TREM2 chimeric antigen receptors and uses thereof

Abstract

Described herein are chimeric antigen receptors (CARs) comprising an extracellular domain comprising a portion of an anti-triggering receptor expressed on myeloid cells 2 (TREM2) scFv for targeting macrophages expressing TREM2. Methods for producing and utilizing cells comprising the CAR are further provided herein.

Description

TREM2 CHIMERIC ANTIGEN RECEPTORS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Nos 63 / 648,504 filed May 16, 2024; 63 / 766,269 filed March 3, 2025; and 63 / 781,181 filed March 31, 2025, the contents of which are incorporated herein by reference in their entirety.TECHNICAL FIELD

[0002] The invention described herein relates to immunotherapy.BACKGROUND

[0003] The tumor microenvironment (TME) for the vast majority, if not all solid tumors recruits tumor-associated macrophages (TAMs) that promote tumor growth and metastasis. TAM-targeting strategies have not yet induced durable clinical responses. This work has been overwhelmed by the success of reinvigorating T cells through immune checkpoint blockade (ICB), but most breast cancer (BC) patients do not yet benefit from ICB, due to low T cell infiltrate. The inventors’ prior work reveals that conversion of TAMs to an anti -tumor phenotype enables T cell recruitment and activation and enhances immuno-therapy. Disclosed herein is a novel TAM-targeting therapy that depletes suppressive TAMs and enables T cell recruitment and activation to enable durable responses to ICB therapy.

[0004] Chimeric Antigen Receptor (CAR)-based cell therapies have demonstrated significant clinical efficacy for hematological cancers, but not in solid tumors. CAR-Macrophages (CAR-Mac) have displayed pre-clinical solid tumor efficacy, attributing to their enhanced infiltration into solid tumors with safe and notable clinical responses. Furthermore, CAR-Monocytes (CAR-Mo), display enhanced infiltration and longevity compared to CAR-Mac, while also being easier and more rapidly produced. CAR-Mac / Mo have not been used to directly target TAMs, which is a novel and innovative strategy disclosed herein and substantially improves clinical responses compared to previous CAR approaches.SUMMARY

[0005] One aspect herein provides a chimeric antigen receptor (CAR) polypeptide comprising: an extracellular domain comprising at least a portion of an anti-triggering receptor expressed on myeloid cells 2 (TREM2) scFv; a leader sequence; a hinge domain; a transmembrane domain; a co-stimulatory domain; and an intracellular signaling domain.

[0006] In one embodiment of any aspect herein, the leader sequences comprises a CD8 leader sequence. In one embodiment of any aspect herein, the CD8 leader sequence comprises the sequence of SEQ ID NO: 1.

[0007] In one embodiment of any aspect herein, the anti-TREM2 antibody is a an anti-TREM2 scFv. The CAR polypeptide of any of the preceding paragraphs, wherein the anti-TREM2 scFv comprises the sequence of SEQ ID NO: 2.

[0008] In one embodiment of any aspect herein, the hinge domain comprises a CD8 hinge domain. In one embodiment of any aspect herein, the CD8 hinge domain comprises the sequence of SEQ ID NO: 3.

[0009] In one embodiment of any aspect herein, the transmembrane domain comprises a CD8 transmembrane domain. In one embodiment of any aspect herein, the CD8 transmembrane domain comprises the sequence of SEQ ID NO: 4.

[0010] In one embodiment of any aspect herein, the intracellular signaling domain comprises a CD3^ signaling domain. In one embodiment of any aspect herein, the CD3^ intracellular signaling domain comprises the sequence of SEQ ID NO: 6.

[0011] In one embodiment of any aspect herein, the co-stimulatory domain is the intracellular domain of 4- IBB. In one embodiment of any aspect herein, the intracellular domain of 4- IBB comprises the sequence of SEQ ID NO: 5.

[0012] One aspect herein provides a chimeric antigen receptor (CAR) polypeptide comprising: an extracellular domain comprising at least a portion of an anti-triggering receptor expressed on myeloid cells 2 (TREM2) scFv; a CD8 leader sequence; a CD8 hinge domain; a CD8 transmembrane domain; a intracellular domain of 4-1BB; and a CD3^ signaling domain.

[0013] One aspect herein provides a CAR polypeptide comprising at least 95% identity with a sequence of SEQ ID NO: 7.

[0014] One aspect herein provides a CAR polypeptide comprising a sequence of SEQ ID NO: 7.

[0015] One aspect herein provides a nucleic acid sequence encoding any of the CAR polypeptides disclosed herein.

[0016] One aspect herein provides a mammalian cell comprising; a CAR polypeptide disclosed herein; or a nucleic acid encoding a CAR polypeptide disclosed herein.

[0017] In one embodiment of any aspect herein, the cell is a human cell.

[0018] In one embodiment of any aspect herein, the cell is a monocyte.

[0019] In one embodiment of any aspect herein, the monocyte is obtained from an individual.

[0020] In one embodiment of any aspect herein, the monocyte is derived from a stem cell selected from the group consisting of: an embryonic stem cell, a mesenchymal stem cell, a hematopoietic stem cell, and an induced pluripotent stem cell.

[0021] One aspect herein provides a method of treating cancer in a subject, the method comprising: engineering a monocyte to comprise any of the CAR polypeptides disclosed herein on the macrophage surface; and administering the engineered monocyte to the subject.

[0022] One aspect herein provides a method of treating cancer in a subject, the method comprising administering any of the cells expressing a CAR polypeptide disclosed herein to the subject.

[0023] In one embodiment of any aspect herein, the cancer is a solid tumor.

[0024] In one embodiment of any aspect herein, the cancer is breast cancer.

[0025] In one embodiment of any aspect herein, the subject is further administered an anti -cancer therapy.

[0026] In one embodiment of any aspect herein, the method further comprises, prior to administering, the step of diagnosing the subject as having cancer.

[0027] In one embodiment of any aspect herein, the method further comprises, prior to administering, receiving results of a diagnostic assay that diagnoses the subject has cancer.

[0028] One aspect herein provides a method of treating cancer in a subject, the method comprising: engineering a monocyte to comprise a CAR polypeptide specifically targeting a monocyte / macrophage cell surface protein, and administering the engineered monocyte to the subject, wherein the engineered monocyte does not bind to or target a cancer cell.

[0029] One aspect herein provides a method of treating cancer in a subject, the method comprising administering a monocyte engineered to comprise a CAR polypeptide specifically targeting a monocyte / macrophage cell surface protein to the subject, wherein the engineered monocyte does not bind to or target a cancer cell.

[0030] One aspect herein provides a composition comprising any of the mammalian cells disclosed herein (i.e., expressing a CAR polypeptide disclosed herein) formulated for the treatment of cancer. In one embodiment of any aspect herein, the composition further comprises a pharmaceutically acceptable carrier.

[0031] Definitions

[0032] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed technology, because the scope of the technology is limited only by the claims. 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 technology belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0033] Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0034] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. Where applicable, a decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0035] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater ascompared to a reference level. In the context of a marker or symptom, an “increase” is a statistically significant increase in such level.

[0036] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include, for example, chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.

[0037] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of disease e.g., cancer. A subject can be male or female, and of any age.

[0038] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having such condition or related complications. For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.

[0039] A “subject in need” of treatment for a particular condition can be a subject having that condition (e.g., cancer), diagnosed as having that condition, or at risk of developing that condition.

[0040] ‘ ‘Cancer” as used herein can refer to a hyperproliferation of cells whose unique trait — loss of normal cellular control — results in unregulated growth, lack of differentiation, local tissue invasion, and metastasis, and can be leukemia, lymphoma, multiple myeloma, or a solid tumor. Non-limiting examples of leukemia include acute myeloid leukemia (AML), Chronic myeloid leukemia (CML), Acute lymphocytic leukemia (ALL), and Chronic lymphocytic leukemia (CLL). In one embodiment, the cancer is ALL or CLL. Non-limiting examples of lymphoma include Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, Chronic lymphocytic leukemia (CLL), Small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), Marginal zone lymphomas, Burkitt lymphoma, hairy cell leukemia (HCL). In one embodiment, the cancer is DLBCL or Follicular lymphoma. Non-limiting examples of solid tumors include Adrenocortical Tumor, Alveolar Soft Part Sarcoma, Carcinoma, Chondrosarcoma, Colorectal Carcinoma, Desmoid Tumors, Desmoplastic Small Round Cell Tumor, Endocrine Tumors, Endodermal Sinus Tumor, Epithelioid Hemangioendothelioma, Ewing Sarcoma, Germ Cell Tumors (Solid Tumor), Giant Cell Tumor ofBone and Soft Tissue, Hepatoblastoma, Hepatocellular Carcinoma, Melanoma, Nephroma, Neuroblastoma, Non-Rhabdomyosarcoma Soft Tissue Sarcoma (NRSTS), Osteosarcoma, Paraspinal Sarcoma, Renal Cell Carcinoma, Retinoblastoma, Rhabdomyosarcoma, Synovial Sarcoma, and Wilms Tumor. Solid tumors can be found in bones, muscles, or organs, and can be sarcomas or carinomas. It is contemplated that any aspect of the invention described herein can be used to treat all types of cancers, including cancers not listed in the instant application. As used herein, the term “tumor” refers to an abnormal growth of cells or tissues, e.g., of malignant type or benign type.

[0041] A “disease” is a state of health of an animal, for example a human, wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated, then the animal's health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

[0042] As used herein, the terms "tumor antigen" and "cancer antigen" are used interchangeably to refer to antigens which are differentially expressed by cancer cells and can thereby be exploited in order to target cancer cells. Cancer antigens are antigens which can potentially stimulate apparently tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, by normal cells. These antigens can be characterized as those which are normally silent (z.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g, activated ras oncogene), suppressor genes (e.g., mutant p53), and fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses. Many tumor antigens have been defined in terms of multiple solid tumors: MAGE 1, 2, & 3, defined by immunity; MART-l / Melan-A, gplOO, carcinoembryonic antigen (CEA), HER2, mucins (z.e., MUC-1), prostate-specific antigen (PSA), and prostatic acid phosphatase (PAP). In addition, viral proteins such as some encoded by hepatitis B (HBV), Epstein-Barr (EBV), and human papilloma (HPV) have been shown to be important in the development of hepatocellular carcinoma, lymphoma, and cervical cancer, respectively.

[0043] As used herein, the term “chimeric” refers to the product of the fusion of portions of at least two or more different polynucleotide molecules. In one embodiment, the term “chimeric” refers to a gene expression element produced through the manipulation of known elements or other polynucleotide molecules.

[0044] As used herein, the terms “specific binding” and “specifically binds” refer to a physical interaction between two molecules, compounds, cells and / or particles wherein the first entity binds to the second, target, entity with greater specificity and affinity than it binds to a third entity which is anon-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second target, entity, which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or more greater than the affinity for the third nontarget entity under the same conditions. A reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized. A non-limiting example includes an antibody, or a ligand, which recognizes and binds with a cognate binding partner protein.

[0045] As used herein, the term “stimulation,” refers to a primary response induced by binding of a stimulatory molecule (e.g., an FcR complex, a TLR complex, or a TCR / CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via Fc receptor machinery or via a synthetic CAR. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-beta, and / or reorganization of cytoskeletal structures, and the like. As used herein, the term “stimulatory molecule,” refers to a molecule of a monocyte, macrophage, or dendritic cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell. In some embodiments, a stimulatory molecule comprises an FcR extracellular domain comprising a CD64 (FcyRI), CD32a (FcyRIIa), CD32b (FcyRIIb), CD32c, CD 16a (FcyRIIIa), CD 16b (FcyRIIIb), FcsRI. FcsRI I. or FcaRI (CD89) domain. In some embodiments, a stimulatory molecule comprises a TLR extracellular domain comprising a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain. As used herein, the term “stimulatory ligand,” refers to a ligand that when present on an antigen presenting cell (e.g., an aAPC, a macrophage, a dendritic cell, a B-cell, and the like) or tumor cell can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a monocyte, macrophage, or dendritic cell thereby mediating a response by the immune cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, inter alia, Toll-like receptor (TLR) ligand, an anti- toll-like receptor antibody, an agonist, and an antibody for a monocyte / macrophage receptor. In addition, cytokines, such as interferon-gamma, are potent stimulants of macrophages.

[0046] A “stimulatory ligand,” as used herein, refers to a ligand that when present on an antigen presenting cell (APC e.g., a macrophage, a dendritic cell, a B-cell, an artificial APC, and the like) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule” or “co-stimulatory molecule”) on a cell, thereby mediating a primary response by the cell, including, but not limited to, proliferation, activation, initiation of an immune response, and the like. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody. Stimulatory ligands are well-known in the art and encompass, inter aha, Toll-like receptor (TLR) ligand, an anti-toll-like receptor antibody, an agonist, and an antibody for amonocyte / macrophage receptor. In addition, cytokines, such as interferon-gamma, are potent stimulants of macrophages.

[0047] A “stimulatory molecule,” as the term is used herein, means a molecule on a cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell.

[0048] As used herein, the term “co -stimulatory ligand” refers to a molecule on an antigen presenting cell (e.g., an APC, dendritic cell, B cell, and the like) that specifically binds a cognate co-stimulatory molecule on a monocyte / macrophage / dendritic cell, thereby providing a signal which mediates a monocyte / macrophage / dendritic cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A co-stimulatory ligand can include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3. A co-stimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a monocyte / macrophage / dendritic cell, such as, but not limited to, CD27, CD28, 4-1BB, 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.

[0049] A “co-stimulatory molecule” refers to a molecule on an innate immune cell that is used to heighten or dampen the initial stimulus. For example, pathogen-associated pattern recognition receptors, such as TLR (heighten) or the CD47 / SIRPa axis (dampen), are molecules on innate immune cells. Co-stimulatory molecules include, but are not limited to TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcRbeta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, other co-stimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a co-stimulatory molecule that has the same functional capability, and any combination thereof.

[0050] In one embodiment, the term “engineered” and its grammatical equivalents as used herein can refer to one or more human-designed alterations of a nucleic acid, e.g., the nucleic acid within an organism's genome. In another embodiment, engineered can refer to alterations, additions, and / or deletion of genes. An “engineered cell” can refer to a cell with an added, deleted and / or altered gene. The term “cell” or “engineered cell” and their grammatical equivalents as used herein can refer to a cell of human or non-human animal origin.

[0051] As used herein, the term “operably linked” refers to a first polynucleotide molecule, such as a promoter, connected with a second transcribable polynucleotide molecule, such as a gene of interest, where the polynucleotide molecules are so arranged that the first polynucleotide molecule affects the function of the second polynucleotide molecule. The two polynucleotide molecules may or may not be part of a single contiguous polynucleotide molecule and may or may not be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.

[0052] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of ordinary skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0053] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g. ligan-mediated receptor activity and specificity of a native or reference polypeptide is retained.

