Monocyte or macrophage compositions with enhanced Anti- tumor activity and uses thereof

Abstract

Provided herein are compositions, kits, and methods for manufacturing cells for adoptive cell therapy comprising engineered monocytes or engineered monocyte-derived macrophages that overexpress ID3 for the treatment of cancer.

Description

Atty. Dkt. No.: 115872-3124 MONOCYTE OR MACROPHAGE COMPOSITIONS WITH ENHANCED ANTI- TUMOR ACTIVITY AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No.63 / 598,474, filed November 13, 2023, the contents of which are incorporated herein by reference in their entireties. TECHNICAL FIELD

[0002] The present technology relates to compositions, kits, and methods for manufacturing cells for adoptive cell therapy comprising engineered monocytes or engineered monocyte-derived macrophages that overexpress ID3 for the treatment of cancer. GOVERNMENT SUPPORT

[0003] This invention was made with government support under CA008748 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0004] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0005] The molecular understanding of mechanisms that control the growth of tumor cells within target tissues help the identification of therapeutic targets and strategies7,10-14. Macrophages are an important component of these niches15-17, which can recognize, bind, and phagocytose tumor cells2,17-19, but frequently fail to do so and can even support tumor growth and dissemination15,16. Macrophage activation and phagocytosis are tightly controlled by a balance of activating and inhibitory receptors which protect normal tissues4,6,8,9,20but allow tumoral cells to escape7,10,14. Binding of the tyrosine-based inhibitory motif (ITIM)-containing SIRPA (inhibitory receptors Signal regulatory protein α)3or Siglec-10 (sialic-acid-binding Ig-like lectin 10)7to their respective ligands CD47 and CD24 on tumor cells prevents activating receptors such as dectin-11, dectin-221, and the calreticulin receptor LRP1 which bind sialoglycoprotein on tumoral cells2,18from initiating macrophage activation.4,5,7The identification of such mechanisms is of general interest to -1- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 clinical tumor immunology, as they could be harnessed for the purpose of novel cellular therapies in cancer. SUMMARY OF THE PRESENT TECHNOLOGY

[0006] In one aspect, the present disclosure provides an engineered monocyte or engineered monocyte-derived macrophage comprising a non-endogenous expression vector including a mammalian ID3 nucleic acid. In some embodiments, the monocyte-derived macrophages or monocytes are CD14+ / CD15-. Additionally or alternatively, in some embodiments, the monocyte-derived macrophages or monocytes are CD163+or mannose receptor+. In some embodiments, the non-endogenous expression vector is a plasmid, a cosmid, a bacmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, or a retroviral vector. The engineered monocyte or engineered monocyte-derived macrophage may be derived from bone marrow or induced pluripotent stem cells. Additionally or alternatively, in certain embodiments, the mammalian ID3 nucleic acid is operably linked to an expression control sequence. The expression control sequence may be an inducible promoter, a constitutive promoter, a native ID3 promoter, or a heterologous promoter. Additionally or alternatively, in some embodiments, the mammalian ID3 nucleic acid comprises the sequence of SEQ ID NO: 3 or wherein the mammalian ID3 nucleic acid encodes an Id3 polypeptide comprising the amino acid sequence of SEQ ID NO: 4. In any of the preceding embodiments, the non-endogenous vector including the mammalian ID3 nucleic acid further comprises at least one of a bioluminescent protein, a fluorescent protein, a chemiluminescent protein, an epitope tag, or a selectable marker.

[0007] Additionally or alternatively, in certain embodiments, the engineered monocyte or engineered monocyte-derived macrophage further comprises a non-endogenous expression vector including a heterologous nucleic acid encoding one or more genes selected from the group consisting of dectin-1, dectin-2, dectin-3, or mincle. In some embodiments, the non-endogenous expression vector including the mammalian ID3 nucleic acid and the non-endogenous expression vector including the heterologous nucleic acid encoding the one or more genes is the same. In other embodiments, the non-endogenous expression vector including the mammalian ID3 nucleic acid and the non-endogenous expression vector including the heterologous nucleic acid encoding the one or more genes are distinct. -2- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0008] Additionally or alternatively, in some embodiments, the engineered monocyte or engineered monocyte-derived macrophage lacks detectable expression or activity of SIRPA, CLEC4a, or Siglec-E. In certain embodiments, the engineered monocyte or engineered monocyte-derived macrophage expresses at least one inhibitory nucleic acid that specifically targets and inhibits expression of one or more of SIRPA, CLEC4a, or Siglec-E. The at least one inhibitory nucleic acid may be an antisense oligonucleotide, a siRNA, a sgRNA or a shRNA. In some embodiments, the engineered monocyte or engineered monocyte-derived macrophage comprises a deletion, insertion, inversion, or frameshift mutation in one or more genes selected from among SIRPA, CLEC4a, or Siglec-E.

[0009] Additionally or alternatively, in some embodiments, the engineered monocyte or engineered monocyte-derived macrophage further comprises a receptor that specifically binds to a tumor antigen and / or a nucleic acid encoding the receptor. The receptor may be a chimeric antigen receptor (CAR). Additionally or alternatively, in some embodiments, the CAR comprises (i) an extracellular antigen binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain, wherein the extracellular antigen binding domain binds to the target antigen. In certain embodiments, the extracellular antigen binding domain comprises a single chain variable fragment (scFv) or a human scFv. The extracellular antigen binding domain may comprise a signal peptide that is covalently joined to the N- terminus of the extracellular antigen binding domain. The transmembrane domain may comprise a CD8 transmembrane domain or a CD28 transmembrane domain. In certain embodiments, the intracellular domain comprises a CD3ζ signaling domain and optionally one or more costimulatory domains selected from a CD28 costimulatory domain, a 4-1BB costimulatory domain, an OX40 costimulatory domain, an ICOS costimulatory domain, a DAP-10 costimulatory domain, a PD-1 costimulatory domain, a CTLA-4 costimulatory domain, a LAG-3 costimulatory domain, a 2B4 costimulatory domain, a BTLA costimulatory domain, or any combination thereof. Additionally or alternatively, in some embodiments of the engineered monocyte or engineered monocyte-derived macrophage, the target antigen comprises a tumor antigen. Examples of tumor antigens include, but are not limited to, 5T4, alpha 5β1-integrin, 707-AP, A33, AFP, ART-4, B7H4, BAGE, Bcl-2, β- catenin, BCMA, Bcr-abl, CA125, CA19-9, CAMEL, CAP-1, CASP-8, CD4, CD5, CD19, CD20, CD21 , CD22, CD25, CDC27 / m, CD33, CD37, CD45, CD52, CD56, CD80, CD123, CDK4 / m, CEA, c-Met, CS-1, CT, Cyp-B, cyclin B1, DAGE, DAM, EBNA, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ephrinB2, estrogen receptor, ETV6-AML1, FAP, ferritin, -3- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 folate-binding protein, GAGE, G250, GD-2, GM2, GnT-V, gp75, gp100 (Pmel 17), HAGE, HER-2 / neu, HLA-A*0201-R170I, HPV E6, HPV E7, Ki-67, HSP70-2M, HST-2, hTERT (or hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR / FUT, LRP, MAGE, MART, MART-1 / melan-A, MART-2 / Ski, MC1R, mesothelin, MUC16, MUM-1 -B, myc, MUM-2, MUM-3, NA88-A, NYESO-1, NY-Eso-B, p53, proteinase-3, p190 minor bcr-abl, Pml / RARα, PRAME, progesterone receptor, PSA, PSCA, PSM, PSMA, ras, RAGE, RU1 or RU2, RORI, SART-1 or SART-3, survivin, TEL / AML1, TGFβ, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, tenascin, TSTA tyrosinase, VEGF, or WT1.

[0010] In any of the preceding embodiments, the engineered monocyte or engineered monocyte-derived macrophage is derived from an autologous donor or an allogenic donor.

[0011] In one aspect, the present disclosure provides a composition comprising an effective amount of any and all embodiments of the engineered monocyte or engineered monocyte-derived macrophage disclosed herein, and a pharmaceutically acceptable carrier.

[0012] In one aspect, the present disclosure provides a method for treating cancer or inhibiting tumor growth in a subject in need thereof comprising administering to the subject an effective amount of any and all embodiments of the ID3-overexpressing the engineered monocyte or engineered monocyte-derived macrophage described herein. In some embodiments, the cancer is selected from the group consisting of adrenal cancers, bladder cancers, blood cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's disease, intestinal cancers, kidney cancers, larynx cancers, acute and chronic leukemias, liver cancers, lymph node cancers, lymphomas, lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, non- Hodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, and metastases thereof. In some embodiments, the cancer is a relapsed or refractory cancer. In some embodiments, the cancer is resistant to one or more cancer therapies, e.g., one or more chemotherapeutic drugs.

[0013] In any and all embodiments of the methods disclosed herein, the engineered monocyte or engineered monocyte-derived macrophage is administered pleurally, -4- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 intravenously, subcutaneously, intranodally, intratumorally, intrathecally, intrapleurally or intraperitoneally. Additionally or alternatively, in some embodiments, the methods of the present technology further comprise administering to the subject an additional anti-cancer therapy. Examples of additional anti-cancer therapies include, but are not limited to, chemotherapeutic agents, adoptive cell therapy, immune checkpoint inhibitors, monoclonal antibodies that specifically target tumor antigens, immune activating agents (e.g., interferons, interleukins, cytokines), oncolytic virus therapy and cancer vaccines. In some embodiments of the methods disclosed herein, the adoptive cell therapy comprises administering to the subject an effective amount of lymphoid effector cells selected from among T cells, B cells, or NK cells. In certain embodiments, the lymphoid effector cells express a native or non-native receptor (e.g., a CAR) that binds to a tumor antigen, and / or are administered intra-arterially.

[0014] In one aspect, the present disclosure provides a method of preparing immune cells for adoptive cell therapy comprising: (a) isolating monocytes or monocyte-derived macrophages from a donor subject, and (b) transducing the isolated monocytes or monocyte-derived macrophages with a nucleic acid encoding ID3 or an expression vector comprising said nucleic acid, optionally wherein the nucleic acid encodes an Id3 polypeptide comprising the amino acid sequence of SEQ ID NO: 4. Additionally or alternatively, in some embodiments, the method further comprises transducing the isolated monocytes or monocyte-derived macrophages with at least one additional nucleic acid encoding one or more of dectin-1, dectin-2, dectin-3, mincle, or a CAR that binds a tumor antigen, or an expression vector comprising the at least one additional nucleic acid. Additionally or alternatively, in certain embodiments, the method further comprises transducing the isolated monocytes or monocyte-derived macrophages with at least one inhibitory nucleic acid that specifically targets and inhibits the expression of one or more of SIRPA, CLEC4a, or Siglec-E. In any of the preceding embodiments, the method further comprises administering the transduced monocytes or monocyte-derived macrophages to a recipient subject. The donor subject and the recipient subject may be the same or different.

[0015] In another aspect, the present disclosure provides a method for treating cancer or inhibiting tumor growth in a subject in need thereof comprising administering to the subject an effective amount of nanoparticles comprising ID3 nucleic acid molecules (e.g., mRNA) or ID3 polypeptides, wherein the nanoparticles are configured to target macrophages or monocytes in the subject. In some embodiments, the nanoparticles target at least one of -5- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 BTK, Siglec-1, TLR, TLR7, mannose receptor, TLR3, VEGF, SHP2, PIGF, CSF-1R or CCR2. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGs.1A-1J Kupffer cells restrict tumor cell engraftment and metastasis. FIGs.1A-1E: Analysis of tumor burden by bioluminescence in liver, lung, spleen, and pancreas of mice 8 weeks after orthotopic pancreas injection of 2×105KPC-2-luciferase cells. FIG.1A: Flt3CreCsf1rf / f(n=12) and control Csf1rf / flittermates (n= 12). FIG.1B: Ccr2- / -(n=12) and Ccr2+ / -littermates (n=11), FIG.1C: Clec4fCreCsf1rf / f(n=19) and Csf1rf / flittermates (n=25). FIG.1D: Clec4fCreSpi1f / f(n=12) and Spi1f / flittermates (n=13). FIG.1E: Clec4fCreR26LSL-DTR(n=26), and R26LSL-DTRlittermates (n=33) received weekly intraperitoneal injection of DT (see methods) from week 1 to 7. Circles represent individual mice, boxes represent 25-75% confidence interval and whisker indicate extreme values. Blue line indicates the median. Green histograms represent background bioluminescence imaging signal from wt C57BL / 6j mice that did not receive tumors (n=3 mice per group). Results from at least 3 independent experiments per genotype. Statistics: Mann-Whitney test, two-tailed, with p<0.05 considered significant. FIG.1F: Schematic of pancreas venous drainage. FIG.1G: Clec4fCreR26LSL-DTR(n=5) and R26LSL-DTRlittermate (n=6) received intra-portal injection of 3x105KPC-2-luciferase cells (d0), and DT injections (d-1, d7 and d14). Survival is analyzed using the Log-rank (Mantel-Cox) test. FIG.1H: Clec4fCreR26LSL-DTR(n=7), and R26LSL-DTRcontrols (n=4) received DT injection 24hrs before intra-portal injection of 1×106KPC-1-luciferase-gfp cells. Numbers of CD45- GFP+tumor cells / g of liver is analyzed 24h later by flow cytometry. FIG.1I: Clec4fCreR26LSL-DTR(n=7) and R26LSL-DTRlittermates (n=8) received 1×106KPC-1-luciferase cells (d0), DT injections (d-1 and d7), and livers are analyzed at d14 by bioluminescence imaging. Representative liver micrographs are shown (right). FIG.1J: As in FIG.1I, with DT injection at d3 and d10 (n=8 and 6). Circles represent individual mice, bars: mean ± sd. Statistics: two-tailed unpaired t-test with p<0.05 considered significant.

[0017] FIGs.2A-2J: Peritumoral niche. FIG.2A: Immunofluorescence staining for F4 / 80, Tim4, Clec4f and CK19 on liver sections from 6-month-old KPC mice (from n=3 independent experiments). FIG.2B: Immunofluorescence (left) and flow cytometry analysis (right) of CD45.1+macrophages in the liver of CD45.2 partner from CD45.1 / CD45.2 parabiotic pairs, 2 weeks after intra-portal injection of 1×106KPC-1-tdT cells in the CD45.2 partner (n=5), or without tumor injection (n=4). FIG.2C: RT-qPCR for -6- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 indicated genes in KC, 2 weeks after intra-portal injection of 1×106KPC-1-tdT cells or in control mice, n=3 mice / group. FIG.2D: immunofluorescence staining and percentage of Tim4+KCs containing tdT in mice from (c), n=4. FIG.2E: tdT expression in LAMP1+(n=713) and LAMP1- (n=237) areas in KC from FIG.2C. FIG.2F: Engulfment of KPC-1- tdT cells by KCs in vivo by intravital imaging. KC and dying cells are labeled by i.v. injection of F4 / 80-AF647 antibodies and CellEvent™Cas3 / 7-green respectively. Arrow: engulfing KC, n= 3. FIG.2G: in vitro analysis of KPC-1 engulfment by KCs in presence of PBS control (n=5 experiments), phosphatidylserine blockade (D89E, n=3) or actin inhibitor (Latrunculin A, n=3). KC and dying cells are labeled as in FIG.2F. Open arrow: engulfing KC, closed arrow: Cas3 / 7 cleavage. Plots represent the % of KCs engulfing Cas3 / 7-green- KPC-1-tdT cells, and the time from stable interaction between individual KC (PBS n=17, D89E=31) and tumor cells to engulfment and Casp.3 / 7 cleavage. FIG.2H: Expression of chemokines / cytokines by Tim4+KCs (n numbers in green) in tumor core and peritumoral liver (0-50mm from tumor and >50mm from tumor) 2 weeks after injection of 1×106KPC- 1-gfp cells. FIGs.2I-2J: representative staining and number of CD8+T cells (i) and Lamp1+Nkp46+cells (j) in liver from (h), n=3 mice. statistics: One-way ANOVA (FIGs. 2B, 2D, 2G, 2I, and 2J), Mann-Whitney test, two-tailed (FIG.2E), Kruskal-Wallis test (FIG.2H), unpaired two-tailed t-test (FIGs.2C and 2G). mean ± sd. ns, not significant.

[0018] FIGs.3A-3K: Id3-expressing KC are required for the restriction of tumor growth. FIG.3A: Analysis of tumor burden by bioluminescence in liver from Id3- / -mice (n=7) and Id3+ / +littermates (n=6) two weeks after intra-portal injection of 1×106KPC-1- luciferase cells, results from 2 independent experiments. FIG.3B: RT-qPCR for Id3 mRNA in macrophage populations from 3 C57BL / 6j mice. FIG.3C: Flow cytometry analysis of tdT expression by Tim4+KCs from Clec4fCre-tdTmice at E15.5 (n=4), E15.5 (n=3), P2 (n=5), P10 (n=5), and 8 week-old (n=3) . FIG.3D: Flow cytometry and immunofluorescence analysis of KCs numbers and morphology in liver from 6 weeks-old Clec4fCre-tdTId3f / fand Id3f / flittermates (n=5 / group). FIG.3E: Flow cytometry analysis of uptake of 2μm beads injected intravenously 2 hrs before analysis by KC from Clec4fCre-tdTId3f / f, and Id3f / flittermates (n=4 / group). FIG.3F: Representative immunofluorescence staining for GFP, Tim4 and F4 / 80 in liver from Clec4fCre-tdTId3f / f(n=4), and Id3f / flittermates (n=5) two weeks after intra-portal injection of 1×106KPC-1-luciferase-gfp cells. FIG.3F: Analysis of tumor burden by bioluminescence in liver from mice in FIG.3F. FIG.3H: Analysis of tumor burden by bioluminescence in Clec4fCreId3f / fmice (n=10) and Id3f / f-7- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 littermates (n=16) 8 weeks after orthotopic pancreas injection of 2×105KPC-2-luciferase cells. i- Number of GFP+CD45-, GFP+CD45-CD47brightand CD9+CD133+tumor cells per liver lobe from the mice in FIGs.3F-3G determined by flow cytometry. FIG.3J: Survival of Clec4fCreId3f / f(n=8) and Id3f / flittermates (n=8) after intra-portal injection of 3x105KPC- 2-luciferase cells (Log-rank (Mantel-Cox) test. FIG.3K: Analysis of tumor burden by bioluminescence in liver from Clec4fCreId3f / fmice (n=4) and Id3f / flittermates (n=4) 2 weeks after received intra-portal injection of 5×105B16F10-luciferase cells, 1×106MC38- luciferase cells, or 1×106LLC1-luciferase cells. Statistics: unpaired two-tailed t-test (FIGs. 3, 3D, 3E, 3G, 3I, and 3K), Mann-Whitney test (FIG.3H). mean ± sd. ns, not significant.

