Modified monocytes / macrophage expressing chimeric antigen receptors and uses thereof

Abstract

To provide methods and compositions for treating cancer, whether a solid tumor or a hematologic malignancy.SOLUTION: Provided is a modified cell comprising a chimeric antigen receptor (CAR). The CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain of a stimulatory and / or co-stimulatory molecule. The cell is a monocyte, macrophage, or dendritic cell that possesses targeted effector activity. Also provided is a pharmaceutical composition comprising the modified cell and a pharmaceutically acceptable carrier. Further provided is a method for treating a disease or condition associated with immunosuppression, such as cancer.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] Cross - Reference to Related Applications This application is entitled to priority under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62 / 197,675, filed Jul. 28, 2015, the entire disclosure of which is hereby incorporated by reference herein in its entirety.

Background Art

[0002] Background of the Invention Cancer immunotherapy has shown remarkable clinical results in the context of numerous solid tumors and hematological malignancies. The endogenous immune system is typically non - responsive to malignant cells or can be actively immunosuppressive in terms of the body's response to the presence of malignant cells. One strategy to improve the treatment of tumors is to force the immune system to recognize tumors through genetic manipulation of white blood cells. T cells can be engineered to express a synthetic immune receptor known as a chimeric antigen receptor (CAR), which contains an extracellularly targeted antibody and an intracellular signaling domain. T cells expressing a CAR directed against CD19 have shown a significant anti - leukemia effect, and complete remission has been achieved in 90% of treated acute lymphoblastic leukemia patients (Maude, et al., NEJM, vol. 371:1507 - 17, 2014 (Non - Patent Document 1)). These results are accompanied by active T - cell proliferation in leukemia patients receiving such treatment and well - documented T - cell infiltration into the tumor site. Despite the high response rates demonstrated in hematological malignancies, the effectiveness of CAR T cells in solid tumors (as well as certain lymphoid tumors) can be limited. Explanations that have been considered in this regard include the possibility that the ability of T cells to infiltrate solid tumors is impaired, poor trafficking, an immunosuppressive tumor microenvironment, and the fact that only a few tumor - specific antigens are expressed on solid tumor cells.

[0003] In the art, there is a need for more effective compositions and methods for treating cancer by improving the specificity for tumor cells and the invasiveness to tumor sites in both solid tumors and hematological malignancies with such compositions. The present invention addresses this need.

PRIOR ART DOCUMENTS

NON-PATENT DOCUMENTS

[0004]

NON-PATENT DOCUMENT 1

SUMMARY OF THE INVENTION

[0005] As disclosed herein, the present invention includes compositions and methods using phagocytes having targeted effector activity.

[0006] In one aspect, the present invention includes modified cells comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain of a stimulatory molecule and / or a costimulatory molecule, and the cells are monocytes, macrophages or dendritic cells having targeted effector activity.

[0007] In another aspect, the present invention includes modified cells comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the nucleic acid sequence comprises a nucleic acid sequence encoding an antigen-binding domain, a nucleic acid sequence encoding a transmembrane domain, and a nucleic acid sequence encoding an intracellular domain of a stimulatory molecule and / or a costimulatory molecule, and the cells are monocytes, macrophages or dendritic cells that express the CAR and have targeted effector activity.

[0008] In yet another aspect, the present invention includes a method of modifying a cell, the method including the step of introducing a chimeric antigen receptor (CAR) into a monocyte, macrophage, or dendritic cell, the CAR including an antigen-binding domain, a transmembrane domain, and an intracellular domain of a stimulatory molecule and / or a co-stimulatory molecule, and the cell being a monocyte, macrophage, or dendritic cell that expresses the CAR and possesses targeted effector activity.

[0009] In still another aspect, the present invention includes a cell modified according to the method described herein.

[0010] In various embodiments of the above-described aspects or any other aspect of the present invention described herein, the antigen-binding domain of the CAR includes an antibody selected from the group consisting of monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, single-domain antibodies, single-chain variable fragments, and antigen-binding fragments thereof. In another embodiment, the antigen-binding domain of the CAR is selected from the group consisting of anti-CD19 antibodies, anti-HER2 antibodies, and fragments thereof. In yet another embodiment, the intracellular domain of the CAR includes a dual-signaling domain.

[0011] In another embodiment, the targeted effector activity is directed against an antigen on a target cell that specifically binds to the antigen-binding domain of the CAR. In yet another embodiment, the targeted effector activity is selected from the group consisting of phagocytosis, targeted cytotoxicity, antigen presentation, and cytokine secretion.

[0012] In another embodiment, the composition further includes an agent selected from the group consisting of nucleic acids, antibiotics, anti-inflammatory agents, antibodies or antibody fragments thereof, growth factors, cytokines, enzymes, proteins, peptides, fusion proteins, synthetic molecules, organic molecules, carbohydrates or analogs, lipids, hormones, microsomes, derivatives or modifications thereof, and any combination thereof.

[0013] In another aspect, the modified cell has at least one upregulated M1 marker and at least one downregulated M2 marker. In yet another aspect, the modified cell is genetically modified to express a CAR. In still another aspect, the targeted effector activity is enhanced by inhibition of CD47 activity or SIRPa activity.

[0014] In another aspect, introduction of a CAR into a cell includes introducing a nucleic acid sequence encoding the CAR, for example, introducing mRNA encoding the CAR by electroporation, or transducing the cell using a viral vector containing the nucleic acid sequence encoding the CAR.

[0015] In another aspect, the targeted effector activity is directed against an antigen on the target cell that specifically binds to the antigen-binding domain of the CAR. In another aspect, the targeted effector activity is selected from the group consisting of phagocytosis, targeted cytotoxicity, antigen presentation, and cytokine secretion.

[0016] In another aspect, the methods described herein further include inhibiting CD47 activity or SIRPα activity to enhance the targeted effector activity, for example, by contacting the cell with an anti-CD47 blocking antibody or an anti-SIRPα blocking antibody. In yet another aspect, the method further includes modifying the cell to deliver an agent to a target, the agent being selected from the group consisting of nucleic acids, antibiotics, anti-inflammatory agents, antibodies or antibody fragments thereof, growth factors, cytokines, enzymes, proteins, peptides, fusion proteins, synthetic molecules, organic molecules, carbohydrates or analogs, lipids, hormones, microsomes, derivatives or modifications thereof, and any combination thereof.

[0017] In one aspect, the invention includes a pharmaceutical composition comprising the cells described herein.

[0018] In another aspect, the present invention includes the use of the modified cells described herein in the manufacture of a medicament for treating an immune response in a subject in need thereof. In yet another aspect, the present invention includes the use of the modified cells described herein in the manufacture of a medicament for treating a tumor or cancer in a subject in need thereof.

[0019] In yet another aspect, the present invention includes a method of treating a disease or condition associated with a tumor or cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified cells described herein.

[0020] In still another aspect, the present invention provides a method of treating a tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified cells described herein.

[0021] In another aspect, the present invention includes a method for stimulating an immune response against target tumor cells or tumor tissue in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified cells described herein. [Invention 1001] A modified cell comprising a chimeric antigen receptor (CAR), the CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain of a stimulatory molecule and / or a co-stimulatory molecule, the cell being a monocyte, macrophage, or dendritic cell having targeted effector activity. [Invention 1002] A modified cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the nucleic acid sequence comprising a nucleic acid sequence encoding an antigen-binding domain, a nucleic acid sequence encoding a transmembrane domain, and a nucleic acid sequence encoding an intracellular domain of a stimulatory molecule and / or a co-stimulatory molecule, the cell being a monocyte, macrophage, or dendritic cell that expresses the CAR and has targeted effector activity. [Invention 1003] The modified cell of the present invention 1001 or 1002, wherein the antigen-binding domain of the CAR comprises an antibody selected from the group consisting of monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, single-domain antibodies, single-chain variable fragments, and antigen-binding fragments thereof. [The present invention 1004] The modified cell of the present invention 1001 or 1002, wherein the antigen-binding domain of the CAR is selected from the group consisting of anti-CD19 antibodies, anti-HER2 antibodies, and fragments thereof. [The present invention 1005] The modified cell of the present invention 1001 or the present invention 1002, wherein the intracellular domain of the CAR comprises a dual signaling domain. [The present invention 1006] The modified cell of the present invention 1001 or 1002, wherein the targeted effector activity is directed against an antigen on a target cell that specifically binds to the antigen-binding domain of the CAR. [The present invention 1007] The modified cell of the present invention 1001 or 1002, wherein the targeted effector activity is selected from the group consisting of phagocytosis, targeted cytotoxicity, antigen presentation, and cytokine secretion. [The present invention 1008] The modified cell of the present invention 1001 or 1002 further comprising an agent selected from the group consisting of nucleic acids, antibiotics, anti-inflammatory agents, antibodies or antibody fragments thereof, growth factors, cytokines, enzymes, proteins, peptides, fusion proteins, synthetic molecules, organic molecules, carbohydrates or analogs, lipids, hormones, microsomes, derivatives or modifications thereof, and any combination thereof. [The present invention 1009] The modified cell of the present invention 1001 or the present invention 1002 having at least one upregulated M1 marker and at least one downregulated M2 marker. [The present invention 1010] The modified cell of the present invention 1001 or the present invention 1002 that has been genetically modified to express the CAR. [The present invention 1011] The modified cell of the present invention 1001 or the present invention 1002, wherein the targeted effector activity is enhanced by inhibition of CD47 activity or SIRPα activity. [The present invention 1012] A pharmaceutical composition comprising the cell of the present invention 1001 or 1002 and a pharmaceutically acceptable carrier. [The present invention 1013] Use of the modified cell of the present invention 1001 or 1002 in the manufacture of a medicament for performing treatment of an immune response in a subject in need thereof. [The present invention 1014] Use of the modified cell of the present invention 1001 or 1002 in the manufacture of a medicament for performing treatment of a tumor or cancer in a subject in need thereof. [The present invention 1015] A method for treating a disease or condition associated with a tumor or cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified cell of the present invention 1001 or 1002. [The present invention 1016] A method for treating a tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified cell of the present invention 1001 or 1002. [The present invention 1017] A method for stimulating an immune response against a target tumor cell or tumor tissue in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified cell of the present invention 1001 or 1002. [The present invention 1018] A step of introducing a chimeric antigen receptor (CAR) into a monocyte, macrophage or dendritic cell, the CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain of a stimulatory molecule and / or a costimulatory molecule, the cell being a monocyte, macrophage or dendritic cell that expresses the CAR and possesses a targeted effector activity. A method for modifying a cell, comprising the step. [The present invention 1019] The method of the present invention 1018, wherein the introduction of the CAR into the cell includes the step of introducing a nucleic acid sequence encoding the CAR. [The present invention 1020] The method of the present invention 1019, wherein the step of introducing the nucleic acid sequence includes introducing mRNA encoding the CAR by electroporation. [The present invention 1021] The method of the present invention 1019, wherein the step of introducing the nucleic acid sequence includes transducing the cell with a viral vector containing a nucleic acid sequence encoding the CAR. [The present invention 1022] The method of the present invention 1018, wherein the targeted effector activity is directed against an antigen on the target cell that specifically binds to the antigen-binding domain of the CAR. [The present invention 1023] The method of the present invention 1018, wherein the targeted effector activity is selected from the group consisting of phagocytosis, targeted cytotoxicity, antigen presentation, and cytokine secretion. [The present invention 1024] The method of the present invention 1018, further comprising the step of inhibiting CD47 activity or SIRPα activity to enhance the targeted effector activity. [The present invention 1025] The method of the present invention 1024, wherein the step of inhibiting CD47 activity or SIRPα activity includes contacting the cell with an anti-CD47 blocking antibody or an anti-SIRPα blocking antibody. [The present invention 1026] The method of the present invention 1018, wherein the intracellular domain of the CAR includes a dual signaling domain. [The present invention 1027] The method of the present invention 1018, wherein the antigen-binding domain of the CAR includes an antibody selected from the group consisting of a synthetic antibody, a human antibody, a humanized antibody, a single-domain antibody, a single-chain variable fragment, and antigen-binding fragments thereof. [The present invention 1028] The method of the present invention 1018, wherein the antigen-binding domain of the CAR is selected from the group consisting of an anti-CD19 antibody, an anti-HER2 antibody, and fragments thereof. [The present invention 1029] Further comprising the step of modifying the cell to deliver an agent, wherein the agent is selected from the group consisting of nucleic acids, antibiotics, anti-inflammatory agents, antibodies or antibody fragments thereof, growth factors, cytokines, enzymes, proteins, peptides, fusion proteins, synthetic molecules, organic molecules, carbohydrates or analogs thereof, lipids, hormones, microsomes, derivatives or modifications thereof, and any combination thereof, the method of the present invention 1018. [The present invention 1030] A composition comprising a cell modified according to the present invention 1018. [Brief Description of the Drawings]

[0022] The following detailed description of the preferred embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For purposes of illustration of the present invention, the presently preferred embodiments are shown in the drawings as examples. However, it should be understood that the present invention is not limited to the exact arrangements and means of the embodiments shown in the drawings.

[0023]

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Modes for Carrying Out the Invention

[0024] Detailed Description Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present invention, but the preferred materials and methods are described herein. The following specialized terms are used in the description and claim of the present invention.

[0025] It should also be understood that the specialized terms used herein are for the purpose of explaining only specific embodiments and are not intended to be limiting.

[0026] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element or more than one element.

[0027] As used herein, when referring to measurable values such as amounts, durations of time, etc., "about" is intended to encompass variations of ±20%, or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from a specified value, as such variations are appropriate for practicing the disclosed methods.

[0028] As used herein, "activation" refers to the state of monocytes / macrophages that have been sufficiently stimulated to induce detectable cell proliferation or have been stimulated to exert their effector functions. Activation can also be accompanied by induced cytokine production, phagocytosis, cell signaling, target cell killing, or antigen processing and presentation. The term "activated monocytes / macrophages" specifically refers to monocytes / macrophages that are undergoing cell division or are exerting effector functions.

[0029] The terms "agent", or "biological agent" or "therapeutic agent" are used herein to refer to molecules that may be expressed, released, secreted, or delivered to a target by the modified cells described herein. Agents include, without limitation, nucleic acids, antibiotics, anti-inflammatory agents, antibodies or antibody fragments thereof, growth factors, cytokines, enzymes, proteins, peptides, fusion proteins, synthetic molecules, organic molecules (e.g., small molecules), carbohydrates or analogs, lipids, hormones, microsomes, derivatives or modifications thereof, and any combination thereof. An agent can bind to any cellular moiety present on or in a target cell, such as a receptor, epitope, or other binding site, etc. An agent may be diffuse or transported into a cell where it may act intracellularly.

[0030] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. The antibody may be an intact immunoglobulin derived from a natural or recombinant source, or it may be an immunoreactive portion of an intact immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies in the present invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab')2, as well as single-chain antibodies (scFv) and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0031] The term "antibody fragment" refers to a portion of an intact antibody and refers to the variable antigen-determining region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0032] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in an entire antibody molecule in its naturally occurring conformation.

[0033] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in an entire antibody molecule in its naturally occurring conformation. Alpha and beta light chains refer to the two major antibody light chain isotypes.

[0034] As used herein, the term "synthetic antibody" means an antibody produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage described herein. This term also refers to an antibody produced by the synthesis of an antibody protein or an antibody-encoding DNA molecule expressing the amino acid sequence defining the antibody, where the DNA sequence or amino acid sequence is obtained using available DNA sequence or amino acid sequence synthesis techniques well known in the art and should be considered to also mean an antibody thus obtained.