[0054] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2)neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into He or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai, into He or into Leu.

[0055] In some embodiments, a polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a fragment or segment of a peptide which retains at least 50% of the wildtype reference polypeptide’s activity according to an assay known in the art or described below herein. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

[0056] In some embodiments, a polypeptide described herein can be a variant of a polypeptide or molecule as described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide -encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains activity of the non-variant polypeptide. A wide variety of PCR-based site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan.

[0057] A variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g. BLASTp or BLASTn with default settings).

[0058] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites permitting ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.Alternatively, oligonucleotide -directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are well established and include, for example, those disclosed by Walder et al. (Gene 42: 133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are herein incorporated by reference in their entireties. Any cysteine residue not involved in maintaining the proper conformation of a polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to a polypeptide to improve its stability or facilitate oligomerization.

[0059] As used herein, the term "DNA" is defined as deoxyribonucleic acid. The term "polynucleotide" is used herein interchangeably with "nucleic acid" to indicate a polymer of nucleosides. Typically a polynucleotide is composed of nucleosides that are naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) joined by phosphodiester bonds. However the term encompasses molecules comprising nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. Where this application refers to a polynucleotide it is understood that both DNA, RNA, and in each case both single- and doublestranded forms (and complements of each single-stranded molecule) are provided. "Polynucleotide sequence" as used herein can refer to the polynucleotide material itself and / or to the sequence information (i.e. the succession of letters used as abbreviations for bases) that biochemically characterizes a specific nucleic acid. A polynucleotide sequence presented herein is presented in a 5' to 3' direction unless otherwise indicated.

[0060] The term " polypeptide " as used herein refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. A peptide is a relatively short polypeptide, typically between about 2 and 60 amino acids in length. Polypeptides used herein typically contain amino acids such as the 20 L-amino acids that are most commonly found in proteins. However, other amino acids and / or amino acid analogs known in the art can be used. One or more of the amino acids in a polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, etc. A polypeptide that has a nonpolypeptide moiety covalently or noncovalently associated therewith is still considered a "polypeptide." Exemplary modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, produced using recombinant DNA technology or synthesized through chemical means such as conventional solid phase peptide synthesis, etc. The term "polypeptide sequence" or "amino acid sequence" as usedherein can refer to the polypeptide material itself and / or to the sequence information (i.e., the succession of letters or three letter codes used as abbreviations for amino acid names) that biochemically characterizes a polypeptide. A polypeptide sequence presented herein is presented in an N-terminal to C-terminal direction unless otherwise indicated.

[0061] In some embodiments, a nucleic acid encoding a polypeptide as described herein (e.g., a TREM2 CAR polypeptide) is comprised by a vector. In some of the aspects described herein, a nucleic acid sequence encoding a given polypeptide as described herein, or any module thereof, is operably linked to a vector. The term "vector," as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, artificial chromosome, virus, virion, etc.

[0062] As used herein, the term "expression vector" refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification. The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g., 5’ untranslated (5’UTR) or "leader" sequences and 3 ’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0063] As used herein, the term “viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain a nucleic acid encoding a polypeptide as described herein in place of non- essential viral genes. The vector and / or particle may be utilized for the purpose of transferring nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0064] By “recombinant vector” is meant a vector that includes a heterologous nucleic acid sequence, or “transgene” that is capable of expression in vivo. It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. Insome embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra-chromosomal DNA thereby eliminating potential effects of chromosomal integration.

[0065] As used herein, the terms "treat,” "treatment," "treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g., cancer. The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. Treatment is generally “effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective" if the progression of a disease is reduced or halted. That is, “treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0066] As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g., a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier in which the active ingredient would not be found to occur in nature.

[0067] As used herein, the term "administering," refers to the placement of a therapeutic or pharmaceutical composition as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising agents as disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.

[0068] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0069] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

[0070] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0071] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0072] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the technology.

[0073] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a nonlimiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0074] In some embodiments of any of the aspects, the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.

[0075] Other terms are defined within the description of the various aspects and embodiments of the technology of the following.BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Fig. 1 presents a schematic of proposed TREM2 CAR-Monocyte mechanism of action.TREM2 CAR-Monocytes are designed to bind and phagocytose TREM2+ cells in the solid tumor microenvironment. TREM2 is a highly restricted gene to the macrophage lineage so the main target of this therapeutic is TREM2+ tumor-associated macrophages (TAMs). TREM2+ TAMs are highly immunosuppressive towards T cells and removing this cell type will decrease TAM-mediated immunosuppression. TREM2 CAR-Monocytes express a chimeric antigen receptor (CAR) with an scFv that can bind TREM2. Binding of TREM2 to the scFv triggers phagocytosis of TREM2expressing cells. TREM2-CAR Monocytes home to tumors where they differentiate into macrophages that facilitate phagocytosis of TREM2+ cells.

[0077] Fig. 2 presents data showing TREM2 expression significantly correlates with poorer survival in Basal breast tumors. Data herein aimed assessed the clinical relevancy of TREM2 on breast cancer patient survival. Showing TCGA BRCA cohort bulk RNA sequencing analysis of TREM2 expression effect on survival in each major PAM50 subtype of breast cancer. TREM2 High and Low groups are designated based on the upper and lower quartile of TREM2 expression respectively. Breast cancer PAM50 molecular subtypes are as defined in the TCGA clinical metadata for each patient.

[0078] Fig. 3 presents data showing TREM2 is expressed by several tumor-associated macrophage (TAM) populations in the breast tumor microenvironment. Data herein assessed the expression level of TREM2 in macrophage subsets within breast tumors. Showing analysis of publicly available single cell RNAseq data from 55 Treatment Naive breast tumors. Data originated from GSE 176078 and GSE161892. Breast tumors in dataset encompass all major breast tumor subtypes. Tumor-associated macrophages (TAMs) identified in data using canonical macrophage markers and re-clustered into 9 populations using the Seurat R package. Graph shows normalized expression of TREM2 in each of the 9 breast TAM subpopulations and monocytes.

[0079] Fig. 4 presents a vector map for human TREM2-CAR lentiviral construct. Vector map generated using Benchling. TREM2 scFv sequence derived from, e.g., Szykowska et al.

[0080] CAR construct utilizes CD8 leader, hinge, and transmembrane domains for assembly. CAR construct utilizes CD3 zeta domain for signaling and 4-1BB domain for co-stimulation. Expression of construct can be determined by GFP fluorescence or anti-HA staining.

[0081] Fig. 5 presents data showing TREM2 expression in human acute monocytic leukemia cell lines. Data herein assessed the levels of TREM2 expression in commercially available monocytic cell lines to identify effector cell line of choice. Showing fluorescence of TREM2-PE antibody on HL-60, U-937, and THP-1 cells that were analyzed on Cytek Aurora flow cytometer. U-937 was chosen to be monocytic effector cell line to be transduced with CAR and THP-1 chosen to be target cell line due to high endogenous TREM2 expression.

[0082] Fig. 6 presents data showing generation of U-937 TREM2 & MSLN CAR-M cell lines. Data herein showed transduction of U-937 with the CAR construct encoding lentiviruses to enable expression of TREM2 and MSLN CARs in the cell line. U-937 cells were transduced with concentrated TREM2 CAR or MSLN CAR lentivirus by spinfection and stained with HA Tag-AF647 antibody for 30 minutes at 4C and analyzed for AF647 and GFP on Cytek Aurora flow cytometer. Showing fluorescence of HA Tag and GFP on U-937 cells post-transduction with lentivirus.GFP+HA+ cells were fluorescently sorted and grown as polyclonal cell lines for following experiments.

[0083] Fig. 7 presents data showing confirmation of no MSLN expression in THP-1 cells. Data herein confirmed that MSLN CARs could be used as an irrelevant CAR control for our experimental validation of the TREM2 CAR. THP-1 cells, the target cell herein, did not express MSLN. THP-1, MCF7 (Breast; MSLN-), and AsPCl (Pancreatic, MSLN+) cells were stained with MSLN-PE antibody for 30 minutes at 4C and analyzed on Cytek Aurora flow cytometer. Showing fluorescence of MSLN-PE antibody. THP-1 cell concluded to be MSLN negative and could be used with U-937 MSLN-CAR cells as a negative control.

[0084] Fig. 8 presents data showing confirmation of U-937 TREM2 CAR efficacy in vitro. Data herein determined if our U-937 TREM2 CAR-M cells had a greater phagocytic affinity for TREM2+ THP-1 cells than U-937 MSLN CAR-M cells had for MSLN negative THP-1 cells. U-937 TREM2 CAR-M cells or U-937 MSLN CAR-M cells were activated with lOng / mL PMA for 48 hours to differentiate into macrophages. 100K GFP+ CAR-Macrophages co-cultured at a 1: 1 ratio with mCherry+ THP-1 target cells for 4 hours at 5% CO2, 37C. All cells harvested and analyzed for GFP and mCherry on Cytek Aurora flow cytometer. GFP+ (CAR) cells analyzed for frequency of mCherry positivity. Increased mCherry fluorescence in GFP+ population (black box) indicates phagocytosis. Data demonstrated superior phagocytic capacity of TREM2 scFv on THP-1 cells compared to MSLN scFv.

[0085] Fig. 9 presents data showing confirmation of murine TREM2 CAR efficacy in vitro. TREM2- / - ER-HoxB8 cells transduced with murine TREM2 CAR retrovirus to generate murine TREM2 CAR-M cells. CAR+ TREM2- / - ER-HoxB8 and CARnegative TREM2- / - ER-HoxB8 cells were differentiated into macrophages with 30ng / mL M-CSF for 7 days. CAR-Macrophages and Macrophages were labeled with cell-trace violet (CTV). 100K CTV+ CAR-Macrophages or CTV+ Macrophages were co-cultured at a 1: 1 ratio with mCherry+ RAW264.7 (TREM2+) or 3T3 (TREM2 negative) target cells for 4 hours at 5% CO2, 37C. All cells harvested and analyzed for CTV and mCherry on Cytek Aurora flow cytometer. CTV+ (CAR) cells analyzed for frequency of mCherry positivity. Increased mCherry fluorescence in CTV+ population (black box) indicates phagocytosis. Data demonstrated superior phagocytic capacity of TREM2 scFv on RAW264.7 cells compared to 3T3 cells and enhanced phagocytic capacity of macrophages expressing the TREM2 CAR.

[0086] Figure 10A and 10B present data showing confirmation of murine TREM2 CAR efficacy in vivo. Data herein assessed if murine TREM2 CAR-monocytes could inhibit breast tumor growth in immunocompetent mice bearing orthotopic breast tumors. (Fig. 10A) le6 CAR+ TREM2- / - ER- HoxB8 cells (CAR) or Untransduced ER-Hoxb8 cells (UTD) were differentiated for 3 days with 30ng / mL M-CSF into monocytes and injected intravenously 3x / week for 2 weeks into EO771 orthotopic-breast tumor bearing mice. (Fig. 10A) The volume of tumors was measured daily by calipers and shown as cubic millimeters. Data demonstrated that TREM2 CAR monotherapysignificantly inhibits breast tumor growth after 14 days of treatment compared to untransduced monocytes.

[0087] Fig. 11A-11D present data showing confirmation of murine TREM2 CAR synergy with anti-PDl in vivo. (Fig. 11A) Data herein assessed if the addition of PD-1 blockade could enhance the 3e6 CAR+ TREM2- / - ER-HoxB8 cells (CAR) or Untransduced ER-Hoxb8 cells (UTD) were differentiated for 3 days with 30ng / mL M-CSF into monocytes and injected intravenously 3x / week for 2 weeks into EO771 orthotopic-breast tumor bearing mice. (Fig. 11A) 200ug of anti-PDl was administered intraperitoneally on days 2,5, &8 after enrollment. (Figs 11B-D) The volume of tumors was measured daily by calipers and shown as cubic millimeters. Data demonstrated that combination therapy significantly extends survival and inhibits tumor growth compared to TREM2 CAR-M monotherapy.

[0088] Fig. 12A and 12B present data showing increased number of CAR-M cells injected achieves significant inhibition of breast tumor growth. Data assessed if an increased number of TREM2 CAR-Monocytes inhibited breast tumor growth relative to a matched number of untransduced monocytes. (Fig. 12A) 5e6 CAR+ TREM2- / - ER-HoxB8 cells (CAR) or Untransduced ER-Hoxb8 cells (UTD) were differentiated for 3 days with 30ng / mL M-CSF into monocytes and injected intravenously 3x / week for 1 week into EO771 orthotopic-breast tumor bearing mice. (Fig. 12B) The volume of tumors was measured daily by calipers and shown as cubic millimeters. Data demonstrated that an increased number of TREM2 CAR monocytes significantly inhibits breast tumor growth after 12 days of treatment compared to untransduced monocytes.

[0089] Fig. 13 presents a schematic of experiment to assess CAR-Mo effect on T cell phenotype.TREM2 CAR-Mo are differentiated into macrophages and co-cultured with the murine macrophage line RAW264.7, which expresses high levels of endogenous TREM2. 24 hours post co-culture, the CM are collected and transferred to naive splenic T cells.DETAILED DESCRIPTIONChimeric Antigen Receptors

[0090] The technology described herein provides improved CARs for use in immunotherapy. The following discusses CARs and the various improvements.

[0091] The terms “chimeric antigen receptor” or “CAR” or “CARs” as used herein refer to engineered cell receptors, which graft a ligand or antigen specificity onto cells (for example, monocytes). CARs are also known as artificial cell receptors, chimeric cell receptors or chimeric immunoreceptors.

[0092] A CAR places a chimeric extracellular target-binding domain that specifically binds a target, e.g., a polypeptide expressed on the surface of a cell to be targeted onto a construct including a transmembrane domain, and intracellular domain(s) (including signaling domains) of a cell receptor molecule. In one embodiment, the chimeric extracellular target-binding domain comprises theantigen-binding domain(s) of an antibody that specifically binds an antigen expressed on a cell to be targeted for a cell response. The properties of the intracellular signaling domain(s) of the CAR can vary as known in the art and as disclosed herein, but the chimeric target / antigen-binding domains(s) render the receptor sensitive to signaling activation when the chimeric target / antigen binding domain binds the target / antigen on the surface of a targeted cell.

[0093] With respect to intracellular signaling domains, so-called “first- generation” CARs include those that solely provide CD3zeta (CD3Q signals upon antigen binding. So-called “second- generation” CARs include those that provide both co-stimulation (e.g., CD28 or CD137) and activation (CD3Q domains, and so-called “third-generation” CARs include those that provide multiple costimulatory (e.g., CD28 and CD137) domains and activation domains (e.g., CD3Q. In various embodiments, the CAR is selected to have high affinity or avidity for the target / antigen - for example, antibody-derived target or antigen binding domains will generally have higher affinity and / or avidity for the target antigen than would a naturally-occurring cell receptor. This property, combined with the high specificity one can select for an antibody provides highly specific cell targeting by CAR cells.

