a full-length TLR transmembrane domain. In some embodiments, the TLR intracellular domain comprises a portion of a full-length TLR intracellular domain.

[0208] Nucleic Acid Constructs The present disclosure provides, inter alia, a nucleic acid molecule encoding at least one CAR or a fragment thereof described herein. An immune cell can comprise a nucleic acid molecule (e.g., a foreign nucleic acid molecule) encoding at least one CAR described herein. In some embodiments, the nucleic acid molecule encoding at least one CAR comprises (a) an extracellular domain (e.g., an extracellular domain described herein), (b) a transmembrane domain (e.g., a transmembrane domain described herein), and (c) an intracellular domain (e.g., an intracellular domain described herein).

[0209] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. A nucleotide sequence encoding a protein or RNA can also include introns, to the extent that a protein-encoding nucleotide sequence may, in some versions, contain intron(s). The term "encoding" refers to the inherent property of a particular nucleotide sequence within a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or other polymers and macromolecules having a defined amino acid sequence in a biological process, as well as the biological properties that result therefrom. Thus, a gene, cDNA, or RNA encodes a protein when transcription and translation of the mRNA corresponding to that gene results in the protein in a cell or other biological system. Both the coding strand (the nucleotide sequence identical to the base sequence of the mRNA, usually shown in a sequence listing) and the non-coding strand (used as a template for transcription of the gene or cDNA) can be said to encode the protein or other product of that gene or cDNA.

[0210] The term "operably linked" or "transcriptional control" refers to a functional linkage between a control sequence and a heterologous nucleic acid sequence that results in the expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Operable linked DNA sequences can be contiguous with each other, and, for example, when two protein coding regions need to be linked, they can be in the same reading frame.

[0211] As used herein, the term "fragment" or "portion" refers to a structure that includes a discrete portion of a whole but lacks one or more portions found in the whole structure. In some embodiments, a fragment includes such a discrete portion. In some embodiments, a fragment consists of or includes a characteristic structural element or portion found in the whole. In some embodiments, a nucleotide fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more monomeric units (e.g., nucleic acid) found throughout the nucleotide sequence. In some embodiments, a nucleotide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the monomeric units (e.g., residues) found in the whole nucleotide. The whole material or entity may be referred to in some embodiments as the whole "parent."

[0212] The nucleic acid molecule encoding at least one CAR or 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 CAR or fragment thereof described herein. In some embodiments, the nucleic acid molecule comprises or is a DNA construct encoding at least one CAR or fragment thereof described herein.

[0213] In some embodiments, all or a fragment of a CAR described herein is encoded by a nucleic acid molecule that has been codon-optimized, e.g., for expression in a cell (e.g., a mammalian cell). Various codon optimization methods are known in the art and are disclosed, for example, in U.S. Patent Nos. 5,786,464 and 6,114,148 (each of which is incorporated herein by reference in its entirety).

[0214] In some embodiments, the vector comprises a nucleic acid molecule encoding at least one CAR or fragment thereof as described herein. In some embodiments, the vector includes a plasmid, a viral vector, a phagemid, a retrotransposon (e.g., piggyback or sleeping beauty), a site-specific insertion vector (e.g., a CRISPR / Cas system for insertion of a template donor DNA comprising a nucleic acid sequence encoding at least one CAR as described herein (e.g., a CRISPR / Cas system comprising one or more of Cas9, Cas12a, or C2c2), a zinc finger nuclease, or a TALEN), a suicide expression vector, or other vector known in the art. The vector may be suitable for replication and integration in eukaryotes. Vectors may include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0215] Vectors can include an origin of replication, a promoter sequence (e.g., a constitutive or inducible promoter), and / or convenient restriction endonuclease sites (see, e.g., WO01 / 96584, WO01 / 29058, and U.S. Pat. No. 6,326,193, each of which is incorporated herein by reference in its entirety). Vectors can also include, for example, a signal sequence to facilitate secretion, a polyadenylation signal, and a transcription terminator (e.g., a bovine growth hormone (BGH) polyadenylation signal), elements allowing episomal replication and replication in prokaryotes (e.g., an SV40 origin and / or ColE1), selection elements (e.g., an ampicillin resistance gene and / or a Zeocin marker), and / or a reporter gene (e.g., luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein).

[0216] Expression of the nucleic acids described herein can be achieved by operably linking a nucleic acid encoding a CAR polypeptide 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 1 alpha promoter (EF-1 alpha), the immediate-early cytomegalovirus (CMV) promoter, the ubiquitin C promoter, the phosphoglycerokinase (PGK) promoter, the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the Moloney murine leukemia virus (MoMuLV) promoter, the avian leukemia virus promoter, the Epstein-Barr virus immediate-early promoter, the Rous sarcoma virus promoter, the actin promoter, the myosin promoter, the hemoglobin promoter, or the creatine kinase promoter. Examples of inducible promoters include, but are not limited to, the metallothionine promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter. The vector may also contain additional promoter elements, such as enhancers, to regulate the frequency of transcription initiation.

[0217] In some embodiments, the vector comprising the nucleic acid molecule encoding at least one CAR or fragment thereof described herein comprises or is a viral vector. Viral vector technology is well known in the art and is described, for example, 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 gamma retroviral vectors). In some embodiments, the vector comprises a lentiviral vector (e.g., as described in U.S. Pat. No. 9,149,519 or International Publication No. WO2017 / 044487 (each of which is incorporated herein by reference in its entirety)).

[0218] In some embodiments, the viral vector comprises an adenovirus vector. Adenoviruses are a large family of viruses that contain double-stranded DNA. Adenoviruses replicate in the nucleus of host cells and use the host's cellular machinery to synthesize viral RNA, DNA, and proteins. Adenoviruses are known in the art to affect both replicating and non-replicating cells, accommodate large transgenes, and encode proteins without integrating into the host cell genome. In some embodiments, the adenovirus vector comprises an Ad2 vector or an Ad5 vector (e.g., an Ad5f35 adenovirus vector, e.g., a helper-dependent Ad5F35 adenovirus vector).

[0219] In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. AAV systems are well known in the art (see, for example, Kelleher and Vos, Biotechniques, 17(6):1110-17(1994); Cotten et al., PNASUSA, 89(13):6094-98(1992); Curiel, Nat Immun, 13(2-3):141-64(1994); Muzyczka, Curr Top Microbiol Immunol, 158:97-129(1992); and Asokan A, et al., Mol. Ther., 20(4):699-708(2012)). Methods for producing and using recombinant AAV (rAAV) vectors are described, for example, in U.S. Patent Nos. 5,139,941 and 4,797,368.

[0220] Several AAV serotypes have been characterized, including AAV1, AAV2, AAV3 (e.g., AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, and variants thereof. Generally, any AAV serotype can be used to deliver at least one CAR described herein. In some embodiments, the AAV serotype has a tropism for a particular tissue.

[0221] In some embodiments, the CRISPR / Cas9 system has recently been shown to facilitate high-level, precise genome editing using adeno-associated virus (AAV) vectors to serve as donor template DNA during homologous recombination (HR).

[0222] In some embodiments, the vector comprises a gammaretroviral vector (e.g., as described in Tobias Maetzig et al., "Gammaretroviral Vectors: Biology, Technology and Application" Viruses. 2011 Jun;3(6):677-713, which is incorporated herein by reference in its entirety). Exemplary gammaretroviral vectors include murine leukemia virus (MLV), spleen-limited focus-forming virus (SFFV), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom.

[0223] In some embodiments, the vector comprises two or more nucleic acid sequences encoding a CAR (e.g., at least one CAR described herein) and a second CAR (e.g., a different CAR described herein). In some embodiments, the two or more nucleic acid sequences encoding the CAR and the second CAR are encoded by a single nucleic molecule, e.g., in the same frame as a single polypeptide chain. In some embodiments, the two or more CARs are separated by one or more cleavage peptide sites (e.g., autocleavage sites or substrates for intracellular proteases). In certain embodiments, the cleavage peptide comprises a porcine teschovirus I (P2A) peptide, a Thosea asigna virus (T2A) peptide, an equine rhinitis A virus (E2A) peptide, a foot-and-mouth disease virus (F2A) peptide, or a variant thereof.

[0224] In some embodiments, the vector comprises at least one nucleic acid sequence encoding a CAR (e.g., at least one CAR described herein) and at least one nucleic acid encoding at least one gene co-expressed with the CAR (e.g., a cytokine described herein (e.g., TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, and / or IL-1), or a stimulatory ligand described herein (e.g., CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, ICOS-L, ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds to a Toll ligand receptor, and / or a B7-H3 ligand)).

[0225] Pharmaceutical Composition The present disclosure provides, inter alia, pharmaceutical compositions comprising a combination of immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0226] When a "therapeutically effective amount," "immune effective amount," "anti-immune response effective amount," or "immune response inhibiting effective amount" is indicated, the exact amount of a pharmaceutical composition comprising immune cells (e.g., macrophages, monocytes, or dendritic cells) 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).

[0227] Pharmaceutical compositions comprising immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein can include a buffer (including neutral buffered saline or phosphate buffered saline (PBS)); a carbohydrate (e.g., glucose, mannose, sucrose, dextran, or mannitol); a protein, polypeptide, or amino acid (e.g., glycine); an antioxidant; a chelating agent (e.g., EDTA or glutathione); an adjuvant (e.g., aluminum hydroxide); and a preservative. In some embodiments, the pharmaceutical composition is substantially free of contaminants, e.g., free of detectable levels of contaminants (e.g., endotoxin).

[0228] The pharmaceutical compositions described herein can be administered in a manner appropriate to the disease, disorder, or condition to be treated or prevented. The amount and frequency of administration will depend on factors such as the condition of the patient and the type and severity of the patient's disease, disorder, or condition, although appropriate dosages may be determined through clinical trials.

[0229] The pharmaceutical compositions described herein can take various forms. Such forms include, for example, liquid, semi-solid, and solid dosage forms, such as solutions (e.g., injection and infusion solutions), dispersions or suspensions, liposomes, and suppositories. Preferred compositions may be injection or infusion solutions. The pharmaceutical compositions described herein can be formulated for intravenous, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intraarterial, or intraperitoneal administration.

[0230] 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, incorporated herein by reference in its entirety).

[0231] As used herein, the terms "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection or infusion, including, but not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, intratumor, and intrasternal injection and infusion.

[0232] The pharmaceutical compositions comprising the immune cells described herein may be administered in an amount of about 10 4 ~about 10 9 cells / kg body weight (e.g., approximately 10 5 ~about 10 6 In some embodiments, the dose of immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein is at least about 1 x 10 cells / kg body weight, including all integer values ​​within these ranges. 6 , about 1.1×10 6 , about 2×10 6 , about 3.6×10 6 , about 5×10 6 , about 1×10 7 , about 1.8×10 7 , about 2×10 7 , about 5×10 7 , about 1×10 8 , about 2×10 8 , about 5×10 8 , about 1×109 , about 2×10 9 , or about 5 × 10 9 The pharmaceutical compositions described herein may also be administered multiple times at a particular dosage. The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the art by monitoring the patient for symptoms of the disease, disorder, or condition and adjusting the treatment accordingly.

[0233] It may be desirable to administer a pharmaceutical composition comprising immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein to a subject, followed by re-drawing blood (or performing apheresis), activating the drawn immune cells, and re-injecting the activated immune cells into the subject. This process may be performed multiple times (e.g., every few weeks). Immune cells (e.g., 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, the methods described herein involving multiple blood draws and re-infusions may select for certain immune cell populations. In some embodiments, pharmaceutical compositions comprising immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein are administered in combination with (e.g., before, simultaneously with, or after) a second therapy. For example, the second therapy can include, but is not limited to, an antiviral therapy (e.g., cidofovir, interleukin-2, cytarabine (ARA-C), or natalizumab), chimeric antigen receptor T cell (CAR-T) therapy, T cell receptor (TCR)-T cell therapy, chemotherapy, radiation, an immunosuppressant (e.g., cyclosporine, azathioprine, methotrexate, mycophenolic acid, FK506 antibody, or glucocorticoid), an antagonist (e.g., a PD-1 antagonist, a PD-L1 antagonist), or a combination thereof. The therapeutic agent may include one or more of an anti-CD52 antibody (e.g., alemtuzumab), an anti-CD3 antibody, cytoxin, fludarivine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, or radiation.

[0234] In some embodiments, a pharmaceutical composition comprising immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein is administered in combination with (e.g., before, concurrently with, or after) bone marrow transplantation or lymphocyte ablative therapy using chemotherapeutic agents (e.g., fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or Rituxan). In certain embodiments, the subject undergoes standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In certain embodiments, after transplantation, the subject receives an infusion of a pharmaceutical composition comprising immune cells described herein. The pharmaceutical compositions described herein can be administered before or after surgery.

[0235] The dosage of any of the aforementioned therapeutic agents to be administered to a subject varies depending on the disease, disorder, or condition being treated and on the particular subject. Scaling of dosages for human administration can be performed according to methods accepted in the art. For example, the dose of alemtuzumab is generally about 1 mg to about 100 mg for an adult, typically administered daily for a period of about 1 to about 30 days, e.g., a daily dose of about 1 mg to about 10 mg per day (e.g., as described in U.S. Pat. No. 6,120,766, incorporated herein by reference in its entirety).

