WO2025024849A1

WO2025024849A1 - Compositions and methods of making and using ipsc-derived car macrophages

Info

Publication number: WO2025024849A1
Application number: PCT/US2024/040031
Authority: WO
Inventors: Jianhua Yu; Michael A. Caligiuri; Zahir Shah
Original assignee: City Of Hope
Priority date: 2023-07-27
Filing date: 2024-07-29
Publication date: 2025-01-30

Abstract

This disclosure relates, inter alia, to compositions comprising and methods making and using iPSC-derived macrophages expressing a chimeric antigen receptor (CAR) targeted one or more tumor antigens (e.g., prostate stem cell antigen (PSCA)).

Description

Attorney Docket No.40056-0101WO1 Compositions and Methods of Making and Using iPSC-derived CAR Macrophages CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application Serial No. 63/516,057, filed on July 27, 2023. The entire contents of the foregoing are incorporated herein by reference. TECHNICAL FIELD This disclosure relates to induced pluripotent stem cells (iPSCs) -derived macrophages expressing a transgene, for example IL-15 or a chimeric antigen receptor (CAR). BACKGROUND Cell-based immunotherapy, such as chimeric antigen receptor (CAR)-T cell therapy, has become a promising approach to augment antitumor immunity and clearance of cancer. CAR-T cells have shown clinical efficacy in numerous hematological malignancies, leading to the U.S. FDA-approval of several CD19 and BCMA targeted CAR-T cell products.¹ However, solid tumors have an immunosuppressive and relatively inaccessible tumor microenvironment (TME), resulting in poor activation and tumor penetration of immune effector cells such as T cells.² Thus, the development of innovative and potent cell therapies capable of overcoming these limitations remains a critical unmet need. SUMMARY This application is based, at least in part, on the discovery of an efficient in vitro myeloid skewed hematopoietic differentiation protocol that can be used to generate iPSC- derived mature macrophages (iMac or iMacs) that were of high yield and purity. This application is also based, inter alia, on the discovery that the iPSCs can be further engineered to express a transgene, for example, a chimeric antigen receptor to create CAR-iPSCs at the single cell level, expanded, and frozen as a source of cells for future use as needed. Attorney Docket No.40056-0101WO1 Described herein, inter alia, are methods for making and using phenotypically defined, functional, and/or expandable macrophages expressing a chimeric antigen receptor. These macrophages are derived from pluripotent stem cells embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). In some embodiments, the cells described herein target a specific predetermined antigen expressed on the cell surface of a target cell (e.g., a cancer cell), possess enhanced functional potential, enhance survival and treatment of cancers and/or targeted diseases, and/or possess anti-tumor activity. The CAR macrophages described herein may be used as “off-the-shelf” cells for administration to multiple recipients. In some embodiments, human blood is starting material for generating iPSC- derived macrophages. In some embodiments, cord blood or peripheral blood mononuclear cells (PBMCs) are the starting material for generating iPSC-derived macrophages. In some embodiments, described herein are methods for preparing a population of macrophages expressing a chimeric antigen receptor (CAR), the methods comprising: (a) isolating a population of CD34⁺ hematopoietic stem and progenitor cells (HSPCs); (b) generating induced pluripotent stem cells (iPSCs) from the HSPCs; (c) introducing a nucleic acid molecule comprising a nucleotide sequence encoding a transgene, for example, a CAR or bound IL-15 into the iPSCs, thereby creating iPSCs harboring a transgene; and (d) selecting a iPSC harboring a transgene and generating a clonal population of a iPSCs from the selected iPSC; and (e) differentiating at least a portion of the clonal population of iPSCs into macrophages expressing a transgene (T-iMacs), for example, a CAR (CAR- iMacs). In some embodiments, the HSPCs are isolated from human blood or human cord blood. In some embodiments, the HSPCs are human. In some embodiments, the nucleic acid molecule encodes both a CAR and bound IL-15 (e.g., membrane bound IL-15). In some embodiments they are co-expressed. In some embodiments the nucleic acid molecule encodes CAR (e.g., a PSCA CAR), at least Attorney Docket No.40056-0101WO1 one ribosomal skip sequence (e.g., T2A or P2A or T2A and P2A) and a membrane-bound IL-15 (e.g., SEQ ID NO: 50). In some embodiments, the HSPCs are reprogrammed to generate iPSCs. In some embodiments, the iPSCs are generated by contacting the HSPCs with one or more of OCT3/4, OCT3, OCT4, SOX2, KLF4, L-MYC, C-MYC, LIN28, or short hairpin RNA targeting TP53 (shRNA-TP53). In some embodiments, the nucleic acid is an RNA (e.g., an mRNA) or a DNA. In some embodiments, the nucleic acid is a vector. In some embodiments, the vector is a viral vector. In some embodiments, viral vector is selected from a baculovirus, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno- associated viral vector, or Herpes viral vector. In some embodiments, the CAR iPSCs also express a membrane-bound interleukin-15 (mIL15 or mIL-15) or soluble IL-15 (sIL15 or sIL-15). In some embodiments, the CAR iPSCs also express a truncated version of EGFR (tEGFR) or a truncated version of CD19. The CAR can be any of the CAR described herein. In some embodiments, the step of differentiating the CAR iPSCs into CAR iMacs comprises: (a) culturing the CAR iPSCs in a hematovascular induction medium comprising Thiazovivin, BMP4, and hVEGF165; (b) replacing the hematovascular induction medium with a hematopoietic specification medium comprising hVEGF₁₆₅, human fms-like tyrosine kinase 3 ligand (Flt3L), human stem cell factor (SCF), Insulin-like growth factor 1(IGF-1), human interleukin 3 (IL-3), StemRegenin 1, and TGF-βRI inhibitor (SB-431542); (c) replacing the hematovascular induction medium with a myeloid hematopoietic differentiation media comprising 10 ng/mL human interleukin 6 (IL-6), and 20 ng/mL human thrombopoietin (TPO); and (d) replacing the myeloid hematopoietic differentiation media with a macrophage differentiation medium comprising L-glutamine, hM-CSF, IL-1β, and IL-6. In some embodiments, the hematovascular induction medium comprises 10 μM Thiazovivin, 10 ng/mL BMP4, and 50 ng/mL hVEGF₁₆₅. In some embodiments, the hematopoietic specification medium comprises 25 ng/mL hVEGF₁₆₅, 20 ng/mL Flt3L, 20 ng/mL SCF, 20 ng/mL IGF-1, 10 ng/mL IL-3, 2 µM StemRegenin 1 (CAS No.1227633- Attorney Docket No.40056-0101WO1 49-9; 4-[2-[[2-Benzo[b]thien-3-yl-9-(1-methylethyl)-9H-purin-6-yl]amino]ethyl]phenol), and TGF-βRI inhibitor (SB-431542; CAS No.301836-41-9; 4-[4-(3,4- Methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide). In some embodiments, the myeloid hematopoietic differentiation medium comprises 10 ng/mL IL-6 and 20 ng/mL TPO. In some embodiments, the macrophage differentiation medium comprises 2mM L-glutamine, 50 ng/mL hM-CSF, 10 ng/mL IL- 1β, and 10 ng/mL IL-6. In some embodiments, the cells are cultured in the macrophage differentiation medium for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days. In some embodiments, the step of differentiating the CAR iPSCs into CAR iMacs comprises using a matrix-based culture system. In some embodiments, the step of generating iPSCs from the HSPCs comprises using a matrix-based culture system. In some embodiments, the CAR iPSCs are cultured in the hematovascular induction medium for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 2426, 27, 28, 29, or 30 days. In some embodiments, the CAR iPSCs are cultured in the hematovascular specification medium for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 2426, 27, 28, 29, or 30 days. In some embodiments, the CAR iPSCs are cultured in the myeloid hematopoietic differentiation medium for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 2426, 27, 28, 29, or 30 days. In some embodiments, the CAR iPSCs are cultured in the macrophage differentiation medium for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 2426, 27, 28, 29, or 30 days. In some embodiments, CAR iPSCs are generated contacting the iPSCs with a nucleic acid or vector encoding a CAR. In some embodiments, the nucleic acid is an RNA (e.g., an mRNA) or a DNA. In some embodiments, the nucleic acid is a vector. In some embodiments, the vector is a viral vector. In some embodiments, viral vector is selected from a baculovirus, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or Herpes viral vector. In some embodiments, transduced CAR iPSCs are cultured for at least 2 passages before single cell sorting and iPSC colonization. In some embodiments, colonized CAR IPSCs are expanded and banked for differentiation. Attorney Docket No.40056-0101WO1 In some embodiments, the CAR iPSCs are differentiated into CAR iMacs cells using a matrix-based culture system. In some embodiments, the CAR-iMacs express CD45, CD14, CD11b, CD86, CD63, CD80, and CD16. In some embodiments, the CAR-iMacs express CD45, CD14, CD11b, CD86, CD63, CD80, and CD16, and have low or no expression of CD206. In some embodiments, the CAR-iMacs express CD45, CD14, CD11b, CD86, CD63, CD80, and CD16, and do not express CD206. In some embodiments, the CAR is specific for a tumor, a cell surface marker, and/or comprise a toxin. In some embodiments, the CAR targets any one or more of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CDS, CD6, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CS1, chlorotoxin receptor, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb- B2,3,4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptors, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), Interleukin-13 receptor subunit alpha-2 (IL- 13Rα2), light chain kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), LI cell adhesion molecule (LICAM), melanoma antigen family A, 1 (MAGE-AI), Mucin 16 (Muc-16), Mucin 1 (Muc-1), Mesothelin (MSLN), NKG2D ligands, cancer- testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor- associated glycoprotein 72 (TAG- 72), vascular endothelial growth factor R2 (VEGF- R2), Wilms tumor protein (WT-1), or combinations thereof. In some embodiments, the CAR is bispecific. In some embodiments, the chimeric antigen receptor comprises: at least one targeting domain, a spacer, a transmembrane domain, a co-stimulatory domain, and a CD3 ζ signaling domain. In some embodiments, a PSCA targeted CAR iMacs co- expressing an IL-15 domain (e.g., a membrane-bound IL-15, at least a portion of IL-15, at least a portion of IL-15Ra, or a fusion protein that includes at least a portion of IL-15 Attorney Docket No.40056-0101WO1 and at least a portion of IL-15Ra) to treat a variety of solid tumors, (e.g., non-small cell lung carcinoma, gall bladder cancer, pancreatic cancer, prostate cancer, and urinary bladder cancer). The PSCA CAR iMacs described herein possess potent antigen-specific anti-tumor efficacy in vitro and in vivo. Described herein, inter alia, are nucleic acid molecules comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a polypeptide, wherein the chimeric antigen receptor or polypeptide comprises: an scFv targeting PSCA, a spacer, a transmembrane domain, a co-stimulatory domain, and a CD3 ζ signaling domain, and a nucleotide sequence encoding a polypeptide comprising an IL-15 domain and a nucleotide sequence encoding a polypeptide comprising a tEGFR domain. In certain embodiments: the scFv comprises the amino acid sequence of SEQ ID NO:1, or 40; the scFv comprises the amino acid sequence of SEQ ID NO:32 and the amino acid sequence of SEQ ID NO:33; the transmembrane domain is selected from: a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, and a NKG2D transmembrane domain; the transmembrane domain is a CD28 transmembrane domain or a NKG2D transmembrane domain; the costimulatory domain is a CD28, a 4-1BB, or a 2B4 costimulatory domain; the costimulatory domain comprises the amino acid sequence of any of SEQ ID NOs:22-25 and 66; the CD3ζ signaling domain comprises the amino acid sequence of SEQ ID NO:21; a linker of 3 to 15 amino acids is located between the costimulatory domain and the CD3 ζ signaling domain or variant thereof; the spacer comprises any one of SEQ ID NOs:2-12 and 44; the CAR or the polypeptide comprises the amino acid sequence of SEQ ID NO:34, 35, 41, or 42; or a variant thereof having 1-5 amino acid modifications; the IL-15 domain comprises a membrane-bound IL-15; the IL-15 domain comprises SEQ ID NO:43, SEQ ID NO:50, SEQ ID NO: 51, SEQ ID NO:52, or SEQ ID NO:53; the IL-15 domain is soluble or membrane-bound IL-15; the nucleic acid molecule encodes a polypeptide comprising tEGFR or tCD19 domain; the nucleic acid molecule is an mRNA molecule or a vector. Also described are expression vectors comprising a nucleic acid molecule described herein and a population of human immune cells (e.g., macrophages or iMacs) transduced by the vector or harboring a nucleic acid molecule (e.g., a vector or mRNA) described herein. Also described are populations of human immune cells (e.g., Attorney Docket No.40056-0101WO1 macrophages or iMacs) expressing the protein encoded by one or more of the nucleic acid molecules described herein. In some embodiments, a population of iMacs expressing a CAR (CAR iMacs), wherein the CAR comprises: a single chain variable fragment (scFv) targeting a cancer cell antigen. In some embodiments, the CAR further comprises a spacer, a transmembrane domain, and at least one intracellular domain. In some embodiments, the CAR iMacs also express mIL15 and/or a tEGFR or a truncated version of CD19 (tCD19). The CAR iMacs described here can be M0, M1, or M2, or a combination thereof. Also described are compositions comprising a population of iPSC-derived macrophages produced by any of the methods described herein. In some embodiments, described herein is a composition comprising the iPSC-derived macrophages (iMacs). In some embodiments, a composition comprising iMacs has enhanced therapeutic properties. In some embodiments, the iMacs demonstrate enhanced functional activity including potent cytokine production, cytotoxicity and cytostatic inhibition of tumor growth, e.g. as activity that reduces the amount of tumor load. In some embodiments, described herein are methods of increasing survival of a subject having cancer comprising administering a population of CAR iMacs described herein. In some embodiments, described herein are methods of treating a cancer in a patient comprising administering a population of CAR iMacs described herein. In some embodiments, described herein are methods of reducing or ameliorating a symptom associated with a cancer in a patient comprising administering a population of CAR iMacs described herein. In some embodiments, a population of CAR iMacs described herein is administered locally or systemically. In some embodiments, a population of CAR iMacs described herein is administered by single or repeat dosing. In some embodiments, a population of CAR iMacs described herein is administered to a patient having a cancer, a pathogen infection, an autoimmune disorder, or an allogeneic transplant. In some embodiments, the cancer is selected from the group consisting of blood cancer, B cell leukemia, multiple myeloma, lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin's lymphoma, ovarian cancer, prostate cancer, pancreatic cancer, lung cancer, breast cancer, and sarcoma, acute myeloid leukemia Attorney Docket No.40056-0101WO1 (AML). In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from the group consisting of pancreatic cancer, prostate cancer, and urinary bladder. In some embodiments, the cancer is any cancer or tumor that comprises a PSCA-positive cell. Also described are methods of treating a solid tumor or cancer in a patient comprising administering a population of autologous or allogeneic human iMacs transduced by a vector comprising the nucleic acid molecule described herein, wherein the solid tumor or cancer comprises cells expressing PSCA. In various embodiments: the population of iMacs expressing the chimeric antigen receptor or the polypeptide is administered locally or systemically; the population of human iMacs expressing the chimeric antigen receptor or the polypeptide is administered by single or repeat dosing. In some embodiments, described herein are methods of treating a solid tumor or a method of reducing or eliminating PSCA-positive cells. In some embodiments, a solid tumor is any one or more of a pancreatic cancer, prostate cancer, bladder cancer, gastric cancer, breast cancer, cervical cancer, endometrial cancer, esophageal cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, etc. or a subpopulation of these or other cancers. Also described herein are methods of treating PSCA-positive cancers or disorders (including, e.g., pancreatic cancer, prostate cancer, and urinary bladder cancer) in a patient comprising administering a population of autologous or allogeneic human iMacs transduced comprising a nucleic acid molecule described herein, wherein the PSCA-positive cancers or disorders comprise cells expressing PSCA, thereby treating the PSCA-positive cancers or disorders. In various embodiments: human iMacs expressing a chimeric antigen receptor or polypeptide described herein are administered locally or systemically; the PSCA-expressing target cells are cancerous cells; and the human iMacs expressing chimeric antigen receptor or polypeptide are administered in a therapeutically effective amount by single or repeat dosing. Chimeric Antigen Receptors The chimeric antigen receptor comprises: an antibody single chain variable fragment (scFv) targeting an antigen (e.g., PSCA), a spacer, a transmembrane domain, a Attorney Docket No.40056-0101WO1 co-stimulatory domain, and a CD3 ζ signaling domain. In some cases, a small spacer is located between the co-stimulatory domain and the CD3 ζ signaling domain. In various embodiments: the transmembrane domain is selected from: a CD4 transmembrane domain or variant thereof having 1-5 amino acid modifications, a CD8 transmembrane domain or variant thereof having 1-5 amino acid modifications, a CD28 transmembrane domain or a variant thereof having 1-5 amino acid modifications, a NKG2D transmembrane domain or a variant thereof having 1-5 amino acid modifications; the spacer comprises 20-150 amino acids and is located between the scFv and the transmembrane domain; the spacer is an IgG4 hinge domain or variant thereof having 1-5 amino acid modifications; the spacer is a IgG1 hinge domain; the chimeric antigen receptor comprises a transmembrane domain selected from: a CD4 transmembrane domain or variant thereof having 1-2 amino acid modifications, a CD8 transmembrane domain or variant thereof having 1-2 amino acid modifications, a CD28 transmembrane domain or a variant thereof having 1-2 amino acid modifications, a NKG2D transmembrane domain or a variant thereof having 1-2 amino acid modifications; the spacer region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-12, 44 or a variant thereof having 1-5 amino acid modifications; the spacer comprises an IgG hinge region; the spacer comprises 10-50 amino acids; the costimulatory domain comprises the amino acid sequence of SEQ ID NO: 22, 23, 24, or 66 or a variant thereof having 1-5 amino acid modifications; the CD3ζ signaling domain comprises the amino acid sequence of SEQ ID NO:21 or a variant thereof having 1-5 amino acid modifications; a linker of 3 to 15 amino acids can be located between the costimulatory domain and the CD3 ζ signaling domain or variant thereof; the CAR or polypeptide comprises the amino acid sequence of SEQ ID NO: 34, 35, 41, or 42 or a variant thereof having 1-5 amino acid modifications; the scFv comprises the amino acid sequence of any of SEQ ID NO:1, 40, or 32 and 33, or a variant thereof having 1-5 amino acid modifications. In another aspect, the nucleic acid molecule encodes an scFv comprising the amino acid sequence of SEQ ID NO: 1, 40, or an equivalent of each thereof, or a variant of each thereof having 1, 2, 3, 4, 5, or 6 amino acid substitutions, wherein the substitutions are conservative and not in the CDRs. Attorney Docket No.40056-0101WO1 In another aspect, the nucleic acid molecule encodes a CAR comprising the amino acid sequence of SEQ ID NO: 34, 35, 41, 42, or an equivalent of each thereof, or a variant of each thereof having 1, 2, 3, 4, 5, or 6 amino acid substitutions, wherein the substitutions are conservative and not in the CDRs. In various embodiments: the chimeric antigen receptor or polypeptide comprises: a PSCA scFv (LH), e.g., an scFv comprising the amino acid sequence DIQLTQSPSTLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWGSSPFTFGQGTKVEIKGSTSGG GSGGGSGGGGSSEVQLVEYGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPG KGLEWVAWIDPENGDTEFVPKFQGRATMSADTSKNTAYLQMNSLRAEDTAVY YCKTGGFWGQGTLVTVSS (SEQ ID NO:1), with up to 5 (e.g., 1, 2, 3, 4, or 5 amino acid substitutions) or up to 10 single amino acid substitutions. In some embodiments, the amino acid substitutions are not in the CDRs. In various embodiments: the chimeric antigen receptor or polypeptide comprises: a PSCA scFv (HL), e.g., an scFv comprising the amino acid sequence EVQLVEYGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDP ENGDTEFVPKFQGRATMSADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQG TLVTVSSGGGSGGGSGGGGSSDIQLTQSPSTLSASVGDRVTITCSASSSVRFIHWY QQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQW GSSPFTFGQGTKVEIKGSTS (SEQ ID NO:40) with up to 5 (e.g., 1, 2, 3, 4, or 5 amino acid substitutions) or up to 10 single amino acid substitutions. In some embodiments, the amino acid substitutions are not in the CDRs. In some embodiments, the PSCA scFv comprises a light chain variable region (VL) that is at least 95% identical to or includes up to 5 single amino acid substitutions (preferably outside the CDRs, underlined) compared to: DIQLTQSPSTLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWGSSPFTFGQGTKVEIKGSTS (SEQ ID NO: 32). In some embodiments, the PSCA scFv comprises a heavy chain variable region (VH) that is at least 95% identical to or includes up to 5 single amino acid substitutions (preferably outside the CDRs, underlined) compared to: Attorney Docket No.40056-0101WO1 EVQLVEYGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDP ENGDTEFVPKFQGRATMSADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQG TLVTVSS (SEQ ID NO: 33). In some embodiments, the PSCA scFv comprises a light chain variable region (VL) that comprises a CDR1 having the sequence SASSSVRFIH (SEQ ID NO: __); a CDR2 having the sequence DTSKLAS (SEQ ID NO: __) and a CDR3 having the sequence QQWGSSPFT (SEQ ID NO: __). In some embodiments, the PSCA scFv comprises a heavy chain variable region (VH) that comprises a CDR1 having the sequence DYYIH (SEQ ID NO: __); a CDR2 having the sequence WIDPENGDTEFVPKFQG (SEQ ID NO: __); and a CDR3 having the sequence GGF (SEQ ID NO: __). The PSCA targeted CAR (also called “PSCA CAR”) or PSCA targeted polypeptide (also called “PSCA polypeptide”) described herein include a PSCA targeting scFv, e.g., a PSCA scFv described above. In some embodiments, an scFv comprising the amino acid sequence: DIQLTQSPSTLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWGSSPFTFGQGTKVEIKGST (SEQ ID NO:32) and the sequence EVQLVEYGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDP ENGDTEFVPKFQGRATMSADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQG TLVTVSS (SEQ ID NO:33) (in either order) joined by a flexible linker. In some embodiments, a useful flexible linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of the sequence GGGS (SEQ ID NO:38). In some embodiments, a useful flexible linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of the sequence GGGGS (SEQ ID NO:39). A useful linker could comprise (G4S)3: GGGGSGGGGSGGGGS (SEQ ID NO:37). A useful PSCA CAR or PSCA polypeptide can consist of or comprises the amino acid sequence of SEQ ID NO:41 or SEQ ID NO: 42 (mature CAR lacking a signal sequence). Any disclosed CAR or polypeptide can be expressed in a form that includes a signal sequence, e.g., a human GM-CSF receptor alpha signal sequence (MLLLVTSLLLCELPHPAFLLIP; SEQ ID NO:36); a IgGk signal peptide Attorney Docket No.40056-0101WO1 (METDTLLLWVLLLWVPGSTG; SEQ ID NO:29); a IgG2 signal peptide (MGWSSIILFLVATATGVH; SEQ ID NO:30); a IL-2 signal peptide (MYRMQLLSCIALSLALVTNS; SEQ ID NO:31). The CAR or polypeptide can be expressed with additional sequences that are useful for monitoring expression, for example, a T2A or P2A skip sequence and a truncated EGFR or truncated CD19 and/or a membrane bound IL-15 (e.g., mIL15). The CAR or polypeptide can comprise an scFv targeted to PSCA. Thus, the CAR or polypeptide can comprise the amino acid sequence of SEQ ID NOs: 1 or 40-42 or can comprise an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 1 or 40-42. The CAR or polypeptide can comprise the amino acid sequence of any of SEQ ID Nos 1 or 40-42 with up to 1, 2, 3, 4 or 5 amino acid changes (preferably conservative amino acid changes). The CAR or polypeptide can comprise SEQ ID NO:32 with up to 1, 2, 3, 4 or 5 amino acid changes (preferably conservative amino acid changes) and SEQ ID NO:33 with up to 1, 2, 3, 4 or 5 amino acid changes (preferably conservative amino acid changes) joined by a flexible linker. The CAR or polypeptide can comprise SEQ ID NO:34 with up to 1, 2, 3, 4 or 5 amino acid changes (preferably conservative amino acid changes) and SEQ ID NO:35 with up to 1, 2, 3, 4 or 5 amino acid changes (preferably conservative amino acid changes) joined by a flexible linker. In some embodiments, the nucleic acid encoding amino acid sequences SEQ ID NOs:1, 32-35, and 40-42 are codon optimized. Spacer Region The CAR or polypeptide described herein can include a spacer located between the PSCA targeting domain (i.e., a PSCA targeted ScFv or variant thereof) and the transmembrane domain. A variety of different spacers can be used. Some of them include at least portion of a human Fc region, for example a hinge portion of a human Fc region or a CH3 domain or variants thereof. Table 1 below provides various spacers that can be used in the CARs described herein. Attorney Docket No.40056-0101WO1 Table 1: Examples of Spacers