[0019] FIGs.4A-4L: Id3-dependent peri-tumoral niche. FIGs.4A-4B: RNAseq analysis of KC from Clec4fCreId3f / f(n=2) and Id3f / flittermates (n=3). Bar graph: pathways downregulated in Clec4fCreId3f / f, heatmap: selected differentially expressed genes. FIG.4C: RT-qPCR for selected genes in KC from Clec4fCreId3f / fand Id3f / flittermates (n=3 / group). FIG.4D: Expression by flow-cytometry of SIRPA and DECTIN1 by KC from Clec4fCreId3f / fand Id3f / flittermates 2 weeks after intraportal injection of 1×106KPC-1-gfp cells or not injected, n=3 / group. FIG.4E: Percentage of Tim4+KCs containing GFP in liver from Clec4fCreId3f / f(n=4) and Id3f / flittermates (n=3) treated as in FIG.4D. FIG.4F: Left: percentage of Tim4+KCs stained with GFP by immunofluorescence in Clec4fCreId3f / fand Id3f / flittermate 24h after intraportal injection of 1×106KPC-1-gfp cells (n=4 / group). Right: number of CD45-GFP+tumor cells / liver lobe by flow-cytometry in the same mice (n=5 / group). FIG.4G: Engulfment of KPC-1-tdT cells by Clec4fCreId3f / for Id3f / fKCs in vitro, as in FIG.2G. Plots represent % of KCs engulfing Cas3 / 7-green- KPC-1-tdT cells (n=3 / group), and the time from stable interaction between KC (Id3f / fn=25, Clec4fCreId3f / fn=7) and tumor cells to engulfment or Casp.3 / 7 cleavage. FIG.4H: Expression of chemokines / cytokines by Tim4+KCs from Id3f / fand Clec4fCreId3f / fmice ) treated as in FIG. 4D, n numbers indicated in green. FIGs.4I-4J: Flow cytometry (FIG.4I) and immunofluorescence analysis (FIG.4J) of CD8+T-cells and Lamp1+Nkp46+cells numbers per g of liver or mm2in mice from (FIG.4H), n=5 mice / group. FIG.4K: IFN-γ and TNF expression by NKp46+and CD8+T cells from (FIGs.4I-4J). FIG.4L: Liver tumor burden by photoradiance 2 weeks after injection of 1×106KPC-1-luciferase cells in Id3f / fmice treated with IgG (n=6) or anti-CD8 / NK1.1 (n=3) and Clec4fCreId3f / fmice treated with IgG or anti-CD8 / NK1.1 (n=4 / group). Statistics: unpaired two-tailed t-test (FIGs.4C, 4D, 4F, -8- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 4G, 4I, 4K, and 4L), One-way ANOVA (FIGs.4E, 4J, and 4L), Kruskal-Wallis test (FIG. 4H), mean ± sd. ns, not significant.

[0020] FIGs.5A-5N: SIRPα and Dectin1 mediates in part ID3 function. FIG.5A: RT-qPCR for Sirpα mRNA in macrophage from 3 C57BL / 6j mice. FIGs.5B-5D: Analysis of Clec4fCreId3f / fand Id3f / flittermates 2 weeks after intra-portal injection of 1×106KPC-1- luciferease cells and treatment with anti-SIRPA or IgG control antibodies. FIG.5B RT- qPCR for indicated genes mRNA in KCs, n=3 mice / group. FIG.5C Photoradiance and histology analysis of liver tumor burden+IgG, Clec4fCreId3f / f+IgG or +anti-SIRPA, n 5 / group, Id3f / f+IgG, Id3f / f+anti-SIRPA, n=4 / group). FIG.5D Percentage of Tim4+KCs containing GFP+ material in the peritumoral niche, Id3f / f / IgG and Clec4fCreId3f / f / IgG, n=4, Clec4fCreId3f / f / aSIRPA, n=3. FIG.5E: In vitro engulfment of live KPC-1-mtdT cells (see FIG.2G) by KCs from Id3f / fwith IgG (n=3 independent experiments) or anti-dectin1 antibodies (n=4) and Clec4fCreId3f / flittermates with IgG or anti-SIRPA (n=3 / group). FIG. 5F: RT-qPCR analysis of mRNA gene expression by wt Kupffer cells cocultured with KPC-1 cells for 12h or not, in the presence of anti-Dectin1 or control IgG, n=3 / group. FIG. 5G-5J: Numbers of CD8+T cells and NK cells by flow cytometry (FIG.5G, n=5 / group), immunofluorescence (FIG.5H, n=4 / group), and production of cytokines by CD8+T cells and NK cells (FIGs.5I-5J, n=4 / group) in tumoral liver from mice treated as in (FIGs.5B- 5D). FIG.5K: Hypothesis for the regulation by ID3 of Sirpα transactivation in macrophages. FIG.5L: CUT&RUN analysis of E2A and ELK1 binding to Sirpα predicted promoter / enhancer regions in KCs from Clec4fCreId3f / fmice and Id3f / flittermates (n=3 / group). FIG.5M: Flow cytometry analysis of SIRPA expression by KCs from Id3+ / +and Id3- / -littermates, expressing scramble, E2A, or ELk1 Sh-RNAs (n=3 / group). FIG.5N: Flow cytometry analysis of SIRPA expression by BMDM expressing lenti-Id3, lenti- control, or indicated Sh-RNAs (n=3 / group). Statistics: One-way ANOVA (FIGs.5B, 5C, 5D, 5E, 5F, 5G, 5H, 5I, 5J, 5M, and 5N), unpaired two-tailed t-test (FIGs.5C and 5L). Dots represent individual mice (FIGs.5A, 5B, 5C, 5D, 5G, 5H, 5I, and 5J). mean ± sd. ns, not significant.

[0021] FIGs.6A-6H: ID3 expression endows macrophages with anti-tumor activity. FIG.6A: Left: RT-qPCR for indicated genes in mouse BMDM expressing lenti-mId3 or lenti-control (left,n=3 independent experiments). Right: in vitro engulfment of Cas3 / 7Green- KPC-1-memtdT cells by mouse BMDM expressing a lenti-mId3 (n=3) or a lenti-control with / without α-SIRPA blocking antibodies (n=3 and 4). FIG.6B: Left: RT- -9- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 qPCR for indicated genes and right: engulfment of Cas3 / 7Green- Panc1-memtdT cells by human iPSC-derived macrophages (hiPSCMacs) expressing lenti-hId3 or lenti-control, n=4 experiments. FIG.6C: RT-qPCR for chemokines and cytokines in hiPSCMacs expressing lenti-hId3 or lenti-control, cultured alone or with Panc1 cells for 48h, n=3 experiments. FIGs.6D and 6E: Flow cytometry analysis of proliferation of human CD8+T cells (FIG. 6D), and IFN-γ expression by human CD8+T cells and CD56+NK cells (FIG.6E) cultured with supernatants from hiPSC-derived macrophages / Panc1 cocultures in FIG.6C, n=3 experiments. FIG.6F: Liver pictures and bioluminescence analysis of liver tumor burden of Clec4fCreId3f / fmice 2 weeks after intra-portal injection of 1×106KPC-1-luciferease cells and intra-portal injection of 1×106BMDM expressing lenti-control(n=4) or lenti-mId3 cells(n=6) at d7. FIG.6G: C57BL / 6j mice received 1x106LLC1-luciferase cells by intra- portal injection at d0, and 1x106BMDM expressing lenti-control, lenti-mId3, or PBS at d7. Left: Bioluminescence analysis of liver tumor burden at d14 (PBS n=4, BMDM / Lenti-ctrl n=5, BMDM / Lenti-mId3 n=6). Right: Survival analysis (Log-rank, Mantel-Cox test (n=6,5,6 mice / group respectively). FIG.6H: C57BL / 6j mice received subcutaneous 1×106B16F10-luci-tdT cells into left and right flanks at d0 and intra-tumor injection of 5×105BMDM expressing lenti-control or lenti-mId3 cells at d7. At d14 flank tumor burden is analyzed by bioluminescence (n=20 mice / group, left), and recruitment of Nkp46+NK and CD8+T cells (n=7 mice / group, center) and IFN-γ production (n=5 mice / group, right) are analyzed by flow-cytometry. Statistic: One-way ANOVA (FIGs.6A, 6C, 6D, 6E, and 6G), Mann-Whitney test, two-tailed (FIG.6H), unpaired two-tailed t-test (FIGs.6A, 6B, 6F, and 6H). mean ± sd. ns, not significant.

[0022] FIGs.7A-7N. Targeting of Kupffer cells in tumor models. FIG.7A: Kupffer cell number (Tim4+F4 / 80+, see methods) analyzed by flow cytometry in 6 to 8 weeks-old C57BL / 6J mice treated with the Csf1r small molecule antagonist PLX5622 food, or control food for 2 weeks, n=5 mice per group. FIG.7B: Photoradiance and histology analysis of liver tumor burden in 6-8 weeks-old C57bl / 6j mice (n=7) pretreated with PLX5622 Csf1r small molecule antagonist food or control food for 2 weeks, followed by intra-portal injection of 1×106cells from the, KPC-1-luciferase(n=9 and 8 for control and PLX respectively), LLC1-luciferase (n=5(n= 5 and 4) tumor cell lines or 3×105cells from the B16F10-luciferase line (n=5 and 5), Results are obtained from 2-3 independent experiments per cell lines. FIG.7C and 7D: Flow cytometry analysis of Tim4+KCs numbers, photoradiance analysis of tumor burden, and representative liver micrographs -10- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 from 6-8 weeks old Flt3CreCsf1rf / fmice (n=4, 13), Csf1rf / flittermates (n=5, 13), or CCR2- / -mice(n=5, 6) and Ccr2+ / -littermates(n=4, 9). two weeks after intra-portal injection of 1×106KPC-1-luciferase cells. Results are obtained from 3 independent experiments. FIG.7E: flow cytometry and cytospin giemsa stain analysis of Kupffer cells (pop1: F4 / 80+Tim4+), and other myeloid cells (pop2 : F4 / 80+Tim4-MHCII+and pop3 : F4 / 80+Tim4-MHCII-) from the liver of C57BL / 6J mice 2 weeks after intra-portal injection of 1×106KPC-1-luci-tdT cells. The bar plot represents the % of cells from each population that are labeled by tdT in the liver of Clec4fCre-tdTmice(n=3 / group) and by YFP in the liver of Flt3CreR26LSL-YFPmice(no tumor n=6, KPC-1 n=4) 2 weeks after intra-portal injection of 1×106KPC-1 cells or in the absence of tumor injection. FIG.7F: Genetic labeling efficiency in 8 weeks old Clec4fCre-tdTR26LSL-YFPmice, %YFP+and % tdT+cells are measured by flow cytometry in liver myeloid cells: Tim4+KCs, F4 / 80+Tim4-MHCII+, F4 / 80+Tim4-MHCII-, cDC1, cDC2), liver CD45- cells, tissue macrophages: kidney macrophages, brain macrophages, lung alveolar macrophages(AM), lung interstitial macrophages(IM), skin macrophages, splenic red pulp macrophages(RPM), bone marrow long term HSCs(LT-HSC), short term HSCs(ST-HSC), multipotent progenitor MPP, blood and spleen CD19+B cells, Ly6G+granulocytes, Ly6C+monocytes, CD3+T cells (see methods). n=3 mice per group. FIG. 7G: Percentage of YFP+cells among CD4+T cells, CD8+T cells, Nkp46+NK cells, Tim4+KCs on liver cryosection from 8 weeks old Clec4fCre-tdTR26LSL-YFPmice. n=3 mice per group. FIG.7H and 7I: Flow cytometry analysis of Tim4+KCs numbers in 6-8 weeks-old Clec4fCreCsf1rf / fmice(n=5) or Csf1rf / flittermates(n=5) (FIG.7H), Clec4fCreSpi1f / fmice(n=4) or Spi1f / flittermates(n=6) (FIG.7I). FIG.7J: Six to 8 weeks-old Clec4fCreR26LSL-DTRmice and R26LSL-DTRmice are injected with DT, liver Tim4+KCs numbers (n=4 / group), lung interstitial macrophages and alveolar macrophages numbers (n=7,5 respectively) are quantified by flow cytometry at indicated time point after DT injection. FIG.7K: Photoradiance analysis of tumor burden in 6-8 weeks old Clec4fCreR26LSL-DTRmice and R26LSL-DTRlittermates 2 weeks after intra-portal injection of 1x106KPC1-luciferase cells, n=4 mice per group. FIG.7L: Representative tSNE and histogram analysis of expression of the markers CD47bright, CD9, and CD133, among GFP+CD45- tumor cells in Clec4fCre-tdTR26LSL-DTR(n=4) and Clec4fCre-tdTlittermates(n=4), treated with DT and that received intra-portal injection of 1×106KPC-1 cells 2 weeks before analysis (see methods). Results from 2 independent experiments. FIG.7M: analysis of metastatic potential of CD47brightCD9+CD133+tumor cells and CD47lowCD9lowCD133lowtumor cells in vivo by bioluminescent analysis, two weeks after intra-portal injection of 5×104cells(n=5 / group) or -11- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 2×105KPC-1-luci-td cells(n=8,7 respectively) (Left, circles represent individual mice), and in vitro clonogenic potential in oncosphere culture(n=561,563 respectively) (right, circles represent individual oncospheres, see Methods). FIG.7N: Plots indicate the number of GFP+CD45- cells and of GFP+CD45-CD47brightCD9+CD133+cells per liver lobe, and the MFI of CD47 in GFP+CD45- cells and GFP+CD45-CD47brightCD9+CD133+cells, in mice from (l). Statistics:One-way ANOVA (FIG.7I) unpaired two-tailed t test (FIGs.7A, 7B, 7C, 7D, 7G, 7H, 7I, 7J, 7K, 7M, and 7N). Mann-Whitney test(two-tailed) (FIG.7M). Dots represent individual mice (FIGs.7A, 7B, 7C, 7D, 7E, 7F, 7H, 7I, 7J, 7K, 7M, and 7N). mean ± sd. ns, not significant.

[0023] FIGs.8A-8O: Anatomical location and turnover of liver macrophages in metastatic liver. FIGs.8A-8G: Representative immunofluorescence staining for Clec4f, F4 / 80, Tim4, tdT, CK19 on frozen liver sections from wt C57BL / 6j mice 8 weeks after pancreatic orthotopic injection of 2×105KPC-2-tdT cells. FIGs.8B-8G: idem, from wt C57BL / 6j mice 2 weeks after intra-portal injection of 1×106KPC-1-tdT cells, 1×106KPC-1 cells, 5×105B16F10-luci-tdT cells, 1×106LLC1-luci-tdT cells, 1×106Pan02-luci-tdT cells, or 1×106MC38 cells. n=3 mice per group. FIG.8H: Flow cytometry analysis of the % of CD45.1+cells among Tim4+CD206+KCs and Tim4+CD206hiKCs in the liver of CD45.1 / CD45.2 parabiotic pairs 2 weeks after intra-portal injection of 106KPC-1-tdT in the CD45.2 partner(n=5),or not injected as control(n=4). FIG.8I: table represents the percentage (mean and sd) of CD45.1+partner-derived cells among Tim4+, Tim4+CD206+, and Tim4+CD206hiKC, and TIM4- TAMs in tumor free and tumor bearing livers from CD45.2 parabionts in (h). FIG.8J: Gating strategy for separation of Tim4+CD206+and Tim4+CD206hiliver KC by flow cytometry. FIG.8K: Bar-plots show the percentage of Tim4+, Tim4+CD206+, Tim4+CD206hiKC, and TIM4- TAMs labeled with GFP in Cxcr4gfp / +mice (n=3 / group) and in Cx3cr1gfp / +mice (n=3 / group), and labeled with tdT in Cxcr4CreERT2;R26LSL-tdTomatomice pulsed with 4OH-TAM at 6 week-old, that have received intra-portal injection of 1×106KPC-1-luci-tdT cells 2 weeks before analysis(n=4), or not injected with tumor cells(n=3 / group), not injected with 4OH-TAM(n=3 / group) as control. FIG.8L: Table represents the percentage (mean and sd) of Tim4+KC, Tim4+CD206+KC, Tim4+CD206hiKC, and TIM4- TAMs in the liver of tumor free and tumor bearing Cxcr4gfp / +mice, Cx3cr1gfp / +mice, and Cxcr4CreERT2;R26LSL-tdTomatomice pulsed at 6 weeks with 4OH- TAM. FIG.8M: Immunofluorescence staining for F4 / 80, Tim4, GFP and tdTomato on frozen liver section from Cx3cr1gfp / +tumor bearing mice in FIG.8K. FIG.8N: -12- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 immunofluorescence staining for F4 / 80, Tim4 and tdTomato on frozen liver section from Cxcr4CreERT2R26LSL-tdTomatotumor bearing mice in FIG.8K. FIG.8O: Cxcr4CreERT2R26LSL-tdTomatomice are pulsed with 4OH-TAM(n=3) or PBS(n=2) at 6 weeks and analyzed 2 weeks later. Bar graphs represent the % of tdT+cells determined by flow cytometry among the indicated cells types. Statistics: One-way ANOVA (FIG.8H and 8K). Dots represent individual mice. mean ± sd. ns, not significant.

[0024] FIG.9A-9H: Kupffer cells engulf tumor cells. FIG.9A: Selected pathways (see Methods) upregulated in RNA-seq analysis of KCs from C57BL / 6j mice 2 weeks after intra-portal injection of 1×106KPC-1 cells, in comparison to no injection, n=3 mice per group, (data not shown). FIG.9B: Scatterplot of differentially expressed genes (adj. p- values are obtained using Benjamini and Hochberg method for multiple testing and considered significant when adj. p<0.05) in KCs from tumor-bearing mice, from the RNA- seq analysis in FIG.9A. Selected genes are indicated in blue (lectins and chemokines) and in pink (cytokeratins, from engulfed tumor cells). FIG.9C: Expression of activating and inhibitory receptors by KCs from RNAseq data (Table 2 and 3) and RTqPCR data (*). Second to 4th column indicates if gene expression is up or down in metastatic liver, fold changes, and adj. p value. Fifth to 7thcolumn ‘Clec4fCreId3f / f’ indicates gene which expression is up or down regulated in Clec4fCreId3f / fmice, fold changes, and adj. p value. Column 8 indicates the presence of ITIM or ITAM motifs. FIG.9D: Heatmap represents the top 100 up and down DEG genes in Tim4+CD206+KC cells and Tim4+CD206hiKC respectively, in RNA-seq analysis from FIG.9A. FIG.9E: Representative immunofluorescence staining for CK19, F4 / 80, Tim4 on frozen liver section from C57BL / 6J mice received 1×106KPC-1 cells through intra-portal injection for 2 weeks. n=3 mice per group. FIG.9F: Representative whole mount immunofluorescence imaging and quantification of tdT+% cells in Tim4+KCs from metastatic liver, from C57BL / 6J mice which received 2×105KPC-2-luci-tdT cells through pancreatic orthotopic injection and are analyzed weekly for 2 months. n=3 mice per group. FIG.9G: Representative immunofluorescence staining for CK19, Tim4, F4 / 80, quantification of CK19 relative MFI in Tim4+KCs on liver samples from KPC mice (p48CreKrasLSL-G12Dp53LSL-R172H) and control mice (KrasLSL-G12Dp53LSL-R172H). Dots represent individual KCs from 3 mice per group. FIG. 9H: Analysis of livers from mice receiving phosphatidylserine blockade D89E or PBS 6 hours before intra-portal injection of 1×106KPC-1-tdT cells and analyzed after 24 hours. Left: Quantification by Immunofluorescence of Tim4+KCs stained with tdT,n=4 / group; -13- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 right: quantification of CD45-GFP+tumor cells by flow cytometry (right). PBS n=4, D89E n=4. Statistics: Kruskal-Wallis test (FIG.9G), unpaired two-tailed t test (FIG.9H), Dots represent individual mice (FIG.9F and 9H). mean ± sd. ns, not significant.

