Chimeric antigen receptor FC-engineered granulocyte-monocyte progenitors for enhanced cancer immunotherapy

Abstract

Provided herein are chimeric antigen receptors, comprising an extracellular domain capable of binding to an antigen, an Fc region, a flexible linker, a transmembrane domain, and may further comprise at least one intracellular domain that is designed to increase the anti-tumor activities of granulocytes, macrophages, and dendritic cells by increasing their phagocytosis and / or proinflammatory cytokines secretion and / or antigen presentation. Provided herein are vectors and nucleic acid molecules encoding any of the chimeric antigen receptors described herein. Provided herein are methods to genetically engineer granulocyte-macrophage progenitors (GMPs) to express the chimeric antigen receptors described herein. The CAR-Fc-GMPs may be induced to differentiate into macrophages or granulocytes. Provided herein are macrophages and granulocytes that express a CAR-Fc prepared by any of the methods described herein. Provided herein is an immunotherapy method for treating a subject having cancer with GMPs or macrophages or granulocytes that express the chimeric antigen receptors described herein.

Description

CHIMERIC ANTIGEN RECEPTOR FC-ENGINEERED GRANULOCYTE-MONOCYTE PROGENITORS FOR ENHANCED CANCER IMMUNOTHERAPYREFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of United States provisional application number 63 / 735,421, filed December 18, 2024, the entire contents of which are incorporated herein by reference.REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on December 16, 2025, is named USCAP0024WO.xml and is 37,629 bytes in size.BACKGROUND1. Field

[0003] The present disclosure relates generally to the field of cancer immunotherapy. More particularly, it concerns chimeric antigen receptors, genetically engineered granulocytemacrophage progenitors (GMPs) expressing chimeric antigen receptors, and uses thereof, including for cancer immunotherapy.2. Description of Related Art

[0004] Granulocytes, macrophages, and dendritic cells are the essential components of the innate immune system in humans. They are the first line of defense against pathogens and also play a central role in maintaining the homeostasis of our body and preventing various diseases including infection, metabolic diseases and cancer. These cells originate from a common progenitor in the bone marrow, the granulocyte-macrophage progenitor (GMP). GMPs have been engineered to express chimeric antigen receptors. However, these cells only promote phagocytosis in the macrophages / DCs that express the CAR-Fc. Means for harnessing the activation of host macrophages and DCs are needed.SUMMARY

[0005] Provided herein are chimeric antigen receptors comprising an extracellular domain capable of binding to an antigen, an Fc region, a linker, a transmembrane domain, and at least one intracellular domain. The intracellular domain may be designed to increase the antitumor activities of granulocytes, macrophages, and dendritic cells by increasing their phagocytosis and / or proinflammatory cytokines secretion and / or antigen presentation. The chimeric antigen receptor may comprise the at least one intracellular domain. The extracellular domain may be a single-chain antibody variable fragment (scFv), a full length heavy chain, a Fab fragment, or a divalent single chain antibody or diabody. The antigen may be CD19, HER2, PD-1, EGFR, EGFRvIII, GPC3, mesothelin, uPAR, CD33, amyloid 0, PSMA, CD117, MPL, or FAP. The Fc region may comprise a CH2CH3 region of IGHG1, or IGHG4. The linker may comprise an amino acid sequence of GGGGS (SEQ ID NO: 1). The transmembrane domain may be derived from CD8, CD28, or FcyR transmembrane domain. The intracellular domain may comprise CD3(^ and / or FcsRIy. The intracellular domain may comprise TIR, ID3, IL-12, and / or NFKB.

[0006] Provided herein are vectors comprising any of the chimeric antigen receptors described herein. The vector may be a viral vector. The viral vector may be replicating or nonreplicating, and may be an adenoviral vector, an adeno-associated virus (AAV) vector, a measles vector, a herpes vector, a retroviral vector, a lentiviral vector, a rhabdoviral vector, a reovirus vector, a Seneca Valley Virus vector, a poxvirus vector, a parvovirus vector, or an alphavirus vector.

[0007] Provided herein are nucleic acid molecules comprising any of the chimeric antigen receptor described herein. The nucleic acid molecule may be a DNA molecule. The nucleic acid molecule may be an RNA molecule. The nucleic acid molecule may be an mRNA molecule.

[0008] Provided herein are methods to genetically engineer granulocyte-macrophage progenitors (GMPs) to express a chimeric antigen receptor comprising: introducing any of the vectors or nucleic acid molecules provided herein into GMPs to form GMPs that express the chimeric antigen receptor (CAR-GMPs, i.e., CAR-Fc-GMPs); and expanding and culturing the CAR-Fc-GMPs for multiple passages in defined culture conditions to generate a population of CAR-Fc-GMPs. The methods may further comprise inducing the population of CAR-Fc-GMPs24915-0966-6435, v. 1to differentiate into granulocytes, macrophages, or dendritic cells in vitro, wherein the granulocytes, macrophages, or dendritic cells express the chimeric antigen receptor. The GMPs may be obtained from stem cells. The stem cells may be hematopoietic stem cells. The hematopoietic stem cells may be isolated from the bone marrow, mobilized peripheral blood, or cord blood of a subject. The subject may be a mammalian subject, and may preferably be a human patient. The defined culture conditions may comprise a basal medium supplemented with human SCF (50 ng / ml), GDC-0879 (1 pM), TN2-30 (5 pM), human IL-3 (20 ng / ml), and human G-CSF (20 ng / ml). The basal medium may comprise IMDM supplemented with human insulin (4 mg / L), human holo-transferrin (20 mg / L), sodium selenite (12.5 pg / L), linoleic acid (1 mg / L), vitamin E (1 mg / L), and human serum albumin (2.5 g / L). Provided herein are GMPs that express a CAR prepared by any of the methods described herein.

[0009] The CAR-Fc-GMPs may be induced to differentiate into macrophages comprising: culturing the CAR-Fc-GMPs with a macrophage differentiation medium comprising macrophage colony-stimulating factor (MCSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF), wherein the macrophages express CAR-Fc. The macrophage differentiation medium may comprise RPMI 1640, fetal bovine serum (FBS) and MCSF or GM-CSF. Provided herein are macrophages that express a CAR-Fc prepared by any of the methods described herein.

[0010] The CAR-Fc-GMPs may be induced to differentiate into granulocytes, comprising: culturing the GMPs with a granulocyte differentiation medium comprising granulocyte colony-stimulating factor (GCSF), wherein the granulocytes express CAR-Fc. The granulocyte differentiation medium may comprise RPMI 1640, FBS and GCSF. Provided herein are granulocytes that express CAR-Fc prepared by any of the methods described herein.

[0011] Provided herein is an immunotherapy method for treating a subject having cancer or an autoimmune disease with macrophages or granulocytes that express a chimeric antigen receptor-Fc (CAR-Fc) comprising administering a composition comprising the GMPs or the macrophages or the granulocytes described herein to the subject having cancer or autoimmune disease. The composition may be administered intravenously or inter-tumoral. The subject may have a cancer selected from adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, anorectal cancer, cancer of the anal canal, appendix cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), biliary cancer, extrahepatic bile duct cancer,34915-0966-6435, v. 1intrahepatic bile duct cancer, bladder cancer, urinary bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, including triple negative breast cancer, bronchial adenomas / carcinoids, carcinoid tumor, gastrointestinal, nervous system cancer, nervous system lymphoma, central nervous system cancer, central nervous system lymphoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary Syndrome, endometrial cancer, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor glioma, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, ocular cancer, islet cell tumors (endocrine pancreas), Kaposi Sarcoma, kidney cancer, renal cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma, Waldenstram macroglobulinemia, medulloblastoma, melanoma, intraocular (eye) melanoma, merkel cell carcinoma, mesothelioma malignant, mesothelioma, metastatic squamous neck cancer, mouth cancer, cancer of the tongue, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, ovarian low malignant potential tumor, pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter, transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, ewing family of sarcoma tumors, soft tissue sarcoma, uterine cancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer44915-0966-6435, v. 1(melanoma), papillomas, actinic keratosis and keratoacanthomas, merkel cell skin carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer, supratentorial primitive neuroectodermal tumors, testicular cancer, throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter and other urinary organs, gestational trophoblastic tumor, urethral cancer, endometrial uterine cancer, uterine sarcoma, uterine corpus cancer, vaginal cancer, vulvar cancer, and Wilm's Tumor. The immunotherapy method may further comprise administering one or more anticancer agents to the subject having cancer. The subject may have an autoimmune disease selected from Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), Type 1 Diabetes (T1D), Sjogren’s syndrome, and Multiple Sclerosis (MS).

[0012] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF DRAWINGS

[0013] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0014] FIG. 1 illustrates an exemplary CAR construct, compared to a CAR-Fc construct design.

[0015] FIG. 2 illustrates an exemplary non-limiting mechanistic illustration of CAR- Fc-GMP interactions with cancer cells.

[0016] FIGS. 3A-3B depict the phagocytosis and digestion of human B-ALL cells by aCD19 CAR-FC human GMP-derived macrophages in vitro. Anti-CD19 CAR-Fc-RFP expressing human GMPs were differentiated into macrophages (red) and cocultured with GFP labeled B-ALL cells (green). (A) Pictures were taken at designed timepoints and (B) FACS was performed 2h after coculturing.54915-0966-6435, v. 1

[0017] FIGS. 4A-4C depict the promotion of nearby WT macrophage phagocytosis by CAR-Fc human GMP-derived macrophages in vitro. (A) RFP control, aCD19 CAR or aCD19 CAR-Fc-RFP human GMP-derived macrophages were mixed with BFP-labeled WT macrophages at a 1 : 1 ratio. GFP-labeled B-ALL cells were added to the culture 24 h later. (B) Representative pictures were taken 1 h after adding cancer cells and (C) cells were collected and analyzed by FACS 2 h after coculturing. The RFP and GFP double positive population indicates phagocytosis by CAR-Fc expressing macrophages, while the BFP and GFP doublepositive population indicates phagocytosis by nearby WT macrophages.

[0018] FIGS. 5A-5D depict the efficacy of allogeneic aCD19 CAR-Fc mGMP in an A20 lymphoma model. (A) Human CD19-luciferase expressing A20 cells were I.V. injected into BALB / c mice at lxl0A5 per mouse 4h after 500 cGy X-ray irradiation. lxlOA7 RFP control-, aCD19 CAR-, or aCD19 CAR-Fc-GMPs derived from C57 mice were infused on days 4, 18, and 30 via I.V. injection. (B-C) Tumor progression was monitored and analyzed by bioluminescent imaging. (D) Survival rate was assessed. (n=10 mice per group).

[0019] FIGS. 6A-6D depict the efficacy of allogeneic aHER2 CAR & CAR-Fc mGMP in 4Tl-hHER2 tumor model. (A) Human HER2 expressing 4T1 cells were injected into the mammary pad of BALB / c mice at 2xlOA5 per mouse 1 day after 450 cGy X-ray irradiation. lxlOA7 Luciferase-RFP control-, aHER2 CAR-Luc-, or aHER2 CAR-Fc-Luc- GMPs derived from C57 mice were infused on days 0, 7, and 14 via I.V. injection. (B) GMP infiltration and biodistribution was monitored and analyzed by bioluminescent imaging. (C) Tumor size was measured by caliper at designed timepoints. (D) Survival rate was assessed. (n=5 mice per group).

[0020] FIGS. 6E-6F depict the trafficking, biodistribution, and differentiation of allogeneic mouse CAR-Fc-GMPs in 4Tl-hHER2 mouse breast cancer model. (E-F) Intratumoral immunostaining for RFP (transplanted cells), CD 11b (monocyte marker) and F4 / 80 (macrophage marker) at different time points suggested that the transplanted CAR-Fc GMPs gave rise to monocytes and infiltrated into the tumor where they further differentiated into macrophages.

[0021] FIGS. 6G-6H depict the trafficking, biodistribution and persistence of allogeneic mouse CAR & CAR-Fc GMPs in 4Tl-hHER2 mouse breast cancer model. (G-H) Intratumoral immunostaining for RFP (transplanted cells) and F4 / 80 (macrophage marker)64915-0966-6435, v. 1revealed that the number of macrophages derived from transplanted CAR-Fc-GMPs was significantly higher than in the CAR and RFP control groups.

[0022] FIGS. 61-6 J depict the intratumoral T-cell infiltration and activation after allogeneic mouse CAR & CAR-Fc GMPs treatment in 4Tl-hHER2 mouse breast cancer model. (I-K) Intratumoral immunostaining for CD3 (pan T cell marker) and Granzyme B (GZB, cytotoxic T cell marker) revealed that the numbers of both pan T cells and cytotoxic T cells in the CAR-Fc-GMPs group were significantly higher than those in the CAR and RFP control groups. These results suggest that CAR-Fc GMP treatment enhances antigen presentation in the tumor.