Attorney Docket No.40056-0101WO1 Some spacer regions include all or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls between the CH1 and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge or a CD8 hinge. Some spacer regions include an immunoglobulin CH3 domain (called CH3 or ΔCH2) or both a CH3 domain and a CH2 domain. The immunoglobulin derived sequences can include one or more amino acid modifications, for example, 1, 2, 3, 4 or 5 substitutions, e.g., substitutions that reduce off-target binding. The spacer region can also comprise an IgG4 hinge region having the sequence ESKYGPPCPSCP (SEQ ID NO:4) or ESKYGPPCPPCP (SEQ ID NO:3). The spacer region can also comprise the hinge sequence ESKYGPPCPPCP (SEQ ID NO:3) followed by the linker sequence GGGSSGGGSG (SEQ ID NO:2) followed by IgG4 CH3 sequence GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:12). Thus, the entire spacer region can comprise the sequence: ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK (SEQ ID NO:11). Transmembrane Domain A variety of transmembrane domains can be used in the CAR. In some cases, the transmembrane domain is a CD28 transmembrane domain that includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:14). In some cases, the CD28 transmembrane domain has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:14. Table 2 includes examples of suitable transmembrane domains. Where a spacer region is present, the transmembrane domain (TM) is located carboxy terminal to the spacer region. Table 2: Examples of Transmembrane Domains

Attorney Docket No.40056-0101WO1

Costimulatory Domain The costimulatory domain can be any domain that is suitable for use with a CD3ζ signaling domain. In some cases, the co-signaling domain is a CD28 co-signaling domain that includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 22). In some cases, the 4-1BB co-signaling domain has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:22. The costimulatory domain(s) are located between the transmembrane domain and the CD3ζ signaling domain. Table 3 includes examples of suitable costimulatory domains together with the sequence of the CD3ζ signaling domain.

Attorney Docket No.40056-0101WO1 Table 3: CD3ζ Domain and Examples of Costimulatory Domains

In various embodiments: the costimulatory domain is selected from the group consisting of: a costimulatory domain depicted in Table 3 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a CD28 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications and an OX40 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications. In certain embodiments, a 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications in present. In some embodiments there are two costimulatory domains, for example a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). In various embodiments the 1-5 (e.g., 1 or 2) amino acid modification are substitutions. The costimulatory domain is amino terminal to the CD3ζ signaling domain and a short linker consisting of 2 – 10, e.g., 3 Attorney Docket No.40056-0101WO1 amino acids (e.g., GGG) can be positioned between the costimulatory domain and the CD3ζ signaling domain. Signaling Domain The CD3ζ signaling domain can be any domain that is suitable for use with a CD3ζ signaling domain. In some cases, the CD3ζ signaling domain includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR (SEQ ID NO:21). In some cases, the CD3ζ signaling has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:21. A CAR or polypeptide can comprise a sequence that is at least 90%, at least 95%, ^{at least 98% identical to or identical to:} DIQLTQSPSTLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWGSSPFTFGQGTKVEIKGSTSGG GSGGGSGGGGSSEVQLVEYGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPG KGLEWVAWIDPENGDTEFVPKFQGRATMSADTSKNTAYLQMNSLRAEDTAVY YCKTGGFWGQGTLVTVSSLEPKSCDKTHTCPPCPDPKGTFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSR VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR (SEQ ID NO:41; PSCA scFv-IGg1 hinge-CD28 TM-CD28 Costim- CD3ζ – Version 1) with up to 5 (e.g., 1, 2, 3, 4, or 5 amino acid substitutions) or up to 10 single amino acid substitutions. In some embodiments, the amino acid substitutions are not in the CDRs. A CAR or polypeptide can comprise a sequence that is at least 90%, at least 95%, ^{at least 98% identical to or identical to:} DIQLTQSPSTLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWGSSPFTFGQGTKVEIKGSTSGG GSGGGSGGGGSSEVQLVEYGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPG KGLEWVAWIDPENGDTEFVPKFQGRATMSADTSKNTAYLQMNSLRAEDTAVY YCKTGGFWGQGTLVTVSSLEPKSCDKTHTCPPCFWVLVVVGGVLACYSLLVTV Attorney Docket No.40056-0101WO1 AFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R (SEQ ID NO:42; PSCA scFv-IGg1 hinge-CD28 TM-CD28 Costim- Version 2)