[0025] FIGs.10A-10B. Kupffer cells engulf live tumor cells in vivo and in vitro. FIG.10A: Schematic of time lapse intravital imaging of in vivo uptake of KPC-1-memtdT by KCs in liver from C57BL / 6j mice 2 weeks after intra-portal injection of 1×106KPC-1- memtdT cells in the presence of iv. injection of Tim4-AF647 antibodies and CellEvent™ Caspase-3 / 7 green reagent. Representative whole mount immunofluorescence imaging and time-lapse images are shown. Sequential images show KC1, KC2, KC3, KC4 engulfing tumor cells (yellow arrows), red arrow shows Cas3 / 7 green signaling. FIG.10B: Representative time-lapse images of in vitro uptake of KPC-1-memtdT by wide type KCs in Matrigel in the presence of F4 / 80-AF647 antibodies, and CellEvent™ Caspase-3 / 7 green reagent, and in the presence of Latrunculin-A or PBS control. In the PBS group, sequential images show a KC engulfing Cas3 / 7 green- tumor cells after 5 hrs of contact, between 12 and 14 hrs of culture (yellow arrows), and Cas3 / 7 green+activation in cellular fragments ~6 hrs later (white arrows).

[0026] FIGs: 11A-11F: Analysis of Id3-deficient mouse models. FIG.11A: Quantification of cytokines / chemokines expression by Tim4+KCs by immunofluorescence on liver tissue sections from KPC (p48CreKrasLSL-G12Dp53LSL-R172H) mice and control (KrasLSL-G12Dp53LSL-R172H) mice. n numbers are indicated in green. Dots represent MFI of individual KCs. FIG.11B: Representative micrographs of immunofluorescence staining for cytokines / chemokines on frozen liver sections from wt mice 2 weeks after intra-portal injection of 1×106KPC-1-gfp cells. n=3 mice per group. FIG.11C: Number of Tim4+cells, Tim4+CD206+cells and Tim4+CD206hicells per gram of liver tissue by flow cytometry in Id3- / -mice, and Id3+ / +littermates, n=5 mice per group. FIG.11D: Flow cytometry quantification Percentage of partner-derived (CD45.1+) wt Tim4+KCs and Tim4- myeloid cells in the liver of CD45.2 Id3- / -mice(n=4), or CD45.2 Id3+ / +mice(n=3) parabiosed with wt CD45.1 mice for 8 weeks. FIG.11E: Representative micrograph of Tim4, F4 / 80, and tdTomato expression by immunofluorescence in liver frozen section from 8 weeks old Id3f / fmice and Clec4fCreId3f / fmice. n=3 mice per group. FIG.11F: (left) Percentage of CD47brightCD9+CD133+cells among GFP+CD45- tumor cells, and relative expression of CD47 among total GFP+CD45- tumor cells and GFP+CD45- CD47brightCD9+CD133+tumor cells in the liver of Id3f / fmice(n=5) and Clec4fCreId3f / f-14- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 mice(n=4) 2 weeks after intra-portal injection of 1×106KPC-1-luci-gfp cells. (right) representative histograms of CD47, CD9, CD133, CD44, and CD24 expression tumor cells from (left). Statistics: unpaired two-tailed t test (FIG.11C and 11F). Mann-Whitney test(two-tailed) (FIG.11A), One-way ANOVA (FIG.11D). Dots represent individual mice (FIGs.11C, 11D, and 11F), mean ± sd. ns, not significant.

[0027] FIGs.12A-12J: Id3-dependent cytokines and chemokines expression and lymphoid cells recruitment and activation in response to tumor. FIG.12A: SIRPA and Dectin-1 expression by flow cytometry of the main (Tim4+CD206+) and minor (Tim4+CD206hi) KCs subsets from the liver of Clec4fCreId3f / fmice and Id3f / flittermates, 2 weeks after intra-portal injection of 1×106KPC-1-luci-gfp cells (KPC +), or from control mice without tumor (KPC -). n=3 mice per group. FIG.12B: Expression of Clec4n, Clec4d, Lrp1, and SiglecG by RT-qPCR by KCs from the liver of Clec4fCreId3f / fmice and Id3f / fmice, n=3 mice per group. FIGs.12C-12D: Representative immunofluorescence micrograph of chemokines (FIG.12C) and cytokines (FIG.12D) expression by KCs of in the liver of Clec4fCreId3f / fmice and Id3f / flittermates 2 weeks after intra-portal injection of 1×106KPC-1-gfp cells. n=3 mice per group. FIGs.12E-12G:- Number of CD8+T cells, Nkp46+NK cells,cells, CD19+B cells, CD4+T cells, Ly6G+granulocytes, Ly6Chimonocytes determined by flow cytometry in blood (FIG.12E), and liver (FIG.12F) and spleen (FIG.12G) from 6-8 weeks old Id3f / fmice and Clec4fCreId3f / fmice (f,g), or from mice 2 weeks after intra-portal injection of 1×106KPC-1-gfp cells (f). n numbers are indicated in green. FIG.12H: Representative immunofluorescence micrograph for Lamp1, Nkp46, CD8, and GFP expression in liver sections from mice in FIG.12C. FIG.12I: Expression of chemokines and cytokines by RT-qPCR by KCs from Clec4fCreId3f / fmice and Id3f / flittermates, cocultured with or without KPC cells for 48h. n=3 per group. FIG.12J: IFN-γ production by flow cytometry by mouse splenic NK cells cultured for 3 days with supernatant from coculture in (h), n=3 per group. Statistics: unpaired two-tailed t test (FIGs. 12B, 12E, 12F, and 12G), One-way ANOVA (FIGs.12A, 12I, and 12J). Dots represent individual mice (FIGs.12A, 12B, 12E, 12F, and 12G). mean ± sd. ns, not significant.

[0028] FIGs.13A-13J: SIRPA blockade and CD47 deficiency rescue Id3 deficient mice. FIG.13A: Dectin1 expression by flow cytometry (dots represent individual mice, n=5 / group), and CCL3, CCL4, and CCL5 expression by immunofluorescence (dots represent individual KCs) by Tim4+KCs in liver from 8-12 weeks-old mice Clec4fCreId3f / fmice and Id3f / flittermates, two weeks after intra-portal injection of 1×106KPC-1-luciferease -15- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 cells, treated with anti-SIRPA antibodies or IgG control antibodies. n numbers are indicated in green. FIGs.13B-13D: Representative immunofluorescence micrograph of CCL3, CCL4, CCL5, by Tim4+ KCs at the boundary of GFP+ tumors in liver sections from mice in FIG.13A. FIG.13E: Number of NKT cells, γδT cells, CD19+B cells, CD4+T cells per gram of tissue by flow cytometry analysis in mice from FIG.13A. n=5 mice per group. FIG.13F: Representative immunofluorescence micrograph of CD8, Lamp1, and Nkp46- expressing cells at the boundary of GFP+ tumors in liver sections from mice in FIG.13A. FIG.13G (left) CD47 expression by flow cytometry of tumor cell lines. (right) bioluminescence imaging and micrograph of liver tumor burden in C57BL / 6J mice 2 weeks after intra-portal injection of 1×106KPC-1-Cd47+ / +-luci-tdT cells or 1×106KPC-1-Cd47- / -- luci-tdT cells, n=7 mice per group from 2 independent experiments. FIG.13H: Competitive in vivo proliferation assay between CD47- / -deficient and Cd47+ / +control KPC1 tumor cells in liver metastasis.7×105KPC-1-Cd47+ / +-gfp tumor cells mixed with 7×105KPC-1- Cd47+ / +-tdT tumor cells or 7×105KPC-1-Cd47- / --tdT tumor cells in a 1:1 ratio are injected into c C57BL / 6J mice through intra-portal injection. Percentage of fluorescent cells expression in the liver and representative immunofluorescence of GFP and tdT performance after 2 weeks are shown. n=5 mice per group. FIG.13I: Photoradiance analysis of liver tumor burden in 8-12 weeks-old Clec4fCreId3f / fmice and Id3f / flittermates two weeks after intra-portal injection of 1×106KPC-1-luciferease-cd47+ / +cells or KPC-1-luciferease-cd47- / -cells, n=4 mice per group. FIG.13J: Numbers of CD8+T cells, Nkp46+NK cells, NKT cells, γδT cells, CD19+B cells, CD4+T cells per gram of tissue in the liver from mice in FIG.13I. n=4 mice per group. Statistics: One-way ANOVA (FIGs.13A, 13E, 13I, and 13J),Kruskal-Wallis test (FIG.13A), unpaired two-tailed t-test (FIGs.13G and 13H). Dots represent individual mice (FIGs.13A, 13E, 13G, 13H, 13I, and 13J). mean ± sd. ns, not significant.

[0029] FIGs.14A-14H: Id3 regulates Sirpα gene expression via Elk1 and E2A. FIG. 14A: Expression of Myc, Hes1, HEB, TCF4, E2A, and Elk1 by KCs from Clec4fCreId3f / fmice(n=2) and Id3f / flittermates(n=3) by RNAseq, circle represent individual mice. FIG.14B: Expression of DAPI and E2A or ELK1 by Tim4+ KC, by immunofluorescence in liver sections from 8 weeks old C57BL / 6J mice, n=3 mice per group. FIG.14C:- Diagram represents candidate binding sites for E2A and Elk1 at the Sirpα locus (see methods). FIG.14D: PWM score and DeepLIFT analysis of candidate Elk1 and E2A binding sites at Sirpα locus in mouse KCs and BMDM. FIGs.14E-14F: -16- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 Binding of E2A and ELK1 to Sirpα promoter / enhancer in KCs from Id3 deficient mice and control littermates 12h after i.p. injected of 2mg / kg LPS or PBS by CUT&RUN analysis. n=3 mice per group. FIG.14G: SIRPA expression by flow cytometry by KCs from Id3 deficient mice and control littermates 12h after i.p. injected of 2mg / kg LPS or PBS. n=6 mice per group. FIG.14H: SIRPA expression by flow cytometry by BMDM expressing Scramble, or E2a sh-RNA, Elk1 Sh-RNA, lenti-mId3 and treated with LPS (50 ng / ml for 6 hrs), or PBS. n=3 per group. Statistics: One-way ANOVA (FIGs.14G-14H), unpaired two- tailed t-test (FIGs.14E-14F). Dots represent individual mice (FIGs.14A and 14G). mean ± sd. ns, not significant.

[0030] FIGs.15A-15H: human KC: anatomical location, engulfment of tumor cells, and gene expression. FIG.15A: Mouse / Human Blastn alignment for Sirpa regulatory elements. FIG.15B: Expression of ID3 and SIRPA gene expression in human cells by scRNAseq, from https: / / www.proteinatlas.org / . FIG.15C: Numbers of Tim4+ CD14+ KC / mm2in tumoral and peri-tumoral areas from metastatic liver samples from PDAC patients, n=3, mean ± sd, unpaired two-tailed t-test. A representative immunofluorescence micrograph of CK19, Tim4, and CD14 expression is also shown (right, bar= 50mM). FIG. 15D: Expression of CK19 by Tim4+ KCs in metastatic liver samples from PDAC patients, each dot represents a KC (left). n numbers are indicated in green. The bar-plot (center,n=3 patients) indicate the % of KC stained for CK19. A representative high power immunofluorescence micrograph of CK19, Tim4, and CD14 expression is also shown (right, bar= 20mM).mean ± sd. FIG.15E: Expression of CCL3, CCL4, CCL5, IL12, IL15, IL18 by Tim4+CD14+KCs from patients in (FIG.15C-15D). Dots represent relative Mean Fluorescence Intensity, of individual KCs, mean ± sd, normalized to background MFI of 1 (green dotted line). n numbers are indicated in green. FIG.15F: ScRNAseq datasets from control liver (1068 cells from one patient, GSE146409) and metastatic liver from colorectal carcinoma (CRC) patients (3182 cells from 3 patients, GSE146409). tSNE in top panels represent clustering of liver cells by cell type (KCs: Kupffer cells, TAM: tumor associated macrophages, HSCs: hepatic stellate cells, LSEC: liver sinusoidal endothelial cells, Cholang: Cholangiocytes LVEC: liver vascular endothelial cells, CAFs: cancer-associated fibroblasts). tSNE in bottom panel represent TIMD4, CD14, CSF1R, SPI1 expression in cell clusters. FIG.15G: % of Tim4+cells among CD14+macrophage clusters (Tim4+CD14+KCs and Tim4- CD14+Trem2+tumor associated macrophages), from the datasets above plus PDAC liver metastasis liver samples (19843 cells, from 3 patients, GSE205013,n=3) and -17- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 control liver samples (8439 cells, from 5 patients, GSE115469,n=5). mean ± sd, Statistics: unpaired two-tailed t-test. FIG.15H: Expression of ID3, TIM4, SIRPA, CCL3, CCL4, and IL18 by ScRNAseq from the CRC and PDAC liver metastasis liver samples above (PDAC: 19843 cells, from 3 patients, GSE205013, CRC: 3182 cells from 3 patients, GSE146409) by the Tim4+CD14+(KCs) and Tim4- CD14+Trem2+ (tumor associated macrophages) clusters. Green line indicates the mean, p-values are obtained using Wilcoxon test(two-tailed). ns, not significant.

[0031] FIG.s 16A-16E: Id3-expressing macrophages control tumor growth. FIG. 16A: Engulfment of KPC-1-memtdT cells (Casp.3 / 7 not cleaved) by BMDM expressing a control lentivirus (n=7) in the presence or absence of α-SIRPA blocking antibody(n=11), or an ID3 lentivirus (n=18). Left: time from stable interaction between macrophages and tumor cells to tumor cells engulfment. Right: time from stable interaction between macrophages and tumor cells and the detection of Casp.3 / 7 cleavage, dots represent individual macrophages. FIG.16B: Production of TNF by Human CD8+T cells stimulated with anti CD3 / CD28 activation beads, with supernatant from hiPSC-mac expressing lenti-control or lenti-hId3, and cocultured with Panc1 cells for 48h or not for 3 days. Human CD8+T cells are treated with cocktail of PMA, ionomycin, brefeldin A and monensin for 6h, and TNF production measured by flow cytometry. n=3 per group. FIG.16C: (left) Numbers of NKT cells, γδT cells, CD19+B cells, and CD4+T cells in the tumors from 8-12 weeks-old C57BL / 6J mice two weeks after subcutaneous injection of 1×106B16F10-luci-tdT cells into left and right flank, followed by intra-tumor injection of 5×105BMDM expressing lenti- control (left flank) or lenti-mId3 (right flank) at day 7 post tumor injection. n=20 mice per group from 3 experiments. (right) production of TNF by CD8+T cells determined by flow cytometry. n=5 mice / group. FIG.16D: Photoradiance analysis of liver tumor burden in 8- 12 weeks-old C57BL / 6j mice two weeks after subcutaneous injection of 1×106B16F10- luci-tdT cells into left and right flank, followed by intra-tumor injection of 5×105BMDM expressing lenti-control(n=4), lenti-mId3 cells(n=5) or not at day7 post tumor injection. Statistics: One-way ANOVA (FIGs.16A, 16B, and 16D). unpaired two-tailed t-test (FIG. 16C). Dots represent individual mice (FIGs.16C-16D). mean ± sd. ns, not significant. FIG. 16E: Schematic shows a hypothesis for the mechanisms that underly Id3-dependent anti- tumor activity of macrophages. -18- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 DETAILED DESCRIPTION

[0032] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0033] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0034] Kupffer cells (KC), the resident macrophages of the liver, are highly phagocytic and are a good candidate to mediate resistance to metastasis17,21,29. As disclosed in the Examples herein, genetic tools were used to selectively target KC and human induced pluripotent stem cell (hiPSC) macrophages, to investigate the role of KC and the KC- specific LDF ID3 (inhibitor of differentiation 3) in cancer. The present disclosure demonstrates that ID3 expression by KC endows them with the ability to orchestrate a potent anti-tumor response, by establishing a peri-tumoral phagocytic and activated lymphoid effector niche. Furthermore, we show that ectopic expression of ID3 in mouse -19- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 bone marrow derived macrophages and human hiPSC-derived macrophages is sufficient to endow them with the ability to orchestrate this vigorous phagocytic and lymphoid anti- tumoral activity in a variety of tumor models in vitro and in vivo. Accordingly, expression of ID3 endows macrophages with the ability to form an efficient anti-tumor niche, which can be harnessed for adoptive cell therapy in cancer. Definitions

[0035] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in the present disclosure. Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

[0036] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0037] As used herein, the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value.

[0038] As used herein, the term “administration” of an agent to a subject includes any route of introducing or delivering the agent to a subject to perform its intended function. Administration can be carried out by any suitable route, including, but not limited to, intravenously, intramuscularly, intraperitoneally, subcutaneously, and other suitable routes as described herein. Administration includes self-administration and the administration by another. “Administration” of a cell or vector or other agent and compositions containing -20- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 same can be performed in one dose, continuously or intermittently throughout the course of treatment. 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, the target cell being treated, 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 or in the case of animals, by the treating veterinarian. In some embodiments, administering or a grammatical variation thereof also refers to more than one doses with certain interval. In some embodiments, the interval is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or longer. In some embodiments, one dose is repeated for once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue. Non-limiting examples of route of administration include oral administration, intraperitoneal, infusion, nasal administration, inhalation, injection, and topical application. In some embodiments, the administration is an infusion (for example to peripheral blood of a subject) over a certain period of time, such as about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours or longer.

[0039] As used herein “adoptive cell therapeutic composition” refers to any composition comprising cells suitable for adoptive cell transfer. In exemplary embodiments, the adoptive cell therapeutic composition comprises peripheral blood mononuclear cells. In one embodiment, the adoptive cell therapeutic composition comprises monocytes or monocyte-derived macrophages.

[0040] The term “amino acid” refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, -21- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine. Amino acid analogs refer to agents that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. In some embodiments, amino acids forming a polypeptide are in the D form. In some embodiments, the amino acids forming a polypeptide are in the L form. In some embodiments, a first plurality of amino acids forming a polypeptide is in the D form and a second plurality is in the L form.

[0041] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter code.

[0042] As used herein, the term “analog” refers to a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule.

[0043] As used herein, the term “antibody” means not only intact antibody molecules, but also fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are regularly employed both in vitro and in vivo. Accordingly, as used herein, the term “antibody” means not only intact immunoglobulin molecules but also the well-known active fragments F(ab')2, and Fab. F(ab')2, and Fab fragments that lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al., J. Nucl. Med.24:316-325 (1983)). Antibodies may comprise whole native antibodies, monoclonal antibodies, human antibodies, humanized antibodies, camelised antibodies, multispecific antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single chain V region fragments (scFv), single domain antibodies (e.g., nanobodies and single domain camelid antibodies), VNAR fragments, Bi-specific T-cell engager (BiTE) antibodies, minibodies, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, intrabodies, fusion polypeptides, unconventional antibodies and antigen binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that -22- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 contain an antigen binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.