[0236] Treatment method The present disclosure provides, inter alia, methods of treating a disease or disorder (e.g., a disease or disorder described herein) in a subject, the method comprising delivering a pharmaceutical composition comprising an immune cell (e.g., a macrophage, monocyte, or dendritic cell) described herein. In some embodiments, a therapeutically effective amount of a pharmaceutical composition described herein is administered to a subject having a disease or disorder. The pharmaceutical compositions described herein can be for use in the manufacture of a medicament for treating a disease or disorder in a subject or for stimulating an immune response in a subject.

[0237] The subject treated with the methods described herein can be a mammal, e.g., a primate, e.g., a human (e.g., a patient having or at risk of having a disease or disorder described herein). In some embodiments, the immune cells (e.g., macrophages, monocytes, or dendritic cells) can be autologous, allogeneic, or xenogeneic to the subject. The pharmaceutical compositions described herein can be administered to a subject alone or in combination with one or more therapeutic agents, procedures, or modalities according to the dosing regimens described herein.

[0238] Pharmaceutical compositions comprising the immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein can be used to treat or prevent tumors or cancers, neurodegenerative diseases or disorders, inflammatory diseases or disorders, cardiovascular diseases or disorders, fibrotic diseases or disorders, diseases associated with amyloidosis, and combinations thereof.

[0239] Methods are provided for treating (e.g., reducing, inhibiting, or delaying the progression of) cancer or tumor in a subject using pharmaceutical compositions comprising the immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein. The subject may have an adult or childhood form of cancer. The cancer may be in early, intermediate, or late stage, or may be metastatic. The cancer may include, but is not limited to, a solid tumor, a blood cancer (e.g., leukemia, lymphoma, or myeloma, e.g., multiple myeloma), or a metastatic lesion. Examples of solid tumors include malignant tumors of various organ systems, such as sarcomas and carcinomas (e.g., adenocarcinomas), including those affecting the lung, breast, ovary, lymphatic system, gastrointestinal (e.g., colon), anus, genital and genitourinary tract (e.g., kidney, urothelium, bladder cells, prostate), pharynx, central nervous system (e.g., brain, nerve, or glial cells), head and neck, skin (e.g., melanoma, e.g., cutaneous melanoma), pancreas, and bone (e.g., chordoma).

[0240] In some embodiments, the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC) (e.g., non-small cell lung cancer (NSCLC) with squamous and / or non-squamous histology, or NSCLC adenocarcinoma), or small cell lung cancer (SCLC)), skin cancer (e.g., Merkel cell carcinoma or melanoma (e.g., advanced melanoma)), ovarian cancer, mesothelioma, bladder cancer, soft tissue sarcoma (e.g., hemangiopericytoma (HPC)), bone cancer (osteosarcoma), kidney cancer (e.g., renal cell carcinoma, etc.), liver cancer (e.g., hepatocellular carcinoma, etc.), cholangiocarcinoma, sarcoma, myelodysplastic syndrome (MDS), prostate cancer, breast cancer (e.g., breast cancer that does not express one, two, or all of estrogen receptors, progesterone receptors, or Her2 / neu (e.g., triple-negative breast cancer)), colon cancer (e.g., recurrent colon cancer, The cancer is selected from cancer or metastatic colorectal cancer, e.g., 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 (e.g., head and neck squamous cell carcinoma (HNSCC)), anal cancer, gastroesophageal cancer, thyroid cancer (e.g., anaplastic thyroid carcinoma), cervical cancer (e.g., cervical squamous cell carcinoma), neuroendocrine tumors (NETs) (e.g., atypical pulmonary carcinoid tumors), lymphoproliferative disorders (e.g., post-transplant lymphoproliferative disorders), lymphomas (e.g., T-cell lymphoma, B-cell lymphoma, or non-Hodgkin's lymphoma), myeloma (e.g., multiple myeloma), or leukemia (e.g., myeloid leukemia or lymphocytic leukemia).

[0241] In some embodiments, the cancer is a brain tumor (e.g., glioblastoma, gliosarcoma, or recurrent brain tumor). In some embodiments, the cancer is pancreatic cancer (e.g., advanced pancreatic cancer). In some embodiments, the cancer is skin cancer (e.g., melanoma (e.g., stage II-IV melanoma, HLA-A2-positive melanoma, unresectable melanoma, or metastatic melanoma), or Merkel cell carcinoma). In some embodiments, the cancer is kidney cancer (e.g., renal cell carcinoma (RCC) (e.g., metastatic renal cell carcinoma)). In some embodiments, the cancer is breast cancer (e.g., metastatic breast cancer or stage IV breast cancer, e.g., triple-negative breast cancer (TNBC)). In some embodiments, the cancer is a virus-associated cancer. In some embodiments, the cancer is anal canal cancer (e.g., squamous cell carcinoma of the anal canal). In some embodiments, the cancer is cervical cancer (e.g., squamous cell carcinoma of the cervix). In some embodiments, the cancer is gastric cancer (e.g., Epstein-Barr virus (EBV)-positive gastric cancer, or gastric or gastroesophageal junction cancer). In some embodiments, the cancer is head and neck cancer (e.g., HPV-positive and -negative squamous cell carcinoma of the head and neck (SCCHN)). In some embodiments, the cancer is nasopharyngeal carcinoma (NPC). In some embodiments, the cancer is colorectal cancer (e.g., recurrent colorectal cancer, metastatic colorectal cancer, e.g., microsatellite-unstable colorectal cancer, microsatellite-stable colorectal cancer, mismatch repair-proficient colorectal cancer, or mismatch repair-deficient colorectal cancer).

[0242] In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is a leukemia (e.g., acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic leukemia, or acute leukemia). In some embodiments, the cancer is a lymphoma (e.g., Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma, lymphocytic lymphoma, or diffuse large B-cell lymphoma (DLBCL) (e.g., relapsed or refractory HL or DLBCL)). In some embodiments, the cancer is a myeloma (e.g., multiple myeloma).

[0243] Pharmaceutical compositions comprising the immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein can be used to enhance or modulate an immune response in a subject. In one embodiment, the pharmaceutical compositions described herein enhance, stimulate, or increase an immune response in a subject (e.g., a subject having or at risk of a disease or disorder described herein). In certain embodiments, the subject is or is at risk of being immunocompromised. For example, the subject is undergoing or has undergone chemotherapy and / or radiation therapy.

[0244] In some embodiments, the subject has or is at risk of developing an inflammatory disorder (e.g., a chronic or acute inflammatory disorder). In some embodiments, the subject has or is at risk of developing an autoimmune disease or disorder. Exemplary autoimmune diseases that can be treated with the methods described herein include, but are not limited to, Alzheimer's disease, asthma (e.g., bronchial asthma), allergies (e.g., atopic allergies), acquired immune deficiency syndrome (AIDS), atherosclerosis, Behcet's disease, celiac disease, cardiomyopathy, Crohn's disease, cirrhosis, diabetes, diabetic retinopathy, eczema, fibromyalgia, fibromyositis, glomerulonephritis, graft-versus-host disease (GVHD), Guillain-Barré syndrome, hemolytic anemia, multiple sclerosis, myasthenia gravis, osteoarthritis, polychondritis, psoriasis, rheumatoid arthritis, sepsis, stroke, vasculitis, ventilator-induced lung injury, transplant rejection, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, ulcerative colitis, uveitis, vitiligo, or Wegener's granulomatosis.

[0245] Administration of the pharmaceutical compositions described herein can be by any convenient method (e.g., injection, ingestion, transfusion, 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 intraarterially, subcutaneously, intravenously, intradermally, intratumorally, intranodally, intramuscularly, or intraperitoneally. In some embodiments, the pharmaceutical compositions described herein are administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or intramuscularly). In some embodiments, the pharmaceutical compositions described herein are administered by intravenous infusion or injection. In some embodiments, the pharmaceutical compositions described herein are administered by intramuscular or subcutaneous injection. The pharmaceutical compositions described herein can be injected directly into a site of inflammation, a site of local disease, a lymph node, an organ, a tumor, or a site of infection in a subject.

[0246] Methods for immune cell modification The methods can include delivering to an immune cell (e.g., a monocyte, macrophage, or dendritic cell) one or more nucleic acid sequences encoding (a) an extracellular domain (e.g., an extracellular domain described herein), (b) a transmembrane domain (e.g., a transmembrane domain described herein), and (c) an intracellular domain (e.g., an intracellular domain described herein), such that the immune cell comprises a CAR comprising (a)-(c). In some embodiments, the nucleic acid construct comprising the one or more nucleic acid sequences further encodes one, two, or three of: (d) an extracellular leader domain (e.g., an extracellular leader domain described herein), (e) an extracellular hinge domain (e.g., an extracellular hinge domain described herein), or (f) an intracellular costimulatory domain (e.g., an intracellular costimulatory domain described herein).

[0247] Nucleic acid constructs comprising one or more nucleic acid sequences encoding at least one CAR described herein can be introduced into immune cells (e.g., monocytes, macrophages, or dendritic cells) by physical, chemical, or biological methods. Physical methods for introducing the nucleic acid constructs described herein into immune cells (e.g., monocytes, macrophages, or dendritic cells) can include electroporation, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, or a combination thereof. Nucleic acid constructs can be introduced into immune cells using commercially available methods, including electroporation (Amaxa Nucleofector-II® (Amaxa Biosystems, Cologne, Germany), ECM 830 BTX (Harvard Instruments, Boston, Mass.), Gene Pulser II® (BioRad, Denver, Colo.), or Multiporator® (Eppendort, Hamburg, Germany)). Nucleic acid constructs can also be introduced into immune cells using mRNA transfection, e.g., cationic liposome-mediated transfection, lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic particle delivery systems such as "gene guns" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001) (incorporated herein by reference in its entirety)).

[0248] Biological methods for introducing the nucleic acid constructs described herein into immune cells (e.g., monocytes, macrophages, or dendritic cells) include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become widely used to insert genes into mammalian cells (e.g., human cells). Viral vectors may also be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses (e.g., Adf535), or adeno-associated viruses (see, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362, incorporated herein by reference in their entireties). Retroviral vectors (e.g., lentiviruses) are suitable tools for achieving long-term gene transfer, allowing transgenes to be stably integrated and transmitted to daughter cells for extended periods of time.

[0249] Chemical means for introducing the nucleic acid constructs described herein into immune cells (e.g., monocytes, macrophages, or dendritic cells) include colloidal dispersion systems, macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems (e.g., oil-in-water emulsions, micelles, mixed micelles, nanoparticles, liposomes, and lipofectamine-nucleic acid complexes).

[0250] Exemplary systems for delivering the nucleic acid constructs described herein include lipid-based systems. The nucleic acid constructs described herein can be encapsulated in the aqueous interior of liposomes, interspersed within lipid bilayers, attached to liposomes via linking molecules, entrapped in liposomes, complexed with liposomes, dispersed in a solution or suspension containing lipids, mixed with lipids, complexed with micelles, or otherwise associated with lipids. Lipids for use in the methods described herein can be natural or synthetic. Lipids can also be obtained from commercial sources. For example, dimyristyl phosphatidylcholine can be obtained from Sigma (St. Louis, MO), dicetyl phosphate can be obtained from K&K Laboratories (Plainview, NY), cholesterol can be obtained from Calbiochem-Behring, and dimyristyl phosphatidylglycerol can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C.

[0251] Various assays can be performed to confirm the presence of the nucleic acid constructs described herein in immune cells (e.g., monocytes, macrophages, or dendritic cells). For example, such assays include molecular biological assays (e.g., Southern and Northern blotting, RT-PCR, and PCR) well known to those skilled in the art, as well as biochemical assays (e.g., assays detecting the presence or absence of specific peptides, e.g., by immunological means (e.g., ELISA and Western blot)).