with up to 5 (e.g., 1, 2, 3, 4, or 5 amino acid substitutions) or up to 10 single amino acid substitutions. In some embodiments, the amino acid substitutions are not in the CDRs. Soluble and Membrane Bound IL-15 The CAR macrophages (CAR-iMacs) can include a nucleic acid molecule that encodes an IL-15 domain, i.e., a domain that includes at least a functional portion of human IL-15 (e.g., amino acids 30-162 human IL-15 isoform I; GenBank NP_0056). In some cases includes a functional portion of human IL-15 receptor alpha subunit isoform I (e.g., amino acids 31-205 of GenBank NP_002180). Thus, the nucleic acid molecule can encode soluble IL-15 (sIL-15), membrane bound IL-15 (mbIL-15 or mIL-15), sIL-15 complex IL-15Rα (sIL-15c), and mbIL-15 complexed with IL-15Rα (mbIL-15c or mIL- 15c), and mimetics thereof. In some cases, the IL-15 domain includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKV TAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEE KNIKEFLQSFVHIVQMFINTS (soluble IL-15; SEQ ID NO:43). In some cases, the IL- 15 domain has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:43. In some embodiments, the IL-15 domain is codon optimized. In some embodiments, the IL-15 domain is membrane-bound and includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVT AMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEK NIKEFLQSFVHIVQMFINTSEQKLISEEDLAVGQDTQEVIVVPHSLPFKVVVISAIL ALVVLTIISLIILIMLWQKKPR (mIL-15; SEQ ID NO:50). In some cases, the mIL-15 domain has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:50. In some embodiments, the mIL-15 domain is codon optimized. Attorney Docket No.40056-0101WO1 In some embodiments, the IL-15 domain includes a transmembrane domain sequence and a soluble IL-15 domain and has a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: VAISTSTVLLCGLSAVSLLACYLGIHVFILGCFSAGLPKTEANWVNVISDLKKIED LIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAN NSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO:BB) In some cases, the transmembrane domain within the IL-15 domain has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:BB. In some embodiments, the IL-15 comprises, or consists essentially of, or yet further consists of an amino acid sequence selected from: GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVT AMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEK NIKEFLQSFVHIVQMFINTSEQKLISEEDLAVGQDTQEVIVVPHSLPFKVVVISAIL ALVVLTIISLIILIMLWQKKPR (SEQ ID NO:50); NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS (SEQ ID NO: 51); GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKV TAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEE KNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 43); MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDL KKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVEN LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 52); or MYRMQLLSCIALSLALVTNSGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQ SMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNS LSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 53); or an equivalent of each thereof. In some cases, one or more self-cleaving peptides are located between the CAR and the IL-15 domain. Non-limiting examples of such include a 2A self-cleaving peptide. A 2A self-cleaving peptide refers to a class of 18–22 aa-long peptides, which can Attorney Docket No.40056-0101WO1 induce the cleaving of the recombinant protein in a cell. In some embodiments, the 2A self-cleaving peptide is selected from P2A, T2A, E2A, F2A and BmCPV2A. See, for example, Wang Y, et al. 2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori. Sci Rep.2015;5:16273. Published 2015 Nov 5. A non- limiting example includes a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical or identical to LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO: 27). Other ribosomal skip sequences useful include T2A and P2A sequences. Thus, a T2A peptide AEGRGSLLTCGDVEENPGPVD (SEQ ID NO: ) or a P2A peptide QLGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: ) or both AEGRGSLLTCGDVEENPGPVDQLGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: __) can be located between the CAR and the IL-15 domain (e.g, SEQ ID NO: 50). Truncated EGFR or truncated CD19 In some embodiments, a CAR or peptide described herein can comprise a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO:27) and a truncated EGFR having a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: LVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHIL PVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGR TKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSG QKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKC NLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKT CPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIA TGMVGALLLLLVVALGIGLFM (SEQ ID NO:28). In some cases, the truncated EGFR has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:28. In some embodiments, a CAR or peptide described herein can comprise a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO:27) and a truncated EGFR having a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDL Attorney Docket No.40056-0101WO1 HILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIR GRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGT SGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVD KCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCV KTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS IATGMVGALLLLLVVALGIGLFM (SEQ ID NO:55). In some cases, the truncated EGFR has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:55. In some embodiments, a CAR or peptide described herein can comprise a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO:27) and a truncated CD19R (also called CD19t) having a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRE SPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPG WTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDR PEIWEGEPPCVPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVH PKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFH LEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKR (SEQ ID NO:26). In some cases, the truncated CD19t has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:26. In some embodiments, a CAR or peptide described herein can comprise a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO:27) and tEGFR having a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDL HILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIR GRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGT SGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVD KCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCV KTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS IATGMVGALLLLLVVALGIGLFM (SEQ ID NO:45). Attorney Docket No.40056-0101WO1 A ribosomal skip sequence useful in a CAR or peptide construct described herein include a T2A. In some embodiments, a T2A comprises a sequence that is at least 95% identical to EGRGSLLTCGDVEENPGP (SEQ ID NO:46) or at least 95% identical to AEGRGSLLTCGDVEENPGPVD (SEQ ID NO:47). A ribosomal skip sequence useful in a CAR or peptide construct described herein include a P2A. In some embodiments, a P2A comprises a sequence that is at least 95% identical to GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:48) or at least 95% identical to QLGSGATNFSLLKQAGDVEENPGP (SEQ ID NO:49). In some cases, the ribosomal skip sequence has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to any one of SEQ ID NO:46-49. An amino acid modification refers to an amino acid substitution, insertion, and/or deletion in a protein or peptide sequence. An “amino acid substitution” or "substitution" refers to replacement of an amino acid at a particular position in a parent peptide or protein sequence with another amino acid. A substitution can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. The following are examples of various groupings of amino acids: 1) Amino acids with nonpolar R groups: Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine; 2) Amino acids with uncharged polar R groups: Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine; 3) Amino acids with charged polar R groups (negatively charged at pH 6.0): Aspartic acid, Glutamic acid; 4) Basic amino acids (positively charged at pH 6.0): Lysine, Arginine, Histidine (at pH 6.0). Another grouping may be those amino acids with phenyl groups: Phenylalanine, Tryptophan, and Tyrosine. In some cases, the PSCA CAR or PSCA polypeptide can be produced using a vector in which the CAR open reading frame is followed by a ribosome skip sequence and a truncated EGFR (EGFRt), which lacks the cytoplasmic signaling tail, or a truncated Attorney Docket No.40056-0101WO1 CD19R. In this arrangement, co-expression of EGFRt provides an inert, non- immunogenic surface marker that allows for accurate measurement of gene modified cells, and enables positive selection of gene-modified cells, as well as efficient cell tracking of the therapeutic iMacs in vivo following adoptive transfer. Efficiently controlling proliferation to avoid cytokine storm and off-target toxicity is an important hurdle for the success of iMac immunotherapy. The EGFRt, or CD19t incorporated in the PSCA CAR lentiviral or retroviral vector can act as suicide gene to ablate the CAR+ iMacs cells in cases of treatment-related toxicity. The CAR or polypeptide described herein can be produced by any means known in the art, though preferably it is produced using recombinant DNA techniques. Nucleic acids encoding the several regions of the chimeric receptor can be prepared and assembled into a complete coding sequence by standard techniques of molecular cloning known in the art (genomic library screening, overlapping PCR, primer-assisted ligation, site-directed mutagenesis, etc.) as is convenient. The resulting coding region is preferably inserted into an expression vector and used to transform a suitable expression host cell line, preferably a macrophage, and most preferably an iMac. The CAR or polypeptide can be transiently expressed in a cell population by an mRNA encoding the CAR or polypeptide. For example, the mRNA can be introduced into the iPSC cells by electroporation (Wiesinger et al. 2019 Cancers (Basel) 11:1198). In some embodiments, administration of any of the cells or compositions described herein can be performed in one dose, continuously or intermittently throughout the course of treatment and an effective amount to achieve the desired therapeutic benefit is provided. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art. In a further aspect, the cells and composition of the disclosure can be administered in combination with other treatments. In some embodiments, the composition or method as disclosed herein can be combined with therapies that may upregulate the expression of a tumor or other antigen Attorney Docket No.40056-0101WO1 to which the CAR binds. In some embodiments, some clinical drugs can increase targeted antigens. In some embodiments, the compositions and therapies described herein can be combined with other therapies, e.g., lymphodepletion chemotherapy followed by infusions (e.g., four weekly infusions) of the therapy, defining one cycle, followed by additional cycles until a partial or complete response is seen or alternatively utilized as a “bridging” therapy to another modality, such as hematopoietic stem cell transplantation or CAR T cell therapy. Also described is: culture medium comprising pluripotent stem cell culture maintenance media (e,g., mTeSR1) supplemented with Thiazovivin,BMP4, and Activin A. In some cases the supplements are 10 μM Thiazovivin, 25 ng/mL BMP4, and 1 ng/mL Activin A; Also described is culture medium comprising pluripotent stem cell culture maintenance media (e,g., mTeSR1) supplemented with 10 μM Thiazovivin, 10 ng/mL BMP4, and 50 ng/mL hVEGF165 (hematovascular induction medium; in some cases the supplements are 10 μM Thiazovivin, 10 ng/mL BMP4, and 50 ng/mL hVEGF165. Also described is a culture medium comprising media for differentiation of iPSC to hematopoietic progenitor cells (e.g., STEMdiff) supplemented with 25 ng/mL hVEGF165, 20 ng/mL human fms-like tyrosine kinase 3 ligand (Flt3L), 20 ng/mL human stem cell factor (SCF), 20 ng/mL Insulin-like growth factor 1 (IGF-1), 10 ng/mL human interleukin 3 (IL-3), 2 µM StemRegenin 1, and 4 μM TGF-βRI inhibitor SB-431542 (“hematopoietic specification medium”). In some cases, the supplements are 25 ng/mL hVEGF165, 20 ng/mL human fms-like tyrosine kinase 3 ligand (Flt3L), 20 ng/mL human stem cell factor (SCF), 20 ng/mL Insulin-like growth factor 1 (IGF-1), 10 ng/mL human interleukin 3 (IL-3), 2 µM StemRegenin 1 (CAS No. 1227633-49-9) and 4 μM TGF-βRI inhibitor SB-431542 (CAS No.301836-41-9). Also described is a culture medium comprising the hematopoietic specification medium of claim 44 or 45 supplemented with human interleukin 6 (IL-6) and human thrombopoietin (TPO) (“myeloid-skewed hematopoietic differentiation media”). In some cases, the supplements are 10 ng/mL human interleukin 6 (IL-6) and 20 ng/mL human thrombopoietin (TPO). Attorney Docket No.40056-0101WO1 Also described is culture medium comprising Iscove's Modified Dulbecco's Medium (e.g., with L-glutamine and without alpha-thioglycerol and 2-mercaptoethanol) supplemented with fetal calf serum (FCS), L-glutamine, hM-CSF, IL-1β, and IL-6). In some cases, the supplements are 10% fetal calf serum (FCS), 2mM L-glutamine, 50 ng/mL hM-CSF, 10 ng/mL IL-1β, and 5 ng/mL IL-6). Also described is a method for differentiating myeloid progenitor cells, comprising culturing the myeloid progenitor cells in this culture medium. Described herein is a method for preparing a population of induced macrophages, the method comprising: providing a population of human induced pluripotent stem cells (iPSCs) comprising a nucleic acid molecule encoding a HLA-E protein (“HLA-modified iPSCs”); transducing at least a portion of the HLA-modified hiPSCs with a viral vector comprising a nucleotide sequence encoding soluble or membrane-bound human IL-15; selecting a transduced HLA-modified hiPSC (“IL-15 HLA-modified hiPSC”) and generating a clonal population from the selected CAR iPSC; and differentiating at least a portion of the clonal population IL-15 HLA-modified hiPSC into macrophage precursors (CAR-iMacs). In various embodiments: the HSPCs are isolated from human blood or human cord blood; the nucleic acid molecule encodes both a CAR and membrane bound IL-15; the iPSCs are generated by contacting the HSPCs with one or more of OCT3/4, OCT3, OCT4, SOX2, KLF4, L-MYC, C-MYC, LIN28, or short hairpin RNA targeting TP53 (shRNA-TP53); the nucleic acid is a vector (e.g., a viral vector such as a baculovirus, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or Herpes viral vector); the step of differentiating the clonal iPSCs into macrophages iMacs) comprises differentiating the clonal iPSCs into monocyte- committed progenitors (iMacP), cryopreserving the iMacP for a period of time, and after a period of time before thawing the iMacP ; iMacs express CD45, CD14, CD11b, CD86, CD63, CD80, and CD16; step of differentiating the CAR iPSCs into CAR iMacs comprises: (a)culturing the iPSCs in a hematovascular induction medium comprising Thiazovivin, BMP4, and hVEGF165; replacing the hematovascular induction medium with a hematopoietic specification medium comprising hVEGF165, human fms-like tyrosine kinase 3 ligand (Flt3L), human stem cell factor (SCF), Insulin-like growth factor Attorney Docket No.40056-0101WO1 1(IGF-1), human interleukin 3 (IL-3), StemRegenin 1 (CAS No.1227633-49-9; 4-[2-[[2- Benzo[b]thien-3-yl-9-(1-methylethyl)-9H-purin-6-yl]amino]ethyl]phenol), and TGF-βRI inhibitor (SB-431542; CAS No.301836-41-9; 4-[4-(3,4-Methylenedioxyphenyl)-5-(2- pyridyl)-1H-imidazol-2-yl]benzamide); the hematovascular induction medium with a myeloid hematopoietic differentiation media comprising 10 ng/mL human interleukin 6 (IL-6), and 20 ng/mL human thrombopoietin (TPO); and replacing the myeloid hematopoietic differentiation media with a macrophage differentiation medium comprising L-glutamine, hM-CSF, IL-1β, and IL-6. In various embodiments of this method: the hematovascular induction medium comprises 10 μM Thiazovivin, 10 ng/mL BMP4, and 50 ng/mL hVEGF165; the hematopoietic specification medium comprises 25 ng/mL hVEGF165, 20 ng/mL Flt3L, 20 ng/mL SCF, 20 ng/mL IGF-1, 10 ng/mL IL-3, 2 µM StemRegenin 1, and 4 μM SB-431542; the myeloid hematopoietic differentiation medium comprises 10 ng/mL IL-6 and 20 ng/mL TPO; the macrophage differentiation medium comprises 2mM L-glutamine, 50 ng/mL hM-CSF, 10 ng/mL IL-1β, and IL-6; the cells are cultured in the macrophage differentiation medium for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days; the step of differentiating the CAR iPSCs into iMacs comprises using a matrix-based culture system; the step of generating iPSCs from the HSPCs comprises using a matrix-based culture system; and the nucleic acid molecule encode a PSCA CAR and membrane bound IL15. In various embodiments the iMacs express a CAR is specific for a tumor and/or toxin. In various embodiments: rein the CAR targets any one or more of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CDS, CD6, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CS1, chlorotoxin receptor, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb- B2,3,4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptors, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), Interleukin-13 receptor subunit alpha-2 (IL-13Rα2), light chain kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), LI cell adhesion molecule Attorney Docket No.40056-0101WO1 (LICAM), melanoma antigen family A, 1 (MAGE-AI), Mucin 16 (Muc-16), Mucin 1 (Muc-1), Mesothelin (MSLN), NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor- associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF- R2), Wilms tumor protein (WT-1), or combinations thereof; the CAR comprises at least one targeting domain, wherein the targeting domain comprises at least one a single chain variable fragment (scFv); and the CAR comprises: at least one targeting domain, a spacer, a transmembrane domain, a co-stimulatory domain, and a CD3 ζ signaling domain. In various embodiments the cells described herein comprise a nucleic acid molecule encoding a polypeptide that includes the amino acid sequence: GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQ EGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDR RFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYL EDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITL TWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLP EPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYYK AEWSDSAQGSESHSL; the cells comprise a transgene encoding a polypeptide comprising: IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSF SKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM. In various embodiments the cells (e.g., the iPSC or iMACs) do not express HLA Class 1 genes. In various embodiments the cells (e.g., the iPSC or iMACs) wherein B2M is knocked out. Also described is a macrophage derived from human iPSC and expressing C-C chemokine receptor type 2 (CCR2) and colony stimulating factor receptor (CSF-R1) (“iMac”). In various embodiments: iMac harbor a nucleic acid molecule encoding soluble or membrane bound IL-15; the iMac harbor a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence: Attorney Docket No.40056-0101WO1 GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQ EGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDR RFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYL EDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITL TWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLP EPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYYK AEWSDSAQGSESHSL; the iMac harbor a nucleic acid molecule encoding a polypeptide comprising: IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSF SKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM; iMac do not express an HLA-1 molecule; the B2M gene is knocked out. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Other features and advantages of the invention will be apparent from the following detailed description and figures and from the claims. DESCRIPTION OF DRAWINGS FIG 1: Reprogramming and differentiation of human cord blood CD34 HSPCs into human iPSCs and subsequent differentiation into macrophages (A) Schematics for cord blood (CB) CD34+ isolated cells, purified by cell-sorting and expanded for 3 days in an expansion medium, followed by reprogramming into CB- 34 iPSCs from days 1 to 21. Attorney Docket No.40056-0101WO1 (B) Reprogrammed CB-34 iPSCs were analyzed by flow cytometric analysis to detect expression of the pluripotency markers CDH1, SSEA4, TRA-1-60, and the differentiation marker CD34. (C) In vivo differentiation potential of CB-34 iPSCs into the three germ layers, including ectoderm, mesoderm, and endoderm, was investigated via a teratoma assay in which we determined the expression of PAX1, VIMENTIN, and SOX17, respectively, for the three germ layers. (D) Harvest of non-adherent CD45+ myeloid progenitors during iPSCs differentiation into macrophages at different time points (n = 2). Cell yield at each harvest isreflected in the y axis. Error bars indicate the standard deviation (SD), and data are represented as mean ± SD. (E) A line graph showing expansion folds of progenitors generated from three clones of CB-34 iPSCs during macrophage differentiation. (F) Comparative analysis of primitive versus definitive hematopoietic cell surface markers between CB-34 iPSC-derived macrophages and PBMC-derived macrophages (PBMC-Macs) by flow cytometric analysis (left). Bar graphs display the percentage (%) of positive cells expressing CCR2 and CSF-1R (right). Data are represented as mean ± SD. n = 3. Statistical analysis was performed using Tukey’s multiple comparisons test in one-way ANOVA: **p = 0.0080, *p = 0.0100, ns, no significance. (G) Comparative characterization of CB-34 iPSC-derived macrophages and PBMC-Macs by assessing standard macrophage cell surface markers using flow cytometric analysis. Solid blue and red peaks on flow graphs represent isotype and surface marker intensity, respectively. See also FIG 6. FIG 2: Phagocytosis and antigen presentation of iPSC-derived CAR macrophages (A) Schematic representation of the mock (top) and CAR (bottom) molecule constructs. (B) Expression of the mock or CAR constructs (represented by tEGFR expression) in undifferentiated CB-34 iPSCs was determined in bulk-sorted CAR+ iPSCs by flow cytometry. Attorney Docket No.40056-0101WO1 (C) Flow-cytometry-based assay to determine phagocytosis of the PSCAhigh (Capan-1 and MIA PaCa-2) and PSCAlow (PANC-1) tumor cells by unsorted mock (mock-iMacs) and CAR macrophages (CAR-iMacs). Left panels: representative data. Right panels: summary data. The percentage of macrophage phagocytosis against carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled tumor cells (CD45+CFSE+) was assessed by flow cytometry. n = 3. Error bars indicate the standard deviation (SD) and data are represented as mean ± SD. Statistical analysis was performed using t test: **p = 0.0019, *p = 0.0118, ns, no significance. (D) The 3D z stack images of Far-red labeled tumor cells phagocytosed/under phagocytosis by GFP+ CAR-iMacs (left), with an overview of the 3D z stack images over a 360 ^ rotation (bottom). The images for tumor cells (Capan-1) alone and CAR- iMac alone are provided in the top left corner for reference. Data represent one of two experiments with similar results. (E) Untrasduced macrophages (UT-iMacs) or CAR-iMacs were cocultured with Capan-1 cells at an effector (E) to target (T) ratio of 2:1 for 20 h. Naive T cells (n = 3 donors) were added to the coculture at a ratio of 2:1:2 for macrophages:tumor cells:naive T cells and incubated for an additional 5 days. Activation of naive T cells was determined by flow cytometry and represented by the percentage of CD3+CD8+CD69+ (CD69 as an activation marker). Data are represented as mean ± SD. Statistical analysis was performed using Tukey’s multiple comparison test in one-way ANOVA: ****p < 0.0001, ns, no significance. (F) UT-iMacs or CAR-iMacs were cocultured with Capan-1 cells at an effector (E) to target (T) ratio of 2:1 for 20 h as in (E). Naive T cells (n = 3 donors) were added to the coculture at a ratio of 2:1:2 for macrophages:tumor cells:T cells, and incubated for an additional 5 days. Cell surface expression of HLA-I molecules on macrophages was analyzed by flow cytometry. Data are represented as mean ± SD. Statistical analysis was performed using Tukey’s multiple comparisons test in one-way ANOVA: ***p = 0.0005, ns, no significance. MFI, median fluorescence intensity. (G) Allogeneic NK cells from healthy donors (n = 3 donors) were cocultured with UT-iMacs or CAR-iMacs for 2–3 h. Activation of NK cells is represented by the percentage of CD3CD56+CD69+ determined by flow cytometry. The statistical data Attorney Docket No.40056-0101WO1 illustrating the percentage of activated NK cells (CD3CD56+CD69+) among different groups are shown. Data are represented as mean ± SD. Statistical analysis was performed using Tukey’s multiple comparisons test in one-way ANOVA: ****p < 0.0001, ns, no significance. (H) UT-iMacs or CAR-iMacs were cocultured overnight with Capan-1 cells. UT- iMacs or CAR-iMacs were sorted as CD45+ cells, and total protein was extracted. Immunoblotting was performed to measure the levels of total and phosphorylated RAC- alpha serine/threonine-protein kinase (AKT). Densitometric analysis was performed using ImageJ software. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (I and J) UT-iMacs or CAR-iMacs were cocultured with Capan-1 cells at an effector (E) to target (T) ratio of 2:1 for 6 h. Macrophages were gated as CD45+ cells, and expression of the indicated cell surface markers was analyzed by flow cytometry (n = 3). Data are represented as mean ± SD. Statistical analysis was performed using student t test: ****p < 0.0001, ***p = 0.0005, **p = 0.0025. MFI, median fluorescence intensity. See also FIGS 7 and 8. FIG 3: CAR-iMacs potently suppress tumor growth in pancreatic cancer mouse models (A) Schematics of the Capan-1Luc-ZsGreen tumor model establishment and treatment strategy of CAR-iMacs administration into NSG-SGM3 mice. (B) Quantification of the bioluminescence images from (A) up to day 57. Error bars indicate the standard deviation (SD) and data are represented as mean ± SD. Statistical analysis was performed on day 35 using Tukey’s multiple comparisons test in one-way ANOVA: **p = 0.0018, *p = 0.021, ns, no significance. (C) Kaplan-Meier survival analysis of mice in the indicated groups from (B) as analyzed using the log-rank test: ***p = 0.0006 comparing the CAR-iMac-treated group with the saline- or mock-treated groups, ns, no significance. (D) Schematics of the Capan-1Luc-ZsGreen tumor model establishment and treatment strategy of CAR-iMacs injection at different time points into NOD/ SCIDIl2rg-/- (NSG) immunodeficient mice. Attorney Docket No.40056-0101WO1 (E) Time-lapse luciferase imaging of the metastatic pancreatic cancer mouse model after indicated treatments in (D). (F) Quantification of the bioluminescence images from (E) up to day 49. Error bars indicate the standard deviation (SD) and data are represented as mean ± SD. Statistical analysis was performed on day 42 using Tukey’s multiple comparisons test in one-way ANOVA: ****p < 0.0001, ***p = 0.0005, ns, no significance. (G) Kaplan-Meier survival analysis of mice in the indicated groups from (F) as analyzed using the log-rank test: **p = 0.0013, ns, no significance. FIG 4: CAR-iMacs possess low toxicity and minimum tissue damage in vivo (A) A humanized mouse model was established by injecting Lin CD34+ cord blood (CB) cells after being expanded in vitro for 3 days.3 months after engraftment with human CB HSPCs, blood draws were conducted to confirm the successful engraftment by flow cytometric analysis. Mice with successful engraftment were implanted with 1x10⁶ Capan-1 Luc-ZsGreen cells. Upon confirmation of a high tumor burden, CAR-iMacs were administered to the mice. (B) Quantitative data for CRS-related factors in the sera from the HuSGM3 model. Sera were collected 2 days after each CAR-iMacs infusion as described in (A). Error bars indicate the standard deviation (SD) and data are represented as mean ± SD. n = 3 mice per group. Statistical analysis was performed using student t test: ns, no significance. (C) Mice from (B) were euthanized at the same time in all groups, i.e., on day 16 or 2 days after the second infusion of CAR-iMacs, and organs were collected for immune toxicity analysis. Slides from the indicated tissues isolated from experimental mice were subjected to H&E staining. (D) Assessment of the CRS-cytokines in the ascites of the mice as described in (A). n = 3 mice per group. Data are represented as mean ± SD. Statistical analysis was performed using Student’s t test: ns, no significance. See also FIG 9. FIG 5: Generation, differentiation, freezing, and functional analysis of the monoclonal cryopreserved CAR-iMac frozen progenitors (FPs) Attorney Docket No.40056-0101WO1 (A) Stepwise procedure for isolation of monoclonal CAR+ iPSCs (monoCAR+ iPSCs). (B) Persistence of CAR expression on monoCAR+ iPSCs during hematopoietic differentiation into CAR-iMacs. (C) Day 10 differentiated progenitors were cryopreserved, thawed 2 months later, and cultured in a macrophage differentiation medium at 37 ^C for 3 h. The viability of progenitors was tested by flow cytometry for fresh (before freezing) (Fresh) and thawed (after freezing) (Thawed). n = 3. Error bars indicate the standard deviation (SD) and data are represented as mean ± SD. (D) Cell surface markers were analyzed by flow cytometry for both fresh- and thawed-iPSC-derived myeloid progenitors, with mature iMacs serving as controls. These markers are indicated on the x axis. (E and F) CAR expression on monoclonal CAR-iMac progenitors (CAR-iMacP) and bulk CAR-iMacP was tested before cryopreservation and post-thawing 2 months later (F). Two different CAR cells featuring the same PSCA CAR part but with different tags (tEGFR or GFP), as depicted in (E), were examined. Fresh and cryopreserved cells were assessed under the same conditions. Data in (F) are from one of three experiments with similar results. (G) Bulk or monoclonal cryopreserved CAR-iMac progenitors were thawed and differentiated into bulk CAR-iMacs and monoclonal CAR-iMacs, respectively. Phagocytosis was assessed after coculturing CFSE-labeled Capan-1 cells with bulk CAR- iMacs or monoclonal CAR-iMacs. The percentage of CAR-iMacs phagocytosis against Capan-1 cells (CD45+CFSE+) was assayed by flow cytometry (n = 3). Error bars indicate the standard deviation (SD) and data are represented as mean ± SD. Statistical analysis was performed using Tukey’s multiple comparisons test in one-way ANOVA: ****p < 0.0001, ***p = 0.0007. See also FIG 10. FIG 6: Characterization of macrophages derived from cord blood CD34+ HSC reprogrammed iPSCs, related to FIG.1. (A) Purity of the isolated cord blood CD34+ (CB-34) donor cells was measured by flow cytometric analysis using CD56, CD34, and CD3 as markers. The CB-34 donor Attorney Docket No.40056-0101WO1 cells were expanded and utilized for somatic cell reprogramming into CB-34 iPSCs. Solid red and blue peaks on flow graphs represent isotypes and an indicated surface marker, respectively. (B) CB-34 iPSCs were investigated for gross chromosomal aberrations by Giemsa (G)-band karyotyping. (C-D) Expression of standard pluripotency markers of CB-34 iPSCs. Immunofluorescence staining of the pluripotency markers OCT3/4 and NANOG in CB- 34 iPSCs; separated (black and white) and merged blue and green channel images are shown in (C). Hoechst 33342 was used for nuclear DNA staining. TRA-1-60-R pluripotency marker expression on several CB-34 iPSC clones was analyzed by flow cytometry (D). (E) The differentiation potential of CB-34 iPSCs into primitive hematopoietic cells through embryoid body and 2-D planar hematopoietic differentiation was determined by detecting the co-expression of CD43 with CD235a and CD34 with CD235a using flow cytometry. (F) Stepwise overview of the entire procedure used for differentiating CB-34 iPSCs into macrophages. (G) The non-adherent CD45+ myeloid progenitors from six iPSC clones were harvested at different time points during myeloid-skewed hematopoietic differentiation. Cell yield at each harvest is reflected in the y-axis. (H) The non-adherent CD45+ myeloid progenitors were collected and differentiated into macrophages using macrophage differentiation media. Expression of lineage markers of mature iPSC-derived untransduced (UT)- iMacs was analyzed and compared with macrophages isolated from PBMCs (PBMC-Macs) via flow cytometry. (I) Bright field images of CD45+ progenitor-derived mature macrophages (left) and May-Grünwald staining of the adherent macrophages (right). (J) Expression of HLA-I and HLA-II molecules on iPSC-iMacs and PBMC-Macs were assessed by flow cytometry. FIG 7: Transduction, differentiation, and phagocytosis of CB-34 iPSC-derived CAR-macrophages (CAR-iMacs), related to FIG. 2. Attorney Docket No.40056-0101WO1 (A) Co-expression of membrane-bound IL-15 (mIL15) and tEGFR, which are in one reading frame with the CAR in our construct, was expressed in undifferentiated CB- 34 iPSCs and assessed by flow cytometry. Soluble IL-15 (sIL15) was used as a staining control. (B) CB-34 iPSCs expressing anti-PSCA CAR were subjected to myeloid-skewed hematopoietic differentiation and macrophage development. CAR expression is indicated by tEGFR intensity and was observed in both mockiMacs (the vector expressing mIL15 and tEGFR but no CAR) and CAR-iMacs using flow cytometry. Iso: isotype staining. (C) The median fluorescence intensity (MFI) of PSCA antigen expression was analyzed in several pancreatic cancer cell lines, including low PSCA-expressing PANC-1 (PANC-1low), high PSCA-expressing MIA PaCa-2 (MIA PaCa-2high), and high PSCA- expressing Capan-1 (Capan-1high) by flow cytometry. A549 lung cancer cells were used as a negative staining control. (D) Microscopic overview of the phagocytosis of far-red labeled CAR-iMacs targeting CFSE-labeled tumor cells. White arrows indicate tumor cells phagocytosed or undergoing phagocytosis. (E-F) Schematics for the coculture experiment to determine naïve T cell activation by CAR-iMacs (E). iPSCderived, untransduced macrophages (UT-iMacs) or CAR-iMacs were cocultured with tumor cells (Capan-1) at an effector (E) to target (T) ratio of 2:1 for 20 hours. Naïve T cells (n = 3 donors) were added to the coculture at a 2:1:2 ratio of macrophages: tumor cells: naïve T cells and incubated for an additional 5 days. Expression of CD69, a marker for T cell activation, was analyzed using flow cytometry (F). (G) Mock-iMacs or CAR-iMacs were cocultured with tumor cells (Capan-1) at a 2:1 effector (E) to target (T) ratio for 20 hours. For the condition with the presence of T cells, T cells isolated from PBMCs (n = 3 donors) were added to the coculture at a 2:1:2 ratio of macrophages: tumor cells: T cells and incubated for an additional 20 hours. Left panels: Representative dot plots showing tumor cell survival (area encircled) after coculture with mock-iMacs or CAR-iMacs in the presence or absence of primed T cells. Right panel: The statistical data illustrating the percentages of viable tumor cells in the coculture system (n = 3 donors). Error bars indicate the standard deviation (SD) and data Attorney Docket No.40056-0101WO1 are represented as mean ± SD. Statistical analysis was performed using student t test: * p = 0.0103 for CAR-iMac group in the absence of T cells versus CAR-iMac group in the presence of T cells. FIG.8: NK cell activation by and signaling involvement and polarization of CAR- iMacs and allorejection, related to FIG: 2. (A-B) Allogeneic NK cells from healthy donors were cocultured with iPSC- derived, untransduced macrophages (UT-iMacs) or CAR-iMacs in 10% FBS- supplemented RPMI 1640 for 2-3 hours (A). Activation of NK cells was assessed with CD3−CD56+CD69+ cell surface markers via flow cytometry (B). Error bars indicate the standard deviation (SD) and data are represented as mean ± SD. Statistical analysis was performed using Tukey’s multiple comparisons test in one-way ANOVA: **** p <0.0001, ** p = 0.0021. (C) UT-iMacs or CAR-iMacs were cocultured with tumor cells overnight. UT- iMacs or CAR-iMacs were sorted as CD45+ cells and total protein was extracted. Immunoblotting assay was conducted to detect phosphorylated and total extracellular signal-regulated kinase (ERK). Densitometric analysis was performed using ImageJ software. GAPDH was used as loading control. (D) UT-iMacs or CAR-iMacs were cocultured with Capan-1 cells at an effector (E) to target (T) ratio of 1:1 overnight. Macrophages were sorted as CD45+ cells and mRNA expressions of tumor-suppressive IL-1β, iNOS, IL-12, and IL-6 genes were analyzed by RT-qPCR (n = 3). Data are represented as mean ± SD. Statistical analysis was performed using Tukey’s multiple comparisons test in one-way ANOVA: * p = 0.0103. ns, no significance. (E) UT-iMacs or CAR-iMacs were cocultured with Capan-1 cells at an effector (E) to target (T) ratio of 1:1 for 48 hours. The expression of HLA-I and HLA-II molecules on macrophages (gating on CD45+) were analyzed by flow cytometry (n = 3). Data are represented as mean ± SD. Statistical analysis was performed using Tukey’s multiple comparisons test in one-way ANOVA:*** p = 0.0003, ** p = 0.0025, * p = 0.0103. ns, no significance. MFI: Median fluorescence intensity. Attorney Docket No.40056-0101WO1 (F) UT-iMacs or CAR-iMacs were plated on the membranes of transwell inserts with a pore size of 8 μm (n = 4). Tumor cells were added to the bottom of the wells of a 24-well plate, and the transwell inserts with UT-iMacs or CAR-iMacs were added to the plate wells and incubated for 48 hours. Data are represented as mean ± SD. Statistical analysis was performed using the student t test. ns, no significance. (G) Schematics for culturing system of UT-iMacs, CAR-iMacs, or PBMC-derived macrophages (PBMC-Macs) with allogeneic NK cells (alloNK) or allogeneic T cells (alloT) isolated from PBMCs of miss-matched donors for48-50 hours. (H-I) UT-iMacs, CAR-iMacs, and PBMC-Macs cocultured with or without alloNK (B) or alloT (C) cells for 48-50 hours. The cocultured cells were harvested after enzymatic digestion with TrypLE, followed by flow cytometric analysis. Representative data (left) and the statistical data (right) showing the percentage (%) of dead macrophages in the coculture system. n = 3 donors. Data are represented as mean ± SD. Statistical analysis was performed using the student t test. ns, no significance. (J) Evaluation of vector copy number in genomic DNA of CAR-iMacs by real- time PCR. PBMC-Macs were used as a negative control (n = 3 donors). Data are represented as mean ± SD. Statistical analysis was performed using the student t test. ** p = 0.0011. FIG 9: Pharmacokinetic (PK) in vivo antitumor activity of CAR-iMacs, and in vivo toxicity, related to FIG 3 and 4. (A) Transgene expression of CAR-iMacs and mock-iMacs prior to infusion into the metastatic pancreatic tumor mouse model. (B) Time-lapse luciferase imaging of FIG. 3B-C. (C) CAR-iMacsLuc-mCherry were injected using both i.v. and i.p. routes into tumor-free NOD/SCIDIl2rg−/− (NSG)(NSG) immunodeficient mice and time-lapse luciferase images were taken at different time points (days 0, 1, 2, 3, 4, and 5). (D) The quantitative data of (C) were analyzed, and the estimated half-life of CAR-iMacs (indicated by a dotted line) was determined. Error bars indicate the standard deviation (SD) and data are represented as mean ± SD. Attorney Docket No.40056-0101WO1 (E) A PK study was conducted using an NSG immunodeficient mouse model with metastatic pancreatic tumors. Tumors were implanted three days prior to treatment initiation (designated as day -3). On day 0, three days following tumor implantation, CAR-iMacs were administered to the tumor-bearing mice via both i.v. and i.p. routes. Euthanasia was performed on days 1, 3, 7, and 14 post CAR-iMac treatment (n = 3 mice for each time point). The immune cells of various tissues, including blood, were collected, counted, and subjected to flow cytometry analysis. The total cell numbers of mouse (m)CD45−human (h)CD45+ (h)CD11b+ cells from the various tissues were calculated. Data are represented as mean ± SD. (F) Time-lapse luciferase imaging of the metastatic pancreatic cancer mouse model following the treatment of saline, CAR-iMacs, or CAR-THP1 via both i.v. and i.p. routes on day 3 post-tumor implantation. (G) Kaplan-Meier survival analysis of mice in the indicated groups of (F) was analyzed using the log-rank test: **** p <0.0001, comparing the CAR-iMac- or CAR- THP1-treated group with the saline. *** p = 0.0007, comparing CAR-iMac with CAR- THP1-treated group, ns, no significance. (H-I) Humanized mouse model was established by injecting Lin−CD34+ HSPCs, sorted from umbilical cord blood. The success of the establishment of humanized mice was determined by detecting human (h)CD45+, hCD4+ and hCD8+ cells three months after CD34+ HSPC engraftment. The representative data of four humanized mice were shown in (H). Subsequently, the pancreatic tumor model was established by injecting 1 × 106 Capan-1Luc-ZsGreen cells into the successfully established humanized mouse model. CAR-iMacs were injected into the mice after confirming a high tumor burden by bioluminescence imaging (BLI) before infusing CAR-iMacs (I). Saline treatment was used as a background control. (J) Body weight and body temperature of the humanized mice bearing pancreatic tumors were monitored daily, commencing from day 0 (before CAR-iMac injection), and tracked for 3 consecutive weeks after CAR iMacs injection. Data are presented as the average of four mice in the format of mean ± SD. (K) Secretome analysis of ascites (abdominal fluid) collected from saline- or CAR-iMac-treated humanized mice bearing pancreatic tumor at the time of euthanasia. Attorney Docket No.40056-0101WO1 Cytokine release syndrome (CRS)-related cytokines are labeled blue in the saline group. Secretome images for three mice in each group are shown. FIG 10: Cryopreservation and functional analysis of monoclonal CAR+ iPSC- derived cryopreserved CAR-iMac progenitors (CAR-iMacFP), related to FIG 5. (A) CAR persistence of monoclonal CAR+ iPSCs (monoCAR+ iPSCs) during hematopoietic differentiation into CAR-iMacs was measured at indicated time points by flow cytometry. (B) CAR expression persistence of monoCAR+ iPSC-derived CAR-iMacs during macrophage differentiation culture condition was analyzed on day 28 by flow cytometry. Mock-iMacs were used as control. (C) Flow cytometry-based phagocytosis assessment of CFSE-labeled Capan-1 cells by monoCAR+ iPSC-derived CAR-iMacs. (D-E) CB-34 iPSCs were subjected to myeloid-skewed hematopoietic differentiation and non-adherent CD45+ myeloid progenitor cells were collected on day 10 of hematopoietic differentiation and then cryopreserved in the indicated freezing media. Cryopreserved progenitors were thawed two months after cryopreservation and cultured in macrophage differentiation medium at 37°C and 5% CO2 for 3 hours. The viability of the progenitors following a single freeze/thaw cycle was assessed by DAPI staining via flow cytometry (D). Expression of cell surface markers was analyzed on the thawed cells using flow cytometry (E). Solid blue and red peaks on flow graphs in (E) represent isotypes and surface markers for progenitors/macrophages, respectively. FIG 11: Disruption of B2M provides enhanced protection to immune cells against allogeneic CD8 T cells. (A) Schematic for generating B2M-knock out (B2MKO) iPSCs. (B-C) Flow cytometry analysis of HLA-ABC expression in parental iPSCs (WT) and a genetically modified B2MKO clones, with or without IFN-γ treatment, (C) as well as their respective differentiated cells. Attorney Docket No.40056-0101WO1 (D) Chromium-release assay evaluating the reactivity of allogeneic CD8+ T cells to WT CD45+ hematopoietic cells and B2MKO CD45+ hematopoietic cells at the indicated ratios (n = 4). FIG 12: B2M deficiency does not affect the morphology and functionality of iPSC- derived macrophages. (A) Immunohistochemistry staining of WT and B2MKO macrophages for HLA- ABC and HLA-DR/DP/DQ molecules. (B) Flow cytometry histograms showing the expression of anti-PSCA CAR (detected by EGFR tag) and membrane-bound IL-15 (mIL15) in comparison WT and B2MKO iPSCs. (C) Flow cytometry-based assay to determine the phagocytosis of PSCAhigh Capan-1 tumor cells by mock (mock-iMacs) and CAR macrophages (CAR-iMacs). The percentage of macrophage phagocytosis against carboxyfluoresceindiacetate succinimidylester (CFSE)-labeled tumor cells (CD45+CFSE+) was assessed by flowcytometry. FIG 13: Forced expression of HLA-E minimizes allorejection of B2M deficient CD45+ hematopoietic cells from allogeneic NK cells. A) Schematic for the generation of HLA-E transduced B2MKO iPSCs. B) Flow cytometry analysis showing that HLA-E expression is maintained throughout the differentiation of iPSCs into CD45+ hematopoietic cells. C) Assessment of NK cell activity through CD107a expression, a marker of immune cell activation and cytotoxic degranulation, in response to WT CD45+ hematopoietic cells and B2MKO CD45+ hematopoietic cells in coculture at the indicated ratios (n = 3). DETAILED DESCRIPTION Monocytes and macrophages are essential participants in immune defense and play an essential role in tissue remodeling