[0044] In certain embodiments, an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant (CH) region. The heavy chain constant region is comprised of three domains, CH1, CH2, and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant CLregion. The light chain constant region is comprised of one domain, CL. The VHand VLregions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VHand VLis composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Cl q) of the classical complement system. As used herein interchangeably, the terms “antigen binding portion”, “antigen binding fragment”, or “antigen binding region” of an antibody, refer to the region or portion of an antibody that binds to the antigen and which confers antigen specificity to the antibody; fragments of antigen binding proteins, for example antibodies, include one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen binding portions encompassed within the term “antibody fragments” of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VHand CH1 domains; a Fv fragment consisting of the VLand VHdomains of a single arm of an antibody; a dAb fragment (Ward et al., Nature 341 : 544-546 (1989)), which consists of a VH domain; and an isolated complementarity determining region (CDR). An “isolated antibody” or “isolated antigen binding protein” is one which has been identified and separated and / or recovered from a component of its natural -23- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 environment. “Synthetic antibodies” or “recombinant antibodies” are generally generated using recombinant technology or using peptide synthetic techniques known to those of skill in the art.

[0045] Antibodies and antibody fragments can be wholly or partially derived from mammals (e.g., humans, non-human primates, goats, guinea pigs, hamsters, horses, mice, rats, rabbits and sheep) or non-mammalian antibody producing animals (e.g., chickens, ducks, geese, snakes, and urodele amphibians). The antibodies and antibody fragments can be produced in animals or produced outside of animals, such as from yeast or phage (e.g., as a single antibody or antibody fragment or as part of an antibody library).

[0046] Furthermore, although the two domains of the Fv fragment, VLand VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules. These are known as single chain Fv (scFv); see e.g., Bird et al., Science 242:423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci.85 : 5879-5883 (1988). These antibody fragments are obtained using conventional techniques known to those of ordinary skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0047] As used herein, the term “single-chain variable fragment” or “scFv” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH::VLheterodimer. The heavy (VH) and light chains (VL) are either joined directly or joined by a peptide- encoding linker (e.g., about 10, 15, 20, 25 amino acids), which connects the N-terminus of the VHwith the C-terminus of the VL, or the C-terminus of the VHwith the N-terminus of the VL. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen binding domain. In certain embodiments, the linker comprises amino acids having GGGGSGGGGSGGGGS (SEQ ID NO: 1). In certain embodiments, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1 is ggcggcggcggatctggaggtggtggctcaggtggcggaggctcc (SEQ ID NO: 2).

[0048] Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH- and VL-encoding sequences -24- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883 (1988)). See, also, U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al., Hybridoma (Larchmt) 27(6):455-51 (2008); Peter et al., J Cachexia Sarcopenia Muscle (2012); Shieh et al., J Imunol 183(4):2277-85 (2009); Giomarelli et al., Thromb Haemost 97(6):955-63 (2007); Fife eta., J Clin Invst 116(8):2252- 61 (2006); Brocks et al., Immunotechnology 3(3): 173-84 (1997); Moosmayer et al., Ther Immunol 2(10):31- 40 (1995). Agonistic scFvs having stimulatory activity have been described (see, e.g., Peter et al., J Biol Chem 25278(38):36740-7 (2003); Xie et al., Nat Biotech 15(8):768-71 (1997); Ledbetter et al., Crit Rev Immunol 17(5-6):427-55 (1997); Ho et al., Bio Chim Biophys Acta 1638(3):257-66 (2003)).

[0049] As used herein, an “antigen” refers to a molecule to which an antibody can selectively bind. The target antigen may be a protein (e.g., an antigenic peptide), carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. An antigen may also be administered to an animal subject to generate an immune response in the subject.

[0050] By “binding affinity” is meant the strength of the total noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Without wishing to be bound by theory, affinity depends on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, and on the distribution of charged and hydrophobic groups. Affinity also includes the term “avidity,” which refers to the strength of the antigen-antibody bond after formation of reversible complexes (e.g., either monovalent or multivalent). Methods for calculating the affinity of an antibody for an antigen are known in the art, comprising use of binding experiments to calculate affinity. The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). A low-affinity complex contains an antibody that generally tends to dissociate readily from the antigen, whereas a high-affinity complex contains an antibody that generally tends to remain bound to the antigen for a longer duration. Antibody activity in functional assays (e.g., flow cytometry assay) is also reflective of antibody affinity. Antibodies and affinities can be phenotypically characterized and compared using functional assays (e.g., flow cytometry assay). -25- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0051] As used herein, “CDRs” are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th U. S. Department of Health and Human Services, National Institutes of Health (1987). Generally, antibodies comprise three heavy chain and three light chain CDRs or CDR regions in the variable region. CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope. In certain embodiments, the CDRs regions are delineated using the Kabat system (Kabat, E. A., et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242(1991)).

[0052] As used herein, the term “cell population” refers to a group of at least two cells expressing similar or different phenotypes. In non-limiting examples, a cell population can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000 cells, at least about 10,000 cells, at least about 100,000 cells, at least about 1×106cells, at least about 1×107cells, at least about 1×108cells, at least about 1×109cells, at least about 1×1010cells, at least about 1×1011cells, at least about 1×1012cells, or more cells expressing similar or different phenotypes.

[0053] As used herein, “complementary” sequences refer to two nucleotide sequences which, when aligned anti-parallel to each other, contain multiple individual nucleotide bases which pair with each other. Paring of nucleotide bases forms hydrogen bonds and thus stabilizes the double strand structure formed by the complementary sequences. It is not necessary for every nucleotide base in two sequences to pair with each other for sequences to be considered “complementary”. Sequences may be considered complementary, for example, if at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the nucleotide bases in two sequences pair with each other. In some embodiments, the term complementary refers to 100% of the nucleotide bases in two sequences pair with each other. In addition, sequences may still be considered “complementary” when the total lengths of the two sequences are significantly different from each other. For example, a primer of 15 nucleotides may be considered “complementary” to a longer polynucleotide containing hundreds of nucleotides if multiple individual nucleotide bases of the primer pair with nucleotide bases in the longer polynucleotide when the primer is aligned anti-parallel to a particular region of the longer -26- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 polynucleotide. Nucleotide bases paring is known in the field, such as in DNA, the purine adenine (A) pairs with the pyrimidine thymine (T) and the pyrimidine cytosine (C) always pairs with the purine guanine (G); while in RNA, adenine (A) pairs with uracil (U) and guanine (G) pairs with cytosine (C). Further, the nucleotide bases aligned anti-parallel to each other in two complementary sequences, but not a pair, are referred to herein as a mismatch.

[0054] As used herein, a “control” is an alternative sample used in an experiment for comparison purpose. A control can be “positive” or “negative.” For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0055] As used herein, the term, “co-stimulatory signaling domain,” or “co-stimulatory domain”, refers to the portion of the CAR comprising the intracellular domain of a co- stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of lymphocytes upon binding to antigen. Examples of such co- stimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2 and a ligand that specifically binds CD83. Accordingly, while the present disclosure provides exemplary costimulatory domains derived from CD28 and 4-1BB, other costimulatory domains are contemplated for use with the CARs described herein. The inclusion of one or more co- stimulatory signaling domains can enhance the efficacy and expansion of immune cells expressing CAR receptors. The intracellular signaling and co-stimulatory signaling domains can be linked in any order in tandem to the carboxyl terminus of the transmembrane domain.

[0056] As used herein, the term “effective amount” or “therapeutically effective amount” refers to a quantity of an agent sufficient to achieve a beneficial or desired clinical result upon treatment. In the context of therapeutic applications, the amount of a therapeutic agent administered to the subject can depend on the type and severity of the disease or condition and on the characteristics of the individual, such as general health, age, sex, body weight, effective concentration of the engineered immune cells administered, and tolerance to drugs. It can also depend on the degree, severity, and type of disease. The -27- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 skilled artisan will be able to determine appropriate dosages depending on these and other factors. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art.

[0057] As used herein, the term “engineered monocyte” or “engineered monocyte- derived macrophage” refers to a monocyte or monocyte-derived macrophage that is genetically modified.

[0058] As used herein, the term “expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the expression level of a gene from one sample can be directly compared to the expression level of that gene from a control or reference sample. In another aspect, the expression level of a gene from one sample can be directly compared to the expression level of that gene from the same sample following administration of the compositions disclosed herein. The term “expression” also refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription) within a cell; (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and / or 3’ end formation) within a cell; (3) translation of an RNA sequence into a polypeptide or protein within a cell; (4) post-translational modification of a polypeptide or protein within a cell; (5) presentation of a polypeptide or protein on the cell surface; and (6) secretion or presentation or release of a polypeptide or protein from a cell.

[0059] As used herein, an "expression vector" includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA -28- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and / or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.

[0060] As used herein, the term “heterologous nucleic acid molecule or polypeptide” refers to a nucleic acid molecule (e.g., a cDNA, DNA or RNA molecule) or polypeptide that is either not normally expressed or is expressed at an aberrant level in a cell or sample obtained from a cell. This nucleic acid can be from another organism, or it can be, for example, an mRNA molecule that is not normally expressed in a cell or sample.

[0061] As used herein, the term “immune cell” refers to any cell that plays a role in the immune response of a subject. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells ; natural killer cells; myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils, and granulocytes. As used herein, the term “engineered immune cell” refers to an immune cell that is genetically modified. As used herein, the term “native immune cell” refers to an immune cell that naturally occurs in the immune system.

[0062] As used herein, "operably linked" with reference to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other. For example, nucleic acid encoding a leader peptide can be operably linked to nucleic acid encoding a polypeptide, whereby the nucleic acids can be transcribed and translated to express a functional fusion protein, wherein the leader peptide effects secretion of the fusion polypeptide. In some instances, the nucleic acid encoding a first polypeptide (e.g., a leader peptide) is operably linked to nucleic acid encoding a second polypeptide and the nucleic acids are transcribed as a single mRNA transcript, but translation of the mRNA transcript can result in one of two polypeptides being expressed. For example, an amber stop codon can be located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, such that, when introduced into a partial amber suppressor cell, the resulting single mRNA transcript can be translated to produce either a fusion protein containing the first and second polypeptides, or can be translated to produce only the first polypeptide. In another example, a promoter can be operably linked to nucleic acid encoding a polypeptide, whereby the promoter regulates or mediates the transcription of the nucleic acid. -29- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0063] The term “myeloid cell” refers to all immature, mature, undifferentiated, and differentiated white blood cell populations that are derived from myeloid progenitors including tissue specific and specialized varieties, and encompasses, by way of non-limiting example, granulocytes (i.e., mast cells, neutrophils, eosinophils and basophils), monocytes, macrophages, and dendritic cells.

[0064] As used herein, the “percent homology” between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions / total # of positions × 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0001] The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol.48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0065] Additionally or alternatively, the amino acids sequences of the presently disclosed subject matter can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215 :403-10. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the specified sequences disclosed herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. -30- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0066] The terms “polynucleotide”, “nucleic acid” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.

[0067] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid, e.g., an amino acid analog. The terms encompass amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0068] As used herein, “expression control sequence” or “regulatory region” of a nucleic acid molecule means a cis- acting nucleotide sequence that influences expression, positively or negatively, of an operatively linked gene. Regulatory regions include sequences of nucleotides that confer inducible (i.e., require a substance or stimulus for -31- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 increased transcription) expression of a gene. When an inducer is present or at increased concentration, gene expression can be increased. Regulatory regions also include sequences that confer repression of gene expression (i.e., a substance or stimulus decreases transcription). When a repressor is present or at increased concentration gene expression can be decreased. Regulatory regions are known to influence, modulate or control many in vivo biological activities including cell proliferation, cell growth and death, cell differentiation and immune modulation. Regulatory regions typically bind to one or more trans-acting proteins, which results in either increased or decreased transcription of the gene.

[0069] Particular examples of gene regulatory regions are promoters and enhancers. Promoters are sequences located around the transcription or translation start site, typically positioned 5' of the translation start site. Promoters usually are located within 1 Kb of the translation start site, but can be located further away, for example, 2 Kb, 3 Kb, 4 Kb, 5 Kb or more, up to and including 10 Kb. Enhancers are known to influence gene expression when positioned 5' or 3' of the gene, or when positioned in or a part of an exon or an intron. Enhancers also can function at a significant distance from the gene, for example, at a distance from about 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb or more.

[0070] Regulatory regions also include, but are not limited to, in addition to promoter regions, sequences that facilitate translation, splicing signals for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and, stop codons, leader sequences and fusion partner sequences, internal ribosome binding site (IRES) elements for the creation of multigene, or polycistronic, messages, polyadenylation signals to provide proper polyadenylation of the transcript of a gene of interest and stop codons, and can be optionally included in an expression vector.

[0071] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

[0072] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days -32- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

[0073] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

[0074] As used herein, the terms “subject,” “individual,” or “patient” are used interchangeably and refer to an individual organism, a vertebrate, or a mammal and may include humans, non-human primates, rodents, and the like (e.g., which is to be the recipient of a particular treatment, or from whom cells are harvested). In certain embodiments, the individual, patient or subject is a human.

[0075] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and / or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. Therapeutic effects of treatment include, without limitation, inhibiting recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. It is also to be appreciated that the various modes of treatment of diseases as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.

[0076] The compositions used in accordance with the disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and / or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose -33- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein. ID3

[0077] An exemplary ID3 nucleic acid sequence (SEQ ID NO: 3) is provided below: 1 gcgttgcagg tcactgtagc gggacttctt ttggttttct ttctctttgg ggcacctctg 61 gactcactcc ccagcatgaa ggcgctgagc ccggtgcgcg gctgctacga ggcggtgtgc 121 tgcctgtcgg aacgcagtct ggccatcgcc cggggccgag ggaagggccc ggcagctgag 181 gagccgctga gcttgctgga cgacatgaac cactgctact cccgcctgcg ggaactggta 241 cccggagtcc cgagaggcac tcagcttagc caggtggaaa tcctacagcg cgtcatcgac 301 tacattctcg acctgcaggt agtcctggcc gagccagccc ctggaccccc tgatggcccc 361 caccttccca tccagacagc cgagctcact ccggaacttg tcatctccaa cgacaaaagg 421 agcttttgcc actgactcgg ccgtgtcctg acacctccag aacgcaggtg ctggcgcccg 481 ttctgcctgg gaccccggga acctctcctg ccggaagccg gacggcaggg atgggcccca 541 acttcgccct gcccacttga cttcaccaaa tcccttcctg gagactaaac ctggtgctca 601 ggagcgaagg actgtgaact tgtggcctga agagccagag ctagctctgg ccaccagctg 661 ggcgacgtca ccctgctccc accccacccc caagttctaa ggtctcttca gagcgtggag 721 gtgtggaagg agtggctgct ctccaaacta tgccaaggcg gcggcagagc tggtcttctg 781 gtctccttgg agaaaggttc tgttgccctg atttatgaac tctataatag agtatatagg 841 ttttgtacct tttttacagg aaggtgactt tctgtaacaa tgcgatgtat attaaacttt 901 ttataaaagt taacattttg cataataaac gatttttaaa cacttgtgta (SEQ ID NO: 3)

[0078] In some embodiments, the engineered monocytes or engineered monocyte- derived macrophages comprise a heterologous nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3. Additionally or alternatively, in some embodiments, the expression levels and / or activity of ID3 in the engineered monocyte or engineered monocyte-derived macrophage is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, -34- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 times higher compared to that observed in a native monocyte or monocyte-derived macrophage.

[0079] In some embodiments, the engineered monocyte or engineered monocyte- derived macrophage further comprises a first regulatory sequence operatively linked to the nucleic acid encoding the ID3 gene. In further embodiments, the first regulatory sequence directs the expression of the ID3 gene. Additionally or alternatively, in some embodiments, the first regulatory sequence comprises, or consists essentially of, or yet further consists of a promoter, for example a constitutive promoter or a conditional promoter. In further embodiments, the conditional promoter is an immune cell specific promoter.

[0080] In one aspect, the engineered monocytes or engineered monocyte-derived macrophages provided herein overexpress ID3 and / or comprise a heterologous nucleic acid encoding the ID3 gene. The engineered monocytes or engineered monocyte-derived macrophages disclosed herein can be generated by in vitro transduction of monocytes or monocyte-derived macrophages with a nucleic acid as disclosed herein.

[0081] An exemplary ID3 amino acid sequence (SEQ ID NO: 4) is provided below:

[0082] NP_002158.3 DNA-binding protein inhibitor ID-3 [Homo sapiens]

[0083] MKALSPVRGCYEAVCCLSERSLAIARGRGKGPAAEEPLSLLDDMNHCYS RLRELVPGVPRGTQLSQVEILQRVIDYILDLQVVLAEPAPGPPDGPHLPIQTAELTPE LVISNDKRSFCH (SEQ ID NO: 4)

[0084] In some embodiments, the engineered monocytes or engineered monocyte- derived macrophages express a heterologous amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4, or a biological equivalent thereof. In further embodiments, the biological equivalent of SEQ ID NO: 4 comprises one or more conservative amino acid substitutions relative to SEQ ID NO: 4, respectively. Additionally or alternatively, in some embodiments, the phagocytic capacity of the biological equivalent is substantially similar to that of the protein of SEQ ID NO: 4. Polynucleotides, Polypeptides and Analogs

[0085] Also included in the presently disclosed subject matter are ID3 polynucleotides and their corresponding polypeptides or fragments, polynucleotides encoding dectin-1, -35- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 dectin-2, dectin-3, or mincle and their corresponding polypeptides or fragments, and / or inhibitory nucleic acids that specifically target SIRPA, CLEC4a, or Siglec-E that may be modified in ways that enhance their functional activity when expressed in an engineered monocyte or engineered monocyte-derived macrophage. The presently disclosed subject matter provides methods for optimizing an amino acid sequence or a nucleic acid sequence by producing an alteration in the sequence. Such alterations can comprise certain mutations, deletions, insertions, or post-translational modifications. The presently disclosed subject matter further comprises analogs of any naturally-occurring polypeptide of the presently disclosed subject matter. Analogs can differ from a naturally-occurring polypeptide of the presently disclosed subject matter by amino acid sequence differences, by post-translational modifications, or by both. Analogs of the presently disclosed subject matter can generally exhibit at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%), about 98%, about 99% or more identity or homology with all or part of a naturally-occurring amino, acid sequence of the presently disclosed subject matter. The length of sequence comparison is at least about 5, about 10, about 15, about 20, about 25, about 50, about 75, about 100 or more amino acid residues. Again, in an exemplary approach to determining the degree of identity, a BLAST program can be used, with a probability score between e-3and e-100indicating a closely related sequence. Modifications comprise in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications can occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Analogs can also differ from the naturally-occurring polypeptides of the presently disclosed subject matter by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethyl sulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2nd ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs which contain residues other than L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., beta (β) or gamma (γ) amino acids.