[0094] One aspect herein provides a chimeric antigen receptor (CAR) polypeptide comprising: an extracellular domain comprising at least a portion of an TREM2 antibody; a leader sequence; a hinge domain; a transmembrane domain; a co-stimulatory domain; and an intracellular signaling domain.

[0095] Another aspect herein provides a chimeric antigen receptor (CAR) polypeptide comprising: an extracellular domain comprising at least a portion of an anti-triggering receptor expressed on myeloid cells 2 (TREM2) scFv; a CD8 leader sequence; a CD8 hinge domain; a CD8 transmembrane domain; a intracellular domain of 4-1BB; and a CD3 signaling domain.

[0096] As used herein, a “CAR M cell” or “CAR-M” refers to a macrophage which expresses a CAR. When expressed in a macrophage cell, CARs have the ability to redirect macrophage -cell specificity and reactivity toward a selected target, exploiting the antigen-binding properties of monoclonal antibodies.

[0097] As used herein, the term “extracellular target binding domain” refers to a polypeptide found on the outside of the cell sufficient to facilitate binding to a target. The extracellular target binding domain will specifically bind to its binding partner. As non-limiting examples, the extracellular targetbinding domain can include an antigen-binding domain of an antibody, or a ligand, which recognizes and binds with a cognate binding partner protein. In this context, a ligand is a molecule which binds specifically to a portion of a protein and / or receptor. The cognate binding partner of a ligand useful in the methods and compositions described herein can generally be found on the surface of a cell. Ligand: cognate partner binding can result in the alteration of the ligand-bearing receptor, or activate a physiological response, for example, the activation of a signaling pathway or cascade. In oneembodiment, the ligand can be non-native to the genome. Optionally, the ligand has a conserved function across at least two species.

[0098] In some embodiments, extra cellular domain is a TLR extracellular domain. In some embodiments, a TLR extracellular domain comprises a full-length TLR extracellular domain. In some embodiments, a TLR extracellular domain comprises a portion of a full-length TLR extracellular domain. In some embodiments, a TLR extracellular domain (or portion thereof) is or comprises a human TLR extracellular domain. In some embodiments, a TLR extracellular domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a TLR extracellular domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a TLR extracellular domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.

[0099] Another aspect of the invention relates to a CAR polypeptide comprising a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with a sequence selected from SEQ ID NO: 7. MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLL IYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRSGLHTFGQGTKLEIKGGGGSGGG GSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSYYYMGWVRQAPGKGLEWVSGISPSSGYTYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYYGYYYSHMDYWGQGTLVTVSSTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL LYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR ( SEQ ID NO : 7 )

[0100] Another aspect of the invention relates to a CAR polypeptide comprising a sequence selected from SEQ ID NO: 7.

[0101] Another aspect of the invention relates to a CAR polypeptide comprising a sequence corresponding to a sequence selected from SEQ ID NO: 7.

[0102] Another aspect of the invention relates to a nucleic acid sequence that encodes any of the CAR polypeptides disclosed herein.

[0103] In one embodiment herein, a CAR described herein comprises at least one domain (e.g., an extracellular domain, a transmembrane domain, and / or an intracellular domain) that inhibits antiphagocytic signaling in an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell). In some embodiments, a CAR described herein improves effector activity of an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell), e.g., by enhancing inhibition of CD47 and / or SIRPa activity, relative to a cell of the same type without a CAR. In some embodiments,a CAR described herein binds CD47, e.g., and serves as a dominant negative receptor, inhibiting SIRPa activity (e.g., a CD47 sink). In some embodiments, a CAR described herein that binds SIRPa, e.g., comprises an activating receptor (e.g., comprises a CD3z intracellular domain). In some embodiments, a CAR described herein inhibits at least one interaction of CD47 and SIRPa. In some embodiments, a CAR is or comprises a phagocytic logic gate.Antibody Reagents

[0104] In various embodiments, the CARs described herein comprise an antibody reagent or an antigen-binding domain thereof as an extracellular target-binding domain.

[0105] As used herein, the term “antibody reagent" refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen. An antibody reagent can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody. In some embodiments of any of the aspects, an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, CDRs, and domain antibody (dAb) fragments (see, e.g. de Wildt et al., Eur J. Immunol. 1996; 26(3):629-39; which is incorporated by reference herein in its entirety)) as well as complete antibodies. An antibody can have the structural features of IgA, IgG, IgE, IgD, or IgM (as well as subtypes and combinations thereof). Antibodies can be from any source, including mouse, rabbit, pig, rat, and primate (human and non-human primate) and primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like. Fully human antibody binding domains can be selected, for example, from phage display libraries using methods known to those of ordinary skill in the art.

[0106] The VH and VL regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" ("FR"). The extent of the framework region and CDRs has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated by reference herein in their entireties). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0107] In one embodiment, the antibody or antibody reagent is not a human antibody or antibody reagent, (i.e., the antibody or antibody reagent is mouse), but has been humanized. A “humanized antibody or antibody reagent” refers to a non-human antibody or antibody reagent that has been modified at the protein sequence level to increase its similarity to antibody or antibody reagent variants produced naturally in humans. One approach to humanizing antibodies employs the grafting of murine or other non-human CDRs onto human antibody frameworks.

[0108] In one embodiment, a CAR’s extracellular target binding domain comprises or consists essentially of a single-chain Fv (scFv) fragment created by fusing the VH and VL domains of an antibody, generally a monoclonal antibody, via a flexible linker peptide. In various embodiments, the scFv is fused to a transmembrane domain and to a cell receptor intracellular signaling domain, e.g., an engineered intracellular signaling domain as described herein.

[0109] Antibody binding domains and ways to select and clone them are well known to those of ordinary skill in the art.

[0110] In one embodiment, the extracellular domain of the CAR polypeptide comprises an anti- TREM2 antibody. In one embodiment, the anti-TREM2 antibody is an anti-TREM2 scFv. In one embodiment, the anti-TREM2 scFv has a sequence comprising a sequence of SEQ ID NO: 2.DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSRSGLHTFGQGTKLEIKGGGGSGGGGSGGGGSEV QLLESGGGLVQPGGSLRLSCAASGFTFSYYYMGWVRQAPGKGLEWVSGISPSSGYTYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYYGYYYSHMDYWGQGTLVTVSS (SEQ ID NO. 2)

[0111] Triggering receptor expressed on myeloid cells (TREM2) is membrane protein that forms a receptor signaling complex with the TYRO protein tyrosine kinase binding protein and functions in the immune system. TREM2 sequences are known for a number of species, e.g., human TREM2, also known as AD17, PLOSL2, TREM-2, Trem2a, Trem2b, and Trem2c, (NCBI Gene ID: 54209) polypeptide (e.g., NCBI Ref Seq NP_001258750.1) and mRNA (e.g., NCBI Ref Seq NM_001271821.2). TREM2 can refer to human TREM2, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, TREM2 can refer to the TREM2 of, e.g., mouse, rat, dog, cat, cow, horse, pig, and the like. Homologs and / or orthologs of human TREM2 are readily identified for such species by one of skill in the art, e.g., using the NCBI ortholog search function or searching available sequence data for a given species for sequence similar to a reference TREM2 sequence.

[0112] In one embodiment, the portion of TREM2 has a sequence corresponding to a sequence selected from SEQ ID NO: 2; or comprises a sequence selected from SEQ ID NO: 2; or comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO 2.Hinge and TM domain

[0113] Each CAR as described herein necessarily includes a hinge domain. As used herein, “hinge domain” refers to an amino acid region that allows for separation and flexibility of the binding moiety and the T cell membrane. The length of the flexible hinges also allow for better binding to relatively inaccessible epitopes, e.g., longer hinge regions are allow for optimal binding. One skilled in the art will be able to determine the appropriate hinge for the given CAR target. In one embodiment, the transmembrane domain or fragment thereof of any of the CAR polypeptides described herein comprises a CD8 hinge domain.

[0114] Each CAR as described herein necessarily includes a transmembrane domain that joins the extracellular target-binding domain to the intracellular signaling domain.

[0115] As used herein, “transmembrane domain” (TM domain) refers to the generally hydrophobic region of the CAR which crosses the plasma membrane of a cell. The TM domain can be the transmembrane region or fragment thereof of a transmembrane protein (for example a Type I transmembrane protein or other transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. While specific examples are provided herein and used in the Examples, other transmembrane domains will be apparent to those of skill in the art and can be used in connection with alternate embodiments of the technology. A selected transmembrane region or fragment thereof would preferably not interfere with the intended function of the CAR. As used in relation to a transmembrane domain of a protein or polypeptide, “fragment thereof’ refers to a portion of a transmembrane domain that is sufficient to anchor or attach a protein to a cell surface.

[0116] In one embodiment, the transmembrane domain or fragment thereof of any of the CAR polypeptides described herein comprises a transmembrane domain of CD8.

[0117] In an alternate embodiment of any aspect, the transmembrane domain or fragment thereof of the CAR described herein comprises a transmembrane domain selected from the transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD9, CD14, CD16, CD22, CD32, CD33, CD37, CD64, CD68, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDld, ITGAE, CD 103, ITGAL, CDla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG(CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In some embodiments, a transmembrane domain comprises a CD28, CD8a, CD64, CD32a, CD32c, CD16a, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD19, CD20, 41BB, CD28, 0X40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, CD3- zeta, FcR y, V / I / LxYxxL / V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPb, CD22, PIR-B, LILRB1, CD36, or Syk transmembrane domain.

[0118] CD8 is an antigen preferentially found on the cell surface of cytotoxic T lymphocytes. CD8 mediates cell-cell interactions within the immune system, and acts as a T cell coreceptor. CD8 consists of an alpha (CD8a) and beta (CD8b) chain. CD8a sequences are known for a number of species, e.g., human CD8a, (NCBI Gene ID: 925) polypeptide (NCBI Ref Seq NP_001139345.1) and mRNA (e.g., NCBI Ref Seq NM_ 000002.12). CD8 can refer to human CD8, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, CD8 can refer to the CD8 of, e.g., dog, cat, cow, horse, pig, and the like.Homologs and / or orthologs of human CD8 are readily identified for such species by one of skill in the art, e.g., using the NCBI ortholog search function or searching available sequence data for a given species for sequence similar to a reference CD 8 sequence.

[0119] In one embodiment, the CD8 hinge sequence corresponds to a sequence selected from SEQ ID NO: 3; or comprises a sequence selected from SEQ ID NO: 3; or comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO: 3.

[0120] Human CD8a Hinge Sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD ( SEQ ID NO : 3 )

[0121] In one embodiment, the CD8 transmembrane sequence corresponds to a sequence selected from SEQ ID NO: 4; or comprises a sequence selected from SEQ ID NO: 4; or comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO: 4.

[0122] Human CD8a Transmembrane Domain Sequence: IYIWAPLAGTCGVLLLSLVITLYC ( SEQ ID NO : 4 )Co-stimulatory Domain

[0123] Each CAR described herein comprises an intracellular domain of a co-stimulatory molecule, or co-stimulatory domain. As used herein, the term “co-stimulatory domain” refers to an intracellularsignaling domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen. Illustrative examples of such costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In some embodiments, a co-stimulatory domain comprises TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcRbeta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8alpha, CD8beta, IL2Rbeta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, other co-stimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a co-stimulatory molecule that has the same functional capability, and any combinations thereof.

[0124] In one embodiment, the intracellular domain is the intracellular domain of 4-1BB.

[0125] 4-1BBL is a type 2 transmembrane glycoprotein belonging to the TNFR / TNF ligand superfamily. 4-1BBL is a co-stimulatory ligand that binds receptor 4-1BB (CD137) expressed on T cell. 4-1BBL is expressed on professional APCs including dendritic cells, macrophages, and activated B cells. 4-1BBL sequences are known for a number of species, e.g., human 4-1BBL, also known as TNFSF9 (NCBI Gene ID: 8744) polypeptide (e.g., NCBI Ref Seq NP_003802.1) and mRNA (e.g., NCBI Ref Seq NM 003811.3). 4-1BBL can refer to human 4-1BBL, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, 4-1BBL can refer to the 4-1BBL of, e.g., dog, cat, cow, horse, pig, and the like. Homologs and / or orthologs of human 4-1BBL are readily identified for such species by one of skill in the art, e.g., using the NCBI ortholog search function or searching available sequence data for a given species for sequence similar to a reference 4-1 BBL sequence.Intracellular Domains

[0126] In one embodiment, the CAR polypeptide further comprises an intracellular domain. As used herein, an “intracellular domain” refers to a nucleic acid fully comprised within a cell. In one embodiment, the intracellular domain refers to the intracellular domain of a receptor. An intracellular domain can interact with the interior of a cell. With respect to the intracellular domain of a receptor, the intracellular domain can function to relay a signal transduced. An intracellular domain of a receptor can comprise enzymatic activity.

[0127] In some embodiments, a CAR comprises one or more intracellular domains. In some embodiments, an intracellular domain is or comprises a human intracellular domain, or portion thereof. In some embodiments, an intracellular domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an intracellular domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an intracellular domain and / or other cytoplasmic domain of a CAR is responsible for activation of the cell in which the CAR is expressed (e.g., an immune cell). In some embodiments, an intracellular domain of a CAR is responsible for signal activation and / or transduction in an immune cell comprising said CAR.

[0128] In some embodiments, an intracellular domain of a CAR includes at least one domain responsible for signal activation and / or transduction. In some embodiments, an intracellular domain is or comprises at least one of a co-stimulatory molecule and a signaling domain. In some embodiments, an intracellular domain of a CAR comprises dual signaling domains. In some embodiments, an intracellular domain of a CAR comprises more than two signaling domains.

[0129] In some embodiments, an intracellular domain comprises a cytoplasmic portion of a surface receptor. In some embodiments, an intracellular domain comprises a co-stimulatory molecule. In some embodiments, an intracellular domain comprises a molecule that acts to initiate signal transduction in an immune cell.

[0130] In some embodiments, an intracellular domain of a CAR includes any portion of one or more co-stimulatory molecules, such as at least one signaling domain from CD3, Fc epsilon RI gamma chain, any derivative or variant thereof, any synthetic sequence thereof that has the same functional capability, and any combination thereof.In one embodiment, the intracellular domain is the intracellular domain of a 4- IBB. In one embodiment, the 4- IBB intracellular domain sequence corresponds to a sequence selected from SEQ ID NO: 5; or comprises a sequence selected from SEQ ID NO: 5; or comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO: 5.

[0131] Human 4-1BB Co-Stimulatory Domain Sequence:KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL ( SEQ ID NO : 5 )Intracellular Signaling Domain

[0132] CARs as described herein comprise an intracellular signaling domain. An “intracellular signaling domain,” refers to the part of a CAR polypeptide that participates in transducing the message of effective CAR binding to a target antigen into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses elicited following antigen binding to the extracellular CAR domain.

