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WO2024173329A2 - Récepteurs antigéniques chimériques comprenant des domaines dap 10 et dap 12 et des cellules immunitaires modifiées - Google Patents

Récepteurs antigéniques chimériques comprenant des domaines dap 10 et dap 12 et des cellules immunitaires modifiées Download PDF

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Publication number
WO2024173329A2
WO2024173329A2 PCT/US2024/015509 US2024015509W WO2024173329A2 WO 2024173329 A2 WO2024173329 A2 WO 2024173329A2 US 2024015509 W US2024015509 W US 2024015509W WO 2024173329 A2 WO2024173329 A2 WO 2024173329A2
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domain
extracellular
cd8a
intracellular
car
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WO2024173329A3 (fr
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Nicholas G. Minutolo
Michael KLICHINSKY
Kevin W. TOSH
Lauren Candice SHAW
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Carisma Therapeutics Inc
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Carisma Therapeutics Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/17—Monocytes; Macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4205—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/22—Intracellular domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present disclosure encompasses, among other things, compositions comprising modified immune cells (e.g., stem cells, macrophages, monocytes, and/or dendritic cells) comprising chimeric antigen receptors (CARs) and methods of producing the same.
  • modified immune cells e.g., stem cells, macrophages, monocytes, and/or dendritic cells
  • CARs chimeric antigen receptors
  • nucleic acid constructs comprising one or more nucleic acid sequences encoding CARs described herein and methods of producing the same.
  • the present disclosure provides, inter alia, CARs comprising one or more intracellular domains comprising a DAP10 domain or a portion thereof or a DAP12 domain or a portion thereof and methods of using and making immune cells comprising said CARs.
  • the present disclosure provides modified immune cells comprising a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) one or more extracellular domains; (b) a transmembrane domain; and (c) one or more intracellular domains comprising: (i) a DAP10 domain or a portion thereof, and/or (ii) a DAP12 domain or a portion thereof; wherein the modified immune cell is or comprises a stem cell, macrophage, monocyte, or dendritic cell.
  • CAR chimeric antigen receptor
  • one or more extracellular domains are or comprise an scFv, VHH antibody, centyrin, darpin, or nanobody.
  • a transmembrane domain is or comprises a DAP 10, DAP12, CD8a, CD28, CD40, MyD88 CD64, CD32a, CD32c, CD16a, CD3zeta, ICOS, Dectin-1, DNGR1, SLAMF7, TRL1, TLR2, TLR3, TRIA, TLR5, TLR6, TLR7, TLR8, or TLR9 transmembrane domain.
  • one or more intracellular domains further comprise one or more of a CD3 ⁇ , FcRy, MyD88, CD40, CD64, CD32a, CD32c, CD16a, CD89, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD 19, CD20, 41BB, CD28, GCSFR (CD114), RAGE, CD30, CD160, DR3, Fnl4, HVEM, CD160, NGFR, RANK, TNFR2, TROY, XEDAR, TRIF, 0X40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, V/I/LxYxxL/V, SI
  • one or more intracellular domains further comprise one or more of a CD20, CD40, Dectin-1, FcRy, MyD88, RAGE, SLAMF7, or TLR2 intracellular domain, a portion of any of the foregoing, or combinations thereof.
  • a CAR further comprises an extracellular leader domain.
  • an extracellular leader domain is or comprises a CD8a extracellular leader domain.
  • a CAR further comprises an extracellular hinge domain.
  • an extracellular hinge domain is or comprises: a DAPIO extracellular hinge domain, a DAP 12 extracellular hinge domain, a CD8a extracellular hinge domain, a CD28 extracellular hinge domain, a DNGR-1 extracellular hinge domain, a Dectin-1 extracellular hinge domain, or an IgG4 extracellular hinge domain.
  • a CAR comprises, from N-terminus to C-terminus: (i) a CD8a extracellular leader domain, an extracellular antigen binding domain, a DAP 10 extracellular hinge domain, a DAP 10 transmembrane domain, and a DAP 10 intracellular domain; (ii) a CD8a extracellular leader domain, an extracellular antigen binding domain, a CD8a extracellular hinge domain, a CD8a transmembrane domain, a DAP10 intracellular domain, and an FcRy intracellular domain; (iii) a CD8a extracellular leader domain, an extracellular antigen binding domain, a CD8a extracellular hinge domain, a CD8a transmembrane domain, an FcRy intracellular domain, and a DAP10 intracellular domain; (iv) a CD8a extracellular leader domain, an extracellular antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and a DAP 10 intracellular domain;
  • a CAR comprises, from N-terminus to C-terminus: (i) a CD8a extracellular leader domain, an extracellular antigen binding domain, a CD8a extracellular hinge domain, a CD8a transmembrane domain, and a DAP 12 intracellular domain; (ii) a CD8a extracellular leader domain, an extracellular antigen binding domain, a CD8a extracellular hinge domain, a CD8a transmembrane domain, a DAP12 intracellular domain, and an FcRy intracellular domain; (iii) a CD8a extracellular leader domain, an extracellular antigen binding domain, a CD8a extracellular hinge domain, a CD8a transmembrane domain, an FcRy intracellular domain, and a DAP12 intracellular domain; (iv) a CD8a extracellular leader domain, an extracellular antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and a DAP 12 intracellular domain; (ii)
  • a CAR has or comprises: (a) an amino acid sequence selected from Table 2; (b) an amino acid sequence that differs from a sequence selected from Table 2 by no more than five substitutions, additions, or deletions; or (c) an amino acid sequence that is at least 80% identical to a sequence selected from Table 2.
  • compositions comprising a modified immune cell as described herein.
  • a pharmaceutical composition comprises a pharmaceutically acceptable carrier.
  • the present disclosure provides a chimeric antigen receptors (CARs) comprising: (a) one or more extracellular domains; (b) a transmembrane domain; and (c) one or more intracellular domains comprising: (i) a DAP10 domain or a portion thereof, or (ii) a DAP 12 domain or a portion thereof.
  • CARs chimeric antigen receptors
  • one or more extracellular domains are or comprise an scFv, VHH antibody, centyrin, darpin, or nanobody.
  • a transmembrane domain is or comprises a DAP 10, DAP12, CD8a, CD28, CD40, MyD88 CD64, CD32a, CD32c, CD16a, CD3zeta, ICOS, Dectin-1, DNGR1, SLAMF7, TRL1, TLR2, TLR3, TRIM, TLR5, TLR6, TLR7, TLR8, or TLR9 transmembrane domain.
  • one or more intracellular domains further comprise one or more of a CD3 FcRy, MyD88, CD40, CD64, CD32a, CD32c, CD16a, CD89, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD 19, CD20, 41BB, CD28, GCSFR (CD114), RAGE, CD30, CD160, DR3, Fnl4, HVEM, CD160, NGFR, RANK, TNFR2, TROY, XEDAR, TRIF, 0X40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, V/I/LxYxxL/V, SIRPa
  • one or more intracellular domains further comprise one or more of a CD20, CD40, Dectin-1, FcRy, MyD88, RAGE, SLAMF7, or TLR2 intracellular domain, a portion of any of the foregoing, or combinations thereof.
  • a CAR further comprises an extracellular leader domain.
  • an extracellular leader domain is or comprises a CD8a extracellular leader domain.
  • a CAR further comprises an extracellular hinge domain.
  • an extracellular hinge domain is or comprises: a DAP10 extracellular hinge domain, a DAP 12 extracellular hinge domain, a CD8a extracellular hinge domain, a CD28 extracellular hinge domain, a DNGR-1 extracellular hinge domain, a Dectin-1 extracellular hinge domain, or an IgG4 extracellular hinge domain.
  • a CAR comprises, from N-terminus to C-terminus: (i) a CD8a extracellular leader domain, an extracellular antigen binding domain, a DAP 10 extracellular hinge domain, a DAP 10 transmembrane domain, and a DAP 10 intracellular domain; (ii) a CD8a extracellular leader domain, an extracellular antigen binding domain, a CD8a extracellular hinge domain, a CD8a transmembrane domain, a DAP10 intracellular domain, and an FcRy intracellular domain; (iii) a CD8a extracellular leader domain, an extracellular antigen binding domain, a CD8a extracellular hinge domain, a CD8a transmembrane domain, an FcRy intracellular domain, and a DAPIO intracellular domain; (iv) a CD8a extracellular leader domain, an extracellular antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and a DAP 10 intracellular domain; (ii)
  • a CAR comprising, from N-terminus to C-terminus: (i) a CD8a extracellular leader domain, an extracellular antigen binding domain, a CD8a extracellular hinge domain, a CD8a transmembrane domain, and a DAP 12 intracellular domain; (ii) a CD8a extracellular leader domain, an extracellular antigen binding domain, a CD8a extracellular hinge domain, a CD8a transmembrane domain, a DAP 12 intracellular domain, and an FcRy intracellular domain; (iii) a CD8a extracellular leader domain, an extracellular antigen binding domain, a CD8a extracellular hinge domain, a CD8a transmembrane domain, an FcRy intracellular domain, and a DAP 12 intracellular domain; (iv) a CD8a extracellular leader domain, an extracellular antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, and a DAP 12 intracellular domain;
  • a CAR has or comprises: (a) an amino acid sequence selected from Table 2; (b) an amino acid sequence that differs from a sequence selected from Table 2 by no more than five substitutions, additions, or deletions; or (c) an amino acid sequence that is at least 80% identical to a sequence selected from Table 2.
  • nucleic acid constructs comprising one or more nucleic acid sequences encoding: (a) one or more extracellular domains; (b) a transmembrane domain; and (c) one or more intracellular domains comprising: (i) a DAP 10 domain or a portion thereof, or (ii) a DAP12 domain or a portion thereof; wherein the nucleic acid construct encodes a chimeric antigen receptor (CAR) comprising (a) through (c).
  • CAR chimeric antigen receptor
  • a nucleic acid construct further comprises one or more nucleic acid sequences encoding: (d) one or more extracellular leader domains, (e) one or more extracellular hinge domains, (f) one or more cleavage peptides, or combinations thereof.
  • a cleavage peptide is or comprises a P2A, F2A, E2A or T2A peptide.
  • a nucleic acid construct has or comprises: (a) a nucleotide sequence selected from Table 3; (b) a nucleotide sequence that differs from a sequence selected from Table 3 by no more than five substitutions, additions, or deletions; or (c) a nucleotide sequence that is at least 80% identical to a sequence selected from Table 3.
  • a nucleic acid construct comprises DNA or messenger RNA (mRNA).
  • mRNA messenger RNA
  • a nucleic acid construct comprises a modification selected from: a modified nucleotide, an alteration to the 5’ untranslated region (UTR), an alteration to the 3’ UTR, a cap structure, a poly(A) tail, or combinations thereof.
  • a cap structure comprises AGCapl, m6AGCapl, or Anti-Reverse Cap Analog (ARCA).
  • a modified nucleotide comprises pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine/pseudouridine (5meC PsU), Nl-methyl-pseudouridine (NlmPsU), or combinations thereof.
  • a nucleic acid construct is a purified nucleic acid construct.
  • a nucleic acid construct is codon-optimized. In some embodiments, a nucleic acid construct is codon-optimized for expression in a stem cell, monocyte, macrophage, or dendritic cell. [0032] In another aspect, the present disclosure provides pharmaceutical compositions comprising a nucleic acid construct as described herein. In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable carrier.
  • the present disclosure provides methods of treating a disease or disorder in a subject, the methods comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition as described herein, wherein at least one sign or symptom of the disease or disorder is improved in the subject after administration.
  • a step of administering is or comprises transarterial, subcutaneous, intravenous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, or intraperitoneal delivery.
  • the present disclosure provides methods of modifying an immune cell, the methods comprising: delivering to the immune cell a nucleic acid construct as described herein, thereby producing a modified immune cell, wherein the modified immune cell is or comprises a macrophage, monocyte, dendritic cell, or stem cell.
  • a nucleic acid construct comprises DNA or messenger RNA (mRNA).
  • a nucleic acid construct comprises a modification selected from: a modified nucleotide, an alteration to the 5’ untranslated region (UTR), an alteration to the 3’ UTR, a cap structure, a poly(A) tail, or combinations thereof.
  • a cap structure comprises AGCapl, m6AGCapl, or Anti-Reverse Cap Analog (ARCA).
  • a modified nucleotide comprises pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine/pseudouridine (5meC PsU), Nl-methyl-pseudouridine (NlmPsU), or combinations thereof.
  • a nucleic acid construct is a purified nucleic acid construct.
  • a purified nucleic acid construct is produced by a method comprising silica membrane purification, high performance liquid chromatography (HPLC), Dynabeads, LiCl precipitation, phenol-chloroform extraction, resin based purification, polyA isolation, RNeasy, or combinations thereof.
  • a nucleic acid construct is codon-optimized.
  • a nucleic acid construct is codon-optimized for expression in a stem cell, monocyte, macrophage, or dendritic cell.
  • delivering comprises electroporation or transfection with the nucleic acid construct.
  • a nucleic acid construct is encapsulated within a delivery vehicle.
  • a delivery vehicle is or comprises a liposome, a lipid nanoparticle, a polymer, an adeno-associated viral (AAV) vector, an adenoviral vector, a retroviral vector or combinations thereof.
  • a liposome or lipid nanoparticle comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, one or more PEG-modified lipids, or combinations thereof.
  • a retroviral vector comprises a lentiviral vector or a gammaretroviral vector.
  • a lentiviral vector is packaged with a Vpx protein.
  • an adenoviral vector comprises an Ad2 vector or an Ad5 vector.
  • an Ad5 vector comprises an Ad5f35 adenoviral vector.
  • methods of the present disclosure further comprise delivering to an immune cell an additional payload.
  • an additional payload is or comprises a pathogen recognition receptor agonist, polyinosinic:poly cytidylic acid (poly EC), a TLR7/8 agonist, a CpG oligodeoxynucleotide, a NOD-like receptor (NLR) agonist, a RIG-I-like receptor (RLR) agonist, a C-type lectins receptor (CLR) agonist, a cytosolic DNA sensing, the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) agonist, an interferon-inducible protein 16 (IFI16) agonist, a DEAD-box helicase 41 (DDX41) agonist, an LRR binding FLII interacting protein 1 (LRRFIP1) agonist, an absent in melanoma 2 (AIM2)
  • the present disclosure provides methods of producing a modified immune cell comprising a chimeric antigen receptor (CAR), the method comprising: administering to a subject a composition comprising: (a) one or more nucleic acid molecules, wherein at least a portion of the one or more nucleic acid molecules encodes the CAR, and (b) a delivery vehicle; wherein following administration of the composition one or more nucleic acid molecules are translated in an immune cell to produce a modified immune cell comprising the CAR, wherein the immune cell is a stem cell, monocyte, macrophage, or dendritic cell in the subject, and wherein the CAR comprises one or more extracellular domains, a transmembrane domain, and one or more intracellular domains comprising: a DAP 10 domain or a portion thereof, and/or a DAP 12 domain or a portion thereof.
  • CAR chimeric antigen receptor
  • Figure 1 shows exemplary illustrations of DAP12- and DAPlO-associated receptors and their native signaling pathways.
  • Figure 2 shows exemplary recovery and viability percentages of macrophages expressing anti-HER2 CAR constructs comprising a DAP 10 domain relative to control constructs and cells, as measured by flow cytometry.
  • Figure 3A and Figure 3B show exemplary CAR expression via histogram (Figure 3A) and quantification by mean fluorescence intensity (Figure 3B) in macrophages expressing anti-HER2 CAR constructs comprising a DAP 10 domain relative to control constructs and cells, as measured by flow cytometry.
  • Figure 4 shows exemplary expression of Ml markers (CD80, CD86) and M2 markers (CD 163, CD206) via quantification by mean fluorescence intensity in macrophages expressing anti-HER2 CAR constructs comprising a DAP 10 domain relative to control constructs and cells, as measured by flow cytometry.
  • Figure 5 shows exemplary tumor cell death of AU565 cells when co-cultured at different ratios with macrophages expressing anti-HER2 CAR constructs comprising a DAP 10 domain relative to control constructs and cells, as measured by GFP intensity from tumor cell expression. Ratios of 1 :4 E:T (20,000 macrophages: 80,000 AU565 cells) and 1 :2 E:T (20,000 macrophages:40,000 AU565 cells) were assessed.
  • Figure 6 shows exemplary tumor cell death of AU565 cells when co-cultured at different ratios with macrophages expressing anti-HER2 CAR constructs comprising a DAP10 domain relative to control constructs and cells, as measured by GFP intensity from tumor cell expression. Ratios of 1 :1 E:T (20,000 macrophages:20,000 AU565 cells) and 2: 1 E:T (20,000 macrophages: 10,000 AU565 cells) were assessed.
  • Figure 7 shows exemplary cytokine secretion in macrophages expressing anti- HER2 CAR constructs comprising a DAP 10 domain relative to control constructs and cells when incubated with PBS, HER2, or Mesothelin for 24 hours. TNFa, IL-8, and IL-6 cytokines were assessed.
  • Figure 8 shows exemplary recovery and viability percentages of macrophages expressing anti-HER2 CAR constructs comprising a DAP12 domain relative to control constructs and cells, as measured by flow cytometry.
  • Figure 9A and Figure 9B show exemplary CAR expression via histogram (Figure 9A) and quantification by mean fluorescence intensity (Figure 9B) in macrophages expressing anti-HER2 CAR constructs comprising a DAP12 domain relative to control constructs and cells, as measured by flow cytometry.
  • Figure 10 shows exemplary expression of Ml markers (CD80, CD86) and M2 markers (CD 163, CD206) via quantification by mean fluorescence intensity in macrophages expressing anti-HER2 CAR constructs comprising a DAP12 domain relative to control constructs and cells, as measured by flow cytometry.
  • Figure 11 shows exemplary tumor cell death of AU565 cells when co-cultured at different ratios with macrophages expressing anti-HER2 CAR constructs comprising a DAP 12 domain relative to control constructs and cells, as measured by GFP intensity from tumor cell expression. Ratios of 1 :4 E:T (20,000 macrophages: 80,000 AU565 cells) and 1 :2 E:T (20,000 macrophages: 40,000 AU565 cells) were assessed.
  • Figure 12 shows exemplary tumor cell death of AU565 cells when co-cultured at different ratios with macrophages expressing anti-HER2 CAR constructs comprising a DAP12 domain relative to control constructs and cells, as measured by GFP intensity from tumor cell expression. Ratios of 1 :1 E:T (20,000 macrophages:20,000 AU565 cells) and 2: 1 E:T (20,000 macrophages: 10,000 AU565 cells) were assessed. [0056] Figure 13 shows exemplary cytokine secretion in macrophages expressing anti- HER2 CAR constructs comprising a DAP12 domain relative to control constructs and cells when incubated with PBS, HER2, or Mesothelin for 24 hours. TNFa, IL-8, and IL-6 cytokines were assessed.
  • Figure 14 shows domains of exemplary DAP 10 CAR constructs (e.g., those as described herein) comprising an anti-HER2 binder.
  • Figure 15 shows exemplary viability and recovery percentages of macrophages expressing anti-HER2 CAR constructs comprising a DAP 10 domain relative to control constructs and cells, as measured by flow cytometry.
  • Figure 16 shows exemplary CAR expression via histogram plot and quantification by percent expressing and mean fluorescence intensity in macrophages expressing anti-HER2 CAR constructs comprising a DAP10 domain relative to control constructs and cells, as measured by flow cytometry.
  • Figure 17 shows exemplary expression of Ml markers (CD80, CD86) and M2 markers (CD 163, CD206) via quantification by mean fluorescence intensity in macrophages expressing anti-HER2 CAR constructs comprising a DAP 10 domain relative to control constructs and cells, as measured by flow cytometry.
  • Figure 18 shows exemplary tumor cell death at different time points of AU565 cells when co-cultured at different ratios with macrophages expressing anti-HER2 CAR constructs comprising a DAPIO domain relative to control constructs and cells, as measured by GFP intensity from tumor cell expression.
  • Figure 19 shows exemplary tumor cell death of AU565 cells when co-cultured at different ratios with macrophages expressing anti-HER2 CAR constructs comprising a DAP10 domain relative to control constructs and cells, as measured by GFP intensity from tumor cell expression.
  • Figure 20 shows exemplary tumor cell death at different time points of Panel and MDA468 cells when co-cultured at 3: 1 E:T (effectortarget) with macrophages expressing anti- HER2 CAR constructs comprising a DAP 10 domain relative to control constructs and cells, as measured by GFP intensity from tumor cell expression.
  • Figure 21 shows exemplary cytokine secretion in macrophages expressing anti- HER2 CAR constructs comprising a DAP 10 domain relative to control constructs and cells when incubated with PBS, HER2, or Mesothelin. TNFa, IL-8, IL6, ILlb, and IL12p70 cytokines were assessed.
  • Figure 22 shows domains of exemplary DAP 10 CAR constructs (e.g., those as described herein) comprising an anti-HER2 binder, and several mutations investigated in certain CAR constructs.
  • Figure 23 shows exemplary experimental timeline for screening mutant DAP10 CAR sequences expressed in Hek Nulll cells.
  • Figure 24 shows exemplary viability percentages of Hek cells expressing anti- HER2 CAR constructs comprising a DAP 10 domain relative to control constructs and cells.
  • Figure 25 shows exemplary CAR expression via quantification by percent expressing and mean fluorescence intensity in Hek cells expressing anti-HER2 CAR constructs comprising a DAP 10 domain relative to control constructs and cells.
  • Figure 26 shows exemplary NFKB activation in HeK cells expressing anti-HER2 CAR constructs comprising a DAP10 domain relative to control constructs and cells when incubated with PBS, HER2, or Mesothelin.
  • Figure 27 shows domains of exemplary DAP10 CAR constructs (e.g., those as described herein) comprising an anti-HER2 binder.
  • Figure 28 shows exemplary viability and recovery percentages of macrophages expressing anti-HER2 CAR constructs comprising a DAP 10 domain relative to control constructs and cells, as measured by flow cytometry.
  • Figure 29 shows exemplary CAR expression via histogram plot and quantification by percent expressing and mean fluorescence intensity in macrophages expressing anti-HER2 CAR constructs comprising a DAP10 domain relative to control constructs and cells, as measured by flow cytometry.
  • Figure 30 shows exemplary expression of Ml markers (CD80, CD86) and M2 markers (CD 163, CD206) via quantification by mean fluorescence intensity in macrophages expressing anti-HER2 CAR constructs comprising a DAP10 domain relative to control constructs and cells, as measured by flow cytometry.
  • Figure 31 shows exemplary tumor cell death at different time points of AU565 cells when co-cultured at a 2: 1 effector to target ratio with macrophages expressing anti-HER2 CAR constructs comprising a DAP10 domain relative to control constructs and cells, as measured by GFP intensity from tumor cell expression.