[0035] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response can include either or both antibody production and activation of specific immunocompetent cells. One of ordinary skill in the art will understand that virtually any macromolecule, including all proteins or peptides, can act as an antigen. Further, an antigen can be derived from recombinant DNA or genomic DNA. One of ordinary skill in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response thus encodes an "antigen" as the term is used herein. Further, one of ordinary skill in the art will understand that an antigen need not be encoded only by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Further, one of ordinary skill in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be made, synthesized or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells or biological fluids.

[0036] As used herein, the term "antineoplastic effect" refers to a biological effect that can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in mean life span, or an improvement in various physiological symptoms associated with cancerous conditions. The "antineoplastic effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention in the prevention of the occurrence of tumors per se.

[0037] As used herein, the term "self-antigen" means any self-antigen that is recognized as foreign by the immune system according to the present invention. Self-antigens include, but are not limited to, cellular proteins, phosphoproteins, cell surface proteins, cell lipids, nucleic acids, and glycoproteins, including cell surface receptors.

[0038] As used herein, the term "autoimmune disease" is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis.

[0039] As used herein, the term "self" is intended to refer to any material derived from the same individual that is later reintroduced into that individual.

[0040] "Allogeneic" refers to a graft derived from different animals of the same species.

[0041] "Xenogeneic" refers to a graft derived from animals of different species.

[0042] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. In certain embodiments, the cancer is medullary thyroid cancer.

[0043] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial T cell surface receptor engineered to be expressed on immune effector cells and that specifically binds to an antigen. CARs can be used as a therapy involving adoptive cell transfer. Monocytes are removed from a patient (blood, tumor, or ascites) and modified to express a receptor specific for a particular form of antigen. In some embodiments, the CAR is expressed, for example, with specificity for a tumor-associated antigen. The CAR can also include an extracellular domain that includes an intracellular activation domain, a transmembrane domain, and a tumor-associated antigen binding region. In some aspects, the CAR includes a fusion of a single-chain variable fragment (scFv)-derived monoclonal antibody fused to the CD3ζ transmembrane and intracellular domains. The specificity of the CAR design can be derived from the ligand (e.g., peptide) of the receptor. In some embodiments, the CAR can target cancer by redirecting monocytes / macrophages that express a CAR specific for a tumor-associated antigen.

[0044] The term "chimeric intracellular signaling molecule" refers to a recombinant receptor that includes one or more intracellular domains of one or more stimulatory and / or costimulatory molecules. The chimeric intracellular signaling molecule is substantially lacking an extracellular domain. In some embodiments, the chimeric intracellular signaling molecule includes additional domains, such as a transmembrane domain, a detectable tag, and a spacer domain, etc.

[0045] As used herein, the term "conservative sequence modification" is intended to mean an amino acid modification that does not significantly affect or change the binding characteristics of an antibody comprising an amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. The modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine) and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family, and the altered antibody can be tested for antigen binding ability using the functional assay methods described herein.

[0046] As used herein, the term "costimulatory ligand" includes molecules on antigen-presenting cells (e.g., aAPC, dendritic cells, B cells, etc.) that specifically bind to cognate costimulatory molecules on monocytes / macrophages, thereby providing signals that mediate monocyte / macrophage responses, including but not limited to proliferation, activation, differentiation, etc. Costimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intracellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands can also include, among others and without limitation, antibodies that specifically bind to costimulatory molecules present on monocytes / macrophages such as CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0047] "Co-stimulatory molecule" refers to a molecule on innate immune cells that is used to enhance or weaken the initial stimulus. For example, pathogen-associated pattern recognition receptors, such as TLR (enhancing) or the CD47 / SIRPα axis (weakening), etc., are molecules on innate immune cells. Co-stimulatory molecules include TCR, CD3ζ, CD3γ, CD3δ, CD3ε, CD86, common FcRγ, FcRβ (FcεR1b), CD79a, CD79b, FcγRIIa, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, other co-stimulatory molecules described herein, any derivatives, variants, or fragments thereof, any synthetic sequences of co-stimulatory molecules having the same functional ability, and any combinations thereof are included without limitation.

[0048] As used herein, the term "co-stimulatory signal" refers to a signal that, in combination with a primary signal, such as activation of a CAR on a macrophage, leads to activation of the macrophage.

[0049] The term "cytotoxic" or "cytotoxicity" refers to killing or damaging cells. In one embodiment, the cytotoxicity of metabolically enhanced cells is, for example, the increased cytolytic activity of macrophages.

[0050] A "disease" is the health state of an animal in which the animal cannot maintain homeostasis and its health continues to deteriorate if the disease is not improved. In contrast, a "disorder" in an animal is a health state in which the animal can maintain homeostasis but is in a less favorable health state than when the animal has no disorder. Left untreated, a disorder does not necessarily cause a further decline in the animal's health state.

[0051] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to the amount of a compound, formulation, material or composition described herein that is effective to achieve a particular biological result or to provide a therapeutic or prophylactic benefit. Such results can include, but are not limited to, antitumor activity as determined by any suitable means in the art.

[0052] "Encoding" refers to the unique property of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, and the biological properties resulting therefrom, which serves as a template for the synthesis of other polymers and macromolecules in a biological process and has either a defined nucleotide (i.e., rRNA, tRNA, and mRNA) sequence or a defined amino acid sequence. Thus, a gene encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, which is identical to the mRNA sequence and is usually the nucleotide sequence shown in the sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode a protein or other product of that gene or cDNA.

[0053] As used herein, "endogenous" refers to any material that is derived from within or produced within an organism, cell, tissue, or system.

[0054] As used herein, the term "exogenous" refers to any material that is introduced from outside or produced outside an organism, cell, tissue, or system.

[0055] As used herein, the term "increasing" refers to an increase in number, such as an increase in the number of monocytes / macrophages. In one embodiment, monocytes / macrophages increased ex vivo have an increased number compared to the number initially present in the culture. In another embodiment, monocytes / macrophages increased ex vivo have an increased number compared to other cell types in the culture. As used herein, the term "ex vivo" refers to cells that are removed from an organism (e.g., a human) and grown outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0056] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0057] An "expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains cis-acting elements sufficient 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 those contained in liposomes) into which the recombinant polynucleotide has been incorporated, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0058] As used herein, "homology" refers to subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When the subunit positions in both of the two molecules are occupied by the same monomeric subunit; for example, if the positions in each of the two DNA molecules are occupied by adenine, they are homologous at that position. The homology between two sequences is a linear function of the number of positions that are identical or homologous; for example, if half of the positions in two sequences (e.g., 5 positions in a 10-subunit polymer) are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are identical or homologous, the two sequences are 90% homologous. When applied to a nucleic acid or protein, "homologous" as used herein refers to a sequence having about 50% sequence identity. More preferably, the homologous sequences have about 75% sequence identity, and even more preferably at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.

[0059] The "humanized" form of a non-human (e.g., murine) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding portion sequences of an antibody) that contains minimal sequences derived from a non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's complementarity-determining regions (CDRs) have been replaced by residues from the CDRs of a non-human species (donor antibody), such as a mouse, rat, or rabbit, that has the desired specificity, affinity, and potency. In some cases, residues in the Fv framework region (FR) of the human immunoglobulin are replaced by the corresponding non-human residues. Additionally, a humanized antibody can contain residues not found in the recipient antibody or in the introduced CDR or framework sequences. These modifications are made to further improve and optimize antibody performance. Generally, a humanized antibody contains substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are of human immunoglobulin sequence. Also, a humanized antibody optimally contains at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0060] "Fully human" refers to an immunoglobulin, such as an antibody, in which the entire molecule is of human origin or consists of the same amino acid sequence as the human form of the antibody.

[0061] As used herein, "identity" refers to subunit sequence identity between two polymer molecules, such as between two polypeptide molecules, particularly between two amino acid molecules. When two amino acid sequences have the same residue at the same position; for example, if the position in each of two polypeptide molecules is occupied by arginine, they are identical at that position. The degree or identity to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a linear function of the number of positions that are identical or the same; for example, if half of the positions in two sequences (e.g., 5 positions in a 10-amino acid polymer) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are identical or the same, the two amino acid sequences are 90% identical.

[0062] "Substantially identical" means a polypeptide molecule or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, more preferably 90%, 95% or even 99% identical to the sequence used for comparison, at the amino acid level or for nucleic acids.

[0063] The guide nucleic acid sequence may be complementary to one strand (nucleotide sequence) of the double-stranded DNA target site. The percentage of complementarity between the guide nucleic acid sequence and the target sequence can be at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. The guide nucleic acid sequence can be at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 nucleotides or longer. In some embodiments, the guide nucleic acid sequence is a continuous stretch of 10-40 nucleotides. The variable targeting domain can be composed of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence (see, e.g., the modifications described herein), or any combination thereof.

[0064] Sequence identity is typically measured using sequence analysis software (e.g., the Sequence Analysis Software Package from Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or the PILEUP / PRETTYBOX program). Such software matches identical or similar sequences by specifying the degree of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In one exemplary approach for determining the degree of identity, the BLAST program can be used, and the probability score between e -3 ~e -100 indicates that the sequences are related.

[0065] As used herein, the terms "immunoglobulin" or "Ig" are defined as classes of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the major antibody present in body secretions such as saliva, tears, breast milk, gastrointestinal tract secretions, and mucus secretions of the respiratory and urogenital tracts. IgG is the most common circulating blood antibody. IgM is the major immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination reactions, complement fixation, and other antibody responses and is important in defense against bacteria and viruses. IgD is an immunoglobulin whose antibody function is unknown but may act as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by causing the release of mediators from mast cells and basophils upon exposure to allergens.

[0066] As used herein, the term "immune response" is defined as the cellular response to an antigen that occurs when lymphocytes identify an antigen molecule as foreign, induce the formation of antibodies, and / or activate lymphocytes to remove the antigen.

[0067] As used herein, "instructional material" includes publications, records, diagrams, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the present invention. The instructional material of the kits of the present invention may, for example, be attached to a container containing the nucleic acids, peptides, and / or compositions of the present invention, or may be shipped together with a container containing the nucleic acids, peptides, and / or compositions. Alternatively, the instructional material may be shipped separately from the container, with the intention that the instructional material and the compound be used jointly by the recipient.

[0068] "Isolated" means changed or removed from its natural state. For example, a nucleic acid or peptide that naturally exists in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from its co-existing substances in its natural state is "isolated". An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-natural environment such as, for example, a host cell.

[0069] As used herein, "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; since they can deliver a significant amount of genetic information into the DNA of host cells, they are one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means for achieving significant levels of gene transfer in vivo.

[0070] As used herein, the term "modified" means an altered state or structure of a molecule or cell of the present invention. A molecule can be modified in many ways, such as chemically, structurally, and functionally. A cell can be modified by the introduction of nucleic acids.

[0071] As used herein, the term "modulate" means to mediate a detectable increase or decrease in the level of response in a subject as compared to the level of response in the subject in the absence of treatment or compound and / or as compared to the level of response in a subject that is otherwise identical but not receiving treatment. This term encompasses disturbing and / or affecting a natural signal or response in a subject, preferably a human, thereby mediating a beneficial therapeutic response.

[0072] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0073] Unless otherwise specified, the term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate to each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding an RNA or protein" also means that the nucleotide sequence encoding the protein may, depending on the type, contain introns to the extent that it may contain introns.

[0074] The term "functionally linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, if a first nucleic acid sequence is placed under the functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is functionally linked to the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is functionally linked to the coding sequence. Generally, functionally linked DNA sequences are continuous and, when it is necessary to join two protein-coding regions, are in the same reading frame.

[0075] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to indicate that the expression of the tumor antigen in cells from a disease area such as a solid tumor within a particular tissue or organ of a patient is at an abnormal level compared to the level of expression in normal cells from that tissue or organ. A patient having a solid tumor or hematological malignancy characterized by overexpression of a tumor antigen can be determined by standard assay methods known in the art.

[0076] "Parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), intratumoral (i.t.) or intraperitoneal (i.p.), or intrasternal injection, or infusion methods.

[0077] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Further, a nucleic acid is a polymer of nucleotides. Thus, the nucleic acids and polynucleotides used herein are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides and that they can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, nucleic acid sequences obtained by any available means in the art, including recombinant means, i.e., conventional cloning techniques and PCR (商標) such as recombinant libraries using such methods or cloning of nucleic acid sequences from cell genomes, as well as all nucleic acid sequences obtained by synthetic means, but are not limited thereto.

[0078] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute the sequence of a protein or peptide. Polypeptides include any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, this term refers to both short chains, which are generally referred to in the art as peptides, oligopeptides and oligomers, and long chains, which are generally referred to in the art as proteins, and there are many types thereof. "Polypeptide" includes, inter alia, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0079] As used herein, the term "promoter" is defined as a DNA sequence recognized by the synthetic machinery of a cell or an introduced synthetic machinery that is necessary to initiate specific transcription of a polynucleotide sequence.

[0080] As used herein, the term "promoter / regulatory sequence" means a nucleic acid sequence required for the expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may include enhancer sequences and other regulatory elements required for the expression of the gene product. The promoter / regulatory sequence may, for example, cause the gene product to be expressed in a tissue-specific manner.

[0081] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced intracellularly under most or all physiological conditions of the cell.

[0082] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced intracellularly only when an inducer substantially corresponding to the promoter is present intracellularly.

[0083] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes the gene product to be produced intracellularly only when the cell is substantially a cell of the tissue type corresponding to the promoter.

[0084] The term "resistant to immunosuppression" means the absence or reduction of suppression of the activity or activation of the immune system.

[0085] "Signal transduction pathway" refers to the biochemical relationships among various signal transduction molecules that play a role in the transmission of signals from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and molecular complexes that can receive signals and transmit signals across the plasma membrane of the cell.

[0086] "Single-chain antibody" refers to an antibody formed by recombinant DNA techniques in which immunoglobulin heavy and light chain fragments are linked to the Fv region via engineered amino acid lengths. Various methods for making single-chain antibodies are known, including those described in U.S. Patent No. 4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041.

[0087] As used herein with respect to an antibody, the term "specifically binds" means an antibody that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may bind to that antigen from one or more species. However, such cross-reactivity by itself does not change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may bind to different genotypes of that antigen. However, such cross-reactivity by itself does not change the classification of the antibody as specific. In some cases, the terms "specific binding" or "specifically binds" may be used in connection with the interaction of an antibody, protein or peptide with a second chemical species, meaning that the interaction depends on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than the entire protein. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody in a reaction containing labeled "A" and that antibody.

[0088] The term "stimulation" means a primary response induced by a stimulatory molecule (e.g., TCR / CD3 complex) binding to its cognate ligand, thereby mediating a signaling event such as, but not limited to, signaling through the Fc receptor mechanism or an artificial CAR. Stimulation can mediate changes in the expression of certain molecules such as downregulation of TGF-β and / or reorganization of the cytoskeletal structure.

[0089] As used herein, the term "stimulatory molecule" means a molecule on a monocyte / macrophage that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0090] As used herein, a "stimulatory ligand" means a ligand that, when present on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.) or a tumor cell, can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule") on a monocyte / macrophage, thereby mediating a response by immune cells, including but not limited to activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, inter alia, Toll-like receptor (TLR) ligands, anti-Toll-like receptor antibodies, agonists, and antibodies against monocyte / macrophage receptors. In addition, cytokines such as interferon-γ are potent stimulants for macrophages.