clearance of apoptotic cells ⁶, and maintaining homeostasis of other immune cells ^7,8. The effector function of Attorney Docket No.40056-0101WO1 monocytes/macrophages is plastic and can change in response to cytokines, pathogen- associated molecular patterns, metabolic cues, cell–cell interactions, and tissue-specific signals ^3,9. It is reported that macrophages can act as a double-edged sword within the TME, depending on whether the macrophages are in an M1 (pro-inflammatory), or M2 (anti-inflammatory) state ^10-12. As a prominent component of tumor stromal cells, M2 macrophages increase the density of microvessels in tumors, induce angiogenesis, and promote tumor invasion ¹³, thus facilitating metastasis and increasing immunosuppression by thwarting natural killer (NK) and T cell function during tumor progression ^14-16. On the other hand, type M1 macrophages can rapidly infiltrate into tumors, phagocytose tumor cells, remodel the TME by engulfing the stromal cells ¹⁷, and initiate as well as potentiate adaptive immune responses via T cell recruitment, antigen presentation, co- stimulation, and cytokine secretion

This polarization/transformation of macrophages from an M2- to M1-phenotype is sufficient to suppress tumors ^17,20, supporting a strategy for using M1-type macrophages in combating solid tumors. In this regard, the feasibility of high-dose autologous monocyte-derived macrophages has been