  • Figure 32 shows exemplary tumor cell death of AU565 cells when co-cultured at different ratios with macrophages expressing anti-HER2 CAR constructs comprising a DAP10 domain relative to control constructs and cells, as measured by GFP intensity from tumor cell expression.
  • Figure 33 shows exemplary cytokine secretion in macrophages expressing anti- HER2 CAR constructs comprising a DAP 10 domain relative to control constructs and cells when incubated with PBS, HER2, or Mesothelin. TNFa, IL-8, and IL6 cytokines were assessed.
  • Figure 34A and Figure 34B show a series of graphs showing exemplary viability percentages and cells recovered of HEK cells expressing anti-FLER2 CAR constructs comprising a DAP 10 domain and Myd88 ICD (labeled as Myd88) described herein (see Table 4-6 in Example 5) relative to control constructs and UTD cells.
  • Figure 35 is a series of graphs showing exemplary CAR expression via quantification by percent expression and mean fluorescence intensity in HEK cells expressing anti-HER2 CAR constructs comprising a DAP10 domain and Myd88 ICD (labelled as M88) described herein (see Table 4-6 in Example 5) relative to control constructs and UTD cells.
  • Figures 36A-B are graphs of Ml (FIG. 36A) and M2 markers (Figure 36B) of CAR macrophages transduced with constructs described herein (see Table 4-6 in Example 5).
  • Figure 37 is a graph showing tumor killing ability of CAR macrophages described herein (see Table 4-6 in Example 5).
  • Figures 38A-C are graphs showing tumor killing ability of CAR macrophages described herein (see Table 4-6 in Example 5) of HER2 expressing (AU562) and HER2 nonexpressing control cells (PANCI).
  • Figures 39A-C are a series of graphs showing exemplary cytokine secretion in macrophages expressing anti-HER2 CAR constructs comprising a DAP 10 domain and Myd88 ICD relative to control constructs and UTD macrophages when incubated with PBS, HER2, or Mesothelin.
  • TNFa Figure 39A
  • IL-6 Figure 39B
  • IL-8 Figure 39C
  • Figure 40 shows domains of exemplary CAR constructs disclosed herein comprising a DAP10 and/or Myd88 co- stimulatory domain.
  • Figure 41 is a graph showing exemplary NFKB activation in HEK cells expressing anti-HER2 CAR constructs comprising a DAP 10 domain, Myd88 ICD (labeled as M88), and FcRy domain (labeled as y) disclosed herein relative to control constructs and UTD cells.
  • Figure 42 are graphs showing exemplary viability percentages and cells recovered of HEK cells expressing anti-HER2 CAR constructs comprising a DAPIO domain, Myd88 ICD (labeled as M88), and FcRy domain (labeled as y) described herein relative to control constructs and UTD cells.
  • Figure 43 is a series of graphs showing exemplary CAR expression via quantification by percent expression and mean fluorescence intensity in HEK cells expressing anti-HER2 CAR constructs comprising a DAP10 domain, Myd88 ICD (labeled as M88), and FcRy domain (labeled as y) described herein relative to control constructs and UTD cells.
  • Figures 44A-B are graphs of Ml (Figure 49A) and M2 markers (Figure 49B) of CAR macrophages transduced with constructs described herein (see Tables 7-9 in Example 7).
  • Figure 45 is a graph showing tumor killing ability of CAR macrophages described herein (see Tables 7-9 in Example 7).
  • Figures 46A-B are a series of graphs showing exemplary cytokine secretion in macrophages expressing anti-HER2 CAR constructs comprising a DAP 10 domain, Myd88 ICD, and FcRy domain relative to control constructs and UTD macrophages when incubated with PBS, HERZ, or Mesothelin.
  • TNFa Figure 46A
  • IL-6 Figure 46B
  • Activation refers to the state of a cell, for example a monocyte, macrophage, or dendritic cell that has been sufficiently stimulated to induce detectable cellular proliferation or has been stimulated to exert its effector function. Activation can also be associated with induced cytokine production, phagocytosis, cell signaling, target cell killing, and/or antigen processing and presentation.
  • activated monocytes/macrophages/dendritic cells refers to, among other things, monocyte/macrophage/dendritic cells that are undergoing cell division or exerting effector function.
  • the term “activated monocytes/macrophages/dendritic cells” refers to, among others thing, cells that are performing an effector function or exerting any activity not seen in the resting state, including phagocytosis, cytokine secretion, proliferation, gene expression changes, metabolic changes, and other functions.
  • agent refers to a molecule that may be expressed, released, secreted or delivered to a target by a modified cell described herein.
  • An agent includes, but is not limited to, a nucleic acid, an antibiotic, an anti-inflammatory agent, an antibody or fragments thereof, an antibody agent or fragments thereof, a growth factor, a cytokine, an enzyme, a protein (e.g., an RNAse inhibitor), a peptide, a fusion protein, a synthetic molecule, an organic molecule (e.g., a small molecule), a carbohydrate, a lipid, a hormone, a microsome, a derivative or a variation thereof, and any combinations thereof.
  • An agent may bind any cell moiety, such as a receptor, an antigenic determinant, or other binding site present on a target or target cell. An agent may diffuse or be transported into a cell, where it may act intracellularly.
  • Antibody refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen.
  • intact antibodies as 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 (about 25 kD each) that associate with each other into what is commonly referred to as a “Y-shaped” structure.
  • Each heavy chain comprises at least four domains (each about 110 amino acids long) - an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CHI, CH2, and the carboxy -terminal CH3 (located at the base of the Y’s stem).
  • VH amino-terminal variable
  • CH2 amino-terminal variable
  • CH3 carboxy -terminal CH3
  • Each light chain comprises two domains - an amino-terminal variable (VL) domain, followed by a carboxyterminal constant (CL) domain, separated from one another by another “switch”.
  • Intact antibody tetramers comprise two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and a tetramer is formed.
  • Naturally-produced antibodies are also glycosylated, typically on the CH2 domain.
  • Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold” formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel beta barrel.
  • Each variable domain contains three hypervariable loops known as “complementarity determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4).
  • CDR1, CDR2, and CDR3 three hypervariable loops known as “complementarity determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4).
  • the Fc region of naturally-occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. Affinity and/or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification.
  • antibodies produced and/or utilized in accordance with the present invention include glycosylated Fc domains, including Fc domains with modified or engineered glycosylation.
  • any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in natural antibodies can be referred to and/or used as an “antibody”, whether such polypeptide is naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology.
  • an antibody is polyclonal.
  • an antibody is monoclonal.
  • an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies.
  • antibody sequence elements are humanized, primatized, chimeric, etc, as is known in the art.
  • an antibody utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPsTM”); single chain or Tandem diabodies (SIPsTM”); single chain or Tandem diabodies (SIPsTM”); single chain or Tandem diabodies (SIPsTM”); single chain or Tandem diabodies (SIPsTM”); single chain or Tandem diabodies (S
  • an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally.
  • an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc.].
  • antibody agent refers to an agent that specifically binds to a particular antigen.
  • the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding.
  • Exemplary antibody agents include, but are not limited to monoclonal antibodies or polyclonal antibodies.
  • an antibody agent may include one or more constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies.
  • an antibody agent may include one or more sequence elements are humanized, primatized, chimeric, etc., as is known in the art.
  • an antibody agent utilized in accordance with the present invention is in
  • an antibody agent may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally.
  • an antibody agent may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc.].
  • an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to one found in a reference antibody.
  • CDR complementarity determining region
  • an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR.
  • an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR.
  • an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR.
  • an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR.
  • an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain.
  • an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain.
  • an antibody agent is not and/or does not comprise a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain.
  • an antibody agent may be or comprise a molecule or composition which does not include immunoglobulin structural elements (e.g., a receptor or other naturally occurring molecule which includes at least one antigen binding domain).
  • antibody fragment refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments and human and humanized versions thereof.
  • Antibody heavy chain refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.
  • Antibody light chain As used herein, the term “antibody light chain” refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.
  • Synthetic antibody refers to an antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein.
  • the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
  • Antigen refers to a molecule that is capable of provoking an immune response. This immune response may involve either antibody production, the activation of specific immunologically-competent cells, or both. A skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA that comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response encodes an “antigen” as that term is used herein.
  • an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
  • Anti-tumor effect refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition.
  • An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of a tumor in the first place.
  • Autologous refers to any material derived from an individual to which it is later to be re-introduced into the same individual.
  • Allogeneic refers to any material (e.g., a population of cells) derived from a different animal of the same species.
  • Xenogenic refers to any material (e.g., a population of cells) derived from an animal of a different species.
  • cancer refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like. In certain embodiments, the cancer is medullary thyroid carcinoma.
  • Conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody compatible with various embodiments by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • Co-stimulatory ligand refers to a molecule on an antigen presenting cell (e.g., an APC, dendritic cell, B cell, and the like) that specifically binds a cognate co-stimulatory molecule on a monocyte/macrophage/dendritic cell, thereby providing a signal which mediates a monocyte/macrophage/dendritic cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
  • an antigen presenting cell e.g., an APC, dendritic cell, B cell, and the like
  • a co- stimulatory ligand can include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3.
  • a co-stimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a monocyte/macrophage/dendritic cell, such as, but not limited to, CD27, CD28, 4-1BB, 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.
  • Cytotoxic refers to killing or damaging cells. In one embodiment, cytotoxicity of the metabolically enhanced cells is improved, e.g., increased cytolytic activity of macrophages.
  • Effective amount As used herein, “effective amount” and “therapeutically effective amount” are interchangeable, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a manufacturing, therapeutic or prophylactic benefit. Such results may include, but are not limited to, anti-tumor activity as determined by any means suitable in the art.
  • effector function refers to a specific activity carried out by an immune cell in response to stimulation of the immune cell. For example, an effector function of macrophages to engulf and digest cellular debris, foreign substances, microbes, cancer cells and other unhealthy cells by phagocytosis.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • Endogenous refers to any material from or produced inside a particular organism, cell, tissue or system.
  • Exogenous refers to any material introduced from or produced outside a particular organism, cell, tissue or system.
  • the term “expand” refers to increasing in number, as in an increase in the number of cells, for example, monocytes, macrophages, and/or dendritic cells.
  • monocytes, macrophages, or dendritic cells that are expanded ex vivo increase in number relative to the number originally present in a culture.
  • monocytes, macrophages, or dendritic cells that are expanded ex vivo increase in number relative to other cell types in a culture.
  • expansion may occur in vivo.
  • expression of a nucleic acid sequence refers to generation of any gene product from a nucleic acid sequence.
  • a gene product can be a transcript.
  • a gene product can be a polypeptide.
  • expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
  • Expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cisacting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).
  • fragment refers to a structure that includes a discrete portion of the whole, but lacks one or more moi eties found in the whole structure. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole.
  • a nucleotide fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more monomeric units (e.g., nucleic acids) as found in the whole nucleotide.
  • monomeric units e.g., nucleic acids
  • a nucleotide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the monomeric units (e.g., residues) found in the whole nucleotide.
  • the whole material or entity may in some embodiments be referred to as the “parent” of the whole.
  • homology refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical.
  • polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions).
  • sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions).
  • a variety of algorithms are available that permit comparison of sequences in order to determine their degree of homology, including by permitting gaps of designated length in one sequence relative to another when considering which residues “correspond” to one another in different sequences.
  • Calculation of the percent homology between two nucleic acid sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-corresponding sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence.
  • the nucleotides at corresponding nucleotide positions are then compared.
  • identity refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules.
  • two amino acid sequences When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an Arginine, then they are identical at that position.
  • the identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage.
  • the identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.
  • Substantial identity refers to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered to be “substantially identical” if they contain identical residues in corresponding positions. As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences.
  • two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over a relevant stretch of residues.
  • the relevant stretch is a complete sequence.
  • the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.
  • reference to “substantial identity” typically refers to a CDR having an amino acid sequence at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to that of a reference CDR.
  • Immune cell refers to a cell that is involved in an immune response, e.g., promotion of an immune response.
  • 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
  • Immune response refers to a cellular and/or systemic response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and/or activate lymphocytes to remove the antigen.
  • Immunoglobulin refers to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as a BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE.
  • IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts.
  • IgG is the most common circulating antibody.
  • IgM is the main immunoglobulin produced in the primary immune response in most subjects.
  • IgD is an immunoglobulin that has no known antibody function, but may serve as an antigen receptor.
  • IgE is an immunoglobulin that mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergen.
  • Isolated refers to something altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • Modified refers to a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.
  • Modulating refers to mediating a detectable increase or decrease in the level of a response and/or a change in the nature of a response in a subject compared with the level and/or nature of a response in the subject in the absence of a treatment or compound, and/or compared with the level and/or nature of a response in an otherwise identical but untreated subject. The term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.
  • nucleic acid refers to a polymer of at least three nucleotides.
  • a nucleic acid comprises DNA.
  • a nucleic acid comprises RNA.
  • a nucleic acid is single stranded.
  • a nucleic acid is double stranded.
  • a nucleic acid comprises both single and double stranded portions.
  • a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages.
  • a nucleic acid comprises a backbone that comprises both phosphodiester and non- phosphodiester linkages.
  • a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and/or one or more peptide bonds, e.g., as in a “peptide nucleic acid”.
  • a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil).
  • a nucleic acid comprises one or more, or all, non-natural residues.
  • a non-natural residue comprises one or more modified sugars (e.g., 2'- fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues.
  • a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide.
  • a nucleic acid has a nucleotide sequence that comprises one or more introns.
  • a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis.
  • enzymatic synthesis e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis.
  • a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long.
  • operably linked refers to functional linkage between, for example, a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • Overexpressed tumor antigen refers to an abnormal level of expression of a tumor antigen in a cell from a disease area like a solid tumor within a specific tissue or organ of the patient relative to the level of expression in a normal cell from that tissue or organ.
  • Patients having solid tumors or a hematological malignancy characterized by overexpression of the tumor antigen can be determined by standard assays known in the art.
  • Polynucleotide refers to a chain of nucleotides.
  • nucleic acids are polymers of nucleotides.
  • nucleic acids and polynucleotides as used herein are interchangeable.
  • nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
  • polypeptide refers to any polymeric chain of residues (e.g., amino acids) that are typically linked by peptide bonds. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature.
  • a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only nonnatural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L- amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids.
  • a polypeptide may include one or more pendant groups or other modifications, e g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof.
  • such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof.
  • a polypeptide may be cyclic, and/or may comprise a cyclic portion.
  • a polypeptide is not cyclic and/or does not comprise any cyclic portion.
  • a polypeptide is linear.
  • a polypeptide may be or comprise a stapled polypeptide.
  • the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides.
  • the present specification provides and/or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family.
  • a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class).
  • a common sequence motif e.g., a characteristic sequence element
  • shares a common activity in some embodiments at a comparable level or within a designated range
  • a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%.
  • a conserved region that may in some embodiments be or comprise a characteristic sequence element
  • Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids.
  • a useful polypeptide may comprise or consist of a fragment of a parent polypeptide.
  • a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and/or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.
  • Protein refers to a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means.
  • Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc.
  • proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.
  • the term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.
  • proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.
  • Signal transduction pathw ay As used herein, the term “signal transduction pathway” refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell.
  • the phrase “cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the plasma membrane of a cell.
  • Single chain antibodies refers to antibodies formed by recombinant DNA techniques in which immunoglobulin heavy and light chain fragments are linked to the Fv region via an engineered span of amino acids.
  • Various methods of generating single chain antibodies are known, including those described in U.S. Pat. No. 4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242: 1038-1041.
  • the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antigen binding domain or antibody agent, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antigen binding domain or antibody agent recognizes and binds to a specific protein structure rather than to proteins generally.
  • an antigen binding domain or antibody agent is specific for epitope “A”
  • the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antigen binding domain or antibody agent will reduce the amount of labeled A bound to the antibody.
  • Stimulation refers to a primary response induced by binding of a stimulatory molecule (e.g., an FcR complex, a TLR complex, or a TCR/CD3 complex), for example, with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via Fc receptor machinery or via a synthetic CAR.
  • a stimulatory molecule e.g., an FcR complex, a TLR complex, or a TCR/CD3 complex
  • Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-beta, and/or reorganization of cytoskeletal structures, and the like.
  • a stimulatory molecule refers to a molecule of a monocyte, macrophage, or dendritic cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell.
  • a stimulatory molecule comprises an FcR extracellular domain comprising a CD64 (FcyRI), CD32a (FcyRIIa), CD32b (FcyRIIb), CD32c, CD 16a (FcyRIIIa), CD 16b (FcyRIIIb), FcsRI, FcsRII, FcaRI (CD89) or CD40 domain.
  • a stimulatory molecule comprises a TLR extracellular domain comprising a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.
  • the term “stimulatory ligand,” refers to a ligand that when present on an antigen presenting cell (e.g., an aAPC, a macrophage, a dendritic cell, a B-cell, and the like) or tumor cell can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a monocyte, macrophage, or dendritic cell thereby mediating a response by the immune cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like.
  • Stimulatory ligands are well-known in the art and encompass, inter alia, Toll-like receptor (TLR) ligand, an anti-toll-like receptor antibody, an agonist, and an antibody for a monocyte/macrophage receptor.
  • TLR Toll-like receptor
  • cytokines such as interferon-gamma, are potent stimulants of macrophages.
  • Subject refers to an organism, for example, a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, or a dog).
  • a human subject is an adult, adolescent, or pediatric subject.
  • a subject is suffering from a disease, disorder or condition, e.g., a disease, disorder, or condition that can be treated as provided herein, e.g., a cancer or a tumor listed herein.
  • a subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and/or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing the disease, disorder, or condition.
  • a subject displays one or more symptoms of a disease, disorder, or condition.
  • a subject does not display a particular symptom (e.g., clinical manifestation of disease) or characteristic of a disease, disorder, or condition.
  • a subject does not display any symptom or characteristic of a disease, disorder, or condition.
  • a subject is a patient.
  • a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
  • substantially purified refers to a cell that is essentially free of other cell types.
  • a substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state.
  • a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state.
  • the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.
  • Target refers to a cell, tissue, organ, or site within the body that is the subject of provided methods, systems, and/or compositions, for example, a cell, tissue, organ or site within a body that is in need of treatment or is preferentially bound by, for example, an antibody (or fragment thereof) or a CAR.
  • Target site refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.
  • T cell receptor refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen.
  • a TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules.
  • a TCR comprises a heterodimer of an alpha (a) and beta (P) chain, although in some cells the TCR comprises gamma and delta (y/8) chains.
  • TCRs may exist in alpha/beta and gamma/delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain comprises two extracellular domains, a variable and constant domain.
  • a TCR may be modified on any cell comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell.
  • therapeutic refers to a treatment and/or prophylaxis.
  • a therapeutic effect is obtained by suppression, remission, or eradication of one or more symptoms or features of a disease state.
  • transfected As used herein, the term “transfected” or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.
  • treat refers to partial or complete alleviation, amelioration, delay of onset of, inhibition, prevention, relief, and/or reduction in incidence and/or severity of one or more symptoms or features of a disease, disorder, and/or condition.
  • treatment may be administered to a subject who does not exhibit signs or features of a disease, disorder, and/or condition (e.g., may be prophylactic).
  • treatment may be administered to a subject who exhibits only early or mild signs or features of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
  • treatment may be administered to a subject who exhibits established, severe, and/or late-stage signs of the disease, disorder, or condition.
  • treating may comprise administering to an immune cell (e.g., a monocyte, macrophage, or dendritic cell) or contacting an immune cell with a modulator of a pathway activated by in vitro transcribed mRNA.
  • an immune cell e.g., a monocyte, macrophage, or dendritic cell
  • Tumor refers to an abnormal growth of cells or tissue.
  • a tumor may comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non- metastatic.
  • a tumor is associated with, or is a manifestation of, a cancer.
  • a tumor may be a disperse tumor or a liquid tumor.
  • a tumor may be a solid tumor.
  • Vector refers to a composition of matter that comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term “vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno- associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • compositions comprising modified immune cells comprising modified immune cells (e.g., stem cells, macrophages, monocytes, and/or dendritic cells) comprising novel chimeric antigen receptors (CARs) described herein and methods of using and producing the same.
  • modified immune cells e.g., stem cells, macrophages, monocytes, and/or dendritic cells
  • CARs of the present disclosure comprise one or more intracellular domains comprising a DAP10 domain or a portion thereof or a DAP12 domain or a portion thereof and methods of using and making immune cells comprising said CARs.
  • DAP 10 is a signaling protein typically associated with the function of natural killer (NK) cells via the NKG2D receptor.
  • NK natural killer
  • DAP 10 function in myeloid cells is not well defined or understood.
  • the main signaling pathway of DAP 10 is thought to be PI3 Kinase. It has been previously shown that PI3K activation in macrophages leads to enhanced phagocytosis.
  • DAP 12 is a signaling protein that associates with a host of receptors, including receptors found on macrophages (e.g., TREM1 and TREM2).
  • the DAP12 signaling domain comprises an immunoreceptor tyrosine-based activation motif (IT AM).
  • a chimeric antigen receptor (CAR) of the present disclosure comprises an extracellular hinge domain comprising a DAP10 domain or a portion thereof.
  • a CAR of the present disclosure comprises a transmembrane domain comprising a DAP 10 domain or a portion thereof.
  • a CAR of the present disclosure comprises an intracellular domain comprising a DAP 10 domain or a portion thereof.
  • a chimeric antigen receptor (CAR) of the present disclosure comprises an extracellular hinge domain comprising a DAP12 domain or a portion thereof.
  • a CAR of the present disclosure comprises a transmembrane domain comprising a DAP 12 domain or a portion thereof.
  • a CAR of the present disclosure comprises an intracellular domain comprising a DAP12 domain or a portion thereof.
  • a chimeric antigen receptor (CAR) of the present disclosure comprises a DAP10 domain (e.g., an extracellular hinge, transmembrane, or intracellular domain) and a DAP 12 domain (e.g., an extracellular hinge, transmembrane, or intracellular domain).
  • DAP10 domain e.g., an extracellular hinge, transmembrane, or intracellular domain
  • DAP 12 domain e.g., an extracellular hinge, transmembrane, or intracellular domain
  • a chimeric antigen receptor (CAR) of the present disclosure comprises an extracellular hinge domain comprising a DAP10 domain or a portion thereof and a transmembrane domain comprising a DAP12 domain or a portion thereof. In some embodiments, a chimeric antigen receptor (CAR) of the present disclosure comprises an extracellular hinge domain comprising a DAP10 domain or a portion thereof and an intracellular domain comprising a DAP 12 domain or a portion thereof.