[0091] The term "subject" is intended to include organisms (e.g., mammals) in which an immune response can be induced. As used herein, a "subject" or "patient" can be a human or a non-human mammal. Non-human mammals include, for example, domestic animals and pets such as sheep, cows, pigs, dogs, cats, and murine mammals. Preferably, the subject is a human.

[0092] As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell that has been separated from other cell types that are normally associated with it in its native state. In one example, a population of substantially purified cells refers to a homogeneous population of cells. In another example, the term simply refers to a cell that has been separated from cells that are normally associated with it in its native state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0093] A "target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0094] "Target" means a cell, organ, or site in the body that requires treatment.

[0095] As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins involved in the activation of T cells in response to antigen presentation. The TCR plays a role in recognizing antigens bound to major histocompatibility complex molecules. The TCR is composed of a heterodimer of alpha (α) and beta (β) chains, although in some cells the TCR consists of gamma and delta (γ / δ) chains. The TCR can exist in α / β and γ / δ forms that are structurally similar but have different anatomical locations and functions. Each chain is composed of two extracellular domains, namely a variable domain and a constant domain. In some embodiments, the TCR can be modified on any cell that contains the TCR (e.g., including helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and γδ T cells).

[0096] As used herein, the term "therapeutic" means treatment and / or prevention. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0097] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is introduced or transferred into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. This includes primary subject cells and their progeny.

[0098] "Treating" a disease, as the term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder that the subject is suffering from.

[0099] As used herein, the term "tumor" means an abnormal growth of tissue that can be benign, pre-cancerous, malignant, or metastatic.

[0100] As used herein, the terms "under transcriptional control" or "functionally linked" mean that the promoter is in the correct position and orientation with respect to the polynucleotide to control the initiation of transcription by RNA polymerase and the expression of the polynucleotide.

[0101] A "vector" is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into the interior of a cell. A number of vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides complexed with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the entry of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

[0102] Range: Throughout this disclosure, various aspects of the invention can be presented in range format. It should be understood that the description in range format is merely for convenience and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, a description of a range should be considered to have specifically disclosed all the sub-ranges and individual numerical values within that range. For example, a range description such as 1 - 6 should be considered to specifically disclose sub-ranges such as 1 - 3, 1 - 4, 1 - 5, 2 - 4, 2 - 6, 3 - 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0103] Description Accumulating evidence suggests that macrophages are present in large numbers in the tumor microenvironment of many cancers, where they can adopt classically activated (M1, antitumor) or alternatively activated (M2, tumor-promoting) phenotypes. Macrophages are powerful effectors of the innate immune system and can perform at least three distinct antitumor functions: phagocytosis, cytotoxicity, and antigen presentation to regulate the adaptive immune response. While T cells require antigen-dependent activation via the T cell receptor or chimeric immune receptor, macrophages can be activated in various ways. Direct macrophage activation is antigen-independent and relies on the recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs). Immune complex-mediated activation is antigen-dependent but requires the presence of antigen-specific antibodies and the absence of inhibitory CD47-SIRPα interactions.

[0104] Tumor-associated macrophages have been shown to be reprogrammable by the tumor microenvironment and to serve as important immunosuppressive actors in the microenvironment. Therefore, if macrophages can be genetically engineered to prevent the occurrence of immunosuppressive gene reprogramming, it would be a vertical advancement in this field.

[0105] The present invention includes compositions and methods for treating malignant tumors in a subject. The present invention includes the expression of a chimeric antigen receptor in monocytes, macrophages or dendritic cells. Such modified cells are mobilized to the tumor microenvironment where they act as potent immune effectors by infiltrating the tumor and killing target cells.

[0106] Chimeric antigen receptor (CAR) In one aspect of the present invention, modified monocytes, macrophages or dendritic cells are produced by expressing a CAR therein. Accordingly, the present invention encompasses CARs and nucleic acid constructs encoding CARs, where the CAR includes an antigen-binding domain, a transmembrane domain, and an intracellular domain.

[0107] In one aspect, the present invention includes a modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain of a co-stimulatory molecule, and the cell is a monocyte, macrophage, or dendritic cell having targeted effector activity. In another aspect, the present invention includes a modified cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the nucleic acid sequence comprising a nucleic acid sequence encoding an antigen-binding domain, a nucleic acid sequence encoding a transmembrane domain, and a nucleic acid sequence encoding an intracellular domain of a co-stimulatory molecule, and the cell is a monocyte, macrophage, or dendritic cell that expresses the CAR and has targeted effector activity. In one embodiment, the targeted effector activity is directed against an antigen on a target cell that specifically binds to the antigen-binding domain of the CAR. In another embodiment, the targeted effector activity is selected from the group consisting of phagocytosis, targeted cytotoxicity, antigen presentation, and cytokine secretion.

[0108] Antigen-binding domain In one embodiment, the CAR of the present invention comprises an antigen-binding domain that binds to an antigen on a target cell. Examples of cell surface markers that can act as antigens that bind to the antigen-binding domain of the CAR include those associated with viral infections, bacterial infections, and parasitic infections, autoimmune diseases, and cancer cells.

[0109] The selection of the antigen-binding domain depends on the type and number of antigens present on the surface of the target cell. For example, the antigen-binding domain may be selected to recognize an antigen that acts as a cell surface marker on a target cell associated with a particular disease state.

[0110] In one aspect, the antigen-binding domain binds to a tumor antigen, such as an antigen specific to a tumor or cancer of interest. In one aspect, the tumor antigen of the present invention comprises one or more antigenic cancer epitopes. Non-limiting examples of tumor-associated antigens include the following: CD19; CD123; CD22; CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319 and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); protease-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface-associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostate acid phosphatase (PAP); elongation factor 2 mutant (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prososome, macropain) subunit, beta type, 9 (LMP2);Glycoprotein 100 (gp100); cancer gene fusion protein (bcr-abl) consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl); tyrosine kinase; Ephrin type-A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor β; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globoH glycosphingolipid (GloboH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR γ-selective reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostain; survivin; telomerase;Prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen recognized by T cell 1 (Melan A or MART1); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein) like (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen recognized by T cell 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1).;

[0111] The antigen-binding domain can include any domain that binds to an antigen, which can include, without limitation, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, and any fragments thereof. Thus, in one aspect, the antigen-binding domain portion includes a mammalian antibody or a fragment thereof. In another aspect, the antigen-binding domain of the CAR is selected from the group consisting of anti-CD19 antibodies, anti-HER2 antibodies, and fragments thereof.

[0112] In some cases, the antigen-binding domain is derived from the same species as that in which the CAR will ultimately be used. For example, for use in humans, the antigen-binding domain of the CAR includes a human antibody, a humanized antibody, or a fragment thereof.

[0113] In some aspects of the present invention, the antigen-binding domain is functionally linked to another domain of the CAR, such as a transmembrane domain or an intracellular domain, for expression in a cell. In one aspect, the nucleic acid encoding the antigen-binding domain is functionally linked to the nucleic acid encoding the transmembrane domain and the nucleic acid encoding the intracellular domain.

[0114] Transmembrane domain With respect to the transmembrane domain, the CAR can be designed to include a transmembrane domain that connects the antigen-binding domain of the CAR to the intracellular domain. In one aspect, the transmembrane domain is naturally associated with one or more of the domains in the CAR. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid the binding of such a domain to the transmembrane domain of the same or different surface membrane proteins in order to minimize the interaction with other members of the receptor complex.

[0115] The transmembrane domain may be derived from either a natural or a synthetic source. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Transmembrane regions that are particularly useful in the present invention can be derived from the α, β, or ζ chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9 (i.e., including at least their transmembrane regions). In some cases, various human hinges, including the human Ig (immunoglobulin) hinge, can also be used.

[0116] In one embodiment, the transmembrane domain may be synthetic, in which case it is thought to mainly contain hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.

[0117] Intracellular domain The intracellular domain of the CAR, or in other words the cytoplasmic domain, contains an intracellular domain similar to or identical to the chimeric intracellular signaling molecules described elsewhere herein, and causes activation of the cells in which the CAR is expressed.

[0118] In one embodiment, the intracellular domain of the CAR contains domains that cause signal activation and / or transduction.

[0119] Examples of intracellular domains for use in the present invention include the cytoplasmic portion of surface receptors, costimulatory molecules, and any molecule that acts cooperatively to elicit signaling in monocytes, macrophages, or dendritic cells, as well as any derivative or mutant of these elements and any synthetic sequence having the same functional capacity, but are not limited thereto.

[0120] Examples of intracellular domains include ligands that specifically bind to TCR, CD3ζ, CD3γ, CD3δ, CD3ε, CD86, common FcRγ, FcRβ (FcεR1b), CD79a, CD79b, FcγRIIa, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other costimulatory molecules described herein, any derivatives, variants or fragments thereof, any synthetic sequences of costimulatory molecules having the same functional ability, and fragments or domains derived from one or more molecules or receptors, including, without limitation, any combinations thereof.

[0121] In one aspect, the intracellular domain of the CAR comprises a dual signaling domain, such as 41BB, CD28, ICOS, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, the β chain of the CD116 receptor, CSF1-R, LRP1 / CD91, SR-A1, SR-A2, MARCO, SR-CL1, SR-CL2, SR-C, SR-E, CR1, CR3, CR4, dectin 1, DEC-205, DC-SIGN, CD14, CD36, LOX-1, CD11b, in any combination, together with any of the signaling domains listed in the above paragraphs. In another aspect, the intracellular domain of the CAR comprises any portion of one or more co-stimulatory molecules, such as at least one signaling domain derived from CD3, the FcεRIγ chain, any derivatives or variants thereof, any synthetic sequences having the same functional ability, and any combination thereof.

[0122] A spacer domain may be incorporated between the antigen-binding domain and the transmembrane domain of the CAR, or between the intracellular domain and the transmembrane domain of the CAR. As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide that serves to link the transmembrane domain to either the antigen-binding domain or the intracellular domain in the polypeptide chain. In one aspect, the spacer domain is composed of up to 300 amino acids, preferably 10 - 100 amino acids, most preferably 25 - 50 amino acids. In another aspect, a short oligopeptide linker or polypeptide linker, preferably one having a length of 2 - 10 amino acids, may form the linkage between the transmembrane domain and the intracellular domain of the CAR. An example of a linker includes a glycine-serine doublet.

[0123] human antibody When using the antigen-binding domain of a CAR, it is considered preferable to use a human antibody or a fragment thereof. For the therapeutic treatment of human subjects, fully human antibodies are particularly desirable. Human antibodies can be produced by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences, in combination with improved methods of these techniques. See also U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT Publication Nos. WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741, each of which is incorporated herein by reference in its entirety.

[0124] In addition, human antibodies can also be produced using transgenic mice that do not have the ability to express functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to the human heavy and light chain genes, human variable regions, constant regions, and diversity regions can also be introduced into mouse embryonic stem cells. The mouse heavy and light chain immunoglobulin genes can be made non-functional separately or simultaneously with the introduction of the human immunoglobulin locus by homologous recombination. For example, homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric mice and germline mutant mice has been described to result in complete inhibition of endogenous antibody production. These modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. Subsequently, the chimeric mice are mated to produce homozygous progeny that express human antibodies. The transgenic mice are immunized in the usual manner with a selected antigen, for example, the whole or a part of the polypeptide of the present invention. Directional antibodies against the selected target can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice are rearranged during B cell differentiation and then undergo class switching and somatic mutations. Therefore, it is possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies, including but not limited to IgG1 (γ1) and IgG3, using such techniques. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (Int. Rev. Immunol, 13:65-93 (1995)).For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies, as well as protocols for producing such antibodies, see, for example, PCT Publication Nos. WO 98 / 24893, WO 96 / 34096 and WO 96 / 33735; and U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, each of which is hereby incorporated by reference in its entirety. In addition, contracts can be made with companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) to obtain human antibodies directed against selected antigens using techniques similar to those described above. For a specific discussion of the transfer of the human germline immunoglobulin gene array into germline mutant mice, which is thought to result in the production of human antibodies by antigen loading stimulation, see, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol, 7:33 (1993); and Duchosal et al., Nature, 355:258 (1992).

[0125] Human antibodies can also be derived from phage display libraries (Hoogenboom et al., J. Mol. Biol, 227:381 (1991); Marks et al., J. Mol. Biol, 222:581-597 (1991); Vaughan et al., Nature Biotech., 14:309 (1996)). Phage display technology (McCafferty et al., Nature, 348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro from the immunoglobulin variable (V) domain gene repertoire of non-immunized donors. According to this approach, the antibody V domain gene is cloned in-frame into the gene of the major coat protein or minor coat protein of filamentous bacteriophage, such as M13 or fd, and presented as a functional antibody fragment on the surface of phage particles. Since the filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in the selection of the gene encoding the antibody exhibiting those properties. Thus, phage mimics some of the properties of B cells. Phage display can be performed in various formats; for an overview, see Johnson, Kevin S, and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small, random combinatorial library of V genes derived from the spleens of immunized mice.Construct a repertoire of V genes from non-immunized human donors and isolate antibodies against diverse arrays of antigens (including autoantigens) essentially according to the methods described in Marks et al., J. Mol. Biol, 222:581-597 (1991), or Griffith et al., EMBO J., 12:725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905, which are hereby incorporated by reference in their entireties.

[0126] Human antibodies can also be made by in vitro activated B cells (see U.S. Patent Nos. 5,567,610 and 5,229,275, which are hereby incorporated by reference in their entireties). Human antibodies can also be made in vitro using hybridoma techniques such as those described by Roder et al. (Methods Enzymol, 121:140-167 (1986)), among others.

[0127] Humanized antibody Alternatively, in some embodiments, non-human antibodies can be humanized, in which case specific sequences or regions of the antibody are modified to enhance similarity to antibodies naturally produced in humans. For example, in the present invention, an antibody or fragment thereof may comprise a non-human mammalian scFv. In one embodiment, the antigen-binding domain portion is humanized.

[0128] Humanized antibodies can be generated using a variety of techniques known in the art, including but not limited to: CDR grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein by reference in its entirety), veneering or resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al., 1994, Proc Natl Acad Sci USA 91:969-973, each of which is incorporated herein by reference in its entirety), chain shuffling (see, e.g., U.S. Patent No. 5,565,332.This is incorporated herein by reference in its entirety), as well as, for example, the methods disclosed in US Patent Application Publication No. US2005 / 0042664, US Patent Application Publication No. US2005 / 0048617, US Patent No. 6,407,213, US Patent No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol, 169: 1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8): 1717-22 (1995), Sandhu J S, Gene, 150(2):409-10(1994), and Pedersen et al., J. Mol. Biol, 235(3):959-73 (1994), each of which is incorporated herein by reference in its entirety. In many cases, framework residues in the framework region are thought to be replaced by the corresponding residues from the CDR donor antibody, preferably to improve antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interaction between the CDR and framework residues to identify framework residues important for antigen binding, and by sequence comparison to identify unusual framework residues at specific positions (see, for example, Queen et al., US Patent No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, each of which is incorporated herein by reference in its entirety).