A CAR-transduced human monocytic cell line, THP-1, has been recently shown to exhibit enhanced phagocytosis and have a sustained pro-inflammatory M1-type macrophage phenotype ²². However, the immortalized nature of THP-1 cells has obvious barriers that precludes their therapeutic use. Alternatively, other studies have utilized cell sources such as bone marrow-derived macrophages (BMDM) engineered with CAR against CD19²³, or cord blood (CB)-derived macrophages engineered with CAR against

vitro antitumor efficacy of CAR-macrophages (CAR-M). Sources of peripheral blood mononuclear cell (PBMC) and CB macrophages are often limited, face donor-to-donor variability, contamination with other cell types, have limited expansion, and exhibit cell exhaustion over time after CAR-engineering or other genetic modifications ^25-29. In addition to these hurdles, ex vivo expansion of macrophages is more challenging compared to T and NK cells, and genetic manipulation of these cells to a high purity has been proven a non-trivial obstacle to overcome. On the contrary, the generation of Attorney Docket No.40056-0101WO1 immune cells from hematopoietic differentiation of human induced pluripotent stem cells (hiPSCs) is gaining traction in clinical trials ^30-32 for both autologous and allogeneic transplantation ^33,34. In addition, hiPSCs provide an amenable source for scalable production of immune cells with a simple platform for gene manipulation at the single- cell level and as a source for off-the-shelf cancer immunotherapy. Recently, hiPSC- derived CAR-macrophages (CAR-iMac) have been reported in solid tumor models^35,36; however, in vivo results only showed moderate antitumor activity of CAR-iMac, partially, if not all, due to low expression of CAR on terminal mature macrophages ³⁷. Thus, the off-the-shelf potential of potent CAR macrophages remains largely unexplored. Here, we reprogrammed expanded CB-isolated CD34⁺ HSPCs into hiPSCs, then introduced prostate stem cell antigen (PSCA)-targeting CAR (PSCA CAR), hereafter referred to as CAR for clarity. Isolated monoclonal CAR-iPSCs were differentiated into mature hiPSC-derived CAR macrophages (CAR-iMac), which retained high CAR expression and expressed standard macrophage cell surface markers. We co-engineered membrane-bound IL-15 (mIL15) and a suicide switch in the form of a truncated version of EGFR (tEGFR) to our CAR construct, in which mIL15 can provide CAR-iMac additional protection against apoptosis ^{38,39, 40} enhance adherence to the tissue ⁴¹, as well as potentiate and initiate adaptive antitumor response by T cells ^{42, 43}. The incorporated tEGFR can provide CAR-iMac detection/tracing and removal of unwanted CAR-iMac in vivo in case of uncontrolled CAR-iMac expansion by using the anti-EGFR antibody cetuximab, as we reported previously for CAR NK cells ¹⁹. Thus, this application provides a proof-of-principle platform using hiPSCs for the generation of CAR-iMac with antigen-dependent functions for adoptive transfer-based immune cell therapies. EXAMPLES The following examples do not limit the scope of the claims. Materials and Methods Cell Lines All the human induced pluripotent stem cell (human iPSC) lines used in this paper were reprogrammed from expanded CD34+ hematopoietic stem and progenitor cells Attorney Docket No.40056-0101WO1 (HSPCs) sourced from cord blood (CB) of a male individual. The iPSC lines were maintained in the undifferentiated state on Matrigel-coated plates (Corning Matrigel) in mTeSR1 medium (STEMCELL Technologies). Further details of reprogramming are provided in the method section. All experiments were conducted under protocols approved by the Institutional Biosafety Committee (IBC) and the Stem Cell Research Oversight (SCRO) Committee of City of Hope. Human PC cell lines (Capan-1, MIA PaCa-2, and PANC-1) were cultured with DMEM supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 mg/ml). All cell lines were routinely tested for the absence of mycoplasma using the MycoAlert Plus Mycoplasma Detection Kitfrom Lonza (Walkersville, MD). Mice All mouse studies were conducted in accordance with national guidelines for the humane treatment of animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at the City of Hope. Male and female NOD/SCIDIl2rg^−/−hIL3- hGMCSF-hSCF (NSG-SGM3), 8-12 weeks old (The Jackson Laboratory, Bar Harbor, ME), were engrafted with the Capan-1 or MIA PaCa-2 pancreatic cancer cell line expressing a luciferase_ZsGreen gene (Capan-1_luc or MIA PaCa-2_luc) by intraperitoneal (i.p.) injection (0.5 × 10⁶ cells/mouse). The engrafted mice were randomly distributed into saline, mock- and CAR-iMac treatment groups and three hours later cells were given to mice by both i.p. (5 × 10⁶ CAR-iMac/dose) and intravenous (i.v.; 2 × 10⁶ CAR-iMac/dose) injection. Reference schematics of FIG 3A for injections of mock- and CAR-iMac in the metastatic pancreatic cancer mouse model. Tumor growth was determined weekly by in vivo biophotonic imaging using a Xenogen IVIS 100. Mice were also monitored for survival, with euthanasia applied according to the American Veterinary Medical Association Guidelines. Cord blood Cord blood (CB) units were obtained from StemCyte under protocols approved by the COH IRB. All donors provided written informed consent, which followed the ethical guidelines of the Declaration of Helsinki. CD34⁺ hematopoietic stem and progenitor cells (HSPCs) were isolated by density gradient centrifugation over Ficoll-Paque (Cat# 17144003, Cytiva) and then underwent sequential rounds of CD34 microbead (Miltenyi Attorney Docket No.40056-0101WO1 Biotec) enrichment and cell sorting for isolating lineage-negative CD34⁺ HSPCs. The sorted CD34⁺ HSPCs were expanded for 2-3 days in StemSpan™ SFEM II (StemCell Technologies) containing hematopoietic stem cell expansion cytokines enlisted in StemSpan™ CD34⁺ expansion supplement (StemCell Technologies). Generation of iPSCs from cord blood CD34⁺ HSPCs The expanded CD34⁺ HSPCs were reprogrammed into human induced pluripotent stem cells (hiPSCs) by transgene- and virus-free Epi5™ episomal iPSC reprogramming kit (Invitrogen) in a feeder-free culture condition. In brief, 1 × 10⁵ CD34⁺ HSPCs were electroporated with 1 μL each of the Epi5™ reprogramming vectors and the Epi5™ p53 & EBNA vectors using the nucleofector 4D electroporation device (Lonza). The plasmid mixture was composed of episomal plasmids encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA for TP53 (Okita, K., Yamakawa, T., Matsumura, Y., Sato, Y., Amano, N., Watanabe, A., Goshima, N., and Yamanaka, S. (2013). An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells 31, 458-466). The transfected cells were cultured onto Geltrex™ matrix-coated plate in expansion medium. Next day, medium was changed to N2B27 supplemented with 100 ng/mL bFGF and followed media change according to the guideline of the reprogramming kit. CD34⁺ HSPCs-reprogrammed hiPSC colonies were visible at days 20-30 of the reprogramming protocol. Discrete colonies were picked under a microscope in aseptic condition for further culture/expansion in cGMP-grade mTeSR1 medium (StemCell Technologies) in Matrigel-coated (Corning) plates. Teratoma assay Human iPSCs culture in the log phase of growth were harvested using TrypLE (Gibco), washed twice with DMEM/F12 and resuspended in PBS supplemented with 30% Matrigel. iPSCs were kept on ice and drawn into 1-mL syringe immediately before injection. Approximately 2-3 × 10⁶ iPSC cells in 100 μL/injection site were used. A total of three NOD scid gamma (NSG) immunodeficient mice (6-8 weeks old) were injected in the dorso-lateral area into the subcutaneous space on both sides as previously reported (Prokhorova, T.A., Harkness, L.M., Frandsen, U., Ditzel, N., Schroder, H.D., Burns, J.S., and Kassem, M. (2009). Teratoma formation by human embryonic stem cells is site Attorney Docket No.40056-0101WO1 dependent and enhanced by the presence of Matrigel. Stem Cells Dev 18, 47-54; Wesselschmidt, R.L. (2011). The teratoma assay: an in vivo assessment of pluripotency. Methods Mol Biol 767, 231-241). After 6-8 weeks, teratomas were harvested in PBS, fixed overnight in 4% paraformaldehyde (Boston BioProducts) at room temperature. Samples were submitted to the City of Hope Histology Core Facility for sectioning and hematoxylin and eosin staining. Sections were examined, interpreted, and photographed microscopically. Unstained sections were processed with PAX1, VIMENTIN, and SOX17 antibodies by Pathology Core at City of Hope Comprehensive Cancer Center. Karyotyping Karyotyping by Giemsa (G)-banding was performed according to the previously reported standard protocol.⁸¹Forty randomly selected metaphase spreads for each cell line were counted and stained using standard G-banding analysis. CAR construction, production, and transduction into iPSCs We modified the PSCA CAR construct from our previous study¹⁹ by replacing soluble IL-15 (sIL15) with membrane-bound IL-15 (mIL15). Briefly, the PSCA CAR was designed to sequentially comprise a signal peptide, anti-PSCA single-chain fragment variable (scFv), human IgG1 hinge region, CD28 transmembrane domain, and CD3z. A codon-optimized mIL15 was incorporated into the PSCA CAR construct and fused to CD3z through a thosea asigna virus 2A-like self-cleaving peptide (T2A). Truncated EGFR (tEGFR), which acts as not only a traceable marker but also a suicide switch in vivo, was linked to mIL15 in the frame via porcine teschovirus-12A (P2A)-like self- cleaving peptide. Control plasmids mIL15_tEGFR were obtained by PCR amplification of the corresponding fragments from PSCA CAR_mIL15_tEGFR plasmid. Control fragment and CAR construct were cloned into the piggyBac construct.⁸² In order to achieve high-level transgene expression, we incorporated a CAG promoter driving our CAR construct in the piggyBac vector. Generation of CAR-positive, clonal iPSC lines Before transduction, iPSC cultures were dissociated with Accutase (ThermoFisher Scientific) treatment, washed with DMEM/F12, and resuspended 2.5 × 10⁵ cells in mTeSR1 medium supplemented with 1 μM Thiazovivin and cultured in 1-well of Matrigel-coated 6-wells plate. The next day, media were replenished with fresh mTeSR1 Attorney Docket No.40056-0101WO1 medium supplemented with 1 μM Thiazovivin and transduced with piggyBac vector and a lower amount of transposase (600 ng and 200 ng, respectively) using the lipofectamine™ 3000 transfection (ThermoFisher Scientific) protocol. The transduced cells were cultured for at least two passages before single cell sorting by flow cytometry and iPSC monoclone islolation. Clonal CAR-positive cells were again expanded in mTeSR1 medium on Matrigel-coated plates and banked for subsequent differentiation. Hematopoietic differentiation of iPSCs into CAR-iMac Prior to the start of hematopoietic differentiation, hiPSCs were subjected to at least three short passages (2-3 days). The cells were typically passaged at 70-80% confluence using TrypLE solution (Life Technologies) at 37°C and minimal digestion time. To initiate hematopoietic development, 1 million hiPSCs at single cell in mTeSR1 medium supplemented with 10 μM Thiazovivin, 25 ng/mL BMP4, and 1 ng/mL Activin were induced to embryoid bodies (EBs) in small-scale suspension culture on an orbital shaker at 70 revolutions per minute (rpm). EBs were collected and transferred to mCollagen IV (Corning) pre-coated 6-wells plate in mTeSR1 medium supplemented with 10 μM Thiazovivin, 10 ng/mL BMP4, and 50 ng/mL hVEGF165 (R&D Systems) and this supplemented mTeSR1 medium was termed as hematovascular induction medium. On day 3 of hematopoietic differentiation, medium was replaced with complete STEMdiff™ hematopoietic medium (StemCell Technologies) containing 25 ng/mL hVEGF165, 20 ng/mL human fms-like tyrosine kinase 3 ligand (Flt3L), 20 ng/mL human stem cell factor (SCF), 20 ng/mL Insulin-like growth factor 1 (IGF-1), 10 ng/mL human interleukin 3 (IL-3), 2 µM StemRegenin 1 (SR1; CAS No.1227633-49-9) and 4 μM TGF-βRI inhibitor SB-431542 (CAS No.301836-41-9; Tocris Bioscience). We termed this complete STEMdiff^TM hematopoietic medium containing the various factors as a hematopoietic specification medium. All cells were incubated at 37°C in a mixture of 5% carbon dioxide and 95% air in a humidified atmosphere. On day 6 of hematopoietic differentiation, the hematopoietic specification medium was replaced with myeloid- skewed hematopoietic differentiation media. The myeloid skewed hematopoietic differentiation medium was prepared from hematopoietic specification medium by supplementing with 10 ng/mL human interleukin 6 (IL-6) and 20 ng/mL human thrombopoietin (TPO). On day 9 of differentiation, non-adherent CD45+ progenitor cells Attorney Docket No.40056-0101WO1 were collected and either expanded in an expansion media (IMDM containing 50 ng/mL SCF, Flt3L, IGF-1, 25 ng/mL GM-CSF, 10 ng/ mL IL-3, 2 mM SR1, and 0.5mM UM729) or subjected to a terminal differentiation (see below) into macrophage using a differentiation medium. Terminal differentiation For further maturation, fresh or expanded progenitor cells were cultured in macrophage differentiation medium IMDM medium supplemented with 10% fetal calf serum (FCS), 2mM L-glutamine, 50 ng/mL hM-CSF, 10 ng/mL IL-1β, and 5 ng/mL IL- 6) for at least 7 days. May-Grunwald staining Sorted iMacs (0.5-1.03105) were washed and resuspended in 100 mL D-PBS. Cells were spun onto polylysine-coated slides at 500 rpm for 5 minutes, air-dried, and stained with May-Gr€unwald stain (Sigma-Aldrich, MG500) according to the manufacturer’s protocol. The slides were washed with distilled (d)H2O, air-dried, and mounted for examination by light microscopy. Flow cytometry-based phagocytosis assay For the phagocytosis assay of human induced pluripotent stem cell-derived macrophages, prostate stem cell antigen (PSCA)-high pancreatic cancer cell lines Capan- 1 and MIA PaCa-2 as well as the PSCA-low cell line PANC-1 prelabeled with CFSE (Thermo Fisher) were cocultured with human macrophages at an effector (E)/target (T) ratio of 1:1 (50,000 PSCA CAR-iMac effector cells with 50,000 tumor cells) for 6 hours at 37C in ultra-low-attachment 96-well U-bottom plates (Corning) in serum-free RPMI 1640 (Life Technologies). The cells were then harvested by centrifugation and stained with anti-human CD45 antibody (BD Biosciences) to distinguish macrophages from tumor cells. All flow cytometry data were collected using a Fortessa X20 flow cytometer (BD Biosciences) and analyzed with Flowjo v10 software. Flow cytometry-based allogeneic protection assay Freshly isolated allogeneic NK (alloNK) or T (alloT) cells were isolated from healthy donors PBMCs. UT-iMacs, CAR-iMacs, and PBMCderived macrophages (PBMC-Macs) were added to a round-bottom 96-wells plate in macrophage differentiation medium, and coculture with or without ^alloNK or ^alloT with IL-15 (10 Attorney Docket No.40056-0101WO1 ng/mL final coculture concentration) and IL-2 (100 IU/mL final coculture concentration). The coculture system was incubated at 37 ^{^}C for 48-50 hours. Next day, cells were harvested and stained for cell surface markers and DAPI. Flow cytometry analysis was carried out to assess the viability percentage of macrophages in the coculture system. Assessment of the expression of cytokine genes in CAR-iMacs after being cocultured with tumor cells Untransduced (UT)-iMacs or CAR-iMacs were mixed with Capan-1 at an effector/target (E/T) ratio of 1:1 (100, 000: 100,000 cells) in 10% FBS-supplemented RPMI 1640 for overnight at 37_C. Macrophages were sorted as CD45+ cells, and mRNA expressions of IL-1beta, iNOS, IL-12, and IL-6 genes were analyzed by qPCR. Assessment of NK cell activation via IL-15 expressed by CAR-iMacs 0.2 x 10⁶ TryPLE-digested iPSC-derived UT-iMacs or CAR-iMacs were added to a round-bottom 96-wells plate in 10% FBS-supplemented RPMI 1640. 13105 freshly isolated NK cells from healthy donors were directly introduced into a 96-well plate containing macrophages at a macrophage: NK ratio of 2:1. The 96-well plate was centrifuged at 1,000 RPM for a minute to bring the cells together in the coculture system. Following incubation at 37 ^C for 2-3 hours, the cells were stained with anti-CD3, anti- CD56, and anti-CD69 antibodies for subsequent flow cytometry analysis. All flow cytometry data were collected using a Fortessa X20 flow cytometer (BD Biosciences) and analyzed with Flowjo v10 software. Flow cytometry-based allogeneic protection assay Freshly isolated allogeneic NK (alloNK) or T (alloT) cells were isolated from healthy donors PBMCs. UT-iMacs, CAR-iMacs, and PBMC derived macrophages (PBMC-Macs) were added to a round-bottom 96-wells plate in macrophage differentiation medium, and coculture with or without alloNK or alloT with IL-15 (10 ng/mL final coculture concentration) and IL-2 (100 IU/mL final coculture concentration). The coculture system was incubated at 37 ^C for 48-50 hours. Next day, cells were harvested and stained for cell surface markers and DAPI. Flow cytometry analysis was carried out to assess the viability percentage of macrophages in the co-culture system. Cell migration assay Attorney Docket No.40056-0101WO1 Cell migration assay was performed for 48 hours at 37 ^C in 5%CO2 in a sterile condition. An equal number of UT-iMacs and CARiMacs were evaluated by cytospin of mixed cells at 100 g for a minute before the experiment and at the starting point before migration. UT-iMacs (13105) and CAR-iMacs (13105) in RPMI-1640 supplemented with 10% FBS were plated on the membranes of transwell inserts with a pore size of 8 mm (Corning, NY). Similarly, tumor cells (Capan-1, 13105) were added to the bottom of the plate in which the transwell was inserted. Migrating cells were detected by Zeiss Light Microscope. Metastatic Pancreatic Cancer Model All mouse studies were conducted in accordance with national guidelines for the humane treatment of animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at the City of Hope. Male and female NOD/SCIDIl2rg−/− hIL3-hGMCSF-hSCF (NSG-SGM3), 8–12 weeks old (The Jackson Laboratory, Bar Harbor, ME), were engrafted with the Capan-1 or MIA PaCa-2 PC cell line expressing a luciferase_ZsGreen gene (Capan-1_luc or MIA PaCa-2_luc) by intraperitoneal (IP) injection (0.5 × 10⁶ cells/mouse). The engrafted mice were randomly distributed into saline, mock- and CAR-iMac treatment groups and three hours later given to mice by both IP (5 × 10⁶ CAR-iMac/dose) and intravenous (IV; 2 × 10⁶ CAR-iMac/dose) injection. Three doses of CAR-iMac, mock-cells, or saline was given started on week 1, between weeks 4^th-5^th, and between weeks 6^th-7^th. All animal experiments were conducted in accordance with Animal Research Reporting In Vivo Experiments (ARRIVE), federal, state, and local guidelines with approval from the City of Hope Animal Care and Use Committee. For the dose rational study, male and female NOD/SCIDIl2rg_/_ (NSG) mice aged 8–12 weeks (The Jackson Laboratory, Bar Harbor, ME) were engrafted with Capan- 1Luc-ZsGreen via i.p. injection (0.53105 cells/mouse) on day 0. On day 0, day 3, or day 7 post-tumor engraftment, the mice received treatment with CAR-iMacs through both i.p. (23106 CAR-iMacs/dose) and i.v. (0.5x10⁶ CAR-iMacs/dose) injection. Saline was injected as a control. Two doses of CAR-iMacs in total were given weekly. Tumor growth was determined weekly by in vivo bioluminescence imaging using Xenogen IVIS 100. Additionally, mice were monitored for survival, and euthanasia was performed at Attorney Docket No.40056-0101WO1 the end time point of the survival in compliance with the guidelines of the American Veterinary Medical Association. For the pharmacokinetic (PK) study on tumor-free mice, CAR-iMacsLuc- mCherry were injected using both i.v. and i.p. routes into NSG immunodeficient mice and time-lapse luciferase imaging was taken on days 0, 1, 2, 3, 4, and 5 following CAR- iMacLuc-mCherry administration. For the PK study on tumor-bearing mice, tumors were implanted on day -3. Three days after the tumor implantation (day 0), CARiMacs were administered via both i.v. and i.p. routes to the tumor-bearing mice. Euthanasia was performed on days 1, 3, 7, and 14 following CAR-iMac treatment. The immune cells of various tissues, including blood, were collected, counted, and subjected to flow cytometry analysis. The total cell number of mouse CD45_ human CD45+ human CD11b+ cells was counted by flow cytometry. For the CAR-THP1 study, THP-1 cells were transfected with the same CAR construct used in CAR-iMacs. The transfected THP1 cells were differentiated into CAR- THP1. Male and female NSG mice, aged 8–12 weeks (The Jackson Laboratory, Bar Harbor, ME), were engrafted with Capan-1Luc-ZsGreen via i.p. injection (0.53105 cells/mouse) on day 0. On day 3 post tumor engraftment, the mice received treatment with CAR-iMacs or CAR-THP1 through both i.p. (23106 cells/dose) and i.v. (0.53106 cells/dose) injection. Saline was injected as a control. Two doses of CAR-cells in total were given weekly. Tumor growth was determined weekly by in vivo bioluminescence imaging using Xenogen IVIS 100. Additionally, mice were monitored for survival, and euthanasia was performed at the end time point of the survival in compliance with the guidelines of the American Veterinary Medical Association. Bioluminescence imaging D-luciferin potassium salt (GoldBio) was dissolved in sterile Dulbecco’s phosphate buffer saline following the manufacturer’s instructions and given to the Capan- 1Luc-ZsGreen-engrafted mice by i.p. injection (150 mg/kg). The mice were anesthetized with 4% isoflurane and oxygen (1 L/min) in an imaging chamber, and luminescence images were captured by Lago-X (Spectral Instruments Imaging) following the manufacturer’s instructions and quantified by Aura Imaging Software (Version 2.2.1.1). May-Grünwald staining Attorney Docket No.40056-0101WO1 Sorted cells (0.5-1.0 × 10⁵) were washed and resuspended in 100 μL 1 × D-PBS. Cells were spun onto polylysine-coated slides at 500 rpm for 5 minutes, air-dried, and stained with May-Grünwald stain (Sigma-Aldrich, MG500) according to the manufacturer's protocol. The slides were washed with distilled (d)H₂O, air-dried, and mounted for examination by light microscopy. Serum collection Blood was collected from mice by tail-clip or retro-orbital bleeding. After the collection, blood was left to clot for 60 min at room temperature. The clotting blood was centrifuged at 6,500 g for 10 min at 4 ^C. Sera were collected and aliquoted into different tubes in order to prevent multiple freeze–thaw cycles and were immediately stored at - 80 ^C until analysis. Assessment of cytokine levels in sera Serum and plasma cytokines were measured by RayBiotech Life company. For measuring SAA3, an ELISA kit (Millipore) was used according to the manufacturer’s instructions. Cryopreservation of CAR-iMacFP Four conditions for cryopreservation of monocyte-committed progenitors (CAR- iMacP) using both commercially available CryoStor® CS5 and CS10 freezing media and macrophage differentiation media (given in “Hematopoietic differentiation of hiPSC into CAR-iMac”) supplemented with either 5 or 10% dimethyl sulfoxide (DMSO). CryoStor® CS5 and CS10 are protein- and animal component-free freezing medium based on the HypoThermosol® formulation and containing 5% and 10% DMSO, respectively. To validate this method, cryotubes were stored for 2 months at -80⁰C before thawing for differentiation and flow cytometry analysis. Quantification and Statistical Analysis Continuous endpoints were presented as mean ± SD and transformed by log2 when running data analysis if the raw data distribution was skewed (e.g. picogram/mL). Data were analyzed by Student’s t-test for 2-group (independent) comparisons, one-way ANOVA model for multiple-group comparisons, linear mixed model or one-way ANOVA with repeated measures for matched-group comparisons or repeated measures over time analysis. For survival data, Kaplan–Meier method and log rank test were used Attorney Docket No.40056-0101WO1 to estimate and compare survival functions. All tests were two-sided. P values were adjusted for multiple comparisons by Holm’s method or Tukey method. A P value of 0.05 or less was considered statistically significant. Statistical software GraphPad and SAS 9.4 were used for the statistical analysis. The statistical details and results were described in the FIG legend. CRISPR-Cas9 genome editing Transfection was performed using the 4D-Nucleofector system (Lonza). To target the B2M gene, approximately 1 x 10⁵ iPSCs were resuspended in 20 µL of P3 Primary Buffer. Subsequently, 5 µL of RNP (ribonucleoprotein) complex, containing 5 µg of recombinant Cas9 (IDT, Cat. No. 1074181) and 1.25 µg of B2M targeted gRNA, was added to the iPSCs. The cells were then electroporated using the CA-137 electroporation program. Following electroporation, the iPSCs were transferred onto Matrigel-coated 6- well plates and cultured in mTeSR1 medium (STEMCELL Technologies) containing 1 μM Thiazovivin. Sub-cloning and genotyping Genome-edited iPSCs were cultured for 48 hours in mTeSR1 containing 20 ng/mL IFN-γ (Peprotech, Cat. No. 300-02) and subsequently stained with HLA-ABC antibodies. Individual iPSC lacking HLA-ABC expression were then sorted using Aria Fusion (BD) cell sorter into wells of a Matrigel-coated 96-well plate containing mTeSR1 medium and 1 μM Thiazovivin. After two weeks, single-cell colonies were picked and transferred to Matrigel-coated 24-well plates for further expansion. These colonies were cultured until they reached a sufficient quantity, producing dozens of millions of cells for freezing and future experiments. Transduction of single-chain HLA-E and subsequent anti-PSCA CAR into B2M^KO iPSCs Single cell B2M^KO expanded iPSCs were transduced with a single-chain (sc) HLA-E fused with G-peptides. Prior to transduction, iPSC cultures were dissociated using Accutase (ThermoFisher Scientific), washed with DMEM/F12, and resuspended in mTeSR1 medium supplemented with 1 μM Thiazovivin at a concentration of 2.5 × 10⁵ cells per well. These cells were then plated in a Matrigel-coated 6-well plate. The following day, the medium was refreshed with mTeSR1 medium containing 1 μM Attorney Docket No.40056-0101WO1 Thiazovivin, and the cells were transduced using the piggyBac vector containing scHLA- E and a reduced amount of transposase (600 ng and 200 ng, respectively) following the lipofectamine™ 3000 transfection protocol (ThermoFisher Scientific). The transduced cells were cultured for at least two passages before undergoing single-cell sorting by flow cytometry to isolate monoclonal human iPSC lines. Clonal iPSCs expressing HLA-E (B2M^KO-E) were further expanded in mTeSR1 medium on Matrigel-coated plates and subsequently banked for differentiation. The expanded B2M^KO-E iPSCs were then transduced with an anti-PSCA CAR using the same procedure as the scHLA-E transduction. Immunocytofluorescence for gene editing studies For immunocytochemical studies, cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature. Following fixation, cells were incubated for 1 hour in the Blocking Buffer (1 × D-PBS, 0.2% Triton X 100, 0.1% Tween-20, 2% goat or donkey serum, and 2% FBS). Primary antibodies were diluted in 1 × D-PBS, 0.1% Tween-20, and applied for 1–3 hours at room temperature or overnight at 4°C. The slides were then washed three times for 3 minutes each with 1 × D-PBS – 0.1% Tween-20. Subsequently, the cells were incubated with secondary antibodies at a 1:1000 dilution. Nuclei were stained with DAPI (Sigma-Aldrich). Finally, the slides were mounted using Vectashield® mounting medium (Vector Laboratories, Burlingame, CA) and examined under the LSM800 Confocal Microscope (Carl Zeiss AG, Oberkochen, Germany). Example 1: Generation of human iPSCs and iPSC-derived macrophages expressing standard markers comparable to PBMC-derived macrophages Human CB-derived CD34+ HSPCs were obtained from healthy donors, expanded, and reprogrammed into human iPSCs using ‘‘Epi5 episomal iPSC reprogramming kit,’’ which provides the optimal system for generating transgene- and virus-free iPSCs in a feeder-free culture condition (FIGS 1A and 6A). The CD34+ HSPC-reprogrammed human iPSCs (CB-34 iPSCs) showed a normal karyotype and expressed standard pluripotency markers, including SSEA4, CDH1, TRA-1-60, OCT3/4, and NANOG, but lost CD34 (FIGS 1B and 6B–6D). The verified CB-34 iPSC clones showed the potential to generate three germ layers, including ectoderm, mesoderm, and endoderm, in a Attorney Docket No.40056-0101WO1 teratoma assay (FIG 1C). The CB-34 iPSCs were assessed for hematopoietic differentiation potential into yolk-sac-derived primitive hematopoietic cells identified as CD34+CD235a+ and CD43+CD235a+ (FIG 6E), following our previously published protocol.43,44 Next, we modified the protocol to differentiate the CB-34 iPSCs into myeloid cells. Briefly, one million CB-34 iPSCs were differentiated to myeloid-skewed hematopoietic progenitors at days 8–10 of differentiation (FIG 6F). The nonadherent progenitors expressing the CD45 pan-leukocyte marker at day 10 of hematopoietic differentiation were collected and expanded throughout days 10–14 in expansion media, followed by maturation into macrophages over an additional 4–7 days (FIG 6F). In comparison with the initial protocol that allowed five harvests (FIG 6G), optimization enabled the harvesting of CD45+ myeloid progenitors for up to eight harvests, facilitating the generation of large numbers of CD45+ myeloid progenitors (FIG 1D). Interestingly, these CD45+ progenitors underwent further expansion and differentiation into mature macrophages, resulting in an over 50-fold expansion from initial CB-34 iPSC source cells (FIG 1E). We and others have previously reported that the TGF-bRI inhibitor SB- 4315⁴², known for inhibiting Activin/Nodal signaling, significantly suppresses primitive hematopoietic cell development (primitive wave).43,45–47 This inhibition biases hematopoietic development toward erythromyeloid-derived cells and more definitive progenitors.^43,46,48 Our human iPSC-derived macrophages expressed C-C motif chemokine receptor 2 (CCR2), which is reported to be expressed by monocytes/macrophages,⁴⁹ as well as colony-stimulating factor 1 receptor (CSF-1R), which is reported to be expressed by primitive/erythromyeloid- derived macrophage.^50,51 Human iPSC-derived macrophages have a comparable expression level of CCR2 when compared with PBMC-Macs by the percentage of positive cells, while human iPSC- derived macrophages express but PBMC derived macrophages (PBMC-Macs) do not express CSF-1R (FIG 1F). This result suggests that our iPSC derived macrophages may fall within the category of erythromyeloid-derived macrophages, originating from intra- embryonic-like hematopoietic progenitors, as recently reported for iPSC-derived macrophages.^52,53 We also assessed other markers and found that CB-34 iPSC-derived macrophages exhibited comparable expression of cell surface markers, including CD45, CD14, CD11b, CD86, CD63, CD80, and CD16 with slightly increased expression of Attorney Docket No.40056-0101WO1 CD206 when compared with PBMC-Macs (FIG 1G). Flow cytometry and May- Gr€unwald staining demonstrated that our protocol yielded highly pure macrophages from CB-34 iPSCs under feeder-free culture conditions with minimum contamination by other cells54 (FIGs 6H and 6I), omission of further purification through CD14 microbeads via magnetic activated cell sorting. Interestingly, CB-34 iPSCderived macrophages (without a CAR) showed lower expression of human leukocyte antigen (HLA)-I and HLA-II compared with PBMC-Macs, suggesting potential advantages in reduced allorejection by the host when infused therapeutically (FIG 6J). Taken together, by employing protocols modified from our previous reports,^43,44 we successfully generated human iPSCderived macrophages with both high yield and purity. These macrophages are likely derived from the erythromyeloid lineage but phenotypically closely resemble their counterparts from PBMCs regarding their morphology and cell surface markers. Example 2: CAR-iMacs exhibit antigen-dependent phagocytosis, activate NK and T cells, and are resistant to allorejection by NK and T cells Previous studies have reported that mIL-15 can trigger monocyte activation and significantly enhance adhesion of monocytes, ⁵⁵ induce anti-apoptotic pathways,^37–39 and act as a potent growth factor enhancing T and NK cell proliferation.7,40,41 Given these studies, we engineered CB-34 iPSCs with a PSCAtargeted specific CAR construct that co-expressed mIL-15 and a suicide switch in the form of tEGFR (referred to as CAR- iMacs; FIG 2A). The rationale for incorporating tEGFR was to use it as a marker for detecting/tracking CAR-iMacs. Additionally, it serves as a suicide switch for the potential removal of unwanted CAR-iMacs in vivo in the event of uncontrolled or unsafe CARiMac expansion, achieved by administering cetuximab, as others and we reported previously for CAR-T cells and CARNK cells, respectively.^19,42 The construct with mIL- 15 and tEGFR but without PSCA CAR was used as a mock control (FIG 2A). The PSCA CAR was transduced into undifferentiated CB-34 iPSCs using transposon mediated gene transfer. Following sorting, the CAR+ iPSCs stably maintained CAR expression during routine human iPSCs passaging (FIG 2B). The co-expression of mIL-15 and tEGFR in CAR-transduced CB-34 iPSCs validated that tEGFR within our CAR construct was in the Attorney Docket No.40056-0101WO1 same reading frame as mIL-15 and hence anti-PSCA (single-chain fragment variable [scFv]), suggesting that the expression of mIL-15 and tEGFR can represent expression of the PSCA CAR (FIG 7A). After differentiation, about half of differentiated human iPSC derived mature macrophages showed stable CAR expression (FIG 7B). The effector potency of unsorted CAR-iMacs and mock-iMacs was investigated by using high PSCA-expressing (PSCAhigh) pancreatic tumor cells Capan-1 and MIA PaCa-2. Low PSCA-expressing (PSCAlow) PANC-1 tumor cells were used as a negative control (FIG 7C). After coculture of the CAR-iMacs or mock-iMacs with pancreatic tumor cells, CARiMacs showed significant CAR-specific phagocytosis of PSCAhigh tumor cells (Capan-1 and MIA PaCa-2) compared with mock-iMacs (FIG 2C). By contrast, both CAR-iMacs and mock-iMacs revealed minimal non-specific phagocytosis of the PSCAlow tumor cells (FIG 2C, PANC-1), confirming CAR- dependent and antigen-specific phagocytosis of pancreatic tumor cells by CAR-iMacs. Consistent with flow-cytometry- based phagocytosis, confocal imaging of CAR-iMacs showed higher phagocytosis of Capan-1 and MIA PaCa-2 than PANC-1 tumor cells (FIG 7D). We further investigated whether the CAR-iMacs are capable of whole-cell phagocytosis or merely stick to tumor cells. For this reason, we undertook 3D confocal images of phagocytosis at higher magnification. An overview of the 3D images at a 360 rotation indicated that the phagocytosed cell remained within the confines of the same CAR-iMac at the same spatial location, strongly suggesting an occurrence of an actual phagocytosis event rather than cell-cell adhesion (FIG 2D). Given the role of macrophages as professional antigen-presenting cells (APCs), a cornerstone of adaptive immunity,56 we evaluated the ability ofmock- andCAR-iMacs to cross-present intracellular tumor antigens acquired through phagocytosis. Following an overnight coculture with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled tumor cells, we introduced freshly isolated naive T cells from PBMCs to the coculture and incubated them for an additional 5 days to facilitate antigen cross-presentation and subsequent activation of naive T cells (FIG 7E). Flow cytometric analysis revealed significantly higher CD69 surface expression (an activation marker) on T cells cocultured with CAR-iMacs compared with other groups (FIGs 2E and 7F). Attorney Docket No.40056-0101WO1 Consistently, we observed a significant upregulation of HLA-I molecules on CAR-iMacs when cocultured with naive T cells in the presence of Capan-1 tumor cells compared with untransduced human iPSC-derived mature macrophages (UT-iMacs) (FIG2F). This supports the notion that the activation of naive T cells in the CAR-iMac group was attributed to antigen cross-presentation by CAR-iMacs to naive T cells. In line with this, when primed T cells were added to the coculture of CAR-iMacs and tumor cells, enhanced tumor cell killing was observed for CAR-iMacs in the presence of the primed T cells when compared with the absence of the T cells (FIG 7G). In our subsequent experiments, we examined the impact ofmIL-15 in our CAR construct on the activation of NK cells. Upon coculture of NK cells (isolated from PBMCs) with UT-iMacs or CAR- iMacs, we observed NK cell activation by CAR-iMacs. This was evident from increased percentages and median fluorescence intensity of