[0252] VPX-mediated viral delivery of nucleic acid encoding CAR Immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein may be resistant to lentiviral transduction due to the expression of the restriction factor SAMHD1, which depletes nucleotide triphosphates available for reverse transcription. For example, SAMHD1 can restrict the replication of human immunodeficiency virus type 1 (HIV-1) by depleting intracellular pools of deoxynucleoside triphosphates. Viral protein X (Vpx), an accessory protein associated with simian immunodeficiency virus (SIV) and HIV-2, induces the degradation of SAMHD1. The use of Vpx can enable viral vectors (e.g., lentiviruses) to contain one or more nucleic acid sequences encoding at least one CAR directed against immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein. In some embodiments, viral vectors comprising one or more nucleic acid sequences encoding at least one CAR and packaged with at least one Vpx protein can increase transfection of immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein, compared to the same type of immune cell transfected with a viral vector comprising one or more nucleic acid sequences encoding at least one CAR that are not packaged with at least one Vpx protein. In some embodiments, Vpx lentiviruses can incorporate CAR sequences into their genomes, enabling long-term, sustained expression of chimeric antigen receptors (CARs). Given that CARs are transmembrane proteins that recycle periodically into the intracellular space, demonstrating sustained cell surface expression of CARs by Vpx-lentiviruses is a notable development in the field. Macrophages transduced with Vpx-lentiviruses are not phenotypically affected by viral transduction, a finding that allows for the production of CAR macrophages with phenotypic plasticity. Whereas other viral vectors (e.g., Ad5f35) induce an M1, pro-inflammatory phenotype, Vpx-lentivirus does not affect the M1 / M2 phenotype, allowing for an M0 CAR macrophage product that may not have pro-inflammatory function / toxicity at baseline.Vpx-lentivirus-transduced CAR macrophages retain phenotypic plasticity and can be further polarized to M1 or M2 phenotypes using cytokines, agonists, peptides, culture media, and other factors. Vpx-lentivirus-transduced CAR macrophages can be exposed to pro-inflammatory signals (e.g., one or more pro-inflammatory cytokines, such as LPS, IFNa, IFNb, IFNγ, CpG, CD40L, GM-CSF, TNFa, IL-6, or STING ligand (STING-L)) and polarized to M1 macrophages. Vpx-lentivirus-transduced CAR macrophages can be exposed to immunosuppressive signals (e.g., one or more immunosuppressive cytokines (e.g., one or more of IL-4, IL-10, IL-13, or TGFb) and / or at least one prostaglandin or at least one adrenocortical hormone), and polarized to M2 macrophages.

[0253] In some embodiments, the lentiviral vector is packaged with a Vpx protein (e.g., as described in International Publication No. 2017 / 044487, incorporated herein by reference in its entirety). In some embodiments, the Vpx comprises a virion-associated protein (e.g., an accessory protein for viral replication). In some embodiments, the Vpx protein is encoded by human immunodeficiency virus type 2 (HIV-2). In some embodiments, the Vpx protein is encoded by simian immunodeficiency virus (SIV). In some embodiments, an immune cell (e.g., monocyte, macrophage, or dendritic cell) described herein is transfected with a lentiviral vector packaged with a Vpx protein. In some embodiments, the Vpx inhibits at least one antiviral factor in an immune cell (e.g., monocyte, macrophage, or dendritic cell) described herein.

[0254] In some embodiments, lentiviral vectors packaged with Vpx proteins exhibit increased transfection efficiency of immune cells (e.g., monocytes, macrophages, or dendritic cells) described herein, e.g., compared to lentiviral vectors not packaged with Vpx proteins. In some embodiments, immune cells (e.g., monocytes, macrophages, or dendritic cells) described herein are electroporated and / or transfected with at least one VPX mRNA prior to transfection with a viral vector (e.g., an adenoviral vector, e.g., an Ad2 vector or an Ad5 vector (e.g., an Ad5f35 adenoviral vector, e.g., a helper-dependent Ad5F35 adenoviral vector)).

[0255] Treatment and culture of immune cells during modification In some embodiments, the methods of the present disclosure include one or more steps of treating immune cells (eg, monocytes, macrophages, or dendritic cells) during the process of modifying the immune cells.

[0256] In some embodiments, the disclosed methods involve treating immune cells (e.g., monocytes, macrophages, or dendritic cells) with a modulator of a pathway activated by in vitro-transcribed mRNA. The in vitro-transcribed (IVT) mRNA is recognized by various endosomal innate immune receptors (Toll-like receptor 3 (TLR3), TLR7, and TLR8) and cytoplasmic innate immune receptors (protein kinase RNA-activated (PKR), retinoic acid-inducible gene I protein (RIG-I), melanoma differentiation-associated protein 5 (MDA5), and 2'-5'-oligoadenylate synthetase (OAS)). Signaling through these different pathways leads to inflammation associated with activation of type 1 interferon (IFN), tumor necrosis factor (TNF), interleukin-6 (IL-6), IL-12, and a cascade of transcriptional programs. Overall, this creates a pro-inflammatory microenvironment poised to induce specific immune responses. Furthermore, downstream effects (e.g., slowing down translation by phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), enhancing RNA degradation by ribonuclease L (RNase L), and overexpression and inhibition of self-amplifying mRNA replication) are relevant to the pharmacokinetics and pharmacodynamics of IVT mRNA.

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

[0258] In some embodiments, treating immune cells (e.g., monocytes, macrophages, or dendritic cells) with an RNaseL inhibitor increases mRNA stability in the modified immune cells compared to mRNA stability in modified immune cells of the same type that are not treated with the RNaseL inhibitor. In some embodiments, treating immune cells (e.g., monocytes, macrophages, or dendritic cells) with an RNaseL inhibitor increases CAR expression in the modified immune cells compared to CAR expression in modified immune cells of the same type that are not treated with the RNaseL inhibitor. In some embodiments, treating immune cells (e.g., monocytes, macrophages, or dendritic cells) with an RNaseL inhibitor increases effector activity in the modified immune cells compared to effector activity in modified immune cells of the same type that are not treated with the RNaseL inhibitor.

[0259] In some embodiments of the present disclosure, the step of treating the immune cells (eg, monocytes, macrophages, or dendritic cells) occurs before the step of delivering the mRNA to the immune cells.

[0260] In some embodiments, the methods of the disclosure include culturing immune cells (e.g., monocytes, macrophages, or dendritic cells) with a cytokine or immunostimulatory recombinant protein. In some embodiments, the cytokine is selected from the group consisting of IFN-α, IFN-β, IFN-γ, TNFα, IL-6, SFNGL, LPS, CD40 agonist, 4-1BB ligand, recombinant 4-1BB, CD19 agonist, TLR agonist (e.g., TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, ​​TLR-7, TLR-8, or TLR-9), TGF-β (e.g., TGF-β1, TGF-β2, or TGF-β3), glucocorticoid, immune complex, insulin, or the like. Interleukin-1 alpha (IL-1α), IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), leukemia inhibitory factor (LIF), oncostatin M (OSM), tumor necrosis factor (TNF-β), CD154, lymphotoxin β (LT-β), and growth-inducing ligand (AGL) ( APRIL), CD70, CD153, glucocorticoid-induced TNF receptor ligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L (CD252), TALL-1 (tumor necrosis factor superfamily member 13B (TNFSF13B)), TNF-related apoptosis-inducing ligand (TRAIL), TNF-related weak inducer of apoptosis (TWEAK), TNF-related activation-inducing cytokine (TRANCE), erythropoietin (Epo), thyroid pelvis oxidase precursor (Tpo), FMS-related tyrosine kinase 3 ligand (FLT-3L), stem cell factor (SCF), macrophage colony-stimulating factor (M-CSF), merozoite surface protein (MSP), nucleotide-binding oligomerization domain-containing protein (NOD) ligand (e.g., NOD1, NOD2, or NOD1 / 2 agonist), RIG-I-like receptor (RLR) ligand (e.g., 5'ppp-dsRNA, 3p-hpRNA, poly(I:C), or poly(dA:dT)),C-type lectin receptor (CLR) ligands (e.g., curdlan, β-glucan, HKCA, laminarin, pustulan, scleroglucan, WGP-dispersible, WGP-soluble, zymosan, zymosan-depleted, furfurman, b-GlcCer, GlcC14C18, HKMT, TDB, TDB-HD15, or TDM), cyclic dinucleotide sensor ligands (e.g., C-Gas agonists or stimulator of interferon genes (STING) ligands), inflammasome inducers (e.g., alum, ATP, CPPD crystals, hemozoin, MSU crystals, nanoSiO2, nigericin, or TDB), aryl hydrocarbon (AhR) ligands (e.g., FICZ, indirubin, ITE, or L-kynurenine), alpha protein kinase 1 (ALPK1) ligands, multi-PRR ligands, NFKB / NFAT activators (e.g., concavalin In some embodiments, the cytokine comprises IFN-β.

[0261] In some embodiments of the present disclosure, the step of culturing immune cells (eg, monocytes, macrophages, or dendritic cells) is performed after the step of delivering mRNA to the immune cells.

[0262] In some embodiments, culturing modified immune cells (e.g., monocytes, macrophages, or dendritic cells) with a cytokine or immunostimulatory recombinant protein increases the viability of the modified immune cells compared to modified immune cells of the same type that are not cultured with the cytokine or immunostimulatory recombinant protein. In some embodiments, culturing modified immune cells (e.g., monocytes, macrophages, or dendritic cells) with a cytokine or immunostimulatory recombinant protein increases protein (e.g., CAR) expression in the modified immune cells compared to modified immune cells of the same type that are not cultured with the cytokine or immunostimulatory recombinant protein. In some embodiments, culturing modified immune cells (e.g., monocytes, macrophages, or dendritic cells) with a cytokine or immunostimulatory recombinant protein increases the longevity of protein (e.g., CAR) expression compared to modified immune cells of the same type that are not cultured with the cytokine or immunostimulatory recombinant protein. In some embodiments, culturing modified immune cells (e.g., monocytes, macrophages, or dendritic cells) with a cytokine or immunostimulatory recombinant protein increases effector activity of the modified immune cells compared to modified immune cells of the same type that are not cultured with the cytokine or immunostimulatory recombinant protein. In some embodiments, culturing modified immune cells (e.g., monocytes, macrophages, or dendritic cells) with a cytokine or immunostimulatory recombinant protein increases M1 polarization of the modified immune cells compared to modified immune cells of the same type that have not been cultured with the cytokine or immunostimulatory recombinant protein.

[0263] All publications, patent applications, patents, and other references mentioned herein, including GenBank accession numbers, are incorporated by reference in their entirety. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in practicing or testing the present invention, suitable methods and materials are described herein.

[0264] The present disclosure is further illustrated by the following examples, which are presented for illustrative purposes only and should not be construed as limiting the scope or content of the disclosure in any way. [Example]

[0265] The following examples are presented to illustrate to one of ordinary skill in the art how to make and use the methods and compositions described herein and are not intended to limit the scope of the disclosure.

[0266] Example 1: Chimeric Antigen Receptor (CAR) Macrophages and Monocytes CAR transgenes can be introduced into monocytes or macrophages by electroporation or transfection with DNA, mRNA, or chemically modified mRNA, or by viral transduction using lentivirus, adenovirus, or alternative viral vectors. CAR expression is confirmed using flow cytometry, real-time PCR, or antigen-specific staining by fluorescence microscopy. These techniques are also used to quantify the intensity and kinetics of CAR expression.

[0267] CAR constructs expressed on the surface of macrophages are tested for activity in tumor phagocytosis and / or tumor killing assays against target-positive cell lines. Constructs that induce phagocytosis and / or killing of target cells are tested for cytokine secretion, chemokine secretion, immune cell recruitment capacity, phenotypic shift (self-polarization to M1 / M2), and T cell stimulation / antigen presentation function.

[0268] Table 1 shows exemplary mRNA and polypeptide sequences used in the CAR components described herein. CAR constructs for exemplary CAR components are shown in Figures 8-117 and 135. [Table 1-1] [Table 1-2]

[0269] Example 2: Viral transduction of primary human macrophages and monocytes To produce lentivirus, HEK293T cells were plated at 3.5 x 106 cells / 10 cm plate in 10 mL of DMEM medium supplemented with 10% FBS and 1% pen / strep. After overnight growth, plates were transfected using calcium phosphate transfection with pVSV-G (2.5 μg), RSV-Rev (2.5 μg), PMDL-Chp6 (10 μg), pVPX (1 μg), and lentiviral transfer plasmids (14 μg) containing the desired insert sequence. 16 hours after transfection, the supernatant was discarded, and 6 mL of DMEM was then added. 48 hours after transfection, the supernatant was harvested, centrifuged at 1,200 x g for 5 minutes, and filtered through a 0.45 mm filter. Virus was stored at -80°C for later use.

[0270] For lentiviral transduction of primary human macrophages, primary human macrophages were plated at a given density and incubated at 37°C for 2 hours. Lentivirus was then added to the macrophage cultures at the indicated multiplicity of infection (MOI). After 48-72 hours, the medium was changed, and the transduced macrophages were used for various assays.

[0271] Example 3: Receptor expression from monocytes and macrophages CD14+ monocyte-derived macrophages were plated in culture medium (10–20% FBS-TEX-MACS and 10 ng / ml GM-CSF) and cultured overnight. They were transfected with 120 nM CAR mRNA and incubated in culture medium at 37°C for 1 day. CAR expression was detected by flow cytometry using rHER2 binding. Approximately 60–70% of CAR- and chimeric FcR (CFR)-transfected cells and 40–60% of toll-like antigen receptor (TAR)-transfected cells survived (Figure 1A). CAR:HER2 expression was positive in 40–60% of CAR-transfected cells and 30–40% of CFR- and TAR-transfected cells (Figure 1B). In primary human macrophages, CAR1 and CAR13 expression was prominent among the other constructs.

[0272] CD14+ monocytes purified with MACS beads were incubated overnight in culture medium. 40 nM CAR mRNA was transfected, and the cells were incubated at 37°C for 3 days. CAR expression was detected by rHER2 binding by flow cytometry. Ad5f35 was used as a positive control. Approximately 50–80% of CAR- and TAR-transfected cells and approximately 40–60% of CFR-transfected cells survived (Figure 2A). CAR:HER2 expression was positive in 15–30% of CAR-, CFR-, and TAR-transfected monocytes (Figure 2B). These results indicate that CAR, CFR, and TAR mRNA can be expressed in primary human monocytes, but the expression intensity is much lower than in macrophages.

