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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
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  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/17—Monocytes; Macrophages
• • A—HUMAN NECESSITIES
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  • 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
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4254—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
  • A61K40/4255—Mesothelin [MSLN]
• • A—HUMAN NECESSITIES
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  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
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  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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  • 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
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
• • 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)
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  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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  • 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
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  • C12N2510/00—Genetically modified cells
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
  • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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  • 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

• compositions comprising modified immune cells (e.g., stem cells, macrophages, monocytes, and/or dendritic cells) comprising anti-mesothelin chimeric antigen receptors (CARs) and methods of producing and using the same.
• modified immune cells e.g., stem cells, macrophages, monocytes, and/or dendritic cells
• CARs anti-mesothelin chimeric antigen receptors
• the present disclosure provides modified immune cells comprising a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) an extracellular domain; (b) a transmembrane domain; and (c) one or more intracellular domains; wherein the extracellular domain is or comprises an anti-mesothelin antigen binding domain comprising an amino acid sequence that is at least 80% identical to a sequence selected from Table 3; and wherein the modified immune cell is or comprises a macrophage, monocyte, dendritic cell, or stem cell.
• CAR chimeric antigen receptor
• an extracellular domain is or comprises an scFv, VHH antibody, centyrin, darpin, or nanobody.
• a transmembrane domain is or comprises a CD8, 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.
• one or more intracellular domains comprise: a CD3 ⁇ FcRy, MyD88, CD40, CD64, CD32a, CD32c, CD16a, CD89, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD19, CD20, 41BB, CD28, GCSFR (CD114), RAGE, CD30, CD 160, DR3, Fnl4, HVEM, CD 160, NGFR, RANK, TNFR2, TROY, XEDAR, TRIF, 0X40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, V/I/LxYxxL/V, SIRPa, CD
• 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.
• 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 a 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.
• Figure 4 shows a graph of exemplary anti-mesothelin mediated phagocytosis by macrophages after electroporation with mRNA encoding 4 different anti-mesothelin binders (Ml 1, M14, M15, and M17) that were used in a CD8 framework CAR.
• Figure 5 shows a graph of exemplary macrophage viability after transduction with varying exemplary MOIs of Ad5f35 vector comprising CTX_269 (an anti-mesothelin CAR).
• Figure 6 shows graphs of exemplary anti-mesothelin CAR expression after transduction of macrophages with varying exemplary MOIs of Ad5f35 vector comprising CTX_269 (an anti-mesothelin CAR).
• Figure 7A, Figure 7B, and Figure 7C shows graphs of exemplary expression of exemplary Ml -associated markers (CD80, CD86, and HLA-DR) after transduction of macrophages with varying exemplary MOIs of Ad5f35 vector comprising CTX_269 (an anti- mesothelin CAR).
• exemplary Ml -associated markers CD80, CD86, and HLA-DR
• CTX_269 an anti- mesothelin CAR
• Figure 9 shows graphs of exemplary M2 -associated marker (CD 163) expression after transduction of macrophages with Ad5f35 vector comprising CTX 269 (an anti-mesothelin CAR).
• Figure 15 shows graphs of exemplary anti-mesothelin mediated killing of mesothelin-expressing ovarian cystadenocarcinoma cells by macrophages after transduction of the macrophages with Ad5f35 vector comprising CTX_269 (an anti-mesothelin CAR).
• Figure 17 shows graphs of exemplary cytokine (TNFa) release after transduction of macrophages with Ad5f35 vector comprising CTX 269 (an anti-mesothelin CAR) and exposure of the macrophages to target cells (A549 lung adenocarcinoma cells or MES-OV ovarian cystadenocarcinoma cells) expressing mesothelin.
• CTX 269 an anti-mesothelin CAR
• Figure 19 shows exemplary graphs of tumor burden in mice treated with macrophages transduced with Ad5f35 vector comprising CTX 269 (an anti-mesothelin CAR).
• Figure 20 shows graphs of exemplary anti-mesothelin CAR expression after transduction of macrophages with lentivirus vector comprising a CD28-based anti-mesothelin CAR or with lentivirus vector comprising a CD8-based anti-mesothelin CAR.
• Figure 21 shows a graph of exemplary target cell killing mediated by macrophages after transduction of macrophages with lentivirus vector comprising a CD28-based anti-mesothelin CAR or with lentivirus vector comprising a CD8-based anti-mesothelin CAR.
• Figure 22 shows graphs of exemplary cytokine (TNFa) release after transduction of macrophages with lentivirus vector comprising a CD28-based anti-mesothelin CAR or with lentivirus vector comprising a CD8-based anti-mesothelin CAR and exposure of the transduced macrophages to mesothelin.
• TNFa cytokine
• Figure 29 shows schematics of exemplary anti-mesothelin CAR constructs comprising an Ml 7 scFv.
• Figure 30 shows graphs of exemplary anti-mesothelin CAR expression on days 2 and 14 after transduction of macrophages with Ad5f35 vector comprising CTX 269 (an anti- mesothelin CAR).
• Figure 31 shows graphs of exemplary anti-mesothelin mediated phagocytosis of K562 cells by macrophages after transduction of macrophages with Ad5f35 vector comprising CTX_269 (an anti-mesothelin CAR).
• 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 carb oxy -terminal CH3 (located at the base of the Y’s stem).
• VH amino-terminal variable
• CH2 amino-terminal variable
• CH3 carb oxy -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. In some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc, as is known in the art.
• 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 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); camel oid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPsTM”); single chain or Tandem diabodies (TandAb®); V
• masked antibodies e.g., Probodies®
• SIPsTM Small Modular ImmunoPharmaceuticals
• 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 As used herein, the term “antigen” or “Ag” refers to a molecule that is capable of provoking an immune response.
• This immune response may involve either antibody production, the activation of specific immunologically-competent cells, or both.
• any macromolecule including virtually all proteins or peptides, can serve as an antigen.
• antigens can be derived from recombinant or genomic DNA.
• any DNA that comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response encodes an “antigen” as that term is used herein.
• an antigen need not be encoded solely by a full length nucleotide sequence of a gene.
• 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.
• 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.
• 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.
• 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.
• 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 moieties found in the whole structure. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole.
• 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
• 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.
• Isolated refers to something altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
• Lentivirus As used herein, the term “lentivirus” refers to a genus of the Retroviridae family.
• 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.
• a polypeptide may comprise D-amino acids, L- amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof.
• a polypeptide may be cyclic, and/or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and/or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides.
• 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 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.
• 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.
• 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.
• modified immune cells described herein comprising or expressing CARs described herein exhibit increased viability, e.g., relative to a modified immune cell of the same type comprising a similar CAR (e.g., a CAR comprising a different anti- mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• a similar CAR e.g., a CAR comprising a different anti- mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR.
• a CAR described herein comprises: (a) an extracellular domain comprising an anti-mesothelin binding domain as described herein; (b) a transmembrane domain (e.g., a CD28 transmembrane domain or CD8 transmembrane domain); and (c) one or more intracellular domains.
• one or more intracellular domains comprise a CD3zeta (CD3Q intracellular domain.
• one or more intracellular domains comprise an FcRy intracellular domain.
• a CAR further comprises one or more extracellular hinge domains.
• one or more extracellular hinge domains comprise a CD28 extracellular hinge domain or a CD8a extracellular hinge domain.
• a CAR described herein comprises, from N-terminus to C-terminus: a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a MyD88 intracellular domain, a CD40 intracellular domain, and a CD3( ⁇ intracellular domain.
• a CAR described herein comprises, from N-terminus to C-terminus: a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a truncated MyD88 intracellular domain, a CD40 intracellular domain, and a CD3 ⁇ intracellular domain.
• a CAR described herein comprises, from N-terminus to C-terminus: a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, an FcRy intracellular domain, a P2A cleavage peptide, and a CD40 ligand (CD40L).
• a CAR described herein comprises, from N-terminus to C-terminus: a CD8a leader domain, an anti-mesothelin antigen binding domain, a CD28 extracellular hinge domain, a CD28 transmembrane domain, a MyD88 intracellular domain, and an FcRy intracellular domain.
• 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)
• PBS phosphate buffered saline
• enrichment of immune cells comprises plastic adherence.
• differentiation of immune cells comprises stimulation with GM-CSF.
• modified immune cells e.g., stem cells, macrophages, monocytes, or dendritic cells
• a modified immune cell described herein exhibits increased anti-tumor activity relative to an unmodified cell of the same type.
• one or more pro-inflammatory agents comprises or is a CD40 agonist (e.g., CD40L).
• one or more pro-inflammatory agents comprises or is a 41BB-ligand agonist (e.g., 4- IBB).
• one or more pro-inflammatory agents comprises or is a CD40 agonist (e.g., CD40L) and a 41BB-ligand agonist (e.g., 4-1BB).