[0129] Humanized antibodies have one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are typically taken from an "import" variable domain. Thus, a humanized antibody contains one or more CDRs from a non-human immunoglobulin molecule and a framework region from a human. Humanization of antibodies is well known in the art and essentially involves substituting the corresponding sequences of a human antibody with rodent CDRs or CDR sequences, i.e., CDR grafting (EP 239,400; PCT Publication No. WO 91 / 09967; and U.S. Patents Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640. The entire contents of these are incorporated herein by reference). In such humanized chimeric antibodies, the human variable domain that is not substantially intact is replaced by the corresponding sequence from a non-human species. In practice, a humanized antibody is typically a human antibody in which some CDR residues and perhaps some framework (FR) residues have been replaced by residues from the analogous sites in a rodent antibody. Also, humanization of an antibody can be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Patent No. 5,565,332), the entire contents of which are incorporated herein by reference).

[0130] The selection of human variable domains of both the light and heavy chains, which is used in the production of humanized antibodies, aims to reduce antigenicity. According to the so-called "best fit" method, the sequences of the variable domains of rodent antibodies are screened against the entire library of known human variable domain sequences. Subsequently, the human sequence closest to that of the rodent is accepted as the human framework (FR) of the humanized antibody (Sims et al., J. Immunol, 151:2296 (1993); Chothia et al., J. Mol. Biol, 196:901 (1987), the contents of which are hereby incorporated by reference in their entirety). In another method, a specific framework derived from the consensus sequence of all human antibodies of a particular subgroup of the light or heavy chain is used. The same framework can be used for several different humanized antibodies ((Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993), the contents of which are hereby incorporated by reference in their entirety).

[0131] The antibody can be humanized, which retains a high affinity for the target antigen and has other advantageous biological properties. According to one aspect of the present invention, a humanized antibody is prepared by a process of analyzing the parental sequence and various conceptual humanized products using three-dimensional models of the parental sequence and the humanized sequence. Three-dimensional models of immunoglobulins are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display the possible three-dimensional conformations for a selected candidate immunoglobulin sequence. Examination of these displays allows for analysis of the putative role of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of the residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so as to achieve desired antibody properties such as increased affinity for the target antigen. Generally, CDR residues are most directly and substantially involved in affecting antigen binding.

[0132] The humanized antibody retains an antigen specificity similar to that of the original antibody. However, using certain humanization methods, the binding affinity and / or specificity of the antibody for the target antigen can be enhanced using a method of "directed evolution" as described by Wu et al., J. Mol. Biol, 294:151 (1999), the entire contents of which are incorporated herein by reference.

[0133] Vector Vectors may be used to introduce CARs into monocytes, macrophages or dendritic cells as described elsewhere herein. In one aspect, the invention includes a vector comprising a nucleic acid sequence encoding a CAR as described herein. In one embodiment, the vector includes a plasmid vector, a viral vector, a retrotransposon (e.g., piggyback, sleeping beauty), a site-specific integration vector (e.g., CRISPR, Zn finger nuclease, TALEN), or a suicide expression vector, or other vectors known in the art.

[0134] All of the constructs described above can be used with a third-generation lentiviral vector plasmid, other viral vectors, or RNA that is approved for use in human cells. In one embodiment, the vector is a viral vector such as a lentiviral vector. In another embodiment, the vector is an RNA vector.

[0135] The production of any of the molecules described herein can be verified by sequencing. The expression of full-length proteins can be verified using immunoblotting, immunohistochemistry, flow cytometry, or other techniques well-known and available in the art.

[0136] The present invention also provides vectors into which the DNA of the present invention has been inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, as they allow for the long-term and stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncoretroviruses such as murine leukemia virus, as they can also transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity in the subjects into which they are introduced.

[0137] The expression of natural or synthetic nucleic acids is typically achieved by operably linking the nucleic acid or a portion thereof to a promoter and incorporating the construct into an expression vector. Vectors are generally replicable in mammalian cells and / or can be integrated into the mammalian cell genome. Typical vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0138] Nucleic acids can be cloned into various types of vectors. For example, nucleic acids can be cloned into vectors including, without limitation, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particularly interesting vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0139] Expression vectors can also be delivered to cells in the form of viral vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), as well as in other manuals of virology and molecular biology. Viruses useful as vectors include, without limitation, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors include an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (see, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0140] Other promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although some promoters have recently been shown to contain functional elements downstream of the start site as well. There is flexibility in the spacing between promoter elements, such that the promoter function is retained even if the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased up to 50 bp without causing a decrease in responsiveness. Depending on the promoter, individual elements may act cooperatively or independently to activate transcription.

[0141] An example of a suitable promoter is the cytomegalovirus (CMV) immediate early promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto. However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, elongation factor 1α promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can activate the expression of a polynucleotide sequence operably linked thereto when such expression is desired and inactivate the expression when it is not desired. Examples of inducible promoters include, but are not limited to, the metallothionine promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0142] For the purpose of evaluating the expression of a CAR polypeptide or a portion thereof, a selection marker gene and / or a reporter gene can be included in the expression vector introduced into the cells to facilitate the identification and selection of the expressing cells from the population of cells to be transfected or infected with a viral vector. In other aspects, the selection marker can be carried on a separate DNA fragment and used in a co-transfection procedure. To enable expression in the host cells, both the selection marker gene and the reporter gene can be adjacent to appropriate regulatory sequences. Useful selection markers include, for example, antibiotic resistance genes such as neo.

[0143] Reporter genes are used to identify cells that may have been transfected and to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that encodes a polypeptide that is not present in or expressed by the recipient organism or tissue and whose expression is manifested by some easily detectable property, such as enzymatic activity. The expression of the reporter gene is assayed at an appropriate time after its DNA has been introduced into the recipient cells. Suitable reporter genes can include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the gene for green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known methods or obtained commercially. Generally, a construct having a minimal 5' flanking region that shows the highest level of expression of the reporter gene is identified as a promoter. Such a promoter region can be ligated to the reporter gene and used to evaluate the ability of an agent to regulate transcription operative by the promoter.

[0144] Introduction of Nucleic Acids In one aspect, the present invention includes a method for modifying a cell, comprising the step of introducing a chimeric antigen receptor (CAR) into a monocyte, macrophage or dendritic cell, the CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain of a co-stimulatory molecule, the cell being a monocyte, macrophage or dendritic cell that expresses the CAR and possesses targeted effector activity. In one embodiment, introduction of the CAR into the cell comprises introducing a nucleic acid sequence encoding the CAR. In another embodiment, introduction of the nucleic acid sequence comprises introducing mRNA encoding the CAR by electroporation.

[0145] Methods for introducing and expressing genes such as CARs into cells are known in the art. In connection with expression vectors, the vectors can be readily introduced into host cells, such as mammalian cells, bacterial cells, yeast cells or insect cells, by any method in the art. For example, the expression vector can be introduced into the host cell by physical, chemical or biological means.

[0146] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1 -4, Cold Spring Harbor Press, NY). Nucleic acids can be introduced into target cells using commercially available methods including electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany)). Nucleic acids can also be introduced into cells using cationic liposome-mediated transfection using lipofection, using polymer encapsulation, using peptide-mediated transfection, or using a microparticle gun particle delivery system such as a "gene gun" (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0147] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. RNA vectors include vectors having an RNA promoter and / or other related domains for producing RNA transcripts. Viral vectors, and particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from, for example, lentivirus, poxvirus, herpes simplex virus, adenovirus, and adeno-associated virus. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0148] Chemical means for introducing a polynucleotide into a host cell include lipid-based systems such as macromolecular complexes, nanocapsules, microspheres, colloidal dispersions such as beads, and water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0149] When using a non-viral delivery system, one exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with the lipid can be encapsulated within the aqueous interior of the liposome, placed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule that binds to both the liposome and the oligonucleotide, entrapped within the liposome, complexed with the liposome, dispersed in a lipid-containing solution, mixed with the lipid, formulated with the lipid, included as a suspension in the lipid, included or complexed in micelles, or otherwise associated with the lipid in other ways. Compositions related to lipids, lipid / DNA or lipid / expression vectors are not limited to any particular structure in solution. For example, they can exist within a bilayer structure, as micelles, or as a "collapsed" structure. They can also simply be dispersed in solution and may form aggregates that are not uniform in size or shape. Lipids are fatty substances that may be natural or synthetic lipids. For example, lipids include lipid droplets that naturally occur in the cytoplasm, as well as classes of compounds that include long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes, etc.

[0150] Suitable lipids can be obtained from commercial sources. For example, dimyristoyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristoyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Since chloroform evaporates more readily than methanol, it is used as the sole solvent. "Liposome" is a general term encompassing various monolayer and multilayer lipid media formed by the generation of sealed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-reorganization before the formation of a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions having structures different from normal vesicular structures in solution are also included. For example, the lipids can assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0151] Regardless of the method used to introduce exogenous nucleic acids into a host cell or to expose cells to the molecules described herein by other means, various assay methods can be carried out to confirm the presence of nucleic acids in the host cell. Such assay methods include, for example, "molecular biology" assay methods well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assay methods for detecting the presence or absence of a specific peptide, for example, by immunological means (ELISA and Western blot) or by the assay methods described herein for identifying agents falling within the scope of the present invention.

[0152] In one embodiment, one or more of the nucleic acid sequences are introduced by a method selected from the group consisting of transducing a population of cells, transfecting a population of cells, and electroporating a population of cells. In one embodiment, the population of cells comprises one or more of the nucleic acid sequences described herein.

[0153] In one embodiment, the nucleic acid introduced into the cell is RNA. In another embodiment, the RNA is mRNA comprising in vitro transcribed RNA or synthetic RNA. The RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The DNA source can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences or any other suitable DNA source. The desired template for in vitro transcription is the CAR.

[0154] A template for in vitro mRNA transcription can be generated using PCR, which is then introduced into cells. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA that will be used as the PCR template. As used herein, "substantially complementary" refers to a nucleotide sequence in which most or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary or mismatched. A substantially complementary sequence can anneal or hybridize to the intended DNA target under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify portions (reading frames) of genes that are normally transcribed in cells, including the 5' and 3' UTRs. Primers can also be designed to amplify a portion of a gene that encodes a particular domain of interest. In one embodiment, the primers are designed to amplify the coding region of human cDNA that includes all or a portion of the 5' and 3' UTRs. Primers useful for PCR are made by synthetic methods well known in the art. A "forward primer" is a primer that includes a region of nucleotides that is substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence to be amplified. As used herein, "upstream" refers to the 5'-side position of the DNA sequence to be amplified relative to the coding strand. A "reverse primer" is a primer that includes a region of nucleotides that is substantially complementary to a double-stranded DNA template that is downstream of the DNA sequence to be amplified. As used herein, "downstream" refers to the 3'-side position of the DNA sequence to be amplified relative to the coding strand.

[0155] A chemical structure having the ability to promote RNA stability and / or translation efficiency may be used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is from 0 to 3000 nucleotides in length. The lengths of the 5' and 3' UTR sequences added to the coding region can be varied by different methods including, but not limited to, designing primers for PCR that anneal to different regions of the UTR. Using this approach, one of ordinary skill in the art can vary the lengths of the 5' and 3' UTRs required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0156] The 5' and 3' UTRs can be the naturally occurring endogenous 5' and 3' UTRs of the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into forward and reverse primers or by any other modification of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for altering the stability and / or translation efficiency of the RNA. For example, AU-rich elements in the 3' UTR sequence are known to be able to decrease mRNA stability. Therefore, the 3' UTR can be selected or designed to increase the stability of the transcribed RNA based on the properties of UTRs well known in the art.

[0157] In one embodiment, the 5' UTR can include the Kozak sequence of an endogenous gene. Alternatively, if a 5' UTR that is not endogenous to the gene of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. The Kozak sequence can enhance the translation efficiency of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for the Kozak sequence for many mRNAs is known in the art. In other embodiments, the 5' UTR can be derived from an RNA genome that is a stable RNA virus intracellularly. In other embodiments, various nucleotide analogs can be used in the 3' or 5' UTR to prevent exonucleolytic degradation of the mRNA.

[0158] To enable synthesis of RNA from a DNA template without the need for gene cloning, the promoter for transcription should be added upstream of the sequence to be transcribed relative to the DNA template. When a sequence that functions as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter will be incorporated into the PCR product upstream of the transcribed reading frame. In one embodiment, the promoter is the T7 polymerase promoter as described elsewhere herein. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences of the T7, T3, and SP6 promoters are known in the art.

[0159] In one aspect, the mRNA has both a 5' cap and a 3' poly(A) tail that determine ribosome binding, initiation of mRNA translation, and stability within the cell. With circular DNA templates, such as plasmid DNA, RNA polymerase produces long-chain products that are not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the end of the 3' UTR results in a normal-sized mRNA that, even when polyadenylated post-transcriptionally, is not effective in eukaryotic transfection.

[0160] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

[0161] The conventional method for incorporating a polyA / T stretch into a DNA template is molecular cloning. However, polyA / T sequences incorporated into plasmid DNA can cause plasmid instability because plasmid DNA templates obtained from bacterial cells are often highly damaged by deletions and other abnormalities. This makes the cloning procedure not only cumbersome and time-consuming but also often unreliable. This is why a method that enables the construction of a DNA template with a polyA / T 3' stretch without cloning is highly desirable.

[0162] The poly A / T segment of the transcribed DNA template can be produced during PCR by using a reverse primer containing a poly T tail such as a 100T tail (the size can be 50 - 5000 T), or after PCR by any other method including, but not limited to, DNA ligation or in vitro recombination. The poly(A) tail also confers stability to the RNA and reduces RNA degradation. Generally, the length of the poly(A) tail has a positive correlation with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is 100 - 5000 adenosines.

[0163] The poly(A) tail of RNA can be further elongated after in vitro transcription using a poly(A) polymerase such as E. coli poly A polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300 - 400 nucleotides increases the translation efficiency of the RNA by about two-fold. Further, the attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachments can include modified / artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using a poly(A) polymerase. ATP analogs can further increase the stability of RNA.

[0164] The 5' cap also confers stability to the RNA molecule. In a preferred embodiment, the RNA produced by the methods disclosed herein includes a 5' cap. 5' caps are known in the art and are provided using the techniques described herein (Cougot, et al., Trends in Biochem. Sci., 29:436 - 444 (2001); Stepinski, et al., RNA, 7:1468 - 95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958 - 966 (2005)).

[0165] The RNA produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to the mRNA and facilitates the start of translation. Any solute suitable for cell electroporation can be included, which can contain factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

[0166] Some in vitro transcribed RNA (IVT-RNA) vectors are known in the literature and are utilized in a standardized manner as templates for in vitro transcription and genetically engineered to produce stabilized RNA transcripts. Currently, the protocols used in the art are based on plasmid vectors having the following structure: a 5' RNA polymerase promoter that enables RNA transcription, followed by a gene of interest flanked by untranslated regions (UTRs) on either the 3' and / or 5' side, and a 3' polyadenylation cassette containing 50 - 70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenylation cassette by a type II restriction enzyme (the recognition sequence corresponds to the cleavage site). Thus, the polyadenylation cassette corresponds to the subsequent poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization and either extend or mask the poly(A) sequence at the 3' end. Whether this non-physiological overhang affects the amount of protein produced intracellularly from such constructs is not clear.