[0023] FIGS. 7A-7H depict CAR-Fc enhancement of allogenic ani-tumor responses. (A) Schematic illustrating the mixed-strain co-culture system experimental design. Engineered BALB / c GMP-derived macrophages were cocultured with wild type (WT) C57BL / 6 macrophages, OT-II macrophages from a C57BL / 6 background, and Py230 tumor cells expressing both hHER2 and thepeptide. OT-II T cell activation would only be enabled by presentation of OVA323-339 peptide on C57BL / 6 MHC II following trans-phagocytosis of Py230 cells. (B) Percent phagocytosis of Py230 cells by engineered BALB / c macrophages (cis-phagocytosis) and WT C57BL / 6 macrophages (trans-phagocytosis) as measured by flow cytometry. (C) Representative images of co-culture conditions show clonal OT-II proliferation only in the presence of CAR-Fc macrophages. Bottom right panel: Percent T cell proliferation indicated by CFSE dilution measured by flow cytometry. (D) Cytokines in co-culture supernatants were elevated only in the presence of CAR-Fc. Top panel: IFNy. Bottom Panel IL-2. (E) Schematic illustrating syngeneic A20 disseminated lymphoma model and experimental design. (F) Schematic illustrating syngeneic hHER2+ 4T1 tumor model and experimental design. (G) Quantification of tumor volume. (H) Survival analysis of mice following 4T1 engraftment.

[0024] FIG. 8 depicts a representative CFSE flow cytometry histograms measuring the percent of CFSElowproliferating OT-II T cells.DETAILED DESCRIPTION

[0025] The present disclosure provides a chimeric antigen receptor architecture in which a human IgGl Fc domain is built directly into the extracellular portion of the receptor. In this design, the Fc region sits downstream of the antigen-binding domain and is preserved74915-0966-6435, v. 1in a fully functional form, capable of engaging Fc receptors (FcR) on host myeloid cells. Without being limited to any one theory, the CAR-Fc configuration allows the engineered cell to behave like a conventional CAR cell, while simultaneously recruiting and activating endogenous FcR on host cells in antigen-dependent manner. This gives the recipient’s own phagocytes and antigen presenting cells direct access to tumor antigens at the time and place where the engineered cell recognizes its target.

[0026] Also provided are methods to amplify the anti-tumor efficacy of chimeric antigen receptor-modified granulocyte-monocyte progenitors (CAR-Fc-GMPs) by integrating an IgGl Fc region and a spacer into the extracellular domain of the CAR construct. The resulting CAR-Fc complex comprises a single-chain variable fragment (scFv), an Fc region, and a flexible spacer, creating a membrane-tethered antibody that can recognize specific tumor antigens through the scFv domain and activate macrophages and dendritic cells (DCs) via Fc receptor binding. Without being limited to any one theory, this combines CAR-targeted specificity with the activation of surrounding immune cells, thereby enhancing the overall antitumor response.

[0027] In a conventional CAR, the extracellular region is limited to an scFv or other ligand-binding domain, followed by a hinge, transmembrane region, and intracellular signaling modules. In the CAR-Fc design, there is an intact human IgGl Fc domain between the ligandbiding domain (e.g., scFv) and a linker (or more generally between the antigen-recognition site and transmembrane region). Without being limited to any one theory, the Fc domain, which is FcR-binding competent, is positioned so that, upon antigen binding, it becomes clustered and displayed in a geometry that efficiently engages FcR on neighboring host myeloid cells. In this way, the Fc region within the CAR-Fc construct can interact with Fc receptors on macrophages and DCs, facilitating antibody-dependent cellular phagocytosis (ADCP) and significantly boosting phagocytic activity. While an IgGl Fc domain is exemplified herein, any domain capable of detection by Fc receptors is conceptually equivalent.

[0028] The intracellular domain of CAR-Fc may incorporate signaling components such as CD3(^, FcRy, Megfl 0, TIR, CD 147, MerTK, or combinations thereof, which further enhance macrophage and DC activation, as well as their phagocytic functions. The intracellular region of the CAR-Fc can be any configuration. The scFv mediates antigen specificity and triggers intracellular CAR signaling.84915-0966-6435, v. 1

[0029] The CAR-Fc-GMP described herein possess a dual functionality: these cells only promote phagocytosis in the macrophages / DCs that express the CAR-Fc but also activate nearby macrophages and DCs through ADCP. When the scFv domain of CAR-Fc binds to a specific antigen on a target cell, such as a cancer cell, the Fc region may become exposed to interact with Fc receptors on neighboring macrophages or DCs. This Fc-FcR interaction triggers signaling cascades in adjacent immune cells, activating them and driving phagocytosis. Collectively, these activated immune cells may cooperate to engulf and eliminate the target cell through ADCP.

[0030] As such, the CAR-Fc design creates two layers of activity. First, the engineered cell retains its intrinsic CAR function. When the scFv binds antigen, the cell signals through its CAR tail and performs its programmed effector activity, e.g., phagocytosis, cytotoxicity, and / or cytokine production. Second, antigen binding exposes and concentrates the Fc domain at the site of engagement. This surface-tethered Fc region can then crosslink FcR on nearby host phagocytes and antigen presenting cells. Thus, host phagocytes and antigen presenting cells are drawn to antigen-positive targets, take up those targets through FcR-mediated phagocytosis, and process and present or cross-present the acquired antigens on their own MHC molecules.

[0031] In some embodiments, this allows tumor antigens to be transferred to host antigen-presenting cells even when the engineered cell is MHC-mismatched with the host. This addresses a longstanding problem in allogeneic myeloid therapies: donor antigen presenting cells cannot prime host T cells in antigen specific manner because they present antigens on mismatched MHC. CAR-Fc bypasses this limitation by enabling FcR-positive host cells to internalize and present or cross-present tumor antigens themselves. As demonstrated herein, CAR-Fc cells were able to drive antigen-specific host T cell proliferation across a full MHC mismatch. Conventional CAR cells, which lack this Fc-mediated handoff mechanism, produced no such response.

[0032] In some embodiments, engaging host dendritic cells through FcR appears to have particular importance in solid tumors. Many solid tumors rely on specialized dendritic cell populations to transport antigens to draining lymph nodes and generate the priming needed for robust T cell responses. Conventional CAR-macrophages may be ineffective in accessing this network. By contrast, CAR-Fc channels antigen into the host’s existing antigen-presenting94915-0966-6435, v. 1system in a highly targeted way. This can initiate T cell responses even in settings where engineered cells alone are insufficient to reshape the tumor microenvironment.

[0033] Although some CARs use IgG-derived “hinge” sequences, those designs typically include mutations that eliminate FcR binding to avoid unwanted myeloid activation. CAR-Fc takes the opposite approach: the FcR interaction is the intended mechanism, not a liability nor incidental. Likewise, monoclonal antibodies rely on soluble Fc domains to recruit myeloid cells, but their distribution and activity depend entirely on pharmacokinetics. CAR-Fc instead localizes Fc engagement to engineered cells that have already recognized antigen. This restricts FcR activation to antigen-positive sites and tightly couples host engagement to the engineered cell’s own recognition event.

[0034] In vitro, CAR-Fc macrophages induced both direct (cis) phagocytosis and robust antigen-dependent phagocytosis by unmodified host macrophages, indicating efficient engagement of FcR. In MHC -mismatched co-cultures, only CAR-Fc cells elicited antigenspecific T cell proliferation, while conventional CAR cells did not. In vivo, CAR-Fc improved tumor control relative to matched conventional CAR designs, increased donor-derived myeloid accumulation in tumors, and enhanced infiltration of endogenous T cells.

[0035] Although the use of myeloid effector cells (macrophages, granulocyte macrophage progenitors (GMPs), GMP-derived cells) was exemplified herein, the CAR-Fc design is not limited to any particular cell type. The same extracellular architecture can, in principle, be applied to T cells, NK cells, iPSC-derived immune cells, or other engineered cell types.

[0036] Additionally, the CAR-Fc-GMPs can be used allogeneically by harnessing Fc- mediated activation of host macrophages and DCs. This Fc interaction supports host T cell activation via antigen presentation, amplifying the overall immune response against tumors.I. Definitions

[0037] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is104915-0966-6435, v. 1therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0038] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0039] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0040] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the inherent variation in the method being employed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value.II. Chimeric Antigen Receptors

[0041] Provided herein are chimeric antigen receptors, comprising an extracellular domain capable of binding to an antigen, an Fc region, a linker, a transmembrane domain, and optionally at least one intracellular domain that, in some embodiments, is designed to increase the anti-tumor activities of granulocytes, macrophages, and dendritic cells by increasing their phagocytosis and / or proinflammatory cytokines secretion. The chimeric antigen receptor may not comprise at least one intracellular domain. The extracellular domain may comprise a singlechain antibody variable fragment (scFv), a full-length heavy chain, a Fab fragment, or a divalent single chain antibody or diabody. The extracellular single-chain antibody variable fragment (scFv) may be directed against a tumor associated antigen, such as, for example, CD19, HER2, EGFR, EGFRvIII, GPC3, mesothelin, uPAR, CD33, amyloid 0, or FAP. The extracellular domain may comprise a means for binding to a tumor associated antigen, such as, for example, CD19, HER2, EGFR, EGFRvIII, GPC3, mesothelin, uPAR, CD33, amyloid 0, or FAP. The Fc region may be a CH2CH3 region of IgGl or IgG4. The linker may be a flexible linker. The transmembrane domain may be derived from a CD8, CD28, or FcyR transmembrane domain. The intracellular domain may comprise CD3(^ and / or FcsRIy. The intracellular domain may comprise TIR, ID3, IL-12, NFKB, and / or other components to regulate macrophage functions. In some embodiments, the chimeric antigen receptor comprises114915-0966-6435, v. 1from an N-terminus to a C-terminus, an extracellular domain, an Fc region, a linker, a transmembrane domain, and an intracellular domain. In some embodiments, the chimeric antigen receptor comprises from an N-terminus to a C-terminus, an scFv, an Fc region, a linker, a transmembrane domain, and an intracellular domain. In some embodiments, the chimeric antigen receptor comprises from an N-terminus to a C-terminus, a CD 19 binding domain, an Fc region, a linker, a transmembrane domain, and an intracellular domain. In some embodiments, the chimeric antigen receptor comprises from an N-terminus to a C-terminus, a HER2 binding domain, an Fc region, a linker, a transmembrane domain, and an intracellular domain. In some instances, the linker is a flexible linker. In some instances, the linker comprises one of the following sequence: GS, GSG, SGG, SG, GGS, SGS, GSS, SSG, or GGGGS (SEQ ID NO: 1), or a combination thereof. Additional contemplated flexible linkers include, but are not limited to, (G)n, (GS)n, (GGSG)n (SEQ ID NO: 21), (GGSGG)n (SEQ ID NO: 22), (GSGSG)n (SEQ ID NO: 23), (GSGGG)n (SEQ ID NO: 24), (GGGSG)n (SEQ ID NO: 25), (GSSSG)n (SEQ ID NO: 26), (GSGGS)n (SEQ ID NO: 27), (GGGS)n (SEQ ID NO: 28), (GGGGS)n (SEQ ID NO: 29), (GGSGGS)n (SEQ ID NO: 30), (SGGGGS)n (SEQ ID NO: 31), (GRAGGGGAGGGG)n (SEQ ID NO: 32), (GRAGGG)n (SEQ ID NO: 33), (GGGGSGGGGSGS)n (SEQ ID NO: 34), and (GGGGSGGGGSGGGGS)n (SEQ ID NO: 35), where n is an integer of at least one.

[0042] An exemplary amino acid sequence of the Fc region is:APELLGGPSVFLFPPKPKDTLMISRTPEVTCIAAIDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRWS' / LTVLHQDWLNGKEYKCR'VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC S 1HEALHNHYTQKSLSLSPGK ( SEQ ID NO : 2 )

[0043] An exemplary amino acid sequence of the transmembrane and intracellular domains is:SASTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI APLAGTCGVILLSLVI TLYCGSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRLKIQVRKAAITSYEKSD GVYTGLSTRNQETYETLKHEKPPQASQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDG PDPAWTSATNFSLLKQAGDVEENPGPASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQT AKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDS SLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKT TYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGA ( SEQ ID NO : 3 )

[0044] An exemplary anti-CD19 CAR-Fc amino acid sequence is:124915-0966-6435, v. 1MALPVTALLLPLALLLHAARPDYKDDDDKGTDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLE ITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVI WGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVS SSREPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGKTSGGGGSGGGGSGGGGSASTTTPAPRPPTPAPTIASQ PLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVILLSLVITLYCGSRVKFSRSADAPAYKQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG KGHDGLYQGLSTATKDTYDALHMQALPPRRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEK PPQASQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWTSATNFSLLKQAGDVE ENPGPASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQ FQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSD GPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLD ITSHNEDYTIVEQYERAEGRHSTGA* ( SEQ ID NO : 4 )

[0045] Another exemplary anti-HER2 CAR-Fc amino acid sequence is:MALPVTALLLPLALLLHAARPDYKDDDDKGTGSDIVLTQTPSSLPVSVGEKVTMTCKSSQTLLYSNNQ KNYLAWYQQKPGQSPKLLISWAFTRKSGVPDRFTGSGSGTDFTLTIGSVKAEDLAVYYCQQYSNYPWT FGGGTRLEIKRGGGGSGGGGSGGGGSEVQLQQSGPEVVKTGASVKISCKASGYSFTGYFINWVKKNSG KSPEWIGHISSSYATSTYNQKFKNKAAFTVDTSSSTAFMQLNSLTSEDSAVYYCVRSGNYEEYAMDYW GQGTSVTVSSSREPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKTSGGGGSGGGGSGGGGSASTTTPAPRPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI APLAGTCGVILLSLVITLYCGSRVKFSR SADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRLKIQVRKAAITSYEKSDGVYTGLSTRNQE TYETLKHEKPPQASQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWTSATNFS LLKQAGDVEENPGPASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLP FAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVK LRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPG AYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGA* ( SEQ ID NO : 5 )