[0086] In addition to full-length polypeptides, the presently disclosed subject matter also provides fragments of any one of the polypeptides or peptide domains of the presently disclosed subject matter. A fragment can be at least about 5, about 10, about 13, or about -36- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 15 amino acids. In some embodiments, a fragment is at least about 20 contiguous amino acids, at least about 30 contiguous amino acids, or at least about 50 contiguous amino acids. In some embodiments, a fragment is at least about 60 to about 80, about 100, about 200, about 300 or more contiguous amino acids. Fragments of the presently disclosed subject matter can be generated by methods known to those of ordinary skill in the art or can result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

[0087] Non-protein analogs have a chemical structure designed to mimic the functional activity of a protein. Such analogs are administered according to methods of the presently disclosed subject matter. Such analogs can exceed the physiological activity of the original polypeptide. Methods of analog design are well known in the art, and synthesis of analogs can be carried out according to such methods by modifying the chemical structures such that the resultant analogs increase the therapeutic activity of the original polypeptide when expressed in an engineered monocyte or engineered monocyte-derived macrophage. These chemical modifications include, but are not limited to, substituting alternative R groups and varying the degree of saturation at specific carbon atoms of a reference polypeptide. The protein analogs can be relatively resistant to in vivo degradation, resulting in a more prolonged therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the Examples below.

[0088] In accordance with the presently disclosed subject matter, the polynucleotides encoding ID3 can be modified by codon optimization. Codon optimization can alter both naturally occurring and recombinant gene sequences to achieve the highest possible levels of productivity in any given expression system. Factors that are involved in different stages of protein expression include codon adaptability, mRNA structure, and various cis- elements in transcription and translation. Any suitable codon optimization methods or technologies that are known to ones skilled in the art can be used to modify the polynucleotides of the presently disclosed subject matter, including, but not limited to, OptimumGene™, Encor optimization, and Blue Heron.

[0089] In some embodiments, a nucleic acid as disclosed herein further comprises a regulatory sequence directing the expression of ID3, dectin-1, dectin-2, dectin-3, mincle, or any receptor (e.g., CAR) disclosed herein, or an inhibitory nucleic acid that specifically targets SIRPA, CLEC4a, or Siglec-E. In certain embodiments, the nucleic acid comprises a -37- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 single regulatory sequence directing the expression of ID3 and (a) dectin-1, dectin-2, dectin- 3, or mincle, (b) the receptor (e.g., CAR) and / or (c) an inhibitory nucleic acid that specifically targets SIRPA, CLEC4a, or Siglec-E. In other embodiments, the nucleic acid comprises a first regulatory sequence directing the expression of ID3 and a second regulatory sequence directing the expression of a) dectin-1, dectin-2, dectin-3, or mincle, (b) the receptor (e.g., CAR) and / or (c) an inhibitory nucleic acid that specifically targets SIRPA, CLEC4a, or Siglec-E. In other embodiments, the first regulatory sequence is the same as the second regulatory sequence. In some embodiments, the first regulatory sequence is different from the second regulatory sequence. Vectors

[0090] Many expression vectors are available and known to those of skill in the art and can be used for nonendogenous expression of ID3, dectin-1, dectin-2, dectin-3, mincle, or any receptor (e.g., CAR), or an inhibitory nucleic acid that specifically targets SIRPA, CLEC4a, or Siglec-E. The choice of expression vector will be influenced by the choice of host expression system. Such selection is well within the level of skill of the skilled artisan. In general, expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational termination signals. Expression vectors that are used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vector in the cells.

[0091] Vectors also can contain additional nucleotide sequences operably linked to the ligated nucleic acid molecule, such as, for example, an epitope tag such as for localization, e.g., a hexa-his tag or a myc tag, hemagglutinin tag or a tag for purification, for example, a GST fusion, and a sequence for directing protein secretion and / or membrane association.

[0092] Expression of the heterologous ID3, dectin-1, dectin-2, dectin-3, mincle, or any receptor (e.g., CAR), or an inhibitory nucleic acid that specifically targets SIRPA, CLEC4a, or Siglec-E can be controlled by any promoter / enhancer known in the art. Suitable bacterial promoters are well known in the art and described herein below. Other suitable promoters for mammalian cells, yeast cells and insect cells are well known in the art and some are exemplified below. Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application and is within the level of skill of the skilled artisan. Promoters which can be used include but are not limited to eukaryotic -38- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 expression vectors containing the SV40 early promoter (Bernoist and Chambon, Nature 290:304-310(1981)), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797(1980)), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 75: 1441-1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)); prokaryotic expression vectors such as the β-lactamase promoter (Jay et al., Proc. Natl. Acad. Sci. USA 75:5543 (1981)) or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 50:21- 25(1983)); see also "Useful Proteins from Recombinant Bacteria": in Scientific American 242:79-94 (1980)); plant expression vectors containing the nopaline synthetase promoter (Herrera- Estrella et al., Nature 505:209-213(1984)) or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., Nucleic Acids Res.9:2871(1981)), and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et al., Nature 510: 115-120(1984)); promoter elements from yeast and other fungi such as the Gal4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline phosphatase promoter, and the following animal transcriptional control regions that exhibit tissue specificity and have been used in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., Cell 55:639-646 (1984); Ornitz et al., Cold Spring Harbor Symp. Quant. Biol.50:399-409(1986); MacDonald, Hepatology 7:425-515 (1987)); insulin gene control region which is active in pancreatic beta cells (Hanahan et al., Nature 515: 115-122 (1985)), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., Cell 55:647-658 (1984); Adams et al., Nature 515:533-538 (1985); Alexander et al., Mol. Cell Biol.7: 1436-1444 (1987)), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., Cell 15:485-495 (1986)), albumin gene control region which is active in liver (Pinckert et al., Genes and Devel.1:268-276 (1987)), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., Mol. Cell. Biol.5:1639-403 (1985)); Hammer et al., Science 255:53-58 (1987)), alpha-1 antitrypsin gene control region which is active in liver (Kelsey et al., Genes and Devel.7:161-171 (1987)), beta globin gene control region which is active in myeloid cells (Magram et al., Nature 515:338-340 (1985)); Kollias et al., Cell 5:89-94 (1986)), myelin basic protein gene control region which is active in oligodendrocyte cells of the brain (Readhead et al., Cell 15:703-712 (1987)), myosin light chain-2 gene control region which is active in skeletal muscle (Shani, Nature 514:283-286 (1985)), and gonadotrophic releasing hormone gene control region which is active in gonadotrophs of the hypothalamus (Mason et al., Science 254: 1372- 1378 (1986)). -39- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0093] In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the ID3 gene in host cells. A typical expression cassette contains a promoter operably linked to the nucleic acid sequence encoding the polypeptide chains of interest and signals required for efficient polyadenylation of the transcript, ribosome binding sites and translation termination. Additional elements of the cassette can include enhancers. In addition, the cassette typically contains a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region can be obtained from the same gene as the promoter sequence or can be obtained from different genes.

[0094] Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a nucleic acid sequence of interest under the direction of the polyhedron promoter or other strong baculovirus promoter.

[0095] Any methods known to those of skill in the art for the insertion of DNA fragments into a vector can be used to construct expression vectors containing a nucleic acid encoding any of the polypeptides provided herein. These methods can include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules can be enzymatically modified. Alternatively, any site desired can be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers can contain specific chemically synthesized nucleic acids encoding restriction endonuclease recognition sequences.

[0096] Exemplary plasmid vectors useful to produce the polypeptides provided herein contain a strong promoter, such as the HCMV immediate early enhancer / promoter or the MHC class I promoter, an intron to enhance processing of the transcript, such as the HCMV immediate early gene intron A, and a polyadenylation (poly A) signal, such as the late SV40 polyA signal. -40- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0097] Genetic modification of engineered monocytes or engineered monocyte-derived macrophages can be accomplished by transducing a substantially homogeneous cell composition with a recombinant DNA or RNA construct. The vector can be a retroviral vector (e.g., gamma retroviral), which is employed for the introduction of the DNA or RNA construct into the host cell genome. For example, a polynucleotide encoding ID3 can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from an alternative internal promoter.

[0098] Non-viral vectors or RNA may be used as well. Random chromosomal integration, or targeted integration (e.g., using a nuclease, transcription activator-like effector nucleases (TALENs), Zinc-finger nucleases (ZFNs), and / or clustered regularly interspaced short palindromic repeats (CRISPRs), or transgene expression (e.g., using a natural or chemically modified RNA) can be used.

[0099] For initial genetic modification of the cells to provide ID3 overexpressing cells, a retroviral vector can be employed for transduction. However, any other suitable viral vector or non-viral delivery system can be used for genetic modification of cells. For subsequent genetic modification of the cells to provide cells comprising an antigen presenting complex comprising at least two co-stimulatory ligands, retroviral gene transfer (transduction) likewise proves effective. Combinations of retroviral vector and an appropriate packaging line are also suitable, where the capsid proteins will be functional for infecting human cells. Various amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller et al. (1985) Mol. Cell. Biol.5:431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol.6:2895-2902); and CRIP (Danos et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non -amphotropic particles are suitable too, e.g., particles pseudotyped with VSVG, RD114 or GALV envelope and any other known in the art.

[0100] Possible methods of transduction also include direct co-culture of the cells with producer cells, e.g., by the method of Bregni, et al., Blood 80: 1418-1422(1992), or culturing with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations, e.g., by the method of Xu, et al., Exp. Hemat. 22:223-230 (1994); and Hughes, et al., J. Clin. Invest.89: 1817 (1992).

[0101] Transducing viral vectors can be used to express a co-stimulatory ligand and / or secretes a cytokine (e.g., 4-1BBL and / or IL-12) in an engineered monocyte or engineered -41- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 monocyte-derived macrophage. In some embodiments, the chosen vector exhibits high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430 (1997); Kido et al., Current Eye Research 15:833-844 (1996); Bloomer et al., Journal of Virology 71 :6641-6649, 1997; Naldini et al., Science 272:263267 (1996); and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A.94: 10319, (1997)). Other viral vectors that can be used include, for example, adenoviral, lentiviral, and adeno- associated viral vectors, vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15- 14, (1990); Friedman, Science 244: 1275-1281 (1989); Eglitis et al., BioTechniques 6:608- 614, (1988); Tolstoshev et al., Current Opinion in Biotechnology 1:55-61(1990); Sharp, The Lancet 337: 1277-1278 (1991); Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322 (1987); Anderson, Science 226:401-409 (1984); Moen, Blood Cells 17:407-416 (1991); Miller et al., Biotechnology 7:980-990 (1989); Le Gal La Salle et al., Science 259:988-990 (1993); and Johnson, Chest 107:77S-83S (1995)). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370 (1990); Anderson et al., U.S. Pat. No.5,399,346).

[0102] In certain non-limiting embodiments, the vector expressing ID3 is a retroviral vector, e.g., an oncoretroviral vector. In some instances, the retroviral vector is a SFG retroviral vector or murine stem cell virus (MSCV) retroviral vector. In certain non-limiting embodiments, the vector expressing an ID3 nucleic acid sequence is a lentiviral vector. In certain non-limiting embodiments, the vector expressing an ID3 nucleic acid sequence is a transposon vector.

[0103] Non-viral approaches can also be employed for the expression of a protein in a cell. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Nat'l. Acad. Sci. U.S.A. 84:7413, (1987); Ono et al., Neuroscience Letters 17:259 (1990); Brigham et al., Am. J. Med. Sci.298:278, (1989); Staubinger et al., Methods in Enzymology 101 :512 (1983)), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263 : 14621 (1988); Wu et al., Journal of Biological Chemistry 264: 16985 (1989)), or by micro- injection under surgical conditions (Wolff et al., Science 247: 1465 (1990)). Other non- viral means for gene transfer include transfection in vitro using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the -42- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 affected tissues of a subject can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue or are injected systemically. Recombinant receptors can also be derived or obtained using transposases or targeted nucleases (e.g., Zinc finger nucleases, meganucleases, or TALE nucleases). Transient expression may be obtained by RNA electroporation.

[0104] cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element or intron (e.g., the elongation factor la enhancer / promoter / intron structure). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

[0105] The resulting cells can be grown under conditions similar to those for unmodified cells, whereby the modified cells can be expanded and used for a variety of purposes.

[0106] In some embodiments, a vector as disclosed herein comprises a regulatory sequence directing the expression of ID3, dectin-1, dectin-2, dectin-3, mincle, or any receptor disclosed herein (e.g., CAR), or an inhibitory nucleic acid that specifically targets SIRPA, CLEC4a, or Siglec-E. In further embodiments, the vector comprises a single regulatory sequence directing the expression of ID3 and (a) dectin-1, dectin-2, dectin-3, or mincle, (b) the receptor (e.g., CAR) and / or (c) an inhibitory nucleic acid that specifically targets SIRPA, CLEC4a, or Siglec-E. In other embodiments, the vector comprises a first regulatory sequence directing the expression of ID3 and a second regulatory sequence directing the expression of (a) dectin-1, dectin-2, dectin-3, or mincle, (b) the receptor (e.g., CAR) and / or (c) an inhibitory nucleic acid that specifically targets SIRPA, CLEC4a, or Siglec-E. In other embodiments, the first regulatory sequence is the same as the second regulatory sequence. In some embodiments, the first regulatory sequence is different from the second regulatory sequence. -43- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 Chimeric Antigen Receptors (CARs)

[0107] In some embodiments, the engineered monocytes or engineered monocyte- derived macrophages provided herein express at least one chimeric antigen receptor (CAR). CARs are engineered receptors, which graft or confer a specificity of interest onto an immune effector cell. For example, CARs can be used to graft the specificity of a monoclonal antibody onto an immune cell, such as a macrophage or monocyte. In some embodiments, transfer of the coding sequence of the CAR is facilitated by nucleic acid vector, such as a retroviral vector.

[0108] There are currently three generations of CARs. In some embodiments, the engineered monocytes or engineered monocyte-derived macrophages provided herein express a “first generation” CAR. “First generation” CARs are typically composed of an extracellular antigen binding domain (e.g., a single-chain variable fragment (scFv)) fused to a transmembrane domain fused to cytoplasmic / intracellular domain of the T cell receptor (TCR) chain. “First generation” CARs typically have the intracellular domain from the CD3ζ chain, which is the primary transmitter of signals from endogenous TCRs. “First generation” CARs can provide de novo antigen recognition and cause activation of both CD4+and CD8+T cells through their CD3ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation.

[0109] In some embodiments, the engineered monocytes or engineered monocyte- derived macrophages provided herein express a “second generation” CAR. “Second generation” CARs add intracellular domains from various co-stimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. “Second generation” CARs comprise those that provide both co- stimulation (e.g., CD28 or 4-1BB) and activation (e.g., CD3ζ).

[0110] In some embodiments, the engineered monocytes or engineered monocyte- derived macrophages provided herein express a “third generation” CAR. “Third generation” CARs comprise those that provide multiple co-stimulation (e.g., CD28 and 4-1BB) and activation (e.g., CD3ζ).

[0111] In accordance with the presently disclosed subject matter, the CARs of the engineered monocytes or engineered monocyte-derived macrophages provided herein comprise an extracellular antigen-binding domain, a transmembrane domain and an intracellular domain. Further, the activity of the engineered monocytes or engineered -44- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 monocyte-derived macrophages can be adjusted by selection of co-stimulatory molecules included in the chimeric antigen receptor.

[0112] Extracellular Antigen-Binding Domain of a CAR. In certain embodiments, the extracellular antigen-binding domain of a CAR specifically binds a tumor antigen. In certain embodiments, the extracellular antigen-binding domain is derived from a monoclonal antibody (mAb) that binds to a tumor antigen. In some embodiments, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the extracellular antigen-binding domain comprises a Fab, which is optionally crosslinked. In some embodiments, the extracellular binding domain comprises a F(ab)2.In some embodiments, any of the foregoing molecules are included in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain comprises a human scFv that binds specifically to a tumor antigen. In certain embodiments, the scFv is identified by screening scFv phage library with a tumor antigen-Fc fusion protein.

[0113] Examples of tumor antigens include, but are not limited to, 5T4, alpha 5β1- integrin, 707-AP, A33, AFP, ART-4, B7H4, BAGE, Bcl-2, β-catenin, BCMA, Bcr-abl, CA125, CA19-9, CAMEL, CAP-1, CASP-8, CD4, CD5, CD19, CD20, CD21 , CD22, CD25, CDC27 / m, CD33, CD37, CD45, CD52, CD56, CD80, CD123, CDK4 / m, CEA, c- Met, CS-1, CT, Cyp-B, cyclin B1, DAGE, DAM, EBNA, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ephrinB2, estrogen receptor, ETV6-AML1, FAP, ferritin, folate- binding protein, GAGE, G250, GD-2, GM2, GnT-V, gp75, gp100 (Pmel 17), HAGE, HER- 2 / neu, HLA-A*0201-R170I, HPV E6, HPV E7, Ki-67, HSP70-2M, HST-2, hTERT (or hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR / FUT, LRP, MAGE, MART, MART-1 / melan-A, MART-2 / Ski, MC1R, mesothelin, MUC16, MUM-1 -B, myc, MUM-2, MUM-3, NA88-A, NYESO-1, NY-Eso-B, p53, proteinase-3, p190 minor bcr-abl, Pml / RARα, PRAME, progesterone receptor, PSA, PSCA, PSM, PSMA, ras, RAGE, RU1 or RU2, RORI, SART-1 or SART-3, survivin, TEL / AML1, TGFβ, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, tenascin, TSTA tyrosinase, VEGF, or WT1. Other examples of tumor antigens include a2b b3 (Glycoprotein IIb / IIIa), a4, a4b7, a4b7 +aEb7, a5, Activin receptor type-2B, ALK1, Alpha-synuclein, amyloid beta, APP, AXL, Blood Group A, CAIX, CCL- 2, CD105 (endoglin), CD115 (CSF1R), CD116a (CSF2Ra), CD123, CD152 (CTLA4), CD184 (CXCR4), CD19, CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD20, CD200, CD22, CD221 (IGF1R), CD248, CD25, CD257 (BAFF), CD26, CD262 (DR5), CD276 -45- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 (B7H3), CD3, CD30 (TNFRSF8), CD319 (SLAMF7), CD33, CD332 (FGFR2), CD350 (FZD10), CD37, CD371 (CLEC12A), CD38, CD4, CD49b (a2), CD51 (a5), CD52, CD56, CD61 (a4b3), CD70, CD73 (NT5E), CD74, CEA, Claudin-18.2, cMET, CRLR, DLL3, DLL4, DNA / histone (H1) complex, EGFR, EpCAM, EGFR- HER3, EGFRvIII, EphA3, ERGT(GalNAc) Tn Antigen, FLT1, FOLR1, frizzled family receptor (FZD), Lewis Y, Lewis X, GCGR, GD2, GD2 α-acetyl, GD3, GM1, GM1 fucosyl, GM2, GPA33, GPNMB, GUCY2C, HER2, HER3, HGFR (cMET), IgHe, IGLF2, Kallikreins, LINGO1, LOXL2, Ly6 / PLAUR domain-containing protein 3, MADCAM1, MAG, Mesothelin, MT1-MMP (MMP14), MUC1, Mucin 5AC, NaPi2b, NeuGc-GM3, notch, NOTCH2 / NOTCH3 receptors, oxLDL, P-selectin, PCSK9, PDGFRA, PDGFRa, phosphatidylserine, polysialic acid, PSMA, PVRL4, RGMA, CD240D Blood group D antigen, root plate-specific spondin 3, serum amyloid P component, STEAP-1, TACSTD2, TGFb, TWEAKR, TYRP1, VEGFR2, VSIR, CD171 (L1CAM), CD19, CD47, pMHC[NY-ESO1], pMHC[MART1], pMHC[MAGEA1], pMHC[Tyrosinase], pMHC[gp100], pMHC[MUC1], pMHC[tax], pMHC[WT-1], pMHC[EBNA-1], pMHC[LMP2], pMHC[hTERT], GPC3, CD80, CD23, and fibronectin extra domain-B.