  • a chimeric antigen receptor (CAR) of the present disclosure comprises a transmembrane domain comprising a DAP10 domain or a portion thereof and an extracellular hinge domain comprising a DAP12 domain or a portion thereof.
  • a chimeric antigen receptor (CAR) of the present disclosure comprises a transmembrane domain comprising a DAP 10 domain or a portion thereof and an intracellular domain comprising a DAP12 domain or a portion thereof.
  • a chimeric antigen receptor (CAR) of the present disclosure comprises an intracellular domain comprising a DAP10 domain or a portion thereof and an extracellular hinge domain comprising a DAP12 domain or a portion thereof.
  • a chimeric antigen receptor (CAR) of the present disclosure comprises an intracellular domain comprising a DAP 10 domain or a portion thereof and a transmembrane domain comprising a DAP 12 domain or a portion thereof.
  • a chimeric antigen receptor (CAR) of the present disclosure comprises an extracellular hinge domain comprising a DAP12 domain or a portion thereof and a transmembrane domain comprising a DAP 10 domain or a portion thereof. In some embodiments, a chimeric antigen receptor (CAR) of the present disclosure comprises an extracellular hinge domain comprising a DAP12 domain or a portion thereof and an intracellular domain comprising a DAP10 domain or a portion thereof.
  • a chimeric antigen receptor (CAR) of the present disclosure comprises a transmembrane domain comprising a DAP12 domain or a portion thereof and an extracellular hinge domain comprising a DAP10 domain or a portion thereof.
  • a chimeric antigen receptor (CAR) of the present disclosure comprises a transmembrane domain comprising a DAP 12 domain or a portion thereof and an intracellular domain comprising a DAP 10 domain or a portion thereof.
  • a chimeric antigen receptor (CAR) of the present disclosure comprises an intracellular domain comprising a DAP12 domain or a portion thereof and an extracellular hinge domain comprising a DAP 10 domain or a portion thereof.
  • a chimeric antigen receptor (CAR) of the present disclosure comprises an intracellular domain comprising a DAP 12 domain or a portion thereof and a transmembrane domain comprising a DAP 10 domain or a portion thereof.
  • modified immune cells e.g., stem cells, macrophages, monocytes, or dendritic cells
  • CAR chimeric antigen receptor
  • modified immune cells e.g., stem cells, macrophages, monocytes, or dendritic cells
  • CAR chimeric antigen receptor
  • modified immune cells comprising a CAR comprising a DAP10 domain do not exhibit reduced viability relative to immune cells of the same type comprising a CAR without a DAP 10 domain as described herein.
  • a modified immune cell comprising a CAR comprising a DAP 10 domain expresses the CAR comprising the DAP 10 domain on the surface of the modified immune cell.
  • a modified immune cell e.g., stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprising a CAR comprising a DAP 10 domain exhibits increased pro- inflammatory (Ml) polarization relative to an immune cell of the same type that does not comprise a CAR.
  • a modified immune cell comprising a CAR comprising a DAP10 domain exhibits decreased anti-inflammatory (M2) polarization relative to an immune cell of the same type that does not comprise a CAR.
  • modified immune cells e.g., stem cells, macrophages, monocytes, or dendritic cells
  • a CAR comprising a DAP 10 domain exhibit enhanced killing function relative to immune cells comprising a CAR without a DAP10 domain as described herein.
  • modified immune cells e.g., stem cells, macrophages, monocytes, or dendritic cells
  • cytokines e.g., TNFa, IL-8, IL-6
  • modified immune cells e g., stem cells, macrophages, monocytes, or dendritic cells
  • CAR chimeric antigen receptor
  • modified immune cells comprising a CAR comprising a DAP12 domain do not exhibit reduced viability relative to immune cells of the same type without a CAR.
  • modified immune cells e.g., stem cells, macrophages, monocytes, or dendritic cells
  • CAR chimeric antigen receptor
  • a modified immune cell comprising a CAR comprising a DAP12 domain expresses the CAR comprising the DAP12 domain on the surface of the modified immune cell.
  • a modified immune cell e.g., stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprising a CAR comprising a DAP12 domain exhibits increased pro- inflammatory (Ml) polarization relative to an immune cell of the same type that does not comprise a CAR.
  • a modified immune cell comprising a CAR comprising a DAP12 domain exhibits decreased anti-inflammatory (M2) polarization relative to an immune cell of the same type that does not comprise a CAR.
  • modified immune cells e.g., stem cells, macrophages, monocytes, or dendritic cells
  • a CAR comprising a DAP12 domain exhibit enhanced killing function relative to immune cells comprising a CAR without a DAP12 domain as described herein.
  • modified immune cells e.g., stem cells, macrophages, monocytes, or dendritic cells
  • cytokines e.g., TNFa, IL-8, IL-6
  • immune cells comprising a CAR without a DAP 12 domain as described herein.
  • chimeric antigen receptor refers to an artificial cell surface receptor that is engineered to be expressed on an immune effector cell and specifically targets a cell and/or binds an antigen.
  • CARs may be used, for example, as a therapy with adoptive cell transfer.
  • immune cells e.g., stem cells, macrophages, monocytes, and/or dendritic cells
  • a patient e.g., from blood, tumor or ascites fluid
  • modified immune cells are then reintroduced to the same or a different subject as a therapeutics.
  • CARs have been expressed with specificity to an antigen, for example, a tumor associated antigen.
  • a CAR comprises an extracellular domain, a transmembrane domain and one or more intracellular moi eties.
  • a modified immune cell for example, a modified stem cell, macrophage, monocyte, or dendritic cell, is generated by expressing a CAR therein.
  • an immune cell comprises a CAR comprising an extracellular domain, a transmembrane domain, and one or more intracellular moieties, wherein the immune cell comprises a stem cell, macrophage, monocyte, or dendritic cell.
  • a CAR may further comprise one or more of one or more extracellular leader domains, one or more extracellular hinge domains and one or more intracellular co-stimulatory domains.
  • a CAR comprises a spacer domain or hinge between an extracellular domain and a transmembrane domain. In some embodiments, a CAR comprises a spacer domain or hinge between one or more intracellular moieties and a transmembrane domain.
  • the term “spacer domain” or “hinge” refers to any oligo- or polypeptide that functions to link a transmembrane domain to either an extracellular domain or to one or more intracellular moieties in a polypeptide chain.
  • a spacer domain or hinge may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • a short oligo- or polypeptide linker may form a linkage between a transmembrane domain and one or more intracellular moieties of a CAR.
  • An example of a linker includes a glycine-serine doublet.
  • an immune cell comprising a CAR
  • a CAR may comprise one or more control systems including, but not limited to: a safety switch (e.g., an on switch, and off switch, a suicide switch), a logic gate, for example an AND gate (e.g., two or more CARs, each of which lacks one or more signaling domains such that activation of both/all CARs is required for full immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) activation or function), an OR gate (e.g., two or more CARs, each with an intracellular domain such as CD3( ⁇ and a co-stimulatory domain), and/or a NOT gate (e.g., two or more CARs, one of which includes an inhibitory domain that antagonizes the function of the other CAR[s]).
  • a safety switch e.g., an on switch, and off switch, a suicide switch
  • a logic gate for example an AND gate (e.g
  • the present disclosure also provides immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) 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 one or more intracellular moieties, wherein the cell is a stem cell, macrophage, monocyte or dendritic cell that expresses the CAR.
  • a nucleic acid sequence e.g., an isolated nucleic acid sequence
  • 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 one or more intracellular moieties
  • the cell is a stem cell, macrophage, monocyte
  • a CAR comprises an extracellular domain that is operably linked to another domain of the CAR, such as a transmembrane domain or one or more intracellular moieties, for expression in an immune cell.
  • a nucleic acid encoding an extracellular domain is operably linked to a nucleic acid encoding a transmembrane domain and the nucleic acid encoding the transmembrane domain is operably linked to a nucleic acid encoding one or more intracellular moieties.
  • an effector activity of an immune cell comprising a CAR is directed against a target cell comprising an antigen that specifically binds an antigen binding domain of the CAR.
  • a targeted effector activity directed against a target cell is or comprises phagocytosis, targeted cellular cytotoxicity, antigen presentation, or cytokine secretion.
  • a CAR described herein comprises at least one domain (e.g., an extracellular domain, a transmembrane domain, and/or an intracellular domain) that inhibits anti -phagocytic signaling in an immune cell described herein (e.g., a stem cell, macrophage, monocyte, or dendritic cell).
  • an immune cell described herein e.g., a stem cell, macrophage, monocyte, or dendritic cell.
  • a CAR described herein improves effector activity of an immune cell described herein (e.g., a stem cell, macrophage, monocyte, or dendritic cell), e.g., by enhancing inhibition of CD47 and/or SIRPa activity.
  • a CAR described herein binds CD47, e.g., and serves as a dominant negative receptor, inhibiting SIRPa activity (e.g., a CD47 sink).
  • a CAR described herein that binds SIRPa e.g., comprises an activating receptor (e.g., comprises a CD3z intracellular domain).
  • a CAR described herein inhibits at least one interaction of CD47 and SIRPa.
  • a CAR is or comprises a phagocytic logic gate.
  • an immune cell described herein comprises or expresses at least one variant or fragment of: SIRPa (e.g., a dominant negative SIRPa or a high-affinity engineered variant of SIRPa (e g., CV1)), 5F9 scFv, B6H12 scFv (e.g., a humanized B6H12 scFv), PD1 (e.g., a dominant negative PD1 or HAC-I), anti-PDl scFv (e.g., E27 or durvalumab), Siglec-10, Siglec-9, Siglec-11, and/or SHP-1.
  • SIRPa e.g., a dominant negative SIRPa or a high-affinity engineered variant of SIRPa (e g., CV1)
  • 5F9 scFv e.g., B6H12 scFv (e.g., a humanized B6H12 scFv)
  • PD1
  • a variant or fragment comprises a mutated intracellular domain.
  • an immune cell described herein e.g., comprising or expressing a CAR as described herein
  • comprises a dominant negative receptor e.g., blocking an inhibitory checkpoint.
  • a CAR described herein further comprises a cleavage peptide (e.g., a P2A, F2A, E2A and/or T2A peptide) and at least one of a second CAR comprising at least one inhibitory domain of anti -phagocytic signaling.
  • at least one second CAR comprises a SIRPa (e.g., a high-affinity engineered variant of SIRPa (e g., CV1)), 5F9 scFv, B6H12 scFv (e.g., a humanized B6H12 scFv), or a CD47 binding extracellular domain or a fragment thereof.
  • At least one second CAR comprises a SIRPa transmembrane domain or a fragment thereof.
  • a second CAR further comprises a hinge domain (e.g., a CD8a hinge domain).
  • at least one second CAR comprises: (i) a leader sequence (e.g., a CD8a leader); ii) an extracellular domain (e.g., a SIRPa, CV1, 5F9 scFv, or B6H12 scFv (e.g., a humanized B6H12 scFv) extracellular domain); and ii) a transmembrane domain (e.g., a SIRPa transmembrane domain).
  • a leader sequence e.g., a CD8a leader
  • an extracellular domain e.g., a SIRPa, CV1, 5F9 scFv, or B6H12 scFv (e.g., a humanized B6H12 scFv)
  • a CAR described herein further comprises a cleavage peptide (e.g., a P2A peptide) and at least one marker protein (e.g., CD20 or a fragment thereof, CD 19 or a fragment thereof, NGFR or a fragment thereof, a synthetic peptide, and/or a fluorescent protein).
  • a cleavage peptide e.g., a P2A peptide
  • at least one marker protein e.g., CD20 or a fragment thereof, CD 19 or a fragment thereof, NGFR or a fragment thereof, a synthetic peptide, and/or a fluorescent protein.
  • an immune cell described herein comprises or expresses one or more phosphatase dead domains (e g. a phosphatase dead Shpl, phosphatase dead 72-5ptase (INPP5E), phosphatase dead Shp2, and/or phosphatase dead SHIP-1 domain) and/or a constitutively active kinase domain (e.g., a constitutively active LYN domain).
  • phosphatase dead domains e g. a phosphatase dead Shpl, phosphatase dead 72-5ptase (INPP5E), phosphatase dead Shp2, and/or phosphatase dead SHIP-1 domain
  • a constitutively active kinase domain e.g., a constitutively active LYN domain
  • a CAR described herein further comprises a cleavage peptide (e.g., a P2A, F2A, E2A and/or T2A peptide) and one or more phosphatase dead domains (e.g. a phosphatase dead Shpl, phosphatase dead 72-5ptase (INPP5E), phosphatase dead Shp2, and/or phosphatase dead SHIP-1 domain) and/or a constitutively active kinase domain (e.g., a constitutively active LYN domain).
  • a CAR of the present disclosure comprises an amino acid sequence at least 80% identical to a sequence selected from Table 2.
  • a CAR of the present disclosure comprises an amino acid sequence at least 85% identical to a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 90% identical to a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 95% identical to a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 96% identical to a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 97% identical to a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 98% identical to a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence at least 99% identical to a sequence selected from Table 2. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence identical to a sequence selected from Table 2.
  • a CAR of the present disclosure is encoded by a nucleic acid sequence at least 80% identical to a sequence selected from Table 3. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 85% identical to a sequence selected from Table 3. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 90% identical to a sequence selected from Table 3. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 95% identical to a sequence selected from Table 3. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 96% identical to a sequence selected from Table 3.
  • a CAR of the present disclosure is encoded by a nucleic acid sequence at least 97% identical to a sequence selected from Table 3. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 98% identical to a sequence selected from Table 3. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence at least 99% identical to a sequence selected from Table 3. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence identical to a sequence selected from Table 3. CAR Extracellular Domains
  • a CAR extracellular domain comprises an Fc receptor (FcR) extracellular domain.
  • a CAR extracellular domain comprises a toll-like receptor (TLR) extracellular domain.
  • a CAR extracellular domain comprises a leader domain.
  • a CAR extracellular domain comprises an antigen binding domain.
  • a CAR extracellular domain comprises a hinge domain.
  • a CAR extracellular domain comprises one or more of an FcR extracellular domain, a TLR extracellular domain, a leader domain, an antigen binding domain and a hinge domain.
  • a CAR extracellular domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a CAR extracellular domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • an extracellular domain is or comprises a DAP10 extracellular domain or a portion thereof.
  • a DAP 10 extracellular domain (or portion thereof) is or comprises a human DAP 10 extracellular domain.
  • a DAP10 extracellular domain (or portion thereof) is or comprises a DAP10 extracellular hinge domain.
  • a DAP 10 extracellular domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 85.
  • a DAP10 extracellular domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 85.
  • a DAP10 extracellular domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 85.
  • a DAP10 extracellular domain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 85. In some embodiments, a DAP10 extracellular domain comprises an amino acid sequence at least 96% identical to SEQ ID NO: 85. In some embodiments, a DAP10 extracellular domain comprises an amino acid sequence at least 97% identical to SEQ ID NO: 85. In some embodiments, a DAP10 extracellular domain comprises an amino acid sequence at least 98% identical to SEQ ID NO: 85. In some embodiments, a DAP10 extracellular domain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 85. In some embodiments, a DAP10 extracellular domain comprises an amino acid sequence identical to SEQ ID NO: 85.
  • a DAP 10 extracellular domain is encoded by a nucleic acid sequence at least 80% identical to SEQ ID NO: 91. In some embodiments, a DAP10 extracellular domain is encoded by a nucleic acid sequence at least 85% identical to SEQ ID NO: 91. In some embodiments, a DAP 10 extracellular domain is encoded by a nucleic acid sequence at least 90% identical to SEQ ID NO: 91. In some embodiments, a DAP10 extracellular domain is encoded by a nucleic acid sequence at least 95% identical to SEQ ID NO: 91.
  • a DAP 10 extracellular domain is encoded by a nucleic acid sequence at least 96% identical to SEQ ID NO: 91 . In some embodiments, a DAP10 extracellular domain is encoded by a nucleic acid sequence at least 97% identical to SEQ ID NO: 91. In some embodiments, a DAP 10 extracellular domain is encoded by a nucleic acid sequence at least 98% identical to SEQ ID NO: 91. In some embodiments, a DAP10 extracellular domain is encoded by a nucleic acid sequence at least 99% identical to SEQ ID NO: 91. In some embodiments, a DAP10 extracellular domain is encoded by a nucleic acid sequence identical to SEQ ID NO: 91.
  • an extracellular domain is or comprises a DAP12 extracellular domain or a portion thereof.
  • a DAP 12 extracellular domain (or portion thereof) is or comprises a human DAP 12 extracellular domain.
  • a DAP12 extracellular domain (or portion thereof) is or comprises a DAP12 extracellular hinge domain.
  • a DAP12 extracellular domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 88.
  • a DAP12 extracellular domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 88.
  • a DAP12 extracellular domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 88.
  • a DAP12 extracellular domain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 88. In some embodiments, a DAP12 extracellular domain comprises an amino acid sequence at least 96% identical to SEQ ID NO: 88. In some embodiments, a DAP12 extracellular domain comprises an amino acid sequence at least 97% identical to SEQ ID NO: 88. In some embodiments, a DAP12 extracellular domain comprises an amino acid sequence at least 98% identical to SEQ ID NO: 88. In some embodiments, a DAP12 extracellular domain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 88. In some embodiments, a DAP12 extracellular domain comprises an amino acid sequence identical to SEQ ID NO: 88.
  • a DAP 12 extracellular domain is encoded by a nucleic acid sequence at least 80% identical to SEQ ID NO: 94. In some embodiments, a DAP12 extracellular domain is encoded by a nucleic acid sequence at least 85% identical to SEQ ID NO: 94. In some embodiments, a DAP12 extracellular domain is encoded by a nucleic acid sequence at least 90% identical to SEQ ID NO: 94. In some embodiments, a DAP12 extracellular domain is encoded by a nucleic acid sequence at least 95% identical to SEQ ID NO: 94.
  • a DAP12 extracellular domain is encoded by a nucleic acid sequence at least 96% identical to SEQ ID NO: 94. In some embodiments, a DAP12 extracellular domain is encoded by a nucleic acid sequence at least 97% identical to SEQ ID NO: 94. In some embodiments, a DAP 12 extracellular domain is encoded by a nucleic acid sequence at least 98% identical to SEQ ID NO: 94. In some embodiments, a DAP12 extracellular domain is encoded by a nucleic acid sequence at least 99% identical to SEQ ID NO: 94. In some embodiments, a DAP12 extracellular domain is encoded by a nucleic acid sequence identical to SEQ ID NO: 94.
  • an FcR extracellular domain comprises a full-length FcR extracellular domain. In some embodiments, an FcR extracellular domain comprises a portion of a full-length FcR extracellular domain. In some embodiments, an FcR extracellular domain (or portion thereof) is or comprises a human FcR extracellular domain. In some embodiments, an FcR extracellular domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an FcR extracellular domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • an FcR extracellular domain comprises a CD64 (FcyRI), CD32a (FcyRIIa), CD32b (FcyRIIb), CD32c, CD16a (FcyRIIIa), CD 16b (FcyRIIIb), FcsRI, FcsRII, or FcaRI (CD89) domain.
  • a TLR extracellular domain comprises a full-length TLR extracellular domain. In some embodiments, a TLR extracellular domain comprises a portion of a full-length TLR extracellular domain. In some embodiments, a TLR extracellular domain (or portion thereof) is or comprises a human TLR extracellular domain. In some embodiments, a TLR extracellular domain may be a domain that is endogenous to a particular immune cell type (e.g, a modified immune cell as provided herein). In some embodiments, a TLR extracellular domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a TLR extracellular domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.
  • a CAR comprises one or more extracellular leader domains.
  • a nucleic acid encoding a CAR comprises a nucleic acid sequence encoding an extracellular leader domain, but the extracellular leader domain is cleaved from the CAR before the CAR is expressed in an immune cell.
  • an extracellular leader domain is or comprises a human extracellular leader domain.
  • an extracellular leader domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • an extracellular leader domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • an extracellular leader domain comprises a CD8a extracellular leader domain.
  • an extracellular leader domain comprises a leader domain from a stimulatory or co-stimulatory domain (e.g, a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD19, CD20, 41BB, CD28, 0X40, GITR, TREM-1, TREM-2, DAP10, DAP12, MR, ICOS, MyD88 domain).
  • a stimulatory or co-stimulatory domain e.g, a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR,
  • a CAR comprises at least one antigen binding domain that binds to an antigen, for example, on a target cell.
  • a CAR comprises an antigen binding domain that binds to an antigen associated with viral infection, bacterial infection, parasitic infection, autoimmune disease, and/or cancer cells.
  • a CAR antigen binding domain recognizes an antigen that acts as a cell surface marker on a target cell associated with a particular disease state.
  • a CAR antigen binding domain binds to a tumor antigen, such as an antigen that is specific for a tumor or cancer of interest.
  • a tumor antigen comprises one or more antigenic cancer epitopes.
  • a tumor antigen comprises CD19; CD123; CD22; CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-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);
  • Lewis(Y) antigen CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stagespecific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2 (Her2/neu); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF -I receptor), carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100 (gplOO); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1
  • an antigen binding domain binds to a misfolded protein antigen or a protein of a protein aggregate, such as a protein that is specific for a disease/disorder of interest.
  • the disease/disorder is a neurodegenerative disease/disorder, an inflammatory disease/disorder, a cardiovascular disease/disorder, a fibrotic disease/disorder, or amyloidosis (e.g., mediated by protein aggregates of immunoglobulin light chains or of transthyretin).
  • the neurodegenerative disease/disorder is selected from the group consisting of tauopathy, asynucleopathy, presenile dementia, senile dementia, Alzheimer's disease (mediated by protein aggregates ofbeta-amyloid), Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), Pick' s disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, Parkinson's disease with dementia, dementia with Lewy bodies, Down syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS), Hallervorden-Spatz syndrome, polyglutamine disease, trinucleotide repeat disease, Familial British dementia, Fatal Familial Insomnia, Gerstmann-Straussler-Scheinker Syndrome, Hereditary cerebral hemorrhage with amyloidosis (Icelandic) (HCHW A-I), Sporadic Fatal Insomnia (
  • an antigen binding domain comprises any domain that binds to an antigen.
  • a CAR antigen binding domain is or comprises a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, or any fragment thereof, for example an scFv.
  • a CAR antigen binding domain is or comprises an aptamer, a darpin, a centyrin, a naturally occurring or synthetic receptor, an affibody, or other engineered protein recognition molecule.
  • a CAR antigen binding domain is or comprises a mammalian antibody or a fragment thereof.
  • a CAR antigen binding domain is derived, in whole or in part, from the same species in which the CAR will ultimately be used.
  • an antigen binding domain of a CAR comprises a human antibody, a humanized antibody, or a fragment thereof (e.g. a scFv).