• CD40 agonist e.g., CD40L
• 41BB-ligand agonist e.g., 4-1BB.
• the disclosure provides methods of treating a disease or disorder in a subject, comprising: delivering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a modified macrophage, monocyte, or dendritic cell described herein.
• the disclosure also provides methods of modifying immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein comprising a CAR described herein, wherein the method comprises treating an immune cell described herein with one or more pro-inflammatory agents, thereby producing a modified immune cell described herein that exhibits increased anti-tumor activity relative to an immune cell of the same type comprising the CAR or a similar CAR that has not been treated with one or more pro-inflammatory agents.
• one or more pro-inflammatory agents comprises or is a CD40 agonist (e.g., CD40L).
• 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.
• a macrophage comprises or is an undifferentiated or MO macrophage.
• a macrophage comprises or expresses one, two, three, four, five, or six of CD14, CD16, CD64, CD68, CD71, or CCR5. Exposure to various stimuli can induce MO macrophages to polarize into several distinct populations, which may be identified by macrophage phenotype markers, cytokine production, and/or chemokine secretion.
• a macrophage comprises or is a polarized macrophage.
• MO macrophages can be exposed to pro-inflammatory signals, such as LPS, IFNy, 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 Thl7 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 at least one CAR 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, IFNa, IFN , IFNy, 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; eg., one of the CX3C chemokines, e.g., one or both of the C chemokines), e.g.,
• a macrophage comprising or expressing at least one CAR described herein stimulates an immune response and/or inflammation, e.g., relative to a macrophage comprising a similar CAR (e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• a similar CAR e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR.
• a macrophage comprising or expressing at least one CAR described herein decreases an immune response in a subject, e.g., relative to a macrophage comprising a similar CAR (e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• 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 at least one CAR described herein exhibits induction of cell survival mechanisms, e.g., relative to a macrophage comprising a similar CAR (e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• a similar CAR e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR.
• a macrophage comprising or expressing at least one CAR described herein exhibits induction of cell death mechanisms, e.g., relative to a macrophage comprising a similar CAR (e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• a similar CAR e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR.
• a monocyte comprises or expresses one or more phenotypic markers.
• 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, CD
• 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 differenti tes into a macrophage.
• a monocyte comprising or expressing at least one CAR 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 comprising a similar CAR (e g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• 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 a similar CAR e
• a monocyte comprising or expressing at least one CAR described herein exhibits increased phagocytosis, e.g., relative to a monocyte comprising a similar CAR (e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• a similar CAR e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR.
• a monocyte comprising or expressing at least one CAR described herein exhibits enhanced differentiation into macrophages (e.g., Ml or M2 macrophages), e.g., relative to a monocyte comprising a similar CAR (e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• macrophages e.g., Ml or M2 macrophages
• a similar CAR e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR.
• a monocyte comprising or expressing at least one CAR described herein exhibits enhanced differentiation into DCs (e.g., resident or migrating DCs and/or in lymphoid and nonlymphoid tissue), e.g., relative to a monocyte comprising a similar CAR (e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• DCs e.g., resident or migrating DCs and/or in lymphoid and nonlymphoid tissue
• a monocyte comprising a similar CAR e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii)
• a monocyte comprising or expressing at least one CAR described herein exhibits increased tumor antigen presentation (e.g., post-phagocytosis presentation) and/or increased antigen processing, e.g., relative to a monocyte comprising a similar CAR (e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• tumor antigen presentation e.g., post-phagocytosis presentation
• antigen processing e.g., relative to a monocyte comprising a similar CAR (e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembr
• a monocyte comprising or expressing at least one CAR described herein exhibits increased production of ROS, e.g., relative to a monocyte without a CAR described herein.
• a monocyte comprising or expressing at least one CAR described herein exhibits metabolic reprogramming, e.g., relative to a monocyte comprising a similar CAR (e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• Exemplarily phenotypic markers for DCs include, but are not limited to, CD11c, CD83, CDla, CDlc, CD141, CD207, CLEC9a, CD123, CD85, CD180, CD187, CD205, CD281, CD282, CD284, CD286 and partially CD206, CD207, CD208 and CD209.
• 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.
• modification of a DC cell to comprise or express at least one CAR described herein can allow mature DCs to simultaneously exhibit increased antigen capture ability and T cell stimulation, e.g., relative to a DC comprising a similar CAR (e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• a similar CAR e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR.
• a DC comprising or expressing at least one CAR described herein mediates tumor antigen presentation, e.g., increased tumor antigen presentation relative to a DC comprising a similar CAR (e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• a similar CAR e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR.
• a DC comprising or expressing at least one CAR described herein exhibits one or both of increased expression of favorable genes or decreased expression of unfavorable genes, e.g., relative to a DC comprising a similar CAR (e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• a similar CAR e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR.
• a DC comprising or expressing at least one CAR described herein exhibits increased production of ROS, e.g., relative to a DC comprising a similar CAR (e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• a similar CAR e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR.
• a DC comprising or expressing at least one CAR described herein exhibits metabolic reprogramming, e.g., relative to a DC comprising a similar CAR (e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• a similar CAR e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR.
• a DC comprising or expressing at least one CAR described herein exhibits induction of cell survival mechanisms, e.g., relative to a DC comprising a similar CAR (e.g., a CAR comprising a different anti- mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• a similar CAR e.g., a CAR comprising a different anti- mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR.
• a DC comprising or expressing at least one CAR described herein exhibits induction of cell death mechanisms, e.g., relative to a DC comprising a similar CAR (e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• a similar CAR e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR.
• a DC comprising or expressing at least one CAR 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 DC without a CAR described herein.
• ECM degrading enzymes e.g., MMPs to degrade tumor ECM and/or exhibit anti fibrotic activity
• a DC comprising or expressing at least one CAR 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 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 an intracellular domain.
• 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 an intracellular domain, 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 (i.e., an extracellular hinge domain). In some embodiments, a CAR comprises a spacer domain or hinge between an intracellular domain and a transmembrane domain (i.e., an intracellular hinge 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 an intracellular domain 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.
• the present disclosure also provides immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a nucleic acid (e g., an isolated nucleic acid) encoding a CAR, wherein the nucleic acid comprises a nucleic acid sequence encoding an extracellular domain, a nucleic acid sequence encoding a transmembrane domain and a nucleic acid sequence encoding an intracellular domain, wherein the cell is a stem cell, macrophage, monocyte or dendritic cell that expresses the CAR.
• a nucleic acid e.g., an isolated nucleic acid
• the nucleic acid comprises a nucleic acid sequence encoding an extracellular domain, a nucleic acid sequence encoding a transmembrane domain and a nucleic acid sequence encoding an intracellular domain
• the cell is a stem cell, macrophage, monocyte or dendritic cell that expresses the CAR.
• a CAR comprises an extracellular domain that is operably linked to another domain of the CAR, such as a transmembrane domain or an intracellular domain, 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 an intracellular domain.
• 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 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. In some embodiments, a variant or fragment does not comprise or express at least one intracellular domain (e.g., an immune cell comprises or expresses an anti-CD47 scFv, CD8 hinge domain, and CD8 transmembrane). In some embodiments, an immune cell described herein (e.g., comprising or expressing at least one CAR described herein) comprises a dominant negative receptor, e.g., blocking an inhibitory checkpoint.
• a payload comprising a CAR described herein further comprises a cleavage peptide (e.g., a P2A, F2A, E2A and/or T2A peptide) and/or at least one 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 CD8 hinge domain).
• at least one second CAR comprises: (i) a leader sequence (e.g., a CD8 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 CD8 leader
• an extracellular domain e.g., a SIRPa, CV1, 5F9 scFv, or B6H12 scFv (e.g., a humanized B6H12 scFv) extracellular domain
• a payload comprising 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 cleavage peptide e.g., a P2A, F2A, E2A and/or T2A peptide
• one or more phosphatase dead domains e.g. a phosphatase dead Shpl, phosphatase dead 72-5ptase (INPP5E), phosphatase dead Shp2, and
• a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 2 by no more than 50 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 2 by no more than 40 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 2 by no more than 30 substitutions, additions, or deletions.
• a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 2 by no more than 20 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 2 by no more than 10 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 2 by no more than 5 substitutions, additions, or deletions.
• a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 2 by no more than 4 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 2 by no more than 3 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 2 by no more than 2 substitutions, additions, or deletions.
• a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 2 by no more than 1 substitution, addition, or deletion. In some embodiments, a CAR of the present disclosure binds to mesothelin and comprises an amino acid sequence that does not have any substitutions, additions, or deletions relative to a sequence selected from Table 2.
• 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 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).
• 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 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, R0R1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD19, CD20, 41BB, CD28, 0X40, GITR, TREM-1, TREM-2, 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, P
• a CAR comprises an 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 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 mesothelin.