cells in the CAR-iMac group compared with the UT-iMac group (FIGs 2G, 8A, and 8B). To delineate the cell signaling pathways that mediate CAR-induced cellular activation, we evaluated the phosphorylation of key macrophage cell signaling mediators, including the phosphatidylinositol-3 kinase (PI3K)/RAC-alpha serine/ threonine-protein kinase (AKT) signaling pathway and the extracellular signal-regulated kinase (ERK) signaling pathway. Analysis of sorted CAR- iMacs after being cocultured with Capan-1 tumor cells (post-phagocytosis) revealed an increased level of total AKT compared with UT-iMacs (FIG 2H). The mechanism behind the observed increase in total AKT levels in CAR-iMacs in the presence of tumor cells remains to be explored. However, we did not observe a substantial difference in the ERK signaling pathway between the two groups (FIG 8C). Next, we examined cytokine gene expression and macrophage polarization in CAR-iMacs when co-cultured with or without tumor cells. Real-time PCR analysis revealed a trend in the upregulated expression of pro-inflammatory cytokines IL-1b and IL-6 in CAR-iMacs compared with UT-iMacs, regardless of the presence or absence of tumor cells. Although the difference did not reach statistical significance, it might result from some tonic signaling in CAR-iMacs (FIG 8D). In addition, both UTiMacs and CAR-iMacs cocultured with tumor cells had upregulated expression of the pro-inflammatory genes IL-12 and iNOS,57,58 compared with the respective effector cells in the Attorney Docket No.40056-0101WO1 absence of tumor cells (FIG 8D). Consistent with the pro-inflammatory cytokines, protein levels analysis of cell surface markers associated with the tumor-suppressive state of macrophages showed significantly elevated levels of activation markers, such as CD86, CD80, and HLA-II,22 among others, in CAR-iMacs compared with UT-iMacs, regardless of tumor presence (FIGs 2I and 8E). Both UT-iMacs and CAR-iMacs demonstrated similar cell migration potential, as observed in a transwell migration assay (FIG 8F). Importantly, tumor-associated macrophage markers CD20659 and CD16360 were significantly downregulated in CAR-iMacs compared with UT-iMacs, regardless of tumor presence (FIG 2J). In exploring allorejection, CAR-iMacs were subjected to coculture with allogeneic NK cells (alloNK) or allogeneic T cells (allot cells), comparing their response with UT-iMacs and PBMC-Macs (FIG 8G). Notably, whether with or without coculture of alloNK cells for 48–50 h, we did not observe significant macrophage cell death (as determined by DAPI) induced by alloNK cells across the tested groups, as assessed by flow cytometry (FIG 8H). Similarly, when CARiMacs were cocultured with alloT cells for 48–50 h (FIG 8G), no detrimental effect on the survival across the tested groups was observed, suggesting that the CAR iMacs could be resistant to allorejection of alloT cells (FIG 8I). Thus, it appears that CAR-iMacs have a potential for evasion of immune-mediated rejection. The integration of CAR-iMacs into the genome was evaluated through vector copy-number (VCN) analysis, with PBMC-Macs as a negative control. The analysis revealed that the VCN of CAR-iMacs was below 3 after we normalized it by the transduction efficiency (FIGs 8J and 10A). Thus, the copy number falls below the FDA-recommended threshold of 5.61 Taken together, the introduction of CAR into human iPSCderived macrophages drives the polarization of macrophage profiles toward a tumor-suppressive phenotype, triggers NK cell activation, and may mediate an adaptive immune response by activating T cells through antigen cross- presentation. These effects may culminate in enhanced CAR-specific antitumor effects. Example 2: CAR-iMacs effectively inhibit tumor growth in mouse models of pancreatic cancer To evaluate the in vivo antitumor efficacy of CAR-iMacs, we established a human xenograft model of pancreatic cancer in non-obese diabetic (NOD)/SCIDIl2rg-/- hIL3 hGMCSF-hSCF (NSG-SGM3) mice expressing human stem cell factor (SCF), Attorney Docket No.40056-0101WO1 granulocyte-macrophage CSF (GM-CSF), and IL-3 (FIG 3A). Capan-1 cells expressing luciferase_ZsGreen (Capan-1Luc- ZsGreen) were intraperitoneally (i.p.) injected into NSG-SGM3 mice. After 3 h, either CAR-iMacs, mock-iMacs, or saline were infused. We adopted the approach of using both i.p. and intravenous (i.v.) routes of administration based on the rationale that alternate routes would enhance CAR-iMacs trafficking to the pancreas as well as inhibit metastasis to other organs, including the lung, liver, and kidney.19 CAR-iMacs were verified to have high levels of CAR expression on the day of CAR-iMac injection (FIG 9A). Tumor growth was monitored using a luciferase based imaging system. It showed that CAR-iMacs effectively repressed tumor growth compared with saline- and mock treated groups, as demonstrated by monitoring bioluminescence imaging (BLI) in live animals over time (FIGs 3B and 9B). Furthermore, CAR-iMac treatment significantly prolongedthe survival of animals compared with both saline and mock treatment (FIG 3C). We next determined the pharmacokinetics (PKs) of CARiMacs in tumor-free and tumor-bearing mouse models. Using both i.v. and i.p. routes of administration, CAR-iMacs expressing both luciferase and mCherry (CAR-iMacLuc-mCherry) were injected into NSG mice without tumor burden. BLI images were taken to analyze the dynamic change of CAR-iMacLuc- mCherry (FIGs 9C and 9D). A PK study was also conducted on tumor- bearing NSG immunodeficient mice.3 days post-tumor implantation, CAR-iMacs were injected via both i.v. and i.p. routes into the mice. Mice were humanely euthanized on days 1, 3, 7, and 14. The total number of human immune cells within the mice was counted at each time point (FIG 9E). Based on the PK data from both tumor-free and tumor-bearing mouse models, we decided to schedule weekly infusions of CARiMacs. We next established a human xenograft model of metastatic pancreatic cancer in NSG mice to determine the rationale for the dosage of cells to be infused (FIG 3D). Tumor cells were implanted on day 0, followed by the administration of CAR-iMacs via both i.p. and i.v. routes on either days 0, 3, or 7, with the second dose a week later. Tumor growth was monitored by BLI in live animals over time. Remarkably, the administration of CARiMacs resulted in a significant reduction in tumor burden and prolonged the survival of tumor-bearing mice when infused on days 0 and 3 in the pancreatic tumor model, compared with saline control (FIGs 3E–3G). However, we did not observe significant Attorney Docket No.40056-0101WO1 tumor reduction in the late metastasized tumor model, where treatment was delayed until day 7 after tumor implantation. This may be attributed to a considerably higher tumor burden relative to the number of administered CAR-iMacs and the more aggressive tumor growth at the late stage compared with the early stage (FIG 3G). An optimized experimental design remains to be explored to successfully treat the pancreatic cancer model 7 days after tumor implantation. Therefore, we currently lack sufficient data to demonstrate the successful targeting of established metastatic pancreatic cancer. Next, we compared the in vivo antitumor capacity of CARiMacs with CAR-transduced THP-1 (CAR-THP-1). To ensure a fair assessment, we evaluated both CAR products in the same in vivo setting with an equal number of CAR-positive cells. Cells were injected 3 days after tumor transplantation following the dosing strategy outlined in FIG 3D. Time-lapse luciferase imaging of the metastatic pancreatic cancer mouse model illustrates that CAR-iMacs have more prompt and potent anti-cancer activity compared with CAR- THP-1 (FIG 9F). Accordingly, survival data indicated that CAR-THP-1 significantly extended the median survival of tumor-bearing mice from 35 days (in saline- treated mice) to 41 days. Remarkably, CAR-iMacs further prolonged the median survival of treated mice to 61 days, surpassingthat of the CAR-THP-1 group (FIG 9G). Example 3: CAR-iMacs possess low toxicity and tissue damage in vivo We next assessed whether treatment with CAR-iMacs can result in tissue damage and/or signs of cytokine release syndrome (CRS). First, we transplanted human CB HSPCs through i.v. injection into irradiated transgenic NSG-SGM3 mice, followed by profiling lympho-hematopoietic reconstitution 3 months later (FIGs 4A and 9H). To investigate toxicity associated with CAR-iMacs, we implanted tumor cells on day 0 into humanized NSG SGM3 (HuSGM3) mice and then infused either saline (control) or CAR-iMacs on days 7 and 14 after confirming the establishment of a high level of tumor burden (FIGs 4A and 9I), as previously reported for CAR-T cells.62,63 Sera were harvested 2 days after each infusion of CAR-iMacs. Cytokine quantification for CRS-related factors in the sera, including human cytokines IL-6, IL-1b, and interferon (IFN)-g as well as the mouse serum amyloid A3 (SAA3), a prominent acute phase reactant, did not reveal significant Attorney Docket No.40056-0101WO1 differences between saline and CAR-iMac treatments (FIG 4B). In line with this, the assessment of daily weight loss and body temperature measurement showed no significant differences between saline and CAR-iMac-treated groups (FIG 9J). Overall, we did not observe upregulation of CRS-related symptoms in the CAR-iMac treated group compared with the saline-treated group, which is consistent with a recent report.64 Organs, including the liver, lung, spleen, and kidney, were harvested 1 week after the second dose of CAR-iMacs was administered. Hematoxylin and eosin (H&E) staining of tissues from mice treated with CAR-iMacs revealed no overt signs of immune toxicity when compared with mice treated with saline (FIG 4C). Cytokine quantification for inflammatory cytokines, including IL-6, IFN-g, IL-1b, tumor necrosis factor alpha (TNF- a), IL-3, IL-2, IL-10, GM-CSF, and IL-8,65 was also determined in the ascites (abdominal fluid) of these mice (FIGs 4D and 9K). Consistent with the results of sera, we did not observe significant differences in inflammatory cytokines between mice treated with CAR-iMacs and mice treated with saline (FIGs 4D and 9K). Example 4: Cryopreservation and characterization of clonal iPSC-derived CAR- iMac progenitors as an off-the-shelf source for functional CAR-iMacs Scalability and off-the-shelf readiness Scalability and off-the-shelf readiness of an allogeneic product are attractive features for a potential CAR-cell-based therapy platform.66 Therefore, we explored whether we could develop such a product using CAR-iMacs. During differentiation into macrophages, bulk-sorted CAR+ iPSCs showed a reduction in CAR expression (FIGs 2B versus FIG 7B). However, when isolated as monoclonal CAR+ iPSCs and differentiated into mature CAR-iMacs, we observed only about 20% loss of CAR expression during the entire 16–28 days differentiation process from monoclonal CAR+ iPSCs to mature CAR-iMacs (FIG 6F), dropping from 100% to about 80% (FIGs 5A, 5B, and 10A). The CAR expression was maintained when CAR- iMacs remained in the culture for about 2 weeks (FIGs 5B, days 16–28, and 10B). Finally, monoclonal CAR-iMacs also showed excellent phagocytosis against Capan-1 target cells (50.5%) (FIG 10C). Attorney Docket No.40056-0101WO1 We next evaluated conditions for the cryopreservation of CD45+ myeloid progenitors (CAR-iMacP) using both commercially available CryoStor CS5 and CS10 freezing media, in addition to home-made freezing media consisting of complete macrophage media supplemented with either 5% or 10% dimethyl sulfoxide (DMSO). After undergoing one freeze thaw cycle, the viability of CAR-iMacP that had been cryopreserved for 2 months (CAR-iMacFPs; FP stands for frozen progenitors) in CS10 freezing media was typically 80%–85% of the viability observed in fresh progenitors, slightly higher than other freezing media (FIGs 5C and 10D). There were no significant surface marker changes between CAR-iMacFP and fresh CARiMacP compared with mature macrophages (FIG 5D). CAR-iMacFP thawed from all conditions of cryopreservation were able to differentiate into mature CAR-iMacs expressing standard macrophage cell surface markers (FIG 10E). Compared with their corresponding fresh counterparts, we demonstrated that in both monoclonal and bulk CAR-iMacFP, a freeze- thaw cycle did not affect their CAR expression (FIGs 5E and 5F). To test whether the CAR-iMacFP retained effector phagocytosis function after a freeze-thaw cycle, we cocultured Capan-1 cells with either the macrophages differentiated from the cryopreserved mock-iMac progenitors, bulk CAR-iMacFP, or monoclonal CAR-iMacFP. The cytolytic function assessed by flow cytometry demonstrated that cryopreserved monoclonal CAR-iMacFP-derived CAR-iMacs retained potent antitumor phagocytosis of Capan-1 cells when compared with mock-iMacs differentiated from the cryopreserved mock progenitors (FIG 5G). Phagocytosis data also highlighted that monoclonal CAR- iMacs with higher transducing efficiency had significantly higher phagocytosis compared with phagocytosis of bulk-iMacs with lower transducing efficiency (FIG 5G), suggesting a CAR-expression level-dependent effect on phagocytosis. These data highlight that CAR-iMacFP can be viably cryopreserved as an off-the shelf source for development of functional CAR-iMacs. Example 5: B2M-knockout iPSC-derived mature cells escape from the allogeneic HLA-I-mediated cytotoxicity induced by allogeneic CD8 T cells Transplanted cells expressing non-self HLA-I molecules trigger an immune response, resulting in their destruction by recipient CD8 T cells ⁴. We confirmed that HLA-I- Attorney Docket No.40056-0101WO1 expressing mature wild-type (WT) cells derived from iPSCs induce an immune reaction to allogeneic CD8 T cells using the mixed lymphocyte reaction (MLR) assay. To evade this reaction, we deleted HLA-I molecules in an iPSC line by knocking out the B2M gene, which is essential for expressing HLA-I molecules on cell surface (FIG 11A) ^5-7. Bi-allelic B2M-knockout (B2M^KO) iPSC clones were identified by flow cytometry (FIG 11B). In addition, no upregulation of HLA-I molecules was detected in B2M^KO iPSCs, regardless of the presence of IFN-γ (FIG 11B). To assess the immunogenicity of B2M^KO cells, B2M^KO iPSCs were differentiated using our serum-free hematopoietic differentiation protocol ^8,9. As a proof-of-principle, we used B2M^KO iPSC-derived hematopoietic cells (CD45⁺), instead of macrophages (FIG 11C). Allorejection of CD45⁺ cells (B2M^KO) were evaluated in ⁵¹Cr-release cytotoxicity assay. Killing of B2M^KO CD45⁺ hematopoietic cells were evaluated by detecting the release of ⁵¹Cr from B2MKO CD45⁺ hematopoietic cells in an 8-hour coculture system. As shown in FIG 1D, significant killing of WT CD45⁺ hematopoietic cells were observed as determine by the release of compared to B2M^KO CD45⁺ cells (FIG 11D). We confirmed this observation in a multiple donor setting, indicating that B2M^KO iPSC-derived hematopoietic cells decreased their HLA-I-related immunogenicity to allogeneic CD8 T cells in a donor- independent manner. Next, we investigated the effect of B2M on the development of macrophages. Compared to WT iPSCs, B2MKO iPSCs successfully developed into morphologically indistinguishable macrophages. Immunohistochemistry staining revealed the absence of HLA-I molecules in B2M^KO macrophages compared to WT macrophages. Expression of HLA-II molecules were similar on both WT and B2M^KO macrophages (FIG 12). B2M^KO anti-PSCA CAR macrophages differentiated normally into CAR-macrophages. Functionality by phagocytosis assay of B2M^KO anti-PSCA CAR- macrophages (B2M^KO) showed that B2M inactivation did not affect the phagocytosis potential of CAR-macrophages compared to mock-control macrophages (FIG 12B and FIG 12C). Example 6: Ectopic expression of HLA-E in B2M^KO cells inhibits the ‘missing-self’ attack by allogeneic NK cells Attorney Docket No.40056-0101WO1 HLA-I molecules play a crucial role as inhibitory ligands for NK cells, with NK cell activity controlled by the balance of inhibitory and activating signals delivered via NK cell surface receptors ^10,11. Therefore, disabling the HLA-I expression in iPSCs could induce unwanted NK cell activation. Previous studies have shown that HLA-E an NK cell-inhibitory ligand, is highly expressed in classical HLA-I-downregulated tissues, such as placenta ¹² and some tumor cells ¹³, thereby suppressing NK cell activation. Furthermore, research has confirmed that the forced expression of HLA-E in human cell lines lacking HLA-I, can inhibit NK cell-mediated lysis ¹⁴. To address this, we transduced a single-chain-trimer HLA-E fusion molecule (scHLA-E) ^15,16 into B2M^KO iPSCs using piggyBac non-viral delivery system, resulting in B2M^KO cell-expressing HLA-E (B2M^KO- ^E FIG 13A). Sequence of a single-chain-trimer HLA-E fusion molecule (scHLA-E): MSRSVALAVLALLSLSGLEAVMAPRTLILGGGGSGGGGSGGGGSIQRTPKIQVYS RHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLY YTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGS GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQ EGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDR RFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYL EDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITL TWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLP EPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYYK AEWSDSAQGSESHSL Regions of scHLA-E B2M signal peptide: MSRSVALAVLALLSLSGL Cloning artifact: EA G-signal peptide: VMAPRTLIL (G4S)3 linker: GGGGSGGGGSGGGGS Attorney Docket No.40056-0101WO1 B2M mature peptide: IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSF SKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (G4S)4 linker: GGGGSGGGGSGGGGSGGGGS HLA-E: GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQ EGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDR RFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYL EDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITL TWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLP EPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYYK AEWSDSAQGSESHSL We also confirmed that B2M^KO-E iPSCs did not lose HLA-E expression during differentiation from iPSCs into CD45⁺ hematopoietic cells (FIG 13B). Using ⁵¹Cr-release cytotoxicity assay, we evaluated that HLA-E expression on B2M^KO-E iPSC-derived mature cells could partially inhibit the attack by NK cells (FIG 13C). Various levels of inhibition of killing HLA-E expressing B2M^KO cells by NK cells was observed among different donors, suggesting that the degree of inhibition is donor-independent. We did not observe complete inhibition because the inhibitory receptor of HLA-E, NKG2A, is only expressed in around 50% of NK cells in healthy individuals ^17,18. In agreement with the cytotoxicity data, in a coculture experiment, we observed less degranulation of NK cells in WT and HLA-E expressed B2M^KO cells compared to B2M^KO and K562 tumor cells (FIG 13D). Collectively, our data demonstrate the benefit of HLA-E incorporation. Materials