[0273] Example 4: CAR macrophage receptor expression and tumor cell killing To generate receptor-expressing macrophages, primary human macrophages were cultured at 1.0x10 in EP buffer containing 50–500 nM mRNA (or the highest possible concentration based on the mRNA stock). 5 ~1.0x10 9 The cells were suspended at a concentration of 1000 cells / mL and electroporated. Cells were removed from the cassette, plated in TexMACS medium containing 20% ​​FBS, and incubated overnight at 37°C and 5% CO2. The MFI and percentage of CAR, CFR, and TAR expression were detected by flow cytometry after 24 hours using rHER2 binding. Live / Dead Aqua was used to detect survival percentages. For killing, macrophages were co-cultured with Her2+ CRL2351-NucGFP tumor cells at a 2:1 E:T ratio and monitored for 72 hours using Incucyte®. Tumor cell death was calculated by the integrated GFP intensity per well relative to time 0.

[0274] Expression and CAR+ percentages varied among CAR constructs (Figures 3A and 3B and Figures 119 and 120). mRNA electroporation did not appear to affect survival percentages (Figures 3C and 3C). Neither the CFR nor the TAR constructs appeared to have any effect on tumor cell killing. Constructs CAR001 (CAR comprising a CD8 leader, HER2 scFv, CD8 hinge, CD8 TM, and CD3z IC), CAR003 (CAR comprising a CD8 leader, HER2 scFv, CD8 hinge, and CD8 TM), CAR007 (CAR comprising a CD8 leader, 4D5 scFv, IgG4 hinge, CD8 TM, and CD3z IC), CAR013 (CAR comprising a CD8 leader, HER2 scFv, CD8 hinge, CD64 TM, and CD64 ICD), CAR017 (CAR comprising a CD8 leader, HER2 scFv, CD8 hinge, CD64 TM, CD64 ICD, P2A, and FCER1G), and CAR039 (CAR comprising a CD8 leader, HER2 scFv, CD8 hinge, TLR4 TM, and TLR4 IC). A CAR containing an ICD was expressed in primary human macrophages and exhibited killing function (Figure 3D).

[0275] Expression and CFR and TAR+ percentages varied between constructs (Figures 4A and B). mRNA electroporation did not appear to affect survival percentages (Figure 4C).

[0276] Example 5: Assessment of CAR expression The duration of CAR expression can be assessed by flow cytometry. CAR macrophages can be plated in wells of a 96-well plate. To measure anti-Her2 CAR expression, His-tagged recombinant Her2 protein is added to the cells along with a buffer (e.g., PBS supplemented with BSA) and incubated for 15 minutes. The cells are then spun down at 300xg for 5 minutes, and the supernatant is removed. Fc receptors are then blocked in PBS for 5 minutes using an Fc blocking solution (e.g., Human TruStain FcX (BioLegend; catalog number 422302)). After Fc blocking, cells can be stained for cell viability and other surface markers. For example, cell viability can be quantified using the LIVE / DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogen; catalog number L34957). An anti-His antibody (e.g., His Tag APC-conjugated Antibody (R&D Systems; catalog number IC050A)) is then added. CAR expression is quantified by flow cytometry by first gating on a single cell population, then selecting live cells, and finally measuring the APC fluorescence of the cells. CAR-expressing cells will be brighter in the APC channel than control cells not exposed to anti-His antibodies. The brightness of CAR-positive cells determines the degree of expression, while repeated measurements over time allow tracking of CAR expression over time.

[0277] For immunofluorescence, cells can be cultured on slides as appropriate. The medium is then removed, and the cells are washed three times with PBS. The cells can then be fixed using 4% paraformaldehyde or methanol. The exact incubation time in the fixative depends on its contents. After the appropriate time, the fixative is removed, and the cells are again washed three times with PBS. If intracellular staining is desired, the cells are now incubated with 1% Triton X-100 in PBS (ThermoFisher; catalog number BP151-100) and then washed three times with PBS. A blocking solution (e.g., BSA in PBS) is then added to the cells and allowed to block for 60 minutes. The blocking solution is then removed, and the cells are washed. Fluorescent dye-conjugated antibodies are diluted according to the manufacturer's instructions and allowed to bind to the cells overnight. The antibody solution is then removed, and the cells are washed. Finally, a mounting solution (e.g., ProLong™ Diamond Anti-Burning Mounting Medium with DAPI (Invitrogen; Cat. No. P36966)) is applied to the cells and a coverslip is placed over them. This is allowed to dry for 24 hours before imaging. Imaging is performed using a fluorescence microscope. Cells expressing CAR will be brighter in the channel appropriate for the fluorescent dye than cells that do not express CAR.

[0278] For RTPCR, macrophages are lysed and RNA is harvested using a one-step RTPCR kit (SuperScript™ III Platinum™ One-Step qRT-PCR Kit; Invitrogen; catalog number 11732-020) according to the manufacturer's instructions. Primers specific for CAR are used in the assay. Macrophages with CAR mRNA will show a signal in the RTPCR assay, whereas non-transduced macrophages will not.

[0279] Example 6: Evaluation of CAR functionality For the cell flow phagocytosis assay, target-positive and target-negative tumor cells were labeled using the CellTrace™ CFSE Cell Proliferation Kit (Invitrogen; catalog number C34554) according to the manufacturer's instructions, or cells were engineered to express a fluorescent protein such as GFP. CAR-expressing and control macrophages were then plated into a U-bottom 96-well plate at a 1:1 macrophage:tumor cell ratio and cultured for 4 hours. After incubation, cells were removed from the wells and stained for flow cytometry. The panel included a viability dye and a macrophage-specific marker (e.g., CD11b). When gating on live cells, cells that were CD11b / CFSE double-positive were identified as macrophages that had presumably phagocytosed the cells. When CAR macrophages were cultured with target-positive tumor cells, the percentage of double-positive cells should be increased compared to macrophages without CAR. Furthermore, the specificity of phagocytosis was demonstrated by the lack of a significant change in the amount of phagocytosis when macrophages were cultured with target-negative cells.

[0280] For bead-based flow phagocytosis assays, polystyrene beads can be functionalized with the CAR target protein or an unrelated protein. Furthermore, the beads are labeled with the pH-sensitive dye pHrodo™ Red, SE (Invitrogen; Catalog No. P36600). Upon acidification, the dye increases its fluorescence level. The beads are then cultured with CAR macrophages and untransduced macrophages. After a period of time, the macrophages can be removed and stained for flow cytometry using a viability dye and a macrophage-specific marker (e.g., CD11b). When gating on live cells, cells that are CD11b / pHrodo double-positive represent macrophages that have presumably phagocytosed the beads. When CAR macrophages are cultured with target-positive beads, the percentage of double-positive cells should be increased compared to macrophages without CAR. Furthermore, the specificity of phagocytosis is demonstrated by the lack of significant changes in the amount of phagocytosis when macrophages are cultured with target-negative beads.

[0281] For Incucyte® cell analysis, target-positive tumor cells expressing a fluorescent protein such as GFP are cultured in 96-well plates with CAR macrophages and untransduced macrophages. The effector macrophage to target tumor cell ratio is varied from 10:1 E:T to 1:10 E:T, with a 0:1 E:T target cell-only control. The number of macrophages is kept constant at 10e3 macrophages per well. Changes in fluorescence over time, measured every 4 hours, can be measured to quantify the amount of tumor cell killing that occurred during culture. Furthermore, image analysis techniques can be used to localize macrophages in the culture and quantify the number of macrophages that also phagocytose tumor cells. CAR macrophages should demonstrate increased colocalization and killing of macrophages and tumor cells compared to untransduced macrophages or tumor cell-only controls.

[0282] For Incucyte® analysis of the beads, pHrodo functionalized beads bearing the protein target of CAR are added to both CAR macrophages and non-transduced macrophages in the wells of a 96-well plate. Macrophages are plated at a concentration of 20e3 per well, and beads are added at a 5:1 bead:macrophage ratio. pHrodo fluorescence is measured for 5 hours, measuring every 30 minutes. The ratio of increase in fluorescence between the initial time point and the 1-hour time point is used to quantify the amount of phagocytosis that has occurred. CAR macrophages are expected to show a larger change in fluorescence compared to non-transduced controls.

[0283] Due to antigen processing and presentation after phagocytosis, T cell expansion, IL2 release, IFNg release, TNFa release, and / or CD69 upregulation can be analyzed. T cell clones known to respond to a particular antigen (e.g., ovalbumin) can be obtained and cultured. Often, these clones are murine cells. Murine CAR macrophages, MHC-matched to T cells, are cultured with cancer cells expressing both the CAR target receptor and the known antigen. Macrophages can then be isolated and cocultured with T cells. Upon antigen presentation by the macrophages, T cells secrete IL2 (which can be measured by cytokine measurement techniques) and proliferate (which can be measured by cell counting or CFSE dilution). Additionally, the cytotoxic function of activated T cells can be confirmed by coculture of T cells with antigen-expressing cell lines. CAR macrophages are expected to induce increased proliferation and cytokine production in T cells compared to untransduced macrophage controls.

[0284] Tumor killing by methods other than phagocytosis can also be evaluated. CAR macrophages and target-positive cells can be co-cultured in conditioned medium for a period of time (e.g., 24 hours) to allow the macrophages to sense and respond to the target cells. The ratio of tumor cells can be varied depending on the expression level of the target protein in the tumor cells. After the desired amount of time has passed, the supernatant is removed from the CAR macrophages, and any cells remaining in the supernatant are removed by filtration through a 0.22 micron filter or centrifugation. The filtered supernatant is then loaded onto other cancer cells that have not been co-cultured with CAR macrophages. The resulting changes in tumor cell viability and proliferation can be measured by cell counting, flow cytometry, MTT / XTT assay, or microscopy. CAR macrophages should kill or reduce the growth rate of tumor cells cultured with the supernatant compared to the supernatant of untransduced macrophages cultured with the same target-positive cell line.

[0285] For target+ / target- / CAR macrophage cocultures, CAR macrophages, target-positive cells, and target-negative cells are cocultured together. There must be some way to distinguish between target-positive and target-negative cells (e.g., expression of different fluorescent proteins). Over time, the relative numbers of different cell types can be quantified by either microscopy or flow cytometry. Comparing the growth of target-negative cells with CAR macrophages in the presence or absence of target-positive cells provides some insight into non-phagocytic tumor cell killing. CAR macrophages would be expected to show increased killing or a reduced growth rate of target-negative cell lines compared to untransduced macrophages. Furthermore, differences are expected between target-negative cells cultured with CAR macrophages depending on the presence or absence of target-positive cells.

[0286] To measure the production of desired cytokines by flow cytometry, CAR macrophages can be co-cultured with target-positive cells or beads and a target-negative control. After a specific amount of time, the culture supernatant can be harvested and filtered or centrifuged to remove scattered cells. The desired cytokines can be measured by measuring the concentration of various cytokines in the supernatant using a flow cytometry-based cytokine bead assay. The beads are stained according to the manufacturer's instructions. An example of such a system is the BioLegend LEGENDplex system. Furthermore, CAR macrophages can be harvested from the culture system and stained for cytokine production using intracellular cytokine staining with flow cytometry. The cells can be fixed, permeabilized, and stained according to the manufacturer's instructions, for example, using the Fixation / Permeabilization Solution Kit (BD; catalog number 554714). CAR macrophages cultured with target-positive cells or beads should exhibit higher levels of pro-inflammatory cytokines compared to non-transduced macrophages or CAR macrophages not stimulated with the target protein.

[0287] For ELISA / MSD analysis, CAR macrophages can be co-cultured with target-positive cells or beads and a target-negative control. After a specific amount of time, the culture supernatant can be harvested and filtered or centrifuged to remove scattered cells. An example would be a 24-hour culture at an 8:1 macrophage:target ratio. After the culture period, the supernatant can be removed and the amount of cytokines present in the solution can be measured using an ELISA cytokine kit (e.g., IL-1 beta Human Instant ELISA™ Kit; Invitrogen; catalog number BMS224INST) according to the manufacturer's instructions. Additionally, cytokines can be measured using the MesoScaleDiscovery QuickPlex system according to the manufacturer's instructions. It is expected that CAR macrophages cultured with target-positive cells or beads should exhibit higher levels of pro-inflammatory cytokines compared to untransduced macrophages or CAR macrophages not stimulated with the target protein.

[0288] To measure ROS production by flow cytometry, CAR macrophages can be co-cultured with target-positive cells or beads and a target-negative control. Non-transduced macrophages can also be cultured under the same conditions. Measurement of reactive oxygen species (ROS) production can be performed using a flow cytometry-based kit (e.g., Total Reactive Oxygen Species (ROS) Assay Kit 520 nm (Invitrogen; catalog number 88-5930-74)). Cells can be prepared according to the manufacturer's instructions. CAR macrophages cultured with target-positive cells or beads are expected to exhibit higher levels of ROS than CAR macrophages cultured with target-negative cells or beads, and non-transduced macrophages cultured with either target-positive or target-negative cells or beads. Further methods are described in Dikalov & Harrison. Antioxid Redox Signal. 2014;20(2):372-382 (incorporated herein by reference in its entirety).