• a CAR antigen binding domain binds to mesothelin and comprises an amino acid sequence that is at least 80% identical to a sequence selected from Table 3. In some embodiments, a CAR antigen binding domain binds to mesothelin and comprises an amino acid sequence that is at least 85% identical to a sequence selected from Table 3. In some embodiments, a CAR antigen binding domain binds to mesothelin and comprises an amino acid sequence that is at least 90% identical to a sequence selected from Table 3. In some embodiments, a CAR antigen binding domain binds to mesothelin and comprises an amino acid sequence that is at least 95% identical to a sequence selected from Table 3.
• a CAR antigen binding domain binds to mesothelin and comprises an amino acid sequence that is at least 96% identical to a sequence selected from Table 3. In some embodiments, a CAR antigen binding domain binds to mesothelin and comprises an amino acid sequence that is at least 97% identical to a sequence selected from Table 3. In some embodiments, a CAR antigen binding domain binds to mesothelin and comprises an amino acid sequence that is at least 98% identical to a sequence selected from Table 3. In some embodiments, a CAR antigen binding domain binds to mesothelin and comprises an amino acid sequence that is at least 99% identical to a sequence selected from Table 3. In some embodiments, a CAR antigen binding domain binds to mesothelin and comprises an amino acid sequence identical to a sequence selected from Table 3.
• a CAR antigen binding domain binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 3 by no more than 10 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 3 by no more than 9 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 3 by no more than 8 substitutions, additions, or deletions.
• a CAR antigen binding domain binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 3 by no more than 7 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 3 by no more than 6 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 3 by no more than 5 substitutions, additions, or deletions.
• a CAR antigen binding domain binds to mesothelin and comprises an amino acid sequence that differs from a sequence selected from Table 3 by no more than 1 substitution, addition, or deletion. In some embodiments, a CAR antigen binding domain binds to mesothelin and comprises an amino acid sequence that does not have any substitutions, additions, or deletions relative to a sequence selected from Table 3.
• 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.
• such 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.
• such 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.
• 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).
• a CAR comprises a transmembrane domain, for example, that connects an extracellular domain to an intracellular domain.
• 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 membranebound 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).
• 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 an FcRy, CD64 (FcyRI), CD32a (FcyRIIa), CD32b (FcyRIIb), CD32c, CD16a (FcyRIIIa), CD16b (FcyRIIIb), FcaRI, FcaRII, or FcaRI (CD89) domain.
• 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 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.
• a CAR intracellular signaling domain is or comprises a human intracellular signaling domain, or portion thereof.
• a CAR 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 CAR 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 CAR intracellular signaling domains comprise a CD3zeta (CD3Q, FcRy, CD64, CD32a, CD32c, CD16a, CD40, 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, CD40, CD 19, CD20, 41BB, CD28, 0X40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, V/I/LxYxxL/V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BT
• 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, IFNa, IFNb, IFN-y, GMCSF, or MCSF), CD40-L, dominant negative SIRPa, dominant negative PD1, dominant negative CD45, dominant negative SIGLEC 10, or dominant negative LILRB.
• 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 binding domains, an FcR extracellular domain, an FcR transmembrane domain, and an FcR intracellular domain.
• a TLR extracellular domain, a TLR transmembrane domain and a TLR intracellular domain together comprise a full-length TLR.
• a TLR extracellular domain, a TLR transmembrane domain and a TLR intracellular domain together comprise portion of a full- length TLR.
• a TLR extracellular domain comprises a portion of a full- length TLR extracellular domain.
• a TLR transmembrane domain comprises a portion of a full-length TLR transmembrane domain.
• a TLR intracellular domain comprises a portion of a full-length TLR intracellular domain.
• 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.
• 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 CD14, 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).
• 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 comprise modified nucleotide comprising pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine/pseudouridine (5meC PsU), Nl-methyl- pseudouridine (NlmPsU), or combinations thereof.
• PsU pseudouridine
• MeC PsU 5-methoxyuridine
• NlmPsU Nl-methyl- pseudouridine
• 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
• a cap structure comprises AGCapl, m6AGCapl, or Anti -Reverse Cap Analog (ARC A).
• 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: 73) (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: 73 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 adeno
• 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: 74) (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 (e.g., with a payload comprising a CAR).
• a composition may comprise one or more additional payloads.
• 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
• 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 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 longevity of protein (e.g., at least one CAR described herein) 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 protein (e.g., at least one CAR described herein) 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., at least one CAR described herein) 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 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
• methods of the present disclosure comprise altering the inflammatory phenotype of a population of cells.
• methods of altering the inflammatory phenotype of a population of cells comprises contacting the population of cells with a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) as described herein.
• a population of cells comprises macrophages, monocytes, dendritic cells, T cells, NK cells, or combinations thereof.
• a modified immune cell of the present disclosure comprises one or more nucleic acids constructs comprising a promoter, a gene of interest, a 3’ untranslated region (UTR), and one or more introns, wherein the one or more introns comprise one or more inhibitory nucleic acids, wherein the one or more inhibitory nucleic acids encode one or more inhibitory RNAs, and, wherein the gene of interest encodes a chimeric antigen receptor (CAR).
• a modified immune cell of the present disclosure comprises a CAR and one or more inhibitory RNAs.
• a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
• 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 mesothelin.
• a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
• a modified immune cell comprising a modified mRNA encoding at least one CAR provided herein exhibits increased viability relative to a modified immune cell of the same type comprising a similar CAR (e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• a similar CAR e.g., a CAR comprising a different anti-mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• a modified immune cell e g., a stem cell, macrophage, monocyte, or dendritic cell
• a CAR as provided herein exhibits increased effector activity relative to a modified immune cell of the same type comprising unmodified mRNA encoding a similar CAR (e.g., a CAR comprising a different anti- mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR).
• a CAR comprising a different anti- mesothelin antigen binding domain and/or without one or both of (i) a CD8 or CD28 extracellular hinge domain, and (ii) a CD8 or CD28 transmembrane domain, but with the other components of the comparator CAR.
• a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
• a CAR as provided 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 provided herein maintains a pro-inflammatory phenotype at least 4 hours, 2 days, 4 days, 7 days, 14 days, 28 days, and/or 40 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 provided 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 provided herein with increasing concentrations of pro-inflammatory cytokines.
• a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
• a modified immune cell comprising a CAR as provided herein has minimal effects on neighboring cells.
• a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
• a modified immune cell comprising a CAR as provided 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 provided 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 provided herein comprises one or more control systems including, but not limited to: a safety switch (e.g., an on switch, an off switch, a suicide switch), transcriptional control (e.g. cell-specific promoters, cell-state specific promoters, promoters downstream of CAR activation, promoters downstream of endogenous signaling pathways, or drug-inducible transcription), post-transcriptional control of CAR mRNA (e g. RNA-based inhibition with endogenous or recombinant miRNA), or post-translational control of CAR structure or stability (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.
• a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
• a modified immune cell comprises one or more inhibitory RNAs selected from the group consisting of antisense RNA (asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), guide RNA (gRNA), long noncoding RNA (IncRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), trans-acting siRNA (tasiRNA), and repeat associated siRNA (rasiRNA).
• asRNA antisense RNA
• cis-NAT CRISPR RNA
• gRNA guide RNA
• IncRNA long noncoding RNA
• miRNA microRNA
• piwi-interacting RNA piRNA
• small interfering RNA small interfering RNA
• shRNA short hairpin RNA
• a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
• a modified immune cell comprises one or more shRNA.
• a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
• an miRNA scaffold comprises an miRNA-155 5’ scaffold, an miRNA-155 3’ scaffold, an miRNA-30 5’ scaffold, an miRNA-30 3’ scaffold, an miRNA- 16 5’ scaffold, an miRNA-16 3’ scaffold, an miRNA-125 5’ scaffold, an miRNA-125 3’ scaffold, an miRNA-223 5’ scaffold, or an miRNA-223 3’ scaffold.
• a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
• a guide strand is about 19-24 bases in length.
• a guide strand has a G/C content of about 36%-50%.
• a guide strand comprises a nucleic acid sequence that is reverse complementary to a target gene transcript comprising a target nucleic acid sequence.
• a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
• a modified immune cell comprises one or more shRNA comprising a passenger strand.
• a passenger strand is 1-2 bases shorter than a corresponding guide strand.
• a passenger strand is not fully complementary to the guide strand.
• a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
• a target gene transcript encoding human ATG7, C/EBP-alpha, C/EBP-beta, CD36, CLEC1A, FATS, GOLM1, HAVCR2, ITGAD, KLF4, KLF6, LILRB2, LILRB4, MAF, Maffi, PD-LI, PIK3CG, PIK3CG, PPARa, PPARy, PTGS2, SIGLEC10, SIRPa, SLC15A3, STAT3, STAT6, TNFRSF1B, TOX, TREM2, YTHDF2, or ZFP36.