[0046] “Chimeric antigen receptor” or “CAR” or “CARs” as used herein refers to engineered receptors, which graft an antigen specificity onto cells (for example GMP cells). CARs are also known as artificial T-cell receptors, chimeric T-cell receptors or chimeric immunoreceptors. The various components of the chimeric antigen receptors contemplated herein are further described in the paragraphs below.A. Extracellular Domains

[0047] The extracellular domain refers to the portion of the CAR that specifically binds the antigen on the target cell. The extracellular domain may comprise a means for binding to the antigen on the target cell. The antigen may be CD 19, HER2, PD-1, EGFR, EGFRvIII, GPC3, mesothelin, uPAR, CD33, amyloid P, PSMA, CD117, MPL, or FAP. Exemplary134915-0966-6435, v. 1extracellular domains may comprise the CDR sequences of tafasitamab (see SEQ ID NOs: 132, 147, 116, 120, 129, and 130 of U.S. Pat. 9,803,020, which is incorporate herein by reference), loncastuximab tesirine (see SEQ ID NOs: 1-6 of U.S. Pat. 9,555,125, which is incorporated herein by reference), FMC63 (see SEQ ID NOs: 5-10 of U.S. Application Publication 2025 / 0339467, which is incorporated by reference herein), trastuzumab (see SEQ ID NOs: 65- 70 of U.S. Pat. 12,240,913, which is incorporated by reference herein), pembrolizumab (see SEQ ID NOs: 1-3 and 6-8 of U.S. Pat. 12,110,330, which is incorporated by reference herein), and nivolumab (see SEQ ID NOs: 57-59 and 60-62 of U.S. Pat. 11,411,489, which is incorporated by reference herein). Exemplary extracellular domains may comprise the variable domain sequences of tafasitamab (see SEQ ID NO: 87 and 106 of U.S. Pat. 9,803,020, which is incorporate herein by reference), loncastuximab tesirine (see SEQ ID NOs: 8 and 7 of U.S. Pat. 9,555,126, which is incorporated herein by reference), FMC63 (see SEQ ID NOs: 3 and 4 of WO 2017 / 015783, which is incorporated by reference herein), trastuzumab (see SEQ ID NOs: 71 and 72 of U.S. Pat. 12,240,913, which is incorporated by reference herein), pembrolizumab (see SEQ ID NOs: 9 and 4 of U.S. Pat. 12,110,330, which is incorporated by reference herein), and nivolumab (see SEQ ID NOs: 54 and 56 of U.S. Pat. 11,411,489, which is incorporated by reference herein).

[0048] The extracellular domain of the CARs may comprise any of the known binding domains used in CAR constructs (see, e.g., PCT / US2017 / 064379) including an antibody or a functional equivalent thereof or a fragment thereof or a derivative thereof. The targeting regions may comprise full length heavy chains, nanobodies (e.g., VHH domains), Fab fragments, single chain Fv (scFv) fragments, divalent single chain antibodies or diabodies, each of which are specific to the target antigen.

[0049] The extracellular domain may comprise an antibody fragment or antigen binding fragment. The term “antibody fragment” or “antigen binding fragment,” as used herein, refer to a protein fragment that comprises only a portion of an intact antibody, or a derivative thereof, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind antigen. Examples of antibody fragments encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VH and CHI domains; (ii) the Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CHI domain; (iii) the FDA fragment having VH and CHI domains; (iv) the Fd' fragment having VH and CHI domains and one or more cysteine residues at the C-terminus of the CHI domain;144915-0966-6435, v. 1(v) the Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) the dAb fragment (Ward et al., Nature 341, 544-546 (1989)) which consists of a VH domain; (vii) isolated CDR regions; (viii) F(ab')2 fragments, a bivalent fragment including two Fab' fragments linked by a disulphide bridge at the hinge region; (ix) single chain antibody molecules (e.g., single chain Fv; scFv) (Bird et al., Science 242:423-426 (1988); and Huston et al., PNAS (USA) 85:5879-5883 (1988)); (x) "diabodies" with two antigen binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); (xi) "linear antibodies" comprising a pair of tandem Fd segments (VH-CHl-VH-CHl) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. Protein Eng. 8(10): 1057- 1062 (1995); and U.S. Pat. No. 5,641,870).B. Fc Regions

[0050] The fragment crystallizable region (Fc region) is the tail region of an antibody that may interact with cell surface receptors (Fc receptors). The Fc region may be involved in activating the immune system. In IgG, IgA, and IgD antibody isotypes, the Fc region comprises two identical protein fragments, derived from the second and third constant domains of the heavy chains. IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2-4). The Fc region can interact with Fc receptors on macrophages and dendritic cells, thereby facilitating antibody-dependent cellular phagocytosis (ADCP) and significantly boosting phagocytic activity.

[0051] The Fc region may be the CH2CH3 region of IgGl, IgG4, or other similar sequences. By way of example, the Fc region may comprise a human IgGl isotype constant region having the amino acid sequence, or an amino acid sequence 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%, or at least 99% identical to the amino acid sequence:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRW SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK (SEQ ID NO: 6), or a portion thereof.154915-0966-6435, v. 1

[0052] The CH2 region of this sequence corresponds to the sequence:APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 7). As such, the Fc region of a CAR as provided herein may comprise an amino acid sequence 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 100% identical to the amino acid sequence of (SEQ ID NO:7).

[0053] The CH3 region of this sequence corresponds to the sequence:GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8). As such, the Fc region of a CAR as provided herein may comprise an amino acid sequence 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 100% identical to the amino acid sequence of (SEQ ID NO:8).

[0054] By way of example, the Fc region may comprise a human IgGl isotype constant region having the amino acid sequence, or an amino acid sequence 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%, or at least 99% identical to the amino acid sequence:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9), or a portion thereof.

[0055] The CH2 region of this sequence corresponds to the sequence:APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 10). As such, the Fc region of a CAR as provided herein may comprise an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least164915-0966-6435, v. 197%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of (SEQ ID NO: 10).

[0056] The CH3 region of this sequence corresponds to the sequence:GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11). As such, the Fc region of a CAR as provided herein may comprise an amino acid sequence 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 100% identical to the amino acid sequence of (SEQ ID NO: H).

[0057] By way of example, the Fc region may comprise a human IgG2 isotype constant region having the amino acid sequence, or an amino acid sequence 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%, or at least 99% identical to the amino acid sequence:ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRWSVLT WHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK (SEQ ID NO: 12), or a portion thereof.

[0058] The CH2 region of this sequence corresponds to the sequence:APPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK (SEQ ID NO: 13). As such, the Fc region of a CAR as provided herein may comprise an amino acid sequence 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 100% identical to the amino acid sequence of (SEQ ID NO: 13).

[0059] The CH3 region of this sequence corresponds to the sequence:GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14). As174915-0966-6435, v. 1such, the Fc region of a CAR as provided herein may comprise an amino acid sequence 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 100% identical to the amino acid sequence of (SEQ ID NO: 14).

[0060] By way of example, the Fc region may comprise a human IgG3 isotype constant region having the amino acid sequence, or an amino acid sequence 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%, or at least 99% identical to the amino acid sequence:ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPP PCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRWSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNI FSCSVMHEALHNRFTQKSLS LSPGK (SEQ ID NO: 15), or a portion thereof.

[0061] The CH2 region of this sequence corresponds to the sequence:APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKPR EEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTK (SEQ ID NO: 16). As such, the Fc region of a CAR as provided herein may comprise an amino acid sequence 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 100% identical to the amino acid sequence of (SEQ ID NO:16).

[0062] The CH3 region of this sequence corresponds to the sequence:GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDG SFFLYSKLTVDKSRWQQGNI FSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 17). As such, the Fc region of a CAR as provided herein may comprise an amino acid sequence 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 100% identical to the amino acid sequence of (SEQ ID NO:17).184915-0966-6435, v. 1

[0063] By way of example, the Fc region may comprise a human IgG4 isotype constant region having the amino acid sequence, or an amino acid sequence 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%, or at least 99% identical to the amino acid sequence:ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVL TVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK (SEQ ID NO: 18), or a portion thereof.

[0064] The CH2 region of this sequence corresponds to the sequence:APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAK (SEQ ID NO: 19). As such, the Fc region of a CAR as provided herein may comprise an amino acid sequence 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 100% identical to the amino acid sequence of (SEQ ID NO:19).

[0065] The CH3 region of this sequence corresponds to the sequence:GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 20). As such, the Fc region of a CAR as provided herein may comprise an amino acid sequence 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 100% identical to the amino acid sequence of (SEQ ID NO:20).C. Linkers

[0066] Chimeric antigen receptors described herein may comprise linkers, preferably flexible linkers. In protein constructs composed of several elements (e.g., extracellular domain, Fc region, transmembrane domain, and intracellular domains), the protein elements may be separated by peptide linker elements. Such elements may be beneficial because they allow for a proper folding of the individual elements and thereby the proper functionality of each194915-0966-6435, v. 1element. Alternatively, the term “spacer” or “peptide spacer” is used herein. When used in the context of the present invention, such linkers or spacers are particularly useful when encoded by a nucleic acid encoding at least two functional protein elements, such as at least one polypeptide, domain, or protein of interest and at least one further protein, domain, or polypeptide element. In that case, the linker is typically located on the polypeptide chain in between the polypeptide of interest and the at least one further protein element. On the nucleic acid level, the coding sequence for such linker is typically placed in the reading frame between coding regions for individual polypeptide domains of a given protein of interest. Peptide linkers are preferably composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. The small size of these amino acids provides flexibility and allows for mobility of the connecting functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduces an interaction between the linker and the protein moieties. A typical sequence of a flexible linker is composed of repeats of the amino acids Glycine (G) and Serine (S). For instance, the linker may have the following sequence: GS, GSG, SGG, SG, GGS, SGS, GSS, SSG, or GGGGS (SEQ ID NO: 1), or a combination thereof. In some instances, the same sequence may be repeated multiple times (e.g. two, three, four, five or six times) to create a longer linker. In some instances, different short linker sequences can be combined to form a linker that is 5 amino acids or more in length. A single amino acid residue such as S or G may be used as a linker. Linkers or spacers may be used as additional elements to promote or improve the activity of the chimeric antigen receptor. On the nucleic acid level, particularly RNA level, any nucleotide sequence moiety can be employed that encodes any of linker or spacer used in the present invention. Owing to the degenerated genetic code, in the case of most polypeptides, more than one nucleic acid sequence is conceivable as encoding the respective polypeptide list. While each and every such nucleic acid may generally be used in the context of the present invention, it is preferable that the nucleic acid sequence that encodes the polypeptide sequence is selected such that its sequence is optimized according to the general guidance provided in this specification. Additional exemplary flexible linkers include, but are not limited to, (G)n, (GS)n, (GGSG)n (SEQ ID NO: 21), (GGSGG)n (SEQ ID NO: 22), (GSGSG)n (SEQ ID NO: 23), (GSGGG)n (SEQ ID NO: 24), (GGGSG)n (SEQ ID NO: 25), (GSSSG)n (SEQ ID NO: 26), (GSGGS)n (SEQ ID NO: 27), (GGGS)n (SEQ ID NO: 28), (GGGGS)n (SEQ ID NO: 29), (GGSGGS)n (SEQ ID NO: 30), (SGGGGS)n (SEQ ID NO: 31), (GRAGGGGAGGGG)n (SEQ ID NO: 32), (GRAGGG)n (SEQ ID NO: 33),204915-0966-6435, v. 1(GGGGSGGGGSGS)n (SEQ ID NO: 34), and (GGGGSGGGGSGGGGS)n (SEQ ID NO: 35), where n is an integer of at least one.D. Transmembrane Domains

[0067] A “transmembrane domain” as used herein refers to the region of the CAR which crosses the plasma membrane. The transmembrane domain of the CAR is the transmembrane region of a transmembrane protein (for example Type I transmembrane proteins), an artificial hydrophobic sequence or a combination thereof. Other transmembrane domains will be apparent to those of skill in the art. The transmembrane domain may comprise transmembrane domain derived or cloned from proteins selected from the transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 160, CD 19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD 100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.E. Intracellular Domains

[0068] An “intracellular domain” as used herein refers to a region that is designed to increase the anti-tumor activities of granulocytes, macrophages, and dendritic cells by increasing their phagocytosis and / or proinflammatory cytokines secretion. The intracellular domain may be human CD3(^ (NM_198053.2, aa52-164)-human Fcerlg (NM_004106.1, aa45- 86)-human CD19 (NM_001178098.1, aa498-544) or a portion, or a derivative thereof. CD3(^ intracellular domain contains the same immunoreceptor tyrosine-based activation motif (IT AM) as FcRy and has been shown to be able to enhance phagocytosis (Isakov, 1997).III. Vectors

[0069] Provided herein are vectors comprising any of the chimeric antigen receptors described herein. The vector may be a viral vector. The viral vector may be replicating or non-214915-0966-6435, v. 1replicating, and may be an adenoviral vector, an adeno-associated virus (AAV) vector, a measles vector, a herpes vector, a retroviral vector, a lentiviral vector, a rhabdoviral vector, a reovirus vector, a Seneca Valley Virus vector, a poxvirus vector, a parvovirus vector, or an alphavirus vector.