[0289] To enhance resistance to phagocytosis checkpoints, CAR macrophages can be cocultured with target-positive beads or cells bearing varying levels of CD47 or other antiphagocytic ligands. The levels of CD47 or other antiphagocytic ligands on beads can be easily altered by varying the amount of protein used for functionalization. Altering the levels of antiphagocytic ligands in cells requires genetic manipulation (e.g., shRNA or CRISPR / Cas9 systems). Phagocytosis can be quantified by either microscopy or flow cytometry, as outlined in the previous section. Both CAR and non-transduced macrophages are expected to exhibit decreased phagocytosis with increasing amounts of CD47 or other antiphagocytic ligands. Macrophages engineered to express dominant-negative receptors (e.g., truncated SIRPa) should exhibit increased levels of phagocytosis compared to macrophages lacking such dominant-negative receptors, both CAR and non-transduced.

[0290] For comparison with CD47 blocking, the action of CD47 can be blocked by using a blocking antibody (e.g., murine clone B6H12). Varying the amount of CD47 on the surface of target-positive or -negative cells allows for different levels of phagocytosis. A dose titration curve of the CD47 blocking antibody can be determined by culturing target-positive and target-negative cells with various amounts of blocking antibody. In phagocytosis assays using both CAR macrophages and non-transduced macrophages, CAR macrophages should exhibit lower levels of inhibition at a given level of CD47 blocking, indicating that they are less sensitive to CD47 than non-transduced cells. In contrast, target-negative cells show no difference between CAR macrophages and non-transduced macrophages.

[0291] The expression of chemokine receptors that support trafficking can be measured by binding specific antibodies to CAR macrophages, which can then be measured using a flow cytometer. CAR macrophages are expected to exhibit higher levels of diverse chemokine receptors than non-transduced macrophages.

[0292] CAR macrophages and non-transduced macrophages can be lysed and RNA extracted for RNA sequencing. By comparing the RNA transcripts expressed within the cells, it is possible to determine which receptors are expressed in the cells. CAR macrophages are expected to express higher levels of a variety of chemokine receptors than non-transduced macrophages.

[0293] For Western blotting, CAR macrophages and non-transduced macrophages can be cultured, lysed, and total protein collected. The proteins are then size-fractionated by gel migration in an electric field and then transferred to a membrane. The membrane can be stained with antibodies specific to a particular protein, and the presence or absence of the protein can be determined by this staining. CAR macrophages are expected to exhibit higher levels of various chemokine receptors than non-transduced macrophages.

[0294] To express chemokines that recruit other immune cells, CAR macrophages can be co-cultured with target-positive cells or beads and a target-negative control. After a specific amount of time, the culture supernatant can be harvested and straggling cells removed by filtration or centrifugation. Desired cytokines can be measured by measuring the concentration of various cytokines in the supernatant using a flow cytometry-based cytokine bead assay. The beads are stained according to the manufacturer's instructions. An example of such a system is the BioLegend LEGENDplex system. Furthermore, CAR macrophages can be harvested from the culture system and stained for cytokine production using intracellular cytokine staining with flow cytometry. Cells can be fixed, permeabilized, and stained according to the manufacturer's instructions, for example, using the Fixation / Permeabilization Solution Kit (BD; catalog number 554714). CAR macrophages cultured with target-positive cells or beads should exhibit higher levels of pro-inflammatory cytokines compared to untransduced macrophages or CAR macrophages not stimulated with the target protein.

[0295] For ELISA / MSD, CAR macrophages can be co-cultured with target-positive cells or beads and a target-negative control. After a specific amount of time, the culture supernatant can be harvested and filtered or centrifuged to remove scattered cells. An example would be a 24-hour culture at an 8:1 macrophage:target ratio. After the culture period, the supernatant can be removed and the amount of cytokine present in the solution can be measured using an ELISA cytokine kit (e.g., IL-1 beta Human Instant ELISA™ Kit; Invitrogen; catalog number BMS224INST) according to the manufacturer's instructions. Additionally, cytokines can be measured using the MesoScaleDiscovery QuickPlex system according to the manufacturer's instructions. It is expected that CAR macrophages cultured with target-positive cells or beads should exhibit higher levels of pro-inflammatory cytokines compared to untransduced macrophages or CAR macrophages not stimulated with the target protein.

[0296] For transwell assays, CAR macrophages can be co-cultured with target-positive cells or target-positive beads and a target-negative control. Additionally, macrophages and target cells can be cultured separately as a control. After sufficient incubation (e.g., 48 hours), the supernatant can be harvested, and remaining cells can be removed by filtration. A subpopulation of purified immune cells (e.g., CD3-positive T cells) can be labeled with CFSE according to the manufacturer's instructions. Transwell inserts (e.g., HTS Transwell®-96 Permeable Support (3.0 μm pore polycarbonate membrane); Corning; Catalog No. 3386) can be functionalized with extracellular matrix proteins (e.g., fibronectin or collagen). The matrix protein is brought into solution and applied to the membrane for 30 minutes at 37°C. The remaining solution is then removed, and the membrane is washed with PBS. CFSE-labeled cells are then resuspended in serum-free medium and placed on the top surface of the insert. These cells are allowed to settle and adhere for 20 minutes. The insert is then moved so that the bottom of the membrane is exposed to the harvested supernatant. The cells are allowed to migrate for at least 2 hours. After the incubation period, the inserts are removed and the fluorescence of the bottom plate wells containing the supernatant is measured. CAR macrophages are expected to exhibit a higher degree of recruitment compared to untransduced controls. Furthermore, CAR macrophages cultured with target-positive cells are expected to recruit labeled immune cells better than CAR macrophages cultured with target-negative cells.

[0297] Macrophages can be labeled using CFSE staining according to the manufacturer's instructions for proliferation by CFSE dilution. These labeled macrophages can then be cultured for an appropriate period of time. The dilution level of intracellular CFSE can be used to quantitate the degree of macrophage proliferation.

[0298] For MTT / XTT assays, plate cells and run the assay according to the manufacturer's instructions for MTT (ThermoFisher; Cat. No. V13154) or XTT (ThermoFisher; Cat. No. X12223) assays.

[0299] To produce macrophages from induced pluripotent stem cells (iPSCs), methods such as those described in Ackermann et al. Nat Commun. 2018;9(1):5088; Zhang et al. Circ Res. 2015;117(1):17-28; Mucci et al. Stem Cell Reports. 2018;11(3):696-710; Shi et al. Curr Protoc Stem Cell Biol. 2019;48(1):e74; and Takata et al. 2017;47(1):183-198.e6; Cao et al. Stem Cell Reports. 2019;12(6):1282-1297 (each of which is incorporated herein by reference in its entirety) can be used. To prepare iPSCs, lentiviral transduction can be performed prior to differentiation.

[0300] Example 7: Priming of macrophages To generate Her2 zeta CAR-expressing macrophages, primary human macrophages were cultured in EP buffer (MaxCyte) containing 300 nM mRNA (TriLink) at 90 x 10 6 The cells were suspended at a concentration of 100 cells / mL. 100 μL of the cell mixture was added to an electroporation cassette (OC100x2; MaxCyte) and electroporated using the Experimental T cell setting of 1. The cells were removed from the cassette and plated in 3 mL of TexMACS medium (Miltenyi Biotech) containing 20% ​​FBS (Gibco) on an UpCell plate (Thermo Scientific) and incubated overnight at 37°C and 5% CO2.

[0301] Recombinant CD40 ligand (Peprotech), 4-1BB ligand (Enzo Life Sciences), and 4-1BB receptor (Peprotech) were resuspended in molecular-grade water to a stock concentration of 100 μg / mL. The stock solutions were then used to create working solutions in PBS ranging from 2 to 0.002 μg / mL. 100 μL of the working solution was added to wells of a 96-well plate and left at room temperature for 4 hours.

[0302] The plates were removed from the incubator and left at room temperature for 30 minutes. Cells were detached from the plates, counted using an NC-200 automated cell counter (Chemomtech), and resuspended in TexMACS medium containing 10% FBS. The protein-coated plates were washed twice with PBS, and then macrophages were added in a final volume of 100 μL of TexMACS medium containing 10% FBS. The plates were incubated at 37°C and 5% CO2 for 3 hours. After 3 hours, 10,000 CRL-2351 cells expressing nuclear GFP were added to each well. The final concentration of GM-CSF was 10 ng / mL in all wells. Cell lysis was detected using an Incucyte® assay (Essen Bioscience). Tumor cell death was calculated based on the integrated GFP intensity per well relative to time 0.

[0303] To detect cell surface proteins, macrophages were plated onto agonist molecule-coated wells in a final volume of 200 μL of TexMACS medium + 10% FBS + 10 ng / mL GM-CSF and incubated at 37°C and 5% CO2 for 3 days. Cells were incubated with 300 μL Accutase (Sigma) for 30 minutes and transferred to a 96-well round-bottom plate for staining. Cells were incubated in FACS buffer containing 20 μg / mL Her2-His for 20 minutes at room temperature, followed by incubation in Human TruStain FcX for 10 minutes at room temperature. Staining for surface proteins was performed using the following panel: CD80-FITC, CD86-PE, CD163-APC-Cy7, CD206-BV421, anti-His-APC, and Aqua Live / Dead. Detection of surface protein expression was completed using an Attune NxT flow cytometer (Thermo Fisher).

[0304] Treatment of CAR macrophages with CD40L significantly enhanced the tumor-killing ability of macrophages and CAR macrophages (Figure 5A). Priming with CD40L also induced an M1 phenotype in mRNA-transduced CAR macrophages (Figure 5B). Treatment with 4-1BB and 4-1BBL produced similar results, but with less efficacy than CD40L (Figures 6A-6B and 7A-7B). These results indicate that pretreatment or priming of CAR macrophages with a CD40 agonist (e.g., CD40L) can increase efficacy, and that combination therapy involving CAR macrophages and a CD40 agonist can increase efficacy.

[0305] Example 8: M1 polarization enhances mRNA persistence and macrophage / CAR-macrophage function Human macrophages were electroporated with HER2 CAR mRNA containing m6AGCap1 and PsU modifications. The cells were cultured with cytokines that induce an M1 phenotype (e.g., IFN-alpha, IFN-beta, IFN-gamma, and lipopolysaccharide (LPS), TNF-alpha, IL-6, or STING ligand (STING-L)) for up to 48 hours, after which the cytokines were washed away and fresh medium was added. CAR expression and M1 marker expression were measured 2 and 7 days after transfection. Two days after transfection, fluorescently labeled HER2+ breast cancer cells (CRL2351) were co-cultured with HER2 CAR macrophages. Cancer cell growth was monitored every 4 hours by their fluorescence using an Incucyte® live imaging microscope. The effector (CAR macrophage):target (cancer cell) ratio was 5:1.

[0306] As shown in Figure 121A, the tested interferon cytokines did not reduce the viability of CAR-transfected macrophages on day 2. Surprisingly, macrophages treated with IFN-β showed higher CAR expression than macrophages treated with control medium, IFN-α, or IFN-γ (Figure 121B). As shown in Figure 121C, treatment of macrophages with interferon cytokines did not reduce the viability of CAR-transfected macrophages on day 7. Surprisingly, macrophages treated with IFN-β showed higher CAR expression than macrophages treated with control medium, IFN-α, or IFN-γ. This demonstrates that IFN-β improves the expression period of mRNA-encoding transgenes (e.g., CAR) in human macrophages (Figure 121D). Furthermore, treatment of mRNA-transfected CAR macrophages with IFN-α, IFN-β, or IFN-γ resulted in the induction of an M1 phenotype (based on CD86 expression; Figure 121E) and a reduction in M2 markers (based on CD163 expression; Figure 121F). IFN-β induced the most potent M1 phenotype of the interferons evaluated, which persisted for at least 7 days after treatment (Figure 121E).

[0307] Example 9: Effect of IFN treatment on CAR expression, CAR macrophage function, M1 phenotype markers, and cytokine production Five different mRNA modifications were also tested to determine whether interferon treatment differentially affected macrophages transfected with mRNA containing different modifications. HER2 CAR mRNA containing different mRNA modifications was electroporated into human macrophages. Four days after electroporation, CAR expression and M1 markers were detected by flow cytometry (Attune). Day-4 CAR macrophages were then co-cultured with Nuc-Light-labeled HER2+ breast cancer cell line (CRL2351) at a 5:1 effector (CAR macrophage):target (cancer cell) ratio. Cancer cell growth was monitored every four hours by fluorescence using an Incucyte® live imaging microscope.

[0308] To further evaluate the effect of IFN-β on CAR persistence, human macrophages electroporated with m6AGCap1 / PsU mRNA encoding the HER2 CAR were evaluated on days 2 and 7. IFN-β treatment significantly improved CAR expression rates on day 7 compared to CAR macrophages not treated with IFN-β (Figure 122).