  • a CAR antigen binding domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • a CAR antigen binding domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • a CAR comprises one or more antigen binding domains. In some embodiments, a CAR comprises two or more antigen binding domains. In some embodiments, a CAR is a bispecific CAR. In some embodiments, an immune cell comprises two or more different CARs comprising one or more antigen binding domains. In some embodiments, an immune cell comprising a bispecific CAR and/or comprising two or more different CARs comprising one or more antigen binding domains can reduce off-target and/or on-target off-tissue effects by requiring that two antigens are present.
  • an immune cell comprises a bispecific CAR and/or comprises two or more different CARs comprising one or more antigen binding domains, wherein the CARs provide distinct signals that in isolation are insufficient to mediate activation of the modified cell, but are synergistic together, stimulating activation of the modified cell.
  • a construct may be referred to as an ‘AND’ logic gate.
  • an immune cell comprising a bispecific CAR and/or comprising two or more different CARs comprising one or more antigen binding domains can reduce off-target and/or on -target off-tissue effects by requiring that one antigen is present and a second, normal protein antigen is absent before the cell’s activity is stimulated.
  • a construct may be referred to as a ‘NOT’ logic gate.
  • NOT gated CAR-modified cells are activated by binding to a single antigen.
  • the binding of a second receptor to the second antigen functions to override the activating signal being perpetuated through the CAR.
  • such an inhibitory receptor would be targeted against an antigen that is abundantly expressed in a normal tissue but is absent in tumor tissue.
  • a CAR comprises one or more extracellular hinge domains.
  • a CAR extracellular hinge domain is or comprises a human extracellular hinge domain.
  • a CAR extracellular hinge domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • a CAR extracellular hinge domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • one or more CAR extracellular hinge domains comprise a DAP 10 extracellular hinge domain, a DAP 12 extracellular hinge domain, a CD8a extracellular hinge domain, a CD28 extracellular hinge domain, a DNGR-1 extracellular hinge domain, a Dectin- 1 extracellular hinge domain, or an IgG4 extracellular hinge domain.
  • one or more CAR extracellular hinge domains comprise a DAP 10 extracellular hinge domain.
  • one or more CAR extracellular hinge domains comprise a DAP 12 extracellular hinge domain.
  • a CAR extracellular hinge domain optimizes the physicochemical parameters of a CAR, e.g., optimal size relative to tumor antigen (e.g., allowing for exclusion of inhibitory molecules), optimal flexibility, optimal protein folding, optimal protein stability, optimal binding, optimal homodimerization, and/or lack of homodimerization.
  • a CAR comprises a transmembrane domain, for example, that connects an extracellular domain to one or more intracellular moieties.
  • a CAR transmembrane domain is naturally associated with one or more other domain(s) of a CAR.
  • a CAR transmembrane domain can be modified to avoid binding to transmembrane domains of other surface membrane proteins, in order to minimize interactions with other members of a receptor complex.
  • a CAR transmembrane domain may be derived either from a naturally-occurring or from a synthetic source.
  • a CAR transmembrane domain is derived from a naturally- occurring membrane-bound or transmembrane protein.
  • a CAR transmembrane domain is or comprises a human transmembrane domain.
  • a CAR transmembrane domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • a CAR transmembrane domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • a CAR transmembrane domain comprises a CD8a, CD64, CD32a, CD32c, CD16a, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, R0R1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD 19, CD20, 41BB, CD28, 0X40, GITR, TREM-1, TREM-2, DAP10, DAP12, MR, ICOS, MyD88, CD3- zeta, FcRy, V/I/LxYxxL/V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b
  • a transmembrane domain is or comprises a DAP 10 transmembrane or a portion thereof.
  • a DAP 10 transmembrane domain (or portion thereof) is or comprises a human DAP 10 transmembrane domain.
  • a DAP 10 transmembrane domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 86.
  • a DAP 10 transmembrane domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 86.
  • a DAP 10 transmembrane domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 86.
  • a DAP10 transmembrane domain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 86. In some embodiments, a DAP10 transmembrane domain comprises an amino acid sequence at least 96% identical to SEQ ID NO: 86. In some embodiments, a DAP 10 transmembrane domain comprises an amino acid sequence at least 97% identical to SEQ ID NO: 86. In some embodiments, a DAP 10 transmembrane domain comprises an amino acid sequence at least 98% identical to SEQ ID NO: 86. In some embodiments, a DAP 10 transmembrane domain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 86. In some embodiments, a DAP 10 transmembrane domain comprises an amino acid sequence identical to SEQ ID NO: 86.
  • a DAP10 transmembrane domain is encoded by a nucleic acid sequence at least 80% identical to SEQ ID NO: 92. In some embodiments, a DAP10 transmembrane domain is encoded by a nucleic acid sequence at least 85% identical to SEQ ID NO: 92. In some embodiments, a DAP10 transmembrane domain is encoded by a nucleic acid sequence at least 90% identical to SEQ ID NO: 92. In some embodiments, a DAP10 transmembrane domain is encoded by a nucleic acid sequence at least 95% identical to SEQ ID NO: 92.
  • a DAP10 transmembrane domain is encoded by a nucleic acid sequence at least 96% identical to SEQ ID NO: 92. In some embodiments, a DAP10 transmembrane domain is encoded by a nucleic acid sequence at least 97% identical to SEQ ID NO: 92. In some embodiments, a DAP10 transmembrane domain is encoded by a nucleic acid sequence at least 98% identical to SEQ ID NO: 92. In some embodiments, a DAP10 transmembrane domain is encoded by a nucleic acid sequence at least 99% identical to SEQ ID NO: 92. In some embodiments, a DAP10 transmembrane domain is encoded by a nucleic acid sequence identical to SEQ ID NO: 92.
  • a transmembrane domain is or comprises a DAP12 transmembrane domain or a portion thereof.
  • a DAP 12 transmembrane domain (or portion thereof) is or comprises a human DAP12 transmembrane domain.
  • a DAP12 transmembrane domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 89.
  • a DAP 12 transmembrane domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 89.
  • a DAP 12 transmembrane domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 89.
  • a DAP12 transmembrane domain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 89. In some embodiments, a DAP12 transmembrane domain comprises an amino acid sequence at least 96% identical to SEQ ID NO: 89. In some embodiments, a DAP12 transmembrane domain comprises an amino acid sequence at least 97% identical to SEQ ID NO: 89. In some embodiments, a DAP12 transmembrane domain comprises an amino acid sequence at least 98% identical to SEQ ID NO: 89. In some embodiments, a DAP12 transmembrane domain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 89. In some embodiments, a DAP12 transmembrane domain comprises an amino acid sequence identical to SEQ ID NO: 89.
  • a DAP12 transmembrane domain is encoded by a nucleic acid sequence at least 80% identical to SEQ ID NO: 95. In some embodiments, a DAP 12 transmembrane domain is encoded by a nucleic acid sequence at least 85% identical to SEQ ID NO: 95. In some embodiments, a DAP 12 transmembrane domain is encoded by a nucleic acid sequence at least 90% identical to SEQ ID NO: 95. In some embodiments, a DAP 12 transmembrane domain is encoded by a nucleic acid sequence at least 95% identical to SEQ ID NO: 95.
  • a DAP12 transmembrane domain is encoded by a nucleic acid sequence at least 96% identical to SEQ ID NO: 95. In some embodiments, a DAP 12 transmembrane domain is encoded by a nucleic acid sequence at least 97% identical to SEQ ID NO: 95. In some embodiments, a DAP12 transmembrane domain is encoded by a nucleic acid sequence at least 98% identical to SEQ ID NO: 95. In some embodiments, a DAP 12 transmembrane domain is encoded by a nucleic acid sequence at least 99% identical to SEQ ID NO: 95. In some embodiments, a DAP12 transmembrane domain is encoded by a nucleic acid sequence identical to SEQ ID NO: 95.
  • an FcR transmembrane domain comprises a full-length FcR transmembrane domain. In some embodiments, an FcR transmembrane domain comprises a portion of a full-length FcR transmembrane domain. In some embodiments, an FcR transmembrane domain is or comprises a human FcR transmembrane domain, or portion thereof. In some embodiments, an FcR transmembrane domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • an FcR transmembrane domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • an FcR transmembrane domain comprises a CD64 (FcyRI), CD32a (FcyRIIa), CD32b (FcyRIIb), CD32c, CD16a (FcyRIIIa), CD16b (FcyRIIIb), FCERI, FCERII, or FcaRI (CD89) domain.
  • a TLR transmembrane domain comprises a full-length TLR transmembrane domain. In some embodiments, a TLR transmembrane domain comprises a portion of a full-length TLR transmembrane domain. In some embodiments, a TLR transmembrane domain is or comprises a human TLR transmembrane domain, or portion thereof. In some embodiments, a TLR transmembrane domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • a TLR transmembrane domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • a TLR transmembrane domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.
  • a CAR comprises one or more intracellular domains.
  • an intracellular domain is or comprises a human intracellular domain, or portion thereof.
  • an intracellular domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • an intracellular domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • an intracellular domain and/or other cytoplasmic domain of a CAR is responsible for activation of the cell in which the CAR is expressed (e.g., an immune cell).
  • an intracellular domain of a CAR is responsible for signal activation and/or transduction in an immune cell comprising said CAR.
  • an intracellular domain of a CAR includes at least one domain responsible for signal activation and/or transduction. In some embodiments, an intracellular domain is or comprises at least one of a co-stimulatory molecule and a signaling domain. In some embodiments, an intracellular domain of a CAR comprises dual signaling domains. In some embodiments, an intracellular domain of a CAR comprises more than two signaling domains.
  • an intracellular domain comprises a cytoplasmic portion of a surface receptor. In some embodiments, an intracellular domain comprises a co-stimulatory molecule. In some embodiments, an intracellular domain comprises a molecule that acts to initiate signal transduction in an immune cell.
  • an intracellular domain of a CAR includes any portion of one or more co-stimulatory molecules, such as at least one signaling domain from CD3, Fc epsilon RI gamma chain, any derivative or variant thereof, any synthetic sequence thereof that has the same functional capability, and any combination thereof.
  • DAP 10 Intracellular Domains include CD3, Fc epsilon RI gamma chain, any derivative or variant thereof, any synthetic sequence thereof that has the same functional capability, and any combination thereof.
  • an intracellular domain is or comprises a DAP 10 intracellular domain or a portion thereof.
  • a DAP 10 intracellular domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 87.
  • a DAP10 intracellular domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 87.
  • a DAP10 intracellular domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 87.
  • a DAP10 intracellular domain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 87.
  • a DAP10 intracellular domain comprises an amino acid sequence at least 96% identical to SEQ ID NO: 87.
  • a DAP10 intracellular domain comprises an amino acid sequence at least 97% identical to SEQ ID NO: 87. In some embodiments, a DAP10 intracellular domain comprises an amino acid sequence at least 98% identical to SEQ ID NO: 87. In some embodiments, a DAP10 intracellular domain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 87. In some embodiments, a DAP10 intracellular domain comprises an amino acid sequence identical to SEQ ID NO: 87.
  • a DAP 10 intracellular domain is encoded by a nucleic acid sequence at least 80% identical to SEQ ID NO: 93. In some embodiments, a DAP 10 intracellular domain is encoded by a nucleic acid sequence at least 85% identical to SEQ ID NO: 93. In some embodiments, a DAP 10 intracellular domain is encoded by a nucleic acid sequence at least 90% identical to SEQ ID NO: 93. In some embodiments, a DAP 10 intracellular domain is encoded by a nucleic acid sequence at least 95% identical to SEQ ID NO: 93.
  • a DAP 10 intracellular domain is encoded by a nucleic acid sequence at least 96% identical to SEQ ID NO: 93. In some embodiments, a DAP 10 intracellular domain is encoded by a nucleic acid sequence at least 97% identical to SEQ ID NO: 93. In some embodiments, a DAP 10 intracellular domain is encoded by a nucleic acid sequence at least 98% identical to SEQ ID NO: 93. In some embodiments, a DAP10 intracellular domain is encoded by a nucleic acid sequence at least 99% identical to SEQ ID NO: 93. In some embodiments, a DAP10 intracellular domain is encoded by a nucleic acid sequence identical to SEQ ID NO: 93.
  • an intracellular domain is or comprises a DAP12 intracellular domain or a portion thereof.
  • a DAP 12 intracellular domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 90.
  • a DAP12 intracellular domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 90.
  • a DAP12 intracellular domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 90.
  • a DAP12 intracellular domain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 90.
  • a DAP12 intracellular domain comprises an amino acid sequence at least 96% identical to SEQ ID NO: 90.
  • a DAP12 intracellular domain comprises an amino acid sequence at least 97% identical to SEQ ID NO: 90. In some embodiments, a DAP12 intracellular domain comprises an amino acid sequence at least 98% identical to SEQ ID NO: 90. In some embodiments, a DAP12 intracellular domain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 90. In some embodiments, a DAP12 intracellular domain comprises an amino acid sequence identical to SEQ ID NO: 90.
  • a DAP 12 intracellular domain is encoded by a nucleic acid sequence at least 80% identical to SEQ ID NO: 96. In some embodiments, a DAP12 intracellular domain is encoded by a nucleic acid sequence at least 85% identical to SEQ ID NO: 96. In some embodiments, a DAP 12 intracellular domain is encoded by a nucleic acid sequence at least 90% identical to SEQ ID NO: 96. In some embodiments, a DAP12 intracellular domain is encoded by a nucleic acid sequence at least 95% identical to SEQ ID NO: 96.
  • a DAP 12 intracellular domain is encoded by a nucleic acid sequence at least 96% identical to SEQ ID NO: 96. In some embodiments, a DAP12 intracellular domain is encoded by a nucleic acid sequence at least 97% identical to SEQ ID NO: 96. In some embodiments, a DAP 12 intracellular domain is encoded by a nucleic acid sequence at least 98% identical to SEQ ID NO: 96. In some embodiments, a DAP12 intracellular domain is encoded by a nucleic acid sequence at least 99% identical to SEQ ID NO: 96. In some embodiments, a DAP12 intracellular domain comprises a nucleic acid sequence identical to SEQ ID NO: 96.
  • an FcR intracellular domain comprises a full-length FcR intracellular domain. In some embodiments, an FcR intracellular domain comprises a portion of a full-length FcR intracellular domain. In some embodiments, an FcR intracellular domain is or comprises a human FcR intracellular domain, or portion thereof. In some embodiments, an FcR intracellular domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an FcR intracellular domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • an FcR intracellular domain comprises a CD64 (FcyRI), CD32a (FcyRIIa), CD32b (FcyRIIb), CD32c, CD16a (FcyRIIIa), CD16b (FcyRIIIb), FceRI, FceRII, or FcaRI (CD89) domain.
  • a TLR intracellular domain comprises a full-length TLR intracellular domain. In some embodiments, a TLR intracellular domain comprises a portion of a full-length TLR intracellular domain. In some embodiments, a TLR intracellular domain is or comprises a human TLR intracellular domain, or portion thereof. In some embodiments, a TLR intracellular domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a TLR intracellular domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a TLR intracellular domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.
  • a CAR comprises one or more intracellular signaling domains.
  • an intracellular signaling domain is or comprises a human intracellular signaling domain, or portion thereof.
  • a signaling domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • a signaling domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • one or more intracellular signaling domains may be or comprise a CD3-zeta, FcR y, CD64, CD32a, CD32c, CD16a, TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD 19, CD20, 4 IBB, CD28, 0X40, GITR, TREM-1, TREM-2, DAP 10, DAP 12, MR, ICOS, MyD88, V/I/LxYxxL/V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD
  • an intracellular domain of a CAR may be or comprise dual signaling domains, such as 41BB, CD28, ICOS, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, CD 116 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, CD1 lb, together with any of the signaling domains listed in the above paragraph in any combination.
  • dual signaling domains such as 41BB, CD28, ICOS, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, CD 116 receptor beta chain, CSF1-R, LRP1/
  • a “co-stimulatory molecule” or “co-stimulatory domain” refers to a molecule in an immune cell that is used to heighten or dampen an initial stimulus.
  • pathogen-associated pattern recognition receptors such as TLR or the CD47/SIRPa axis, are molecules on immune cells that, respectively, heighten or dampen an initial stimulus.
  • a CAR co-stimulatory domain comprises TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma RIIa, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 127, CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamm
  • a co-stimulatory domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a co-stimulatory domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).
  • a “co-stimulatory signal” refers to a signal, which in combination with a primary signal, such as activation of a CAR on an immune cell, leads to activation of the immune cell.
  • a cleavage peptide refers to a peptide that can induce the cleaving of a recombinant protein in a cell.
  • a cleavage peptide is a 2A peptide.
  • a cleavage peptide is or comprises a P2A, F2A, E2A or T2A peptide.
  • a nucleic acid as described herein comprises one or more nucleic acid sequences encoding one or more cleavage peptides.
  • a nucleic acid comprising a nucleic acid sequence encoding a cleavage peptide also comprises one or more nucleic acid sequences encoding one or more intracellular domains and one or more nucleic acid sequences comprising one or more peptide agents, wherein translation of the nucleic acid results in a protein comprising one or more intracellular domains separated from one or more peptide agents by a cleavage peptide.
  • a first promoter is operably linked to one or more nucleic acids encoding a CAR and a second promoter is operably linked to one or more nucleic acids encoding a peptide agent.
  • a nucleic acid sequence comprising a CAR, and optionally one or more peptide agents further comprises an internal ribosome entry site (IRES) sequence.
  • IRES sequence may be any viral, chromosomal or artificially designed sequence that initiates cap-independent ribosome binding to mRNA facilitates the initiation of translation.
  • a CAR peptide agent refers to a peptide co-expressed with a CAR in an immune cell.
  • a CAR peptide agent is co-expressed with a CAR to ensure stoichiometric balance and optimal signaling of a CAR.
  • a CAR peptide agent forms a homodimer with an identical peptide agent.
  • a CAR peptide agent forms a heterodimer with a different peptide agent.
  • a nucleic acid as described herein comprises one or more nucleic acid sequences encoding one or more CAR peptide agents.
  • a CAR peptide agent is or comprises an FcR gamma chain.
  • a CAR peptide agent comprises any peptide, protein, receptor, secreted antibody or a fragment thereof (e.g., an scFv, Fab, Fab', F(ab')2, Fc, or nanobody).
  • a CAR peptide agent comprises one or more cytokines (e g., one or more of IL-1, IL-2, IL-6, IL-8, TNF-a, IFN-a, IFN-p, IFN-y, GMCSF, or MCSF), CD40- L, dominant negative SIRPa, dominant negative PD1, dominant negative CD45, dominant negative SIGLEC 10, or dominant negative LILRB.
  • FcR Fc Receptors
  • a CAR comprises one or more antigen binding domains and an FcR extracellular domain, and/or the transmembrane domain of the CAR comprises an FcR transmembrane domain, and/or the intracellular domain of the CAR comprises an FcR intracellular domain.
  • a CAR comprises, from N-terminus to C-terminus, one or more extracellular domains, an FcR extracellular domain, an FcR transmembrane domain, and an FcR intracellular domain.
  • one or more of the FcR extracellular domain, the FcR transmembrane domain and the FcR intracellular domain is or comprises a human FcR domain.
  • an FcR extracellular domain, an FcR transmembrane domain and an FcR intracellular domain together comprise a full-length FcR. In some embodiments, an FcR extracellular domain, an FcR transmembrane domain and an FcR intracellular domain together comprise a portion of a full-length FcR. In some embodiments, an FcR extracellular domain comprises a portion of a full-length FcR extracellular domain. In some embodiments, an FcR transmembrane domain comprises a portion of a full-length FcR transmembrane domain. In some embodiments, an FcR intracellular domain comprises a portion of a full-length FcR intracellular domain.
  • TLR Toll-Like Antigen Receptors
  • a CAR comprises one or more antigen binding domains and a toll-like receptor (TLR) extracellular domain and/or the transmembrane domain of the CAR comprises a TLR transmembrane domain and/or the intracellular domain of the CAR comprises a TLR intracellular domain.
  • a CAR comprises, from N- terminus to C-terminus, one or more extracellular domains, a TLR extracellular domain, a TLR transmembrane domain, and a TLR intracellular domain.
  • one or more of the TLR extracellular domain, the TLR transmembrane domain and the TLR intracellular domain is or comprises a human TLR domain.
  • a TLR extracellular domain, a TLR transmembrane domain and a TLR intracellular domain together comprise a full- length TLR. In some embodiments, a TLR extracellular domain, a TLR transmembrane domain and a TLR intracellular domain together comprise portion of a full-length TLR. In some embodiments, a TLR extracellular domain comprises a portion of a full-length TLR extracellular domain. In some embodiments, a TLR transmembrane domain comprises a portion of a full- length TLR transmembrane domain. In some embodiments, a TLR intracellular domain comprises a portion of a full-length TLR intracellular domain.
  • modified immune cells e.g., stem cells, macrophages, monocytes, or dendritic cells
  • CAR chimeric antigen receptor
  • a population of immune cells as described herein comprises stem cells, monocytes, macrophages, dendritic cells, and/or precursors thereof.
  • a population of immune cells comprises a substantially purified population of stem cells, monocytes, macrophages, or dendritic cells, or a cell line.
  • an immune cell is activated, e.g., an immune cell exhibits increased cytokine production, chemokine production, phagocytosis, cell signaling, target cell killing, and/or antigen presentation in response to a particular stimuli, e.g., relative to an inactive cell.
  • an activated immune cell exhibits changes in gene expression, e.g., an induction of pro-inflammatory gene expression, e.g., relative to an inactive cell.
  • an activated immune cell exhibits changes in gene expression, e.g., an induction of anti-inflammatory gene expression, e.g., relative to an inactive cell.
  • activated immune cells are undergoing cell division.
  • targeted effector activity of an immune cell is enhanced by inhibition of CD47 and/or SIRPa activity.
  • CD47 and/or SIRPa activity may be inhibited by treating an immune cell with an anti-CD47 or anti- SIRPa antibody or by any method known to those skilled in the art.
  • immune cells e.g., stem cells, macrophages, monocytes, or dendritic cells
  • Immune cells may be autologous or sourced from allogeneic or universal donors.
  • Cells can be obtained from a number of sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, tumors, and/or induced pluripotent stem cells, such as embryonic stem cells (ESCs).
  • ESCs embryonic stem cells
  • cells can be obtained from a unit of blood collected from a subject using any number of separation techniques known to a skilled artisan, such as Ficoll separation.
  • cells from circulating blood of a subject are obtained by apheresis or leukapheresis.
  • Cells collected by apheresis may be washed to remove a plasma fraction and resuspended in a variety of buffers (e.g., phosphate buffered saline (PBS)) or culture media).
  • buffers e.g., phosphate buffered saline (PBS)
  • enrichment of immune cells comprises plastic adherence.