[0114] In certain embodiments, the extracellular antigen-binding domain of a presently disclosed CAR has a high binding specificity and high binding affinity to a tumor antigen. For example, in some embodiments, the extracellular antigen-binding domain of the CAR (embodied, for example, in a human scFv or an analog thereof) binds to a particular tumor antigen with a dissociation constant (Kd) of about 1 × 10-5M or less. In certain embodiments, the Kd is about 5 × 10-6M or less, about 1 × 10-6M or less, about 5 × 10-7M or less, about 1 × 10-7M or less, about 5 × 10-8M or less, about 1 × 10-8M or less, about 5 × 10-9or less, about 4 × 10-9or less, about 3 × 10-9or less, about 2 × 10-9or less, or about 1 × 10-9M or less. In certain non-limiting embodiments, the Kd is from about 3 × 10-9M or less. In certain non-limiting embodiments, the Kd is from about 3 × 10-9to about 2 × 10-7.

[0115] Binding of the extracellular antigen-binding domain (embodiment, for example, in an scFv or an analog thereof) of a tumor antigen-specific CAR can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detect the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody, or an scFv) specific for the complex of interest. For example, the scFv can be radioactively labeled and used in a -46- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a γ counter or a scintillation counter or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of the tumor antigen-specific CAR is labeled with a fluorescent marker. Non- limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet). In certain embodiments, the scFv of a tumor antigen-specific CAR is labeled with GFP.

[0116] In some embodiments, the extracellular antigen-binding domain of the expressed CAR binds to a tumor antigen that is expressed by a tumor cell. In some embodiments, the extracellular antigen-binding domain of the expressed CAR binds to a tumor antigen that is expressed on the surface of a tumor cell.

[0117] In certain embodiments, the extracellular antigen-binding domain (e.g., human scFv) comprises a heavy chain variable (VH) region and a light chain variable (VL) region, optionally linked with a linker sequence, for example a linker peptide (e.g., SEQ ID NO: 1), between the heavy chain variable (VH) region and the light chain variable (VL) region.

[0118] In certain non-limiting embodiments, an extracellular antigen-binding domain of the presently disclosed CAR can comprise a linker connecting the heavy chain variable (VH) region and light chain variable (VL) region of the extracellular antigen-binding domain. As used herein, the term “linker” refers to a functional group (e.g., chemical or polypeptide) that covalently attaches two or more polypeptides or nucleic acids so that they are connected to one another. As used herein, a “peptide linker” refers to one or more amino acids used to couple two proteins together (e.g., to couple VHand VLdomains). In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO: 1. In certain embodiments, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 is set forth in SEQ ID NO: 2.

[0119] Additionally or alternatively, in some embodiments, the extracellular antigen- binding domain can comprise a leader or a signal peptide sequence that directs the nascent protein into the endoplasmic reticulum. The signal peptide or leader can be essential if the -47- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 CAR is to be glycosylated and anchored in the cell membrane. The signal sequence or leader sequence can be a peptide sequence (about 5, about 10, about 15, about 20, about 25, or about 30 amino acids long) present at the N-terminus of the newly synthesized proteins that direct their entry to the secretory pathway.

[0120] In certain embodiments, the signal peptide is covalently joined to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide comprises a human CD8 signal polypeptide comprising amino acids having the sequence set forth in SEQ ID NO: 5 as provided below: MALPVTALLLPLALLLHAARP (SEQ ID NO: 5).

[0121] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5 is set forth in SEQ ID NO: 6, which is provided below: ATGGCCCTGCCAGTAACGGCTCTGCTGCTGCCACTTGCTCTGCTCCTCCATGCAG CCAGGCCT (SEQ ID NO: 6).

[0122] In certain embodiments, the signal peptide comprises a human CD8 signal polypeptide comprising amino acids having the sequence set forth in SEQ ID NO: 7 as provided below: MALPVTALLLPLALLLHA (SEQ ID NO: 7).

[0123] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 7 is set forth in SEQ ID NO: 8, which is provided below: ATGGCTCTCCCAGTGACTGCCCTACTGCTTCCCCTAGCGCTTCTCCTGCATGCA (SEQ ID NO: 8).

[0124] In certain embodiments, the signal peptide comprises a mouse CD8 signal polypeptide comprising amino acids having the sequence set forth in SEQ ID NO: 9 as provided below: MASPLTRFLSLNLLLLGESII (SEQ ID NO: 9).

[0125] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9 is set forth in SEQ ID NO: 10, which is provided below:

[0126] ATGGCCAGCCCCCTGACCAGGTTCCTGAGCCTGAACCTGCTGCTGCT GGGCGAGAGCATCATC (SEQ ID NO: 10).

[0127] In certain embodiments, the signal peptide comprises a mouse CD8 signal polypeptide comprising amino acids having the sequence set forth in SEQ ID NO: 11 as provided below: MASPLTRFLSLNLLLLGE (SEQ ID NO: 11).

[0128] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 11 is -48- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 set forth in SEQ ID NO: 12, which is provided below: ATGGCCAGCCCCCTGACCAGGTTCCTGAGCCTGAACCTGCTGCTGCTGGGCGAG (SEQ ID NO: 12).

[0129] Transmembrane Domain of a CAR. In certain non-limiting embodiments, the transmembrane domain of the CAR comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stability. After antigen recognition, receptors cluster and a signal is transmitted to the cell. In accordance with the presently disclosed subject matter, the transmembrane domain of the CAR can comprise a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a synthetic peptide (e.g., a transmembrane peptide not based on a protein associated with the immune response), or a combination thereof.

[0130] In certain embodiments, the transmembrane domain of a presently disclosed CAR comprises a CD28 polypeptide. The CD28 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous to the sequence having a UniProtKB Reference No: P10747 or NCBI Reference No: NP006130 (SEQ ID NO: 13), or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 13 which is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length. Additionally or alternatively, in non- limiting various embodiments, the CD28 polypeptide has an amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, or 200 to 220 of SEQ ID NO: 13. In certain embodiments, the CAR of the present disclosure comprises a transmembrane domain comprising a CD28 polypeptide, and optionally an intracellular domain comprising a co-stimulatory signaling region that comprises a CD28 polypeptide. In certain embodiments, the CD28 polypeptide comprised in the transmembrane domain and the intracellular domain has an amino acid sequence of amino acids 114 to 220 of SEQ ID NO: 13. In certain embodiments, the CD28 polypeptide comprised in the transmembrane domain has an amino acid sequence of amino acids 153 to 179 of SEQ ID NO: 13.

[0131] SEQ ID NO: 13 is provided below: -49- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNALSCKYSYNLFSREFRASLHKG LDSAVEVCWYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYQTDIYFCKIEV MYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVA FIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 13)

[0132] In accordance with the presently disclosed subject matter, a “CD28 nucleic acid molecule” refers to a polynucleotide encoding a CD28 polypeptide. In certain embodiments, the CD28 nucleic acid molecule encoding the CD28 polypeptide comprised in the transmembrane domain (and optionally the intracellular domain (e.g., the co- stimulatory signaling region)) of the presently disclosed CAR (e.g., amino acids 114 to 220 of SEQ ID NO: 13 or amino acids 153 to 179 of SEQ ID NO: 13) comprises at least a portion of the sequence set forth in SEQ ID NO: 14 as provided below. attgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagt cccctatttcccggaccttctaagcccttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcct ttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccaccc gcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctcc (SEQ ID NO: 14)

[0133] In certain embodiments, the transmembrane domain comprises a CD8 polypeptide. The CD8 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%) homologous to SEQ ID NO: 15 (homology herein may be determined using standard software such as BLAST or FASTA) as provided below, or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 15 which is at least 20, or at least 30, or at least 40, or at least 50, and up to 235 amino acids in length. Additionally or alternatively, in various embodiments, the CD8 polypeptide has an amino acid sequence of amino acids 1 to 235, 1 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 235 of SEQ ID NO: 15.

[0134] MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSN PTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDF RRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEAC RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRP WKSGDKPSLSARYV (SEQ ID NO: 15) -50- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0135] In certain embodiments, the transmembrane domain comprises a CD8 polypeptide comprising amino acids having the sequence set forth in SEQ ID NO: 16 as provided below:

[0136] PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCN (SEQ ID NO: 16)

[0137] In accordance with the presently disclosed subject matter, a “CD8 nucleic acid molecule” refers to a polynucleotide encoding a CD8 polypeptide. In certain embodiments, the CD8 nucleic acid molecule encoding the CD8 polypeptide comprised in the transmembrane domain of the presently disclosed CAR (SEQ ID NO: 16) comprises nucleic acids having the sequence set forth in SEQ ID NO: 17 as provided below.

[0138] CCCACCACGACGCCAGCGCCGCGACCACCAACCCCGGCGCCCACGAT CGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGG GCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGC CCCTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTG CAAC (SEQ ID NO: 17)

[0139] In certain non-limiting embodiments, a CAR can also comprise a spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The spacer region can be flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition while preserving the activating activity of the CAR. In certain non-limiting embodiments, the spacer region can be the hinge region from IgGl, the CH2CH3 region of immunoglobulin and portions of CD3, a portion of a CD28 polypeptide (e.g., SEQ ID NO: 13), a portion of a CD8 polypeptide (e.g., SEQ ID NO: 15), a variation of any of the foregoing which is at least about 80%, at least about 85%, at least about 90%, or at least about 95% homologous thereto, or a synthetic spacer sequence. In certain non-limiting embodiments, the spacer region may have a length between about 1-50 (e.g., 5-25, 10-30, or 30-50) amino acids.

[0140] Intracellular Domain of a CAR. In certain non-limiting embodiments, an intracellular domain of the CAR can comprise a CD3ζ polypeptide, which can activate or stimulate a cell. CD3ζ comprises 3 ITAMs, and transmits an activation signal to the cell after antigen is bound. The CD3ζ polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to the sequence having a NCBI Reference No: NP_932170 (SEQ -51- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 ID NO: 18), or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0141] In certain embodiments, the CD3ζ polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 19 which is at least 20, or at least 30, or at least 40, or at least 50, and up to 164 amino acids in length. Additionally or alternatively, in various embodiments, the CD3ζ polypeptide has an amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 100 to 150, or 150 to 164 of SEQ ID NO: 19. In certain embodiments, the CD3ζ polypeptide has an amino acid sequence of amino acids 52 to 164 of SEQ ID NO: 19.

[0142] SEQ ID NO: 19 is provided below: MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 19)

[0143] In certain embodiments, the CD3ζ polypeptide has the amino acid sequence set forth in SEQ ID NO: 20, which is provided below: RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR (SEQ ID NO: 20)

[0144] In certain embodiments, the CD3ζ polypeptide has the amino acid sequence set forth in SEQ ID NO: 21, which is provided below: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR (SEQ ID NO: 21)

[0145] In accordance with the presently disclosed subject matter, a “CD3ζ nucleic acid molecule” refers to a polynucleotide encoding a CD3ζ polypeptide. In certain embodiments, the CD3ζ nucleic acid molecule encoding the CD3ζ polypeptide (SEQ ID NO: 20) comprised in the intracellular domain of the presently disclosed CAR comprises a nucleotide sequence as set forth in SEQ ID NO: 22 as provided below. AGAGTGAAGTTCAGCAGGAGCGCAGAGCCCCCCGCGTACCAGCAGGGCCAGAA CCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGG ACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAA -52- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 CCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCT ACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGG CCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACAT GCAGGCCCTGCCCCCTCGCG (SEQ ID NO: 22)

[0146] In certain embodiments, the CD3ζ nucleic acid molecule encoding the CD3ζ polypeptide (SEQ ID NO: 21) comprised in the intracellular domain of the presently disclosed CAR comprises a nucleotide sequence as set forth in SEQ ID NO: 23 as provided below. AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAA CCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGG ACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAA CCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCT ACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGG CCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACAT GCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO: 23)

[0147] In certain non-limiting embodiments, an intracellular domain of the CAR further comprises at least one signaling region. The at least one signaling region can include a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP- 10 polypeptide, a PD-1 polypeptide, a CTLA-4 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a synthetic peptide (not based on a protein associated with the immune response), or a combination thereof.

[0148] In certain embodiments, the signaling region is a co-stimulatory signaling region.

[0149] In certain embodiments, the co-stimulatory signaling region comprises at least one co-stimulatory molecule, which can provide optimal lymphocyte activation. As used herein, “co-stimulatory molecules” refer to cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of lymphocytes to antigen. The at least one co-stimulatory signaling region can include a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof. The co-stimulatory molecule can bind to a co-stimulatory ligand, which is a protein expressed on cell surface that upon binding to its receptor produces a co- stimulatory response, i.e., an intracellular response that effects the stimulation provided -53- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 when an antigen binds to its CAR molecule. Co-stimulatory ligands, include, but are not limited to CD80, CD86, CD70, OX40L, 4-1BBL, CD48, TNFRSF14, and PD- Ll. As one example, a 4-1BB ligand (i.e., 4-1BBL) may bind to 4-1BB (also known as “CD 137”) for providing an intracellular signal that in combination with a CAR signal induces an effector cell function of the CAR+expressing cell. CARs comprising an intracellular domain that comprises a co-stimulatory signaling region comprising 4-1BB, ICOS or DAP-10 are disclosed in U.S.7,446,190, which is herein incorporated by reference in its entirety. In certain embodiments, the intracellular domain of the CAR comprises a co-stimulatory signaling region that comprises a CD28 polypeptide. In certain embodiments, the intracellular domain of the CAR comprises a co-stimulatory signaling region that comprises two co-stimulatory molecules: CD28 and 4-1BB or CD28 and OX40.

[0150] 4-1BB can act as a tumor necrosis factor (TNF) ligand and have stimulatory activity. The 4-1BB polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous to the sequence having a UniProtKB Reference No: P41273 or NCBI Reference No: NP_001552 (SEQ ID NO: 24) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0151] SEQ ID NO: 24 is provided below: MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAG GQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQEL TKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLGTKERDWCGPSPADLSPGAS SVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSWKRGRKKLLYIFKQPFMR PVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 24)

[0152] In certain embodiments, the 4-1BB co-stimulatory domain has the amino acid sequence set forth in SEQ ID NO: 25, which is provided below: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 25)

[0153] In accordance with the presently disclosed subject matter, a “4-1BB nucleic acid molecule” refers to a polynucleotide encoding a 4-1BB polypeptide. In certain embodiments, the 4-1BB nucleic acid molecule encoding the 4-1BB polypeptide (SEQ ID NO: 25) comprised in the intracellular domain of the presently disclosed CAR comprises a nucleotide sequence as set forth in SEQ ID NO: 26 as provided below. AAACGGGGCAGAAAGAAGCTCCTGTATATATTCAAACAACCATTTATGAGACC -54- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 AGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAG AAGAAGGAGGATGTGAACTG (SEQ ID NO: 26)

[0154] An OX40 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous to the sequence having a UniProtKB Reference No: P43489 or NCBI Reference No: NP_003318 (SEQ ID NO: 27), or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0155] SEQ ID NO: 27 is provided below: MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVS RCSRSQNTVCRPCGPGFYNDWSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCR AGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAIC EDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGL VLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 27)

[0156] In accordance with the presently disclosed subject matter, an “OX40 nucleic acid molecule” refers to a polynucleotide encoding an OX40 polypeptide.

[0157] An ICOS polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous to the sequence having a NCBI Reference No: NP_036224 (SEQ ID NO: 28) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0158] SEQ ID NO: 28 is provided below: MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLL KGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSI FDPPPFKVTLTGGYLHIYESQLCCQLKFWLPIGCAAFVWCILGCILICWLTKKKYSSS VHDPNGEYMFMRATAKKSRLTDVTL (SEQ ID NO: 28)

[0159] In accordance with the presently disclosed subject matter, an “ICOS nucleic acid molecule” refers to a polynucleotide encoding an ICOS polypeptide.

[0160] CTLA-4 is an inhibitory receptor expressed by activated T cells, which when engaged by its corresponding ligands (CD80 and CD86; B7-1 and B7-2, respectively), mediates activated T cell inhibition or anergy. In both preclinical and clinical studies, CTLA-4 blockade by systemic antibody infusion, enhanced the endogenous anti-tumor -55- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 response albeit, in the clinical setting, with significant unforeseen toxicities.

[0161] CTLA-4 contains an extracellular V domain, a transmembrane domain, and a cytoplasmic tail. Alternate splice variants, encoding different isoforms, have been characterized. The membrane-bound isoform functions as a homodimer interconnected by a disulfide bond, while the soluble isoform functions as a monomer. The intracellular domain is similar to that of CD28, in that it has no intrinsic catalytic activity and contains one YVKM motif (SEQ ID NO: 29) able to bind PI3K, PP2A and SHP-2 and one proline-rich motif able to bind SH3 containing proteins. One role of CTLA-4 in inhibiting T cell responses seem to be directly via SHP-2 and PP2A dephosphorylation of TCR-proximal signaling proteins such as CD3 and LAT. CTLA-4 can also affect signaling indirectly via competing with CD28 for CD80 / 86 binding. CTLA-4 has also been shown to bind and / or interact with PI3K, CD80, AP2M1, and PPP2R5A.

[0162] In accordance with the presently disclosed subject matter, a CTLA-4 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to UniProtKB / Swiss- Prot Ref. No.: P16410.3 (SEQ ID NO: 30) (homology herein may be determined using standard software such as BLAST or FASTA) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0163] SEQ ID NO: 30 is provided below: MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAWLASSRGIASFV CEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQL TIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSS GLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 30)

[0164] In accordance with the presently disclosed subject matter, a “CTLA-4 nucleic acid molecule” refers to a polynucleotide encoding a CTLA-4 polypeptide.