[0167] In certain instances, the RNA constructs are delivered into cells by electroporation. See, for example, the formulations and methodologies for electroporation of nucleic acid constructs into mammalian cells as taught in U.S. Patent No. 2004 / 0014645, U.S. Patent No. 2005 / 0052630A1, U.S. Patent No. 2005 / 0070841A1, U.S. Patent No. 2004 / 0059285A1, and U.S. Patent No. 2004 / 0092907A1. See also various parameters including the electric field strength required for electroporation of any known cell type, which are generally known in the relevant research literature as well as in numerous patents and applications in the art. See, for example, U.S. Patent No. 6,678,556, U.S. Patent No. 7,171,264, and U.S. Patent No. 7,173,116. Devices for the therapeutic application of electroporation are commercially available, such as the MedPulser (商標) DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.), and are described in patents such as U.S. Patent No. 6,567,694; U.S. Patent No. 6,516,223, U.S. Patent No. 5,993,434, U.S. Patent No. 6,181,964, U.S. Patent No. 6,241,701, and U.S. Patent No. 6,233,482; electroporation can also be used for transfection of cells in vitro as described, for example, in U.S. Patent No. 20070128708A1. Electroporation can also be utilized to deliver nucleic acids to cells in vitro. Thus, electroporation-mediated administration of nucleic acids, including expression constructs that utilize any of a number of available devices and electroporation systems known to those of skill in the art, represents an excellent new means for delivering the RNA of interest to target cells.

[0168] Cell source In one aspect, phagocytes are used in the compositions and methods described herein. Sources of phagocytes such as monocytes, macrophages and / or dendritic cells are obtained from a subject. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is human. The cells can be obtained from several sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, and tumors. In certain aspects, any number of monocytes, macrophages, dendritic cells or progenitor cell lines available in the art can be used. In certain aspects, T cells can be obtained from units of blood collected from a subject using any number of techniques known to those of skill in the art such as Ficoll separation. In one aspect, cells from an individual's circulating blood are obtained by apheresis or leukapheresis. Apheresis products typically contain lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, erythrocytes, and platelets. The cells collected by apheresis are washed to remove the plasma fraction and placed in a washing solution that may be calcium-free and magnesium-free or may lack many divalent cations, but not all, such as phosphate-buffered saline (PBS) for subsequent processing steps. After washing, the cells can be resuspended in various biocompatible buffers such as calcium-free, magnesium-free PBS. Alternatively, unwanted components of the apheresis sample are removed and the cells can be resuspended directly in the medium.

[0169] In another aspect, the cells are isolated from peripheral blood by lysing erythrocytes and depleting lymphocytes and erythrocytes by centrifugation through a gradient, e.g., PERCOLL (商標) Alternatively, the cells can be isolated from the umbilical cord. In any case, specific subpopulations of monocytes, macrophages and / or dendritic cells can be further isolated by positive or negative selection techniques.

[0170] The isolated mononuclear cells can deplete cells expressing specific antigens, including but not limited to CD34, CD3, CD4, CD8, CD14, CD19 or CD20. Depletion of these cells can be achieved using isolated antibodies, biological samples containing antibodies such as ascites, antibodies bound to physical supports, and cell-binding antibodies.

[0171] Enrichment of monocyte, macrophage and / or dendritic cell populations by negative selection can be achieved using a combination of antibodies directed against surface markers specific to the negatively selected cells. Preferred methods are negative magnetic immunoadhesion or cell sorting and / or selection by flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, enrichment by negative selection of a cell population of monocytes, macrophages and / or dendritic cells can be achieved using a cocktail of monoclonal antibodies typically including antibodies against CD34, CD3, CD4, CD8, CD14, CD19 or CD20.

[0172] During isolation of the desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between the beads and cells. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, more than 100 million cells / ml are used. In a further embodiment, cell concentrations of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml are used. In yet another embodiment, cell concentrations of 75, 80, 85, 90, 95, or 100 million cells / ml are used. In a further embodiment, concentrations of 125 or 150 million cells / ml can be used. The use of high cell concentrations can result in increased cell yield, cell activation, and cell expansion.

[0173] In one embodiment, the cell population comprises monocytes, macrophages, or dendritic cells of the present invention. Examples of cell populations include, but are not limited to, peripheral blood mononuclear cells, cord blood cells, populations of purified monocytes, macrophages or dendritic cells, and cell lines. In another embodiment, the peripheral blood mononuclear cells comprise a population of monocytes, macrophages or dendritic cells. In yet another embodiment, the purified cells comprise a population of monocytes, macrophages or dendritic cells.

[0174] In another embodiment, the cells have upregulated M1 markers and downregulated M2 markers. For example, at least one M1 marker, such as HLA DR, CD86, CD80, and PDL1, etc., is upregulated in phagocytes. In another example, at least one M2 marker, such as CD206, CD163, etc., is downregulated in phagocytes. In one embodiment, the cells have at least one upregulated M1 marker and at least one downregulated M2 marker.

[0175] In yet another embodiment, the targeted effector activity in phagocytes is enhanced by inhibition of either CD47 activity or SIRPα activity. CD47 activity and / or SIRPα activity can be inhibited by treating phagocytes with an anti-CD47 antibody or an anti-SIRPα antibody. Alternatively, CD47 activity or SIRPα activity can also be inhibited by any method known to those skilled in the art.

[0176] Cell expansion In one embodiment, a cell or cell population comprising monocytes, macrophages or dendritic cells is cultured for expansion. In another embodiment, a cell or cell population comprising progenitor cells is cultured for the differentiation and expansion of monocytes, macrophages or dendritic cells. The present invention includes expanding monocytes, macrophages or dendritic cells comprising a chimeric antigen receptor as described herein.

[0177] As demonstrated by the data disclosed herein, the step of increasing cells by the method disclosed herein can increase the cells by about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or more, and any and all whole and partial integer multiples therebetween. In one embodiment, the cells are increased in the range of about 20-fold to about 50-fold.

[0178] After culturing, the cells are incubated in a cell culture medium in a culture device for a period of time or until the cells reach confluence or a high cell density for optimal passage, and then passaged to another culture device. The culture device may be any culture device commonly used for culturing cells in vitro. Preferably, the confluence level before passage of the cells to another culture device is 70% or higher. More preferably, the confluence level is 90% or higher. The period may be any time suitable for culturing cells in vitro. The replacement of the culture medium may be performed at any point during the culturing of the cells. Preferably, the culture medium is replaced every about 2 to 3 days. Subsequently, the cells are harvested from the culture device and can be used immediately thereon or stored for later use.

[0179] The culturing stage (contact with the agent described herein) described in this specification may be very short, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours, etc., and may be less than 24 hours. The culturing stage (contact with the agent described herein) further described in this specification may be longer, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days.

[0180] In one embodiment, the cells can be cultured for several hours (about 3 hours) to about 14 days, or any integer value of time unit therebetween. Conditions suitable for cell culture can include factors necessary for growth and survival, including serum (e.g., fetal bovine serum or human serum), L-glutamine, insulin, M-CSF, GM-CSF, IL-10, IL-12, IL-15, TGF-β, and TNF-α, or any other additive known to those skilled in the art for cell growth, in a suitable medium (e.g., macrophage complete medium, DMEM / F12, DMEM / F12-10 (Invitrogen)). Other additives for cell growth include, but are not limited to, surfactants, plasma protein fraction, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. The medium can be RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer supplemented with amino acids, sodium pyruvate, and vitamins, and can be either serum-free or supplemented with an appropriate amount of serum (or plasma) or defined hormone groups, and / or an amount of cytokines sufficient for cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells intended for injection into a subject. The target cells are maintained under conditions necessary to support growth, such as a suitable temperature (e.g., 37°C) and atmosphere (e.g., 5% CO2 in addition to air).

[0181] The medium used for culturing cells may contain an agent capable of activating the cells. For example, an agent known in the art to be capable of activating monocytes, macrophages or dendritic cells is included in the medium.

[0182] Therapeutic method The modified cells described herein can be included in a composition for the treatment of a subject. In one aspect, the composition may include modified cells comprising a chimeric antigen receptor described herein. The composition can include a pharmaceutical composition and may further include a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the modified cells can be administered.

[0183] In one aspect, the present invention is a method for treating a disease or condition associated with a tumor or cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified cells described herein. In another aspect, the present invention is a method for treating a solid tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified cells described herein. In another aspect, the present invention is a method for stimulating an immune response against target tumor cells or tumor tissue in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified cells described herein. In yet another aspect, the present invention includes the use of the modified cells described herein in the manufacture of a medicament for performing an immunotherapy in a subject in need thereof. In still another aspect, the present invention includes the use of the modified cells described herein in the manufacture of a medicament for treating a tumor or cancer in a subject in need thereof.

[0184] The modified cells produced as described herein possess targeted effector activity. In one aspect, the modified cells have targeted effector activity that is directed against an antigen on a target cell, for example, through specific binding to the antigen-binding domain of a CAR. In another aspect, targeted effector activity includes, without limitation, phagocytosis, targeted cytotoxicity, antigen presentation, and cytokine secretion.

[0185] In another aspect, the modified cells described herein have the ability to deliver an agent, biological agent, or therapeutic agent to a target. The cells can be modified to deliver an agent to a target, where the agent is selected from the group consisting of nucleic acids, antibiotics, anti-inflammatory agents, antibodies or antibody fragments thereof, growth factors, cytokines, enzymes, proteins, peptides, fusion proteins, synthetic molecules, organic molecules, carbohydrates or analogs, lipids, hormones, microsomes, derivatives or variants thereof, and any combination thereof. As a non-limiting example, macrophages modified with a CAR targeting a tumor antigen can secrete an agent such as a cytokine or antibody to assist macrophage function. Antibodies such as anti-CD47 / anti-SIRPα mAB can also assist macrophage function. In yet another example, macrophages modified with a CAR targeting a tumor antigen are engineered to encode an siRNA that assists macrophage function by downregulating an inhibitory gene (i.e., SIRPα). In another example, CAR macrophages are engineered to express a dominant negative (or otherwise mutated) version of a receptor or enzyme that assists macrophage function.

[0186] In one aspect, macrophages are modified by multiple genes, where at least one gene contains a CAR and at least one other gene contains a factor that enhances CAR macrophage function. In another aspect, macrophages are modified by multiple genes, where at least one gene contains a CAR and at least one other gene aids or reprograms the function of other immune cells (such as T cells in the tumor microenvironment).

[0187] Furthermore, the modified cells can be administered to an animal, preferably a mammal, more preferably a human, to suppress immune responses such as those common to diabetes, psoriasis, rheumatoid arthritis, multiple sclerosis, GVHD, enhanced induction of allogeneic immune tolerance, transplant rejection, etc. In addition, the cells of the present invention can also be used for the treatment of any condition where attenuation or inhibition in other ways of an immune response, particularly a cell-mediated immune response, is desirable for the treatment or alleviation of a disease. In one aspect, the present invention includes treating a condition such as an autoimmune disease in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the cell population described herein. In addition, the cells of the present invention can also be administered as a pretreatment or preconditioning prior to treatment with alternative anti-cancer immunotherapies, including but not limited to CAR T cells, tumor-infiltrating lymphocytes, or checkpoint inhibitors.

[0188] Examples of autoimmune diseases include acquired immunodeficiency syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behçet's disease, cardiomyopathy, celiac disease - dermatitis herpetiformis; chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Dego's disease, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia - fibromyositis, Graves' disease, Guillain - Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin - dependent diabetes, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Ménière's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), which is also known as systemic sclerosis (SS)), Sjögren's syndrome, stiff - man syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vitiligo as well as Wegener's granulomatosis, but are not limited thereto.

[0189] The cells can also be used to treat inflammatory disorders. Examples of inflammatory disorders include, but are not limited to, chronic and acute inflammatory disorders. Examples of inflammatory disorders include Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft - versus - host disease, hemolytic anemia, osteoarthritis, sepsis, stroke, tissue and organ transplantation, vasculitis, diabetic retinopathy as well as ventilator - induced lung injury.

[0190] The cells of the present invention can be used for treating cancer. Cancer includes tumors that have no developed blood vessels or have not yet substantially developed blood vessels, as well as tumors with developed blood vessels. Cancer may include non-solid tumors (hematological tumors such as leukemia and lymphoma, etc.), or may also include solid tumors. The types of cancer to be treated by the cells of the present invention include carcinomas, blastomas and sarcomas, certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors such as sarcomas, carcinomas and melanomas, which are non-limitingly included. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0191] A solid tumor is an abnormal tumor of tissue that usually does not include cysts or liquid regions. Solid tumors can be either benign or malignant. Various types of solid tumors are named according to the type of cells that form them (such as sarcomas, carcinomas and lymphomas, etc.). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, sebaceous gland carcinoma of chromaffin cell type, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, and CNS tumors (such as gliomas (such as brainstem gliomas and mixed gliomas, etc.), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germ cell tumor, medulloblastoma, schwannoma, craniopharyngioma, epithelioma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma and brain metastases, etc.) are included.

[0192] Hematological malignancies are cancers of the blood or bone marrow. Examples of hematological (or hematopoietic) malignancies include leukemia, acute leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic leukemia and erythroleukemia, etc.), chronic leukemia (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia, etc.), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin lymphoma (asymptomatic and high-grade types), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0193] The cells of the present invention can be administered in dosages, routes and times determined in appropriate preclinical and clinical experiments and tests. The cell compositions can be administered multiple times at dosages within these ranges. Administration of the cells of the present invention may be combined with other methods useful for treating the desired disease or condition determined by those skilled in the art.

[0194] The cells of the present invention administered can be autologous, allogeneic or xenogeneic with respect to the subject being treated.

[0195] Administration of the cells of the present invention can be carried out in any convenient manner known to those skilled in the art. The cells of the present invention can be administered to the subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein can be administered to the patient arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullarily, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In other examples, the cells of the present invention are directly injected into the site of inflammation in the subject, the local disease site in the subject, lymph nodes, organs, tumors, etc.

[0196] Pharmaceutical composition The pharmaceutical composition of the present invention may comprise the cells described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0197] The pharmaceutical composition of the present invention may be administered in a manner appropriate to the disease being treated (or prevented). Appropriate dosages may be determined by clinical trials, but the amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease.

[0198] When an "immunologically effective amount", "amount effective against an immune response", "effective amount to inhibit an immune response" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician taking into account individual differences in the age, weight, immune response, and condition of the patient (subject). The pharmaceutical composition comprising the cells described herein can be administered at a dosage of 10 4 ~10 9 cells / kg body weight, preferably 10 5 ~10 6 cells / kg body weight, and it can generally be said that it can be administered at all integer values within those ranges. The cell compositions described herein can also be administered multiple times at these dosages. The cells can be administered by using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment plan for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0199] In certain embodiments, it may be desirable to administer to monocytes, macrophages, or dendritic cells, then re-collect blood (or perform apheresis), and then activate the monocytes, macrophages, or dendritic cells according to the present invention and re-infuse these activated cells into the patient. This process can be performed multiple times every few weeks. In certain embodiments, the cells can be activated from a blood collection of 10 ml to 400 ml. In certain embodiments, the cells are activated from a blood collection of 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml or 100 ml. Without being bound by theory, certain populations of cells can be selected using this multiple blood collection / multiple re-infusion protocol.