[0133] CD3 is a T cell co-receptor that facilitates T lymphocytes activation when simultaneously engaged with the appropriate co-stimulation (e.g., binding of a co-stimulatory molecule). A CD3 complex consists of 4 distinct chains; mammal CD3 consists of a CD3y chain, a CD35 chain, and two CD3s chains. These chains associate with a molecule known as the T cell receptor (TCR) and the CD3^ to generate an activation signal in T lymphocytes. A complete TCR complex comprises a TCR, CD3^, and the complete CD3 complex.

[0134] In some embodiments, a CAR comprises one or more intracellular signaling domains. In some embodiments, an intracellular signaling domain is or comprises a human intracellular signaling domain, or portion thereof. In some embodiments, a signaling domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a signaling domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).

[0135] In some embodiments, one or more intracellular signaling domains comprise a CD3-zeta, FcRy, CD64, CD32a, CD32c, CD16a, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, R0R1, ROS1, RYK, TIE2, TRIF, STING, RIG-I, MDA5, MERTK, TYRO, TRK, VEGFR, CD40, CD19, CD20, 41BB, CD28, 0X40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, V / I / LxYxxL / V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPb, CD22, PIR-B, LILRB1, Syk, 41BB ligand (41BBL; TNFSF9), CD27, OX40L, CD32b, CD1 lb, ITGAM, SLAMF7, CD206, CD163, CD209, Dectin-2, or one or more cytokine receptor signaling domains (e.g., an IL1R, an IL2R, an IL3R, an IL4R, an IL5R, an IL6R, an IL7R, an IL8R, an IL9R, an IL10R, an IL11R, an IL12R, an IL13R, an IL14R, an IL15R, an IL17R, an IFNaR, an IFNgR, an TNFR, an CSF1R, an CSF2R, DaplO, CD36, Dectin-1, or ICOSL intracellular signaling domain).

[0136] In some embodiments, an intracellular domain of a CAR comprises dual signaling domains, such as 41BB, CD28, ICOS, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, CD 116 receptor beta chain, CSF1-R, LRP1 / CD91, SR-A1, SR-A2, MARCO, SR-CL1, SR- CL2, SR-C, SR-E, CR1, CR3, CR4, dectin 1, DEC-205, DC-SIGN, CD14, CD36, LOX-1, CDl lb, together with any of the signaling domains listed in the above paragraph in any combination.

[0137] In some embodiments, an FcR intracellular domain comprises a full-length FcR intracellular domain. In some embodiments, an FcR intracellular domain comprises a portion of a full-length FcR intracellular domain. In some embodiments, an FcR intracellular domain is or comprises a human FcR intracellular domain, or portion thereof. In some embodiments, an FcR intracellular domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an FcR intracellular domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an FcR intracellular domain comprises a CD64 (FcyRI), CD32a (FcyRIIa), CD32b (FcyRIIb), CD32c (Fc gamma Rile), CD 16a (FcyRIIIa), CD 16b (FcyRIIIb), FcsRI. FcsRI I. or FcaRI (CD89) domain.

[0138] In some embodiments of any aspect, a CAR polypeptide described herein comprises an intracellular signaling domain that comprises a CD3 zeta (CD3Q intracellular signaling sequence.

[0139] In one embodiment, the CD3^ intracellular signaling sequence corresponds to a sequence selected from SEQ ID NO: 6; or comprises a sequence selected from SEQ ID NO: 6; or comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO: 6.

[0140] Human CD3z Signaling Domain:RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRC7 ( SEQ ID NO : 6 )

[0141] A more detailed description of CARs and CAR T cells can be found in Maus et al. Blood 2014 123:2624-35; Reardon et al. Neuro-Oncology 2014 16: 1441-1458; Hoyos et al. Haematologica 2012 97: 1622; Byrd et al. J Clin Oncol 2014 32:3039-47; Maher et al. Cancer Res 2009 69:4559- 4562; and Tamada et al. Clin Cancer Res 2012 18:6436-6445; each of which is incorporated by reference herein in its entirety.Leader Sequence

[0142] The CAR polypeptide disclosed herein comprises a leader sequence. As used herein, a “leader sequence”, also known as leader RNA, refers to a region of an mRNA that is directly upstream of the initiation codon. A leader sequence can be important for the regulation of translation of a transcript.

[0143] In one embodiment, the leader sequence is a CD8 leader sequence. In one embodiment, the CD8 leader sequence corresponds to a sequence selected from SEQ ID NO: 1; or comprises a sequence selected from SEQ ID NO: 1; or comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO: 1.

[0144] Human CD8a Leader Sequence:MALPVTALLLPLALLLHAARP ( SEQ ID NO : 1 )Linker

[0145] In one embodiment, the CAR further comprises a linker domain. As used herein “linker domain” refers to an oligo- or polypeptide region from about 2 to 100 amino acids in length, which links together any of the domains / regions of the CAR as described herein. In some embodiment, linkers can include or be composed of flexible residues such as glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another. Linkers may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (for example T2A), 2A-like linkers or functional equivalents thereof and combinations thereof. In one embodiment, the linker region is T2A derived from Thosea asigna virus. Non-limiting examples of linkers include linkers derived from Thosea asigna virus, and a linker derived from the internal ribosomal entry site (IRES) sequence.Reporter Molecule

[0146] In one embodiment, a CAR as described herein further comprises a reporter molecule, e.g., to permit for non-invasive imaging (e.g., positron-emission tomography PET scan). In a bispecific CAR that includes a reporter molecule, the first extracellular binding domain and the second extracellular binding domain can include different or the same reporter molecule. In a bispecific CAR cell, the first CAR and the second CAR can express different or the same reporter molecule. In another embodiment, a CAR as described herein further comprises a reporter molecule (for example hygromycin phosphotransferase (hph)) that can be imaged alone or in combination with a substrate or chemical (for example 9-[4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG)). In another embodiment, a CAR as described herein further comprises nanoparticles at can be readily imaged using non-invasive techniques (e.g., gold nanoparticles (GNP) functionalized with64Cu2+). Labeling of CAR T cells for non-invasive imaging is reviewed, for example in Bhatnagar P, et al. Integr Biol. (Camb). 2013 Jan; 5(1): 231-238, and Keu KV, et al. Sdci Transl Med. 2017 Jan 18; 9(373), which are incorporated herein by reference in their entireties.

[0147] GFP are demonstrated herein as fluorescent tags useful for imaging a CAR expressed on a cell (e.g., a CAR cell). It is expected that essentially any fluorescent protein known in the art can be used as a fluorescent tag for this purpose. For clinical applications, the CAR need not include a fluorescent tag or fluorescent protein.CAR-Expressing Cells

[0148] Another aspect of the invention relates to a mammalian cell comprising any of the CAR polypeptides described herein; or a nucleic acid encoding any of the CAR polypeptides described herein. In one embodiment, the mammalian cell comprises an antibody, antibody reagent, antigenbinding portion thereof, or any of the CAR polypeptides described herein, or a nucleic acid encoding such an antibody, antibody reagent, antigen-binding portion thereof, or any of the CAR polypeptides described herein. The mammalian cell or tissue can be of human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog or cat origin, but any other mammalian cell may be used. In a preferred embodiment of any aspect, the mammalian cell is human.

[0149] In one embodiment, the cell is an immune cell. In one embodiment, the cell is a monocyte. In one embodiment, the cell is a macrophage.

[0150] The present disclosure, among other things, provides modified immune cells (e.g., monocyes or macrophages) comprising at least one CAR (e.g., a TREM2 CAR) as described herein. As used herein, the term “immune cell,” refers to a cell that is involved in an immune response, e.g., promotion of an immune response. Examples of immune cells include, but are not limited to, macrophages, monocytes, dendritic cells, neutrophils, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, T-lymphocytes, or B-lymphocytes. A source of immune cells (e.g., macrophages, monocytes, or dendritic cells) can be obtained from a subject. Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof.

[0151] In some embodiments, a population of immune cells as described herein comprises monocytes, macrophages, and / or precursors thereof. In some embodiments, a population of immune cells comprises a purified population of monocytes or macrophages.

[0152] In some embodiments, an immune cell is activated, e.g., an immune cell exhibits increased cytokine production, chemokine production, phagocytosis, cell signaling, target cell killing, and / or antigen presentation, e.g., relative to an inactive cell. In some embodiments, an activated immune cell exhibits changes in gene expression, e.g., an induction of pro-inflammatory gene expression (e.g., one, two, three, four, five, six, or seven of TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, or IL-1), e.g., relative to an inactive cell. In one embodiment, the immune cell is activated after it has been engineered to express any of the CARs disclosed herein. In one embodiment, the immune cell is activated prior to being engineered to express any of the CARs disclosed herein.

[0153] In some embodiments, immune cells (e.g., monocytes or macrophages) are obtained (e.g., isolated) from a subject. Immune cells may be autologous or sourced from allogeneic or universal donors. Cells can be obtained from a number of sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, tumors, and / or induced pluripotentstem cells, such as embryonic stem cells (ESCs). In certain embodiments, cells can be obtained from a unit of blood collected from a subject using any number of separation techniques known to a skilled artisan, such as Ficoll separation. In some embodiments, cells from circulating blood of a subject are obtained by apheresis or leukapheresis. Cells collected by apheresis may be washed to remove a plasma fraction and resuspended in a variety of buffers (e.g., phosphate buffered saline (PBS)) or culture media. In some embodiments, enrichment of immune cells (e.g. monocytes) comprises plastic adherence. In some embodiments, following enrichment, differentiation of immune cells (e.g. monocytes) comprises stimulation with GM-CSF. In some embodiments, a composition comprising blood cells (e.g., monocytes, lymphocytes, platelets, plasma, and / or red blood cells), such as a leukapheresis composition (e.g., a leukopak) is used for enrichment. In some embodiments, a leukapheresis composition (e.g., a leukopak) comprises a sample from a healthy human donor. In certain embodiments, apheresis of immune cells (e.g. monocytes) is followed by mobilization with GM-CSF. In certain embodiments, selection of immune cells (e,g, monocytes) comprises CD 14 positive selection using microbeads (e.g., MACS® MicroBeads on a CliniMACS Prodigy device). In some embodiments, an immune cell precursor (e.g., precursors to macrophages, monocytes, or dendritic cells) is used in compositions and methods described herein. Immune cell precursors may be differentiated in vivo or ex vivo into immune cells. Non-limiting examples of precursor immune cells include hematopoietic stem cells, common myeloid progenitors, myeloblasts, monoblasts, promonocytes, or intermediates thereof. For example, induced pluripotent stem cells may be used to generate monocytes, macrophages, and / or dendritic cells. Induced pluripotent stem cells (iPSCs) may be derived from normal human tissue, such as peripheral blood, fibroblasts, skin, keratinocytes, or renal epithelial cells. Autologous, allogeneic, or universal donor iPSCs could be differentiated toward a myeloid lineage (e.g., a monocytes or macrophages, or precursor thereof).

[0154] Immune cells (e.g., monocytes or macrophages) as described herein can be isolated from peripheral blood, for example, by lysing red blood cells and depleting lymphocytes and red blood cells, such as by centrifugation through a PERCOLL™ gradient. Alternatively, immune cells can be isolated from umbilical cord tissue. A specific subpopulation of immune cells can be further isolated by positive or negative selection techniques. In some embodiments, immune cells can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD3, CD4, CD8, CD56, CD66b, CD 19, or CD20. In some embodiments, enrichment of an immune cell population, for example, by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. By way of non-limiting example, cell selection can also comprise negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on negatively selected cells.

[0155] During isolation of a desired population of immune cells (e.g., monocytes or macrophages) as described herein by positive or negative selection, immune cell concentration and surface (e.g.,particles, such as beads) can be varied. It may be desirable to significantly decrease volume in which beads and cells are mixed together to ensure maximum contact area of cells and beads.

[0156] Monocytes are multipotent cells that circulate in the blood, bone marrow, and spleen, and generally do not proliferate when in a steady state. Monocytes can vary in size significantly in the range of about 10-30 pm in diameter. A ratio of nucleus to cytoplasm for a monocyte can range from about 2: 1 to about 1: 1. Typically, monocytes comprise chemokine receptors and pathogen recognition receptors that mediate migration from blood to tissues, such as during an infection. Monocytes can produce inflammatory cytokines, take up cells and / or toxic molecules, and differentiate into dendritic cells or macrophages. Exemplarily phenotypic markers for human monocyte cells include, but are not limited to, CD9, CDl lb, CDl lc, CDwl2, CD13, CD15, CDwl7, CD31, CD32, CD33, CD35, CD36, CD38, CD43, CD49b, CD49e, CD49f, CD63, CD64, CD65s, CD68, CD84. CD85, CD86, CD87, CD89, CD91, CDw92, CD93, CD98, CD101, CD102, CD111, CD112, CD115, CD116, CD119, CDwl21b, CDwl23, CD127, CDwl28, CDwl31, CD147, CD155, CD156a, CD157, CD162 CD163, CD164, CD168, CD171, CD172a, CD180, CD206, CD131al, CD213 2, CDw210, CD226, CD281, CD282, CD284, and CD286. Exemplarily phenotypic markers for mouse monocyte cells include, but are not limited to, CDl la, CDl lb, CD16, CD18, CD29, CD31, CD32, CD44, CD45, CD49d, CD115, CD116, Cdwl31, CD281, CD282, CD284, CD286, F4 / 80, and CD49b. In certain embodiments monocytes comprises one, two, or three of CD1 lb, CD14, or CD16. In certain embodiments, monocytes comprises CD14+CD16-monocytes, CD 14+CD 16+ monocytes, or CD14-CD16+ monocytes.

[0157] Macrophages are immune cells specialized for detection, phagocytosis, and destruction of target cells, such as pathogens or tumor cells. Macrophages are potent effectors of the innate immune system and are capable of at least three distinct anti-tumor functions: phagocytosis of dead and dying cells, microorganisms, cancer cells, cellular debris, or other foreign substances; cytotoxicity against tumor cells; and presentation of tumor antigens to orchestrate an adaptive anti-tumor immune response.

[0158] In one embodiment, the cell is obtained from an individual having or diagnosed as having cancer.

[0159] A cell, for example a monocyte cell, can be engineered to comprise any of the CAR polypeptides described herein; or a nucleic acid encoding any of the CAR polypeptides described herein. In one embodiment, a CAR polypeptide described herein is comprised in a lentiviral vector. The lentiviral vector is used to express the CAR polypeptide in a cell using infection standard techniques.

[0160] Accordingly, one aspect provided herein provides an engineered monocyte expressing any of the CAR polypeptides, or nucleic acid sequence that encodes any of the CAR polypeptides disclosed herein.