[0309] To verify the effect of IFN-β treatment on improving CAR expression and optimize the IFN-β concentration, macrophages transfected with M6AGCap1 / PsU-modified mRNA were treated with 0, 3, 10, 30, or 100 ng / mL of IFN-β for 4 hours, and viability, CAR percentage, and CAR MFI were assessed 4 and 7 days after electroporation. A dose-dependent effect of IFN-β was observed on CAR expression by human macrophages (Figure 123A). As described in Example 4, treatment of CAR macrophages with IFN-β induces an M1 phenotype. Therefore, further experiments were performed to determine whether this effect was dose-dependent. As shown in Figure 123B, induction of M1 markers CD80, CD86, and HLA-DR was IFN-β dose-dependent.

[0310] Considering that macrophage phenotype is thought to be plastic and that immunosuppressive cytokines such as IL-10 are known to induce an M2 phenotype, we evaluated the effect of IL-10 treatment on HER2 CAR mRNA-transfected macrophages treated with or without IFN-β. IFN-β-treated CAR macrophages were resistant to the effects of IL-10 and did not express the M2 marker CD163. Instead, they maintained expression of the M1 marker CD86 48 hours (Figure 124A) and 7 days (Figure 124B) after IL-10 treatment. IFN-β-primed CAR macrophages were also resistant to other M2 inducers.

[0311] To evaluate the antitumor function of macrophages transfected with mRNA encoding a HER2 CAR with or without IFN-β priming, untransduced (UTD) or CAR macrophages were primed with 0, 3, 10, 30, or 100 ng / mL of IFN-β for 4 or 20 hours. These effector cells were cocultured with the HER2+ breast cancer cell line CRL2351-GFP at an effector:target ratio of 3:1 or 1.5:1, and antitumor activity was measured based on GFP expression using an Incucyte® live imaging microscope. IFN-β priming improved the ability of CAR macrophages to kill cancer cells (Figure 125A). To evaluate whether IFN-β treatment improved the antitumor activity of CAR macrophages bearing mRNA containing the modification, the antitumor activity of macrophages electroporated with mRNA containing the unique modification was evaluated with or without IFN-β treatment. IFN-β treatment improved the antitumor activity of all CAR macrophages except for those transfected with 5moU mRNA (Figure 125B). To assess whether the improved antitumor effect of mRNA-transfected CAR macrophages was universal across all interferons or only interferon beta, macrophages electroporated with M6AGCap1 / PsU mRNA were treated with IFN alpha, beta, or gamma, and their ability to kill cancer cells was assessed. IFN-β-treated CAR macrophages were more effective than IFN-α or IFN-γ, resulting in the best cancer cell killing (Figure 125C).

[0312] To assess whether interferon treatment of macrophages improves other antitumor functions, cytokine secretion was assessed in mRNA-transfected HER2 CAR macrophages treated with or without interferon after co-culture with HER2+ breast cancer cells. Human macrophages were electroporated with 150 nM HER2 CAR mRNA containing m6AGCap1 and PsU modifications. HER2+ breast cancer cells (CRL2351) were co-cultured with CAR mRNA-transfected macrophages at a 3:1 effector (CAR macrophage):target (cancer cell) ratio. Supernatants were collected 48 hours after co-culture of cancer cells and macrophages, and cytokine levels were measured using a Meso Scale Discovery (MSD) instrument. As shown in Figure 126, treatment of macrophages with IFN-α, IFN-β, or IFN-γ increased secretion of the cytokines IL-6, IL-8, and TNFα from macrophages.

[0313] Additional experiments were performed to determine whether treatment with interferon further improved CAR mRNA persistence within macrophages and extended the functionality of CAR macrophages. Human macrophages were electroporated with 300 nM CAR mRNA containing m6AGCap1 and PsU modifications. Cells were cultured with 20 ng / mL IFN for 24 hours, then washed to remove cytokines. CAR expression was detected by flow cytometry (Attune) two days after transfection. The cells were then co-cultured with a Nuc-Light-labeled HER2+ breast cancer cell line (CRL2351) at a 3:1 effector:target ratio. Cancer cell growth was monitored by fluorescence using an Incucyte® live imaging microscope. 7 days after transfection, CAR expression and M1 markers were detected in macrophages by flow cytometry (Attune).

[0314] As shown in Figure 127A, two days after CAR mRNA transfection, CAR macrophage viability and CAR expression were very high, except in macrophages treated with IFN-γ. Furthermore, as shown in Figures 127B and 127C, IFN treatment enhanced the target cell killing activity of CAR macrophages. Figure 127C shows target cell killing after 72 hours of coculture of cancer cells and macrophages. IFN treatment also affected macrophage viability, CAR expression, M1 marker expression, and CAR macrophage functionality. As shown in Figure 128A, treatment of transfected macrophages with IFN-β increased cell viability, HER2 CAR expression, and expression of M1 markers CD80, CD86, and HLA-DR at the later 7-day time point compared to macrophages not treated with interferon. Figure 128A shows that all macrophages had high viability at day 7, but IFN-β-treated macrophages expressed CAR at the highest level by a significant margin. Figure 128B shows that of all CAR macrophages tested in the cancer cell killing assay 7 days after electroporation, IFN-β-treated macrophages produced the highest level of cancer killing (greatest reduction in tumor growth). Seven days after electroporation, the macrophages were co-cultured with target cancer cells for 72 hours. As shown in Figure 128C, all interferons improved the cancer cell killing activity of CAR macrophages compared to CAR macrophages not treated with interferon.

[0315] Example 10: Transfected macrophages are sensitive to IFNγ Human macrophages were transfected with mCherry mRNA containing m6AGCap1 and PsU or N1mPsU modifications and cultured with different doses of IFN-γ for 1 day. mCherry expression was monitored using an Incucyte® live imaging microscope. As shown in Figure 129, IFN-γ reduced mCherry mRNA expression when macrophages were transfected with the mRNA.

[0316] It has previously been shown that in vitro-transcribed mRNA is recognized by various endosomal innate immune receptors (e.g., Toll-like receptor (TLR) 3, TLR7, and TLR8) as well as cytoplasmic innate immune receptors (protein kinase RNA-activated (PKR), retinoic acid-inducible gene I protein (RIG-I), melanoma differentiation-associated protein 5 (MDA5), and 2'-5'-oligoadenylate synthetase (OAS)). Signaling through these distinct pathways leads to inflammation associated with the activation of type 1 interferon (IFN), tumor necrosis factor (TNF), interleukin-6 (IL-6), IL-12, and a cascade of transcriptional programs. Overall, this creates a pro-inflammatory microenvironment poised to induce specific immune responses. Furthermore, downstream effects (e.g., decreased translation via phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), enhanced RNA degradation by ribonuclease L (RNase L), and overexpression and inhibition of self-amplifying mRNA replication) are all relevant to the pharmacokinetics and pharmacodynamics of IVT mRNA.

[0317] Activation of IFN-γ through the TLR pathway can activate 2'-5'-oligoadenylate synthetase (OAS), which produces 2'-5'-oligoadenylate (2-5A), which in turn can activate RNase L, leading to RNA degradation and apoptosis.

[0318] Example 11: Effects of RNaseL inhibitors, sunitinib, and ABCE1 on CAR macrophages To examine whether RNase L inhibitors can rescue IFN-γ-induced instability of transfected mRNA, human macrophages were treated with 1 μM sunitinib (RNase L inhibitor) and then transfected with mCherry mRNA containing m6AGCap1 and PsU modifications 2 hours later. The transfected cells were then cultured with different doses of IFN-γ for 1 day. mCherry expression was monitored using an Incucyte® live imaging microscope. As shown in Figure 130A, sunitinib rescued IFN-γ-induced degradation of the mRNA.

[0319] To assess whether RNAse L inhibition could improve the antitumor function of mRNA-transfected CAR macrophages, macrophages were transfected with modified mRNA and pretreated with sunitinib before being evaluated in a cancer cell killing assay as effector cells. CAR macrophages pretreated with 1 nM sunitinib resulted in greater cancer cell killing than CAR macrophages not pretreated with sunitinib or untransfected control macrophages treated with or without sunitinib (Figure 130B). The improved cancer killing ability of sunitinib-primed CAR macrophages in a 48-hour CRL2351 breast cancer cell killing assay is shown in Figure 130C.

[0320] The effects of alternative RNase L inhibitors (RLI or ABCE1) were also tested to further validate the concept. Human macrophages were co-transfected with mRNA encoding mCherry containing m6AGCap1 and PsU modifications and mRNA encoding ABCE1.

[0321] As shown in Figure 131, co-expression of ABCE1 significantly improved the expression of the mRNA-encoding transgene of interest 48 hours after electroporation. The viability of macrophages co-transfected with ABCE1 was not affected and remained high. ABCE1 co-transfection increased mCherry expression by nearly two-fold, and pretreatment with sunitinib further enhanced this effect.

[0322] Example 12: Receptor expression and tumor cell killing of CAR macrophages containing the CD28 hinge domain In this example, a CAR construct was generated containing a CD8 leader, 4D5 scFv, a CD28 hinge domain, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain (CTX_219) (Figure 132). The expression of CTX_219 was then compared to an identical CAR construct (CTX_001; Figure 8) that had a CD8 hinge domain and a CD8 transmembrane domain instead of the CD28 hinge domain and CD28 transmembrane domain. The tumor-killing ability of CTX_219 was compared to CTX_001 and also to a CAR construct identical to CTX_001 but lacking the intracellular signaling domain (CTX_003).

[0323] Primary human macrophages were cultured at 1.0 x 10 in EP buffer containing 50–500 nM mRNA. 5 ~1.0×10 9 The cells were suspended at a concentration of 1000 cells / mL and electroporated. The cells were removed from the cassette, plated, and cultured overnight at 37°C and 5% CO2. CAR expression MFI and percentage were detected after 24 hours by flow cytometry using rHER2 binding. After 24 hours, survival percentage was detected using Live / Dead Aqua. For killing, macrophages were co-cultured with Her2+ CRL2351-NucGFP tumor cells at a 2:1 E:T ratio or a 1:1 E:T ratio and monitored for 72 hours using Incucyte®. Tumor cell death was calculated by the integrated GFP intensity per well relative to time 0.

[0324] Expression and CAR+ percentages were comparable for the CAR construct containing the CD28 hinge domain (CTX_219), the CAR construct containing the CD8 hinge domain (CTX_001), and the CAR construct lacking the intracellular signaling domain (CTX_003; see Figures 133A-133C). Survival percentages were also comparable for CTX_219 and CTX_001 (Figure 133D). Surprisingly, the CAR construct containing the CD28 hinge domain (CTX_219) exhibited enhanced killing function compared to the CTX_001 and CTX_003 controls (Figure 133E). These data indicate that the CAR construct containing the CD28 hinge domain resulted in improved tumor cell killing by macrophages compared to the CAR construct containing the CD8 hinge domain.

[0325] TNFα and IL6 secretion by macrophages containing CTX_219 was quantified compared with CTX_001 and CTX_003. Effector cells were cocultured with HER2+ target cells at a 1:1 E:T ratio for 24 hours. Supernatants were collected, and TNFα and IL6 concentrations were measured using the MSD Pro-Inflammatory Panel 1 (Meso Scale Discovery) according to the manufacturer's instructions. Surprisingly, the CAR construct containing the CD28 hinge domain (CTX_219) showed a significant increase in TNFα secretion (approximately 5-fold compared with CTX_001) without further increasing IL6 production (Figure 134). These data indicate that the CAR construct containing the CD28 hinge domain results in improved secretion of the tumor-killing cytokine TNFα compared with the CAR construct containing the CD8 hinge domain, without increasing the production of the pro-inflammatory cytokine IL6.

[0326] Example 13: VPX-mediated lentiviral transduction of macrophages with CAR For lentiviral transduction of macrophages for CAR expression, macrophages were thawed and plated in 10 mL complete medium (CM) at a density of 5-10e6 cells per 10 cm. After 2-3 hours of incubation, VPX lentiviral particles were diluted in CM and added to the macrophages at the indicated MOI. 24 hours after adding the lentivirus, the medium was completely changed.

[0327] To harvest macrophages for the experiments described herein, 10 cm plates were removed from the incubator and placed at 4°C for 30 minutes. Cells were removed from the plates, pelleted, and resuspended in CM. A cell count was performed, and macrophages were either used for experiments or replated for later time points.

[0328] Functional assays for evaluating CAR macrophages For killing assays, macrophages were plated in 96-well plates in 100 μL of CM at a defined density to achieve a specific macrophage:target ratio (E:T). After a 1-2 hour incubation period, 10,000 tumor cells expressing nucLight GFP were resuspended in 100 μL of CM and added to each well. The plates were placed in an Incucyte® chamber and imaged every 4 hours, starting approximately 1 hour after tumor cell addition. Killing was defined as the GFP integrated intensity relative to the time 0 scan. The tumor cells used in these assays were AU565 HER2+ breast cancer cells and SKOV3 ovarian cancer cells.