[0200] In certain embodiments of the invention, the cells are modified using the methods described herein or other methods known in the art for expanding cells to therapeutic levels and are administered to a patient (e.g., before, simultaneously with, or after) in conjunction with many related treatment methods including, but not limited to, antiviral therapies, drugs such as cidofovir and interleukin-2, cytarabine (also known as ARA-C), or treatment with natalizumab for MS patients or treatment for PML patients. In further embodiments, the cells of the invention may be used in conjunction with CART cell therapy, chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunodepleting agents such as the anti-CD52 antibody alemtuzumab (CAMPATH), anti-CD3 antibodies or other antibody therapies, cytotoxins, fludaribine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase important for signal transduction induced by growth factors (rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). In further embodiments, the cell compositions of the invention are administered to a patient (e.g., before, simultaneously with, or after) in conjunction with bone marrow transplantation, lymphodepleting therapy using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, rituxan, or antibodies such as OKT3 or CAMPATH. For example, in one embodiment, a subject may undergo peripheral blood stem cell transplantation after standard treatment with high-dose chemotherapy. In certain embodiments, after transplantation, the subject receives an infusion of the cells of the invention. In further embodiments, the cells may be administered before or after surgery.

[0201] The dosage of the treatment to be administered to a subject will vary depending on the condition being treated and the exact nature of the recipient of the treatment. Scaling of dosage for human administration can be done according to practices recognized in the art. The dosage of CAMPATH, for example, generally ranges from 1 to about 100 mg for adult patients and is usually administered daily for 1 to 30 days. A preferred daily dosage is 1 to 10 mg / day, although in some cases higher dosages up to 40 mg / day may be used (as described in U.S. Patent No. 6,120,766).

[0202] It should be understood that the methods and compositions useful in the present invention are not limited to the specific formulations described in the examples. The following examples are set forth to provide those skilled in the art with a complete disclosure and description of how to make and use the cells, methods of expansion and culture, and methods of treatment of the present invention and are not intended to limit the scope of what the inventors regard as their invention.

[0203] In the practice of the present invention, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology are utilized, which are well within the purview of those skilled in the art. Such techniques are well described in the literature such as "Molecular Cloning: A Laboratory Manual", fourth edition (Sambrook, 2012); "Oligonucleotide Synthesis" (Gait, 1984); "Culture of Animal Cells" (Freshney, 2010); "Methods in Enzymology", "Handbook of Experimental Immunology" (Weir, 1997); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Short Protocols in Molecular Biology" (Ausubel, 2002); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting", (Babar, 2011); "Current Protocols in Immunology" (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the present invention and are thus contemplated in the making and practice of the present invention. Techniques particularly useful for certain embodiments are discussed in the following sections.

Examples

[0204] Experimental Examples The present invention will be described in more detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should not be construed as limited to the following examples, but rather should be construed to include any and all variations that become apparent as a result of the teachings provided herein.

[0205] Without further elaboration, it is believed that one of ordinary skill in the art can make and utilize the compounds of the present invention and practice the methods described in the claims using the above description and the following examples. Therefore, the following examples are specific illustrations of preferred embodiments of the present invention and should not be construed as limiting the remainder of the present disclosure in any way.

[0206] The materials and methods used in these experiments are described below.

[0207] Cell culture: THP1, K562, SKOV3, SKBR3, HDLM2, MD468, and all cell lines were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and penicillin / streptomycin at 37°C under 5% CO2. The THP1 mRFP+ subline (Wt) was generated by lentiviral transduction and FACS purification of the mRFP+ cell line. Using the THP1 mRFP+ subline, THP1 mRFP+ CAR19z+ (CAR19z; CARMA19z), THP1 mRFP+ CAR19Δz+ (CAR19Δz; CARMA19Δz), THP1 mRFP+ MesoZ+ and THP1 mRFP+ CARHer2z+ (CARHer2z; CARMAHer2z) sublines were generated. Monocyte differentiation was induced by culturing the cells for 48 hours with 1 ng / mL phorbol 12-myristate 13-acetate in the medium.

[0208] Primary human macrophages: Primary human monocytes were purified from apheresis products of normal donors using Miltenyi CD14 MicroBeads (Miltenyi, 130-050-201). Monocytes were cultured for 7 days in MACS GMP Cell Differentiation Bags (Miltenyi, 170-076-400) with penicillin / streptomycin, glutamax, and 10 ng / mL recombinant human GM-CSF (PeproTech, 300-03) in X-Vivo medium supplemented with 5% human AB serum or RPMI 1640 supplemented with 10% fetal bovine serum. Macrophages were harvested on day 7 and cryopreserved in FBS + 10% DMSO until later use.

[0209] Phagocytosis assay: Wt or CARMA mRFP+ THP1 sublines were differentiated with 1 ng / mL phorbol 12-myristate 13-acetate over 48 hours. GFP+ antigen-bearing tumor sublines, namely K562 CD19+ GFP+ cells, were added to the differentiated THP1 macrophages at a 1:1 ratio after PMA washout. Macrophages were co-cultured with target tumor cells for 4 hours, and phagocytosis was quantified by fluorescence microscopy using an EVOS FL Auto Cell Imaging System. The average of three fields was considered as n, and quantification was performed three times for all conditions. FACS-based phagocytosis was analyzed on a BD LSR-Fortessa. Flow cytometry data were analyzed using FlowJo (Treestar, Inc.). mRFP / GFP double-positive events by viable singlet gating were considered phagocytosis. Blockade of the CD47 / SIRPα axis was performed by addition of blocking monoclonal antibodies (mouse anti-human CD47 clone B6H12, eBioscience #14-0479-82; mouse anti-human CD47 clone 2D3 as negative control, eBioscience #14-0478-82; mouse anti-human SIRPα clone SE5A5, BioLegend #323802) at the indicated concentrations at the start of co-culture. TLR co-stimulation was performed by addition of TLR1-9 agonists (human TLR 1-9 agonist kit; Invivogen #tlrl-kit1hw) at the time of co-culture.

[0210] In vitro killing assay: Wt macrophages or CAR-bearing macrophages were co-cultured with antigen-bearing or control comet luciferase (CBG) / green fluorescent protein (GFP)-positive target tumor cells at various effector-target ratios (starting at 30:1 and decreasing as 3-fold dilutions). Bioluminescence imaging was utilized to determine tumor burden using an IVIS Spectrum Imaging System (Perkin Elmer). Percent specific lysis was calculated as follows: Percent specific lysis = ((treated well - tumor-only well) / (maximum kill - tumor-only well) * 100)

[0211] Time-lapse microscopy: Fluorescent time-lapse video microscopy of CAR-mediated phagocytosis was performed using an EVOS FL Auto Cell Imaging System. Images were captured every 40 seconds over 18 hours. Image analysis was performed using FIJI imaging software.

[0212] Lentivirus production and transfection: The chimeric antigen receptor construct was newly synthesized by GeneArt (Life Technologies) and cloned into a lentiviral vector as previously described. Concentrated lentivirus was produced using HEK293T cells as previously described.

[0213] Adenovirus production and transfection: Ad5f35 chimeric adenoviral vectors encoding GFP, CAR, or no transgene under the CMV promoter were produced and titrated according to standard molecular biology procedures. Primary human macrophages were transduced at various multiplicities of infection and continuously imaged for GFP expression and viability using an EVOS FL Auto Cell Imaging System. CAR expression was evaluated by FACS analysis of surface CAR expression using His-tagged antigen and anti-His-APC secondary antibody (R&D Biosystems Clone AD1.1.10).

[0214] Flow cytometry: FACS was performed on a BD LSR Fortessa. Surface CAR expression was evaluated using biotinylated Protein L (GenScript M00097) and streptavidin APC (BioLegend, #405207), or His-tagged antigen and anti-His-APC secondary antibody (R&D Biosystems Clone AD1.1.10). Fc receptors were blocked with human TruStain FcX (BioLegend, #422301) before staining. CD47 expression was determined using mouse anti-human CD47 APC (eBioscience #17-0479-41) together with a mouse IgG1κ APC isotype control for background determination. Calreticulin expression was determined using mouse anti-calreticulin PE clone FMC75 (Abcam #ab83220). For all flow results, gating on viable (Live / Dead Aqua Fixable Dead Cell Stain, Life Technologies L34957) single cells was performed.

[0215] Imagestream cytometry: FACS by single-cell fluorescence imaging was performed on an ImageStream MarkII Imaging Flow Cytometer (EMD Millipore). Briefly, mRFP+ macrophages or macrophages stained with DiI (CAR or control) were co-cultured with GFP+ tumor cells for 4 hours, followed by fixation and ImageStream data collection. Data were analyzed using ImageStream software (EMD Millipore).

[0216] RNA Electroporation: Using standard molecular biology techniques, the CAR construct was cloned into an in vitro transcription plasmid under the control of the T7 promoter. CAR mRNA was transcribed in vitro using the mMessage mMachine T7 Ultra In Vitro Transcription Kit (Thermo Fisher), purified using the RNEasy RNA Purification Kit (Qiagen), and electroporated into human macrophages using a BTX ECM850 electroporator (BTX Harvard Apparatus). CAR expression was assayed using FACS analysis at various time points after electroporation.

[0217] TLR / Dectin-1 Priming Stimulation: TLR or Dectin-1 priming stimulation in Wt macrophages or CAR macrophages was performed by pre-incubating the cells with the recommended doses of TLR 1-9 agonists (Human TLR1-9 Agonist Kit, Invivogen) or β-glucan (MP Biomedicals, LLC) for 30 minutes before in vitro phagocytosis assay or killing assay, followed by co-culture. The in vitro functions of Wt macrophages or CAR macrophages were compared between non-primed and primed conditions.

[0218] Macrophage / Monocyte Phenotype: The following surface markers were evaluated as part of a macrophage / monocyte immunophenotype FACS panel for M1 / M2 discrimination: CD80, CD86, CD163, CD206, CD11B, HLA-DR, HLA-A / B / C, PDL1 and PDL2 (BioLegend). TruStain FcX was used for Fc receptor blockade before immunostaining. Before phenotype determination, macrophages / monocytes were exposed to activation conditions, i.e., Ad5f35 transduction was performed for 48 hours or not.

[0219] Seahorse assay: The metabolic phenotype and oxygen consumption of macrophages were determined using the Seahorse assay (Seahorse XF, Agilent). Prior to analysis, control macrophages or CAR macrophages were exposed to medium control or immunosuppressive cytokines for 24 hours. Throughout the Seahorse assay, cells were sequentially treated with oligomycin, FCCP, and rotenone. The assay was performed six times for each condition.

[0220] In vivo assay: NOD-scid IL2Rg-null-IL3 / GM / SF, NSG-SGM3 (NSGS) mice were used for the human xenograft model. Mice engrafted with CBG-luciferase-positive human SKOV3 ovarian cancer cells were left untreated or administered non-transduced human macrophages, empty Ad5f35-transduced human macrophages, or Ad5f35 CAR-HER2-transduced human macrophages at various doses. Tumor burden was monitored using continuous bioluminescence imaging (IVIS Spectrum, Perkin Elmer). Organs and tumors were harvested for FACS analysis after sacrifice. Overall survival was monitored and compared using Kaplan-Meier analysis.

[0221] The results of the experiments are described below.

[0222] Figure 1A is a series of images showing a conceptual diagram of a chimeric antigen receptor (CAR) composed of a gene / gene product containing an extracellular domain, a hinge domain, a transmembrane domain, an intracellular signaling domain, and / or 2A (P2A, T2A) with targeting function for the stoichiometric co-expression of additional gene products, which may be secretory or non-secretory, including but not limited to cytokines, monoclonal antibodies, antibody fragments, single-chain variable fragments, enzymes, additional receptors, dominant-negative receptors, tumor-associated antigens, and any combination thereof. In addition, the CAR construct may include co-delivery of CRISPR / Cas9 gene editing materials or may be introduced in the context of cells pre-edited by CRISPR / Cas9. Specific examples of CAR constructs including CARMA-ζ, CARMA-γ, and CARMA-dectin, which include an antigen-specific scFv domain, a CD8 hinge domain, a CD8 transmembrane domain, and a CD3ζ domain, an FcεRI common γ subunit domain, or an intracellular domain of dectin-1, respectively, are modeled in Figure 1B.

[0223] Figure 2A is a graph showing CAR19z expressed on the surface of myeloid cells after lentiviral transduction. The dose setting of the CAR19z lentivirus was performed at 3-fold dilution and used for the transduction of 1e5 cells / 0.1 mL of mRFP+ THP1 cells. mRFP is a reporter gene (red fluorescent protein) expressed by lentiviral transduction of the myeloid cell line THP1. These cells can be induced to differentiate into macrophages by exposure to chemical PMA. THP1 cells were harvested 24 hours after transduction and stained for CAR surface expression by using biotinylated protein L followed by streptavidin-APC. The transduced THP1 cells were expanded and sorted by FACS to generate a 100% CAR19z-positive mRFP+ THP1 subline (Figure 2B). Figure 2C demonstrates the expression of anti-CD19, anti-HER2, and anti-mesothelin lentiviral CAR constructs on THP1 macrophages, with CAR(+) events shown in the upper right quadrant.

[0224] Figure 3A is a flowchart showing the generation of CARMA sublines using the THP1 macrophage model, differentiation with 1 ng / mL phorbol 12-myristate 13-acetate (PMA), and in vitro phagocytosis assays. Anti-CD19 CAR macrophages, anti-HER2 CAR macrophages, and anti-mesothelin CAR macrophages, unlike wild-type (Wt) macrophages, were demonstrated by phagocytosis assays using a fluorescence microscope to phagocytose K562 tumor cells expressing CD19, HER2, or mesothelin, respectively (Figures 3B - 3D). CARMA tumor phagocytosis was further verified by a flow cytometry-based assay in which mRFP+ CARMA against CD19 was co-cultured with CD19+ GFP+ K562 cells to quantify double-positive events (representative FACS plots are shown - Figure 3E). The standard 10x fields used in the graphical representation of the CARMA phagocytosis function are shown as mRFP only (Figure 3F) or overlaid (Figure 3G). FACS-based mRFP / GFP double-positive events were defined as phagocytosis events and verified by, for example, Amnis Imagestream FACS analysis. The events shown were gated for double-positive events and are in order from high to low by the Amnis Imagestream phagocytosis - erosion algorithm (Figure 3H). Phagocytosis of tumor cells by mRFP+ CARMA in the THP-1 cell line model was further demonstrated by confocal microscopy, and it was demonstrated via three-dimensional confocal z-stack reconstruction that GFP+ tumor cells were completely enclosed within phagosomes (Figures 3I and 3J). Figure 3K demonstrates the time-dependent fate of a single CARMA cell (contact and immunological synapse formation being the first step, leading to phagocytic engulfment, tumor degradation using the disappearance of GFP as a marker of cell death, phagosome collapse, and phagosome repair), demonstrating that CARMA survives after tumor cell phagocytosis. Figure 3L demonstrates the ability of CARMA to phagocytose multiple tumor cells at once.

[0225] Anti-CD19 CAR macrophages were examined using an in vitro phagocytosis assay against CD19+ (target) or CD19− (control) GFP+ K562 tumor cells. To demonstrate the antigen specificity of CARMA, only antigen-bearing tumor cells were phagocytosed (Figure 4A). To demonstrate that the intracellular signaling domain is required for CARMA function, the CAR19-Δζ construct (lacking the intracellular signaling domain) was utilized. CAR19-Δζ macrophages were unable to phagocytose tumor cells and significantly reduced antitumor function via a specific lysis assay using luciferase in vitro (Figures 4B and 4C). The in vitro CARMA phagocytosis assay was performed in the presence of R406 (Syk inhibitor), cytochalasin D (actin polymerization inhibitor), or blebbistatin (non-muscle myosin IIA inhibitor). R406, cytochalasin D, and blebbistatin independently inhibited the phagocytosis function of CARMA, indicating that CAR signaling in macrophages is Syk-dependent and results in actin polymerization and NMIIA-mediated phagocytosis function (Figures 4D-4F).