• a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
• a target gene transcript encoding human SIRPa In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a target gene transcript encoding human PD-L 1.
• 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 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.
• 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
• the effect of a modified immune cell e.g., a stem cell, macrophage, monocyte, or dendritic cell
• an 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 e.g., a stem cell, macrophage, monocyte, or dendritic cell
• 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
• a CAR as described herein has significant cytotoxic effects on neighboring cells (e g., cancer cells).
• 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
• an effector cell i.e., a modified immune cell of the present disclosure (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a nucleic acid construct as described herein secretes one or more inhibitory RNAs of the present disclosure.
• one or more inhibitory RNAs are packaged within an extracellular vesicle.
• one or more inhibitory RNAs are packaged within an exosome.
• secreted inhibitory RNA affects the phenotype of bystander cells.
• bystander cells are bystander macrophages, bystander monocytes, bystander dendritic cells, or bystander stem cells. In some embodiments, bystander cells are skewed to an anti-tumor phenotype. In some embodiments, bystander cells are skewed to an antiinflammatory phenotype. In some embodiments, bystander macrophages are skewed to an Ml phenotype. In some embodiments, bystander macrophages are skewed to an M2 phenotype.
• a variety of assays may be performed to confirm the presence of a nucleic acid construct described herein and/or the presence of a protein (e.g., a CAR) in 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 (ELISAs and Western blots).
• Other assays of the present disclosure include, for example, fluorescence-activated cell sorting (FACS), immunofluorescent microscopy, MSD cytokine analysis, mass spectrometry (MS
• 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
• 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).
• all or a fragment of a protein (e.g., at least one 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.
• EF-lot 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.
• CMV immediate early cytomegalovirus
• PGK phosphoglycerokinase
• SV40 simian virus 40
• MMTV mouse mammary tumor virus
• HSV human immunodeficiency virus
• LTR human immunodeficiency virus
• 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 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).
• 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., at least one 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 a CAR, 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 CAR, e.g., at least one CAR described herein, and at least one nucleic acid encoding at least one gene co-expressed with a CAR, e.g., a cytokine described herein (e.g., TNF, IL- 12, IFN, GM-CSF, G-CSF, M-CSF, and/or IL-1) or a stimulatory ligand described herein (e.g., CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, ICOS-L, ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor, and/
• a CAR of the present disclosure binds to mesothelin and is encoded by a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 4. In some embodiments, a CAR of the present disclosure binds to mesothelin and is encoded by a nucleic acid sequence that is at least 85% identical to a sequence selected from Table 4. In some embodiments, a CAR of the present disclosure binds to mesothelin and is encoded by a nucleic acid sequence that is at least 90% identical to a sequence selected from Table 4.
• a CAR of the present disclosure binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 4 by no more than 40 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 4 by no more than 30 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 4 by no more than 20 substitutions, additions, or deletions.
• a CAR of the present disclosure binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 4 by no more than 10 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 4 by no more than 9 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 4 by no more than 8 substitutions, additions, or deletions.
• a CAR of the present disclosure binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 4 by no more than 7 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 4 by no more than 6 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 4 by no more than 5 substitutions, additions, or deletions.
• a CAR of the present disclosure binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 4 by no more than 4 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 4 by no more than 3 substitutions, additions, or deletions. In some embodiments, a CAR of the present disclosure binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 4 by no more than 2 substitutions, additions, or deletions.
• a CAR of the present disclosure binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 4 by no more than 1 substitution, addition, or deletion. In some embodiments, a CAR of the present disclosure binds to mesothelin and is encoded by a nucleic acid sequence that does not have any substitutions, additions, or deletions relative to a sequence selected from Table 4.
• a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 5. In some embodiments, a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that is at least 85% identical to a sequence selected from Table 5. In some embodiments, a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that is at least 90% identical to a sequence selected from Table 5.
• a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that is at least 95% identical to a sequence selected from Table 5. In some embodiments, a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that is at least 96% identical to a sequence selected from Table 5. In some embodiments, a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that is at least 97% identical to a sequence selected from Table 5.
• a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that is at least 98% identical to a sequence selected from Table 5. In some embodiments, a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that is at least 99% identical to a sequence selected from Table 5. In some embodiments, a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that is identical to a sequence selected from Table 5.
• a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 50 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 40 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 30 substitutions, additions, or deletions.
• a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 20 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 10 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 9 substitutions, additions, or deletions.
• a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 8 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 7 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 6 substitutions, additions, or deletions.
• a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 2 substitutions, additions, or deletions. In some embodiments, a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that differs from a sequence selected from Table 5 by no more than 1 substitution, addition, or deletion. In some embodiments, a CAR antigen binding domain binds to mesothelin and is encoded by a nucleic acid sequence that does not have any substitutions, additions, or deletions relative to a sequence selected from Table 5.
• the present disclosure provides nucleic acid molecules comprising a promoter, a gene of interest, a 3’ untranslated region (UTR), and one or more introns.
• one or more introns of the present disclosure comprise one or more inhibitory nucleic acids of the present disclosure.
• one or more inhibitory nucleic acids of the present disclosure encode one or more inhibitory RNAs of the present disclosure.
• a gene of interest encodes at least one chimeric antigen receptor (CAR) described herein or a fragment thereof.
• a modified immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure can comprise a nucleic acid molecule (e.g., an exogenous nucleic acid molecule) comprising one or more introns of the present disclosure and encoding at least one protein (e.g., a CAR of the present disclosure) described herein.
• the present disclosure provides nucleic acid molecules wherein expression of a CAR is increased relative to a reference nucleic acid molecule that lacks one or more introns.
• one or more introns of the present disclosure comprise one, two, or three introns. In some embodiments, one or more introns of the present disclosure are located downstream of a promoter. In some embodiments, one or more introns of the present disclosure are located within a gene of interest. In some embodiments, one or more introns of the present disclosure are located within a 3’ UTR. In some embodiments, one or more introns of the present disclosure are located within a gene of interest and within a 3’ UTR. In some embodiments, one or more introns of the present disclosure comprise three inhibitory nucleic acids. In some embodiments, three inhibitory nucleic acids encode three inhibitory RNAs.
• Inhibitory nucleic acids of the present disclosure comprise any nucleic acids that can bind a target messenger RNA (mRNA).
• mRNA target messenger RNA
• an inhibitory nucleic acid of the present disclosure comprises RNA.
• an inhibitory nucleic acid of the present disclosure comprises DNA.
• an inhibitory nucleic acid of the present disclosure is a DNA/RNA hybrid.
• an inhibitory nucleic acid of the present disclosure is an inhibitory RNA.
• an inhibitory RNA silences expression of a target gene via RNA interference.
• an inhibitory RNA is selected from the group consisting of antisense RNA (asRNA), cis-natural antisense transcript (cis-NAT), CRISPRRNA (crRNA), guide RNA (gRNA), long noncoding RNA (IncRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), trans-acting siRNA (tasiRNA), and repeat associated siRNA (rasiRNA).
• an inhibitory RNA is an shRNA.
• an shRNA of the present disclosure comprises a stem-loop structure. In some embodiments, an shRNA of the present disclosure comprises a stem-loop structure flanked by at least 10 bases on either side of the stem. In some embodiments, a stem is double-stranded RNA structure. In some embodiments, a stem is about 30-40 bases in length on each side. In some embodiments, a stem is about 30-39 bases in length on each side. In some embodiments, a stem is about 30-38 bases in length on each side. In some embodiments, a stem is about 30-37 bases in length on each side. In some embodiments, a stem is about 30-36 bases in length on each side. In some embodiments, a stem is about 30-35 bases in length on each side.
• a stem is about 30-34 bases in length on each side. In some embodiments, a stem is about 30-33 bases in length on each side. In some embodiments, a stem is about 30-32 bases in length on each side. In some embodiments, a stem is about 30-31 bases in length on each side. In some embodiments, a stem is about 31-40 bases in length on each side. In some embodiments, a stem is about 32-40 bases in length on each side. In some embodiments, a stem is about 33-40 bases in length on each side. In some embodiments, a stem is about 34-40 bases in length on each side. In some embodiments, a stem is about 35-40 bases in length on each side. In some embodiments, a stem is about 36-40 bases in length on each side.
• a stem is about 37-40 bases in length on each side. In some embodiments, a stem is about 38-40 bases in length on each side. In some embodiments, a stem is about 39-40 bases in length on each side. In some embodiments, a stem is 35 bases in length on each side.
• an shRNA of the present disclosure comprises a scaffold. In some embodiments, an shRNA of the present disclosure comprises a 5’ scaffold. In some embodiments, an shRNA of the present disclosure comprises a 3’ scaffold. In some embodiments, an shRNA of the present disclosure comprises a guide strand. In some embodiments, an shRNA of the present disclosure comprises a passenger strand.