  • differentiation of immune cells comprises stimulation with GM-CSF.
  • a composition comprising blood cells (e.g., monocytes, lymphocytes, platelets, plasma, and/or red blood cells), such as a leukapheresis composition (e.g., a leukopak) is used for enrichment.
  • a leukapheresis composition e.g., a leukopak
  • a leukapheresis composition comprises a sample from a healthy human donor.
  • apheresis of immune cells e.g. monocytes
  • GM-CSF GM-CSF
  • selection of immune cells comprises CD14 positive selection using microbeads (e.g., MACS® MicroBeads on a CliniMACS Prodigy device).
  • microbeads e.g., MACS® MicroBeads on a CliniMACS Prodigy device.
  • an immune cell precursor e.g., precursors to macrophages, monocytes, or dendritic cells including, but not limited to induced pluripotent stem cells, or iPSCs
  • Immune cell precursors may be differenti ted in vivo or ex vivo into immune cells.
  • Non-limiting examples of precursor immune cells include hematopoietic stem cells, common myeloid progenitors, myeloblasts, monoblasts, promonocytes, or intermediates thereof.
  • induced pluripotent stem cells may be used to generate monocytes, macrophages, and/or dendritic cells.
  • Induced pluripotent stem cells may be derived from normal human tissue, such as peripheral blood, fibroblasts, skin, keratinocytes, or renal epithelial cells. Autologous, allogeneic, or universal donor iPSCs could be differentiated toward a myeloid lineage (e.g., a monocyte, macrophage, dendritic cell, or precursor thereof).
  • Immune cells e g., stem cells, macrophages, monocytes, or dendritic cells
  • peripheral blood for example, by lysing red blood cells and depleting lymphocytes and red blood cells, such as by centrifugation through a PERCOLLTM gradient.
  • immune cells can be isolated from umbilical cord tissue.
  • a specific subpopulation of immune cells can be further isolated by positive or negative selection techniques.
  • immune cells can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD3, CD4, CD8a, CD56, CD66b, CD19, or CD20.
  • enrichment of an immune cell population for example, by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • cell selection can also comprise negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on negatively selected cells.
  • immune cell concentration and surface e.g., particles, such as beads
  • immune cell concentration and surface can be varied. It may be desirable to significantly decrease volume in which beads and cells are mixed together to ensure maximum contact area of cells and beads.
  • immune cells e.g., stem cells, macrophages, monocytes, or dendritic cells
  • a pro-inflammatory agent e.g., M2
  • a pro-inflammatory agent comprises or is a pro- inflammatory cytokine.
  • a pro-inflammatory agent comprises or is IFN-a, IFN-P, IFN-X, IFN-y, TNF-a, IL-6, IL-lb, IL-2, IL-8, IL-12, IL-15, IL-18, IL-17, IL-la, IL-3, IL-21, IL-33, IL-23, IL-37, or IL-36.
  • a pro-inflammatory agent comprises or is an IFN-y agonist (e.g., IFN-y).
  • immune cells e.g., stem cells, macrophages, monocytes, or dendritic cells
  • an anti-inflammatory agent e.g., treatment with an anti-inflammatory agent promotes a pro-inflammatory (i.e., Ml) phenotype in immune cells as described herein.
  • an anti-inflammatory agent comprises or is an anti-inflammatory cytokine.
  • an anti-inflammatory agent comprises or is IL- 10, TGF- , IL-4, IL- 13, IL-11, or IL-35.
  • an anti-inflammatory agent comprises or is an IL-10 agonist (e.g., IL-10).
  • immune cells e.g., stem cells, macrophages, monocytes, or dendritic cells
  • a pro-inflammatory agent e.g., stem cells, macrophages, monocytes, or dendritic cells
  • a pro-inflammatory agent comprises or is an IFN-y agonist (e.g., IFN-y).
  • immune cells e.g., stem cells, macrophages, monocytes, or dendritic cells
  • a subject in combination with an anti-inflammatory agent.
  • immune cells e.g., stem cells, macrophages, monocytes, or dendritic cells
  • administration with an anti-inflammatory agent increases anti-tumor activity of immune cells as described herein.
  • an anti-inflammatory agent promotes a pro-inflammatory (i.e., Ml) phenotype in immune cells as described herein.
  • an anti-inflammatory agent comprises or is an IL-10 agonist (e.g., IL-10).
  • immune cells are modified in a subject (e.g., via in vivo delivery mechanisms).
  • the present disclosure provides modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in a subject, wherein the subject is administered a composition as described herein comprising: (a) one or more nucleic acid molecules, wherein at least a portion of the one or more nucleic acid molecules encodes a CAR and/or CAR peptide agent, and (b) a delivery vehicle.
  • the one or more nucleic acid molecules are translated in an immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) to produce a modified immune cell comprising the CAR and/or CAR peptide agent.
  • an immune cell e.g., stem cell, macrophage, monocyte, or dendritic cell
  • the modified immune cell comprising the CAR and/or CAR peptide agent possesses targeted effector activity.
  • Macrophages are immune cells specialized for detection, phagocytosis, and destruction of target cells, such as pathogens or tumor cells. Macrophages are potent effectors of the innate immune system and are capable of at least three distinct anti-tumor functions: 1) phagocytosis of dead and dying cells, microorganisms, cancer cells, cellular debris, or other foreign substances; 2) cytotoxicity against tumor cells; and 3) presentation of tumor antigens to orchestrate an adaptive anti-tumor immune response.
  • TAMs tumor-associated macrophages
  • a macrophage comprises or is an undifferentiated or MO macrophage.
  • a macrophage comprises or expresses one, two, three, four, five, or six of CD 14, CD 16, CD64, CD68, CD71, or CCR5. Exposure to various stimuli can induce MO macrophages to polarize into several distinct populations, which may be identified by macrophage phenotype markers, cytokine production, and/or chemokine secretion.
  • a macrophage comprises or is a polarized macrophage.
  • MO macrophages can be exposed to pro-inflammatory signals, such as LPS, IFN-y, and GM-CSF, and polarize into pro-inflammatory (i.e., Ml) macrophages.
  • pro-inflammatory (Ml) macrophages are associated with pro- inflammatory immune responses, such as Thl and Thl 7 T cell responses. Exposure to other stimuli can polarize macrophages into a diverse group of “alternatively activated” or antiinflammatory (i.e., M2) macrophages.
  • a macrophage comprises or is a pro-inflammatory (Ml) macrophage.
  • a macrophage expresses one or more markers of pro- inflammatory (Ml) macrophages (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of CD86, CD80, MHC II, IL-1R, TLR2, TLR4, iNOS, SOCS3, CD83, PD-L1, CD69, MHC I, CD64, CD32, CD16, IL1R, a IFIT family member, or an ISG family member).
  • Ml pro-inflammatory
  • a macrophage comprising or expressing a CAR as described herein secretes relatively high levels of one or more inflammatory cytokines (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of IL-1, TNF, IL-12, IL-18, IL-23, IFN-a, IFN-0, IFN-y, IL-2, IL- 6, IL-8, or IL33) or chemokines (e.g., one or both of CC or CXC chemokines) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the CXC chemokines; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 of the CC chemokines; e.g., one of the CX3C chemokines, e.g., one or both of the C chemokines), e
  • a macrophage comprises or is an anti-inflammatory (M2) macrophage (e.g., an M2a, M2b, M2c, and M2d macrophage).
  • M2a macrophage can be induced by IL-4, IL-13, and/or fungal infection.
  • An M2b macrophage can be induced by IL-1R ligands, an immune complex, and/or LPS.
  • An M2c macrophage can be induced by IL-10 and/or TGFp.
  • An M2d macrophage can be induced by IL-6 and/or adenosine.
  • a macrophage comprising or expressing a CAR as described herein decreases an immune response in a subject, e.g., relative to a macrophage without a CAR as described herein.
  • a macrophage expresses one or more markers of anti-inflammatory (M2) macrophages (e.g., one, two, or three of CD206, CD163, or CD209).
  • M2 markers of anti-inflammatory
  • a macrophage comprising or expressing a CAR as described herein exhibits increased secretion of one or more anti-inflammatory cytokines (e.g., one or both of IL- 10 or TGFP), e g., relative to a macrophage without a CAR as described herein.
  • a macrophage comprises at least one upregulated pro- inflammatory (Ml) marker and/or at least one downregulated anti-inflammatory (M2) marker as compared to a control macrophage that does not comprise a CAR as described herein and/or the same macrophage before delivery of a CAR as described herein.
  • at least one pro-inflammatory (Ml) marker e.g., HLA DR, CD86, CD80, PD-L1, CD83, CD69, MHC I, CD64, CD32, CD 16, IL1R, an IFIT family member, and/or an ISG family member
  • M2 marker e.g., CD206, CD163, and/or CD209 is downregulated in a macrophage.
  • a macrophage comprising or expressing a CAR as described herein exhibits increased phagocytosis, e.g., relative to a macrophage without a CAR as described herein. In some embodiments, a macrophage comprising or expressing a CAR as described herein exhibits increased cytotoxicity against a tumor cell, e.g., relative to a macrophage without a CAR as described herein.
  • a macrophage comprising or expressing a CAR as described herein exhibits increased tumor antigen presentation (e.g., post-phagocytosis presentation) and/or increased antigen processing, e.g., relative to a macrophage without a CAR as described herein.
  • a macrophage comprising or expressing at a CAR as described herein exhibits increased tumor killing (e.g., by phagocytosis, lysis, apoptosis, or production of tumor killing cytokines (e.g., TNFa)), e.g., relative to a macrophage without a CAR as described herein.
  • a macrophage comprising or expressing a CAR as described herein exhibits one or both of increased expression of one or more genes typically associated with increased effector function (e.g., phagocytosis, targeted cellular cytotoxicity, antigen presentation, or cytokine secretion) (e.g., CD80, CD86, MHC-I, MHC-II, CD40, 41BBL, TNF, IFN-a, IFN- , IFN-y, IL2, IL12, IL6, IL8, ILlb, and/or CXCL12) or decreased expression of one or more genes typically associated with decreased effector function (e.g., phagocytosis, targeted cellular cytotoxicity, antigen presentation, or cytokine secretion) (e.g., CD 163, CD206, TGF0, IL-10, and/or IL4), e.g., relative to a macrophage without a CAR as described herein.
  • genes typically associated with increased effector function
  • a macrophage comprising or expressing a CAR as described herein exhibits increased production of ROS, e.g., relative to a macrophage without a CAR as described herein.
  • a macrophage comprising or expressing a CAR as described herein exhibits metabolic reprogramming (e.g., of an interferon signaling pathway, TH1 pathway, PTEN signaling, PI3K signaling, MTOR signaling, TLR signaling, CD40 signaling, 4 IBB signaling, 41BBL signaling, macrophage maturation signaling, dendritic cell maturation signaling, CD3-zeta signaling, FcR y signaling, CD64 signaling, CD32a signaling, CD32c signaling, CD 16a signaling, TLR1 signaling, TLR2 signaling, TLR3 signaling, TLR4 signaling, TLR5 signaling, TLR6 signaling, TLR7 signaling, TLR8 signaling, TLR9 signaling,
  • metabolic reprogramming
  • a macrophage comprising or expressing a CAR as described herein exhibits induction of cell survival mechanisms, e.g., relative to a macrophage without a CAR as described herein. In some embodiments, a macrophage comprising or expressing a CAR as described herein exhibits induction of cell death mechanisms, e.g., relative to a macrophage without a CAR as described herein.
  • a macrophage comprising or expressing a CAR as described herein exhibits one, two, three, four, or five of increased resistance to phagocytic checkpoints, increased expression of chemokine receptors to aid in trafficking, increased expression of chemokines to recruit other immune cells, increased expression of ECM degrading enzymes (e.g., MMPs to degrade tumor ECM and/or exhibit anti fibrotic activity), and/or increased proliferation, e.g., relative to a macrophage without a CAR as described herein.
  • ECM degrading enzymes e.g., MMPs to degrade tumor ECM and/or exhibit anti fibrotic activity
  • a macrophage comprising or expressing a CAR as described herein exhibits one, two, three, or four of improved duration of CAR expression, improved stability of the CAR on the cell surface, increased level of CAR expression, and/or decreased background activity of the CAR, e g., relative to a macrophage without a CAR as described herein.
  • a macrophage comprising or expressing a CAR as described herein decreases one or more signs and/or symptoms of an infection (e.g., of an infectious agent) in a subject, e g., relative to a macrophage without a CAR as described herein.
  • an infectious agent comprises or is a virus, a protozoa (e.g., trypanosome, malaria, or toxoplasma), a bacteria (e.g., mycobacterium, salmonella, or listeria), a fungi (e.g., Candida), or a combination thereof.
  • a virus comprises hepatitis virus (e.g., hepatitis A, hepatitis B, hepatitis C, or hepatitis E), retrovirus, human immunodeficiency virus (e.g., HIV1 or HIV2), T cell leukemia virus, a Lymphotropic virus (e.g., HTLV1 or HTLV2), herpes simplex virus (e.g., herpes simplex virus type 1 or type 2), Epstein-Barr virus, cytomegalovirus, varicella-zoster virus, poliovirus, measles virus, Rubella virus, Japanese encephalitis virus, mumps virus, influenza virus, adenovirus, enterovirus, rhinovirus, coronavirus (e g., severe acute respiratory syndrome (SARS) virus, Middle East respiratory syndrome (MERS) virus, or severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)), Ebola virus, West Nile virus, or a variant or
  • a macrophage comprising or expressing a CAR as described herein decreases formation and/or degrades existing aggregates via phagocytosis of at least one protein aggregate in a subject (e.g., a subject having a neurodegenerative disease, an inflammatory disease, a cardiovascular disease, a fibrotic disease, amyloidosis, or a combination thereof), e.g., relative to a macrophage without a CAR as described herein.
  • a subject e.g., a subject having a neurodegenerative disease, an inflammatory disease, a cardiovascular disease, a fibrotic disease, amyloidosis, or a combination thereof
  • a neurodegenerative disease is selected from the group consisting of tauopathy, a- synucleopathy, presenile dementia, senile dementia, Alzheimer's disease, progressive supranuclear palsy (PSP), Pick's disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, dementia with Lewy bodies, Down's syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS), Hallervorden-Spatz syndrome, polyglutamine disease, trinucleotide repeat disease, and prion disease.
  • tauopathy tauopathy
  • a- synucleopathy presenile dementia
  • senile dementia Alzheimer's disease
  • PPP progressive supranuclear palsy
  • Pick's disease primary progressive aphasia
  • frontotemporal dementia corticobasal dementia
  • Parkinson's disease dementia with Lewy bodies
  • Down's syndrome multiple system atrophy
  • an inflammatory disease is selected from the group consisting of systemic lupus erythematosus, vasculitis, rheumatoid arthritis, periodontitis, ulcerative colitis, sinusitis, asthma, tuberculosis, Crohn’s disease, chronic infection, hereditary periodic fever, a malignancy, systemic vasculitides, cystic fibrosis, bronchiectasis, epidermolysis bullosa, cyclic neutropenia, an immunodeficiency, Muckle-Wells (MWS) disease, and Familiar Mediterranean Fever (FMF).
  • systemic lupus erythematosus vasculitis, rheumatoid arthritis, periodontitis, ulcerative colitis, sinusitis, asthma, tuberculosis, Crohn’s disease, chronic infection, hereditary periodic fever, a malignancy, systemic vasculitides, cystic fibrosis, bronchiectasis,
  • amyloidosis is selected from the group consisting of Primary Amyloidosis (AL), Secondary Amyloidosis (AA), Familial Amyloidosis (ATTR) , Beta-2 Microglobulin Amyloidosis, Localized Amyloidosis, Heavy Chain Amyloidosis (AH), Light Chain Amyloidosis (AL), Primary Systemic Amyloidosis, ApoAI Amyloidosis, ApoAII Amyloidosis, ApoAIV Amyloidosis, Apolipoprotein C2 Amyloidosis, Apolipoprotein C3 Amyloidosis, Corneal lactoferrin amyloidosis, Transthyretin-Related Amyloidosis, Dialysis amyloidosis, Fibrinogen amyloidosis, Lect2 amyloidosis (ALECT2), and Lysozyme amyloidosis.
  • AL Primary Amyloidosis
  • AA Secondary Amyloidosis
  • ARR Famili
  • a 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.
  • a fibrotic disease is selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis, cirrhosis, cystic fibrosis, scleroderma, cardiac fibrosis, radiation-induced lung injury, steatohepatitis, glomerulosclerosis, interstitial lung disease, liver fibrosis, mediastinal fibrosis, retroperitoneal cavity fibrosis, bone marrow fibrosis, and skin fibrosis.
  • Monocytes are multipotent cells that circulate in the blood, bone marrow, and spleen, and generally do not proliferate when in a steady state. Monocytes can vary in size significantly in the range of about 10-30 pm in diameter. A ratio of nucleus to cytoplasm for a monocyte can range from about 2: 1 to about 1 :1. Typically, monocytes comprise chemokine receptors and pathogen recognition receptors that mediate migration from blood to tissues, such as during an infection. Monocytes can produce inflammatory cytokines, take up cells and/or toxic molecules, and differentiate into dendritic cells or macrophages. [0245] In some embodiments, a monocyte comprises or expresses one or more phenotypic markers.
  • Exemplarily phenotypic markers for human monocyte cells include, but are not limited to, CD9, CDl lb, CDl lc, CDwl2, CD13, CD15, CDwl7, CD31, CD32, CD33, CD35, CD36, CD38, CD43, CD49b, CD49e, CD49f, CD63, CD64, CD65s, CD68, CD84, CD85, CD86, CD87, CD89, CD91, CDw92, CD93, CD98, CD101, CD102, CD111, CD112, CD115, CD116, CD119, CDwl21b, CDwl23, CD127, CDwl28, CDwl31, CD147, CD155, CD156a, CD157, CD162 CD163, CD164, CD168, CD171, CD172a, CD180, CD206, CD131al, CD213 2, CDw210, CD226, CD281, CD282, CD284, and CD286.
  • Exemplarily phenotypic markers for mouse monocyte cells include, but are not limited to, CD1 la, CD1 lb, CD16, CD18, CD29, CD31, CD32, CD44, CD45, CD49d, CD115, CD116, Cdwl31, CD281, CD282, CD284, CD286, F4/80, and CD49b.
  • monocytes comprise one, two, or three of CD1 lb, CD14, or CD16.
  • monocytes comprise CD14+ CD16- monocytes, CD 14+ CD 16+ monocytes, or CD 14- CD 16+ monocytes.
  • a monocyte differentiates into a macrophage.
  • a monocyte differentiates into a dendritic cell (DC).
  • Monocytes can be differentiated into macrophages or DCs by any technique known in the art. For example, differentiation of monocytes into macrophages can be induced by macrophage colony stimulating factor (M-CSF). Differentiation of monocytes into DCs can be induced by granulocyte-macrophage colony stimulating factor (GM-CSF) in combination with IL-4.
  • M-CSF macrophage colony stimulating factor
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • a monocyte comprising or expressing a CAR as described herein exhibits increased secretion of one or more cytokines (e.g., one, two, three, four, five, six, or seven of TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, or IL-1), e.g., relative to a monocyte without a CAR as described herein.
  • 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
  • a monocyte comprising or expressing a CAR as described herein exhibits increased phagocytosis, e.g., relative to a monocyte without a CAR as described herein.
  • a monocyte comprising or expressing a CAR as described herein exhibits enhanced survival, e g., relative to a monocyte without a CAR as described herein.
  • a monocyte comprising or a CAR as described herein exhibits enhanced differentiation into macrophages (e.g., Ml or M2 macrophages), e.g., relative to a monocyte without a CAR as described herein.
  • a monocyte comprising or expressing a CAR as described herein as described herein exhibits enhanced differentiation into DCs (e.g., resident or migrating DCs and/or in lymphoid and non-lymphoid tissue), e.g., relative to a monocyte without a CAR as described herein.
  • a monocyte comprising or expressing a CAR as described herein exhibits increased cytotoxicity against a tumor cell, e.g., relative to a monocyte without a CAR as described herein.
  • a monocyte comprising or expressing a CAR as described herein exhibits increased tumor antigen presentation (e.g., post-phagocytosis presentation) and/or increased antigen processing, e.g., relative to a monocyte without a CAR as described herein.
  • a monocyte comprising or expressing a CAR as described herein exhibits increased tumor killing (e.g., by phagocytosis, lysis, apoptosis, or production of tumor killing cytokines (e.g., TNFa), e.g., relative to a monocyte without a CAR as described herein.
  • a monocyte comprising or expressing a CAR as described herein exhibits one or both of increased expression of one or more genes typically associated with increased effector function (e.g., phagocytosis, targeted cellular cytotoxicity, antigen presentation, or cytokine secretion) or decreased expression of one or more genes typically associated with decreased effector function (e.g., phagocytosis, targeted cellular cytotoxicity, antigen presentation, or cytokine secretion), e.g., relative to a monocyte without a CAR as described herein.
  • increased effector function e.g., phagocytosis, targeted cellular cytotoxicity, antigen presentation, or cytokine secretion
  • decreased expression of one or more genes typically associated with decreased effector function e.g., phagocytosis, targeted cellular cytotoxicity, antigen presentation, or cytokine secretion
  • a monocyte comprising or expressing a CAR as described herein exhibits increased production of ROS, e.g., relative to a monocyte without a CAR as described herein.
  • a monocyte comprising or expressing a CAR as described herein exhibits metabolic reprogramming, e.g., relative to a monocyte without a CAR as described herein.
  • a monocyte comprising or expressing a CAR as described herein exhibits induction of cell survival mechanisms, e.g., relative to a monocyte without a CAR as described herein.
  • a monocyte comprising or expressing a CAR as described herein exhibits induction of cell death mechanisms, e.g., relative to a monocyte without a CAR as described herein.
  • a monocyte comprising or expressing a CAR as described herein exhibits one, two, three, four, or five of increased resistance to phagocytic checkpoints, increased expression of chemokine receptors to aid in trafficking, increased expression of chemokines to recruit other immune cells, increased expression of ECM degrading enzymes (e.g., MMPs to degrade tumor ECM and/or exhibit anti fibrotic activity), or increased proliferation, e.g., relative to a monocyte without a CAR as described herein.
  • ECM degrading enzymes e.g., MMPs to degrade tumor ECM and/or exhibit anti fibrotic activity
  • a monocyte comprising or expressing a CAR as described herein exhibits one, two, three, or four of improved duration of CAR expression, improved stability of the CAR on the cell surface, increased level of CAR expression, and/or decreased background activity of the CAR, e.g., relative to a monocyte without a CAR as described herein.
  • Dendritic cells are bone marrow-derived, specialized antigen presenting cells that are involved in initiating immune responses and maintaining tolerance of the immune system to self-antigens. Dendritic cells may be found in both lymphoid and non-lymphoid organs and are generally thought to arise from lymphoid or myeloid lineages.