[0226] Figure 5A is a flow cytometry graph showing the expression of CD47 on the target tumor cell line in comparison to an isotype control. K562 and K562-CD19+ (K19) were used in these experiments, and both are CD47 high-expression cell lines.

[0227] Figure 5B is a graph showing that the addition of anti-CD47 monoclonal antibody selectively enhanced CAR macrophage-mediated phagocytosis of target antigen-bearing tumor cells, unlike Wt macrophage-mediated phagocytosis. Wt macrophages or CAR19ζ macrophages were incubated with CD19+ K562 tumor cells and anti-CD47 monoclonal antibody at either 0, 0.01, 0.10, 1.00, or 10.0 mcg / mL.

[0228] Figure 5C is a graph showing that the CAR macrophage-mediated phagocytosis of target antigen-bearing tumor cells is selectively enhanced by the addition of anti-SIRPα monoclonal antibody, unlike the Wt macrophage-mediated phagocytosis. Wt macrophages or CAR19ζ macrophages were incubated with CD19+ K562 tumor cells together with anti-SIRPα monoclonal antibody at either 0, 0.01, 0.10, 1.00 or 10.0 mcg / mL.

[0229] Figure 5D is a graph demonstrating that the blockade of the CD47 / SIRPα axis by anti-SIRPα monoclonal antibody enhances the hyperphagocytosis (defined as macrophages that phagocytosed two or more tumor cells at once) by CAR macrophages.

[0230] As a control for the opsonization effect exerted by the CD47 / SIRPα blocking monoclonal antibody, a control anti-CD47 monoclonal antibody (clone 2D3) that binds to CD47 but does not block it against the SIRPα binding site was used in the in vitro phagocytosis assay. Only the clone that blocked the binding site (anti-CD47, clone B6H12) or the blockade of the SIRPα receptor directly led to the enhancement of CARMA tumor phagocytosis (Figure 5E).

[0231] To examine whether the blockade of the CD47 / SIRPα axis on CAR macrophages leads to the loss of antigen specificity, in vitro phagocytosis against antigen-negative (CD19-negative) tumor cells was performed in the presence of anti-CD47 or anti-SIRPα monoclonal antibody, and no observable phagocytosis was seen (Figure 5F).

[0232] The specificity of the enhanced CARMA phagocytosis in the presence of the SIRPα-blocking monoclonal antibody was examined by knocking out the SIRPα receptor on THP1 macrophages and comparing the tumor phagocytosis by CARMA or SIRPα-KO CARMA in the absence or presence of the anti-SIRPα antibody. CRISPR / Cas9 was used for SIRPα deletion, and the cells were sorted for SIRPα negativity prior to the functional assay. The knockout of SIRPα enhanced CARMA function, and adding anti-SIRPα again to the knockout cells could not further enhance phagocytosis (Figure 5G).

[0233] Figure 6A is a graph showing the specific lysis of CD19+ GFP+ luciferase+ K562 cells by CAR19ζ CARMA macrophages, different from Wt macrophages (using the THP-1 macrophage model), in a dose-dependent manner at the 48-hour time point in a luciferase-based killing assay in vitro.

[0234] Figure 6B is a graph demonstrating the specific lysis of tumor cells by CAR19ζ or Wt THP-1 monocytes (undifferentiated and thus serving as a model for monocytes rather than macrophages) in a dose-dependent manner at the 48-hour time point in a luciferase-based killing assay in vitro.

[0235] Figure 6C is a set of images showing the bioluminescence brought about by luciferase from luciferase-positive CD19+ K562 tumor cells after 48-hour co-culture with Wt macrophages or CAR19ζ macrophages in vitro in the absence or presence of 10 mcg / mL anti-SIRPα monoclonal antibody. Figure 6D is a graph demonstrating the specific lysis of Wt macrophages or CAR19ζ macrophages in the presence or absence of anti-SIRPα monoclonal antibody.

[0236] A CAR construct with the intracellular domain of the FcεRI common γ (CAR19γ, CARMA19γ) subunit was prepared, packaged in lentivirus, and used for the transduction of THP-1 myeloid cells as a 3-fold serial virus dilution. CAR19γ was expressed on THP-1 macrophages (Figure 7A).

[0237] CAR19γ macrophages or CAR19ζ macrophages were sorted for 100% CAR positivity and used for in vitro functional characterization. Both CAR19ζ macrophages and CAR19γ macrophages phagocytosed CD19+ tumor cells and both exhibited synergy with the blockade of the CD47 / SIRPα axis by the addition of anti-SIRPα monoclonal antibody (Figure 7B).

[0238] As demonstrated in the R406 Syk inhibition in vitro phagocytosis assay, both CAR19ζ macrophages and CAR19γ macrophages activated tumor phagocytosis by signaling through Syk (Figure 7C).

[0239] In an in vitro luciferase-based specific lysis assay, after 24-hour co-culture at various E:T ratios, both CAR19ζ macrophages and CAR19γ THP1 macrophages, unlike Wt THP1 macrophages, efficiently killed CD19+ tumor cells (Figure 7D).

[0240] As leukocytes of the innate immune system, macrophages respond to conserved molecular triggers of infection, such as pathogen-associated molecular patterns, via constitutively expressed pathogen recognition receptors. Toll-like receptors are the best-characterized pathogen recognition receptors and are known to activate macrophages.

[0241] To enhance the tumor phagocytosis function of CARMA, an in vitro phagocytosis assay was performed using CAR macrophages that were independently primed with ligands for TLR1-9 or a medium control. Ligands for TLR1, 2, 4, 5, and 6 enhanced the phagocytosis function of CARMA (Figure 8A). This suggests that TLR ligands can be used to prime CARMA during production, or that TLR signaling domains can be encoded in the CAR construct as a novel second-generation / next-generation CARMA construct to enhance CAR signaling and downstream effector functions.

[0242] Figures 8B and 8C show that the difference between TLR ligands that enhanced or did not enhance the tumor cell phagocytosis of CARMA was maintained at TLR3 or TLR6 ligand concentrations within a certain range.

[0243] One of the enzyme products, β-glucan, binds to dectin-1 on the surface of macrophages, resulting in activation and effector functions. To examine the ability of β-glucan to enhance CARMA function, an in vitro tumor phagocytosis assay was performed in the absence or presence of 5 mcg / mL β-glucan. β-glucan enhanced the phagocytosis ability of CAR macrophages but not that of Wt macrophages (Figure 9A).

[0244] To examine the ability of β-glucan to enhance CARMA tumor killing, a specific lysis assay using luciferase was performed in vitro at various effector (E):target (T) ratios in the presence of 0, 0.5, 5, or 50 mcg / mL β-glucan. β-glucan enhanced the specific lysis of antigen-bearing tumor cells by CAR THP-1 macrophages but not by Wt THP-1 macrophages (Figure 9B). These results indicate that β-glucan can be used as an adjuvant during the production of CARMA, or that the dectin-1 intracellular signaling domain can be encoded in the CAR transgene.

[0245] Considering that β-glucan enhanced the function of CARMA, a CAR construct composed of the intracellular signaling domain of dectin-1 was generated (Figure 10A). These constructs were packaged into lentiviruses and used for the transduction of THP-1 myeloid cells as 3-fold serial dilutions of lentiviral titers. CAR was detected on the surface in both the CD8TM-dectin 1 CAR construct and the dectinTM-dectin 1 CAR construct (Figures 10B and 10C). Cells were sorted for 100% positivity and used for downstream in vitro functional experiments.

[0246] CD8TM-dectin 1 CAR and dectinTM-dectin 1 CAR macrophages were examined in an in vitro luciferase killing assay. Both constructs demonstrated specific lysis of tumor cells (10D).

[0247] When dectin 1-CAR macrophages were examined in an in vitro tumor phagocytosis assay against K562 (control) or K19 (target) tumor cells, dectin 1-CAR macrophages selectively phagocytosed cognate-antigen-bearing tumor cells (Figure 10E). Dectin-1 CAR macrophages demonstrated the ability against the phagocytosis of multiple tumor cells (Figure 10F).

[0248] In the in vitro tumor phagocytosis assay, dectin 1-CAR macrophages demonstrated a synergistic effect with the initial stimulation by SIRPα blockade or TLR ligands (Figure 10G).

[0249] Figure 11A is a graph showing calreticulin levels in three different CD19+ target cell lines compared to an isotype control. Figure 11B is a graph showing the normalized mean fluorescence intensity of calreticulin expression in three different CD19+ target cell lines.

[0250] Figure 11C is a graph showing that low levels of calreticulin moderately protected target cells, specifically the Nalm6 cell line and the JEKO cell line, from CAR19z macrophage phagocytosis. These data suggest that the use of calreticulin deposition / induction can be employed as an additional strategy to enhance CARMA effector function.

[0251] To verify and examine the function of CAR in macrophages derived from primary human monocytes, several gene delivery approaches were investigated. In Figure 12A, an anti-HER2 CAR construct was cloned into an mRNA expression plasmid, transcribed in vitro, and mRNA electroporation was directly performed on primary human monocytes. Figure 13A demonstrates a transfection efficiency of 84.3% compared to cells that underwent gating strategy, viability, and mock electroporation. Figure 12B shows that the efficiency of anti-HER2 CAR mRNA electroporation into macrophages (fully differentiated) derived from primary human monocytes is 79.7%.

[0252] Figure 12C is a graph demonstrating that while mRNA electroporation results in high CAR transfection efficiency in both monocytes and macrophages, CAR expression is transient due to mRNA degradation, with a peak on the second day after in vitro electroporation and disappearing by the seventh day.

[0253] For NSG mice, IE6 SKOV3 CBG / GFP+ human ovarian cancer cells, which are a model of metastatic intraperitoneal carcinomatosis of HER2+ ovarian cancer, were injected via IP injection. The mice were co-injected with either mock electroporated or electroporated primary human monocytes or primary human macrophages (E:T ratio 1:1) with anti-HER2 CAR mRNA, and tumor burden was imaged. CAR macrophages (Figure 13A) and CAR monocytes (Figure 13B) demonstrated a slight decrease in tumor growth over approximately two weeks. The first time point at which tumor burden was bioluminescently quantified was 24 hours post-administration, demonstrating that CAR monocytes and CAR macrophages were active during the first 24 hours.

[0254] Lentiviral delivery of the CAR transgene to macrophages derived from primary human monocytes was investigated using multiple CAR constructs. In Figure 14A, CAR19 was delivered to human macrophages via lentiviral transduction, demonstrating transduction efficiencies of 4.27% and 38.9% respectively in comparison between the control group and the CAR19 lentivirus (MOI 10) group. The FACS gating strategy is shown.

[0255] Figure 14B is a representative FACS plot showing the expression of anti-HER2 CAR in primary human macrophages, with transduction efficiencies of 1.47% and 18.1% respectively between the control condition and the MOI 10 CAR LV condition.

[0256] Monocyte-derived macrophages were generated by differentiating selected CD14+ cells (derived from apheresis products of normal donors) in GM-CSF conditioned medium for 7 days. To optimize the delivery of CAR via lentiviral transduction, anti-CD19 and anti-HER2 lentiviruses were used to transduce macrophages at various time points during the differentiation process from monocytes to macrophages. For both anti-CD19 and anti-HER2 CAR constructs, it has been shown that the transduction efficiency peaked at the midpoint of transduction (day 4) (Figures 15A and 15B). After defining CD11b+ / GFP+ events, anti-CD19 CAR primary human macrophages were used in an in vitro FACS-based phagocytosis assay against CD19+ GFP+ K562 tumor cells. Macrophage transduction at various time points as shown in Figure 15A was used in this assay. Figure 15C demonstrates that the effectiveness of phagocytosis is proportional to the CAR transduction efficiency and that macrophage transduction peaks on day 4 of the differentiation process.

[0257] Considering that mRNA electroporation was transient and the efficiency of lentivirus was only moderate and required high titers, alternative transduction approaches for introducing transgenes into primary human macrophages were investigated. Adenovirus (recombinant, replication-deficient) was identified as an efficient approach for primary human macrophage transduction. When the expression of coxsackievirus adenovirus receptor (docking protein for Ad5) and CD46 (docking protein for Ad35) was examined in comparison to isotype controls on primary human macrophages, CD46 was highly expressed while the coxsackievirus adenovirus receptor was not detected (Figure 16A). Therefore, chimeric Ad5f35 adenovirus was utilized for primary human macrophage transduction and engineered via standard molecular biology techniques to express a chimeric antigen receptor against HER2 (using GFP and empty Ad5f35 virus as controls).

[0258] Figure 16B shows that Ad5f35 efficiently delivered the transgene (using GFP as a model transgene) into human macrophages at an MOI of 1000, and the expression increased over time as monitored by GFP signal quantification with an IVIS Spectrum. Figure 16C shows a comparison of the transduction kinetics of primary human macrophages at various time points over a wide range of MOIs (up to 10,000).

[0259] Figure 16C shows a series of representative FACS plots of anti-HER2 CAR expression on Ad5f35-transduced human macrophages at 48 hours post-transduction at a wide range of viral MOIs.

[0260] Figure 16D shows a series of representative fluorescence microscopy images of Ad5f35-GFP-transduced primary human macrophages, and the maximum transduction efficiency was demonstrated at an MOI of 1000.

[0261] Primary human CARMA was examined in an in vitro phagocytosis assay via FACS analysis. Macrophages (non-transduced or anti-HER2 CAR) were stained with DiI prior to co-culture with GFP+ SKOV3 ovarian cancer cells. Phagocytosis defined by DiI / GFP double-positive events was measured at levels of 26.6% in the CAR group and 4.55% in the control group (Figure 17A). To demonstrate that DiI / GFP double-positive events were phagocytosis events and not doublets, cytochalasin D (a phagocytosis inhibitor) was added to one arm of the experiment, and CAR-mediated phagocytosis was sufficiently inhibited to 1.74%. To further demonstrate that primary human CAR macrophages could phagocytose tumor cells, double-positive events were gated with an Amnis Imagestream FACS and sorted from high to low by the Amnis phagocytosis-eradication algorithm, and it was visually demonstrated that these double-positive events represented phagocytosis (Figure 17B). In addition, DiI-stained CAR-HER2 macrophages were co-cultured with SKOV3-GFP and imaged by confocal microscopy, and phagocytosis was demonstrated.

[0262] Anti-HER2 CAR primary human macrophages were generated using Ad5f35-CAR transduction of monocytes-derived macrophages. These cells (or control non-transduced cells) were utilized as effectors against SKBR3 human breast cancer cells in an in vitro FACS-based phagocytosis assay. Figure 18 demonstrates that CAR human macrophages, unlike UTD human macrophages, phagocytose breast cancer cells. Additionally, the addition of an anti-SIRPα monoclonal antibody enhanced the phagocytosis of CARMA macrophages against breast cancer cells, but not for UTD macrophages. These results demonstrate that the synergistic blockade of the CD47 / SIRPα axis observed with CARMA in the THP-1 model also applies to primary human macrophage assays.

[0263] Macrophages are white blood cells of the innate immune system and thus have surveillance antibacterial properties. To demonstrate that CAR macrophages are still functional innate immune cells in an antibacterial sense and have not lost the ability to respond to infection stimuli, control non-transduced macrophages or CAR macrophages were used in a FACS-based Escherichia coli phagocytosis assay. Figure 19 is a representative FACS plot showing that CARMA exhibits complete phagocytosis of pH-Rodo Green E. coli particles.