[0161] Retroviruses, such as lentiviruses, provide a convenient platform for delivery of nucleic acid sequences encoding a gene, or chimeric gene of interest. A selected nucleic acid sequence can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells, e.g. in vitro or ex vivo. Retroviral systems are well known in the art and are described in, for example, U.S. Pat. No. 5,219,740; Kurth and Bannert (2010) “Retroviruses: Molecular Biology, Genomics and Pathogenesis" Calster Academic Press (ISBN:978-l-90455-55-4); and Hu and Pathak Pharmacological Reviews 2000 52:493-512; which are incorporated by reference herein in their entirety. Lentiviral system for efficient DNA delivery can be purchased from OriGene; Rockville, MD. In alternative embodiments, the CAR polypeptide of any of the CARs described herein are expressed in the mammalian cell via transfection or electroporation of an expression vector comprising nucleic acid encoding the CAR. Transfection or electroporation methods are known in the art.

[0162] Efficient expression of the CAR polypeptide of any of the CAR polypeptides described herein can be assessed using standard assays that detect the mRNA, DNA, or gene product of the nucleic acid encoding the CAR. For example, RT-PCR, FACS, northern blotting, western blotting, ELISA, or immunohistochemistry.

[0163] In one embodiment, the CAR polypeptide of any of the CAR polypeptides described herein is constitutively expressed. In one embodiment, the CAR polypeptide of any of the CAR polypeptides described herein is encoded by recombinant nucleic acid sequence.Methods of Treatment

[0164] One aspect of the invention described herein relates to a method to a method of treating cancer in a subject, the method comprising: engineering a cell to comprise any of the CAR polypeptides described herein on the cell surface; administering the engineered cell to the subject. In one embodiment, the cell is an immune cell. In one embodiment, the cell is a monocyte.

[0165] Another aspect of the invention described herein relates to a method of treating cancer in a subject, the method comprising administering a cell (e.g., a monocyte) comprising any of the CAR polypeptides described herein, or a nucleic acid encoding any of the CAR polypeptides described herein, such that the cell expresses the CAR polypeptide.

[0166] Another aspect provided herein provides a method of treating cancer in a subject, the method comprising engineering a monocyte to comprise a CAR polypeptide that targets a monocyte surface protein, and administering the engineered monocyte to the subject, wherein the engineered monocyte does not bind to or target a cancer cell.

[0167] Another aspect provided herein provides a method of treating cancer in a subject, the method comprising administering an monocyte engineered to comprise a CAR polypeptide that targets amonocyte surface protein to the subject, wherein the engineered monocyte does not bind to or target a cancer cell.

[0168] The data presented herein demonstrate that engineered monocytes can specifically target monocytes and macrophages within the tumor microenvironment (TME), effectively addressing challenges associated with CAR-T cell therapies in solid tumors. CAR-T cells have shown limited efficacy in solid tumors due to factors such as poor infiltration into tumor sites, the immunosuppressive nature of the TME, and the scarcity of tumor-specific antigens, leading to potential off-target effects and reduced therapeutic outcomes. In contrast, the engineered monocytes described herein exhibit selective targeting capabilities, focusing exclusively on monocytes and macrophages within the TME without affecting other cell types, including cancer cells themselves. This targeted approach minimizes off-target cytotoxicity and enhances the potential for modulating the TME to support anti-tumor immunity.

[0169] Accordingly, in one embodiment, any of the engineered monocytes disclosed herein do not target any other cell type. In one embodiment, any of the engineered monocytes disclosed herein do not target a cancer cell.

[0170] In one embodiment, the method further comprises, prior to administering, the step of diagnosing the subject as having cancer.

[0171] In one embodiment, the method further comprises, prior to administering, receiving results of a diagnostic assay that diagnoses the subject has cancer. Diagnostic assays for diagnosing cancer are well known in the art and can be performed by a skilled practitioner. Assays that may aid in a diagnosis of cancer include but are not limited to, blood screening, MRI scan, tissue biopsy, and bone marrow testing, and are known in the art for a given condition. A family history for a condition, or exposure to risk factors for a condition can also aid in determining if a subject is likely to have the condition or in making a diagnosis of the condition.

[0172] Methods and compositions described herein are mainly directed at the treatment of solid tumors. In one embodiment, the cancer is a solid tumor. As used herein, the term “tumor” refers to an abnormal growth of cells or tissues, e.g., of malignant type or benign type. In one embodiment, the solid tumor is a carcinoma, sarcoma, central nervous system tumor, germ cell tumor, neuroendocrine tumor, melanoma, or mesothelioma. Non-limiting examples of solid tumors include Adrenocortical Tumor, Alveolar Soft Part Sarcoma, Carcinoma, Chondrosarcoma, Colorectal Carcinoma, Desmoid Tumors, Desmoplastic Small Round Cell Tumor, Endocrine Tumors, Endodermal Sinus Tumor, Epithelioid Hemangioendothelioma, Ewing Sarcoma, Germ Cell Tumors (Solid Tumor), Giant Cell Tumor of Bone and Soft Tissue, Glioblastoma, Hepatoblastoma, Hepatocellular Carcinoma, Melanoma, Nephroma, Neuroblastoma, Non-Rhabdomyosarcoma Soft Tissue Sarcoma (NRSTS), Osteosarcoma, Paraspinal Sarcoma, Renal Cell Carcinoma, Retinoblastoma, Rhabdomyosarcoma, Synovial Sarcoma, and Wilms Tumor. Solid tumors can be found in bones, muscles, the brain, ororgans (i.e., breast cancer), and can be sarcomas or carinomas, where the technology described herein can overcome barriers to solid tumor treatment with CAR cells. It is contemplated that aspects of the technology described herein can be used to treat all types of solid tumor cancers, including cancers not listed in the instant specification.

[0173] In one embodiment, the cancer is any cancer having a TME comprising TREM2 -expressing cells, e.g., monocytes or macrophages. In one embodiment, the cancer is any cancer having a TME comprising TREM2 -expressing monocytes or macrophages.

[0174] In one embodiment, the cancer is breast cancer. Exemplary breast cancers include, but are not limited to, non-invasive (in situ) breast cancers, including ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (LCIS); invasive (infiltrating) breast cancers including invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC); subtypes of invasive breast cancer including medullary carcinoma, mucinous (colloid) carcinoma, Tubular carcinoma, papillary carcinoma, and cribriform carcinoma; inflammatory breast cancer (IBC), Paget’s disease of the breast; Triple-negative breast cancer (TNBC); HER2 -positive breast cancer; male breast cancer; and metaplastic breast cancer (MpBC).Administration

[0175] In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having cancer with a mammalian cell (e.g., a monocyte cell) comprising any of the CAR polypeptides described herein, or a nucleic acid encoding any of the CAR polypeptides described herein. As used herein, a “CAR cell as described herein” refers to a mammalian cell comprising any of the CAR polypeptides described herein, or a nucleic acid encoding any of the CAR polypeptides described herein. As used herein, a “condition” refers to a cancer, a plasma cell disease or disorder, or an autoimmune disease or disorder. Subjects having a condition can be identified by a physician using current methods of diagnosing the condition. Symptoms and / or complications of the condition, which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, fatigue, persistent infections, and persistent bleeding. Tests that may aid in a diagnosis of, e.g. the condition, but are not limited to, blood screening and bone marrow testing, and are known in the art for a given condition.

[0176] The compositions described herein can be administered to a subject having or diagnosed as having cancer. In some embodiments, the methods described herein comprise administering an effective amount of activated CAR cells described herein to a subject in order to alleviate a symptom of the condition. As used herein, "alleviating a symptom of the condition" is ameliorating any condition or symptom associated with the condition, i.e., cancer. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering thecompositions described herein to subjects are known to those of skill in the art. In one embodiment, the compositions described herein are administered systemically or locally. In a preferred embodiment, the compositions described herein are administered intravenously. In another embodiment, the compositions described herein are administered at the site of the tumor.

[0177] The term “effective amount" as used herein refers to the amount of CAR cells needed to alleviate at least one or more symptom of the cancer, and relates to a sufficient amount of the cell preparation or composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of activated CAR cells that is sufficient to provide a particular anticondition effect when administered to atypical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a condition), or reverse a symptom of the condition. Thus, it is not generally practicable to specify an exact “effective amount". However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0178] Effective amounts, toxicity, and therapeutic efficacy can be evaluated by standard pharmaceutical procedures in cell cultures or experimental animals. The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (z.e., the concentration of CAR cells, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for bone marrow testing, among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0179] In one aspect of the invention, the technology described herein relates to a pharmaceutical composition comprising activated CAR cells as described herein, and optionally a pharmaceutically acceptable carrier. The active ingredients of the pharmaceutical composition at a minimum comprise activated CAR cells as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist essentially of CAR cells as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of CAR cells as described herein. Pharmaceutically acceptable carriers for cell-based therapeutic formulation include saline and aqueous buffer solutions, Ringer's solution, and serum component, such as serum albumin, HDL and LDL. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein.

[0180] In some embodiments, the pharmaceutical composition comprising CAR cells as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, the components apart from the CAR cells themselves are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. Any of these can be added to the CAR cells preparation prior to administration.

[0181] Suitable vehicles that can be used to provide parenteral dosage forms of CAR cells as disclosed within are well known to those skilled in the art. Examples include, without limitation: saline solution; glucose solution; aqueous vehicles including but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.Dosage

[0182] " Unit dosage form" as the term is used herein refers to a dosage for suitable one administration. By way of example a unit dosage form can be an amount of therapeutic disposed in a delivery device, e.g., a syringe or intravenous drip bag. In one embodiment, a unit dosage form is administered in a single administration. In another, embodiment more than one unit dosage form can be administered simultaneously.

[0183] In some embodiments, the CAR cells described herein are administered as a monotherapy, i.e., another treatment for the condition is not concurrently administered to the subject.

[0184] A pharmaceutical composition comprising the cells described herein can generally be administered at a dosage of 104to 109cells / kg body weight, in some instances 105to 106cells / kg body weight, including all integer values within those ranges. If necessary, cell compositions can also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).

[0185] Modes of administration can include, for example intravenous (i.v.) injection or infusion. The compositions described herein can be administered to a patient transarterially, intratumorally, intranodally, or intramedullary. In some embodiments, the compositions of CAR cells may be injected directly into a tumor, lymph node, or site of infection. In one embodiment, the compositions described herein are administered into a body cavity or body fluid (e.g., ascites, pleural fluid, peritoneal fluid, or cerebrospinal fluid).

[0186] The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices.

[0187] In some embodiments, a single treatment regimen is required. In others, administration of one or more subsequent doses or treatment regimens can be performed. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. In some embodiments, no additional treatments are administered following the initial treatment.

[0188] The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to administer further cells, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosage should not be so large as to cause adverse side effects, such as cytokine release syndrome. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.Combinational therapy

[0189] The CAR cells described herein can be used in combination with other known agents and therapies, e.g., anti -cancer agents and therapies. In one embodiment, the subject is further administered an anti-cancer agent or therapy. Administered "in combination", as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery". In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partiallyadditive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered. The CAR cells described herein and the at least one additional agent or therapy can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the CAR-expressing cells described herein can be administered first, and the additional anti -cancer agent can be administered second, or the order of administration can be reversed. The CAR cell therapy and / or other agent or therapy, procedures or modalities can be administered during periods of active disorder, or during a period of remission or less active disease. The CAR cell therapy can be administered before another treatment, concurrently with the treatment, post-treatment, or during remission of the disorder.

[0190] When administered in combination, the CAR cells and the additional anti -cancer agent (e.g., second or third agent), or all, can be administered in an amount or dose that is higher, lower or the same as the amount or dosage of each agent used individually, e.g., as a monotherapy. In certain embodiments, the administered amount or dosage of the CAR cells, the additional agent (e.g., second or third agent), or all, is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually. In other embodiments, the amount or dosage of the CAR cells, the additional agent (e.g., second or third agent), or all, that results in a desired effect (e.g., treatment of cancer) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dosage of each agent individually required to achieve the same therapeutic effect. In further embodiments, the CAR cells described herein can be used in a treatment regimen in combination with surgery, chemotherapy, radiation, an mTOR pathway inhibitor, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, or a peptide vaccine, such as that described in Izumoto et al. 2008 J Neurosurg 108:963- 971.

[0191] In one embodiment, the CAR cells described herein can be used in combination with a checkpoint inhibitor. Exemplary checkpoint inhibitors include anti-PD-1 inhibitors (Nivolumab, MK- 3475, Pembrolizumas, Pidilizumab, AMP -224, AMP-514), anti-CTLA4 inhibitors (Ipilimumab and Tremelimumab), anti-PDLl inhibitors (Atezolizumab, Avelomab, MSB0010718C, MEDI4736, and MPDL3280A), anti-LAG3 inhibitors, and anti-TIM3 inhibitors.