• an shRNA of the present disclosure comprises a loop connecting the guide strand and the passenger strand.
• a loop is about 8-20 bases in length. In some embodiments, a loop is about 8-19 bases in length. In some embodiments, a loop is about 8-18 bases in length. In some embodiments, a loop is about 8-17 bases in length. In some embodiments, a loop is about 8-16 bases in length. In some embodiments, a loop is about 8-15 bases in length. In some embodiments, a loop is about 8-14 bases in length. In some embodiments, a loop is about 8-13 bases in length. In some embodiments, a loop is about 8-12 bases in length. In some embodiments, a loop is about 8-11 bases in length.
• a loop is about 8-10 bases in length. In some embodiments, a loop is about 8-9 bases in length. In some embodiments, a loop is about 9-20 bases in length. In some embodiments, a loop is about 10-20 bases in length. In some embodiments, a loop is about 11- 20 bases in length. In some embodiments, a loop is about 12-20 bases in length. In some embodiments, a loop is about 13-20 bases in length. In some embodiments, a loop is about 14- 20 bases in length. In some embodiments, a loop is about 15-20 bases in length. In some embodiments, a loop is about 16-20 bases in length. In some embodiments, a loop is about 17- 20 bases in length.
• a loop is about 18-20 bases in length. In some embodiments, a loop is about 19-20 bases in length. In some embodiments, the sections of the stem closest to the loop comprises a guide strand and a passenger strand. In some embodiments, the sections of the stem furthest from the loop comprises a 5’ scaffold and a 3’ scaffold. In some embodiments, an shRNA of the present disclosure comprises, from 5’ to 3’, a 5’ scaffold, a guide strand, a loop, a passenger strand, and a 3’ scaffold. In some embodiments, an shRNA of the present disclosure comprises, from 5’ to 3’, a 5’ scaffold, a passenger strand, a loop, a guide strand, and a 3’ scaffold.
• a scaffold of the present disclosure comprises an miRNA scaffold.
• an miRNA scaffold comprises an miRNA-155 5’ scaffold, an miRNA-155 3’ scaffold, an miRNA-30 5’ scaffold, an miRNA-30 3’ scaffold, an miRNA-16 5’ scaffold, an miRNA-16 3’ scaffold, an miRNA-125 5’ scaffold, an miRNA-125 3’ scaffold, an miRNA-223 5’ scaffold, or an miRNA-223 3’ scaffold.
• a guide strand of the present disclosure is about 19-24 bases in length. In some embodiments, a guide strand of the present disclosure is 19-24 bases in length. In some embodiments, a guide strand is 18 bases in length. In some embodiments, a guide strand is 19 bases in length. In some embodiments, a guide strand is 20 bases in length. In some embodiments, a guide strand is 21 bases in length. In some embodiments, a guide strand is 22 bases in length. In some embodiments, a guide strand is 23 bases in length. In some embodiments, a guide strand is 24 bases in length. In some embodiments, a guide strand is 25 bases in length.
• a passenger strand of the present disclosure is about 17-24 bases in length. In some embodiments, a passenger strand of the present disclosure is about 17- 22 bases in length. In some embodiments, a passenger strand of the present disclosure is 17-22 bases in length. In some embodiments, a passenger strand is 17 bases in length. In some embodiments, a passenger strand is 18 bases in length. In some embodiments, a passenger strand is 19 bases in length. In some embodiments, a passenger strand is 20 bases in length. In some embodiments, a passenger strand is 21 bases in length. In some embodiments, a passenger strand is 22 bases in length. In some embodiments, a passenger strand is 23 bases in length.
• a passenger strand is 22 bases in length. In some embodiments, a passenger strand is 24 bases in length. In some embodiments, a passenger strand is 1-2 bases shorter than a corresponding guide strand. In some embodiments, a passenger strand is 1 base shorter than a corresponding guide strand. In some embodiments, a passenger strand is 2 bases shorter than a corresponding guide strand. In some embodiments, a passenger strand is the same length as a corresponding guide strand.
• a guide strand of the present disclosure has a G/C content of about 36%-50%. In some embodiments, a guide strand of the present disclosure has a G/C content of 36%-50%. In some embodiments, a guide strand has a G/C content of 35%. In some embodiments, a guide strand has a G/C content of 36%. In some embodiments, a guide strand has a G/C content of 37%. In some embodiments, a guide strand has a G/C content of 38%. In some embodiments, a guide strand has a G/C content of 39%. In some embodiments, a guide strand has a G/C content of 40%.
• a guide strand has a G/C content of 41%. In some embodiments, a guide strand has a G/C content of 42%. In some embodiments, a guide strand has a G/C content of 43%. In some embodiments, a guide strand has a G/C content of 44%. In some embodiments, a guide strand has a G/C content of 45%. In some embodiments, a guide strand has a G/C content of 46%. In some embodiments, a guide strand has a G/C content of 47%. In some embodiments, a guide strand has a G/C content of 48%. In some embodiments, a guide strand has a G/C content of 49%. In some embodiments, a guide strand has a G/C content of 50%. In some embodiments, a guide strand has a G/C content of 51%.
• a passenger strand of the present disclosure has a G/C content of about 36%-50%. In some embodiments, a passenger strand of the present disclosure has a G/C content of 36%-50%. In some embodiments, a passenger strand has a G/C content of 35%. In some embodiments, a passenger strand has a G/C content of 36%. In some embodiments, a passenger strand has a G/C content of 37%. In some embodiments, a passenger strand has a G/C content of 38%. In some embodiments, a passenger strand has a G/C content of 39%. In some embodiments, a passenger strand has a G/C content of 40%.
• a passenger strand has a G/C content of 41%. In some embodiments, a passenger strand has a G/C content of 42%. In some embodiments, a passenger strand has a G/C content of 43%. In some embodiments, a passenger strand has a G/C content of 44%. In some embodiments, a passenger strand has a G/C content of 45%. In some embodiments, a passenger strand has a G/C content of 46%. In some embodiments, a passenger strand has a G/C content of 47%. In some embodiments, a passenger strand has a G/C content of 48%. In some embodiments, a passenger strand has a G/C content of 49%. In some embodiments, a passenger strand has a G/C content of 50%. In some embodiments, a passenger strand has a G/C content of 51%.
• a stemloop formed by a 5' scaffold, a guide strand, a passenger strand, and a 3' scaffold is recognized by Drosha.
• Drosha cleaves 10-15 bases above the base of a stemloop.
• a Drosha-cleaved stemloop will be recognized by Dicer.
• Dicer cleaves 20-24 bases from the 5' and 3' ends of the stemloop.
• a guide strand of the present disclosure comprises a nucleic acid sequence that is reverse complementary to a target gene transcript comprising a target nucleic acid sequence.
• a passenger strand of the present disclosure comprises a nucleic acid sequence that is not fully complementary to a corresponding guide strand.
• a passenger strand comprises a nucleic acid sequence that is not fully complementary to a corresponding guide strand by one nucleotide.
• a passenger strand of the present disclosure comprises a nucleic acid sequence that is not fully complementary to a corresponding guide strand by two nucleotides.
• a passenger strand of the present disclosure comprises a nucleic acid sequence that is not fully complementary to a corresponding guide strand by three nucleotides. In some embodiments, a passenger strand of the present disclosure comprises a nucleic acid sequence that is not fully complementary to a corresponding guide strand by four nucleotides.
• an shRNA of the present disclosure comprises: (a) a nucleic acid sequence selected from Table 6a, Table 6b, Table 6c, Table 6d, or Table 6e; (b) a nucleic acid sequence that differs from a sequence selected from Table 6a, Table 6b, Table 6c, Table 6d, or Table 6e by no more than 5 substitutions, additions, or deletions; or (c) a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 6a, Table 6b, Table 6c, Table 6d, or Table 6e.
• an shRNA of the present disclosure comprises a nucleic acid sequence selected from Table 6a, Table 6b, Table 6c, Table 6d, or Table 6e. In some embodiments, an shRNA of the present disclosure comprises a nucleic acid sequence that differs from a sequence selected from Table 6a, Table 6b, Table 6c, Table 6d, or Table 6e by no more than 5 substitutions, additions, or deletions. In some embodiments, an shRNA of the present disclosure comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 6a, Table 6b, Table 6c, Table 6d, or Table 6e.
• an inhibitory RNA of the present disclosure is or comprises an miRNA.
• an miRNA of the present disclosure comprises: (a) a nucleic acid sequence selected from Table 7; (b) a nucleic acid sequence that differs from a sequence selected from Table 7 by no more than 5 substitutions, additions, or deletions; or (c) a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 7.
• an miRNA of the present disclosure comprises a nucleic acid sequence selected from Table 7.