[0165] PD-1 is a negative immune regulator of activated T cells upon engagement with its corresponding ligands PD-L1 and PD-L2 expressed on endogenous macrophages and dendritic cells. PD-1 is a type I membrane protein of 268 amino acids. PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. The protein's structure comprises an extracellular IgV domain followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located in an -56- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine- based switch motif, that PD-1 negatively regulates TCR signals. SHP- I and SHP-2 phosphatases bind to the cytoplasmic tail of PD-1 upon ligand binding. Upregulation of PD-L1 is one mechanism tumor cells may evade the host immune system. In pre-clinical and clinical trials, PD-1 blockade by antagonistic antibodies induced anti -tumor responses mediated through the host endogenous immune system. In accordance with the presently disclosed subject matter, a PD-1 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to NCBI Reference No: NP_005009.2 (SEQ ID NO: 31) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0166] SEQ ID NO: 31 is provided below: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLWTEGDNATFTCSF SNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVV RARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQT LVVGWGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYG ELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPED GHCSWPL (SEQ ID NO: 31)

[0167] In accordance with the presently disclosed subject matter, a “PD-1 nucleic acid molecule” refers to a polynucleotide encoding a PD-1 polypeptide.

[0168] Lymphocyte-activation protein 3 (LAG-3) is a negative immune regulator of immune cells. LAG-3 belongs to the immunoglobulin (Ig) superfamily and contains 4 extracellular Ig-like domains. The LAG3 gene contains 8 exons. The sequence data, exon / intron organization, and chromosomal localization all indicate a close relationship of LAG3 to CD4. LAG3 has also been designated CD223 (cluster of differentiation 223).

[0169] In accordance with the presently disclosed subject matter, a LAG-3 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to UniProtKB / Swiss- Prot Ref. No.: P18627.5 (SEQ ID NO: 32) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0170] SEQ ID NO: 32 is provided below: MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPWWAQEGAPAQLPCSPTIPLQDLSLL -57- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 RRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRS GRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRL GQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLA ESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVG LPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHI HLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSF SGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAG HLLLFLILGVLSLLLLVTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEP EPEPEPEPEPEPEPEPEQL (SEQ ID NO: 32)

[0171] In accordance with the presently disclosed subject matter, a “LAG-3 nucleic acid molecule” refers to a polynucleotide encoding a LAG-3 polypeptide.

[0172] Natural Killer Cell Receptor 2B4 (2B4) mediates non-MHC restricted cell killing on NK cells and subsets of T cells. To date, the function of 2B4 is still under investigation, with the 2B4-S isoform believed to be an activating receptor, and the 2B4-L isoform believed to be a negative immune regulator of immune cells.2B4 becomes engaged upon binding its high-affinity ligand, CD48. 2B4 contains a tyrosine-based switch motif, a molecular switch that allows the protein to associate with various phosphatases. 2B4 has also been designated CD244 (cluster of differentiation 244).

[0173] In accordance with the presently disclosed subject matter, a 2B4 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to UniProtKB / Swiss-Prot Ref. No.: Q9BZW8.2 (SEQ ID NO: 33) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0174] SEQ ID NO: 33 is provided below: MLGQWTLILLLLLKVYQGKGCQGSADHWSISGVPLQLQPNSIQTKVDSIAWKKLLP SQNGFHHILKWENGSLPSNTSNDRFSFIVKNLSLLIKAAQQQDSGLYCLEVTSISGK VQTATFQVFVFESLLPDKVEKPRLQGQGKILDRGRCQVALSCLVSRDGNVSYAWY RGSKLIQTAGNLTYLDEEVDINGTHTYTCNVSNPVSWESHTLNLTQDCQNAHQEFR FWPFLVIIVILSALFLGTLACFCVWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHE QEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYE VIGKSQPKAQNPARLSRKELENFDVYS (SEQ ID NO: 33)

[0175] In accordance with the presently disclosed subject matter, a “2B4 nucleic acid -58- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 molecule” refers to a polynucleotide encoding a 2B4 polypeptide.

[0176] B- and T-lymphocyte attenuator (BTLA) expression is induced during activation of T cells, and BTLA remains expressed on Thl cells but not Th2 cells. Like PD1 and CTLA4, BTLA interacts with a B7 homolog, B7H4. However, unlike PD-1 and CTLA-4, BTLA displays T-Cell inhibition via interaction with tumor necrosis family receptors (TNF- R), not just the B7 family of cell surface receptors. BTLA is a ligand for tumor necrosis factor (receptor) superfamily, member 14 (TNFRSF14), also known as herpes virus entry mediator (HVEM). BTLA-HVEM complexes negatively regulate T-cell immune responses. BTLA activation has been shown to inhibit the function of human CD8+cancer-specific T cells. BTLA has also been designated as CD272 (cluster of differentiation 272).

[0177] In accordance with the presently disclosed subject matter, a BTLA polypeptide can have an amino acid sequence that is at least about 85%>, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to UniProtKB / Swiss- Prot Ref. No.: Q7Z6A9.3 (SEQ ID NO: 34) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0178] SEQ ID NO: 34 is provided below: MKTLPAMLGTGKLFWVFFLIPYLDIWNIHGKESCDVQLYIKRQSEHSILAGDPFELE CPVKYCANRPHVTWCKLNGTTCVKLEDRQTSWKEEKNISFFILHFEPVLPNDNGSY RCSANFQSNLIESHSTTLYVTDVKSASERPSKDEMASRPWLLYRLLPLGGLPLLITTC FCLFCCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYD NDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYA SICVRS (SEQ ID NO: 34)

[0179] In accordance with the presently disclosed subject matter, a “BTLA nucleic acid molecule” refers to a polynucleotide encoding a BTLA polypeptide. Engineered Monocytes or Engineered Monocyte-Derived Macrophages

[0180] Engineered immune cells can be generated from peripheral donor lymphocytes, e.g., those disclosed in Sadelain, M., et al., Nat Rev Cancer 3 :35-45 (2003), in Morgan, R.A. et al. (2006) Science 314: 126-129, in Panelli et al. (2000) J Immunol 164:495-504; Panelli et al. (2000) J Immunol 164:4382-4392 (2000), and in Dupont et al. (2005) Cancer Res 65:5417-5427; Papanicolaou et al. (2003) Blood 102:2498-2505. The engineered immune cells of the present technology (e.g., monocyte-derived macrophages, monocytes) -59- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor or stem cells.

[0181] The unpurified source of immune cells can be any known in the art, such as the bone marrow, fetal, neonate or adult or other hematopoietic cell source, e.g., fetal liver, peripheral blood or umbilical cord blood. Various techniques can be employed to separate the cells. For instance, negative selection methods can remove non-immune cell initially. Monoclonal antibodies are particularly useful for identifying markers associated with particular cell lineages and / or stages of differentiation for both positive and negative selections. In some embodiments, the monocyte-derived macrophages or monocytes are CD14+ / CD15-. Additionally or alternatively, in some embodiments, the monocyte-derived macrophages or monocytes are CD163+or mannose receptor+.

[0182] A large proportion of terminally differentiated cells can be initially removed by a relatively crude separation. For example, magnetic bead separations can be used initially to remove large numbers of irrelevant cells. In some embodiments, at least about 80%, usually at least 70% of the total hematopoietic cells will be removed prior to cell isolation.

[0183] Procedures for separation include, but are not limited to, density gradient centrifugation; resetting; coupling to particles that modify cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; cytotoxic agents joined to or used in conjunction with a mAb, including, but not limited to, complement and cytotoxins; and panning with antibody attached to a solid matrix, e.g., plate, chip, elutriation or any other convenient technique.

[0184] Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels.

[0185] The cells can be selected against dead cells, by employing dyes associated with dead cells such as propidium iodide (PI). In some embodiments, the cells are collected in a medium comprising 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA) or any other suitable, preferably sterile, isotonic medium.

[0186] Monocytes or Monocyte-Derived Macrophages Overexpressing ID3

[0187] The presently disclosed subject matter provides engineered monocytes or engineered monocyte-derived macrophages that overexpress a mammalian ID3 polypeptide. In certain embodiments, monocytes or monocyte-derived macrophages can be transduced -60- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 with a vector comprising nucleic acid sequences that encode a mammalian ID3 polypeptide. In some embodiments, the mammalian ID3 polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

[0188] The engineered monocytes or engineered monocyte-derived macrophages of the presently disclosed subject matter can express non-endogenous levels of ID3 for the treatment of cancer. Such engineered monocytes or engineered monocyte-derived macrophages can be administered to a subject (e.g., a human subject) in need thereof for the treatment of cancer.

[0189] In certain embodiments, the presently disclosed engineered monocytes or engineered monocyte-derived macrophages expresses from about 1 to about 5, from about 1 to about 4, from about 2 to about 5, from about 2 to about 4, from about 3 to about 5, from about 3 to about 4, from about 4 to about 5, from about 1 to about 2, from about 2 to about 3, from about 3 to about 4, or from about 4 to about 5 vector copy numbers per cell of a ID3 heterologous nucleic acid.

[0190] Additionally or alternatively, in some embodiments, the engineered monocytes or engineered monocyte-derived macrophages of the present technology further comprise a vector that overexpresses one or more of dectin-1, dectin-2, dectin-3, or mincle. In some embodiments, the vector comprising nucleic acid sequences encoding the mammalian ID3 polypeptide and the vector comprising nucleic acid sequences encoding one or more of dectin-1, dectin-2, dectin-3, or mincle are the same. In other embodiments, the vector comprising nucleic acid sequences encoding the mammalian ID3 polypeptide and the vector comprising nucleic acid sequences encoding one or more of dectin-1, dectin-2, dectin-3, or mincle are distinct.

[0191] Additionally or alternatively, in some embodiments, the engineered monocytes or engineered monocyte-derived macrophages may further comprise an engineered receptor (e.g., a CAR) or other ligand that comprises an extracellular antigen-binding domain, a transmembrane domain and an intracellular domain, where the extracellular antigen-binding domain specifically binds a tumor antigen, including a tumor receptor or ligand. Examples of tumor antigens include, but are not limited to, 5T4, alpha 5β1-integrin, 707-AP, A33, AFP, ART-4, B7H4, BAGE, Bcl-2, β-catenin, BCMA, Bcr-abl, CA125, CA19-9, CAMEL, CAP-1, CASP-8, CD4, CD5, CD19, CD20, CD21 , CD22, CD25, CDC27 / m, CD33, CD37, CD45, CD52, CD56, CD80, CD123, CDK4 / m, CEA, c-Met, CS-1, CT, Cyp-B, cyclin B1, -61- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 DAGE, DAM, EBNA, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ephrinB2, estrogen receptor, ETV6-AML1, FAP, ferritin, folate-binding protein, GAGE, G250, GD-2, GM2, GnT-V, gp75, gp100 (Pmel 17), HAGE, HER-2 / neu, HLA-A*0201-R170I, HPV E6, HPV E7, Ki-67, HSP70-2M, HST-2, hTERT (or hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR / FUT, LRP, MAGE, MART, MART-1 / melan-A, MART-2 / Ski, MC1R, mesothelin, MUC16, MUM-1 -B, myc, MUM-2, MUM-3, NA88-A, NYESO-1, NY-Eso-B, p53, proteinase-3, p190 minor bcr-abl, Pml / RARα, PRAME, progesterone receptor, PSA, PSCA, PSM, PSMA, ras, RAGE, RU1 or RU2, RORI, SART-1 or SART-3, survivin, TEL / AML1, TGFβ, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, tenascin, TSTA tyrosinase, VEGF, or WT1. Other examples of tumor antigens include a2b b3 (Glycoprotein IIb / IIIa), a4, a4b7, a4b7 +aEb7, a5, Activin receptor type-2B, ALK1, Alpha-synuclein, amyloid beta, APP, AXL, Blood Group A, CAIX, CCL-2, CD105 (endoglin), CD115 (CSF1R), CD116a (CSF2Ra), CD123, CD152 (CTLA4), CD184 (CXCR4), CD19, CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD20, CD200, CD22, CD221 (IGF1R), CD248, CD25, CD257 (BAFF), CD26, CD262 (DR5), CD276 (B7H3), CD3, CD30 (TNFRSF8), CD319 (SLAMF7), CD33, CD332 (FGFR2), CD350 (FZD10), CD37, CD371 (CLEC12A), CD38, CD4, CD49b (a2), CD51 (a5), CD52, CD56, CD61 (a4b3), CD70, CD73 (NT5E), CD74, CEA, Claudin-18.2, cMET, CRLR, DLL3, DLL4, DNA / histone (H1) complex, EGFR, EpCAM, EGFR- HER3, EGFRvIII, EphA3, ERGT(GalNAc) Tn Antigen, FLT1, FOLR1, frizzled family receptor (FZD), Lewis Y, Lewis X, GCGR, GD2, GD2 α-acetyl, GD3, GM1, GM1 fucosyl, GM2, GPA33, GPNMB, GUCY2C, HER2, HER3, HGFR (cMET), IgHe, IGLF2, Kallikreins, LINGO1, LOXL2, Ly6 / PLAUR domain-containing protein 3, MADCAM1, MAG, Mesothelin, MT1-MMP (MMP14), MUC1, Mucin 5AC, NaPi2b, NeuGc-GM3, notch, NOTCH2 / NOTCH3 receptors, oxLDL, P-selectin, PCSK9, PDGFRA, PDGFRa, phosphatidylserine, polysialic acid, PSMA, PVRL4, RGMA, CD240D Blood group D antigen, root plate-specific spondin 3, serum amyloid P component, STEAP-1, TACSTD2, TGFb, TWEAKR, TYRP1, VEGFR2, VSIR, CD171 (L1CAM), CD19, CD47, pMHC[NY-ESO1], pMHC[MART1], pMHC[MAGEA1], pMHC[Tyrosinase], pMHC[gp100], pMHC[MUC1], pMHC[tax], pMHC[WT-1], pMHC[EBNA-1], pMHC[LMP2], pMHC[hTERT], GPC3, CD80, CD23, and fibronectin extra domain-B.

[0192] Additionally or alternatively, in some embodiments, the engineered monocytes or engineered monocyte-derived macrophages lack detectable expression or activity of a wild-type SIRPA, CLEC4a, or Siglec-E. An engineered monocyte or macrophage may -62- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 comprise one or more disruptions in endogenous genes encoding a SIRPA, CLEC4a, or Siglec-E polypeptide (e.g., CRISPR knockouts) and / or an inhibitory RNA (e.g., shRNA, siRNA) targeting SIRPA, CLEC4a, or Siglec-E).

[0193] Gene suppression can be performed in a number of ways. For example, gene expression can be suppressed by knock out, altering a promoter of a gene, and / or by inhibiting transcriptional or translational activity. This can be done at an organism level or at a tissue, organ, and / or cellular level. Sometimes a stop codon can be inserted or created (e.g., by nucleotide replacement), in one or more genes, which can result in a nonfunctional transcript or protein (sometimes referred to as knockout). For example, if a stop codon is created within the middle of one or more genes, the resulting transcription and / or protein can be truncated, and can be nonfunctional.

[0194] Alternatively, one or more genes can be suppressed by administering inhibitory nucleic acids, e.g., siRNA, shRNA, antisense or microRNA. For example, an inhibitory nucleic acid (e.g., siRNA, shRNA, antisense or microRNA) can be stably transfected into a cell to knock down expression. Alternatively, an inhibitory nucleic acid (e.g., siRNA, shRNA, antisense or microRNA) can be integrated into the genome of a monocyte or macrophage, thus knocking down a gene within the monocyte or macrophage.

[0195] Also provided herein are monocytes or macrophages comprising one or more transgenes that encode one or more inhibitory nucleic acids that can suppress genetic expression, e.g., can knockdown a gene. RNAs that suppress genetic expression can comprise, but are not limited to, antisense, shRNA, siRNA, RNAi, and microRNA. For example, transgenes encoding siRNA, RNAi, and / or microRNA can be delivered to a macrophage or monocyte to suppress genetic expression. For example, an engineered macrophage or monocyte may comprise a transgene encoding an inhibitory nucleic acid (e.g., siRNA, RNAi, antisense etc.) that specifically targets and inhibits the expression of a nucleic acid encoding SIRPA, CLEC4a, or Siglec-E. An engineered macrophage or monocyte may comprise about 1, 2, 3, 4, 5, or more transgenes encoding one or more inhibitory nucleic acids that suppress the activity and / or expression of SIRPA, CLEC4a, or Siglec-E.

[0196] Transgenes of the present technology can be incorporated into a cell. A transgene may be inserted within a coding genomic region or a noncoding genomic region. A transgene may be inserted into a genome with or without homologous recombination. -63- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0197] One or more of the transgenes disclosed herein can be derived from different species. For example, one or more transgenes can comprise a human gene, a mouse gene, a rat gene, a pig gene, a bovine gene, a dog gene, a cat gene, a monkey gene, a chimpanzee gene, or any combination thereof. For example, a transgene can be from a human, having a human genetic sequence. One or more transgenes can comprise human genes.

[0198] A transgene of the present technology can be inserted into a genome of a macrophage or monocyte in a random or site-specific manner. For example, a transgene can be inserted to a random locus in a genome of a macrophage or monocyte. A transgene can include its own promoter or can be inserted into a position where it is under the control of an endogenous promoter. Alternatively, a transgene can be inserted into a gene, such as an intron of a gene or an exon of a gene, a promoter, or a non-coding region. A transgene can be inserted such that the insertion disrupts a gene, e.g., an endogenous gene. A transgene insertion can be guided by recombination arms that can flank a transgene. Sometimes, more than one copy of a transgene can be inserted into a random locus in a genome. For example, multiple copies can be inserted into a random locus in a genome. This can lead to increased overall expression than if a transgene was randomly inserted once. Alternatively, a copy of a transgene can be inserted into a gene, and another copy of a transgene can be inserted into a different gene. A transgene can be targeted so that it could be inserted to a specific locus in a genome of a macrophage or monocyte.

[0199] Expression of any transgene disclosed herein can be controlled by one or more promoters. A promoter can be an ubiquitous promoter, a constitutive promoter, a tissue- specific promoter or an inducible promoter. Expression of a transgene that is inserted adjacent to or near a promoter can be regulated. For example, a transgene can be inserted near or next to a ubiquitous promoter. Examples of ubiquitous promoters include, but are not limited to, a CAGGS promoter, an hCMV promoter, a PGK promoter, an SV40 promoter, or a ROSA26 promoter. A promoter may be endogenous or exogenous. For example, one or more transgenes can be inserted adjacent or near to an endogenous or exogenous ROSA26 promoter. Tissue specific promoter or cell-specific promoters can be used to control the location of expression. Inducible promoters can be used as well. These inducible promoters can be turned on and off when desired, by adding or removing an inducing agent. Examples of inducible promoters include, but are not limited to, Lac, tac, trc, trp, araBAD, phoA, recA, proU, cst-1, tetA, cadA, nar, PL, cspA, T7, VHB, Mx, and / or Trex. -64- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0200] In another aspect, a macrophage or monocyte can be engineered to knock out endogenous genes. For example, knocking out one or more genes may comprise deleting one or more genes from a genome of a macrophage or monocyte (e.g., SIRPA, CLEC4a, or Siglec-E). Knocking out can also comprise removing all or a part of a gene sequence (e.g., deletion) from a macrophage or monocyte (e.g., SIRPA, CLEC4a, or Siglec-E). It is also contemplated that knocking out can comprise replacing all or a part of a gene in a genome of a macrophage or monocyte with one or more nucleotides. Knocking out one or more genes can also comprise inserting a sequence in one or more genes (e.g., insertion), thereby disrupting expression of the one or more genes. For example, inserting a sequence can generate a stop codon in the middle of one or more genes (e.g., nonsense mutation). Inserting a sequence can also shift the open reading frame of one or more genes (e.g., frameshift mutation).