  • a DC comprises or expresses one or more phenotypic markers.
  • phenotypic markers for DCs include, but are not limited to, CD11c, CD83, CDla, CDlc, CD141, CD207, CLEC9a, CD123, CD85, CD180, CD187, CD205, CD281, CD282, CD284, CD286 and partially CD206, CD207, CD208 and CD209.
  • Immature DCs can be characterized by a high capacity for antigen capture, but relatively low T cell stimulatory capability. Inflammatory mediators promote DC maturation. Once DCs reach the mature stage, there is a dramatic change in properties relative to immature DCs, such as a decrease in antigen capture ability and/or an increased ability to stimulate T cells.
  • a DC comprises or is an immature DC. In other embodiments, a DC comprises or is a mature DC.
  • a DC comprising or expressing a CAR as described herein mediates tumor antigen presentation, e.g., increased tumor antigen presentation relative to a DC without a CAR as described herein.
  • a DC comprising or expressing a CAR as described herein mediates tumor T cell stimulation, e.g., increased T cell stimulation relative to a DC without a CAR as described herein.
  • a DC comprising or expressing a CAR as described herein exhibits increased secretion of one or more cytokines (e.g., one, two, three, four, five, six, or seven of TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, or IL-1), e.g., relative to a DC without a CAR as described herein.
  • 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
  • a DC comprising or expressing a CAR as described herein exhibits increased phagocytosis, e.g., relative to a DC without a CAR as described herein.
  • a DC comprising or expressing a CAR as described herein exhibits increased tumor antigen presentation (e.g., post-phagocytosis presentation), increased antigen processing, increased antigen cross presentation, increased T cell priming, and/or stimulation of T cells, e.g., relative to a DC without a CAR as described herein.
  • tumor antigen presentation e.g., post-phagocytosis presentation
  • antigen processing e.g., increased antigen processing
  • increased antigen cross presentation e.g., increased T cell priming
  • stimulation of T cells e.g., relative to a DC without a CAR as described herein.
  • a DC comprising or expressing a CAR as described herein exhibits one or both of increased expression of favorable genes or decreased expression of unfavorable genes, e.g., relative to a DC without a CAR as described herein.
  • a DC comprising or expressing a CAR as described herein exhibits increased production of ROS, e.g., relative to a DC without a CAR as described herein.
  • a DC comprising or expressing a CAR as described herein exhibits metabolic reprogramming, e.g., relative to a DC without a CAR as described herein.
  • a DC comprising or expressing a CAR as described herein exhibits induction of cell survival mechanisms, e.g., relative to a DC without a CAR as described herein.
  • a DC comprising or expressing a CAR as described herein exhibits induction of cell death mechanisms, e.g., relative to a DC without a CAR as described herein.
  • a DC comprising or expressing a CAR as described herein exhibits one, two, three, four, or five of increased resistance to phagocytic checkpoints, increased expression of chemokine receptors to aid in trafficking, increased expression of chemokines to recruit other immune cells, increased expression of ECM degrading enzymes (e.g., MMPs to degrade tumor ECM and/or exhibit anti fibrotic activity), or increased proliferation, e.g., relative to a DC without a CAR as described herein.
  • ECM degrading enzymes e.g., MMPs to degrade tumor ECM and/or exhibit anti fibrotic activity
  • a DC comprising or expressing a CAR as described herein exhibits one, two, three, or four of improved duration of CAR expression, improved stability of the CAR on the cell surface, increased level of CAR expression, and/or decreased background activity of the CAR, e.g., relative to a DC without a CAR as described herein.
  • the present disclosure provides, among other things, methods for modifying an immune cell (e.g., a stem cell, monocyte, macrophage, or dendritic cell) comprising delivering to the immune cell a nucleic acid construct comprising one or more nucleic acids encoding a CAR as described herein or a fragment thereof into an immune cell.
  • an immune cell e.g., a stem cell, monocyte, macrophage, or dendritic cell
  • Methods can comprise delivering to an immune cell (e.g., a stem cell, monocyte, macrophage, or dendritic cell), a nucleic acid construct comprising one or more nucleic acids encoding: a CAR comprising an extracellular domain, a transmembrane domain and one or more intracellular domains comprising a DAP 10 domain or a portion thereof, or a DAP 12 domain or a portion thereof.
  • an immune cell e.g., a stem cell, monocyte, macrophage, or dendritic cell
  • a nucleic acid construct comprising one or more nucleic acids encoding: a CAR comprising an extracellular domain, a transmembrane domain and one or more intracellular domains comprising a DAP 10 domain or a portion thereof, or a DAP 12 domain or a portion thereof.
  • a nucleic acid construct of the present disclosure further comprises one or more nucleic acids encoding a CAR.
  • the present disclosure provides methods of producing a modified immune cell (e.g., a stem cell, monocyte, macrophage, or dendritic cell) in a subject comprising administering to a subject a composition as described herein comprising: (a) one or more nucleic acid molecules, wherein at least a portion of one or more nucleic acid molecules encodes a CAR and/or CAR peptide agent, and (b) a delivery vehicle.
  • a modified immune cell e.g., a stem cell, monocyte, macrophage, or dendritic cell
  • nucleic acid molecules are translated in an immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) to produce a modified immune cell comprising the CAR and/or CAR peptide agent.
  • an immune cell e.g., stem cell, macrophage, monocyte, or dendritic cell
  • the modified immune cell comprising the CAR and/or CAR peptide agent possesses targeted effector activity.
  • a nucleic acid construct comprising one or more nucleic acid sequences encoding at least one CAR described herein, can be introduced into an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) by physical, chemical, or biological methods.
  • an immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • the present disclosure provides methods for modifying an immune cell comprising producing a modified immune cell (e.g., a stem cell, monocyte, macrophage, or dendritic cell) ex vivo.
  • the present disclosure provides methods for modifying an immune cell comprising producing a modified immune cell (e.g., a stem cell, monocyte, macrophage, or dendritic cell) in a subject (i.e., in vivo).
  • certain provided methods of modifying immune cells include delivering compositions comprising one or more nucleic acid molecules, wherein at least a portion of the one or more nucleic acid molecules encodes the CAR, and a delivery vehicle to the subject.
  • Physical methods for introducing a nucleic acid construct as described herein into an immune cell can comprise electroporation, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, or a combination thereof.
  • a nucleic acid construct can be introduced into immune cells using commercially available methods, including electroporation (Amaxa Nucleofector-II® (Amaxa Biosystems, Cologne, Germany), ECM 830 BTX (Harvard Instruments, Boston, Mass.) Gene Pulser II® (BioRad, Denver, Colo.), or Multiporator® (Eppendort, Hamburg Germany)).
  • a nucleic acid construct can also be introduced into 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), which is hereby incorporated by reference in its entirety).
  • 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), which is hereby incorporated by reference in its entirety).
  • Biological methods for introducing a nucleic acid construct as described herein into an immune cell include use of DNA and RNA vectors.
  • a vector comprises a plasmid vector, a viral vector, a transposon, a retrotransposon (e.g., piggyback, sleeping beauty), a site directed insertion vector (e.g., CRISPR, Zn finger nucleases, TALEN), suicide expression vector, or another vector known in the art.
  • Viral vectors, and especially retroviral vectors have become widely used for inserting genes into mammalian cells (e.g., human cells).
  • Viral vectors can also be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses (e.g. Ad5f35), or adeno-associated viruses (See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362, which are hereby incorporated by reference in their entirety).
  • Retroviral vectors such as lentivirus, are suitable tools to achieve long-term gene transfer that allow for long-term, stable integration of a transgene and its propagation in daughter cells.
  • a lentiviral vector is packaged with a Vpx protein (e.g., as described in International Publication No.
  • Vpx comprises a virion- associated protein (e.g., an accessory protein for viral replication).
  • a Vpx protein is encoded by human immunodeficiency virus type 2 (HIV-2).
  • a Vpx protein is encoded by simian immunodeficiency virus (SIV).
  • an immune cell as described herein e g., a stem cell, macrophage, monocyte, or dendritic cell
  • a lentiviral vector packaged with a Vpx protein is transfected with a lentiviral vector packaged with a Vpx protein.
  • Vpx inhibits at least one antiviral factor of an immune cell as described herein (e.g., a stem cell, macrophage, monocyte, or dendritic cell).
  • a lentiviral vector packaged with a Vpx protein exhibits increased transfection efficiency of an immune cell as described herein (e.g., a stem cell, macrophage, monocyte, or dendritic cell), e.g., relative to a lentiviral vector not packaged with a Vpx protein.
  • an immune cell as described herein e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a viral vector e.g., an adenoviral vector, e.g., an Ad2 vector or an Ad5 vector (e.g., Ad5f35 adenoviral vector, e.g., a helper-dependent Ad5F35 adenoviral vector)
  • Ad5f35 adenoviral vector e.g., a helper-dependent Ad5F35 adenoviral vector
  • Chemical means for introducing a nucleic acid construct as described herein into an immune cell include colloidal dispersion systems, macromolecule complexes, nanocapsules, microspheres, beads, and lipid- based systems (e.g., oil-in-water emulsions, micelles, mixed micelles, nanoparticles, liposomes, and lipofectamine-nucleic acid complexes).
  • An exemplary system for delivery of a nucleic acid construct as described herein is a lipid-based system.
  • a nucleic acid construct as described herein may be encapsulated in an aqueous interior of a liposome, interspersed within a lipid bilayer, attached to a liposome via a linking molecule, attached to a lipid nanoparticle (LNP) via a linking molecule, entrapped in a liposome, entrapped in an LNP, complexed with a liposome, complexed with an LNP, dispersed in a solution or suspension comprising a lipid, mixed with a lipid, complexed with a micelle, or otherwise associated with a lipid.
  • LNP lipid nanoparticle
  • Lipids for use in methods described herein may be naturally occurring or synthetic lipids. Lipids can also be obtained from commercial sources. For example, dimyristyl phosphatidylcholine can be obtained from Sigma (St. Louis, MO); dicetyl phosphate can be obtained from K & K Laboratories (Plainview, NY); cholesterol can be obtained from Calbiochem-Behring; and dimyristyl phosphatidylglycerol can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL.). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20°C.
  • a lipid-based system may comprise one or more lipids that facilitate targeting of the composition to a desired cell type or cell types (e.g., stem cells, monocytes, macrophages, or dendritic cells).
  • a delivery vehicle allows a composition to be preferentially taken up (e.g. endocytosed, phagocytosed) by an immune cell (e.g., stem cell, monocyte, macrophage, or dendritic cell) relative to a composition that does not comprise the delivery vehicle.
  • a delivery vehicle may comprise one or more targeting moieties.
  • a targeting moiety may facilitate passive targeting of a composition to a desired target.
  • a targeting moiety may facilitate active targeting of a composition to a desired target.
  • a targeting moiety may be or comprise one of more of an antibody (e.g., a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody) or any fragment thereof, for example an scFv, an aptamer, a darpin, a centyrin, a naturally occurring or synthetic receptor, an affibody, or other engineered protein recognition molecule, for example, to bind to one or more of CD 14, CD1 lb, CD163, CD206, CD33, CD209.
  • a targeting moiety may be or comprise a small molecule.
  • a targeting moiety may be or comprise a particular lipid or combination of hydrophobic entities, for example, present in or forming an exterior surface of a liposome or lipid nanoparticle (e.g., for targeting to a particular cell type or cell types).
  • one or more nucleic acid molecules are or comprise DNA.
  • one or more nucleic acid molecules are or comprise messenger RNA (mRNA).
  • mRNA messenger RNA
  • mRNA according to the present disclosure may be synthesized as unmodified or modified mRNA.
  • mRNAs are modified to enhance stability. Modifications of mRNA can include, for example, modifications of the nucleotides of the RNA.
  • a modified mRNA according to the present disclosure can thus include, for example, backbone modifications, sugar modifications or base modifications.
  • a step of modifying an mRNA comprises causing the mRNA to include a modified nucleotide, an alteration to the 5’ or 3’ untranslated region (UTR), a cap structure, and/or a poly(A) tail.
  • mRNAs of the present disclosure may contain RNA backbone modifications.
  • a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are modified chemically.
  • Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g. cytidine 5'-O-(l- thiophosphate)), boranophosphates, positively charged guanidinium groups etc., which comprises replacing the phosphodi ester linkage by other anionic, cationic or neutral groups.
  • mRNAs of the present disclosure may contain sugar modifications.
  • a typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 2'-deoxy-2'-fluoro-oligoribonucleotide (2'- fluoro-2'-deoxycytidine 5 '-triphosphate, 2'-fluoro-2'-deoxyuridine 5 '-triphosphate), 2'-deoxy-2'- deamine-oligoribonucleotide (2'-amino-2'-deoxy cytidine 5'-triphosphate, 2'-amino-2'- deoxyuridine 5'-triphosphate), 2'-O-alkyloligoribonucleotide, 2'-deoxy-2'-C- alkyloligoribonucleotide (2'-O-methylcytidine 5'-triphosphat
  • mRNAs of the present disclosure comprise modified nucleotide comprising pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine/pseudouridine (5meC PsU), Nl-methyl-pseudouridine (NlmPsU), or combinations thereof.
  • PsU pseudouridine
  • 5moU 5-methoxyuridine
  • meC PsU 5-methylcytidine/pseudouridine
  • NlmPsU Nl-methyl-pseudouridine
  • mRNAs of the present disclosure may contain modifications of the bases of the nucleotides (base modifications).
  • base modifications A modified nucleotide which contains a base modification is also called a base-modified nucleotide.
  • mRNA synthesis includes the addition of a “cap” on the N-terminal (5’) end, and a “tail” on the C-terminal (3’) end.
  • the presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells.
  • the presence of a “tail” serves to protect the mRNA from exonuclease degradation.
  • mRNAs of the present disclosure include a 5’ cap structure.
  • a 5’ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5’ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5’ triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase.
  • GTP guanosine triphosphate
  • cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G.
  • a cap comprises a CapO structure.
  • a capO structures lack a 2'-O-methyl residue of the ribose attached to bases 1 and 2.
  • a cap comprises an AGCapl structure.
  • An AGCapl structures has a 2'-O-methyl residue at base 2.
  • a cap comprises a Cap2 structure. Cap2 structures have a 2'-O-methyl residue attached to both bases 2 and 3.
  • a cap structure comprises AGCapl, m6AGCapl, or Anti-Reverse Cap Analog (ARCA).
  • a modified mRNA of the present disclosure comprises an m6AGCapl and modified nucleotides comprising pseudouridine (PsU).
  • mRNAs of the present disclosure include a 3’ poly(A) tail structure.
  • a poly(A) tail on the 3' terminus of mRNA typically includes about 10 to 400 adenosine nucleotides (SEQ ID NO: 100) (e.g., about 100 to 400 adenosine nucleotides, about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides).
  • SEQ ID NO: 100 e.g., about 100 to 400 adenosine nucleotides, about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine
  • mRNAs include a 3’ poly(C) tail structure.
  • a suitable poly(C) tail on the 3' terminus of mRNA typically include about 10 to 200 cytosine nucleotides (SEQ ID NO: 101) (e g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides).
  • a poly(C) tail may be added to a poly(A) tail or may be a substitute for the poly(A) tail.
  • mRNAs of the present disclosure include a 5’ and/or 3’ untranslated region.
  • a 5’ untranslated region includes one or more elements that affect an mRNA’s stability or translation, for example, an iron responsive element.
  • a 5’ untranslated region may be between about 50 and 500 nucleotides in length.
  • a 3’ untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA’s stability of location in a cell, or one or more binding sites for miRNAs.
  • a 3’ untranslated region may be between 50 and 500 nucleotides in length or longer.
  • methods of the present disclosure comprise one or more steps of treating an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) during the process of modifying the immune cell.
  • methods of the present disclosure comprise one or more steps of administering to a subject an additional payload for modulating an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) during the process of modifying the immune cell.
  • a composition may comprise one or more additional payloads.
  • a composition may comprise one or more additional payloads in the same delivery vehicle as one or more nucleic acid molecules.
  • a composition may comprise one or more additional payloads in a different delivery vehicle than the one used with one or more nucleic acid molecules.
  • methods of the present disclosure comprise a step of treating an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) with a modulator of a pathway activated by in vitro transcribed mRNA.
  • an immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • an additional payload may be or comprise a modulator of a pathway activated by in vitro transcribed mRNA.
  • IVT hi vitro transcribed mRNA is recognized by various endosomal innate immune receptors (Toll-like receptor 3 (TLR3), TLR7 and TLR8) and cytoplasmic innate immune receptors (protein kinase RNA-activated (PKR), retinoic acid-inducible gene I protein (RIG-I), melanoma differentiation- associated protein 5 (MDA5) and 2'-5'-oligoadenylate synthase (OAS)).
  • PLR protein kinase RNA-activated
  • RAG-I retinoic acid-inducible gene I protein
  • MDA5 melanoma differentiation- associated protein 5
  • OF 2'-5'-oligoadenylate synthase
  • eukaryotic translation initiation factor 2a eukaryotic translation initiation factor 2a
  • RNaseL ribonuclease L
  • overexpression and inhibition of replication of self-amplifying mRNA are of relevance for the pharmacokinetics and pharmacodynamics of IVT mRNA.
  • a modulator of a pathway activated by in vitro transcribed mRNA comprises an RNase inhibitor. In some embodiments, a modulator of a pathway activated by in vitro transcribed mRNA comprises an RNaseL, RNase T2 or RNasel inhibitor. In some embodiments, a modulator of a pathway activated by in vitro transcribed mRNA comprises an RNaseL inhibitor. In some embodiments, an RNaseL inhibitor comprises sunitinib. In some embodiments, an RNaseL inhibitor comprises ABCE1.
  • treating an immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • an RNaseL inhibitor increases mRNA stability in a modified immune cell relative to mRNA stability in a modified immune cell of the same type that was not treated with an RNaseL inhibitor.
  • treating an immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • treating an immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • an RNaseL inhibitor increases effector activity in a modified immune cell relative to effector activity in a modified immune cell of the same type that was not treated with an RNaseL inhibitor.
  • administering to a subject an RNaseL inhibitor increases mRNA stability in a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) relative to mRNA stability in a modified immune cell of the same type in a subject that that was not administered an RNaseL inhibitor.
  • administering to a subject an RNaseL inhibitor increases CAR expression in a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) relative to CAR expression in a modified immune cell of the same type in a subject that was not administered an RNaseL inhibitor.
  • administering to a subject an RNaseL inhibitor increases effector activity in a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) relative to effector activity in a modified immune cell of the same type in a subject that was not administered an RNaseL inhibitor.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a step of treating an immune cell occurs before a step of delivering an mRNA to the immune cell.
  • a step of administering an additional payload to a subject occurs before a step of administering a composition comprising an mRNA to the subject.
  • methods of the present disclosure comprise a step of culturing an immune cell (e g., a stem cell, macrophage, monocyte, or dendritic cell) with a cytokine or immune stimulating recombinant protein.
  • methods of the present disclosure comprise a step of administering to a subject a cytokine or immune stimulating recombinant protein.
  • a cytokine comprises IFN-a, IFN-0, IFN-y, TNFa, IL-6, STNGL, LPS, a CD40 agonist, a 4-1BB ligand, recombinant 4-1BB, a CD19 agonist, a TLR agonist (e.g., TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8 or TLR-9), TGF-0 (e g., TGF-01, TGF- 02, or TGF-03), a glucocorticoid, an immune complex, interleukin-1 alpha (IL-1 a), IL-10, 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 colon
  • a step of culturing an immune cell occurs after a step of delivering an mRNA to the immune cell.
  • a step of administering to a subject a cytokine or immune stimulating recombinant protein occurs after a step of administering a composition comprising an mRNA to the subject.
  • culturing a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a cytokine or immune stimulating recombinant protein increases the viability of the modified immune cell relative to a modified immune cell of the same type that was not cultured with the cytokine or immune stimulating recombinant protein.
  • culturing a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a cytokine or immune stimulating recombinant protein increases protein (e.g., CAR) expression in the modified immune cell relative to a modified immune cell of the same type that was not cultured with the cytokine or immune stimulating recombinant protein.
  • culturing a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a cytokine or immune stimulating recombinant protein increases longevity of protein (e.g., CAR) expression relative to a modified immune cell of the same type that was not cultured with the cytokine or immune stimulating recombinant protein.
  • culturing a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a cytokine or immune stimulating recombinant protein increases effector activity of the modified immune cell relative to a modified immune cell of the same type that was not cultured with the cytokine or immune stimulating recombinant protein.
  • culturing a modified immune cell e g., a stem cell, macrophage, monocyte, or dendritic cell
  • a cytokine or immune stimulating recombinant protein increases pro-inflammatory (Ml) polarization of the modified immune cell relative to a modified immune cell of the same type that was not cultured with the cytokine or immune stimulating recombinant protein.
  • administering to a subject a cytokine or immune stimulating recombinant protein increases the viability of a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulating recombinant protein.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • administering to a subject a cytokine or immune stimulating recombinant protein increases protein (e.g., CAR) expression of a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulating recombinant protein.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • administering to a subject a cytokine or immune stimulating recombinant protein increases longevity of protein (e.g., CAR) expression in a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulating recombinant protein.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • administering to a subject a cytokine or immune stimulating recombinant protein increases effector activity of a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulating recombinant protein.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • administering to a subject a cytokine or immune stimulating recombinant protein increases pro- inflammatory (Ml) polarization of a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulating recombinant protein.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) is made by methods of the present disclosure.
  • a modified immune cell comprises a CAR.
  • a modified immune cell comprises one or more nucleic acids encoding a CAR.
  • a CAR comprises an extracellular domain, a transmembrane domain and one or more intracellular domains.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprises at least one CAR described herein comprising one or more extracellular domains; a transmembrane domain; and one or more intracellular domains comprising: a DAP10 domain or a portion thereof, or a DAP12 domain or a portion thereof; wherein the modified immune cell is or comprises a stem cell, macrophage, monocyte, or dendritic cell.
  • a modified immune cell e g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR comprising a DAP 10 intracellular domain or a portion thereof.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR comprising a DAP12 intracellular domain or a portion thereof.