• an miRNA of the present disclosure comprises a nucleic acid sequence that differs from a sequence selected from Table 7 by no more than 5 substitutions, additions, or deletions.
• an miRNA of the present disclosure comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 7
• inhibitory nucleic acids of the present disclosure regulate gene expression in modified immune cells of the present disclosure via RNA interference (RNAi).
• RNAi RNA interference
• inhibitory nucleic acids of the present disclosure target mRNA comprising a complementary sequence.
• inhibitory nucleic acids of the present disclosure induce degradation of target mRNA.
• inhibitory nucleic acids of the present disclosure repress translation of target mRNA.
• a target gene transcript is mammalian. In some embodiments, a target gene transcript is human. In some embodiments, contacting a target gene transcript with one or more inhibitory nucleic acids (e.g., RNAs) reduces translation of the target gene transcript. In some embodiments, a target gene transcript is expressed in an immune cell. In some embodiments, an immune cell is a stem cell, macrophage, monocyte, or dendritic cell. In some embodiments, a target gene transcript is expressed in a macrophage. In some embodiments, a target gene transcript is expressed in a monocyte. In some embodiments, reduced translation of a target gene transcript is associated with an Ml phenotype.
• reduced translation of a target gene transcript is associated with an M2 phenotype.
• increased expression of an inhibitory nucleic acid (e.g., RNA) of the present disclosure is associated with an Ml phenotype.
• increased expression of an inhibitory nucleic acid (e.g., RNA) of the present disclosure is associated with an M2 phenotype.
• a target gene transcript of the present disclosure comprises: (a) a target nucleic acid sequence selected from Table 8; (b) a target nucleic acid sequence that differs from a sequence selected from Table 8 by no more than 5 substitutions, additions, or deletions; or (c) a target nucleic acid sequence that is at least 80% identical to a sequence selected from Table 8.
• a target gene transcript of the present disclosure comprises a target nucleic acid sequence selected from Table 8.
• a target gene transcript of the present disclosure comprises a target nucleic acid sequence that differs from a sequence selected from Table 8 by no more than 5 substitutions, additions, or deletions.
• a target gene transcript of the present disclosure comprises a target nucleic acid sequence that is at least 80% identical to a sequence selected from Table 8.
• a target gene transcript of the present disclosure encodes human ATG7, C/EBP-alpha, C/EBP-beta, CD36, CLEC1A, FATS, GOLM1, HAVCR2, ITGAD, KLF4, KLF6, LILRB1, LILRB2, LILRB4, MAF, Maffi, PD-L1, PIK3CG, PIK3CG, PPARa, PPARy, PTGS2, SIGLEC7, SIGLEC10, SIRPa, SLC15A3, STAT3, STAT6, TNFRSF1B, TOX, TREM2, YTHDF2, or ZFP36.
• a target gene transcript of the present disclosure encodes human SIRPa.
• a target gene transcript of the present disclosure encodes human PD-1 or PD-L1.
• an inhibitory nucleic acid of the present disclosure comprises one or more inhibitory nucleic acids.
• one or more inhibitory nucleic acids comprise one inhibitory nucleic acid.
• one or more inhibitory nucleic acids comprise at least two, three, four or five inhibitory nucleic acids.
• one or more inhibitory nucleic acids comprise two inhibitory nucleic acids.
• one or more inhibitory nucleic acids comprise three inhibitory nucleic acids.
• one or more inhibitory nucleic acids comprise four inhibitory nucleic acids.
• one or more inhibitory nucleic acids comprise five inhibitory nucleic acids.
• At least two, three, four, or five inhibitory nucleic acids are in tandem. In some embodiments, at least two, three, four, or five inhibitory nucleic acids comprise identical sequences. In some embodiments, at least two, three, four, or five inhibitory nucleic acids comprise at least two different sequences. In some embodiments, at least three, four, or five inhibitory nucleic acids comprise at least three different sequences. In some embodiments, at least four or five inhibitory nucleic acids comprise at least four different sequences. In some embodiments, at least five inhibitory nucleic acids comprise at least five different sequences.
• At least two, three, four, or five inhibitory nucleic acids encode inhibitory RNAs comprising nucleic acid sequences that are reverse complementary to the same target gene transcript. In some embodiments, at least two, three, four, or five inhibitory nucleic acids encode inhibitory RNAs comprising nucleic acid sequences that are reverse complementary to different target gene transcripts.
• At least two, three, four, or five inhibitory nucleic acids encode inhibitory RNAs comprising identical miRNA scaffolds. In some embodiments, at least two, three, four, or five inhibitory nucleic acids encode inhibitory RNAs comprising at least two different miRNA scaffolds. In some embodiments, at least three, four, or five inhibitory nucleic acids encode inhibitory RNAs comprising at least three different miRNA scaffolds. In some embodiments, at least four or five inhibitory nucleic acids encode inhibitory RNAs comprising at least four different miRNA scaffolds. In some embodiments, at least five inhibitory nucleic acids encode inhibitory RNAs comprising at least five different miRNA scaffolds. In some embodiments, at least two different miRNA scaffolds are selected from the group consisting of an miRNA- 155 5’ scaffold, an miRNA- 155 3’ scaffold, an miRNA-30 5’ scaffold, and an miRNA-30 3’ scaffold.
• the present disclosure also provides methods of designing inhibitory nucleic acids of the present disclosure.
• the present disclosure provides methods of designing inhibitory RNA of the present disclosure.
• the present disclosure provides methods of designing shRNA of the present disclosure.
• methods of designing an shRNA comprising a guide strand, a passenger strand, and a loop comprise the steps of: (a) selecting a target region of a target gene transcript, (b) designing the guide strand, and (c) designing the passenger strand.
• a guide strand is a reverse complement of a target region.
• a guide strand comprises, from 5’ to 3’, an adenine or uracil at position 1, an adenine, guanine, or cytosine at position 10, and a G/C content between 36% and 45% that is increases from 5’ to 3’.
• a guide strand comprises a higher G/C content of the 10 bases at the 3’ end of the guide strand relative to the G/C content of the 10 bases at the 5’ end of the guide strand.
• a passenger strand comprises the sequence of a target region, from 5’ to 3’, with the following variations: (i) deletion of a nucleotide at position 7 relative to the loop, (ii) deletion of a nucleotide at position 11 relative to the loop, and (iii) mutation of an adenine to guanine and/or mutation of a cytosine to uracil near the deletions at positions 7 and 11, wherein the mutations create two partial G-U base pairs between the passenger strand and the guide strand.
• a passenger strand is 2 base pairs shorter than a guide strand.
• methods of designing an shRNA comprising a guide strand, a passenger strand, and a loop comprise the steps of: (a) selecting a target region of a target gene transcript, (b) designing the guide strand, wherein the guide strand is a reverse complement of the target region, and wherein the guide strand comprises, from 5’ to 3’, an adenine or uracil at position 1, an adenine, guanine, or cytosine at position 10, and a G/C content between 36% and 45% that is increases from 5’ to 3’; and (c) designing the passenger strand, wherein the passenger strand comprises the sequence of the target region, from 5’ to 3’, with the following variations: (i) deletion of a nucleotide at position 7 relative to the loop, (ii) deletion of a nucleotide at position 11 relative to the loop, (iii) mutating an adenine to guanine and/or mutating a cyto
• the present disclosure provides pharmaceutical compositions comprising modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising one or more of CARs described herein in combination 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 one or more CARs 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.
• 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 intrasternal injection and infusion.
• compositions comprising modified immune cells 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 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 10 7 , about 2 x 10 7 , about 5 x 10 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.
• 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
• 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 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, mycophenolate, 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.
• following transplant subjects receive an infusion of a pharmaceutical composition comprising immune cells 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.
• 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 delivering a pharmaceutical composition described herein.
• a therapeutically effective amount of a pharmaceutical composition described herein is administered to a subject having a disease or disorder.
• Pharmaceutical compositions 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 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.
• 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 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
• a solid tumor e.g., leukemia, lymphoma, or myeloma, e.g., multiple myeloma
• 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, e.g., aden
• 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 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.
• 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.
• a subject is undergoing or has undergone a chemotherapeutic treatment and/or radiation therapy.
• compositions described herein may be carried out in any convenient manner (e.g., injection, ingestion, transfusion, inhalation, implantation, or transplantation). Tn some embodiments, a pharmaceutical compositions described herein is administered by injection or infusion. Pharmaceutical compositions described herein may be administered to a patient transarterially, subcutaneously, intravenously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, or intraperitoneally. In some embodiments, a pharmaceutical composition described herein is administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or intramuscularly). In some embodiments, a pharmaceutical composition described herein is administered by intravenous infusion or injection.
• a 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.
• Table 1 includes exemplified CAR constructs described herein.
• Figures 28 and 29 show schematics of these exemplary CAR constructs.