[0070] The term “vector” refers to a vector comprising a recombinant polynucleotide optionally comprising expression control sequences, operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses etc.) that incorporate the recombinant polynucleotide.

[0071] The term “lentiviral vector” refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include but are not limited to, e.g., the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.

[0072] The term “retroviral vector” refers to a vector derived from at least a portion of a retrovirus genome. Examples of retrovirus vectors include MSCVneo, MSCV-pac (or MSCV-puro), MSCV-hygro as available from Addgene or Clontech.IV. Genetic Engineering of GMPs

[0073] Provided herein are methods to genetically engineer granulocyte-macrophage progenitors (GMPs) to express a chimeric antigen receptor comprising: introducing any of the vectors provided herein into GMPs to form GMPs that express the chimeric antigen receptor (CAR-Fc-GMPs); expanding and culturing the CAR-Fc-GMPs for multiple passages in defined culture conditions to generate a population of CAR-Fc-GMPs; and inducing the population of CAR-Fc-GMPs to differentiate into granulocytes, macrophages, or dendritic cells in vitro, wherein the granulocytes, macrophages, or dendritic cells express the chimeric antigen receptor. The GMPs may be obtained from stem cells. The stem cells may be hematopoietic224915-0966-6435, v. 1stem cells. The hematopoietic stem cells may be isolated from the bone marrow of a subject. The hematopoietic stem cells may be isolated from the blood of a subject. The subject may be a mammalian subject, and may preferably be a human patient. The defined culture conditions may comprise a culture medium comprising at least two or more components selected from a group consisting of: (i) a growth factor; (ii) a B-Raf kinase inhibitor; (iii) optionally, an agent that inhibits the mitogen-activated kinase interacting protein kinases 1 and 2 (Mnkl / 2); (iv) optionally, an agent that inhibits the PI3K pathway; (v) optionally, one or more serum components; and (vi) TN2-30. The defined culture conditions may comprise a basal medium supplemented with human SCF (50 ng / ml), GDC-0879 (1 pM), TN2-30 (5 pM), human IL-3 (20 ng / ml), and human G-CSF (20 ng / ml). The basal medium may comprise IMDM supplemented with human insulin (4 mg / L), human holo-transferrin (20 mg / L), sodium selenite (12.5 pg / L), linoleic acid (1 mg / L), vitamin E (1 mg / L), and human serum albumin (2.5 g / L). The TN2-30 may be partially substituted by PY-60, a small-molecule activator of the transcriptional coactivator Yes-associated protein (YAP).

[0074] The one or more agents that inhibit the mitogen-activated protein kinase interacting protein kinases 1 and 2 (Mnkl / 2) may be selected from CGP-57380, cercosporamide, BAY 1143269, tomivosertib, ETC-206, SLV-2436 and any combination thereof. The one or more agents that inhibit the PI3K pathway may be selected from 3- methyladenine, LY294002, alpelisib, wortmannin, quercetin, hSMG-1 inhibitor l lj, zandelisib, alpelisib hydrochloride, idelalisib, buparlisib, copanlisib, IPI549, dactolisib, pictilisib, SAR405, duvelisib, fimepinostat, GDC-0077, PI-103, YM-20163, PF-04691502, Taselisib, omipalisib, samotolisib, isorhamnetin, ZATK474, parsaclisib, rigosertib, AZD8186, GSK2636771, disitertide, TGI 00-115, AS-605240, PI3K-IN-1, dactolisib tosylate, gedatolisib, TGX-221, umbralisib, AZD 6482, serabelisib, bimiralisib, apitolisib, alpha-linolenic acid, Vps34-PIK-III, PIK-93, Vps34-IN-1, CH5132799, leniolisib, voxtalisib, GSK1059615, sonolisib, PKI-402, PI4KIIIbeta-IN-9, HS-173, BGT226 maleate, pictilisib dimethane sulfonate, VS-5584, IC-87114, quercetin dihydrate, CNX-1351, SF2523, GDC-0326, seletalisib, acalisib, SAR-260301, ZAD-8835, GNE-317, AMG319, nemiralisib, IITZ-01, PI- 103 hydrochloride, oroxin B, pilaralisib, AS-252424, cpanlisib dihydrochloride, AMG 511, disitertide TFA, PIK-90, tenalisib, esculetin, CGS 15943, GNE-477, PI-3065, A66, AZD3458, ginsenoside Rkl, sophocarpine, buparlisib hydrochloride, Vps34-IN-2, linperlisib, amicolide D, KP372-1, CZC24832, PF-4989216, (R)-Duvelisib, PQR530, P118-IN-1, umbralisib hydrochloride, MTX-211, PI3K / mT0R Inhibitor-2, LX2343, PF-04979064, polygalasaponin234915-0966-6435, v. 1F, glaucocalyxin A, NSC781406, MSC2360844, CAY10505, IPI-3063, TG 100713, BEBT- 908, PI-828, brevianamide F, ETP-46321, PIK-294, SRX3207, sophocarpine monohydrate, AS-604850, desmethylglycitein, SKI V, WYE-687, NVP-QAV-572, GNE-493, CAL-130 hydrochloride, GS-9901, BGT226, H4MT-PI3K6-372, PI3Ka-IN-4, parsaclisib hydrochloride, PF-06843195, PI3K-IN-6, (S)-PI3Ka-IN-4, PI3K(gamma)-IN-8, BAY1082439, CYH33, PI3Ky inhibitor 2, PI3K8 inhibitor 1, PARP / PI3K-IN-1, LAS 191954, PI3K-IN-9, CHMFL- PI3KD-317, PI3K / HDAC-IN-1, MSC2360844 hemifumarate, PI3K-IN-2, PI3K / mT0R Inhibitor- 1, PI3K6-IN-1, euscaphic acid, KU-0060648, AZD 6482, WYE-687 dihydrochloride, GSK2292767, (R)-Umbralisib, PIK-293, idelalisib D5, PIK-75, hirsutenone, quercetin D5, PIK-108, hSMG-1 inhibitor l ie, PI3K-IN-10, NVP-BAG956, PI3Ky inhibitor 1, CAL-130, ON 146040, PI3k6 inhibitor 1, PI3Ka / mT0R-IN-l, and any combination thereof. The B-Raf kinase inhibitor may be selected from GDC-0879, PLX4032, GSK2118436, BMS-908662, LGX818, PLX3603, RAF265, RO5185426, vemurafenib, PLX8394, SB590885 and any combination thereof. The B-Raf kinase inhibitor is preferably GDC-0879. TN2-30 is:TN2-30

[0075] A “growth factor” refers to a substance, e.g., a compound or molecule, that is effective to promote the growth of cells, e.g., stem cells, and which, unless added to the culture medium as a supplement, is not otherwise a component of the basal medium. Growth factors include, but are not limited to, stem cell factor (SCF), interlukine-3 (IL-3), granulocyte colonystimulating factor (G-CSF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), insulinlike growth factor-II (IGF-II), platelet-derived growth factor- AB (PDGF), and vascular endothelial cell growth factor (VEGF), activin-A, Wnt and bone morphogenic proteins (BMPs), insulin, cytokines, chemokines, morphogens, neutralizing antibodies, other proteins, and small molecules. Exogenous growth factors may also be added to a medium according to the disclosure to assist in the maintenance of cultures of GMPs in a substantially undifferentiated state. Such factors and their effective concentrations can be identified as described elsewhere herein or using techniques known to those of skill in the art of culturing cells. The GMPs may be cultured in a culture medium which comprises SCF.244915-0966-6435, v. 1

[0076] The disclosure provides methods to genetically modify the GMPs disclosed herein using genetic engineering techniques. In particular it was shown herein that the GMPs of the disclosure are susceptible to genetic modification techniques, thereby allowing for the use of the GMPs in basic scientific research and clinical therapeutic applications. Thus, expanded and genetically modified GMPs can be readily translated into broad clinical applications. Accordingly, the disclosure further provides methods to genetically modify GMPs disclosed herein. Such methods can include the step of genetically engineering modifications into GMPs by using a gene editing system, homologous recombination, or site directed mutagenesis. Particular examples of gene editing systems include zing finger nucleases, TALEN and CRISPR.

[0077] The CRISPR system may be a type II CRISPR system and the Cas enzyme may be Cas9, which catalyzes DNA cleavage. Enzymatic action by Cas9 derived from Streptococcus pyogenes or any closely related Cas9 generates double stranded breaks at target site sequences which hybridize to 20 nucleotides of the guide sequence and that have a protospacer-adjacent motif (PAM) sequence (examples include NGG / NRG or a PAM that can be determined as described herein) following the 20 nucleotides of the target sequence. CRISPR activity through Cas9 for site-specific DNA recognition and cleavage is defined by the guide sequence, the tracr sequence that hybridizes in part to the guide sequence and the PAM sequence. More aspects of the CRISPR system are described in Karginov and Hannon, The CRISPR system: small RNA-guided defense in bacteria and archaea, Mole Cell 2010, January 15; 37(1): 7.

[0078] The type II CRISPR locus from Streptococcus pyogenes SF370, which contains a cluster of four genes Cas9, Casl, Cas2, and Csnl, as well as two non-coding RNA elements, tracrRNA and a characteristic array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers, about 30 bp each). In this system, targeted DNA double-strand break (DSB) is generated in four sequential steps. First, two non-coding RNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the direct repeats of pre-crRNA, which is then processed into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to target sequences comprising the protospacer and the corresponding PAM via heteroduplex formation between the spacer region of the crRNA and the protospacer DNA. Finally, Cas9 mediates cleavage of target sequence of PAM to create a DSB within the254915-0966-6435, v. 1protospacer. The RNA polymerase Ill-based U6 promoter may be used to drive the expression of tracrRNA.

[0079] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. Without wishing to be bound by theory, the tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. One or more vectors driving expression of one or more elements of a CRISPR system may be introduced into a host cell (e.g., a GMP or stem cell) such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to (“upstream” of) or 3' with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. A single promoter may drive expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequences, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). The CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence may be operably linked to and expressed from the same promoter.

[0080] A CRISPR expression vector may comprise one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In some embodiments, one or more insertion sites (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8,264915-0966-6435, v. 19, 10, or more insertion sites) are located upstream and / or downstream of one or more sequence elements of one or more vectors. A vector may comprise an insertion site upstream of a tracr mate sequence, and optionally downstream of a regulatory element operably linked to the tracr mate sequence, such that following insertion of a guide sequence into the insertion site and upon expression the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell (e.g., a GMP or stem cell). A vector may comprise two or more insertion sites, each insertion site being located between two tracr mate sequences so as to allow insertion of a guide sequence at each site. In such an arrangement, the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or combinations of these. When multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell. For example, a single vector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide sequences. About or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containing vectors may be provided, and optionally delivered to a cell.

[0081] A vector may comprise a regulatory element operably linked to an enzymecoding sequence encoding a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof. The unmodified CRISPR enzyme may have DNA cleavage activity, such as Cas9. The CRISPR enzyme may direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. The CRISPR enzyme may direct cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. A vector may encode a CRISPR enzyme that is mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that render Cas9a nickase include, without limitation, H840A, N854A, and N863 A. As a further example,274915-0966-6435, v. 1two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III or the HNH domain) may be mutated to produce a mutated Cas9 substantially lacking all DNA cleavage activity. A D10A mutation may be combined with one or more of H840A, N854A, or N863A mutations to produce a Cas9 enzyme substantially lacking all DNA cleavage activity. A CRISPR enzyme may be considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutated enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or lower with respect to its non-mutated form. Where the enzyme is not SpCas9, mutations may be made at any or all residues corresponding to positions 10, 762, 840, 854, 863 and / or 986 of SpCas9 (which may be ascertained for instance by standard sequence comparison tools. In particular, any or all of the following mutations are preferred in SpCas9: D10A, E762A, H840A, N854A, N863 A and / or D986A; as well as conservative substitution for any of the replacement amino acids is also envisioned. The same (or conservative substitutions of these mutations) at corresponding positions in other Cas9s are also indicated.

[0082] Indicated orthologs are also described herein. A Cas enzyme may be identified Cas9 as this can refer to the general class of enzymes that share homology to the biggest nuclease with multiple nuclease domains from the type II CRISPR system. Most preferably, the Cas9 enzyme is from, or is derived from, spCas9 or saCas9. By derived, it is meant that the derived enzyme is largely based, in the sense of having a high degree of sequence homology with, a wildtype enzyme, but that it has been mutated (modified) in some way as described herein.

[0083] It will be appreciated that the terms Cas and CRISPR enzyme are generally used herein interchangeably, unless otherwise apparent. As mentioned above, many of the residue numberings used herein refer to the Cas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes. However, it will be appreciated that this disclosure includes many more Cas9s from other species of microbes, such as SpCas9, SaCa9, StlCas9 and so forth.