[0192] In one embodiment, the CAR cells described herein can be used in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include an anthracy cline (e.g., doxorubicin (e.g., liposomal doxorubicin)), a vinca alkaloid (e.g., vinblastine, vincristine, vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), an immune cell antibody (e.g., alemtuzamab, gemtuzumab, rituximab, tositumomab), an antimetabolite (including, e.g., folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors (e.g., fludarabine)), an mTOR inhibitor, a TNFR glucocorticoidinduced TNFR related protein (GITR) agonist, a proteasome inhibitor (e.g., aclacinomycin A, gliotoxin or bortezomib), an immunomodulator such as thalidomide or a thalidomide derivative (e.g., lenalidomide). General chemotherapeutic agents considered for use in combination therapies include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5- deoxy-5 -fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC- Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5- fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine (difluorodeoxy citidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®),6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90 / MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®). Exemplary alkylating agents include, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes): uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide (Mitoxana®), melphalan (Alkeran®), Chlorambucil (Leukeran®), pipobroman (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, Temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and Dacarbazine (DTIC-Dome®). Additional exemplary alkylating agents include, without limitation, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® and Temodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®); Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran®); Altretamine (also known as hexamethylmelamine (HMM), Hexalen®); Carmustine (BiCNU®); Bendamustine (Treanda®); Busulfan (Busulfex® and Myleran®); Carboplatin (Paraplatin®); Lomustine (also known as CCNU, CeeNU®); Cisplatin (also known as CDDP, Platinol® and Platinol®-AQ); Chlorambucil (Leukeran®); Cyclophosphamide (Cytoxan® and Neosar®); Dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC-Dome®); Altretamine (also known ashexamethylmelamine (HMM), Hexalen®); Ifosfamide (Ifex®); Prednumustine; Procarbazine (Matulane®); Mechlorethamine (also known as nitrogen mustard, mustine and mechloroethamine hydrochloride, Mustargen®); Streptozocin (Zanosar®); Thiotepa (also known as thiophosphoamide, TESPA and TSPA, Thioplex®); Cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®); and Bendamustine HC1 (Treanda®). Exemplary mTOR inhibitors include, e.g., temsirolimus; ridaforolimus (formally known as deferolimus, (lR,2R,45)-4-[(2R)-2 [(lR,95,125,15R,16E,18R,19R,21R,235,24E,26E,28Z,305,325,35R)-l,18-dihydroxy-19,30- dimethoxy-15,17,21,23, 29,35- hexamethyl-2,3,10,14,20-pentaoxo-l l,36-dioxa-4- azatricyclo [30.3.1.04'9] hexatriaconta- 16,24,26,28-tetraen- 12-yl] propyl] -2 -methoxy cyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03 / 064383); everolimus (Afinitor® or RADOO1); rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3); emsirolimus, (5-{2,4-Bis[(35,)-3-methylmorpholin-4-yl]pyrido[2,3- (i]pyrimidin-7-yl}-2- methoxyphenyl)methanol (AZD8055); 2-Amino-8-[iraw5,-4-(2- hydroxyethoxy)cyclohexyl]-6- (6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-JJpyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and N2-[l,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-l-benzopyran-2- yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-a-aspartylL-serine- (SEQ ID NO: 84), inner salt (SF1126, CAS 936487-67-1), and XL765. Exemplary immunomodulators include, e.g., afutuzumab (available from Roche®); pegfdgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®); thalidomide (Thalomid®), actimid (CC4047); and IRX-2 (mixture of human cytokines including interleukin 1, interleukin 2, and interferon y, CAS 951209-71-5, available from IRX Therapeutics). Exemplary anthracyclines include, e.g., doxorubicin (Adriamycin® and Rubex®); bleomycin (lenoxane®); daunorubicin (dauorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, Cerubidine®); daunorubicin liposomal (daunorubicin citrate liposome, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®, Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin; and desacetylravidomycin. Exemplary vinca alkaloids include, e.g., vinorelbine tartrate (Navelbine®), Vincristine (Oncovin®), and Vindesine (Eldisine®)); vinblastine (also known as vinblastine sulfate, vincaleukoblastine and VLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®). Exemplary proteosome inhibitors include bortezomib (Velcade®); carfilzomib (PX- 171-007, (5)-4-Methyl-N-((5)-l-(((5)-4-methyl-l-((R)-2-methyloxiran-2-yl)-l -oxopentan-2- yl)amino)- l-oxo-3-phenylpropan-2-yl)-2-((5,)-2-(2-morpholinoacetamido)-4- phenylbutanamido)-pentanamide); marizomib (NPT0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O-Methyl-N- [(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O- methyl-N-[(llS')-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-l- (phenylmethyl)ethyl]- L-serinamide (ONX-0912).

[0193] One of skill in the art can readily identify a chemotherapeutic agent of use (e.g. see Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones &Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in Abeloff s Clinical Oncology, 2013 Elsevier; and Fischer D S (ed): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).

[0194] In an embodiment, CAR cells described herein are administered to a subject in combination with a second anti-cancer cellular therapy known in the art, e.g., a CAR-T therapy, a tumorinfiltrating lymphocyte (TIL) therapy, or a CAR-NK therapy.

[0195] In an embodiment, CAR cells described herein are administered to a subject in combination with a molecule that decreases the activity and / or level of a molecule targeting GITR and / or modulating GITR functions, a molecule that decreases the Treg cell population, an mTOR inhibitor, a GITR agonist, a kinase inhibitor, a non-receptor tyrosine kinase inhibitor, a CDK4 inhibitor, and / or a BTK inhibitor.Efficacy

[0196] The efficacy of CAR cells in, e.g. the treatment of cancer, or to induce a response as described herein (e.g. a reduction in tumor size) can be determined by the skilled clinician. However, a treatment is considered “effective treatment," as the term is used herein, if one or more of the signs or symptoms of a condition (i.e., cancer) described herein is altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and / or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate. Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g., by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.

[0197] Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and / or are described herein.

[0198] Treatment includes any treatment of a disease in an individual or an animal (some nonlimiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. It is well within the ability of one skilled in the art to monitor efficacy of administration and / or treatment by measuringany one of such parameters, or any combination of parameters. Efficacy of a given approach can be assessed in animal models of a condition described herein, for example treatment of breast cancer. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed.

[0199] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0200] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0201] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0202] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting

[0203] Some embodiments of the invention described herein can be defined according to any of the following numbered paragraphs:1. A chimeric antigen receptor (CAR) polypeptide comprising: a. an extracellular domain comprising at least a portion of an anti-triggering receptor expressed on myeloid cells 2 (TREM2) scFv; b. a leader sequence; c. a hinge domain; d. a transmembrane domain; e. a co-stimulatory domain; and f. an intracellular signaling domain.2. The CAR polypeptide of paragraph 1, wherein the leader sequences comprises a CD8 leader sequence.3. The CAR polypeptide of any of the preceding paragraphs, wherein the CD8 leader sequence comprises the sequence of SEQ ID NO: 1.4. The CAR polypeptide of any of the preceding paragraphs, wherein the anti-TREM2 antibody is a an anti-TREM2 scFv.5. The CAR polypeptide of any of the preceding paragraphs, wherein the anti-TREM2 scFv comprises the sequence of SEQ ID NO: 2.6. The CAR polypeptide of any of the preceding paragraphs, wherein the hinge domain comprises a CD8 hinge domain.7. The CAR polypeptide of any of the preceding paragraphs, wherein the CD8 hinge domain comprises the sequence of SEQ ID NO: 3.8. The CAR polypeptide of any of the preceding paragraphs, wherein the transmembrane domain comprises a CD8 transmembrane domain.9. The CAR polypeptide of any of the preceding paragraphs, wherein the CD8 transmembrane domain comprises the sequence of SEQ ID NO: 4.10. The CAR polypeptide of any of the preceding paragraphs, wherein the intracellular signaling domain comprises a CD3^ signaling domain.11. The CAR polypeptide of any of the preceding paragraphs, wherein the CD3^ intracellular signaling domain comprises the sequence of SEQ ID NO: 6.12. The CAR polypeptide of any of the preceding paragraphs, wherein the co-stimulatory domain is the intracellular domain of 4- IBB.13. The CAR polypeptide of any of the preceding paragraphs, wherein the intracellular domain of 4-1BB comprises the sequence of SEQ ID NO: 5.14. A chimeric antigen receptor (CAR) polypeptide comprising:a. an extracellular domain comprising at least a portion of an anti-triggering receptor expressed on myeloid cells 2 (TREM2) scFv; b. a CD8 leader sequence; c. a CD8 hinge domain; d. a CD 8 transmembrane domain; e . a intracellular domain of 4- 1 BB ; and f. a CD3 > signaling domain. A CAR polypeptide comprising at least 95% identity with a sequence selected from SEQ ID NO: 7. A CAR polypeptide comprising a sequence selected from SEQ ID NO: 7. A nucleic acid sequence encoding a CAR polypeptide of any of the preceding paragraphs. A mammalian cell comprising; a. a CAR polypeptide of any of the preceding paragraphs; or b. a nucleic acid encoding a CAR polypeptide of any of the preceding paragraphs. The cell of any of the preceding paragraphs, wherein the cell is a human cell. The cell of any of the preceding paragraphs, wherein the cell is a monocyte. The cell of any of the preceding paragraphs, wherein the monocyte is obtained from an individual. The cell of any of the preceding paragraphs, wherein the monocyte is derived from a stem cell selected from the group consisting of: an embryonic stem cell, a mesenchymal stem cell, a hematopoietic stem cell, and an induced pluripotent stem cell. A method of treating cancer in a subject, the method comprising: a. engineering a monocyte to comprise a CAR polypeptide of any of the preceding paragraphs on the macrophage surface; b. administering the engineered monocyte to the subject. A method of treating cancer in a subject, the method comprising administering a cell of any of any of the preceding paragraphs to the subject. The method of any of the preceding paragraphs, wherein the cancer is a solid tumor. The method of any of the preceding paragraphs, wherein the cancer is breast cancer. The method of any of the preceding paragraphs, wherein the subject is further administered an anti -cancer therapy. The method of any of the preceding paragraphs, further comprising, prior to administering, the step of diagnosing the subject as having cancer. The method of any of the preceding paragraphs, further comprising, prior to administering, receiving results of a diagnostic assay that diagnoses the subject has cancer. A method of treating cancer in a subject, the method comprising:a. engineering a monocyte to comprise a CAR polypeptide specifically targeting a monocyte cell surface protein, and b. administering the engineered monocyte to the subject, wherein the engineered monocyte does not bind to or target a cancer cell.31. A method of treating cancer in a subject, the method comprising administering a monocyte engineered to comprise a CAR polypeptide specifically targeting a monocyte cell surface protein to the subject, wherein the engineered monocyte does not bind to or target a cancer cell.32. A composition comprising the mammalian cells of any of the preceding paragraphs formulated for the treatment of cancer.33. The composition of any of the preceding paragraphs, further comprising a pharmaceutically acceptable carrier.EXAMPLESEXAMPLE 1

[0204] INTRODUCTION & BACKGROUND

[0205] Disclosed herein is a novel and innovative therapeutic that, for the first time, uses chimeric antigen receptor (CAR) expressing monocytes to directly target and eliminate suppressive tumor associated macrophages (TAMs) in solid tumors.

[0206] Solid tumors recruit TAMs, which can account for up to 50% of solid tumors.6,7TAMs promote angiogenesis, metastasis and tumor growth; and their presence is highly correlated with drug resistance.1,2,6 17The inventors’ work has shown that removal or conversion of TAMs to an anti-tumor phenotype enhances chemo- and immuno-therapy1,5, and establishes TAMs as targets for anti -cancer therapy; however clinically effective strategies to target TAMs are lacking.

[0207] CAR-expressing T cells have revolutionized treatment of hematological malignancies but have been ineffective in solid tumors due to poor ability to traffic to solid tumors and inability to survive the harsh immunosuppressive tumor microenvironment (TME) imposed, largely in part, by TAMs. In addition, there are only a handful of tumor antigens to make CARs against to target on the surface of cancer cells and tumors can escape from CAR therapy due to lack of homogenous antigen expression throughout. The technology disclosed herein overcome these limitations by utilizing CAR- expressing monocytes (CAR-M) to eliminate suppressive TAMs in the TME to enable T cell recruitment and activation leading to durable anti -cancer responses in solid tumors. CAR- Macrophages have been used to target HER2+ breast cancer notable clinical responses. Data disclosed herein shows for the first time a CAR-M product will be used to target its same lineage and is extraordinarily innovative (Fig. 1). CAR-M have great potential to infiltrate solid tumors and targeting suppressive TAMs hold immense pan-cancer therapeutic potential.

[0208] The inventors performed single cell RNA-sequencing on over 50 breast tumors encompassing all subtypes of breast cancer and identified TREM2 to be upregulated on the most suppressive subsets of TAMs defined by their lipid metabolic phenotype and PD-L1 expression, among others (Fig. 2 and 3). The inventors and others have demonstrated the highly suppressive function of TREM2+ lipid TAMs across most solid tumors.5 18-20TREM2 is highly expressed on TAMs in most human and murine solid tumors21. Non-depleting antagonistic antibodies or genetic deletion of Trem2 in mouse models significantly modifies the TME allowing for increased CD8+ T cell infiltration and synergy with anti-PDl treatment.21Depleting TREM2 antibodies also demonstrated significant reduction in tumor growth of orthotopic ovarian, breast, and colorectal models22justifying the initiation of a phase 1 clinical trial investigating anti-TREM2 for cancer therapy (NCT04691375, NCT06877533). Targeting TREM2+ TAMs holds great promise as anti-tumor TAMs are spared which can promote endogenous anti-tumor T cell immune responses.

[0209] As shown herein, TREM2+ TAMs are immune suppressive cells that promote tumorigenesis, in part, by limiting T cell infiltration and function, and can be eliminated using CAR-M, which will enable T cell infiltration and activation, leading to durable anti -cancer responses.

[0210] SIGNIFICANCE

[0211] The tumor microenvironment (TME), devoid of nutrients and oxygen, recruits TAMs that promote angiogenesis, metastasis and tumor growth. Our work has shown that removal or conversion of TAMs to an anti -tumor phenotype enhances chemo- and immuno-therapy and establishes TAMs as targets for anti-cancer therapy. While there is tremendous rationale to target TAMs, to date, TAM- targeting strategies have not yet induced durable anti-cancer responses in the clinic. This work has been overwhelmed with the success of reinvigorating T cells through immune checkpoint blockade (ICB). ICB is now a pillar of cancer care but despite the encouraging success of ICB for some cancers, the majority of patients with solid tumors do not yet benefit from ICB. Because ICB agents work by reinvigorating pre-existing T cells, it is less likely that these agents will be effective in tumors with low levels of T cell infiltrate. Other ways to induce cancer cell killing by T cells is through the use of CAR expressing T cells, which has revolutionized treatment of hematological malignancies. However, CAR T cells have been largely ineffective in solid tumors. CAR T cells have demonstrated poor ability to traffic to solid tumors and are subject to the harsh immunosuppressive tumor microenvironment imposed, in part, by tumor macrophages. In addition, there are only a handful of tumor antigens to target on the surface of cancer cells in solid tumors. This proposal overcomes these limitations by testing how CAR-expressing monocytes (CAR-Mo) can be utilized to eliminate suppressive TAMs in the TME to enable T cell recruitment and activation leading to durable anti -cancer responses. The clinical path for this work is established as tumor targeted CAR- Macrophages have entered the clinic with notable responses. This work proposed here goes well beyond what has been previously tested and even considered for CAR therapy. This is the first time aCAR-M product has been used to target its same lineage and is extraordinarily innovative. The inventors have established the CAR-M product and demonstrated anti-tumor responses in mice. Selectively targeting suppressive TAMs hold immense pan-cancer therapeutic potential. These data will contribute to revolutionizing the field of cancer immunotherapy by generating a new paradigm for the treatment of cancer through selective targeting of TAMs.

[0212] RESULTS

[0213] Human and murine TREM2- CAR-Mo were generated that robustly induce killing / phagocytosis of target TREM2+ TAMs ex vivo (Fig. 7-9). Human TREM2 -CAR-M were transfected to express GFP and were cultured with TREM2+ TAMs (m Cherry positive). Phagocytosis was measured by identifying GFP+ effector cells having mCherry expression by flow cytometry. A mesothelin (MSLN) CAR-M was used as a negative control and demonstrated minimal ability to phagocytose TREM2+ TAMs. Similar data was obtained for murine TREM2-CAR-M (Figure 9). Murine TREM2-CAR-M were assessed in murine models of breast cancer for anti -tumor activity. Immune-competent mice are fully implanted with EO771 TNBC tumors. When tumors reached 200mm3they were enrolled into treatment groups. Mice were treated IV every 3 days with le6 untransduced monocytes (UND) or TREM2-CAR-Mo (CAR). (Figs 10A-10C). These data indicated that the TREM2-CAR-M have single agent activity against EO771 murine breast tumors.