• the present Example assesses 20 different anti-mesothelin binders (M1-M20) that were used in a CD8 framework CAR. All 20 CARs were manufactured as 5-methoxyuridine (5moU) mRNA. The CAR mRNA was electroporated into human primary macrophages using a MaxCyte Atx instrument. 24 hours post-electroporation, CAR expression was evaluated via binding of biotinylated mesothelin followed by APC-streptavidin.
• 5moU 5-methoxyuridine
• CAR001 an anti-HER2 CAR
• mRNA was used as a positive control for electroporation and mRNA quality.
• the present Example assesses four different anti-mesothelin binders (Ml 1, M14, Ml 5, and Ml 7) that were used in a CD8 framework CAR. All four CARs were manufactured as 5-methoxyuridine (5moU) mRNA. The CAR mRNA was electroporated into human primary macrophages using a MaxCyte ATx instrument. 24 hours post-electroporation, cytokine release was evaluated using a 24 hour co-incubation with K562 WT (mesothelin negative) or K562 MESO (mesothelin positive) cells at a 1: 1 ratio of effector cells to target cells (E:T).
• K562 WT messagesothelin negative
• K562 MESO meothelin positive
• Example 3 Phagocytosis Screening of Macrophages Expressing Anti-Mesothelin Binders
• the present Example assesses four different anti-mesothelin binders (Ml 1 , M14, Ml 5, and Ml 7) that were used in a CD8 framework CAR. All four CARs were manufactured as 5-methoxyuridine (5moU) mRNA. The CAR mRNA was electroporated into human primary macrophages using a MaxCyte ATx instrument.
• 5moU 5-methoxyuridine
• the present Example assesses the phenotype of macrophages transduced with CTX 269.
• Previously frozen macrophages from three donors were thawed and transduced with varying exemplary MOIs of Ad5f 5 vector comprising CTX_269. After 48 hours, cells were analyzed via FACS for viability, CAR expression, and M1/M2 polarization.
• the present Example assesses the resistance of macrophages transduced with CTX_269 to M2 cytokines.
• Previously frozen macrophages from three donors were thawed and transduced with 3000 MOI of Ad5f35 vector comprising CTX 269.
• An anti-HER2 CAR delivered via Ad5f35 (CT0508) at an MOI of 3000 served as a positive control.
• Ad5f35 virus was removed from the macrophage cultures.
• 72 hours post-transduction media containing 10 ng/mL IL 10 was added to the macrophages.
• cytokine (IL 10) addition macrophages were phenotyped for M1/M2 marker expression.
• macrophages transduced with CTX 269 exhibited a reduced upregulation of CD163 relative to untransduced (UTD) controls when exposed to IL10, indicating a less M2-like phenotype.
• macrophages transduced with CTX 269 exhibited a reduced downregulation of CD86 relative to untransduced (UTD) controls when exposed to IL10, suggesting maintenance of the Ml phenotype.
• the present Example assesses the changes in M1ZM2 polarization of macrophages transduced with CTX 269 after exposure to mesothelin.
• Previously frozen macrophages from two donors were thawed and transduced with 3000 MOI of Ad5f35 vector comprising CTX_269.
• Macrophages were plated on plates containing exemplary titrations of mesothelin. After 24 hours, macrophages were lifted and analyzed for M1/M2 polarization via flow cytometry.
• the present Example assesses the phagocytosis ability of macrophages transduced with CTX_269.
• Previously frozen macrophages from two donors were thawed and transduced with 3000 MOI of Ad5f35 vector comprising CTX 269. After 48 hours, the macrophages were lifted and placed onto a 96 well plate (50,000 cells/well). Wild-type (no mesothelin expression) and mesothelin-expressing target cells were stained with pHrodo (a pH sensitive dye). Macrophages and target cells were mixed at a 1 : 1 E:T ratio. Fluorescence was tracked in an Incucyte S3 Live-Cell Analysis System for 24 hours, with imaging occurring every hour. Phagocytosis was determined by calculating the area under the curve (AUC) of the fluorescence values at each time point.
• AUC area under the curve
• CTX_269 transduced macrophages only show increased phagocytosis of mesothelin-positive cells, indicating that phagocytosis of A549 lung adenocarcinoma cells by macrophages transduced with CTX 269 involved mesothelin expression by the target cells.
• Figure 13 shows that, in the phagocytosis assay with MES_OV ovarian cystadenocarcinoma cells, there was increased phagocytosis of target cells expressing mesothelin by macrophages transduced with CTX 269 relative to untransduced (UTD) control macrophages. Such increased phagocytosis was not seen in wild-type ovarian cystadenocarcinoma that did not express mesothelin.
• the present Example assesses the killing ability of macrophages transduced with CTX 269.
• Previously frozen macrophages from three donors were thawed and transduced with 3000 MOI of Ad5f35 vector comprising CTX_269.
• the macrophages were lifted and placed into a 96 well plate at various E:T ratios.
• Mesothelin/mKate2-expressing target cells were plated at a constant 10,000 cells per well. Fluorescence was tracked on an Incucyte S3 Live-Cell Analysis System for 72 hours, with imaging occurring every 4 hours. Killing of target cells was determined by calculating the fluorescence at 72 hours divided by the initial fluorescence.
• CTX 269 transduced macrophages exhibited enhanced killing of mesothelin-expressing A549 lung adenocarcinoma cells compared to transduced (UTD) macrophages. Additionally, Figure 15 shows that CTX 269 transduced macrophages exhibited enhanced killing of mesothelin-expressing MES OV ovarian cystadenocarcinoma cells compared to UTD macrophages.
• Example 9A Cytokine Release from Macrophages Transduced with an Anti-Mesothelin Binder after Exposure to Mesothelin
• the present Example assesses the cytokine release of macrophages transduced with CTX 269 after exposure to mesothelin.
• Previously frozen macrophages from three donors were thawed and transduced with 3000 MOI of Ad5f35 vector comprising CTX 269. After 48 hours, the macrophages were placed on plates functionalized with titrations of mesothelin. After 24 hours, supernatants were harvested and analyzed using Meso Scale Discovery QuickPlex SQ 120.
• Example 9B Cytokine Release from Macrophages Transduced with an Anti-Mesothelin Binder after Exposure to Cell Lines Expressing Mesothelin
• the present Example assesses the cytokine release of macrophages transduced with CTX_269 after contact with cell lines expressing mesothelin.
• Previously frozen macrophages from two donors were thawed and transduced with 3000 MOI of Ad5f 5 vector comprising CTX_269.
• the macrophages were plated with cell lines expressing mesothelin in a 1: 1 E:T ratio.
• supernatants were harvested and analyzed using Meso Scale Discovery QuickPlex SQ 120.
• Figure 18 illustrates the experimental design described above.
• Figure 19 shows that a significant reduction in tumor burden was observed in mice treated with macrophages transduced with anti-mesothelin CARs, compared to untreated or UTD macrophage controls.
• the present Example assesses the expression of an exemplary CD28-based CAR in macrophages.
• Previously frozen untransduced (UTD) macrophages from two donors were thawed and transduced with lentivirus vector comprising a CD28-based anti-mesothelin CAR at an MOI of 5.
• the macrophages were harvested, tested for expression of anti- mesothelin CAR, and used for a killing assay.
• Mesothelin/mKate2-expressing target cells were plated at a constant 10,000 cells per well. Fluorescence was tracked on an Incucyte S3 Live Cell Analysis System for 72 hours, with imaging occurring every 4 hours. Killing was determined by calculating the fluorescence at 72 hours divided by the initial fluorescence.
• the CD28-based anti-mesothelin CAR expresses similarly or better than an exemplary CD8-based anti-mesothelin CAR. Indeed, the CD28-based anti- mesothelin CAR expressed better than the CD8-based anti-mesothelin CAR at the same MOI. Additionally, as shown in Figure 21, macrophages transduced with the CD28-based anti- mesothelin CAR exhibited enhanced effector function (killing of target cells) compared to macrophages transduced with the CD8-based anti-mesothelin CAR across cell lines. Without wishing to be bound to a theory, the enhanced killing function of the macrophages transduced with the CD28-based anti-mesothelin CAR was likely due to higher levels of CAR expression.
• Example 12 Cytokine Release from Macrophages Transduced with a CD28-Based Anti- Mesothelin CAR after Exposure to Mesothelin
• the present Example assesses the cytokine release of macrophages transduced with an exemplary CD28-based anti-mesothelin CAR after exposure to mesothelin.
• Previously frozen UTD macrophages from two donors were thawed and transduced with lentivirus vector comprising a CD28-based anti-mesothelin CAR at an MOI of 5.
• the macrophages were harvested and plated on titrations of mesothelin. After 24 hours, supernatants were harvested and analyzed using a Meso Scale Discovery QuickPlex SQ 120.