[0329] For cytokine secretion testing, soluble Her2-His was dissolved in molecular-grade water at a concentration of 0.2 μg / μL. Her2-His was then diluted in PBS at the indicated concentrations. 100 μL of the Her2-His dilution was added to wells of a 96-well plate. The plate was stored at 4°C overnight and then washed twice with PBS. 50,000 macrophages were added to each well in a final volume of 200 μL of CM. Approximately 24 hours after macrophage addition, the supernatant was collected and stored at -20°C for further evaluation. Cytokine detection was performed using the MSD U-Plex kit.

[0330] To detect cell surface protein and CAR expression, cells were incubated in FACS buffer containing Human TruStain FcX for 10 minutes at room temperature. Surface protein staining was performed using the following panel: CD80-FITC, CD86-PE, CD163-APC-Cy7, CD206-BV421, anti-trastuzumab-APC, and Aqua Live / Dead. Detection of surface protein expression was completed using an Attune NxT flow cytometer (Thermo Fisher) and analyzed using FlowJo.

[0331] To detect CAR expression comparing VPX and non-VPX lentiviruses, cells were incubated in FACS buffer containing 20 μg / mL Her2-His for 20 minutes at room temperature, followed by incubation in Human TruStain FcX for 10 minutes at room temperature. Surface protein staining was performed using the following panel: CD80-FITC, CD86-PE, CD163-APC-Cy7, CD206-BV421, anti-His-APC, and Aqua Live / Dead. Detection of surface protein expression was completed using an Attune NxT flow cytometer (Thermo Fisher) and analyzed using FlowJo.

[0332] Results of lentiviral transduction of CAR macrophages with VPX The addition of VPX significantly increased the expression of a CAR construct (CTX_001) in macrophages (Figure 136). CTX_001 contains an anti-HER2 scFv, a CD8 hinge, a CD8 transmembrane domain, and a CD3 zeta intracellular signaling domain (Figure 8). VPX-based transduction increased CAR (%) by more than 40% at an MOI of 5, more than 60% at an MOI of 10, more than 80% at an MOI of 50, and more than 90% at an MOI of 50. In contrast, the same CAR (CTX_001) transduced without VPX only increased CAR (%) by approximately 20% at MOIs of 5 to 50.

[0333] Next, viability and CAR expression were assessed over time for VPX-lentiviral transduction using MOI titration. CAR-M transduced with VPX-lentivirus were shown to have high viability and express CTX-001 for over 30 days (Figure 137). CAR-M transduced with VPX-lentivirus at MOIs of 1, 5, and 10 survived for a time period of approximately 30 days with survival percentages ranging from approximately 80% to approximately 100%. CAR expression percentage increased from MOI 1 to MOIs 5 and 10. Untransduced (UTD) macrophages were used as a control.

[0334] CAR expression in CTX_001 macrophages transduced with VPX-lentivirus was also assessed using α-trastuzumab-AF647 for surface protein analysis over time. CAR cell surface expression was demonstrated at MOI: 1, MOI: 5, and MOI: 10, with increased expression at MOI: 5 and 10 on days 4, 7, 15, 22, and 29 (Figure 138). UTD macrophages were used as a control.

[0335] The mean fluorescence intensity (MFI) of CAR expression in CTX_001 in VPX-lentivirus-transduced macrophages was also assessed using α-trastuzumab-AF647 for surface protein analysis over time. VPX-lentivirus-transduced CAR macrophages were shown to be highly viable and express CTX-001 over a 30-day time period (Figure 139). Thus, VPX-lentivirus-transduced CAR macrophages maintained CAR expression for at least 29 days.

[0336] We also evaluated the killing function of CTX_001 macrophages transduced with VPX lentivirus. The effector macrophage:target tumor cell ratios (E:T) were 4:1, 2:1, 1:1, and 1:2. Integrated intensity indicates tumor burden. UTD macrophages and AU565 cells (a HER2+ breast cancer cell line) without macrophages were used as controls. The killing function of CTX_001 macrophages was evident at all E:T ratios and MOIs of 1, 5, and 10 after 4 days and was maintained at 7 days (Figure 140). Notably, CTX_001 macrophages at MOIs of 5 and 10 showed similar levels of killing function at all E:T ratios. Antitumor activity after 72 hours of coculture was plotted.

[0337] Next, we investigated the MOI range for clinical transduction. We investigated CARs with (CTX_001) and without (CTX_003; a CAR containing an anti-HER2 scFv, a CD8 hinge, and a CD8 transmembrane domain; Figure 10). At MOI: 2 and MOI: 5, CAR cell surface expression of CTX_001 and CTX_003 was obtained in macrophages transduced with VPX lentivirus (Figure 141A). VPX-lentivirus-transduced CTX_001 and CTX_003 macrophages were shown to be viable and express the respective CARs compared with UTD macrophages (Figure 141B). VPX-lentivirus-transduced CTX_001 macrophages were shown to express higher CAR levels at MOI: 5 than at MOI: 2 (Figure 142). VPX lentiviral transduction also resulted in a moderate, dose-dependent M1 polarization of CTX_001 and CTX_003 macrophages, based on increased expression of CD80 and CD86 and decreased expression of CD163 and CD206 (Figure 143).

[0338] We also performed experiments to compare the polarization of CTX_001 macrophages transduced with VPX-lentivirus to that of adeno-associated virus transduction with Ad5f35. Transduction with Ad5f35, but not VPX-lentivirus, induced the M1 markers CD80 and CD86 (Figure 144). VPX-lentivirus-transduced CTX_001 macrophages also demonstrated sustained antitumor activity compared with Ad5f35-transduced CTX_001 macrophages at 7 and 21 days post-transduction (Figure 145). These results suggest that, in some cases, when using VPX-lentivirus, a different viral vector may be used to express CAR equally in macrophages without M1 induction, allowing for the maintenance of macrophage plasticity and subsequent phenotypic control. Therefore, VPX-lentivirus can be used to generate M0 CAR macrophages. Subsequently, Vpx lentivirus-derived M0 CAR macrophages can be polarized to M1 or M2 to generate M1 or M2 polarized engineered macrophages or CAR macrophages. Such CAR macrophages could also incorporate signaling domains encoded within the CAR construct to induce a phenotypic shift. These CAR macrophages could be primed with M1 cytokines (e.g., IFNα, IFNb, IFNg, LPS, or TLR agonists) or M2 cytokines (e.g., IL4, IL10, IL13, or TGFb) to generate CAR macrophages with a selected phenotype. These CAR macrophages could be engineered to induce an M1 or M2 phenotype using transcription factors, immune ligands, or secreted cytokines.

[0339] The killing function of CTX_001 and CTX_003 macrophages transduced with VPX lentivirus was also evaluated in AU565 and SKOV3 cells at MOIs of 2 and 5. The E:T ratio was 4:1, and for AU565, it was 1:2. Seven days after transduction with VPX lentivirus, CTX_001 macrophages transduced with MOIs of 2 and 5 showed similar killing ability to control UTD and AU565 cells at E:T ratios of 4:1 and 2:1 (Figure 146). For SKOV3, the E:T ratio was 4:1. Seven days after transduction with VPX lentivirus, CTX_001 macrophages transduced with MOIs of 2 and 5 showed similar killing ability to control UTD and SKOV3 cells at E:T ratios of 4:1 (Figure 147).

[0340] We also examined the production of inflammatory cytokines in CTX_001 and CTX_003 macrophages after transduction with VPX lentivirus. CTX_001 and CTX_003 macrophages were transduced with VPX lentivirus and cultured for 24 hours with or without plate-bound HER2. Seven days after transduction with VPX lentivirus, CTX_001 macrophages transduced at MOI: 2 and MOI: 5 showed differences in inflammatory cytokine secretion, with increased production of TNFα and IL-6 in the presence of HER2 (Figure 148).

[0341] Next, viability and CAR expression were assessed over time for VPX-lentiviral transduction of CTX_001 and CTX_003 macrophages at MOIs of 2 and 5. VPX-lentiviral transduced CTX_001 and CTX_003 macrophages demonstrated high viability and CTX-001 and CTX_003 expression over a 21-day time period (Figure 149). UTD macrophages were used as a control. The MFI of CAR expression in CTX-001 and CTX_003 macrophages transduced with VPX-lentivirus was also assessed using α-trastuzumab-AF647 for analysis of cell surface proteins over time. VPX-lentivirus transduced CTX_001 and CTX_003 macrophages showed high viability and expression of CTX-001 and CTX-003 over a 21 day time period (Figure 150).

[0342] The killing function of VPX lentiviral-transduced CTX_001 and CTX_003 macrophages was also assessed over time in AU565 and SKOV3 cells at MOIs of 2 and 5. Killing assays were initiated 21 days post-transduction. The E:T ratio was 4:1 for VPX lentiviral transduction and 1:2 for AU565. 21 days post-transduction with VPX lentiviral transduction, CTX_001 macrophages transduced at MOIs of 2 and 5 exhibited similar killing abilities to control UTD and AU565 cells over 72 hours at E:T ratios of 4:1 and 2:1 (Figure 151). For SKOV3, the E:T ratio was 4:1. Twenty-one days after transduction with VPX lentivirus, transduced CTX_001 macrophages at MOI:2 and MOI:5 showed similar killing ability to control UTD and SKOV3 cells at an E:T ratio of 4:1 (Figure 152). We also examined the expression of CTX_001 (Figure 8) and viability in monocytes after transduction with VPX lentivirus. CD14+ human monocytes were thawed, washed, and counted. Cells were plated at 3e6 cells / well in 6-well Upcell plates at UTD or with lentivirus at the appropriate MOI. Cells were fed on day 5 and harvested for flow cytometry analysis on day 7. CAR macrophages and CAR monocytes transduced with Ad5f35 transduction were used as controls. CAR monocytes were viable after transduction with VPX lentivirus at MOIs of 2, 5, or 10 when Ad5f35-transduced CAR monocytes and CAR macrophages were used as viability controls (Figure 15A). CAR monocytes transduced with VPX lentivirus at MOIs of 2, 5, and 10 showed an increase in percentage (%) of CAR expression of 27.6% at MOI 2, 51.3% at MOI 5, and 66.3% at MOI 10 when Ad5f35-transduced CAR monocytes and CAR macrophages were used as viability controls (Figure 153B). CAR monocytes transduced with VPX lentivirus at MOIs of 2, 5, and 10 also showed an increase in mean fluorescence intensity (MFI) of CAR expression with increasing MOI when Ad5f35-transduced CAR monocytes and CAR macrophages were used as comparators (Figure 153C). CAR expression histograms of CD14+ monocytes transduced with VPX lentivirus at MOIs of 2, 5, and 10 also showed an increase in CAR expression with increasing MOI when Ad5f35-transduced CAR monocytes and CAR macrophages were used as comparators (Figure 153D). UTD macrophages were also used as a control for CAR expression histograms. These results demonstrate that monocytes expressed CAR after VPX lentiviral transduction and that monocytes maintain CAR expression at the macrophage stage.

[0343] Example 14: SIRPα knockout for enhanced phagocytosis of CAR macrophages The interaction of the immune checkpoint SIRPα with CD47 inhibits macrophage phagocytosis. Thus, CAR-expressing macrophages receive opposing pro- and anti-phagocytic signals from tumors. The results presented herein demonstrate that knockout of SIRPα improved the killing and phagocytosis of CAR macrophages.

[0344] Ribonucleoprotein (RNP) formation was performed and mixed with macrophages one day after thawing. Purified Cas9 protein was incubated with a cocktail of three gRNAs at room temperature for 10–20 minutes. The gRNA sequences used were those from Synthego's "Gene Knockout Kit" for human SIRPα (Table 2). A 1:3 Cas9:total gRNA ratio (thus, a 1:1 ratio of Cas9:individual gRNAs in the cocktail) was used. RNPs were mixed with macrophages in EP buffer to a final concentration of 2.5 μM Cas9 RNP for 3e7–8e7 cells / mL. Macrophages were electroporated using a MaxCyte electroporation cassette. After recovery culture, cells were analyzed at least 3 days after electroporation to allow the gene knockout to have an effect at the protein expression level. SIRPα expression was then analyzed, and functional assays were performed. [Table 2]

[0345] Fresh macrophages from two different donors (HCC163264 and HCC151560) were obtained 1 day before the start of the experiment. On day 0, macrophages were electroporated with the indicated RNP concentrations of Cas9, and SIRPα expression was assessed on day 3. SIRPα immunostaining was performed on the macrophage surface using flow cytometry. Results demonstrated dose-dependent knockout of SIRPα in macrophages from both donors (Figures 154A-B). Results also demonstrated two distinct CAR macrophage populations in the flow cytometry plot at 312 nM RNP (Figure 154C). This supports the all-or-none nature of Cas9-mediated gene knockout, leading to a binary population rather than a gradient of SIRPα expression, whether the SIRPα gene was knocked out or not. Importantly, SIRPα knockout did not affect cell viability (Figure 154D).