[0084] The gene editing systems (e.g., zing finger nucleases, CRISPR and TALEN) can be used to genetically engineer modifications into the GMP or stem cells, such as replacing or disrupting an existing gene found in the GMP or stem cell (knockout). As shown in the Examples presented herein, the GMPs of the disclosure are particular susceptible to knockout mutations. Moreover, it is expected that additional knockouts could be easily created from the GMPs of the disclosure such as SIRPa gene knockouts and / or a PI3Ky gene knockouts. Alternatively, the same editing systems (e.g., CRISPR and TALEN) can be used to alter a284915-0966-6435, v. 1genetic locus to contain sequence information not found at the genetic locus (a knock-in mutation). Such modifications, can be used to create GMP’s that have “gained a function.” Such modified GMPs are particularly useful for mimicking a disease state, e.g., by expressing biomolecules associated with a disease or disorder.V. Macrophages, Granulocytes, and Stem Cells

[0085] The CAR-Fc-GMPs may be induced to differentiate into macrophages with a method comprising: culturing the CAR-Fc-GMPs with a macrophage differentiation medium comprising macrophage colony-stimulating factor (MCSF) or GM-CSF, wherein the macrophages express a CAR, such as a CAR-Fc provided elsewhere herein. The macrophage differentiation medium may comprise RPMI 1640, fetal bovine serum (FBS), and MCSF or GM-CSF. Provided herein are macrophages that express a CAR-Fc prepared by any of the methods described herein.

[0086] The CAR-Fc-GMPs may be induced to differentiate into granulocytes, using a method comprising: culturing the GMPs with a granulocyte differentiation medium comprising granulocyte colony-stimulating factor (GCSF), wherein the granulocytes express CAR-Fc. The granulocyte differentiation medium may comprise RPMI 1640, FBS and GCSF. Provided herein are granulocytes that express CAR-Fc prepared by any of the methods described herein.

[0087] Granulocytes and macrophages are the two major cell types of the innate immune system. Without being limited to any one theory, they are the first line of defense against pathogens and also play a central role in maintaining the homeostasis of our bodies and preventing infections and various diseases, including metabolic diseases and cancers. Granulocytes and macrophages engulf and digest invading microorganisms in a process called phagocytosis. Besides phagocytosis, macrophages also play a critical role as antigen presenters, initiating specific defense mechanisms (adaptive immunity) by recruiting other immune cells such as lymphocytes. Recently, macrophages have also become an attractive therapeutic target to combat cancer. Dendritic cells (DCs) are major antigen-presenting cells (APCs) that capture and display antigens to adaptive immune system cells, for example T cells, which helps to trigger and regulate immune responses. DCs play a role in many diseases, including cancer, autoimmune disease, viral infections, and transplantation. Macrophage mediated clearance may be initiated through antibody-dependent cellular phagocytosis (ADCP), in which Fc receptor engagement promotes internalization of antibody- or Fc fusion protein bound targets294915-0966-6435, v. 1by professional phagocytes, resulting in selective elimination of opsonized cells or particles. In some embodiments, the efficiency of ADCP may be influenced by one or more of Fc glycosylation, Fey receptor expression levels, and intracellular ITAM signaling, which collectively govern phagosome formation and lysosomal degradation. Dendritic cells may also perform Fc receptor mediated antigen uptake, followed by processing in the endosomal compartment and MHC class I or class II presentation to T cell subsets, thereby linking innate recognition to adaptive immune activation. Despite their huge therapeutic potential, historically, there has been no effective method to expand and genetically modify granulocytes, macrophages, and dendritic cells, greatly limiting their clinical application. Existing challenges include limited ex vivo proliferation capacity, poor transduction efficiency using viral or nonviral delivery systems, and rapid terminal differentiation following cytokine exposure, which may hinder stable genetic modification of myeloid-lineage cells. Accordingly, improved strategies that enable targeted Fc receptor engagement, controlled phagocytic activation, and efficient genetic manipulation of granulocytes, macrophages, and DCs may offer significant clinical benefit in inflammatory disease, oncology, vaccine design, and immune-cell engineering applications.

[0088] One promising approach is to expand and genetically modify the granulocytemonocyte progenitor (GMP) cells, which is the common progenitor in the bone marrow from which granulocytes and macrophages originate. As used herein, the GMPs can be derived from or obtained from a mammalian species (e.g., bovine, canine, equine, feline, human, murine, primate, rat etc.). Ex vivo expanded GMPs could allow the ability to generate ample macrophages and dendritic cells for therapeutic applications. Expanded GMPs could also be induced to differentiate into the most abundant type of granulocyte, the neutrophil, which could then be infused into blood circulation to fight infection in patients with neutropenia or neutrophil dysfunction. More importantly, GMPs could easily be modified to generate genetically engineered macrophages with enhanced antitumor or antimicrobial activity. Macrophages engulf and digest any foreign particles including the genetic materials used to engineer them, but GMPs do not have phagocytic activity, making them a much more favorable target. However, despite decades of intensive studies, the long-term ex vivo expansion of GMPs, as well as other stem / progenitor cells of the hematopoietic system, is yet to be realized.

[0089] The cells of the hematopoietic system are organized in a hierarchy with hematopoietic stem cells (HSCs) at the top, various mature blood cells at the bottom, and304915-0966-6435, v. 1intermediate hematopoietic stem and progenitor cells (HSPCs) such as GMPs in between. Lured by the great therapeutic potential of HSPCs, numerous groups have attempted to develop culture conditions for their ex vivo expansion in the past two decades. So far, all the conditions have a fundamental limitation: they are unable to continuously and exponentially expand homogeneous populations of any type of HSPC.

[0090] A unique challenge has been the difficulty of distinguishing and separating one type of HSPC from another, and particularly from its immediate upstream progenitors and downstream progeny. In fact, the conventional immunophenotypic analysis cannot distinguish one HSPC type from its immediate upstream progenitors and downstream progeny. At the clonal level, prospectively purified HSCs are still highly heterogeneous, containing cells with diverse gene expression patterns and distinct cellular functions. This is expected, because HSPCs span a continuum of cells with somewhat similar cell surface marker expression, but heterogeneous functions. Within this heterogenous cell population, it is technically very challenging to identify growth factors / cytokines and small molecules that can promote the expansion of a single stem / progenitor cell type.

[0091] Granulocytes, macrophages, and dendritic cells originate from a common progenitor in the bone marrow, the granulocyte-macrophage progenitor (GMP). Despite the immense therapeutic potential of innate immune cells, their application in the clinic has been greatly limited because of the current inability to effectively expand and genetically modify these cells or their progenitors GMPs. Provided herein are methods for the long-term expansion of GMPs. Ex vivo expanded GMPs can efficiently differentiate into mature and functional granulocytes, macrophages, and dendritic cells both in vitro and in vivo. These ex vivo expanded GMPs can also be genetically modified. The methods disclosed herein for the production of GMPs, and the GMPs produced therefrom, have great utility because: (1) longterm expansion of GMPs provide unlimited homogenous cell populations for both basic research and clinical applications; (2) long-term expansion of GMPs allows for the studying the regulation of an immune response by modifying GMP genes, and their expression thereof; and (3) ex vivo expanded GMPs can be used for clinical applications, including transplantation. For example, ex vivo expanded GMPs can readily be used to treat neutropenia. Moreover, the disclosure also provides for the genetic modification of GMPs (e.g., knockout SIRPa and / or PI3Ky gene; overexpression of angiotensin converting enzyme), which can be further induced to differentiate into macrophages and dendritic cells. In the studies presented herein these314915-0966-6435, v. 1engineered macrophages and dendritic cells exhibit enhanced antitumor effects and can be used clinically to treat cancer, either as monotherapy or combination therapy with other immunological agents, such as anti-PD-l / PD-Ll antibodies and chimeric antigen receptor T (CAR-T) cells. GMPs were also engineered to produce CAR-macrophages. These CAR- macrophages can be used treat cancer and other diseases.

[0092] Macrophages display divergent phenotypes that were originally classified as Ml or M2 polarity. Ml polarized macrophages display the capacity to present antigen, produce IL-12, IL-23, interferon gamma (IFNy), and reactive oxygen species (ROS). Ml macrophages are more effective at antitumor and skewing T cell responses toward a T helper type 1 (Thl) or cell mediated immune response. In contrast, M2 macrophages produce IL- 10 and TGF-P and participate in tissue remodeling, have immunosuppressive qualities, and promote Th2 or antibody mediated immune responses. However, the M1 / M2 binary model represents a simplified framework. Macrophage activation states in vivo rarely conform to a single discrete phenotype, and instead reflect a dynamic continuum of transcriptional programs, cytokine response profiles, and metabolic states that change over time in response to tissue-specific cues, chemokines, stromal interactions, and environmental stimuli. Contemporary transcriptomic studies, including single-cell RNA sequencing, have demonstrated that macrophages exist across a spectrum of functional states rather than two mutually exclusive categories. Mixed phenotypes expressing both inflammatory and immunoregulatory markers are frequently observed, and polarization outcomes are strongly influenced by multiple simultaneous signals rather than a single Thl or Th2 stimulus. Tumor-associated macrophages (TAMs) constitute a major component of the tumor microenvironment. These cells are predominant M2 phenotype macrophages which promote tumor immunosuppression. Yet even within the TAM compartment, multiple distinct clusters have been identified, including inflammatory TAMs, angiogenic TAMs, lipid-associated TAMs, interferon-responsive TAMs, and regulatory TAMs, demonstrating considerable cellular heterogeneity. TAM phenotypes may shift across spatial regions of a tumor, across disease stage, or in response to therapeutic intervention, and may simultaneously express markers traditionally associated with both Ml and M2 activation, further illustrating the limitations of the binary polarization schema. The ability of macrophages to undergo repolarization within the tumor microenvironment has emerged as an attractive therapeutic strategy. Pharmacologic intervention, cytokine modulation, and receptor- directed engineering have shown potential to convert immunosuppressive TAMs toward pro- inflammatory, antitumor phenotypes. This capacity for repolarization supports the324915-0966-6435, v. 1development of engineered macrophage platforms, including macrophages expressing CARs, which may be programmed to recognize tumor antigens while simultaneously adopting sustained antitumor activation states, thereby providing a promising modality for targeted cancer immunotherapy. Recent studies support their contribution to the suppression of T cell function, which is not abolished by the use of Immune checkpoint blockage. Macrophages have therefore become an attractive therapeutic target to combat cancer. Despite the huge therapeutic potential of macrophages, their application in clinic has been greatly limited because currently there is no effective method to expand and genetically modify macrophages or their progenitors GMPs. Long-term expansion of GMPs allows for genetic modification to make these cells more therapeutically applicable.

[0093] “Macrophage colony-stimulating factor” or “MCSF” (also known as colonystimulating factor 1 (CSF 1)) and granulocyte-macrophage colony-stimulating factor (GM- CSF) are involved in the proliferation, differentiation, and survival of monocytes, macrophages, and bone marrow progenitor cells. The gene sequence, protein sequence and orthologs across various species are known in the art (see, e.g., NCBI Reference Sequence: NP_000748.4, which is incorporated herein by reference).

[0094] Stem cells are cells capable of differentiation into other cell types, including those having a particular, specialized function (e.g., tissue specific cells, parenchymal cells and progenitors thereof). Progenitor cells (i.e., "multipotent") are cells that can give rise to different terminally differentiated cell types, and cells that are capable of giving rise to various progenitor cells. Cells that give rise to some or many, but not all, of the cell types of an organism are often termed "pluripotent" stem cells, which are able to differentiate into any cell type in the body of a mature organism, although without reprogramming they are unable to dedifferentiate into the cells from which they were derived. As will be appreciated, "multipotent" stem / progenitor cells (e.g., granulocyte / macrophage progenitor cells (GMPs)) have a narrower differentiation potential than do pluripotent stem cells. Prior to derivation into GMPs, the stem cells disclosed herein can be genetically modified by use of any number of genetic engineering techniques, e.g., such as gene therapy, gene editing systems, homologous recombination, etc. Such modified stem cells may provide for enhanced therapies (e.g., see Nowakowski et al., Acta Neurobiol Exp (Wars) 73(1): 1-18 (2013)). A stem cell or progenitor cell may be engineered to express, or contain a polynucleotide encoding, a chimeric antigen receptor (CAR).334915-0966-6435, v. 1

[0095] The GMPs disclosed herein may be derived from stem cells. Stem cells can include embryonic stem cells, induced pluripotent stem cells, non-embryonic (adult) stem cells, and cord blood stem cells. Stem cell types that can be cultured using the media of the disclosure include stem cells derived from any mammalian species including humans, mice, rats, monkeys, and apes (see, e.g., Nature 448:313-318, July 2007; and Takahashi et al., Cell 131 (5): 861 -872; which are incorporated herein by reference).