• the present Example assesses the resistance of macrophages transduced with CTX_269 or CTX_293 to M2 cytokines.
• Previously frozen macrophages from three donors were thawed and transduced with 3000 MOI of Ad5f35 vector comprising CTX_269 or CTX_293.
• Ad5f35 virus was removed from the macrophage cultures.
• 72 hours post-transduction media containing 10 ng/mL IL- 10 was added to the macrophages.
• IL-10 cytokine
• the present Example assesses the effect of transduction of monocytes with an anti-mesothelin CAR.
• Previously frozen CD 14+ monocytes from two donors were thawed and transduced with 3000 MOI of Ad5f35 vector comprising CTX_001 or CTX_269. Additional monocytes were cultured in GMCSF for differentiation into macrophages.
• untransduced monocytes now macrophages
• CAR monocytes and macrophages were harvested, phenotyped via flow cytometry, and used in a killing assay.
• CTX 269 CD8-based anti-mesothelin CAR monocytes exhibited a similar phenotype to CTX 001 (anti-HER2 CAR) monocytes.
• the groups shown in Figures 27A-F include: untransduced monocytes (UTD Mono), monocytes transduced with CTX_001 (CAR Mono 001), monocytes transduced with CTX_269 (CAR Mono 269), untransduced monocytes that were differentiated into macrophages (UTD-M), monocytes transduced with CTX_001 and then differentiated into macrophages (cmdCAR-M 001), monocytes transduced with CTX 0269 and then differentiated into macrophages (cmdCAR-M 269), macrophages transduced with CTX_001 after differentiation from monocytes (cmdCAR-M 001), and macrophages transduced with CTX_0269 after differentiation from monocytes (cmdCAR-M 269).
• monocytes transduced with a CAR vector and then differentiated into macrophages cmdCAR-M
• macrophages transduced with a CAR vector after differentiation from monocytes exhibited similar levels of effector function (killing of target cells).
• Example 16 Effect of Macrophages Transduced with an Anti-Mesothelin Binder in an in vitro Model
• the present Example assesses the phenotype, phagocytosis ability, and cytokine release of macrophages transduced with CTX 269 in an in vitro model.
• Previously frozen macrophages from two donors were thawed and transduced with 3000 MOI of Ad5f35 vector comprising CTX_269.
• Cells were maintained in TexlO (TexMACS + 10% FBS + 1% pen/strep) for the indicated number of days.
• TexlO TexMACS + 10% FBS + 1% pen/strep
• CTX_269 anti-mesothelin CAR (CTX_269) expression was robust for at least two weeks.
• CTX_269 transduced macrophages only show increased phagocytosis of mesothelin-positive cells, indicating that phagocytosis of K562 cells by macrophages transduced with CTX_269 involved mesothelin expression by the target cells.
• the observed increase in mesothelin-specific phagocytosis was present at days 2 and 14, indicating that macrophages transduced with CTX_269 maintain their killing activity for at least two weeks.
• macrophages transduced with CTX_269 exhibited cytokine (TNFa) release when target cells (K562 cells) expressed mesothelin.
• TNFa cytokine
• Example 17 Effect of Macrophages Transduced with an Anti-Mesothelin Binder in an in vivo Model
• CTX_269 in an in vivo model.
• mice were challenged with 0.5e6 A549 lung adenocarcinoma cells expressing mesothelin and mKate2/CBG via tail vein injection.
• previously frozen untransduced (UTD) macrophages from one donor were thawed and transduced with 3000 MOI of Ad5f35 vector comprising CTX_269.
• mice were treated with the transduced macrophages via tail vein injection, treated with UTD macrophage controls, or not treated (saline injections without macrophages). Tumor burden in the mice was measured via BLI 2-3 times per week.
• mice were euthanized and lung tissue was harvested for sectioning and IHC staining (Histowiz) with anti-human mesothelin antibody (Origene, catalog number TA805169BM). Images were analyzed using HALO software for number of nodules per given area (converted to nodules/cm 2 ) and ratio of tumor area to total area of sample.
• Figure 18 illustrates the experimental design described above.
• Figure 33 shows representative tissue sections of murine lung tumor nodules IHC stained for human mesothelin.
• Figure 34 shows that a significant reduction in lung tumor nodules was observed in mice treated with macrophages transduced with anti-mesothelin C ARs, compared to untreated or UTD macrophage controls.
• Example 18 Expression of a CD28-Based Anti-Mesothelin CAR and Viability of CAR Monocytes and Macrophages
• the present Example assesses the expression of an exemplary CD28-based CAR in monocytes and macrophages.
• Previously frozen CD 14+ monocytes from eight donors were transduced with Ad5f35 virus containing either CTX_964 or CTX_1461 at 5 IFU/cell for 1 hour at a concentration of 3e6 cells/mL in TexlO media containing lOng/mL hGM-CSF. Cells were then cultured for two days in order to produce CAR monocytes or seven days in order to produce CAR monocyte-derived CAR macrophages. Viability and CAR expression were assessed via flow cytometry. Each point in Figure 35A and Figure 35B represents the average of technical triplicates for each donor.
• the present Example assesses the expression of SIRPa in monocytes and macrophages expressing exemplary CAR constructs.
• Previously frozen CD 14+ monocytes from eight donors were transduced with Ad5f35 virus containing either CTX 964 or CTX 1461 (CTX964 + intronic shRNA against SIRPa) at 5 IFU/cell for 1 hour at a concentration of 3e6 cells/mL in TexlO media containing lOng/mL hGM-CSF. Cells were then cultured for two days in order to produce CAR monocytes or seven days in order to produce CAR monocyte-derived CAR macrophages. SIRPa expression was measured via flow cytometry.
• SIRPa expression normalized to untransduced control cells was calculated by determining the fold change in fluorescence when using a SIRPa targeting mAb compared to an isotype control, and then normalizing to UTD values.
• Each point in Figure 36A and Figure 36B represents the average of technical triplicates for each donor.
• Example 20 Cytokine Release from Cells Transduced with a CD28-Based Anti-Mesothelin CAR after Exposure to Mesothelin
• the present Example assesses the cytokine release of cells transduced with an exemplary CD28-based anti-mesothelin CAR after exposure to mesothelin.
• Previously frozen CD 14+ monocytes from eight donors were transduced with Ad5f35 virus containing either CTX_964 or CTX_1461 at 5 IFU/cell for 1 hour at a concentration of 3e6 cells/mL in TexlO media containing lOng/mL hGM-CSF. Cells were then cultured for two days in order to produce CAR monocytes or seven days in order to produce CAR monocyte-derived CAR macrophages.
• monocytes or macrophages transduced with the CTX_964 or CTX_1461 anti-mesothelin CAR constructs exhibited enhanced cytokine (TNFa) release compared to UTD cells, even in the presence of CD47 (MSLN+CD47).
• the present Example assesses the killing ability of cells transduced with CTX_964 or CTX_1461.
• Previously frozen CD14+ monocytes were transduced with Ad5f 5 virus containing either CTX 964 or CTX 1461 at 5 IFU/cell for 1 hour at a concentration of 3e6 cells/mL in TexlO media containing lOng/mL hGM-CSF. Cells were then cultured for seven days in order to produce CAR monocyte-derived CAR macrophages.
• CAR macrophages were plated at 20e3 viable cells per well in a 96 well plate along with target cells expressing fluorescent protein in a 1: 1 E:T (effector to target) ratio.
• Example 22 Effect of Cells Transduced with an Anti-Mesothelin Binder in an in vivo Model
• the present Example assesses the effect of cells transduced with CTX_964 or CTX_1461 in an in vivo model.
• Previously frozen CD 14+ monocytes were transduced with Ad5f35 virus containing either CTX_964 or CTX_1461 at 5 IFU/cell for 1 hour at a concentration of 3e6 cells/mL with lOng/mL hGM-CSF. Cells were then cultured for seven days in order to produce CAR monocyte-derived CAR macrophages.
• NSG-S mice were injected with SKOV3-CBG/GFP-CTX1698-Meso cells IP, le5 cells/mouse.
• mice were imaged with IVIS, randomized based on whole body bioluminescence signal and injected with CAR monocyte- derived CAR macrophages 5e6 cells/mouse within ⁇ 3-4 hours after tumor cell injection. Tumor burden was assessed every 3-4 days with IVIS bioluminescence imaging.
• CAR monocyte-derived CAR macrophages expressing CTX 964 or CTX 1461 were able to suppress tumor growth in an in vivo xenograft model.

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  • 2023-10-02 WO PCT/US2023/075737 patent/WO2024076927A2/en not_active Ceased
  • 2023-10-02 KR KR1020257014572A patent/KR20250080888A/ko active Pending
  • 2023-10-02 CN CN202380082690.7A patent/CN120302988A/zh active Pending

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