  • one or more extracellular domains are or comprise an scFv, VHH antibody, centyrin, darpin, or nanobody.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR comprising a transmembrane domain comprising a DAP10, DAP12, CD8a, CD28, CD40, MyD88 CD64, CD32a, CD32c, CD16a, CD3zeta, ICOS, Dectin-1, DNGR1, SLAMF7, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, or TLR9 transmembrane domain.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR comprising a DAP 10 transmembrane domain.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR comprising a DAP 12 transmembrane domain.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR comprising one or more intracellular domains further comprising one or more of a CD3( ⁇ , FcRy, MyD88, CD40, CD64, CD32a, CD32c, CD16a, CD89, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD19, CD20, 41BB, CD28, GCSFR (CD1 14), RAGE, CD30, CD160, DR3, Fnl4, HVEM, CD160, NGFR, RANK, TNFR2, TROY, XEDAR,
  • one or more intracellular domains further comprise one or more of a CD20, CD40, DAP10, DAP12, Dectin-1, FcRy, MyD88, RAGE, SLAMF7, or TLR2 intracellular domain, a portion of any of the foregoing, or combinations thereof.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR further comprising an extracellular leader domain.
  • an extracellular leader domain is or comprises a CD8a extracellular leader domain.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR further comprising an extracellular hinge domain.
  • an extracellular hinge domain is or comprises: a DAP10 extracellular hinge domain, a DAP12 extracellular hinge domain, a CD8a extracellular hinge domain, a CD28 extracellular hinge domain, a DNGR-1 extracellular hinge domain, a Dectin-1 extracellular hinge domain, or an IgG4 extracellular hinge domain.
  • an extracellular hinge domain is or comprises a DAP 10 extracellular hinge domain.
  • an extracellular hinge domain is or comprises a DAP12 extracellular hinge domain.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR comprising, from N- terminus to C-terminus: (i) a CD8a extracellular leader domain, an extracellular antigen binding domain, a DAP 10 extracellular hinge domain, a DAP 10 transmembrane domain, and a DAP 10 intracellular domain; (ii) a CD8a extracellular leader domain, an extracellular antigen binding domain, a CD8a extracellular hinge domain, a CD8a transmembrane domain, a DAP10 intracellular domain, and an FcRy intracellular domain; (iii) a CD8a extracellular leader domain, an extracellular antigen binding domain, a CD8a extracellular hinge domain, a CD8a transmembrane domain, an FcRy intracellular domain, and a DAPIO intracellular domain; (iv) a CD8a extracellular leader domain
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR comprising, from N- terminus to C-terminus: (i) a CD8a extracellular leader domain, an extracellular antigen binding domain, a CD8a extracellular hinge domain, a CD8a transmembrane domain, and a DAP 12 intracellular domain; (ii) a CD8a extracellular leader domain, an extracellular antigen binding domain, a CD8a extracellular hinge domain, a CD8a transmembrane domain, a DAP 12 intracellular domain, and an FcRy intracellular domain; (iii) a CD8a extracellular leader domain, an extracellular antigen binding domain, a CD8a extracellular hinge domain, a CD8a transmembrane domain, an FcRy intracellular domain, and a DAP12 intracellular domain; (iv) a CD8a extracellular leader domain
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR comprising (a) an amino acid sequence selected from Table 2; an amino acid sequence that differs from a sequence selected from Table 2 by no more than five substitutions, additions, or deletions; or (c) an amino acid sequence that is at least 80% identical to a sequence selected from Table 2.
  • a modified immune cell e g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprises a CAR comprising an amino acid sequence at least 80% identical to a sequence selected from Table 2.
  • a modified immune cell e g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprises a CAR comprising an amino acid sequence at least 85% identical to a sequence selected from Table 2.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprises a CAR comprising an amino acid sequence at least 95% identical to a sequence selected from Table 2.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprises a CAR comprising an amino acid sequence at least 96% identical to a sequence selected from Table 2.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprises a CAR comprising an amino acid sequence at least 98% identical to a sequence selected from Table 2.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprises a CAR comprising an amino acid sequence at least 99% identical to a sequence selected from Table 2.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR comprising an amino acid sequence that differs from a sequence selected from Table 2 by no more than five substitutions, additions, or deletions.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR comprising an amino acid sequence that differs from a sequence selected from Table 2 by no more than four substitutions, additions, or deletions.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR comprising an amino acid sequence that differs from a sequence selected from Table 2 by no more than three substitutions, additions, or deletions.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR comprising an amino acid sequence that differs from a sequence selected from Table 2 by no more than two substitutions, additions, or deletions.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR comprising an amino acid sequence that differs from a sequence selected from Table 2 by no more than one substitution, addition, or deletion.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR comprising an amino acid sequence that differs from a sequence selected from Table 2 by zero substitutions, additions, or deletions.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprises a CAR encoded by a nucleic acid sequence at least 80% identical to a sequence selected from Table 3.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprises a CAR encoded by a nucleic acid sequence at least 90% identical to a sequence selected from Table 3.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprises a CAR encoded by a nucleic acid sequence at least 96% identical to a sequence selected from Table 3.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprises a CAR encoded by a nucleic acid sequence at least 98% identical to a sequence selected from Table 3.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprises a CAR encoded by a nucleic acid sequence identical to a sequence selected from Table 3.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR comprising an extracellular domain that binds a tumor antigen, such as an antigen that is specific for a tumor or cancer of interest.
  • a tumor antigen comprises one or more antigenic cancer epitopes.
  • a tumor antigen comprises CD19; CD123; CD22;
  • CD30 CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule- 1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvlIl); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2- 3)bDGalp(l-4)bDGlcp(l-l)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-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
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprising a modified mRNA encoding a CAR as described herein exhibits increased viability relative to a modified immune cell of the same type comprising unmodified mRNA encoding a CAR as described herein.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprising a modified mRNA encoding a CAR as described herein exhibits increased expression of an mRNA encoding a CAR as described herein relative to a modified immune cell of the same type comprising unmodified mRNA encoding the CAR.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified mRNA encoding a CAR as described herein exhibits increased CAR expression relative to a modified immune cell of the same type comprising unmodified mRNA encoding the CAR.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprising a modified mRNA encoding a CAR as described herein exhibits increased longevity of a mRNA encoding a CAR as described herein relative to a modified immune cell of the same type comprising unmodified mRNA encoding the CAR.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified mRNA encoding a CAR as described herein exhibits increased longevity of the CAR relative to a modified immune cell of the same type comprising unmodified mRNA encoding the CAR.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprising a modified mRNA encoding a CAR as described herein exhibits increased effector activity relative to a modified immune cell of the same type comprising unmodified mRNA encoding the CAR.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • Ml pro-inflammatory
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR as described herein maintains a pro-inflammatory phenotype over time.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR as described herein maintains a pro-inflammatory phenotype at least 4 hours, 2 days, 4 days, 7 days, 14 days, and/or 28 days after an immune cell is modified with a nucleic acid encoding the CAR.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprising a CAR as described herein maintains an antiinflammatory phenotype over time.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR as described herein maintains an anti-inflammatory phenotype at least 4 hours, 2 days, 4 days, 7 days, 14 days, and/or 28 days after an immune cell is modified with a nucleic acid encoding the CAR.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR as described herein maintains a pro-inflammatory phenotype and/or otherwise resists subversion when challenged by anti-inflammatory cytokines.
  • the sensitivity of a modified immune cell to environmental cytokines is measured by generating a dose-response curve of pro-inflammatory markers by treating modified immune cells comprising a CAR as described herein with increasing concentrations of antiinflammatory cytokines.
  • the sensitivity of a modified immune cell to environmental cytokines is measured by generating a dose-response curve of pro-inflammatory markers by treating modified immune cells comprising a CAR as described herein with increasing concentrations of pro-inflammatory cytokines.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a CAR as described herein maintains an antiinflammatory phenotype and/or otherwise resists subversion when challenged by pro- inflammatory cytokines.
  • the sensitivity of a modified immune cell to environmental cytokines is measured by generating a dose-response curve of anti-inflammatory markers by treating modified immune cells comprising a CAR as described herein with increasing concentrations of pro-inflammatory cytokines.
  • the sensitivity of a modified immune cell to environmental cytokines is measured by generating a doseresponse curve of anti-inflammatory markers by treating modified immune cells comprising a CAR as described herein with increasing concentrations of anti-inflammatory cytokines.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • a modified immune cell comprising a CAR as described herein has minimal effects on neighboring cells.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • unmodified cell e.g., an immune cell that doesn’t comprise a CAR as described herein
  • modified immune cells and unmodified immune cells can be co-cultured in a culture dish where the modified immune cells and unmodified immune cells contact each other.
  • modified immune cells and unmodified immune cells can be co-cultured in a culture dish where the modified immune cells and unmodified immune cells are separated by a transwell assay membrane.
  • a modified immune cell comprising a CAR as described herein has minimal cytotoxic effects on neighboring cells.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • modifying an immune cell to comprise a CAR as described herein is not cytotoxic to the modified immune cell.
  • RNAseq data from modified immune cells are examined to determine if upregulation of genes indicative of cytotoxic effects is present.
  • a modified immune cell comprising a CAR as described herein may comprise one or more control systems including, but not limited to: a safety switch (e.g., an on switch, an off switch, a suicide switch), transcriptional control (e.g. cell-specific promoters, cell-state specific promoters, promoters downstream of CAR activation, promoters downstream of endogenous signaling pathways, or drug-inducible transcription), post-transcriptional control of CAR mRNA (e.g.
  • a safety switch e.g., an on switch, an off switch, a suicide switch
  • transcriptional control e.g. cell-specific promoters, cell-state specific promoters, promoters downstream of CAR activation, promoters downstream of endogenous signaling pathways, or drug-inducible transcription
  • post-transcriptional control of CAR mRNA e.g.
  • RNA-based inhibition with endogenous or recombinant miRNA or post-translational control of a CAR’s structure or stability (e.g. a CAR whose intracellular domain conditionally associates with the full structure by drug/light-inducible association (to allow signaling) or dissociation (to inhibit signaling), or whose stability is drug-regulated for inducible stabilization (to allow signaling) or degradation (to inhibit signaling)).
  • a CAR whose intracellular domain conditionally associates with the full structure by drug/light-inducible association (to allow signaling) or dissociation (to inhibit signaling), or whose stability is drug-regulated for inducible stabilization (to allow signaling) or degradation (to inhibit signaling)).
  • AND gate e.g. a CAR with a CAR-inducible promoter and cytosolic domain that associates in a drug-dependent manner, thus requiring CAR activation and the presence of a small molecule
  • an OR gate e.g.
  • a CAR under control of a promoter that is transcriptionally active following CAR activation or small molecule addition and/or a NOT gate (e.g. a CAR whose mRNA is degraded by endogenous miRNAs expressed in natural immune cell signaling states (such as miRNAs upregulated by a particular cytokine signaling pathway, thus only expressing CAR in the absence of this cytokine)).
  • a NOT gate e.g. a CAR whose mRNA is degraded by endogenous miRNAs expressed in natural immune cell signaling states (such as miRNAs upregulated by a particular cytokine signaling pathway, thus only expressing CAR in the absence of this cytokine).
  • a variety of assays may be performed to confirm the presence of a nucleic acid construct as described herein and/or the presence of a protein (e.g., a CAR) in an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell).
  • a protein e.g., a CAR
  • an immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell.
  • assays include molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR, and PCR; and biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELlSAs and Western blots).
  • FACS fluorescence-activated cell sorting
  • MSD MSD cytokine analysis
  • MS mass spectrometry
  • RNA-Seq RNA-Seq and functional assays.
  • a variety of assays may be performed to determine various characteristics of a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell), such as, but not limited to, immune cell viability, nucleic acid expression, nucleic acid longevity, protein (e.g., CAR) expression, protein (e.g., CAR) longevity, effector activity, and pro-inflammatory (Ml) polarization.
  • a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
  • immune cell viability e.g., nucleic acid expression, nucleic acid longevity, protein (e.g., CAR) expression, protein (e.g., CAR) longevity, effector activity, and pro-inflammatory (Ml) polarization.
  • assays include flow cytometry, quantitative PCR, and in vitro functional assays such as cytokine/chemokine secretion, phagocytosis, and specific lysis assay
  • the present disclosure provides nucleic acid molecules encoding at least one CAR described herein or a fragment thereof.
  • An immune cell e.g., stem cell, macrophage, monocyte, or dendritic cell
  • can comprise a nucleic acid molecule e.g., an exogenous nucleic acid molecule
  • at least one protein e.g., a CAR of the present disclosure
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase “nucleotide sequence that encodes a protein or an RNA” may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
  • encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene, cDNA, or RNA encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the heterologous nucleic acid sequence.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.
  • Nucleic acid molecules encoding at least one protein (e.g., a CAR of the present disclosure) described herein or a fragment thereof can be a DNA molecule, an RNA molecule, or a combination thereof.
  • a nucleic acid molecule comprises or is a messenger RNA (mRNA) transcript encoding at least one protein (e.g., a CAR of the present disclosure) described herein or a fragment thereof.
  • mRNA messenger RNA
  • a nucleic acid molecule comprises or is a DNA construct encoding at least one protein (e.g., a CAR of the present disclosure) described herein or a fragment thereof.
  • all or a fragment of a protein (e.g., a CAR of the present disclosure) described herein is encoded by a codon optimized nucleic acid molecule, e.g., for expression in a cell (e.g., a mammalian cell).
  • a codon optimized nucleic acid molecule e.g., for expression in a cell (e.g., a mammalian cell).
  • a variety of codon optimization methods are known in the art, e.g., as disclosed in US Patent Nos. 5,786,464 and 6,114,148, each of which is hereby incorporated by reference in its entirety.
  • Expression of nucleic acids as described herein may be achieved by operably linking a nucleic acid encoding a protein (e.g., a CAR of the present disclosure) or fragment thereof to a promoter in an expression vector.
  • Exemplary promoters include, but are not limited to, an elongation factor- lot promoter (EF-la) promoter, immediate early cytomegalovirus (CMV) promoter, ubiquitin C promoter, phosphoglycerokinase (PGK) promoter, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Moloney murine leukemia virus (MoMuLV) promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, or a creatine kinase promoter.
  • EF-la elongation factor- lot promoter
  • CMV immediate early cytomegalovirus
  • PGK phosphoglycerokinase
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • a vector can also comprise additional promoter elements, e.g., enhancers, to regulate the frequency of transcriptional initiation.
  • a vector comprising a nucleic acid molecule encoding a protein (e.g., a CAR of the present disclosure) or fragment thereof comprises or is a viral vector.
  • Viral vector technology is well known in the art and is described (e.g., in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY).
  • examples of viral vectors include, but are not limited to, adenoviral vectors, adeno- associated viral vectors, or retroviral vectors (e.g., a lentiviral vector or a gammaretroviral vector).
  • a vector comprises a lentiviral vector (e.g., as described in US Patent No. 9,149,519 or International Publication No. WO 2017/044487, each of which is hereby incorporated by reference in its entirety).
  • a viral vector comprises an adenoviral vector.
  • Adenoviruses are a large family of viruses containing double stranded DNA. They replicate within the nucleus of a host cell, using the host’s cell machinery to synthesize viral RNA, DNA and proteins. Adenoviruses are known in the art to affect both replicating and non-replicating cells, to accommodate large transgenes, and to code for proteins without integrating into the host cell genome.
  • an adenoviral vector comprises an Ad2 vector or an Ad5 vector (e.g., Ad5f35 adenoviral vector, e.g., a helper-dependent Ad5F35 adenoviral vector).
  • a viral vector is an adeno-associated virus (AAV) vector.
  • AAV systems are generally well known in the art (see, e.g., Kelleher and Vos, Biotechniques, 17(6): 1110-17 (1994); Cotten et al., P.N.A.S. U.S.A., 89(13):6094-98 (1992); Curiel, Nat Immun, 13(2-3): 141-64 (1994); Muzyczka, Curr Top Microbiol Immunol, 158:97-129 (1992); and Asokan A, et al., Mol. Ther., 20(4):699-708 (2012)).
  • Methods for generating and using recombinant AAV (rAAV) vectors are described, for example, in U.S. Pat. Nos. 5,139,941 and 4,797,368.
  • AAV serotypes have been characterized, including AAV1, AAV2, AAV3 (e.g., AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, as well as variants thereof.
  • AAV serotype may be used to deliver a protein (e.g., a CAR of the present disclosure) or fragment thereof described herein.
  • an AAV serotype has a tropism for a particular tissue.
  • CRISPR/Cas9 system has recently been shown to facilitate high levels of precise genome editing using adeno associated viral (AAV) vectors to serve as donor template DNA during homologous recombination (HR).
  • AAV adeno associated viral
  • a vector comprises a gammaretroviral vector (e.g., as described in Tobias Maetzig et al., “Gammaretroviral Vectors: Biology, Technology and Application” Viruses. 2011 Jun; 3(6): 677-713, which is hereby incorporated by reference in its entirety).
  • exemplary gammaretroviral vectors include Murine Leukemia Virus (MLV), Spleen- Focus Forming Virus (SFFV), and Myeloproliferative Sarcoma Virus (MPSV), and vectors derived therefrom.
  • a vector comprises two or more nucleic acid sequences encoding proteins, e.g., at least one CAR described herein, and a second CAR, e.g., a different CAR described herein.
  • two or more nucleic acid sequences encoding a CAR and a second CAR are encoded by a single nucleic molecule, e.g., in same frame and as a single polypeptide chain.
  • two or more CARs are separated by one or more cleavage peptide sites (e.g., an auto-cleavage site or a substrate for an intracellular protease).
  • a cleavage peptide comprises a porcine teschovirus-1 (P2A) peptide, Thosea asigna virus (T2A) peptide, equine rhinitis A virus (E2A) peptide, foot-and- mouth disease virus (F2A) peptide, or a variant thereof.
  • P2A porcine teschovirus-1
  • T2A Thosea asigna virus
  • E2A equine rhinitis A virus
  • F2A foot-and- mouth disease virus
  • a vector comprises at least one nucleic acid sequence encoding a protein, e.g., at least one CAR described herein, and at least one nucleic acid encoding at least one gene co-expressed with a second protein, e.g., a cytokine described herein (e.g., TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, and/or IL-1) or a stimulatory ligand described herein (e.g, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, ICOS-L, ICAM, CD30L, CD40, CD40L, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor,
  • the present disclosure provides pharmaceutical compositions comprising modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a CAR as described herein with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.
  • modified immune cells e.g., stem cells, macrophages, monocytes, or dendritic cells
  • nucleic acids encoding a CAR as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.
  • a therapeutically effective amount “an immunologically effective amount,” “an anti-immune response effective amount,” or “an immune response-inhibiting effective amount” is indicated, a precise amount of a pharmaceutical composition described herein can be determined by a physician with consideration of individual differences in age, weight, immune response, and condition of the patient (subject).
  • compositions described herein may comprise buffers, such as neutral buffered saline or phosphate buffered saline (PBS); carbohydrates, such as glucose, mannose, sucrose, dextrans, or mannitol; proteins, polypeptides, or amino acids (e.g., glycine); antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); serum and preservatives, such as cryoprotectant.
  • a pharmaceutical composition is substantially free of contaminants, e.g., there are no detectable levels of a contaminant (e.g., an endotoxin).
  • compositions described herein may be administered in a manner appropriate to the disease, disorder, or condition to be treated or prevented. Quantity and frequency of administration will be determined by such factors as condition of a patient, and type and severity of a patient’s disease, disorder, or condition, although appropriate dosages may be determined by clinical trials.
  • compositions described herein may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. Preferred compositions may be injectable or infusible solutions. Pharmaceutical compositions described herein can be formulated for administration intravenously, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, transarterially, or intraperitoneally.
  • a pharmaceutical composition described herein is formulated for parenteral (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular) administration. In some embodiments, a pharmaceutical composition described herein is formulated for intravenous infusion or injection. In some embodiments, a pharmaceutical composition described herein is formulated for intramuscular or subcutaneous injection. Pharmaceutical compositions described herein can be formulated for administered by using infusion techniques that are commonly known in immunotherapy (See, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988, which is hereby incorporated by reference in its entirety).
  • parenteral administration and “administered parenterally” refer to modes of administration other than enteral and topical administration, usually by injection or infusion, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intratumoral, and intrastemal injection and infusion.
  • compositions comprising modified immune cells as described herein may be administered at a dosage of about 10 4 to about 10 9 cells/kg body weight (e.g., about 10 5 to about 10 6 cells/kg body weight), including all integer values within those ranges.
  • a dose of immune cells as described herein comprises at least about 1 x 10 6 , about 1.1 x 10 6 , about 2 x 10 6 , about 3.6 x 10 6 , about 5 x 10 6 , about 1 x 10 7 , about 1.8 x IO 7 , about 2 x IO 7 , about 5 x IO 7 , about 1 x 10 8 , about 2 x 10 8 , about 5 x 10 8 , about 1 x 10 9 , about 2 x 10 9 , or about 5 x 10 9 cells.
  • Pharmaceutical compositions described herein may also be administered multiple times at a certain dosage. An optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art by monitoring a patient for signs of a disease, disorder, or condition and adjusting treatment accordingly.
  • Immune cells e.g., stem cells, macrophages, monocytes, or dendritic cells
  • blood draws of from about 10 cc to about 400 cc.
  • immune cells e.g., macrophages, monocytes, or dendritic cells
  • blood draws 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.
  • methods comprising multiple blood draw and reinfusions as described herein may select for certain immune cell populations.
  • compositions described herein are administered in combination with (e.g., before, simultaneously, or following) a second therapy.
  • a second therapy can include, but is not limited to 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 immunosuppressive agent (e.g., cyclosporin, azathioprine, methotrexate, my cophenolate, FK506 antibody, or glucocorticoids), an antagonist (e.g., one or more of a PD-1 antagonist, a PD-L1 antagonist, CTLA4 antagonist, CD47 antagonist, SIRPa antagonist, CD40 agonists, CSF1/CSF1R antagonist, or a STING agonist), or an immunoablative agent (e.g., an antiviral therapy (e.g.,
  • compositions described herein are administered in combination with (e.g., before, simultaneously, or following) bone marrow transplantation or lymphocyte ablative therapy using a chemotherapy agent (e.g., fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or Rituxan).
  • a chemotherapy agent e.g., fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or Rituxan
  • subjects undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • subjects receive an infusion of a pharmaceutical composition comprising immune cells as described herein.
  • Pharmaceutical compositions described herein may be administered before or following surgery.
  • a dosage of any aforementioned therapy to be administered to a subject will vary with a disease, disorder, or condition being treated and based on a specific subject. Scaling of dosages for human administration can be performed according to art-accepted practices.
  • a dose of alemtuzumab will generally be about 1 mg to about 100 mg for an adult, usually administered daily for a period of between about 1 day to about 30 days, e.g., a daily dose of about 1 mg to about 10 mg per day (e.g., as described in U.S. Patent No. 6,120,766, which is hereby incorporated by reference in its entirety).
  • the present disclosure provides methods of treating a disease or disorder (e g., a disease or a disorder described herein) in a subject comprising providing (e.g. delivering) a modified cell, for example, in a pharmaceutical composition described herein.
  • a disease or disorder e.g., a disease or a disorder described herein
  • methods of treating a disease or disorder e.g., a disease or a disorder described herein in a subject comprising providing (e.g. delivering) nucleic acids encoding a CAR as described herein, for example, in a pharmaceutical composition described herein.