[0214] TREM2 CAR-Mo synergy with anti-PDl in vivo. 3e6 CAR+ TREM2- / - ER- HoxB8 cells (CAR) or Untransduced ER-Hoxb8 cells (UTD) were differentiated for 3 days with 30ng / mL M-CSF to monocytes and injected intravenously 3x / week for 2 weeks into EO771 orthotopic-breast tumor bearing mice. 200ug of anti-PDl was administered intraperitoneally on days 2,5, &8 after enrollment. Data demonstrates that combination therapy significantly extends survival and inhibits tumor growth compared to TREM2 CAR- M monotherapy (Figure 11 A-D).

[0215] Increased number of CAR-M cells injected achieves significant inhibition of breast tumor growth. The inventors sough to determine if increased number of TREM2 CAR-Monocytes inhibited breast tumor growth relative to a matched number of untransduced monocytes. 5e6 CAR+ TREM2- / - ER-HoxB8 cells (CAR) or Untransduced ER-Hoxb8 cells (UTD) were differentiated for 3 days with 30ng / mL M-CSF and injected intravenously 3x / week for 1 week into EO771 orthotopic-breast tumor bearing mice. The volume of tumors was measured daily by calipers and shown as cubic millimeters. Data demonstrated that an increased number of TREM2 CAR monocytes significantly inhibits breast tumor growth after 12 days of treatment compared to untransduced monocytes.

[0216] TREM2 CAR-M depletes endogenous TREM2+ TAMs and induce an endogenous T cell response. It is specifically contemplated herein that TREM2 CAR-M will selectively eliminateTREM2+ TAMs which will lessen the immune-suppressive TME allowing T cell infiltration and activation of CD8+ T cells that will mediate durable anti-cancer responses. C57B1 / 6 mice are inoculated with EO771 breast tumor cells. When tumor reach 100-200mm3they are treated with le6 TREM2 CAR-M or Mesothelin-CAR-M (tumor cells are Mesothelin negative, this is used as a negative control) every 3 days (n=8 mice / group). On day 7, non-draining and tumor draining lymph nodes, spleens and tumors are harvested for immune-phenotyping by spectral flow cytometry and spatial analysis by immunohistochemistry and multi-plex, single cell cyclic immunofluorescence (CyCIF) which enables repeated cycles of immunofluorescence staining and imaging (typically 30-40 plex).23-25TREM2+ TAM depletion and T cell phenotyping are assessed along with other immune subsets. Macrophage subsets include, but are not limited to, Viable, CD45+ myeloid (CD1 lb); dendritic cell (CD11c and subsets of CD103) subsets: macrophage (F480); anti-tumor markers (CD80, CD86, CD40, pTBKl, IRF3, MHCII; pSTATl); protumor markers (CD206, PD-L1, CSF-1R); neutrophil (Grl+); Lipid TAMs (CD36, TREM2, SPP1); MDSCs (CD45+CDl lb+Grl+); PMN- MDSCs (CD45+CD1 lb+Ly6G+Ly6C-); M-MDSCs (CD45+CD1 lb+Ly6G-Ly6C+); NK (NKp46); B cell (CD19, CD20) endothelial (CD31); and fibroblast (Thyl). T cell subsets include, but are not limited to, Viable, CD45+CD3+CDl lb- subsets of T cells: Treg (CD4+CD24+Foxp3+), Thl7 (CD4+Rorgt+Foxp3-), Thl (CD4+Tbet+Foxp3-), Th2 (CD4+GATA3+Foxp3-), Tem (CD8+CCR71o+CD62Llo+CD271o+CD127hi+), Tcm: (CD8+CCR7hi+CD62Lhi+CD27hi+CD127hi+). Absolute number of cells in organs, phenotype and proportions are calculated. CyCIF image processing will be performed with MCMICRO26(in-house, open-source image analysis pipeline for segmentation and quantification). Spatial proximity, neighborhood analysis, and cell interactions will be assessed focusing on relationships of T cells and TREM2+ TAMs.

[0217] TREM2 CAR-M enhances chemo- and immuno-therapy. It is specifically contemplated herein that targeting TREM2+ TAMs with CAR-M will synergize with standard therapies leading to durable anti-tumor responses. Figs 11A-1 ID demonstrated TREM2-CAR-Mo enhance anti-PD-1 therapy. Chemo- and immuno-therapy are added to the treatment strategy in long-term efficacy experiments to enhance this response. C57B1 / 6 mice are inoculated with EO771 breast tumor cells as described above. Tumors are measured every 3 days and OS will be recorded. Cured mice are rechallenged with tumor cells to test memory T cell functions. Efficacy experiments are repeated twice. Combinations of chemo- or immuno-therapy with TREM2 CAR-M that enhance OS compared to monotherapy are profiled as disclosed herein above to determine mechanistic basis for response. Drug combinations with Mesothelin-CAR-M are used as negative controls to confirm TREM2 dependence of response. Anti-CD8 IgG is used to deplete T cells to confirm CD8+ T cell dependence.

[0218] Clinical grade TREM2 CAR-M cell product. It is specifically contemplated herein that TREM2 CAR-M therapeutics will be most effective clinically as an induced pluripotent stem cell(iPSC) or hematopoietic stem cell (HSC) derived product. Unlike T cell CAR products, primary myeloid cells are highly resistant to manipulation and are lowly proliferative, leading to issues with scaling. However, iPSC are highly amenable to manipulation and scaling and HSC / iPSC-derived CAR-M products have been demonstrated to display robust efficacy in pre-clinical xenograft models.27Human TREM2 CAR-M are developed using standard techniques know in the are. The efficacy of the product is assessed using TREM2+ acute myeloid leukemia xenograft models in vivo, solid tumor models in humanized mice, and patient-derived TREM2+ TAM phagocytosis assays ex vivo.References for Example 11 Guerriero, J. L., Sotayo, A., Ponichtera, H. E., Castrillon, J. A., Pourzia, A. L., Schad, S., Johnson, S. F., Carrasco, R. 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D., Marks, A., Belabed, M., Hamon, P, Chin, T, Troncoso, L., Lee, J. J., Ahimovic, D., Bale, M., Chung, G., D’souza, D., Angeliadis, K., Dawson, T, Kim-Schulze, S., Flores, R. M., Kaufman, A. J., Ginhoux, F., Josefowicz, S. Z., Ma, S., Tsankov, A. M., Marron, T. U., Brown,B. D. & Merad, M. Myeloid progenitor dysregulation fuels immunosuppressive macrophages in tumors. bioRxiv, 2024.2006.2024.600383, doi: 10. 1101 / 2024.06.24.600383 (2024).20 Yofe, I., Shami, T, Cohen, N., Landsberger, T, Sheban, F., Stoler-Barak, L., Yalin, A., Phan, T. S., Li, B., Monteran, L., Scharff, Y, Giladi, A., Elbaz, M., David, E., Gurevich-Shapiro, A., Gur,C., Shulman, Z., Erez, N. & Amit, I. Spatial and Temporal Mapping of Breast Cancer Lung Metastases Identify TREM2 Macrophages as Regulators of the Metastatic Boundary. Cancer Discov 13, 2610-2631, doi: 10.1158 / 2159-8290. CD-23-0299 (2023).21 Molgora, M., Esaulova, E., Vermi, W., Hou, J., Chen, Y, Luo, J., Brioschi, S., Bugatti, M., Omodei, A. S., Ricci, B., Fronick, C., Panda, S. K., Takeuchi, Y, Gubin, M. M., Faccio, R., Celia, M., Gilfdlan, S., Unanue, E. R., Artyomov, M. N., Schreiber, R. D. & Colonna, M. TREM2 Modulation Remodels the Tumor Myeloid Landscape Enhancing Anti -PD-1 Immunotherapy. Cell 182, 886-900 e817, doi:10.1016 / j.cell.2020.07.013 (2020). PMC7485282.22 Binnewies, M., Pollack, J. L., Rudolph, J., Dash, S., Abushawish, M., Lee, T, Jahchan, N. S., Canaday, P., Lu, E., Nomg, M., Mankikar, S., Liu, V. M., Du, X., Chen, A., Mehta, R., Palmer, R., Juric, V., Liang, L., Baker, K. P., Reyno, L., Krummel, M. F., Streuli, M. & Sriram, V. Targeting TREM2 on tumor-associated macrophages enhances immunotherapy. Cell Rep 37, 109844, doi: 10.1016 / j.celrep.202L 109844 (2021).23 Lin, J. R., Izar, B., Wang, S., Yapp, C., Mei, S., Shah, P. M., Santagata, S. & Sorger, P. K. Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes. eLife 7, doi: 10.7554 / eLife.31657 (2018). PMC6075866.24 Lin, J. R., Fallahi-Sichani, M., Chen, J. Y. & Sorger, P. K. Cyclic Immunofluorescence (CycIF), A Highly Multiplexed Method for Single-cell Imaging. Curr Protoc Chem Biol 8, 251-264, doi: 10.1002 / cpch,14 (2016). PMC5233430.25 Lin, J. R., Fallahi-Sichani, M. & Sorger, P. K. Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method. Nat Commun 6, 8390, doi: 10.1038 / ncomms9390 (2015). PMC4587398.26 Schapiro, D., Sokolov, A., Yapp, C., Chen, Y. A., Muhlich, J. L., Hess, J., Creason, A. L., Nirmal, A. J., Baker, G. J., Nariya, M. K., Lin, J. R., Maliga, Z., Jacobson, C. A., Hodgman, M. W., Ruokonen, J., Farhi, S. L., Abbondanza, D., McKinley, E. T, Persson, D., Betts, C., Sivagnanam, S., Regev, A., Goecks, J., Coffey, R. J., Coussens, L. M., Santagata, S. & Sorger, P. K. MCMICRO: a scalable, modular image-processing pipeline for multiplexed tissue imaging. Nat Methods 19, 311- 315, doi: I0.1038 / s41592-021-01308-y (2022). PMC8916956.27 Shah, Z., Tian, L., Li, Z., Jin, L., Zhang, J., Li, Z., Barr, T, Tang, H., Feng, M., Caligiuri, M. A. & Yu, J. Human anti-PSCA CAR macrophages possess potent antitumor activity against pancreatic cancer. Cell Stem Cell 31, 803-817.e806, doi: 10.1016 / j.stem.2024.03.018 (2024). PMC11162318.EXAMPLE 2

[0219] Background

[0220] TAMs are a heterogeneous population of cells whose phenotype is regulated by the TME.27To identify potential CAR targets on immunosuppressive TAM populations, the inventor analyzed scRNASeq data from 55 treatment-naive human breast tumors, that was inclusive of all BC subtypes. They identified 9 distinct clusters of BC TAMs and revealed TREM2 to be highly expressed in multiple clusters. TREM2+ TAMs have been recently described in solid tumor types to be immunosuppressive and enable resistance to ICB.28 30TCGA analysis of TREM2 expression in breast tumors revealed a significantly lower survival rate in high TREM2 -expressing. TREM2 targeting therapies have demonstrated tremendous pre-clinical efficacy28-30, but have not been fully evaluated in BC despite the abundance of TREM2+ TAMs in murine and human BC.31Data provided assess whether TREM2+ TAMs limit T cell infiltration and function in the breast TME and can be therapeutically targeted with CAR-Mo to enhance response to ICB; monocytes expressing TREM2 CAR (TREM2 CAR-Mo) eliminates suppressive TREM2+ TAMs and enables T cell recruitment, activation, and subsequent response to ICB in BC.

[0221] Methods

[0222] CAR-Mo: To test the effects of TREM2 CAR-Mo on the endogenous immune response, inventors generated a murine primary cell line capable of generating TREM2 CAR-Mo reproducibly, at scale. This was achieved by isolating myeloid progenitors derived from TREM2- / - (C57B1 / 6) and Balb / c (TREM2 CRISPR KO) bone marrow and stably maintaining multipotency using a conditional ER-Hoxb8 retroviral method that has been widely utilized.32They generated retroviruses encoding a CAR construct utilizing a murine scFv Trem2 sequence. The TREM2 CAR was transduced into ER- Hoxb8 myeloid progenitors to create a stable source of TREM2 CAR-Mo progenitors, overcoming asignificant hurdle of low viral transduction efficiency in differentiated primary myeloid cells. TREM2 CAR progenitors were differentiated into CAR-Mo using M-CSF on ultra-low attachment plates for 3 days (in vivo) or into CAR-Mac for 6 days (in vitro', Figs 8 and 9). Additionally, inventors generated human TREM2 CAR-Mo by generating a CAR-containing lentivirus utilizing a human TREM2 scFv sequence.33The inventors transduced the human CAR into U-937 monocytic leukemia cells to create a TREM2 CAR-Mo cell line, which can be differentiated with PMA for 24 hours into human TREM2 CAR-Mac (Figs 8 and 9). Using this same approach, human MSLN CAR-Mo were also generated in U-937 and murine MSUN CAR-Mo inER-Hoxb8 myeloid progenitors as non-targeting CAR controls.

[0223] Murine tumor models: Two syngeneic orthotopic models of BC, EO771 and 4T1 were used, in which Luciferase was expressed to enable imaging of primary and metastatic tumors. Inventors evaluated 4T1, EO771, and EMT6 orthotopic primary breast tumors by FACS for the presence of TREM2+ TAMs and found EO771 and 4T1 tumors to have the largest and fewest abundance of TREM2+ TAMs respectively. Syngeneic tumors are implanted orthotopically with le5cells into the 4thmammary fat pad. When tumors reach ~200mm3,mice will be randomly enrolled into treatment groups and treated intravenously twice weekly with le6CAR-Mo or Untransduced-Mo.

[0224] Cyclic Immunofluorescence (CyCIF) analysis'. Formalin-fixed and paraffin embedded (FFPE) murine tumor sections are stained and imaged at the Harvard Laboratory of Systems Pharmacology (LSP) using the Rarecyte Orion.18Lymphocyte lineages are identified with: CD3, CD8, CD4, CD19, FoxP3, and NKp46. Myeloid lineages will be identified with: CD1 lb, F4 / 80, Ly6G, Ly6C, MHC II, and CD11c. Additional lineages and cell states will include but are not limited to using pan- cytokeratin, CD31, aSMA, TREM2, PD-1, PD-L1, Cleaved-Caspase 3, and Granzyme B. Data analysis is performed using the in-house MCMICRO pipeline built at HMS.34

[0225] Flow cytometry. Tumor immune infiltrate is profiled using spectral flow cytometry at the HMS Flow Cytometry Core. Lymphocyte and myeloid cell lineages are analyzed using the same markers as CyCIF, and additional antibodies disclosed herein.