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European journal of immunology 30, 1623-1631, doi:10.1002/1521- 4141(200006)30:6<1623::Aid-immu1623>3.0.Co;2-m (2000). 13. Liu, X. et al. Immune checkpoint HLA-E:CD94-NKG2A mediates evasion of circulating tumor cells from NK cell surveillance. Cancer Cell 41, 272-287.e279, doi:10.1016/j.ccell.2023.01.001 (2023). 14. Han, X. et al. Generation of hypoimmunogenic human pluripotent stem cells. Proceedings of the National Academy of Sciences of the United States of America 116, 10441-10446, doi:10.1073/pnas.1902566116 (2019). 15. Crew, M. D., Cannon, M. J., Phanavanh, B. & Garcia-Borges, C. N. An HLA-E single chain trimer inhibits human NK cell reactivity towards porcine cells. Molecular immunology 42, 1205- 1214, doi:10.1016/j.molimm.2004.11.013 (2005). 16. Gornalusse, G. G. et al. HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells. Nature biotechnology 35, 765-772, doi:10.1038/nbt.3860 (2017). 17. Cooley, S. et al. A subpopulation of human peripheral blood NK cells that lacks inhibitory receptors for self-MHC is developmentally immature. Blood 110, 578-586, doi:10.1182/blood- 2006-07-036228 (2007). 18. Sun, C. et al. High NKG2A expression contributes to NK cell exhaustion and predicts a poor prognosis of patients with liver cancer. Oncoimmunology 6, e1264562, doi:10.1080/2162402x.2016.1264562 (2017). 19. Zijlstra, M. et al. Beta 2-microglobulin deficient mice lack CD4-8+ cytolytic T cells. Nature 344, 742-746, doi:10.1038/344742a0 (1990). Attorney Docket No.40056-0101WO1 20. Koller, B. H., Marrack, P., Kappler, J. W. & Smithies, O. Normal Development of Mice Deficient in β2M, MCClass I Proteins, and CD8+ T Cells. Science 248, 1227-1230, doi:10.1126/science.2112266 (1990). OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No.40056-0101WO1 1. A method for preparing a population of macrophages expressing a chimeric antigen receptor (CAR), the method comprising: (a) isolating a population of CD34⁺ hematopoietic stem and progenitor cells (HSPCs); (b) generating induced pluripotent stem cells (iPSCs) from the HSPCs; (c) introducing a nucleic acid molecule comprising a nucleotide sequence encoding a CAR or membrane bound IL-15 into the iPSCs, thereby creating CAR iPSCs; (d) selecting a CAR iPSC and generating a clonal population of a CAR iPSCs from the selected CAR iPSC; and (e) differentiating at least a portion of the clonal population of CAR iPSCs into CAR macrophages (CAR-iMacs). 2. The method of claim 1, wherein the HSPCs are isolated from human blood or human cord blood. 3. The method of claim 1, wherein the nucleic acid molecule encodes both a CAR and membrane bound IL-15. 4. The method of claim 1, wherein the iPSCs are generated by contacting the HSPCs with one or more of OCT3/4, OCT3, OCT4, SOX2, KLF4, L-MYC, C-MYC, LIN28, or short hairpin RNA targeting TP53 (shRNA-TP53). 5. The method of claim 1, wherein the nucleic acid is a vector. 6. The method of claim 5, wherein the vector is a viral vector. 7. The method of claim 6, the viral vector is selected from a baculovirus, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or Herpes viral vector. Attorney Docket No.40056-0101WO1 8. The method of claim 1, wherein the step of differentiating the clonal CAR iPSCs into CAR macrophages (CAR-iMacs) comprises differentiating the clonal iPSCs into monocyte-committed progenitors (CAR-iMacP), cryopreserving the CAR-iMacP for a period of time, and after a period of time thawing the CAR-iMacP . 9. The method of claim 1, wherein CAR-iMacs express CD45, CD14, CD11b, CD86, CD63, CD80, and CD16. 10. The method of claim 1, wherein the step of differentiating the CAR iPSCs into CAR iMacs comprises: (a) culturing the CAR iPSCs in a hematovascular induction medium comprising Thiazovivin, BMP4, and hVEGF₁₆₅; (b) replacing the hematovascular induction medium with a hematopoietic specification medium comprising hVEGF₁₆₅, human fms-like tyrosine kinase 3 ligand (Flt3L), human stem cell factor (SCF), Insulin-like growth factor 1(IGF-1), human interleukin 3 (IL-3), StemRegenin 1 (CAS No.1227633-49-9; 4-[2-[[2-Benzo[b]thien-3- yl-9-(1-methylethyl)-9H-purin-6-yl]amino]ethyl]phenol), and TGF-βRI inhibitor (SB- 431542; CAS No.301836-41-9; 4-[4-(3,4-Methylenedioxyphenyl)-5-(2-pyridyl)-1H- imidazol-2-yl]benzamide); (c) replacing the hematovascular induction medium with a myeloid hematopoietic differentiation media comprising 10 ng/mL human interleukin 6 (IL-6), and 20 ng/mL human thrombopoietin (TPO); and (d) replacing the myeloid hematopoietic differentiation media with a macrophage differentiation medium comprising L-glutamine, hM-CSF, IL-1β, and IL-6. 11. The method of claim 10, wherein (a) the hematovascular induction medium comprises 10 μM Thiazovivin, 10 ng/mL BMP4, and 50 ng/mL hVEGF₁₆₅; (b) the hematopoietic specification medium comprises 25 ng/mL hVEGF₁₆₅, 20 ng/mL Flt3L, 20 ng/mL SCF, 20 ng/mL IGF-1, 10 ng/mL IL-3, 2 µM StemRegenin 1, and 4 μM SB-431542; Attorney Docket No.40056-0101WO1 (c) the myeloid hematopoietic differentiation medium comprises 10 ng/mL IL-6 and 20 ng/mL TPO; and (d) the macrophage differentiation medium comprises 2mM L-glutamine, 50 ng/mL hM-CSF, 10 ng/mL IL-1β, and IL-6. 12. The method of claim 10, wherein the cells are cultured in the macrophage differentiation medium for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days. 13. The method of claim 1, wherein the step of differentiating the CAR iPSCs into CAR iMacs comprises using a matrix-based culture system. 14. The method of claim 1, wherein the step of generating iPSCs from the HSPCs comprises using a matrix-based culture system. 15. The method of claim 1 wherein the nucleic acid molecule encode a PSCA CAR and membrane bound IL15. 16. The method of any one of the preceding claims, wherein the CAR is specific for a tumor and/or toxin. 17. The method of any one of the preceding claims, wherein the CAR targets any one or more of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CDS, CD6, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CS1, chlorotoxin receptor, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb- B2,3,4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptors, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), Interleukin-13 receptor subunit alpha-2 (IL- 13Rα2), light chain kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y Attorney Docket No.40056-0101WO1 (LeY), LI cell adhesion molecule (LICAM), melanoma antigen family A, 1 (MAGE-AI), Mucin 16 (Muc-16), Mucin 1 (Muc-1), Mesothelin (MSLN), NKG2D ligands, cancer- testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor- associated glycoprotein 72 (TAG- 72), vascular endothelial growth factor R2 (VEGF- R2), Wilms tumor protein (WT-1), or combinations thereof. 18. The method of any one of the preceding claims, wherein the CAR comprises at least one targeting domain, wherein the targeting domain comprises at least one a single chain variable fragment (scFv). 19. The method of any one of the preceding claims, wherein the CAR comprises: at least one targeting domain, a spacer, a transmembrane domain, a co-stimulatory domain, and a CD3 ζ signaling domain. 20. A composition comprising the iPSC-derived macrophages produced by the method of any one of the preceding claims. 21. A population of iPSC-induced macrophages expressing a CAR (CAR iMacs), wherein the CAR comprises: a single chain variable fragment (scFv) targeting a cancer cell antigen. 22. The population of claim 21, wherein the CAR further comprises a spacer, a transmembrane domain, and at least one intracellular domain. 23. The population of claim 21 or 22, wherein the CAR iMacs also express mIL15. 24. The population of any one of claims 21-23, wherein the CAR iMacs also express a tEGFR or a truncated version of CD19 (tCD19). Attorney Docket No.40056-0101WO1 25. The population of any one of claims 21-24, wherein the CAR targets any one or more of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CDS, CD6, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CS1, chlorotoxin receptor, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb- B2,3,4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptors, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), Interleukin-13 receptor subunit alpha-2 (IL- 13Rα2), light chain kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), LI cell adhesion molecule (LICAM), melanoma antigen family A, 1 (MAGE-AI), Mucin 16 (Muc-16), Mucin 1 (Muc-1), Mesothelin (MSLN), NKG2D ligands, cancer- testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor- associated glycoprotein 72 (TAG- 72), vascular endothelial growth factor R2 (VEGF- R2), Wilms tumor protein (WT-1), or combinations thereof. 26. The population of any one of claims 21-25, wherein the CAR iMacs are M0, M1, or M2, or a combination thereof. 27. A composition comprising the population of any one of claims 21-26. 28. A method of increasing survival of a patient having a cancer comprising administering the composition of claim 20 or 27 or the population of any one of claims 21-26 to the patient. 29. A method of reducing or ameliorating a symptom associated with a cancer in a patient comprising administering the composition of claim 20 or 27 or the population of any one of claims 21-26 to the patient. Attorney Docket No.40056-0101WO1 30. A method for treating a cancer in a patient in need thereof, comprising administering the patient the composition of claim 20 or 27 or the population of any one of claims 21-26, wherein the CAR scFv targets an antigen expressed by the cancer, thereby treating the cancer. 31. A method of reducing or eliminating cells expressing an antigen in a patient comprising administering the composition of claim 20 or 27 or the population of any one of claims 21-26 to the patient, wherein the CAR scFv targets the antigen expressed on the cells. 32. A method of reducing or eliminating cells expressing an antigen in a patient comprising administering the composition of claim 20 or 27 or the population of any one of claims 21-26 to the patient, wherein the CAR scFv targets the antigen expressed on the cells. 33. The method of claim 32, wherein the antigen is any one or more of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CDS, CD6, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CS1, chlorotoxin receptor, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb- B2,3,4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptors, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), Interleukin-13 receptor subunit alpha-2 (IL-13Rα2), light chain kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), LI cell adhesion molecule (LICAM), melanoma antigen family A, 1 (MAGE-AI), Mucin 16 (Muc-16), Mucin 1 (Muc-1), Mesothelin (MSLN), NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor- associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF- R2), and Wilms tumor protein (WT-1). 34. The method of claim 32, wherein the cells are cancer cells. Attorney Docket No.40056-0101WO1 35. The method of any one of claims 28-32, wherein the population of CAR iMacs is autologous to the patient being treated. 36. The method of any one of claims 28-32, wherein the population of CAR iMacs is allogeneic to the patient being treated. 37. The method of any one of claims 28-30 and 34, wherein the cancer is any one or more of: a breast cancer, a brain cancer, a blood cancer, a bone cancer, a head or neck cancer, a liver cancer, a lung cancer, a lymphoma, a leukemia, a gall bladder cancer, a gall bladder adenocarcinoma, an oral cancer, an ovary cancer, a pancreatic cancer, a prostate cancer, a skin cancer, a urinary bladder cancer, a cervical cancer, an esophageal cancer, a colorectal cancer, or a gastric cancer. 38. The method of any one of claims 28-37, wherein the composition or the population of CAR iMacs is administered locally or systemically. 39. The method of any one of claims 28-38, wherein the composition or the population of CAR iMacs is administered by single or repeat dosing. 40. A culture medium comprising pluripotent stem cell culture maintenance media (e,g., mTeSR1) supplemented with Thiazovivin,BMP4, and Activin A. 41. The culture medium of claim 40, wherein the supplements are 10 μM Thiazovivin, 25 ng/mL BMP4, and 1 ng/mL Activin A. 42. A culture medium comprising pluripotent stem cell culture maintenance media (e,g., mTeSR1) supplemented with 10 μM Thiazovivin, 10 ng/mL BMP4, and 50 ng/mL hVEGF165 )hematovascular induction medium) 43. The hematovascular induction medium of claim 42, wherein the supplements are 10 μM Thiazovivin, 10 ng/mL BMP4, and 50 ng/mL hVEGF165. 44. A culture medium comprising media for differentiation of iPSC to hematopoietic progenitor cells (e.g., STEMdiff) supplemented with 25 ng/mL hVEGF165, 20 ng/mL human fms-like tyrosine kinase 3 ligand (Flt3L), 20 ng/mL human stem cell factor (SCF), Attorney Docket No.40056-0101WO1 20 ng/mL Insulin-like growth factor 1 (IGF-1), 10 ng/mL human interleukin 3 (IL-3), 2 µM StemRegenin 1, and 4 μM TGF-βRI inhibitor SB-431542 (“hematopoietic specification medium”). 45. The hematopoietic specification medium of claim 44 wherein the supplements are 25 ng/mL hVEGF165, 20 ng/mL human fms-like tyrosine kinase 3 ligand (Flt3L), 20 ng/mL human stem cell factor (SCF), 20 ng/mL Insulin-like growth factor 1 (IGF-1), 10 ng/mL human interleukin 3 (IL-3), 2 µM StemRegenin 1 (CAS No.1227633-49-9) and 4 μM TGF-βRI inhibitor SB-431542 (CAS No. 301836-41-9). 46. A culture medium comprising the hematopoietic specification medium of claim 44 or 45 supplemented with human interleukin 6 (IL-6) and human thrombopoietin (TPO) (“myeloid-skewed hematopoietic differentiation media”). 47. The myeloid skewed hematopoietic differentiation medium of claim 46 wherein the supplements are 10 ng/mL human interleukin 6 (IL-6) and 20 ng/mL human thrombopoietin (TPO). 48. A culture medium comprising Iscove's Modified Dulbecco's Medium (e.g., with L-glutamine and without alpha-thioglycerol and 2-mercaptoethanol) supplemented with fetal calf serum (FCS), L-glutamine, hM-CSF, IL-1β, and IL-6). 49. The culture medium of claim 48 wherein the supplements are 10% fetal calf serum (FCS), 2mM L-glutamine, 50 ng/mL hM-CSF, 10 ng/mL IL-1β, and 5 ng/mL IL-6). 50. A method for differentiating myeloid progenitor cells, comprising culturing the cells in the culture medium of claim 48 or 49. 51. A method for preparing a population of induced macrophages, the method comprising: providing a population of human induced pluripotent stem cells (iPSCs) comprising a nucleic acid molecule encoding a HLA-E protein (“HLA-modified iPSCs”); Attorney Docket No.40056-0101WO1 transducing at least a portion of the HLA-modified iPSCs with a viral vector comprising a nucleotide sequence encoding soluble or membrane-bound human IL-15; selecting a transduced HLA-modified hiPSC (“IL-15 HLA-modified hiPSC”) and generating a clonal population from the selected iPSC; and differentiating at least a portion of the clonal population IL-15 HLA-modified hiPSC into macrophage precursors (iMacs-P). 52. The method of claim 51, wherein the HSPCs are isolated from human blood or human cord blood. 53. The method of claim 51, wherein the viral vector encodes both a CAR and membrane bound IL-15. 54. The method of claim 51, wherein the iPSCs are generated by contacting the HSPCs with one or more of OCT3/4, OCT3, OCT4, SOX2, KLF4, L-MYC, C-MYC, LIN28, or short hairpin RNA targeting TP53 (shRNA-TP53). 55. The method of claim 51, wherein the nucleic acid is a vector. 56. The method of claim 55, wherein the vector is a viral vector. 57. The method of claim 56, the viral vector is selected from a baculovirus, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or Herpes viral vector. 58. The method of claim 51, wherein the step of differentiating the clonal iPSCs into iMacs comprises differentiating the clonal iPSCs into monocyte-committed progenitors (iMacP), cryopreserving the iMacP for a period of time, and after a period of time thawing the iMacP. 59. The method of claim 51, wherein iMacs express CD45, CD14, CD11b, CD86, CD63, CD80, and CD16. Attorney Docket No.40056-0101WO1 60. The method of claim 51, wherein the step of differentiating the iPSCs into iMacs comprises: (a) culturing the iPSCs in a hematovascular induction medium comprising Thiazovivin, BMP4, and hVEGF165; (b) replacing the hematovascular induction medium with a hematopoietic specification medium comprising hVEGF165, human fms-like tyrosine kinase 3 ligand (Flt3L), human stem cell factor (SCF), Insulin-like growth factor 1(IGF-1), human interleukin 3 (IL-3), StemRegenin 1 (CAS No. 1227633-49-9; 4-[2-[[2-Benzo[b]thien-3- yl-9-(1-methylethyl)-9H-purin-6-yl]amino]ethyl]phenol), and TGF-βRI inhibitor (SB- 431542; CAS No. 301836-41-9; 4-[4-(3,4-Methylenedioxyphenyl)-5-(2-pyridyl)-1H- imidazol-2-yl]benzamide); (c) replacing the hematovascular induction medium with a myeloid hematopoietic differentiation media comprising 10 ng/mL human interleukin 6 (IL-6), and 20 ng/mL human thrombopoietin (TPO); and (d) replacing the myeloid hematopoietic differentiation media with a macrophage differentiation medium comprising L-glutamine, hM-CSF, IL-1β, and IL-6. 61. The method of claim 60, wherein (a) the hematovascular induction medium comprises 10 μM Thiazovivin, 10 ng/mL BMP4, and 50 ng/mL hVEGF165; (b) the hematopoietic specification medium comprises 25 ng/mL hVEGF165, 20 ng/mL Flt3L, 20 ng/mL SCF, 20 ng/mL IGF-1, 10 ng/mL IL-3, 2 µM StemRegenin 1, and 4 μM SB-431542; (c) the myeloid hematopoietic differentiation medium comprises 10 ng/mL IL-6 and 20 ng/mL TPO; and (d) the macrophage differentiation medium comprises 2mM L-glutamine, 50 ng/mL hM-CSF, 10 ng/mL IL-1β, and IL-6. Attorney Docket No.40056-0101WO1 62. The method of claim 60, wherein the cells are cultured in the macrophage differentiation medium for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days. 63. The method of claim 51, wherein the step of differentiating the iPSCs into iMacs comprises using a matrix-based culture system. 64. The method of claim 51, wherein the step of generating iPSCs from the HSPCs comprises using a matrix-based culture system. 65. The method of claim 51, wherein the vecotr encodes a PSCA CAR and membrane bound IL15. 66. The method of any one of the preceding claims, wherein the CAR is specific for a tumor and/or toxin. 67. The method of any one of the preceding claims, wherein the CAR targets any one or more of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CDS, CD6, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CS1, chlorotoxin receptor, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb- B2,3,4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptors, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), Interleukin-13 receptor subunit alpha-2 (IL- 13Rα2), light chain kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), LI cell adhesion molecule (LICAM), melanoma antigen family A, 1 (MAGE-AI), Mucin 16 (Muc-16), Mucin 1 (Muc-1), Mesothelin (MSLN), NKG2D ligands, cancer- testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor- associated glycoprotein 72 (TAG- 72), vascular endothelial growth factor R2 (VEGF- R2), Wilms tumor protein (WT-1), or combinations thereof. Attorney Docket No.40056-0101WO1 68. The method of any one of the preceding claims, wherein the CAR comprises at least one targeting domain, wherein the targeting domain comprises at least one a single chain variable fragment (scFv). 69. The method of any one of the preceding claims, wherein the CAR comprises: at least one targeting domain, a spacer, a transmembrane domain, a co-stimulatory domain, and a CD3 ζ signaling domain. 70. The method of claim 51, wherein the HLA-E protein comprises the amino acid sequence: GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQ EGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDR RFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYL EDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITL TWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLP EPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYYK AEWSDSAQGSESHSL. 71. The method of claim 70, wherein the HLA modified iPSCs comprise a transgene encoding a polypeptide comprising: IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSF SKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM. 72. The method of claim any of the forgoing claims wherein the cells (e.g., the iPSC or iMACs) do not express HLA Class 1 genes and/or the B2M gene is knocked out. 73. A macrophage derived from human iPSC and expressing C-C chemokine receptor type 2 (CCR2) and colony stimulating factor receptor (CSF-R1) (“iMac”). 74. The iMac of claim 73, wherein the iMac harbor a nucleic acid molecule encoding soluble or membrane bound IL-12. 75. The iMac of claim 73 or 74, harboring a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence: Attorney Docket No.40056-0101WO1 GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQ EGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDR RFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYL EDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITL TWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLP EPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYYK AEWSDSAQGSESHSL. 76. The iMac of any of claims 73-75, wherein the iMac harbor a nucleic acid molecule encoding a polypeptide comprising: IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSF SKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM. 77. The iMac of any of claims 74-75 wherein the iMac do not express an HLA-1 molecule. 78. The iMac of claims of claims 74-77 wherein the B2M gene is knocked out.