[0096] The GMPs of the disclosure may be derived from induced pluripotent stem cells (iPSs, or iPSCs). iPSCs are a type of pluripotent stem cell obtained from non-pluripotent cells by selective gene expression (of endogenous genes) or by transfection with a heterologous gene. Induced pluripotent stem cells are described by Shinya Yamanaka's team at Kyoto University, Japan. Yamanaka et al. had identified genes that are particularly active in embryonic stem cells, and used retroviruses to transfect mouse fibroblasts with a selection of those genes. Eventually, four key pluripotency genes essential for the production of pluripotent stem cells were isolated: Oct-3 / 4, SOX2, c-Myc, and Klf4. More recent research has provided the fewer of these factors in combination with certain culture conditions as well as additional factors can induce pluripotent stem cells. Cells were isolated by antibiotic selection for Fbxl5+ cells. The same group published a study along with two other independent research groups from Harvard, MIT, and the University of California, Los Angeles, showing successful reprogramming of mouse fibroblasts into iPS and even producing a viable chimera.

[0097] The GMPs disclosed herein may be derived from embryonic stem cells (ESCs). ESCs are stem cells derived from the undifferentiated inner mass cells of a human embryo. ESCs are pluripotent, meaning they are able to grow (i.e. differentiate) into all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. Pluripotency distinguishes embryonic stem cells from adult stem cells found in adults; while embryonic stem cells can generate all cell types in the body, adult stem cells are multipotent and can produce only a limited number of cell types. Additionally, under defined conditions, embryonic stem cells are capable of propagating themselves indefinitely. This allows embryonic stem cells to be employed as useful tools for both research and regenerative medicine, because they can produce limitless numbers of themselves for continued research or clinical use.

[0098] The GMPs disclosed herein may be derived from cord blood stem cells. Umbilical cord blood is the blood left over in the placenta and in the umbilical cord after the birth of the baby. The cord blood is composed of all the elements found in whole blood. It344915-0966-6435, v. 1contains red blood cells, white blood cells, plasma, platelets and is also rich in hematopoietic stem cells. Hematopoietic stem cells can be isolated from cord blood using any number of isolation methods taught in the art, including those taught in Chularoj montri et al., J Med Assoc Thai 92(3):S88-94 (2009). Moreover, commercial kits are available for isolation CD34+ cells (i.e., hematopoietic stem cells) from human umbilical cord blood from multiple vendors, including STEMCELL Technologies, Thermo Fisher Scientific, Zen-Bio, etc.

[0099] The GMPs disclosed herein may be derived from non-embryonic stem cells. The non-embryonic stem cell can renew itself and can differentiate to yield some or all of the major specialized cell types of the tissue or organ. The primary roles of non-embryonic stem cells in a living organism are to maintain and repair the tissue in which they are found. Scientists also use the term somatic stem cell instead of non-embryonic stem cell, where somatic refers to cells of the body (not the germ cells, sperm or eggs). Non-embryonic stem cells have been identified in many organs and tissues, including brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, gut, liver, ovarian epithelium, and testis. They are thought to reside in a specific area of each tissue (called a “stem cell niche”). In a living animal, non-embryonic stem cells are available to divide for a long period, when needed, and can give rise to mature cell types that have characteristic shapes and specialized structures and functions of a particular tissue.

[0100] The GMPs disclosed herein may be derived from hematopoietic stem cells (HSCs). HSCs can easily be isolated from umbilical cord blood, mobilized peripheral blood, and bone marrow. Such isolation protocols are known in the art and typically use CD34+ as a cell selection marker for the isolation of HSCs (e.g., see Lagasse et al., Nat Med. 6: 1229 - 1234(2000)).VI. Pharmaceutical Compositions

[0101] Provided herein is an immunotherapy method for treating a subject having cancer or an autoimmune disease with macrophages or granulocytes that express a chimeric antigen receptor (CAR) comprising administering a composition comprising the macrophages or the granulocytes described herein to the subject having cancer or an autoimmune disease. The composition may be administered intravenously or inter-tumoral.

[0102] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions,354915-0966-6435, v. 1suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the macrophages or granulocytes described herein, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, intra-rectal, vaginal, topical or delivered by mechanical ventilation.

[0103] The pharmaceutical compositions may comprise pharmaceutically acceptable salts. Pharmaceutically acceptable salts include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0104] Generally, the ingredients of compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0105] The compositions of the disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.364915-0966-6435, v. 1

[0106] Pharmaceutical compositions described herein may exhibit anti-cancer effects. The anti -cancer effect may have an IC50 of about 1 pM to about 20 pM. The anti-cancer effect may have an IC50 of less than 20 pM.

[0107] An autoimmune disease may refer to a non-malignant disease or disorder arising from and directed against an individual’s own (self) antigens and / or tissues. Exemplary autoimmune diseases include Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), Type 1 Diabetes (T1D), Sjogren’s syndrome, idiopathic thrombocytopenia purpura (ITP), and Multiple Sclerosis (MS). Further exemplary autoimmune diseases include allogenic islet graft rejection, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison’s disease, antineutrophil cytoplasmic autoantibodies (ANCA), autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, autoimmune urticaria, Behcet’s disease, bullous pemphigoid, cardiomyopathy, Castleman’s syndrome, celiac spruce-dermatitis, chronic fatigue immune disfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Churg- Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn’s disease, dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyositis, glomerulonephritis, Grave’s disease, Guillain-Barre, Goodpasture’s syndrome, graft-versus-host disease (GVHD), Hashimoto's thyroiditis, hemophilia A, idiopathic pulmonary fibrosis, IgA neuropathy, IgM polyneuropathies, immune mediated thrombocytopenia, juvenile arthritis, Kawasaki's disease, lichen plantus, lupus erthematosis, Meniere’s disease, mixed connective tissue disease, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobinulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Reynauld’s phenomenon, Reiter’s syndrome, sarcoidosis, scleroderma, solid organ transplant rejection, stiff-man syndrome, takayasu arteritis, temporal arteristis / giant cell arteritis, thrombotic thrombocytopenia purpura, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, and Wegner’s granulomatosis.

[0108] Cancer results from the outgrowth of a clonal population of cells from tissue. The development of cancer, referred to as carcinogenesis, can be modeled and characterized in a number of ways. An association between the development of cancer and inflammation has374915-0966-6435, v. 1long-been appreciated. The inflammatory response is involved in the host defense against microbial infection, and also drives tissue repair and regeneration. Considerable evidence points to a connection between inflammation and a risk of developing cancer, i.e., chronic inflammation can lead to dysplasia.

[0109] Cancer cells to which the methods of the present disclosure can be applied include but are not limited to cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, pancreas, testis, tongue, cervix, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these:

[0110] The subject may have a cancer selected from adrenocortical carcinoma, AIDS- related cancers, AIDS-related lymphoma, anal cancer, anorectal cancer, cancer of the anal canal, appendix cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), biliary cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, urinary bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, including triple negative breast cancer, bronchial adenomas / carcinoids, carcinoid tumor, gastrointestinal, nervous system cancer, nervous system lymphoma, central nervous system cancer, central nervous system lymphoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary Syndrome, endometrial cancer, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor glioma, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, ocular cancer, islet cell tumors (endocrine pancreas), Kaposi Sarcoma, kidney cancer, renal cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, lung cancer, non-small cell lung cancer, small cell lung384915-0966-6435, v. 1cancer, AIDS-related lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma, Waldenstram macroglobulinemia, medulloblastoma, melanoma, intraocular (eye) melanoma, merkel cell carcinoma, mesothelioma malignant, mesothelioma, metastatic squamous neck cancer, mouth cancer, cancer of the tongue, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, ovarian low malignant potential tumor, pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter, transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, ewing family of sarcoma tumors, soft tissue sarcoma, uterine cancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), papillomas, actinic keratosis and keratoacanthomas, merkel cell skin carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer, supratentorial primitive neuroectodermal tumors, testicular cancer, throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter and other urinary organs, gestational trophoblastic tumor, urethral cancer, endometrial uterine cancer, uterine sarcoma, uterine corpus cancer, vaginal cancer, vulvar cancer, and Wilm’s Tumor. The immunotherapy method may further comprise administering one or more anticancer agents to the subject having cancer.

[0111] Such compositions comprise a prophylactically or therapeutically effective amount of macrophages or granulocytes described herein, and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be394915-0966-6435, v. 1employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0112] In addition, the methods of the disclosure can be applied to a wide range of species, e.g., humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. Cancers may also be recurrent, metastatic and / or multi -drug resistant, and the methods of the present disclosure may be particularly applied to such cancers so as to render them resectable, to prolong or re-induce remission, to inhibit angiogenesis, to prevent or limit metastasis, and / or to treat multi-drug resistant cancers. At a cellular level, this may translate into killing cancer cells, inhibiting cancer cell growth, or otherwise reversing or reducing the malignant phenotype of tumor cells.

[0113] The term “effective amount” or “therapeutically effective amount” as used herein refers to the amount of a composition comprising GMPs (macrophages or granulocytes derived therefrom) that have been engineered to express a CAR, to decrease at least one or more symptom of the disease or disorder and relates to a sufficient amount of the composition to provide the desired effect.

[0114] The phrase “therapeutically effective amount” as used herein means a sufficient amount of the composition to treat a disorder, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0115] A therapeutically or prophylactically significant reduction in a symptom is, e.g. at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150% or more in a measured parameter as compared to a control or non-treated subject or the state of the subject prior to administering the cellular compositions described herein. Measured or measurable parameters include clinically detectable markers of disease, for example, elevated or depressed levels of a biological marker. The exact amount required will vary depending on factors such as the type of disease being treated, gender, age, and weight of the subject.404915-0966-6435, v. 1

[0116] The administering in any of the methods described herein may reduce the number of cancerous cells in the patient, may reduce and / or eliminate the tumor burden in the patient and / or may result in an enhanced cancer treatment.

[0117] Also, compositions of the present disclosure may be used in vivo to treat cancers. Thus, compositions can be formulated for parenteral administration, e.g., formulated for injection via the intradermal, intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Administration by the intradermal and intramuscular routes are specifically contemplated. The compositions can also be administered by a topical route directly to the mucosa, for example by nasal drops or mist, inhalation, or by nebulizer.

[0118] Some variation in dosage and regimen will necessarily occur depending on the age and medical condition of the subject being treated, as well as the route chosen. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. In many instances, it will be desirable to have multiple administrations. Thus, the compositions of the disclosure may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. Administrations will normally be at from one to twelve-week intervals, more usually from one to six-week intervals. Periodic re-administration will be desirable with recurrent exposure to the pathogen.

[0119] The administration may use various “unit doses.” Unit dose is defined as containing a predetermined quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts.

[0120] The CAR-Fc-GMPs can be further administered in combination with one or more anticancer agents to treat a subject with cancer. Examples, of anticancer agents that can be used with the CAR-Fc-GMPs disclosed herein include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and tiimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly414915-0966-6435, v. 1cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; vinca alkaloids; epipodophyllotoxins; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall; L-asparaginase; anthracenedione substituted urea; methyl hydrazine derivatives; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitiaerine; pentostatin; phenamet; pirarubicin; losoxantione; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,22"-trichlorotiiethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g.,424915-0966-6435, v. 1TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® (docetaxel) (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO); retinoids such as retinoic acid; capecitabine; leucovorin (LV); irenotecan; adrenocortical suppressant; adrenocorticosteroids; progestins; estrogens; androgens; gonadotropin-releasing hormone analogs; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included anticancer agents are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4- hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON- toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASL® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARTMIDEX® anastrozole; and anti -androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3- dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as a VEGF-A expression inhibitor (e.g., ANGIOZYME® ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rJL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELLX® rmRH; antibodies such as trastuzumab and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0121] The second anti-cancer therapy may comprise surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.434915-0966-6435, v. 1

[0122] To kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis or otherwise reverse or reduce the malignant phenotype of tumor cells, using the methods and compositions of the present disclosure, one would generally contact a “target” cell with a composition according to the present disclosure and at least one other agent. These compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with a composition according to the present disclosure and the other agent(s) or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes a composition according to the present disclosure and the other includes the other agent.

[0123] Alternatively, the immunotherapy of the present disclosure may precede or follow the other agent treatment by intervals ranging from minutes to weeks. When the other agent and the immunotherapy of the present disclosure are applied separately to the cell, one would generally ensure that a significant period of time did not expire between each delivery, such that the agent and composition according to the present disclosure would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one would contact the cell with both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

[0124] It also is conceivable that more than one administration of either a composition of the present disclosure or the other agent will be desired. Various combinations may be employed, where a therapeutic composition according to the present disclosure therapy is “A” and the other therapy is “B”, as exemplified below:

[0125] A / B / A B / A / B B / B / A A / A / B B / A / A A / B / B B / B / B / A B / B / A / B

[0126] A / A / B / B A / B / A / B A / B / B / A B / B / A / A B / A / B / A B / A / A / B B / B / B / A

[0127] A / A / A / B B / A / A / A A / B / A / A A / A / B / A A / B / B / B B / A / B / B B / B / A / B444915-0966-6435, v. 1

[0128] Other combinations are contemplated. Again, to achieve cell killing, both agents are delivered to a cell in a combined amount effective to kill the cell. Agents or factors suitable for cancer therapy include any chemical compound or treatment method that induces damage when applied to a cell. Such agents and factors include radiation and waves that induce DNA damage such as irradiation, microwaves, electronic emissions, and the like. A variety of chemical compounds, also described as “chemotherapeutic” or “genotoxic agents,” may be used. This may be achieved by irradiating the localized tumor site; alternatively, the tumor cells may be contacted with the agent by administering to the subject a therapeutically effective amount of a pharmaceutical composition. A combination therapy may also include surgery. Various modes of these therapies are discussed below.