[0346] We also demonstrated that SIRPα knockout promotes CAR macrophage phagocytosis in SKOV-3 ovarian cancer cells. Macrophages were thawed and transduced with adenovirus encoding CTX_001 (CAR comprising an anti-HER2 scFv, CD8 hinge, CD8 transmembrane domain, and CD3 zeta intracellular signaling domain; Figure 8) one day before electroporation with Cas9 RNP. Three days later, SIRPα expression was assessed, and phagocytosis assays using SKOV-3 cells were initiated. These results demonstrate SIRPα knockout in CTX_001 macrophages with Cas9 and gRNA targeting SIRPα (Figure 155A). CTX_001 was expressed in transduced macrophages (Figure 155B). CTX_001 macrophages exhibited phagocytosis over a 12-hour period, as measured by pHrodo-green signaling using Incucyte® analysis (Figure 155C). Total phagocytosis measured over 12 hours was quantified as the area under the curve (Figure 155D). SIRPα knockout and CAR expression in macrophages maximized phagocytosis, whereas SIRPα knockout alone did not significantly enhance phagocytosis in UTD macrophages.

[0347] SIRPα KO was also shown to enhance CAR macrophage phagocytosis in HCC-1954 human breast cancer cells. Macrophages were thawed, transduced with adenovirus encoding CTX_001, and one day later, electroporated with Cas9 RNP. Three days later, SIRPα expression was assessed, and phagocytosis assays using HCC-1954 cells were initiated. These results demonstrate SIRPα knockout in CTX_001 macrophages with Cas9 and gRNA targeting SIRPα (Figure 156A). CTX_001 was expressed in transduced macrophages (Figure 156B). CTX_001 macrophages exhibited phagocytosis over a 12-hour period, as measured by pHrodo-green signal using Incucyte® analysis. The lower plot, rescaled on the Y-axis, shows that CTX_001 macrophages exhibited greater phagocytosis than all UTD groups (Figure 156C). Total phagocytosis measured over 12 hours was quantified as the area under the curve (Figure 156D). These results indicate that although control gRNA increased phagocytosis, SIRPα knockout and CAR expression in macrophages remained the greatest producers of phagocytic signals.

[0348] SIRPα knockout was also shown to enhance CAR macrophage killing in SKOV-3 ovarian cancer cells using macrophages from a different donor cell line than in the previous experiments. Macrophages were thawed one day before electroporation with Cas9 RNP and transduced with adenovirus encoding CTX_001 three days after electroporation. Two days later, SIRPα expression was assessed, and phagocytosis assays with SKOV-3 cells were initiated. These results demonstrate SIRPα knockout in CTX_001 macrophages with Cas9 and gRNA targeting SIRPα (Figure 157A). CTX_001 was expressed in the transduced macrophages (Figure 157B). SKOV-3 killing was measured as the growth of SKOV-3 cells expressing nuclight green fluorescent protein, as measured by Incucyte® analysis over a 24-hour period (Figure 157C). SIRPα knockout in human CAR macrophages increased tumor killing kinetics based on the time required for 50% clearance of target tumor cells (Figure 157D). Total phagocytosis was measured over 12 hours (Figure 157E). SIRPα knockout and CAR expression in macrophages maximized phagocytosis, whereas SIRPα knockout alone did not significantly enhance phagocytosis in UTD macrophages. Phagocytosis results using a second donor were consistent. SIRPα knockout CAR macrophages appeared to kill more rapidly than CAR macrophages without SIRPα knockout. These results presented herein demonstrate highly efficient SIRPα knockout using the CRISPR / Cas system in primary human CAR macrophages and demonstrate that SIRPα knockout using the CRISPR / Cas system can be consistently performed in primary human CAR macrophages. These results are paired with functional data showing increased phagocytosis and killing by SIRPα knockout CAR macrophages compared to CAR macrophages without SIRPα knockout. In two separate donors and experimental runs, SIRPα knockout increased phagocytosis of SKOV-3 cells by CAR macrophages. Notably, SIRPα knockout in macrophages that do not express CAR did not increase phagocytosis or tumor killing. Therefore, this effect can be considered synergistic.

[0349] equivalent It should be understood by those skilled in the art that various changes, modifications, and improvements to the present disclosure will be readily apparent to those skilled in the art. Such changes, modifications, and improvements are intended to be part of this disclosure and to be within the spirit and scope of the present invention. Therefore, the foregoing description and drawings are by way of example only, and any inventions described in this disclosure are further described in detail by the following claims.

[0350] Those of skill in the art will understand the typical standards of deviation or error attributable to values ​​obtained in the assays or other processes described herein. Publications, websites, and other reference materials referred to herein to describe the background of the invention and to provide further details regarding its practice are hereby incorporated by reference in their entirety. The present invention provides, for example, the following items. (Item 1) (a) an extracellular domain; (b) a transmembrane domain; (c) an intracellular domain. An immune cell comprising a chimeric antigen receptor (CAR), wherein the immune cell comprises a macrophage, monocyte, or dendritic cell. (Item 2) 2. The immune cell of item 1, wherein the extracellular domain of the CAR comprises one or more antigen-binding domains and an FcR extracellular domain, and / or the transmembrane domain of the CAR comprises an FcR transmembrane domain, and / or the intracellular domain of the CAR comprises an FcR intracellular domain. (Item 3) 3. The immune cell of item 2, wherein the CAR comprises, in N-terminal to C-terminal direction, one or more extracellular binding domains, FcR extracellular domains, FcR transmembrane domains, and FcR intracellular domains. (Item 4) 4. The immune cell of claim 2, wherein one or more of the FcR extracellular domain, the FcR transmembrane domain, and the FcR intracellular domain are or comprise human FcR domains. (Item 5) 5. The immune cell according to any one of items 2 to 4, wherein the FcR extracellular domain, the FcR transmembrane domain, and the FcR intracellular domain together constitute a full-length FcR. (Item 6) 6. The immune cell of any one of Items 2 to 5, wherein the FcR extracellular domain comprises a CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c, CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, or FcαRI (CD89) domain. (Item 7) 7. The immune cell of any one of items 2 to 6, wherein the FcR transmembrane domain comprises a CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c, CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, or FcαRI (CD89) domain. (Item 8) 8. The immune cell of any one of items 2 to 7, wherein the FcR intracellular domain comprises a CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c, CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, or FcαRI (CD89) domain. (Item 9) 2. The immune cell of item 1, wherein the extracellular domain of 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. (Item 10) 10. The immune cell of item 9, wherein the CAR comprises, in N-terminal to C-terminal direction, one or more extracellular binding domains, TLR extracellular domains, TLR transmembrane domains, and TLR intracellular domains. (Item 11) 11. The immune cell of claim 9 or 10, wherein one or more of the TLR extracellular domain, the TLR transmembrane domain, and the TLR intracellular domain are or comprise human TLR domains. (Item 12) 12. The immune cell according to any one of items 9 to 11, wherein the TLR extracellular domain, the TLR transmembrane domain, and the TLR intracellular domain together constitute a full-length TLR. (Item 13) 13. The immune cell of any one of items 9 to 12, wherein the TLR extracellular domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain. (Item 14) 14. The immune cell of any one of items 9 to 13, wherein the TLR transmembrane domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain. (Item 15) 15. The immune cell according to any one of items 9 to 14, wherein the TLR intracellular domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain. (Item 16) The chimeric antigen receptor (d) one or more extracellular reader domains; (e) one or more extracellular hinge domains, and (f) The immune cell of item 1, further comprising one or more of one or more intracellular costimulatory domains. (Item 17) 17. The immune cell of item 16, wherein the one or more extracellular leader domains comprise a CD8 extracellular leader domain. (Item 18) 18. The immune cell of any one of items 1 to 17, wherein the one or more extracellular antigen-binding domains comprise an scFv, centyrin, or darpin. (Item 19) 19. The immune cell of any one of items 16 to 18, wherein the one or more extracellular hinge domains comprise a CD28 extracellular hinge domain, a CD8a extracellular hinge domain, or an IgG4 extracellular hinge domain. (Item 20) 20. The immune cell of any one of items 1 to 19, wherein the transmembrane domain comprises CD28, CD8a, CD64, CD32a, CD32c, CD16a, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, or TLR9. (Item 21) 21. The immune cell according to any one of items 1 to 20, wherein the intracellular domain comprises one or more intracellular signaling domains. (Item 22) the one or more intracellular signaling domains are selected from the group consisting of CD3...

Claims

1. (a) an extracellular domain; (b) an extracellular hinge domain derived from CD28; and (c) a CD28 transmembrane domain; and (d) an intracellular domain; A macrophage or monocyte comprising a chimeric antigen receptor (CAR) comprising: The macrophages or monocytes exhibit increased tumor-killing ability compared to macrophages or monocytes that do not have the CD28 extracellular hinge and CD28 transmembrane domain.

2. The macrophage or monocyte (i) exhibits increased TNFα production compared to macrophages or monocytes that do not possess the CD28 extracellular hinge and CD28 transmembrane domain; or (ii) do not exhibit increased IL6 production compared to macrophages or monocytes lacking the CD28 extracellular hinge and CD28 transmembrane domain; The macrophage or monocyte of claim 1.

3. (i) the extracellular domain comprises an Fc receptor (FcR) extracellular domain; and / or (ii) the intracellular domain comprises an FcR intracellular domain; The macrophage or monocyte of claim 1.

4. (i) the extracellular domain comprises a Toll-like receptor (TLR) extracellular domain, and / or (ii) the intracellular domain comprises a TLR intracellular domain; The macrophage or monocyte of claim 1.

5. The macrophage or monocyte of claim 1, wherein the CAR further comprises one or more extracellular leader domains.

6. 6. The macrophage or monocyte of claim 5, wherein the one or more extracellular leader domains comprise or are a CD8 extracellular leader domain.

7. The macrophage or monocyte of claim 1 , wherein the extracellular domain comprises or is an scFv, centyrin, or darpin.

8. The transmembrane domain is selected from the group consisting of CD28, CD8a, CD64, CD32a, CD32c, CD16a, TLR1, 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 zeta, FcRγ, 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, CD36, or Syk transmembrane domain.

9. The one or more intracellular domains are selected from the group consisting of CD3 zeta, FcRγ, CD64, CD32a, CD32c, CD16a, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphA1, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, and TRK. , VEGFR, CD40, CD19, CD20, 41BB, CD28, 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, SI RPb, CD22, PIR-B, LILRB1, 41BBL (TNFSF9), CD27, OX40L, CD32b, CD11b, ITGAM, SLAMF7, CD206, CD163, CD 209, Dectin-2, IL1R, IL2R, IL3R, IL4R, IL5R, IL6R, IL7R, IL8R, IL9R, IL10R, IL11R, IL12R, IL13R, IL14 2. The macrophage or monocyte of claim 1, comprising or being one or more of the following intracellular signaling domains: R, IL15R, IL17R, IFNaR, IFNgR, TNFR, CSF1R, CSF2R, Dap10, CD36, Dectin-1, ICOSL, CD2, CD7, CD96, CRTAM, DC-SIGN, NKG2D, NTB-A, CD30, or Syk intracellular signaling domains.

10. 10. The macrophage or monocyte of claim 9, wherein the one or more intracellular domains comprise or are a CD3 zeta intracellular signaling domain.

11. A pharmaceutical composition comprising the macrophages or monocytes of claim 1 and a pharmaceutically acceptable carrier.

12. 12. The pharmaceutical composition of claim 11 for treating a subject having a disease, disorder, or condition.

13. (a) an extracellular domain; (b) the CD28 extracellular hinge domain; and (c) a CD28 transmembrane domain; and (d) an intracellular domain; A nucleic acid construct comprising one or more nucleic acid sequences encoding a chimeric antigen receptor (CAR) comprising: A nucleic acid construct, wherein macrophages or monocytes comprising the CAR exhibit increased tumor-killing ability compared to macrophages or monocytes not having the CAR.

14. (e) one or more extracellular leader domains; (f) one or more intracellular costimulatory domains; (g) a truncated peptide, and / or (h) one or more peptide drugs 14. The nucleic acid construct of claim 13, further comprising one or more nucleic acid sequences encoding:

15. 15. The nucleic acid construct of claim 14, wherein the truncation peptide comprises or is a P2A, F2A, E2A, or T2A peptide.

16. 1. A composition comprising a nucleic acid construct for use in a method of modifying macrophages or monocytes, the method comprising: (a) an extracellular domain; (b) an extracellular hinge domain derived from CD28; and (c) a CD28 transmembrane domain; and (d) an intracellular domain; to said macrophages or monocytes, thereby producing modified macrophages or monocytes that exhibit increased tumor-killing ability compared to unmodified macrophages or monocytes.

17. 17. The composition of claim 16, wherein the nucleic acid construct comprises or is a viral vector.

18. 18. The composition of claim 17, wherein at least one Vpx protein is also delivered to the macrophage or monocyte.

19. 19. The composition of claim 18, wherein the modified macrophages or monocytes exhibit increased CAR expression compared to macrophages or monocytes comprising a CAR delivered by a viral vector but not delivered at least one Vpx.

20. 18. The composition of claim 17, wherein the viral vector comprises or is a lentiviral vector.

21. The method comprises: (i) a CD40 agonist, or (ii) 4-1BB ligand agonist 17. The composition of claim 16, further comprising treating the modified macrophages or monocytes with one or more of:

22. 22. The composition of claim 21, wherein the modified macrophages or monocytes exhibit increased polarization towards an M1 phenotype compared to unmodified macrophages or monocytes.

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