  • a therapeutically effective amount of a modified cell and/or pharmaceutical composition described herein is provided (e.g., administered) to a subject having a disease or disorder.
  • Pharmaceutical compositions as described herein can be for use in the manufacture of a medicament for treating a disease or disorder in a subject or stimulating an immune response in a subject.
  • a subject to be treated with 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).
  • modified immune cells e.g., stem cells, macrophages, monocytes, or dendritic cells
  • Pharmaceutical compositions as described herein can be administered to a subject in accordance with a dosage regimen described herein, alone or in combination with one or more therapeutic agents, procedures, or modalities.
  • Modified cells and/or pharmaceutical compositions described herein can be used to treat or prevent a disease associated with a tumor or cancer, a neurodegenerative disease or disorder, an inflammatory disease or disorder, a cardiovascular disease or disorder, a fibrotic disease or disorder, a disease associated with amyloidosis, and a combination of thereof.
  • a method of treating e g., one or more of reducing, inhibiting, or delaying progression of) a cancer or a tumor in a subject with a modified cell and/or pharmaceutical composition described herein is provided.
  • a subject can have an adult or pediatric form of cancer.
  • a cancer may be at an early, intermediate, or late stage, or a metastatic cancer.
  • a cancer can include, but is not limited to, a solid tumor, a hematological cancer (e.g., leukemia, lymphoma, or myeloma, e.g., multiple myeloma), or a metastatic lesion.
  • solid tumors include malignancies, e.g., sarcomas and carcinomas, e.g., adenocarcinomas of the various organ systems, such as those affecting the lung, breast, ovarian, lymphoid, gastrointestinal (e.g., colon), anal, genitals and genitourinary tract (e.g., renal, urothelial, bladder cells, prostate), pharynx, CNS (e.g., brain, neural or glial cells), head and neck, skin (e.g., melanoma, e.g., a cutaneous melanoma), pancreas, and bones (e.g., a chordoma).
  • malignancies e.g., sarcomas and carcinomas
  • carcinomas e.g., adenocarcinomas of the various organ systems, such as those affecting the lung, breast, ovarian, lymphoid, gastrointestinal (e.g., colon),
  • a cancer is selected from a lung cancer (e.g., a non-small cell lung cancer (NSCLC) (e.g., a non-small cell lung cancer (NSCLC) with squamous and/or non-squamous histology, or a NSCLC adenocarcinoma), or a small cell lung cancer (SCLC)), a skin cancer (e.g., a Merkel cell carcinoma or a melanoma (e.g., an advanced melanoma)), an ovarian cancer, a mesothelioma, a bladder cancer, a soft tissue sarcoma (e.g., a hemangiopericytoma (HPC)), a bone cancer (a bone sarcoma), a kidney cancer (e.g., a renal cancer (e.g., a renal cell carcinoma)), a liver cancer (e.g., a hepatocellular carcinoma), a lung cancer (e.g.,
  • a cancer is a brain tumor, e.g., a glioblastoma, a gliosarcoma, or a recurrent brain tumor.
  • a cancer is a pancreatic cancer, e.g., an advanced pancreatic cancer.
  • a cancer is a skin cancer, e.g., a melanoma (e.g., a stage II-IV melanoma, an HLA-A2 positive melanoma, an unresectable melanoma, or a metastatic melanoma), or a Merkel cell carcinoma.
  • a cancer is a renal cancer, e.g., a renal cell carcinoma (RCC) (e.g., a metastatic renal cell carcinoma).
  • RCC renal cell carcinoma
  • a cancer is a breast cancer, e.g., a metastatic breast carcinoma or a stage IV breast carcinoma, e.g., a triple negative breast cancer (TNBC).
  • TNBC triple negative breast cancer
  • a cancer is a virus-associated cancer.
  • a cancer is an anal canal cancer (e.g., a squamous cell carcinoma of the anal canal).
  • a cancer is a cervical cancer (e.g., a squamous cell carcinoma of the cervix).
  • a cancer is a gastric cancer (e.g., an Epstein Barr Virus (EBV) positive gastric cancer, or a gastric or gastro-esophageal junction carcinoma).
  • a cancer is a head and neck cancer (e.g., an HPV positive and negative squamous cell cancer of the head and neck (SCCHN)).
  • a cancer is a nasopharyngeal cancer (NPC).
  • a cancer is a colorectal cancer, e.g., a relapsed colorectal cancer, a metastatic colorectal cancer, e g., a microsatellite unstable colorectal cancer, a microsatellite stable colorectal cancer, a mismatch repair proficient colorectal cancer, or a mismatch repair deficient colorectal cancer.
  • a cancer is a hematological cancer.
  • a cancer is a leukemia, e.g., acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic leukemia, or acute leukemia.
  • a cancer is a lymphoma, e.g., Hodgkin lymphoma (HL), non-Hodgkin's lymphoma, lymphocytic lymphoma, or diffuse large B cell lymphoma (DLBCL) (e.g., a relapsed or refractory HL or DLBCL).
  • a cancer is a myeloma, e.g., multiple myeloma.
  • Modified cells and/or pharmaceutical compositions described herein can be used to enhance or modulate an immune response in a subject.
  • a pharmaceutical composition described herein enhances, stimulates, or increases an immune response in a subject (e.g., a subject having, or at risk of, a disease or disorder described herein).
  • a subject is, or is at risk of being, immunocompromised. For example, a subject is undergoing or has undergone a chemotherapeutic treatment and/or radiation therapy.
  • a subject has, or is at risk of, developing an inflammatory disorder (e.g., a chronic or acute inflammatory disorder). In some embodiments, a subject has, or is at risk, of developing an autoimmune disease or disorder.
  • an inflammatory disorder e.g., a chronic or acute inflammatory disorder.
  • a subject has, or is at risk, of developing an autoimmune disease or disorder.
  • autoimmune diseases that can be treated with methods described herein include, but are not limited to, Alzheimer's disease, asthma (e.g., bronchial asthma), an allergy (e.g., an atopic allergy), Acquired Immunodeficiency Syndrome (AIDS), atherosclerosis, Behcet's disease, celiac, cardiomyopathy, Crohn's disease, cirrhosis, diabetes, diabetic retinopathy, eczema, fibromyalgia, fibromyositis, glomerulonephritis, graft vs.
  • asthma e.g., bronchial asthma
  • an allergy e.g., an atopic allergy
  • AIDS Acquired Immunodeficiency Syndrome
  • GVHD host disease
  • GVHD host disease
  • multiple sclerosis multiple sclerosis
  • myasthenia gravis osteoarthritis
  • polychondritis psoriasis
  • rheumatoid arthritis sepsis
  • stroke vasculitis
  • ventilator-induced lung injury transplant rejection
  • Raynaud's phenomena Reiter's syndrome
  • rheumatic fever sarcoidosis
  • scleroderma Sjogren's syndrome
  • ulcerative colitis uveitis
  • vitiligo vitiligo
  • Wegener's granulomatosis granulomatosis
  • modified cells and/or pharmaceutical compositions described herein may be carried out in any convenient manner (e.g., injection, ingestion, transfusion, inhalation, implantation, or transplantation).
  • a modified cell and/or pharmaceutical compositions described herein is administered by injection or infusion.
  • Modified cells and/or pharmaceutical compositions described herein may be administered to a patient transarterially, subcutaneously, intravenously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, or intraperitoneally.
  • a modified cell and/or pharmaceutical composition described herein is administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or intramuscularly).
  • a modified cell and/or pharmaceutical composition described herein is administered by intravenous infusion or injection. In some embodiments, a modified cell and/or pharmaceutical composition described herein is administered by intramuscular or subcutaneous injection. Pharmaceutical compositions described herein may be injected directly into a site of inflammation, a local disease site, a lymph node, an organ, a tumor, or site of infection in a subject.
  • the present Example assesses cell viability, CAR expression, M1/M2 phenotypic marker expression, tumor killing function, and cytokine secretion in macrophages expressing anti-HER2 CAR constructs comprising a DAP 10 domain.
  • Macrophages were thawed and plated at a density of 2e6 cells per well in 6-well UpCell plates in 2 mL TexMacs + 10% FBS + l%Pen/Strep + 10 ng/ml GM-CSF (complete media; CM). After 2-3 hours or rest, VPX lentivirus particles were diluted into CM and added to macrophages at specified MOIs. Media was fully exchanged 24 hours post-lentivirus addition. Cells were tested 6-7 days post-transduction.
  • culture plates were placed at 4 °C for 30 minutes. Cells were removed from the plate, pelleted, and resuspended in CM. Cell counts were taken using an NC-200 or Vi-Cel Blue and macrophages were used for experiments.
  • M1/M2 phenotypic marker expression in macrophages expressing DAP 10 CAR constructs is shown in Figure 4. No differences were observed in M1/M2 skewing of macrophages expressing DAP 10 CAR constructs relative to control constructs and cells. Without wishing to be bound by any particular theory, the present application puts forth that increases in Ml phenotypic marker expression may be attributed to, at least in part, lentiviral addition and not CAR construct expression alone.
  • tumor killing function e.g., cytotoxicity
  • macrophages were plated at different concentrations in 100 pL TexlO in wells of a 96 well flat-bottom dish. Macrophages were incubated for 30-45 minutes at 37 °C, followed by addition of tumor cells at different concentrations in equivalent volumes. Final volume for all wells was 200 pL TexlO.
  • Tumor cells e.g., AU565 cells
  • HER2 and Mesothelin antigens were diluted in PBS to a concentration of 28 nM. 100 pL of antigen solution or control PBS was added to wells and incubated at 4 °C for 24 hours so that the antigens passively bound to the plate. Wells were then washed twice with PBS and 50,000 macrophages were added to each well. Plates were incubated for 24 hours, followed by centrifugation at 300xg for 5 minutes. Supernatant was removed and stored at -20 °C. Cytokine levels were assessed using Proinflammatory Human Kit and Chemokine Human Kit from MSD following standard protocol instructions.
  • CARs including a DAP 10 intracellular domain are able to be expressed in immune cells, enhance tumor cell killing, and modulate the expression of pro-inflammatory cytokines from modified cells.
  • Example 2 Functional effects of DAP12 CAR constructs in macrophages
  • the present Example assesses cell viability, CAR expression, M1/M2 phenotypic marker expression, tumor killing function, and cytokine secretion in macrophages expressing anti-HER2 CAR constructs comprising a DAP 12 domain.
  • Macrophages were thawed and plated at a density of 2e6 cells per well in 6-well UpCell plates in 2 mL TexMacs + 10% FBS + l%Pen/Strep + 10 ng/ml GM-CSF (complete media; CM).
  • VPX lentivirus particles were diluted into CM and added to macrophages at specified MOIs. Media was fully exchanged 24 hours post-lentivirus addition. Cells were tested 6-7 days post-transduction.
  • culture plates were placed at 4 °C for 30 minutes. Cells were removed from the plate, pelleted, and resuspended in CM. Cell counts were taken using an NC-200 or Vi-Cel Blue and macrophages were used for experiments.
  • FIG. 8 Cell recovery and viability are shown in Figure 8. Recovery is defined as number of live cells post-lifting on day 6 or 7 divided by total number of cells initially seeded for transduction with lentivirus at day 0. Viability is defined as percentage of events captured via flow cytometry that are negative for viability dye. DAP 12 CAR constructs were not found to have major impact on macrophage recovery or viability. Expression of DAP12 CAR constructs is shown via histograms in Figure 9A. Certain DAP12 CAR constructs (e.g., DAP12 H/TM/ICD) were observed to have a better defined peak relative to other standard CAR constructs. Quantification of expression of DAP 12 CAR constructs, both in terms of percentage of cells expressing CAR construct and mean fluorescence intensity, is shown in Figure 9B. DAP 12 CAR constructs were found to express at macrophage cell surface.
  • DAP 12 CAR constructs were found to express at macrophage cell surface.
  • M1/M2 phenotypic marker expression in macrophages expressing DAP12 CAR constructs is shown in Figure 10. No differences were observed in M1/M2 skewing of macrophages expressing DAP 12 CAR constructs relative to control constructs and cells. Without wishing to be bound by any particular theory, the present application puts forth that increases in Ml phenotypic marker expression may be attributed to, at least in part, lentiviral addition and not CAR construct expression alone.
  • tumor killing function e.g., cytotoxicity
  • macrophages were plated at different concentrations in 100 pL TexlO in wells of a 96 well flat-bottom dish. Macrophages were incubated for 30-45 minutes at 37 °C, followed by addition of tumor cells at different concentrations in equivalent volumes. Final volume for all wells was 200 pL TexlO.
  • Tumor cells e.g., AU565 cells
  • HER2 and Mesothelin antigens were diluted in PBS to a concentration of 28 nM. 100 pL of antigen solution or control PBS was added to wells and incubated at 4 °C for 24 hours so that the antigens passively bound to the plate. Wells were then washed twice with PBS and 50,000 macrophages were added to each well. Plates were incubated for 24 hours, followed by centrifugation at 300xg for 5 minutes. Supernatant was removed and stored at -20 °C. Cytokine levels were assessed using Proinflammatory Human Kit and Chemokine Human Kit from MSD following standard protocol instructions.
  • CARs including a DAP 12 intracellular domain are able to be expressed in immune cells, enhance tumor cell killing, and modulate the expression of pro-inflammatory cytokines from modified cells.
  • the present Example assesses cell viability, CAR expression, M1/M2 phenotypic marker expression, tumor killing function, and cytokine secretion in macrophages expressing anti-HER2 CAR constructs comprising a DAP10 domain with costimulation domains (e.g., CD40 and/or Myd88 domains).
  • costimulation domains e.g., CD40 and/or Myd88 domains.
  • Macrophages were thawed and plated at a density of 2e6 cells per well in 6-well UpCell plates in 2 mL TexMacs + 10% FBS + l%Pen/Strep + 10 ng/ml GM-CSF (complete media; CM).
  • VPX lentivirus particles were diluted into CM and added to macrophages at specified MOIs. Media was fully exchanged 24 hours post-lentivirus addition. Cells were tested 6-7 days post-transduction.
  • Cell viability and recovery are shown in Figure 15 and Figure 28. Viability is defined as percentage of events captured via flow cytometry that are negative for viability dye. Recovery is defined as number of live cells post-lifting on day 6 or 7 divided by total number of cells initially seeded for transduction with lentivirus at day 0. DAP10 CAR constructs were not found to have major impact on macrophage recovery or viability. Expression of DAP10 CAR constructs is quantified and shown in Figure 16 and Figure 29.
  • FIG. 17 and Figure 30 Representative M1/M2 phenotypic marker expression in macrophages expressing DAP10 CAR constructs is shown in Figure 17 and Figure 30.
  • CAR constructs comprising a Myd88 domain affected an Ml-skewed phenotype (e.g., as characterized by increased expression of Ml markers and decreased expression of M2 markers).
  • tumor killing function e.g., cytotoxicity
  • macrophages were plated at different concentrations in 100 pL TexlO in wells of a 96 well flat-bottom dish.
  • Macrophages were incubated for 30-45 minutes at 37 °C, followed by addition of tumor cells at different concentrations in equivalent volumes. Final volume for all wells was 200 pL TexlO.
  • Tumor cells e.g., AU565, Panel, or MDA468 cells
  • AU565 cells express HER2 protein whereas Panel and MDA468 cells do not express HERZ protein. Plates were then incubated at room temperature for 15 minutes after addition of tumor cells, followed by 45 minutes incubation at 37°C. GFP expression was then assessed in these co-cultures using an Incucyte.
  • HERZ and Mesothelin antigens were diluted in PBS to a concentration of 28 nM. 100 pL of antigen solution or control PBS was added to wells and incubated at 4°C for 24 hours so that the antigens passively bound to the plate. Wells were then washed twice with PBS and 50,000 macrophages were added to each well. Plates were incubated for 24 hours, followed by centrifugation at 300xg for 5 minutes. Supernatant was removed and stored at -20°C. Cytokine levels were assessed using Proinflammatory Human Kit and Chemokine Human Kit from MSD following standard protocol instructions.
  • CTX 1364 (DAP10-M88-CD40) and CTX 1366 (DAP10-M88) expression led to
  • CTX_1364 Ml polarization. Robust tumor cell killing by macrophages expressing CTX_1364 was observed at low E:T ratios but tapered off at very low E:T ratios (e.g., 1 : 16). CTX_1364 had some activity against HERZ- Panel cells but not HERZ- MDA468 cells. CTX 1364 exhibited increased target-specific cytokine secretion, but might also possess tonic activity. CTX_1366 also shows increased cytokine secretion but to a lesser extent.
  • the present Example assesses cell viability, CAR expression, and CAR activity with DAP10 CAR constructs comprising Myd88 domains containing different mutations. Exemplary CAR constructs, and mutations investigated, utilized in experiments described herein are shown in Figure 22.
  • Cell viability is shown in Figure 24. Viability is defined as percentage of events captured via flow cytometry that are negative for viability dye. Expression of DAP 10 CAR constructs is quantified and shown in Figure 25.
  • NFKB activation is shown in Figure 26. Mutant Myd88 domains were observed to reduce tonic signaling in Hek reporter cells.
  • Example 5 Viability, Receptor Expression, and M1/M2 Surface Expression of DAP10 CAR Macrophages comprising a Myd88 ICD mutant variant
  • the present Example assesses tumor killing function and cytokine secretion in macrophages expressing anti-HER2 CAR constructs comprising a DAP 10 extracellular hinge domain and transmembrane domain with costimulation domains, such as a Myd88 mutant variant ICD. Macrophages were transduced with CAR constructs as described in Tables 4-6.
  • Macrophages were thawed and plated at a density of 2 x 10 6 cells per well in 6- well UpCell plates in 2 mL TexMacs with 10% FBS, 1% Pen/Strep, 10 ng/ml GM-CSF. After 2- 3 hours of rest, VPX lentivirus particles were diluted into TexMacs media and added to macrophages at specified multiplicity of infection (MOIs). Media was fully exchanged 24 hours post-1 enti virus addition. Cells were then tested 6-7 days post-transduction.
  • MOIs multiplicity of infection
  • the desired number of macrophages were plated in a volume of 100 pL TexlO in a 96 well flat-bottom dish. These macrophages were then incubated for a duration of 30-45 minutes at 37°C, followed by addition of the desired number of tumor cells in a volume of 100 pL TexlO, resulting in a final volume for all wells of 200 pL TexlO.
  • the tumor cells (AU565 and Panel) were engineered prior to the experiment to express nuclear localized GFP.
  • DAP10 CAR constructs comprising a Myd88 ICD mutation e.g., CTX_1504, CTX_1505, CTX_1510, CTX_1511, CTX 519, CTX_1522, and CTX 523) showed diminished tumor burden relative to their non-mutated counterparts ( Figure 37). These results demonstrate how DAP 10 CAR constructs comprising Myd88 mutated variants are capable of diminishing tumor burden and in some cases are likely superior to their non-mutated counterparts.
  • HER2 and Mesothelin antigens were diluted in PBS to a concentration of 28 nM, then 100 pL of antigen solution or control PBS was added to wells and incubated at 4°C for 24 hours so that the antigens passively bound to the plate. Wells were then washed twice with PBS, and 50,000 macrophages were added to each well. Plates were incubated for 24 hours, followed by centrifugation at 300xg for 5 minutes. Supernatant was removed and stored at -20 °C. Cytokine levels were assessed using Proinflammatory Human Kit and Chemokine Human Kit (Meso Scale Discovery) following standard protocol instructions.
  • Example 7 Tonic Signaling of CAR constructs comprising a Myd88 mutant variant ICD and FcRy ICD [0400]
  • CAR-expressing HEK cells were generated using the methods described in Example 4 for analysis of tonic signaling, except for the plates used were not coated with an antigen.
  • HEK Nulll reporter cells were lipofected with CAR constructs described above and shown in Tables 7-9.
  • NFkb activation is shown in Figure 41. Mutant Myd88 domains were observe to reduce tonic signaling in HEK reporter cells.
  • Example 8 Viability, Receptor Expression, and M1/M2 Surface Expression of CAR Macrophages comprising a Myd88 mutant variant ICD and FcRy
  • macrophages with CARs comprising a CD8 or CD28 hinge and transmembrane with a Myd88 mutant variant ICD and FcRy (also referred to herein as “FCER1GIC”) ICD were evaluated for CAR expression, viability, and surface marker phenotype.
  • human macrophages underwent culturing conditions and lentiviral transduction, as explained above. Transduction of macrophages was performed with the CAR constructs shown in Table 7-9.
  • DAP 10 CAR constructs comprising a Myd88 mutant variant ICD were compared to CAR macrophages with a CD8 or CD28 hinge and transmembrane domains for their tumor cell killing function and cytokine release.
  • the desired number of macrophages were plated in a volume of 100 pL Texl 0 in a 96 well flatbottom dish. These macrophages were then incubated for a duration of 30-45 minutes at 37°C, followed by addition of the desired number of tumor cells in a volume of 100 pL TexlO, resulting in a final volume for all wells of 200 pL TexlO.
  • the tumor cells, AU565, were engineered prior to the experiment to express nuclear localized GFP.
  • a cytokine release assay was performed to compare the effects of these CAR constructs.
  • a HER2 or mesothelin antigen was resuspended in PBS to a concentration of 28 nM.
  • 100 pL of either antigen solution was added to wells in a 96-well flat bottom plate and incubated at 37°C for 2 hours or 4°C overnight.
  • PBS control wells that contained only PBS were also incubated. After incubation, wells were washed two times with 200 pL PBS, followed by the addition of 50,000 macrophages from each condition to a final volume of 200 pL TexlO.
  • CAR constructs with a Myd88 truncated mutant variant of R32A or R32K (e.g., CTX_1506, CTX_1507, CTX_1704, CTX_1711, and CTX_1713) all had notably high cytokine secretion of TNFa and IL-8 ( Figures 46A-B).
  • CAR constructs comprising a DAP10, CD8, or CD28 hinge and transmembrane domain with a Myd88 R32A or R32K mutant variant domain in maintaining high potentiation post-activation without the hindrance of background cytokine secretion.

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Abstract

La présente divulgation concerne des cellules immunitaires modifiées comprenant des récepteurs antigéniques chimériques (CAR) comprenant un ou plusieurs domaines intracellulaires comprenant un domaine DAP 10 ou une partie de celui-ci ou un domaine DAP 12 ou une partie de celui-ci et des procédés d'utilisation et de fabrication de cellules immunitaires comprenant lesdits CAR.
PCT/US2024/015509 2023-02-14 2024-02-13 Récepteurs antigéniques chimériques comprenant des domaines dap 10 et dap 12 et des cellules immunitaires modifiées Ceased WO2024173329A2 (fr)

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