[0129] The term “chemotherapy” refers to the use of drugs to treat cancer. A “chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.

[0130] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially454915-0966-6435, v. 1calicheamicin gammall and calicheamicin omegall; dynemicin, including dynemicin A uncialamycin and derivatives thereof; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin,464915-0966-6435, v. 1doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, paclitaxel, docetaxel, gemcitabien, navelbine, famesyl- protein tansferase inhibitors, transplatinum, 5 -fluorouracil, vincristin, vinblastin and methotrexate and pharmaceutically acceptable salts, acids or derivatives of any of the above. Radiotherapy, also called radiation therapy, is the treatment of cancer and other diseases with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated by damaging their genetic material, making it impossible for these cells to continue to grow. Although radiation damages both cancer cells and normal cells, the latter are able to repair themselves and function properly.

[0131] Radiation therapy used according to the present disclosure may include, but is not limited to, the use of y-rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors induce a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[0132] Radiotherapy may comprise the use of radiolabeled antibodies to deliver doses of radiation directly to the cancer site (radioimmunotherapy). Antibodies are highly specific proteins that are made by the body in response to the presence of antigens (substances recognized as foreign by the immune system). Some tumor cells contain specific antigens that trigger the production of tumor-specific antibodies. Large quantities of these antibodies can be made in the laboratory and attached to radioactive substances (a process known as radiolabeling). Once injected into the body, the antibodies actively seek out the cancer cells, which are destroyed by the cell-killing (cytotoxic) action of the radiation. This approach can minimize the risk of radiation damage to healthy cells.

[0133] Conformal radiotherapy uses the same radiotherapy machine, a linear accelerator, as the normal radiotherapy treatment but metal blocks are placed in the path of the x-ray beam to alter its shape to match that of the cancer. This ensures that a higher radiation dose is given to the tumor. Healthy surrounding cells and nearby structures receive a lower dose of radiation, so the possibility of side effects is reduced. A device called a multi-leaf474915-0966-6435, v. 1collimator has been developed and may be used as an alternative to the metal blocks. The multileaf collimator consists of a number of metal sheets which are fixed to the linear accelerator. Each layer can be adjusted so that the radiotherapy beams can be shaped to the treatment area without the need for metal blocks. Precise positioning of the radiotherapy machine is very important for conformal radiotherapy treatment and a special scanning machine may be used to check the position of internal organs at the beginning of each treatment.

[0134] High-resolution intensity modulated radiotherapy also uses a multi -leaf collimator. During this treatment the layers of the multi-leaf collimator are moved while the treatment is being given. This method is likely to achieve even more precise shaping of the treatment beams and allows the dose of radiotherapy to be constant over the whole treatment area.

[0135] Although research studies have shown that conformal radiotherapy and intensity modulated radiotherapy may reduce the side effects of radiotherapy treatment, it is possible that shaping the treatment area so precisely could stop microscopic cancer cells just outside the treatment area being destroyed. This means that the risk of the cancer coming back in the future may be higher with these specialized radiotherapy techniques.

[0136] Scientists also are looking for ways to increase the effectiveness of radiation therapy. Two types of investigational drugs are being studied for their effect on cells undergoing radiation. Radiosensitizers make the tumor cells more likely to be damaged, and radioprotectors protect normal tissues from the effects of radiation. Hyperthermia, the use of heat, is also being studied for its effectiveness in sensitizing tissue to radiation.

[0137] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present disclosure, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and / or alternative therapies.

[0138] Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs’ surgery).484915-0966-6435, v. 1It is further contemplated that the present disclosure may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.

[0139] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.

[0140] After removal of the tumor, an adjuvant treatment with an agent of the present disclosure is believed to be particularly efficacious in reducing the recurrence of the tumor. Additionally, the compounds of the present disclosure can also be used in a neoadjuvant setting.

[0141] It also should be pointed out that any of the foregoing therapies may prove useful by themselves in treating cancer. The skilled artisan is directed to “Remington’s Pharmaceutical Sciences” 15th Edition, Chapter 33, in particular pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

[0142] There are several advantages of this GMP -based cancer immunotherapy over other types of cellular immunotherapies, such as CAR-T therapy. Long-term expansion of GMPs offers the opportunity for producing off-the-shelf CAR-macrophages for immunotherapy. A major hurdle in the clinical application of off-the-shelf CAR-macrophages is human leukocyte antigen (HLA) compatibility. The ability to long-term expand and genetically engineer GMPs allows the establishment of a master cell bank of GMPs collected from healthy donors and / or umbilical cord blood for generating off-the-shelf CAR- macrophages for immunotherapy. Alternatively, HLA-universal GMPs can be generated using gene modification techniques as described above. Long-term expansion of GMPs allows for sophisticated and multiplex genetic engineering on GMPs to render these cells more therapeutically applicable. For example, the signal-regulatory protein-a (SIRPa) and phosphatidylinositol 3-kinase-y (PI3Ky) genes can be knocked out to further enhance the antitumor activity of GMP-derived CAR-macrophages. SIRPa knockout in macrophages is494915-0966-6435, v. 1expected to enhance their antitumor activity by disrupting the CD47-SIRPa interaction between tumor cells and macrophages. PI3Ky is abundantly expressed in macrophages and directly controls a macrophage switch between immune stimulation (Ml macrophage) and suppression (M2 macrophage). Activation of PI3Ky in macrophages induces a transcriptional program that promotes immune suppression during inflammation and tumor growth, whereas inactivation of macrophage PI3Ky promotes an immunostimulatory transcriptional program. It is expected that PI3Ky- / - macrophages will have enhanced antitumor activity by polarizing to an immune stimulatory Ml phenotype.

[0143] Adoptive transfer of genetically engineered GMPs has the potential to reverse the immunosuppressive tumor microenvironment (TME). Tumor-associated macrophages (TAMs) constitute a major component of the TME. Experimental and clinical studies have found that the majority of TAMs are immunosuppressive M2 macrophages that prevent tumor cells from being attacked by natural killer (NK) and T cells. These observations suggest a need to target TAMs in combination with other immunotherapies in order to achieve maximal antitumor effect. One strategy is to replenish immunosuppressive M2 macrophages with immunostimulatory Ml macrophages that have antitumor activity. This can be achieved by depleting TAMs followed by adoptive transfer of immunostimulatory Ml macrophages generated from PI3Ky- / - GMPs or GMPs overexpressing IL-12. Monocytes and macrophages expressing IL- 12 have been shown to change the TME from immunosuppressive to immunostimulatory.

[0144] GMPs can be engineered to produce macrophages with the potential to mount more complete and robust immune responses than CAR-T cells. Macrophages exhibit their antitumor activity through the secretion of inflammatory cytokines, the phagocytosis of cancer cells, and more importantly, the processing and presentation of cancer antigens to NK and T cells. Macrophages are professional antigen-presenting cells (APCs). Endogenous NK and T cells activated by macrophages are likely to mount an immune response with high selectivity and efficiency. Therefore, harnessing the power of GMPs / macrophages through genetic engineering represents a promising approach for developing the next-generation cancer immunotherapy.504915-0966-6435, v. 1VII. Examples

[0145] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1 - CAR-Fc GMP Design

[0146] The CAR-Fc construct comprises an extracellular single-chain antibody variable fragment (scFv), an Fc region, a flexible linker, a transmembrane domain, and intracellular domains to promote phagocytosis. To target different antigens, the scFv can be directed against CD 19, HER2, EGFR, EGFRvIII, GPC3, mesothelin, uPAR, CD33, amyloid P, FAP, and other antigens. The Fc can be the CH2CH3 region of IgGl, IgG4, or other similar sequences. The flexible linker can consist of several repeats of GGGGS (SEQ ID NO: 1). The transmembrane domain (TM) can be derived from CD8, CD28, FcyR, or other transmembrane proteins. The intracellular domains contain CD3(^ and FcsRIy to trigger phagocytosis in macrophages. The intracellular domains can also include TIR, ID3, IL-12, NFKB, and other components to regulate macrophage functions. Additionally, the intracellular domains can be missing to form a membrane-tethered antibody that attains similar functions. An exemplary CAR-Fc construct, compared to a CAR construct, is illustrated in FIG. 1.

[0147] FIG. 2 illustrates an exemplary, non-limiting mechanism for CAR-Fc targeting cancer cells. As shown, when coculturing cancer cells with CAR-Fc expressing macrophages, the scFv region recognizes and binds to antigens on cancer cells to trigger intracellular phagocytosis machinery. At the same time, the Fc region is recognized by macrophages through Fc receptors. This interaction activates the antibody -dep endent cellular phagocytosis (ADCP) pathway, resulting in an enhanced phagocytosis of cancer cells. When CAR-Fc- expressing macrophages are utilized for allogeneic cancer therapy, the CAR-Fc guides these macrophages to the tumor site, where they accumulate. This accumulation not only facilitates the phagocytosis of CAR-Fc-expressing macrophages but also activates recipient macrophages514915-0966-6435, v. 1to engage in phagocytosis. This process can further activate recipient T cells through antigen presentation.Example 2 - Anti-CD19 CAR-Fc human GMP-derived macrophages phagocytose and digest human B-ALL cells in vitro

[0148] Macrophages are an attractive therapeutic target to treat cancer. Macrophages exhibit their anticancer effect through phagocytosis of cancer cells and subsequent presentation of cancer antigens to T cells. GMPs were genetically engineered to specifically target human B cell lymphoma. Chimeric antigen receptor (CAR) T cell therapies have been approved by the U.S Food and Drug Administration (FDA) to treat B cell lymphoma. More recently, studies have demonstrated that macrophage-mediated phagocytosis of cancer cells can be enhanced through engineering macrophages to express a CAR for phagocytosis. A CAR-Fc construct, as described in FIG. 1, was generated containing the extracellular single-chain antibody variable fragment (scFv) that recognizes human B cell antigen CD19 (aCD19 scFv). This CAR-Fc transgene was linked to the Red Fluorescent Protein (RFP) to facilitate monitoring of transgene expression.

[0149] After introducing CAR-Fc into SCF / 2i mGMPs or SCF / 2i-IG human GMPs by lentiviral infection, RFP positive GMPs were sorted and expanded in SCF / 2i (for mGMPs) or SCF / 2i-IG (for human GMPs). To evaluate phagocytosis, macrophages were generated from CAR-Fc GMPs and co-cultured with GFP-labeled human B-cell acute lymphoblastic leukemia (B-ALL) cells.

[0150] As shown in FIG. 3, anti-CD19 CAR-Fc-RFP expressing human GMPs were differentiated into macrophages (red) and cocultured with GFP labeled B-ALL cells (green). Anti-CD19 CAR-Fc human GMP-derived macrophages efficiently phagocytosed and digested human B-ALL cells.Example 3 - CAR-Fc human GMP-derived macrophages promote nearby WT macrophage phagocytosis in vitro

[0151] aCD19 CAR-Fc-RFP human macrophages were mixed with BFP -labeled WT macrophages at a 1 : 1 ratio. GFP-labeled B-ALL cells were added to the culture 24 h later. Shown in FIG. 4 are representative pictures that were taken 1 h after adding cancer cells and FACS analysis of cells that were collected and analyzed 2 h after coculturing. The RFP and524915-0966-6435, v. 1GFP double-positive population indicate phagocytosis by CAR-Fc expressing macrophages, while the BFP and GFP double-positive population indicates phagocytosis by nearby WT macrophages. As shown in FIG. 4, CAR-Fc human GMP-derived macrophages, but not CAR- RFP human GMP-derived macrophages, promoted nearby WT macrophage phagocytosis in vitro.Example 4 - Efficacy of allogeneic anti-CD19 CAR-Fc mGMP in A20 lymphoma model

[0152] Human CD19-luciferase expressing A20 cells were I V. injected into BALB / c mice at lxl0A5 per mouse 4 h after 500 cGy X-ray irradiation. lxlOA7 RFP control-, aCD19 CAR-, or aCD19 CAR-Fc-GMPs derived from C57 mice were infused on days 4, 18, and 30 via I. V. injection. Tumor progression was monitored and analyzed by bioluminescent imaging, and survival rates were assessed. (n=10 mice per group). As shown in FIG. 5, CAR-Fc-GMPs were the most effective at preventing tumor progression and increasing survival rates.Example 5 - Efficacy of allogeneic anti-Her2 CAR and CAR-Fc mGMP in 4Tl-hHer2 tumor model

[0153] Human HER2 expressing 4T1 cells were injected into the mammary pad of BALB / c mice at 2xl05per mouse 1 day after 450 cGy X-ray irradiation. IxlO7Luciferase-RFP control-, aHer2 CAR-Luc-, or aHER2 CAR-Fc-Luc-GMPs derived from C57 mice were infused on days 0, 7, and 14 via I.V. injection. GMP infiltration and biodistribution was monitored and analyzed by bioluminescent imaging (FIG. 6A). Tumor size was measured by caliper at designed timepoints, and survival rate was assessed. (n=5 mice per group). As shown in FIGS. 6B-6D, the Her2 CAR-Fc was most effective at reducing tumor size and had the highest rate of survival.