[0226] Statistics & Rigor. Two-tailed t-tests are used to compare TREM2 CAR vs control and Two- way ANOVA with a two-sided a = 0.05 for experiments involving combination therapy, followed by multiple comparisons testing. Statistical differences between murine growth curves will utilize nonparametric Mann-Whitney-Wilcoxon testing. Administered treatments are blinded in all experiments. Breast cancer occurs predominantly in the female sex (99% of all cases); therefore, all studies are performed using female mice. All experiments are repeated to ensure reproducibility and will be conducted using at least 2 cell lines to alleviate cell line specific conclusions.

[0227] TREM2 CAR-Mo activates an endogenous T cell response and response to ICB. Murine TAM-targeting (F4 / 80 or FR[3) CAR-T cells have been shown to indirectly reduce tumor burden through elimination of TAMs, which correlated with the activation of endogenous tumor-reactive T cells in multiple solid tumor types.35,36The inventors murine TREM2 CAR-Mo also significantlydelays tumor growth, however, the mechanism by which TREM2 CAR-Mo reduces tumor burden is not known (Figs 10A-10C). It is specifically contemplated herein that TREM2 CAR-Mo elimination of suppressive TREM2+ TAMs lessens the immune-suppressive BC TME allowing increased T cell infiltration, activation, and subsequent synergy with ICB (Fig. 1). The inventors novel murine TREM2 CAR-Mo is used to test for synergy in mouse models of BC. Efficacy, survival, and T cell phenotyping by flow cytometry and scRNAseq are performed.

[0228] To assess CAR-Mo activation directly on T cell phenotype, co-culture and conditioned media (CM) transfer assays are used in vitro (Fig. 13). Murine TREM2 CAR-Mo are differentiated into macrophages and co-cultured with the murine macrophage line RAW264.7, which expresses high levels of endogenous TREM2 and not MSLN. Controls include differentiated MSLN CAR-Mo and CM from RAW264.7 cells cultured alone. 24 hours after co-culture, the CM are collected and transferred to naive splenic T cells in the presence of aCD3 + aCD28 for 48 hours. CM are analyzed by ELISA for IL- ip, IL-6, IL- 12, TNFa, and IL-18. T cells are analyzed via flow cytometry for CD4, CD8, CFSE dilution, IFNy, TNFa, and Granzyme B. These experiments are recapitulated with human TREM2 CAR-Mo (U-937), PMA-differentiated THP-1 macrophages, and T cells isolated from donors.

[0229] EO771 and 4T1 tumors are established, and CAR-Mo are administered as disclosed herein in Methods. Tumors are dissociated, and T cells are quantified and profiled for markers of activation by spectral flow cytometry (Methods; n=8 / group). T cells from tumors with or without TREM2 CAR-Mo therapy are further analyzed by scRNAseq (n=3 / group). T cells are isolated from tumors by FACS and sorted into individual wells for scRNASeq by the DFCI Center for Cancer Genomics. Single cell data are analyzed using the Seurat R package. TCR sequencing analysis is used determine the clonality of intratumoral T cells induced by CAR-Mo.

[0230] TREM2 CAR-Mo are administered to mice bearing EO771 and 4T1 orthotopic tumors. EO771 tumors display sensitivity to aPD-1 monotherapy, whereas 4T1 tumors do not.37,38Tumors are established, and CAR-Mo are administered as in Methods herein able (n=10 / group). 0.2mg / kg aPD-1 or control IgG will be given intraperitoneally 2x / week beginning at enrollment for 4 total doses. Overall survival and tumor burden are assessed.

[0231] TREM2 CAR-Mo transforms the molecular and spatial context of the metastatic TME. TREM2+ TAMs localize to the margins of human and murine lung metastases derived from primary breast tumors and correlate with the exclusion of T cells from the metastatic core26,39,40. Furthermore, murine TREM2+ metastatic breast TAMs significantly inhibit T cell activation ex vivo.26Therapeutic targeting of metastatic TREM2+ TAMs from primary breast tumors has not been previously tested and proves to be highly beneficial to enabling T cell infiltration and subsequent ICB efficacy in metastatic disease. It is specifically contemplated herein that depletion of metastatic TREM2+ TAMs via TREM2 CAR-Mo leads to a significant remodeling of the metastatic microenvironment that willincrease T cell infiltration and enhance overall survival (Fig. 1). TREM2 CAR-Mo are assessed in murine lung metastases generated from primary breast tumors. Spatial immunophenotyping by CyCIF, spatial transcriptomics, spectral flow cytometry, and survival analysis are performed to test my hypothesis.

[0232] 4T1 and EO771 BC lines have demonstrated have spontaneous metastases to the lung.26-39Mice are injected with le5Luciferase-expressing EO771 or 4T1 cells (n=10 per group). On day 14 primary breast tumors are resected. Lung metastatic outgrowth is confirmed by weekly bioluminescent imaging (BLI) and le6TREM2 CAR-Mo are injected IV (Figs 11A-11D). Experiments are repeated in combination with 0.2mg / kg aPD-1 or IgG twice weekly to test synergy.

[0233] Metastases is generated, and CAR-Mo are administered disclosed herein above (n=10 / group, CAR-Mo and untreated). Two-weeks post CAR-Mo administration, lungs are obtained and split in half for FFPE whole tissue analysis and for flow cytometry analysis. FFPE sections are subject to IHC for CD3, and individual metastases are analyzed for # of CD3+ cells / mm3. Phenotype and absolute number of T cells in the lung are analyzed by flow cytometry (Methods).

[0234] Metastases is generated, and CAR-Mo are administered disclosed herein above (n=10 / group, CAR-Mo and untreated). Lungs are harvested at 1- and 2-weeks post CAR-Mo and used for spatial tissue analysis using both antibody-based CyCIF and transcript-based spatial transcriptomics (Visium HD). FFPE sections are subject to CyCIF imaging (Methods) to identify both the frequency and localization of cell types. Spatial transcriptomics data are analyzed for hallmark genes that define unique cell populations, signaling pathways and differentially expressed genes between CAR-Mo and control lung cell populations or within treatment groups across timepoints. These data reveal spatial changes within the metastases after CAR-Mo treatment, providing novel insights into the spatial relationship and potential signaling pathways of suppressive TAMs and T cell populations in metastatic lesions.

[0235] Experiments disclosed herein define how TREM2 CAR-Mo modulates both the primary and metastatic BC TME and subsequently affects the efficacy of ICB.References for Example 21 Curiel, T. J. et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 10, 942-949, doi: 10.1038 / nml093 (2004).2 Condeelis, J. & Pollard, J. W. Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell 124, 263-266, doi: 10.1016 / j .cell.2006.01.007 (2006).3 DeNardo, D. G. et al. Leukocyte complexity predicts breast cancer survival and functionally regulates response to chemotherapy. Cancer Di scov 1, 54-67, doi: 10.1158 / 2159-8274. CD-10-0028 (2011).4 DeNardo, D. G. & Coussens, L. M. Inflammation and breast cancer. Balancing immune response: crosstalk between adaptive and innate immune cells during breast cancer progression. 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Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol 38, 947-953, doi: 10.1038 / s41587-020-0462-y (2020).20 Greiner, D. et al. CSPG4-targeting CAR-macrophages inhibit melanoma growth. bioRxiv, doi : 10. 1101 / 2024.06.04.597413 (2024) .21 Guan, L. et al. GPC3-targeted CAR-M cells exhibit potent antitumor activity against hepatocellular carcinoma. Biochem Biophys Rep 39, 101741, doi: 10.1016 / j.bbrep.2024.101741 (2024).22 Shah, Z. et al. Human anti-PSCA CAR macrophages possess potent antitumor activity against pancreatic cancer. Cell Stem Cell 31, 803-817 e806, doi: 10.1016 / j.stem.2024.03.018 (2024).23 Shen, J. et al. Activating innate immune responses repolarizes hPSC-derived CAR macrophages to improve anti-tumor activity. Cell Stem Cell 31, 1003-1019 el009, doi : 10. 1016 / j .stem .2024.04.012 (2024) .24 Pierini, S. et al. Chimeric antigen receptor macrophages (CAR-M) sensitize HER2+ solid tumors to PD1 blockade in pre-clinical models. Nat Commun 16, 706, doi: 10.1038 / s41467-024- 55770-1 (2025).25 Reiss, K. A. et al. CAR-macrophage therapy for HER2 -overexpressing advanced solid tumors: a phase 1 trial. Nat Med, doi: 10.1038 / s41591-025-03495-z (2025).26 Yofe, I. et al. Spatial and temporal mapping of breast cancer lung metastases identify TREM2 macrophages as regulators of the metastatic boundary. Cancer Discov. doi: 10.1158 / 2159-8290. CD- 23-0299 (2023).27 DeNardo, D. G. & Ruffell, B. Macrophages as regulators of tumour immunity and immunotherapy. Nat Rev Immunol 19, 369-382, doi: 10.1038 / s41577-019-0127-6 (2019).28 Park, M. D. et al. TREM2 macrophages drive NK cell paucity and dysfunction in lung cancer. Nat Immunol 24, 792-801, doi: 10.1038 / s41590-023-01475-4 (2023).29 Molgora, M. et al. TREM2 Modulation Remodels the Tumor Myeloid Landscape Enhancing Anti-PD-1 Immunotherapy. Cell 182, 886-900 e817, doi: 10.1016 / j.cell.2020.07.013 (2020).30 Binnewies, M. et al. Targeting TREM2 on tumor-associated macrophages enhances immunotherapy. Cell Reports 37, doi: 10.1016 / j.celrep.2021. 109844 (2021).31 Nalio Ramos, R. et al. Tissue-resident FOLR2(+) macrophages associate with CD8(+) T cell infiltration in human breast cancer. Cell 185, 1189-1207 ell25, doi: 10.1016 / j .cell.2022.02.021 (2022).32 Wang, G. G. et al. Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8. Nature Methods 3, 287-293, doi: 10.1038 / nmeth865 (2006).33 Szykowska, A. et al. Selection and structural characterization of anti-TREM2 scFvs that reduce levels of shed ectodomain. Structure 29, 1241-1252 el245, doi: 10. 1016 / j. str.2021.06.010 (2021).34 Schapiro, D. et al. MCMICRO: a scalable, modular image-processing pipeline for multiplexed tissue imaging. Nature Methods 19, 311-315, doi: 10.1038 / s41592-021-01308-y (2021).35 Sanchez-Paulete, A. R. et al. 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Claims

What is claimed herein is:

1. A chimeric antigen receptor (CAR) polypeptide comprising: g. an extracellular domain comprising at least a portion of an anti -triggering receptor expressed on myeloid cells 2 (TREM2) scFv; h. a leader sequence; i. a hinge domain; j . a transmembrane domain; k. a co-stimulatory domain; and l. an intracellular signaling domain.

2. The CAR polypeptide of claim 1, wherein the leader sequences comprises a CD8 leader sequence.

3. The CAR polypeptide of any of claims 1-2, wherein the CD8 leader sequence comprises the sequence of SEQ ID NO: 1.

4. The CAR polypeptide of any of claims 1-3, wherein the anti-TREM2 antibody is a an anti- TREM2 scFv.

5. The CAR polypeptide of any of claims 1-4, wherein the anti-TREM2 scFv comprises the sequence of SEQ ID NO: 2.

6. The CAR polypeptide of any of claims 1-5, wherein the hinge domain comprises a CD8 hinge domain.

7. The CAR polypeptide of any of claims 1-6, wherein the CD8 hinge domain comprises the sequence of SEQ ID NO: 3.

8. The CAR polypeptide of any of claims 1-7, wherein the transmembrane domain comprises a CD 8 transmembrane domain.

9. The CAR polypeptide of any of claims 1-8, wherein the CD8 transmembrane domain comprises the sequence of SEQ ID NO: 4.

10. The CAR polypeptide of any of claims 1-9, wherein the intracellular signaling domain comprises a CD3^ signaling domain.

11. The CAR polypeptide of any of claims 1-10, wherein the CD3^ intracellular signaling domain comprises the sequence of SEQ ID NO: 6.

12. The CAR polypeptide of any of claims 1-11, wherein the co-stimulatory domain is the intracellular domain of 4-1BB.

13. The CAR polypeptide of any of claims 1-12, wherein the intracellular domain of 4- IBB comprises the sequence of SEQ ID NO: 5.

14. A chimeric antigen receptor (CAR) polypeptide comprising:g. an extracellular domain comprising at least a portion of an anti -triggering receptor expressed on myeloid cells 2 (TREM2) scFv; h. a CD8 leader sequence; i. a CD8 hinge domain; j . a CD 8 transmembrane domain; k. a intracellular domain of 4-1BB; and l. a CD3^ signaling domain.

15. A CAR polypeptide comprising at least 95% identity with a sequence selected from SEQ ID NO: 7.

16. A CAR polypeptide comprising a sequence selected from SEQ ID NO: 7.

17. A nucleic acid sequence encoding a CAR polypeptide of any of claims 1-16.

18. A mammalian cell comprising; c. a CAR polypeptide of any of claims 1-16; or d. a nucleic acid encoding a CAR polypeptide of claim 17.

19. The cell of claim 18, wherein the cell is a human cell.

20. The cell of any of claims 18-19, wherein the cell is a monocyte.

21. The cell of claim 20, wherein the monocyte is obtained from an individual.

22. The cell of claim 20, wherein the monocyte is derived from a stem cell selected from the group consisting of: an embryonic stem cell, a mesenchymal stem cell, a hematopoietic stem cell, and an induced pluripotent stem cell.

23. A method of treating cancer in a subject, the method comprising: c. engineering a monocyte to comprise a CAR polypeptide of any of claims 1-16 on the macrophage surface; d. administering the engineered monocyte to the subject.

24. A method of treating cancer in a subject, the method comprising administering a cell of any of claims 18-22 to the subject.

25. The method of any of claims 23-24, wherein the cancer is a solid tumor.

26. The method of any of claims 23-25, wherein the cancer is breast cancer.

27. The method of any of claims 23-26, wherein the subject is further administered an anti -cancer therapy.

28. The method of any of claims 23-27, further comprising, prior to administering, the step of diagnosing the subject as having cancer.

29. The method of any of claims 23-28, further comprising, prior to administering, receiving results of a diagnostic assay that diagnoses the subject has cancer.

30. A method of treating cancer in a subject, the method comprising:a. engineering a monocyte to comprise a CAR polypeptide specifically targeting a monocyte cell surface protein, and b. administering the engineered monocyte to the subject, wherein the engineered monocyte does not bind to or target a cancer cell.

31. A method of treating cancer in a subject, the method comprising administering a monocyte engineered to comprise a CAR polypeptide specifically targeting a monocyte cell surface protein to the subject, wherein the engineered monocyte does not bind to or target a cancer cell.

32. A composition comprising the mammalian cells of claims 18-22 formulated for the treatment of cancer.

33. The composition of claim 32, further comprising a pharmaceutically acceptable carrier.

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