[0264] The primary human anti-HER2 CARMA was examined as an effector cell in a luciferase-based killing assay in vitro. Anti-HER2 CARMA macrophages induced specific lysis of HER2+ K562 cells after 48 hours of co-culture, but not of control K562 cells lacking HER2 expression, and neither control UTD macrophages did so (Figure 20A). To demonstrate that CARMA killing also applies to tumor cells expressing HER2 at physiological levels (in contrast to K562-HER2 transduced lentivirally to overexpress HER2), SKBR3 breast cancer cells and SKOV3 ovarian cancer cells were used as targets. CARMA had significant antitumor activity against both models after 48 hours of co-culture, but neither control UTD nor control empty Ad5f35-transduced macrophages did (Figures 20B and 20C). For the purpose of examining the synergy between blockade of the CD47 / SIRPα axis in the killing assay, SKOV3 ovarian cancer cells were co-cultured with medium, control non-transduced macrophages, anti-HER2 CARMA, anti-HER2 CARMA + anti-CD47 mAb (10 mcg / mL) or anti-HER2 CARMA + anti-SIRPα (10 mcg / mL), and the luciferase signal was measured continuously. CARMA led to complete tumor eradication by day 13, but the kinetics of tumor eradication were even more rapid in the presence of blockade of the CD47 / SIRPα axis (Figure 20D). When the synergy with β-glucan demonstrated in the THP-1 macrophage CARMA model was examined in a similar experiment, the initial β-glucan stimulation of CARMA led to enhanced tumor killing kinetics (Figure 20E). Exposure of CARMA to LPS (one of the TLR-4 ligands) or poly-IC (one of the TLR-3 ligands) led to changes in the antitumor effect (Figure 20F).

[0265] In the luciferase assays in FIGS. 20A - 20F, the ability of primary human CARMA to eliminate tumors in vitro was demonstrated. To prove these results, GFP+ SKOV3 ovarian cancer cells were co - cultured with control UTD macrophages, control UTD macrophages + 10 mcg / mL trastuzumab, control empty Ad5f35 virus - transduced macrophages, or anti - HER2 primary human CARMA. CARMA was able to eliminate tumor cells, unlike the control conditions (FIG. 21).

[0266] Macrophages are plastic cells in terms of phenotype and can have diverse functional characteristics, which are generally classified into M1 and M2 macrophage subsets, where M1 is inflammatory / activating and M2 is immunosuppressive / tumor - promoting. Forty - eight hours after transduction of primary human macrophages with Ad5f35 CAR virus, dose - dependent up - regulation of the M1 marker CD80 / CD86 and dose - dependent down - regulation of the M2 marker CD163 were measured by FACS (FIG. 22A). To examine whether this effect was a result of CAR expression or Ad5f35 transduction, macrophage transduction was performed using nothing, empty Ad5f35, or anti - HER2 Ad5f35, and empty / CAR Ad5f35 showed the same pattern of phenotypic shift (FIG. 22B).

[0267] The solid tumor microenvironment is generally immunosuppressive and can induce macrophage polarization into the M2 state. To examine whether CARMA polarized to M1 due to viral transduction is resistant to conversion to M2 mediated by immunosuppressive cytokines, control non-transduced human macrophages or anti-HER2 CAR human macrophages were exposed to IL-4, IL-10, or IL-13 for 24 hours and then co-cultured with SKOV3 ovarian cancer cells. Control UTD macrophages conditioned by inhibitory cytokines caused enhanced tumor growth, while CARMA exposed to inhibitory cytokines maintained their killing activity at the 48-hour time point in an in vitro specific lysis assay using luciferase (Figure 22C).

[0268] To further examine the resistance of human CAR macrophages to immunosuppression, macrophages transduced with control UTD, empty Ad5f35, or anti-HER2 CAR Ad5f35 were exposed to 10 ng / mL of IL-4, a standard M2-inducing cytokine, or cancer cells (SKOV3, an ovarian cancer cell line; HDLM2, a Hodgkin lymphoma cell line) that have previously been shown to convert macrophages to M2 during co-culture. Control UTD macrophages upregulated CD206, an M2 marker that specifically responds to IL-4 stimulation via STAT6 phosphorylation. Empty Ad5f35 exhibited resistance to disruption to the IL-4- and tumor-induced M2 phenotype, and CAR-Ad5f35-transduced macrophages were even more so (Figure 22D).

[0269] To further characterize the phenotype of CAR macrophages, the metabolic phenotype was investigated using a Seahorse assay to measure oxygen consumption. M2 macrophages had a higher basal oxygen consumption rate than M0 or M1 macrophages because of their greater dependence on oxidative phosphorylation for ATP production. Control UTD or anti-HER2 CAR macrophages were exposed to IL-4 for 24 hours to polarize them to M2 (or not), and the oxygen consumption rate was measured. Control UTD macrophages demonstrated increased basal oxygen consumption characteristics characteristic of M2 macrophages, while CARMA did not respond to IL-4, suggesting that it is resistant to M2 conversion (Figure 22E). Collectively, these data tell the story of CARMA's resistance to M2 conversion using phenotypic, metabolic, and functional assays.

[0270] Primary human normal donor monocytes (purified via CD14 positive selection) were transduced with Ad5f35-CAR-HER2 at an MOI ranging from 0 (UTD) to 1000. CAR expression was measured via FACS 48 hours after transduction. CAR monocytes were efficiently generated by Ad5f35, and expression peaked at an MOI of 1000 (Figures 23A and 23B). Monocytes maintained high viability (measured by FACS Live / Dead Aqua analysis) at an MOI up to 1000 (Figure 23C). CAR human monocytes upregulated M1 activation markers (Figure 23D) and downregulated M2 markers (Figure 23E) in FACS analysis, while untransduced (UTD) human monocytes did not, demonstrating an M1 monocyte phenotype at 48 hours after transduction.

[0271] Anti-HER2 CAR monocyte killing was evaluated in vitro via a luciferase-based killing assay at a range of effector:target (E:T) ratios. Non-transduced (UTD) or CAR-HER2-ζ (CAR) monocytes were co-cultured in vitro with either HER2+ SKBR3 (human breast cancer) or HER2+ SKOV3 (human ovarian cancer) cells. Specific lysis was calculated at 24, 48, and 96 hours after the start of co-culture. CAR monocytes, unlike UTD monocytes, lysed both breast cancer and ovarian cancer cells in vitro (Figures 24A and 24B).

[0272] NOD-scid IL2Rg-null-IL3 / GM / SF, NSG-SGM3 (NSGS) mice were used to model human HER2(+) ovarian cancer xenografts in vivo. On day 0, mice were injected intraperitoneally (IP) with 7.5E5 comet luciferase (CBG luc) positive / green fluorescent protein (GFP) positive SKOV3 ovarian cancer cells as a model of intraperitoneal carcinomatosis, an essentially metastatic model of solid malignant tumors with high malignancy. Mice were either left untreated (tumor only) or injected IP on day 0 with a single dose of 4E6 non-transduced (UTD) human macrophages or CAR-HER2 (CARMA) human macrophages (schematic, Figure 25A). Mice were continuously imaged using bioluminescence (total flux; photons / sec) as a surrogate for tumor burden. In mice receiving CARMA, an approximate two-digit reduction in tumor burden was seen (Figures 25B and 25C). Mice receiving CARMA had a 30-day survival benefit (p = 0.018) compared to untreated mice or mice receiving UTD macrophages (Figure 25D). To demonstrate the transport of macrophages into solid tumor nodules, tumors were harvested from mice that died on day 36 and the presence of adoptively transferred human macrophages was evaluated via human CD45 expression by FACS analysis (Figure 25E).

[0273] Human macrophages were either not transduced (UTD), or transduced at a multiplicity of infection of 1000 with empty Ad5f35 virions lacking the transgene (empty) or Ad5f35-CAR-HER2-ζ (CARMA). Surface CAR expression was verified by FACS analysis at 48 hours post-transduction (Figure 26A). To demonstrate M1 macrophage polarization in cells transduced with either empty Ad5f35 or CAR-HER2-ζ Ad5f35, surface markers were evaluated. M1 markers (HLA DR, CD86, CD80, PDL1) were upregulated, while M2 markers (CD206, CD163) were downregulated (Figure 26B). NSGS mice were again used as an IP model of HER2+ metastatic ovarian cancer and stratified into four treatment arms (n = 5 per arm). Mice were either left untreated or received an IP injection of 1E7 non-transduced macrophages, empty-Ad5f35 transduced macrophages, or CAR-HER2-ζ transduced macrophages on day 0 (Figure 26C). Tumor burden was monitored via continuous bioluminescence imaging, and representative data shown at day 27 post-tumor engraftment are presented (Figures 26D and 26E). In mice receiving CARMA, tumor burden at day 20 post-administration was approximately 1 / 2400 that of untreated mice.

[0274] NSGS mice were used as an IP model of HER2+ metastatic ovarian cancer and stratified into four treatment arms (n = 5 per arm) consisting of untreated, and IP administration of either 3E6, 1E7 or 2E7 CAR-HER2-ζ human macrophages on day 0 (Figure 27A). When tumor burden was monitored by continuous bioluminescence imaging, a dose response dependent on the number of macrophages was observed in this model (Figure 27B). A single administration of CAR-HER2 macrophages at 3E6, 1E7 or 2E7 macrophages per mouse led to dose-dependent tumor eradication (compared to untreated mice) by day 36 post-engraftment (Figure 27C).

[0275] Figure 28 is an explanatory diagram of a therapeutic approach proposed for CARMA. Briefly, patient monocytes are selected from peripheral blood, differentiated ex vivo, transduced to express CAR, and co-stimulated with a synergistic compound (or not), and then injected back into the patient by any one of intravenous, intraperitoneal, intratumoral, interventional radiology procedures, or other routes. Note that it is also possible to transduce monocytes without the differentiation process and inject them back into the patient. The source of monocytes may be a donor with HLA matching.

[0276] Other aspects The description of the list of elements in the definition of a variable herein includes the definition of that variable as a single element or a combination (or partial combination) of the listed elements. The description of an aspect herein includes that aspect as a single aspect or in combination with other aspects or a part thereof.

[0277] The disclosures of each patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety. Although the invention is disclosed in connection with specific aspects, it is apparent that other aspects and variations of the invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to cover all such aspects and equivalent variations.

Claims

**Claim 1** A modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain of a stimulatory molecule and / or a costimulatory molecule, and the cell is a monocyte, macrophage or dendritic cell having targeted effector activity. **Claim 2** A modified cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the nucleic acid sequence comprises a nucleic acid sequence encoding an antigen-binding domain, a nucleic acid sequence encoding a transmembrane domain, and a nucleic acid sequence encoding an intracellular domain of a stimulatory molecule and / or a costimulatory molecule, and the cell is a monocyte, macrophage or dendritic cell that expresses the CAR and has targeted effector activity. **Claim 3** The modified cell according to claim 1 or 2, wherein the antigen-binding domain of the CAR comprises an antibody selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a single-domain antibody, a single-chain variable fragment, and antigen-binding fragments thereof. **Claim 4** The modified cell according to claim 1 or 2, wherein the antigen-binding domain of the CAR is selected from the group consisting of an anti-CD19 antibody, an anti-HER2 antibody, and fragments thereof. **Claim 5** The modified cell according to claim 1 or claim 2, wherein the intracellular domain of the CAR comprises a dual signaling domain. **Claim 6** The modified cell according to claim 1 or 2, wherein the targeted effector activity is directed against an antigen on a target cell that specifically binds to the antigen-binding domain of the CAR. **Claim 7** The modified cell according to claim 1 or 2, wherein the targeted effector activity is selected from the group consisting of phagocytosis, targeted cytotoxicity, antigen presentation, and cytokine secretion. **Claim 8** The modified cell according to claim 1 or 2, further comprising an agent selected from the group consisting of a nucleic acid, an antibiotic, an anti-inflammatory agent, an antibody or an antibody fragment thereof, a growth factor, a cytokine, an enzyme, a protein, a peptide, a fusion protein, a synthetic molecule, an organic molecule, a carbohydrate or analogue, a lipid, a hormone, a microsome, a derivative or modification thereof, and any combination thereof. **Claim 9** The modified cell according to claim 1 or claim 2, having at least one upregulated M1 marker and at least one downregulated M2 marker.

10. The modified cell according to claim 1 or claim 2, which is genetically modified to express the CAR.

11. The modified cell according to claim 1 or claim 2, wherein the targeted effector activity is enhanced by inhibition of CD47 activity or SIRPα activity.

12. A pharmaceutical composition comprising the cell according to claim 1 or 2 and a pharmaceutically acceptable carrier.

13. Use of the modified cell according to claim 1 or 2 in the manufacture of a medicament for use in treating an immune response in a subject in need thereof.

14. Use of the modified cell according to claim 1 or 2 in the manufacture of a medicament for use in treating a tumor or cancer in a subject in need thereof.

15. A method of treating a disease or condition associated with a tumor or cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified cell according to claim 1 or 2.

16. A method of treating a tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified cell according to claim 1 or 2.

17. A method for stimulating an immune response against target tumor cells or tumor tissue in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified cell according to claim 1 or 2.

18. Introducing a chimeric antigen receptor (CAR) into a monocyte, macrophage or dendritic cell, the CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain of a stimulatory molecule and / or a costimulatory molecule, the cell being a monocyte, macrophage or dendritic cell that expresses the CAR and possesses targeted effector activity comprising the step of

19. The method according to claim 18, wherein introducing the CAR into the cell comprises introducing a nucleic acid sequence encoding the CAR.

20. The method according to claim 19, wherein introducing the nucleic acid sequence comprises introducing an mRNA encoding the CAR by electroporation.

21. The method according to claim 19, wherein the step of introducing the nucleic acid sequence comprises transducing a cell using a viral vector comprising a nucleic acid sequence encoding a CAR.

22. The method according to claim 18, wherein the targeted effector activity is directed against an antigen on a target cell that specifically binds to the antigen-binding domain of the CAR.

23. The method according to claim 18, wherein the targeted effector activity is selected from the group consisting of phagocytosis, targeted cytotoxicity, antigen presentation, and cytokine secretion.

24. The method according to claim 18, further comprising the step of inhibiting CD47 activity or SIRPα activity to enhance the targeted effector activity.

25. The method according to claim 24, wherein the step of inhibiting CD47 activity or SIRPα activity comprises contacting the cell with an anti-CD47 blocking antibody or an anti-SIRPα blocking antibody.

26. The method according to claim 18, wherein the intracellular domain of the CAR comprises a dual signaling domain.

27. The method according to claim 18, wherein the antigen-binding domain of the CAR comprises an antibody selected from the group consisting of a synthetic antibody, a human antibody, a humanized antibody, a single-domain antibody, a single-chain variable fragment, and antigen-binding fragments thereof.

28. The method according to claim 18, wherein the antigen-binding domain of the CAR is selected from the group consisting of an anti-CD19 antibody, an anti-HER2 antibody, and fragments thereof.

29. The method according to claim 18, further comprising the step of modifying the cell to deliver an agent to a target, the agent being selected from the group consisting of a nucleic acid, an antibiotic, an anti-inflammatory agent, an antibody or an antibody fragment thereof, a growth factor, a cytokine, an enzyme, a protein, a peptide, a fusion protein, a synthetic molecule, an organic molecule, a carbohydrate or analog, a lipid, a hormone, a microsome, a derivative or modification thereof, and any combination thereof.

30. A composition comprising a cell modified according to claim 18.

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