[0154] Furthermore, intratumoral immunostaining for RFP (transplanted cells), CD1 lb (monocyte marker) and F4 / 80 (macrophage marker) at different time points suggested that the transplanted CAR-Fc GMPs gave rise to monocytes and infiltrated into the tumor where they further differentiated into macrophages (FIGS. 6E and 6F).

[0155] Intratumoral immunostaining for RFP (transplanted cells) and F4 / 80 (macrophage marker) revealed that the number of macrophages derived from transplanted CAR-Fc GMPs was significantly higher than in the CAR and RFP control groups (FIGS. 6G and 6H)534915-0966-6435, v. 1

[0156] Intratumoral immunostaining for CD3 (pan T cell marker) and Granzyme B (GZB, cytotoxic T cell marker) revealed that the numbers of both pan T cells and cytotoxic T cells in the CAR-Fc GMPs group were significantly higher than those in the CAR and RFP control groups. These results suggest that CAR-Fc GMP treatment enhances antigen presentation in the tumor (FIGS. 6I-6K)Example 6 -CAR-Fc enables antigen presentation in MHC-mismatched settings

[0157] A central limitation of engineered myeloid therapies is that donor macrophages do not engage the host’s existing antigen presenting cell (APC) network, and in allogeneic contexts, non-self MHC restricts the ability to activate host neoantigen-specific T cells. To address this, the CAR was modified to include an extracellular IgGl Fc domain (CAR-Fc; FIG. 1), allowing donor CARs to drive FC-receptor engagement on host phagocytes This architecture preserved direct, or cis, phagocytosis by the CAR-expressing cell while also enabling Fc-dependent trans phagocytosis by adjacent host macrophages (FIG. 2). CAR-Fc was evaluated in a mixed-strain co-culture system that modeled interactions between allogeneic donor CAR-Fc-GMP-derived macrophages and host APCs and T cells (FIG. 7A). In this assay, BALB / c-derived CAR-Fc-GMP macrophages served as donor cells, and C57BL / 6-derived macrophages and OT-II CD4 T cells represented the host compartment. Tumor cells expressed HER2 and the peptide OVA323-339, which is specifically recognized by OT-II T cells only when presented on matched C57BL / 6 MHC-II (PMID: 9553774). This configuration ensured that antigen-specific T-cell activation could occur only through host macrophages. Both aHER2 CAR and CAR-Fc macrophages performed cis phagocytosis, but only CAR-Fc induced Fc-dependent trans phagocytosis by host macrophages (FIG. 7B). This distinction dictated downstream immune activation. Control and conventional CAR cultures failed to activate OT-II cells, whereas CAR-Fc cultures induced robust OT-II proliferation and cytokine production (FIGS. 7C and 7D and 8). Thus, CAR-Fc preserved intrinsic phagocytic function while enabling antigen transfer to MHC-compatible host APCs, restoring antigen-specific T- cell engagement across MHC barriers and providing a mechanistic basis for evaluating allogeneic CAR-Fc in vivo.Example 7 - CAR-Fc enhances allogeneic anti-tumor activity in vivo

[0158] Having shown that CAR-Fc restores antigen presentation and T-cell engagement across MHC barriers in vitro, its therapeutic efficacy was next assessed in vivo. Mouse GMPs expressing conventional CAR or CAR-Fc constructs were tested in two544915-0966-6435, v. 1syngeneic tumor models to determine whether Fc-mediated host engagement augments antitumor responses in allogeneic contexts. CD19+A20 disseminated lymphoma was used for the first model (FIG. 7E). Similar to aHER2, aCD19 CAR-FC efficiently induced both cis and trans phagocytosis of CD 19+ A20 cells, while aCD19 CAR only induced cis phagocytosis (FIGS. 4B and 4C). Mice receiving control (Null CAR) GMPs developed rapid lymphoma burden, while aCD19 CAR GMPs modestly delayed tumor progression whereas aCD19 CAR- Fc GMPs achieved dramatic suppression in most mice (FIGS. 5B and 5C). In a similar manner, aCD19 CAR modestly extended survival, while aCD19 CAR achieved durable disease control with four of five mice surviving beyond day 60 (FIG. 5D). To evaluate efficacy in a solid tumor setting, aHER2 CAR and CAR-Fc GMPs were compared in the orthotopic 4T1 carcinoma model (FIG. 7F). Regardless of CAR construct, CD1 lb+ cells were abundant in the tumors on day 8 that had transitioned into F4 / 80+ macrophages by day 11 (FIGS. 6E and 6F). Tumors from CAR-Fc-treated mice contained higher densities of both CD3+and granzyme+T cells relative to CAR or control groups (FIGS. 6I-6K). These coordinated cellular responses correlated with reduced tumor burden (FIG. 7G) and prolonged survival (FIG. 7H). Collectively, these findings indicate that Fc incorporation enhances myeloid persistence, promotes host immune activation, and confers superior allogeneic anti-tumor activity across both hematologic and solid tumor models.* * *

[0159] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.554915-0966-6435, v. 1

Claims

CLAIMS1. A chimeric antigen receptor comprising an extracellular domain capable of binding to an antigen, an Fc region, a linker, a transmembrane domain, and an intracellular domain.

2. The chimeric antigen receptor of claim 1, comprising the intracellular domain that is designed to increase the anti-tumor activities of granulocytes, macrophages, and dendritic cells by increasing their phagocytosis and / or proinflammatory cytokines secretion and / or antigen presentation.

3. The chimeric antigen receptor of claim 1 or 2, wherein the extracellular domain is a single-chain antibody variable fragment (scFv), a full-length heavy chain, a Fab fragment, or a divalent single chain antibody or diabody.

4. The chimeric antigen receptor of any one of claims 1-3, wherein the antigen comprises CD19, HER2, PD-1, EGFR, EGFRvIII, GPC3, mesothelin, uPAR, CD33, amyloid 0, PSMA, CD 117, MPL, or FAP.

5. The chimeric antigen receptor of any one of claims 1-4, wherein the Fc region comprises a CH2CH3 region of IgGl, or IgG4.

6. The chimeric antigen receptor of any one of claims 1-5, wherein the linker comprises an amino acid sequence of GGGGS (SEQ ID NO: 1).

7. The chimeric antigen receptor of any one of claims 1-3, wherein the transmembrane domain is derived from CD8, CD28, or FcyR transmembrane domain.

8. The chimeric antigen receptor of claim 1, wherein the intracellular domain comprises CD3(^ and / or FcsRIy.

9. The chimeric antigen receptor of claim 1, wherein the intracellular domain comprises TIR, ID3, IL- 12, and / or NFKB.

10. A vector comprising the chimeric antigen receptor of any one of claims 1-9.

11. The vector of claim 10, wherein the vector is a viral vector.

12. The vector of claim 11, wherein the viral vector is replicating or non-replicating, and is an adenoviral vector, an adeno-associated virus (AAV) vector, a measles vector, a herpesvector, a retroviral vector, a lentiviral vector, a rhabdoviral vector, a reovirus vector, a Seneca Valley Virus vector, a poxvirus vector, a parvovirus vector, or an alphavirus vector.

13. The vector of claim 12, wherein the viral vector is a lentiviral vector.

14. A nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 1-9.

15. The nucleic acid molecule of claim 14, wherein the nucleic acid molecule is an RNA.

16. A method to genetically engineer granulocyte-macrophage progenitors (GMPs) to express a chimeric antigen receptor comprising: introducing the vector of any one of claims 10-13 or the nucleic acid molecule of claim 14 or 15 into GMPs to form GMPs that express the chimeric antigen receptor (CAR- Fc-GMPs); and expanding and culturing the CAR-Fc-GMPs for multiple passages in defined culture conditions to generate a population of CAR-Fc-GMPs.

17. The method of claim 16, further comprising inducing the population of CAR-Fc-GMPs to differentiate into granulocytes, macrophages, or dendritic cells in vitro, wherein the granulocytes, macrophages, or dendritic cells express the chimeric antigen receptor.

18. The method of claim 16, wherein the GMPs are obtained from stem cells.

19. The method of claim 18, wherein the stem cells are hematopoietic stem cells.

20. The method of claim 19, wherein the hematopoietic stem cells are isolated from the bone marrow or mobilized peripheral blood of a subject.

21. The method of claim 20, wherein the subject is a mammalian subject.

22. The method of claim 21, wherein the subject is a human patient.

23. The method of any one of claims 16-22, wherein the defined culture conditions include culturing the CAR-Fc-GMPs in a culture medium comprising at least two components selected from the group consisting of:574915-0966-6435, v. 1(i) a growth factor,(ii) a B-Raf kinase inhibitor, and(iii) a TN2-30.

24. The method of claim 23, wherein the culture medium comprises IMDM.

25. The method of claim 23 or 24, wherein the culture medium comprises one or more supplements selected from the group consisting of insulin, holo-transferrin, sodium selenite, linoleic acid, vitamin E, and human serum albumin.

26. The method of any one of claims 23 to 25, wherein the growth factor comprises stem cell factor (SCF).

27. The method of any one of claims 23 to 26, wherein the B-Raf kinase inhibitor is selected from the group consisting of GDC-0879, PLX4032, GSK2118436, BMS-908662, LGX818, PLX3603, RAF265, RO5185426, vemurafenib, PLX8394, SB590885 and any combination thereof.

28. The method of any one of claims 23 to 27, wherein the B-Raf kinase inhibitor is GDC- 0879.

29. The method of any one of claims 23 to 28, wherein the culture medium further comprises human IL-3.

30. The method of any one of claims 23-29, wherein the culture medium further comprises human G-CSF.

31. The method of any one of claims 23-30, wherein the culture medium further comprises PY-60.

32. The method of any one of claims 16-31, wherein the CAR-Fc-GMPs are induced to differentiate into a population of macrophages comprising: culturing the CAR-Fc-GMPs with a macrophage differentiation medium comprising GM-CSF, wherein the macrophages express the CAR-Fc.584915-0966-6435, v.

133. The method of claim 32, wherein the macrophage differentiation medium comprises RPMI 1640, fetal bovine serum (FBS) and MCSF.

34. The method of any one of claims 16 to 31, further comprising differentiating the CAR- Fc-GMPs into a population of granulocytes comprising: culturing the GMPs with a granulocyte differentiation medium comprising granulocyte colony-stimulating factor (GCSF), wherein the granulocytes express the CAR-Fc.

35. The method of claim 34, wherein the granulocyte differentiation medium comprises RPMI 1640, FBS and GCSF.

36. A population of granulocyte-macrophage progenitors (GMPs) that express a CAR-Fc, wherein the GMPs are prepared by the method of any one of claims 16-31.

37. A population of macrophages that express a CAR-Fc, wherein the macrophages are prepared by the method of claim 32 or claim 33.

38. A population of granulocytes that express a CAR-Fc, wherein the granulocytes are prepared by the method of claim 34 or claim 34.

39. An immunotherapy method for treating a subject having a cancer or an autoimmune disease with macrophages or granulocytes that express a chimeric antigen receptor (CAR-Fc): administering a composition comprising the GMPs of claim 36 or the macrophage of claim 37 or the granulocyte of claim 38 to the subject having the cancer or the autoimmune disease.

40. The immunotherapy method of claim 39, wherein the composition is administered intravenously or inter-tumoral.

41. The immunotherapy method of claim 39 or 40, wherein the subject has a cancer selected from adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, anorectal cancer, cancer of the anal canal, appendix cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), biliary cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, urinary bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brain stem glioma, cerebellar astrocytoma, cerebral594915-0966-6435, v. 1astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, including triple negative breast cancer, bronchial adenomas / carcinoids, carcinoid tumor, gastrointestinal, nervous system cancer, nervous system lymphoma, central nervous system cancer, central nervous system lymphoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary Syndrome, endometrial cancer, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor glioma, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, ocular cancer, islet cell tumors (endocrine pancreas), Kaposi Sarcoma, kidney cancer, renal cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, AIDS-related lymphoma, nonHodgkin lymphoma, primary central nervous system lymphoma, Waldenstram macroglobulinemia, medulloblastoma, melanoma, intraocular (eye) melanoma, merkel cell carcinoma, mesothelioma malignant, mesothelioma, metastatic squamous neck cancer, mouth cancer, cancer of the tongue, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, ovarian low malignant potential tumor, pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter, transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, ewing family of sarcoma tumors, soft tissue sarcoma, uterine cancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), papillomas, actinic keratosis and keratoacanthomas, merkel cell skin carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer, supratentorial primitive neuroectodermal604915-0966-6435, v. 1tumors, testicular cancer, throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter and other urinary organs, gestational trophoblastic tumor, urethral cancer, endometrial uterine cancer, uterine sarcoma, uterine corpus cancer, vaginal cancer, vulvar cancer, and Wilm's Tumor.

42. The immunotherapy method of any one of claims 39-41, further comprising administering one or more anticancer agents to the subject having cancer.

43. The immunotherapy method of claim 39, wherein the subject has an autoimmune disease selected from Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), Type 1 Diabetes (T1D), Sjogren’s syndrome, and Multiple Sclerosis (MS).614915-0966-6435, v. 1

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