[0201] By way of example only, one or more endogenous genes may be knocked out using an endonuclease selected from the group consisting of a CRISPR system (e.g., a Cas endonuclease), TALEN, Zinc Finger, transposon-based, ZEN, meganuclease, Mega-TAL, and any combination thereof.

[0202] CRISPR System. Methods described herein can take advantage of a CRISPR system. There are at least five types of CRISPR systems which all incorporate RNAs and Cas proteins. Types I, III, and IV assemble a multi-Cas protein complex that is capable of cleaving nucleic acids that are complementary to the crRNA. Types I and III both require pre-crRNA processing prior to assembling the processed crRNA into the multi-Cas protein complex. Types II and V CRISPR systems comprise a single Cas protein complexed with at least one guiding RNA.

[0203] The general mechanism and recent advances of CRISPR system is discussed in Cong, L. et al., Science, 339 (6121): 819-823 (2013); Fu, Y. et al., Nature Biotechnology, 31, 822-826 (2013); Chu, V T et al., Nature Biotechnology 33, 543-548 (2015); Shmakov, S. et al., Molecular Cell, 60, 1-13 (2015); Makarova, K S et al., Nature Reviews Microbiology, 13, 1-15 (2015). Site-specific cleavage of a target DNA occurs at locations determined by both 1) base-pairing complementarity between the guide RNA and the target DNA (also called a protospacer) and 2) a short motif in the target DNA referred to as the protospacer adjacent motif (PAM). For example, an engineered cell can be generated using a CRISPR system, e.g., a type II CRISPR system. A Cas enzyme used in the methods disclosed herein can be Cas9, which catalyzes DNA cleavage. Enzymatic action by Cas9 -65- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 derived from Streptococcus pyogenes or any closely related Cas9 can generate double stranded breaks at target site sequences which hybridize to about 20 nucleotides of a guide sequence and that have a protospacer-adjacent motif (PAM) following the about 20 nucleotides of the target sequence.

[0204] a. Cas Protein. A vector can be operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein (CRISPR-associated protein). Non- limiting examples of Cas proteins can include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1, c2c1, c2c3, Cas9HiFi, homologues thereof, or modified versions thereof. In some embodiments, the Cas protein is Cas9. A Cas9 endonuclease may create a double strand break in at least one gene (e.g., SIRPA, CLEC4a, or Siglec-E). In some cases, a double strand break can be repaired using homology directed repair (HDR), non- homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or any combination or derivative thereof.

[0205] An unmodified CRISPR enzyme can have DNA cleavage activity, such as Cas9. A CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within a target sequence and / or within a complement of a target sequence. For example, a CRISPR enzyme can direct cleavage of one or both strands within or within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. A vector that encodes a CRISPR enzyme that is mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence can be used. A Cas protein can be a high fidelity cas protein such as Cas9HiFi.

[0206] A vector that encodes a CRISPR enzyme comprising one or more nuclear localization sequences (NLSs), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs can be used. For example, a CRISPR enzyme can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, or at or near the carboxyl-terminus, or any combination of these (e.g., one or more NLS at the amino-terminus and one or more NLS at the carboxyl terminus). When more than one NLS is present, each can be selected independently of others, such that a single NLS can be present in more than one copy and / or in combination with one or more other NLSs present in one or more copies. -66- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0207] Cas9 can refer to a polypeptide with at least about 50%, 60%, 70%, 80%, 90%, 100% sequence identity and / or sequence similarity to a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from S. pyogenes). Cas9 can refer to a polypeptide with at most about 50%, 60%, 70%, 80%, 90%, 100% sequence identity and / or sequence similarity to a wild-type exemplary Cas9 polypeptide (e.g., from S. pyogenes). Cas9 can refer to the wild- type or a modified form of the Cas9 protein that can comprise an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof.

[0208] A polynucleotide encoding an endonuclease (e.g., a Cas protein such as Cas9) can be codon optimized for expression in particular cells, such as eukaryotic cells. This type of optimization can entail the mutation of foreign-derived (e.g., recombinant) DNA to mimic the codon preferences of the intended host organism or cell while encoding the same protein.

[0209] CRISPR enzymes used in the methods can comprise NLSs. The NLS can be located anywhere within the polypeptide chain, e.g., near the N- or C-terminus. For example, the NLS can be within or within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 amino acids along a polypeptide chain from the N- or C-terminus. Sometimes the NLS can be within or within about 50 amino acids or more, e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 amino acids from the N- or C-terminus.

[0210] An endonuclease can comprise an amino acid sequence having at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, amino acid sequence identity to the nuclease domain of a wild-type exemplary site-directed polypeptide (e.g., Cas9 from S. pyogenes). In some cases, a different non-Cas9 endonuclease may be used to target certain genomic targets. In some cases, synthetic SpCas9-derived variants with non-NGG PAM sequences may be used.

[0211] Additionally, other Cas9 orthologues from various species have been identified and these “non-SpCas9s” bind a variety of PAM sequences that could also be useful for the present technology (e.g., Staphylococcus aureus Cas9 (SaCas9)).

[0212] Alternatives to S. pyogenes Cas9 may include RNA-guided endonucleases from the Cpf1 family that display cleavage activity in mammalian cells. Unlike Cas9 nucleases, the result of Cpf1-mediated DNA cleavage is a double-strand break with a short 3′ overhang. Cpf1's staggered cleavage pattern may open up the possibility of directional gene -67- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 transfer, analogous to traditional restriction enzyme cloning, which may increase the efficiency of gene editing. Like the Cas9 variants and orthologues described above, Cpf1 may also expand the number of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack the NGG PAM sites favored by SpCas9.

[0213] Any functional concentration of Cas protein can be introduced to a cell. For example, 15 micrograms of Cas mRNA can be introduced to a cell. In other cases, a Cas mRNA can be introduced from 0.5 micrograms to 100 micrograms. A Cas mRNA can be introduced from 0.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 micrograms.

[0214] b. Guide RNA. A method disclosed herein also can comprise introducing into a cell (e.g., a macrophage or monocyte) at least one guide RNA or nucleic acid, e.g., DNA encoding at least one guide RNA. A guide RNA can interact with a RNA-guided endonuclease to direct the endonuclease to a specific target site, at which site the 5′ end of the guide RNA base pairs with a specific protospacer sequence in a chromosomal sequence.

[0215] A guide RNA may comprise a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA). A guide RNA can sometimes comprise a single-guide RNA (sgRNA) formed by fusion of a portion (e.g., a functional portion) of crRNA and tracrRNA. A guide RNA can also be a dual RNA comprising a crRNA and a tracrRNA. A guide RNA can comprise a crRNA and lack a tracrRNA. In some embodiments, a crRNA can hybridize with a target DNA or protospacer sequence.

[0216] A guide RNA can be an expression product. For example, a DNA that encodes a guide RNA can be a vector comprising a sequence coding for the guide RNA. A guide RNA can be transferred into a cell or organism by transfecting the cell or organism with an isolated guide RNA or plasmid DNA comprising a sequence coding for the guide RNA and a promoter. A guide RNA can also be transferred into a cell or organism in other way, such as using virus-mediated gene delivery. In other embodiments, a guide RNA can be isolated. For example, a guide RNA can be transfected in the form of an isolated RNA into a cell or organism. A guide RNA can be prepared by in vitro transcription using any in vitro transcription system. A guide RNA can be transferred to a cell in the form of isolated RNA rather than in the form of plasmid comprising encoding sequence for a guide RNA.

[0217] A guide RNA can comprise a DNA-targeting segment and a protein binding segment. A DNA-targeting segment (or DNA-targeting sequence, or spacer sequence) -68- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 comprises a nucleotide sequence that can be complementary to a specific sequence within a target DNA (e.g., a protospacer). A protein-binding segment (or protein-binding sequence) can interact with a site-directed modifying polypeptide, e.g. an RNA-guided endonuclease such as a Cas protein. By “segment” it is meant a segment / section / region of a molecule, e.g., a contiguous stretch of nucleotides in an RNA. A segment can also mean a region / section of a complex such that a segment may comprise regions of more than one molecule. For example, in some cases a protein-binding segment of a DNA-targeting RNA is one RNA molecule and the protein-binding segment therefore comprises a region of that RNA molecule. In other cases, the protein-binding segment of a DNA-targeting RNA comprises two separate molecules that are hybridized along a region of complementarity.

[0218] A guide RNA can comprise two separate RNA molecules or a single RNA molecule. An exemplary single molecule guide RNA comprises both a DNA-targeting segment and a protein-binding segment. An exemplary two-molecule DNA-targeting RNA can comprise a crRNA-like (“CRISPR RNA” or “targeter-RNA” or “crRNA” or “crRNA repeat”) molecule and a corresponding tracrRNA-like (“trans-acting CRISPR RNA” or “activator-RNA” or “tracrRNA”) molecule. A first RNA molecule can be a crRNA-like molecule (targeter-RNA), that can comprise a DNA-targeting segment (e.g., spacer) and a stretch of nucleotides that can form one half of a double-stranded RNA (dsRNA) duplex comprising the protein-binding segment of a guide RNA.

[0219] A second RNA molecule can be a corresponding tracrRNA-like molecule (activator-RNA) that can comprise a stretch of nucleotides that can form the other half of a dsRNA duplex of a protein-binding segment of a guide RNA. In other words, a stretch of nucleotides of a crRNA-like molecule can be complementary to and can hybridize with a stretch of nucleotides of a tracrRNA-like molecule to form a dsRNA duplex of a protein- binding domain of a guide RNA. As such, each crRNA-like molecule can be said to have a corresponding tracrRNA-like molecule. A crRNA-like molecule additionally can provide a single stranded DNA-targeting segment, or spacer sequence. Thus, a crRNA-like and a tracrRNA-like molecule (as a corresponding pair) can hybridize to form a guide RNA. A subject two-molecule guide RNA can comprise any corresponding crRNA and tracrRNA pair.

[0220] A DNA-targeting segment or spacer sequence of a guide RNA can be complementary to sequence at a target site in a chromosomal sequence, e.g., protospacer sequence) such that the DNA-targeting segment of the guide RNA can base pair with the -69- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 target site or protospacer. In some cases, a DNA-targeting segment of a guide RNA can comprise from about 10 nucleotides to from about 25 nucleotides or more. For example, a region of base pairing between a first region of a guide RNA and a target site in a chromosomal sequence can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25 nucleotides in length. Sometimes, a first region of a guide RNA can be about 19, 20, or 21 nucleotides in length.

[0221] A guide RNA can target a nucleic acid sequence of about 20 nucleotides. A target nucleic acid can be less than about 20 nucleotides. A target nucleic acid can be at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. A target nucleic acid can be at most about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. A target nucleic acid sequence can be about 20 bases immediately 5′ of the first nucleotide of the PAM.

[0222] A guide nucleic acid, for example, a guide RNA, can refer to a nucleic acid that can hybridize to another nucleic acid, for example, the target nucleic acid or protospacer in a genome of a cell. A guide nucleic acid can be RNA. A guide nucleic acid can be DNA. The guide nucleic acid can be programmed or designed to specifically bind to a sequence at a nucleic acid site. A guide nucleic acid can comprise a polynucleotide chain and can be called a single guide nucleic acid. A guide nucleic acid can comprise two polynucleotide chains and can be called a double guide nucleic acid.

[0223] A guide nucleic acid can comprise one or more chemical or physical modifications. A guide nucleic acid can comprise a nucleic acid affinity tag. A guide nucleic acid may comprise one or more synthetic nucleotides, synthetic nucleotide analogs, nucleotide derivatives, and / or modified nucleotides. A guide nucleic acid can comprise a nucleotide sequence (e.g., a spacer), for example, at or near the 5′ end or 3′ end, that can hybridize to a sequence in a target nucleic acid (e.g., a protospacer). A spacer of a guide nucleic acid can interact with a target nucleic acid in a sequence-specific manner via hybridization (i.e., base pairing). A spacer sequence can hybridize to a target nucleic acid that is located 5′ or 3′ to a protospacer adjacent motif (PAM). The length of a spacer sequence can be at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. The length of a spacer sequence can be at most about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides.

[0224] A guide RNA may also comprise a dsRNA duplex region that forms a secondary -70- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 structure. For example, a secondary structure formed by a guide RNA can comprise a stem (or hairpin) and a loop. A length of a loop and a stem can vary. For example, a loop can range from about 3 to about 10 nucleotides in length, and a stem can range from about 6 to about 20 base pairs in length. A stem can comprise one or more bulges of 1 to about 10 nucleotides. The overall length of a second region can range from about 16 to about 60 nucleotides in length. For example, a loop can be about 4 nucleotides in length and a stem can be about 12 base pairs. A dsRNA duplex region can comprise a protein-binding segment that can form a complex with an RNA-binding protein, such as a RNA-guided endonuclease, e.g., Cas protein.

[0225] A guide RNA can also comprise a tail region at the 5′ or 3′ end that can be single-stranded. For example, a tail region is sometimes not complementarity to any chromosomal sequence in a cell of interest and is sometimes not complementarity to the rest of a guide RNA. Further, the length of a tail region can vary. A tail region can be more than about 4 nucleotides in length. For example, the length of a tail region can range from about 5 to about 60 nucleotides in length.

[0226] A guide RNA can be introduced into a cell or embryo as an RNA molecule. For example, a RNA molecule can be transcribed in vitro and / or can be chemically synthesized. A guide RNA can then be introduced into a cell or embryo as an RNA molecule. A guide RNA can also be introduced into a cell or embryo in the form of a non-RNA nucleic acid molecule, e.g., DNA molecule. For example, a DNA encoding a guide RNA can be operably linked to promoter control sequence for expression of the guide RNA in a cell of interest. A DNA molecule encoding a guide RNA may be linear or circular.

[0227] When both a RNA-guided endonuclease and a guide RNA are introduced into a cell as DNA molecules, each can be part of a separate molecule (e.g., one vector containing the RNA-guided endonuclease coding sequence and a second vector containing the guide RNA coding sequence) or both can be part of a same molecule (e.g., one vector containing coding (and regulatory) sequence for both a RNA-guided endonuclease and a guide RNA).

[0228] A Cas protein, such as a Cas9 protein or any derivative thereof, can be pre- complexed with a guide RNA to form a ribonucleoprotein (RNP) complex. The RNP complex can facilitate homology directed repair (HDR). The RNP complex can be introduced into primary macrophages or monocytes. Introduction of the RNP complex can -71- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 be timed. The cell can be synchronized with other cells at G1, S, and / or M phases of the cell cycle. The RNP complex can be delivered at a cell phase such that HDR is enhanced.

[0229] A guide RNA can also be modified. The modifications can comprise chemical alterations, synthetic modifications, nucleotide additions, and / or nucleotide subtractions. The modifications can also enhance CRISPR genome engineering. A modification can alter chirality of a gRNA. In some cases, chirality may be uniform or stereopure after a modification. A guide RNA can be synthesized. The synthesized guide RNA can enhance CRISPR genome engineering. A guide RNA can also be truncated. Truncation can be used to reduce undesired off-target mutagenesis. The truncation can comprise any number of nucleotide deletions. For example, the truncation can comprise 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more nucleotides. A guide RNA can comprise a region of target complementarity of any length. For example, a region of target complementarity can be less than 20 nucleotides in length. A region of target complementarity can be more than 20 nucleotides in length.

[0230] In some cases, a modification is on a 5′ end, a 3′ end, from a 5′ end to a 3′ end, a single base modification, a 2′-ribose modification, or any combination thereof. A modification can be selected from a group consisting of base substitutions, insertions, deletions, chemical modifications, physical modifications, stabilization, purification, and any combination thereof. In some embodiments, a modification is a chemical modification. A modification can be selected from 5′ adenylate, 5′ guanosine-triphosphate cap, 5′N7- Methylguanosine-triphosphate cap, 5′ triphosphate cap, 3′ phosphate, 3′ thiophosphate, 5′ phosphate, 5′ thiophosphate, Cis-Syn thymidine dimer, trimers, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9,3′-3′ modifications, 5′-5′ modifications, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-Biotin, dual biotin, PC biotin, psoralen C2, psoralen C6, TINA, 3′DABCYL, black hole quencher 1, black hole quencer 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linkers, 2′ deoxyribonucleoside analog purine, 2′ deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2′-O-methyl ribonucleoside analog, sugar modified analogs, wobble / universal bases, fluorescent dye label, 2′ fluoro RNA, 2′ O-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5′-triphosphate, 5-methylcytidine-5′- -72- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 triphosphate, 2-O-methyl 3phosphorothioate or any combinations thereof. A modification may be a pseudouride modification. In some cases, a modification is a 2-O-methyl 3 phosphorothioate addition. A 2-O-methyl 3 phosphorothioate addition can be performed from 1 base to 150 bases. A 2-O-methyl 3 phosphorothioate addition can be performed from 1 base to 4 bases. A 2-O-methyl 3 phosphorothioate addition can be performed on 2 bases. A 2-O-methyl 3 phosphorothioate addition can be performed on 4 bases. A modification can also be a truncation. A truncation can be a 5 base truncation. In some cases, a 5 base truncation can prevent a Cas protein from performing a cut.

[0231] In some cases, a dual nickase approach may be used to introduce a double stranded break. Cas proteins can be mutated at known amino acids within either nuclease domains, thereby deleting activity of one nuclease domain and generating a nickase Cas protein capable of generating a single strand break. A nickase along with two distinct guide RNAs targeting opposite strands may be utilized to generate a DSB within a target site (often referred to as a “double nick” or “dual nickase” CRISPR system). This approach may dramatically increase target specificity, since it is unlikely that two off-target nicks will be generated within close enough proximity to cause a DSB.

[0232] A gRNA can be introduced at any functional concentration. For example, a gRNA can be introduced to a cell at 10 micrograms. In other cases, a gRNA can be introduced from 0.5 micrograms to 100 micrograms. A gRNA can be introduced from 0.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 micrograms.

[0233] A DNA sequence encoding a guide RNA or transgene disclosed herein can also be part of a vector. Some examples of vectors include plasmid vectors, phagemids, cosmids, artificial / mini-chromosomes, transposons, and viral vectors. Further, a vector can comprise additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences, etc.), selectable marker sequences (e.g., antibiotic resistance genes), origins of replication, and the like.

[0234] Additionally or alternatively, in some embodiments, the ID3-overexpressing engineered macrophage or monocyte of the present technology lacks detectable expression or activity of a SIRPA, CLEC4a, or Siglec-E polypeptide. In some embodiments, the engineered macrophage or monocyte expresses an inhibitory nucleic acid that specifically targets and inhibits the expression of a SIRPA, CLEC4a, or Siglec-E nucleic acid sequence. -73- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 The inhibitory nucleic acid may be an antisense oligonucleotide, a siRNA, a sgRNA or a shRNA. Additionally or alternatively, in some embodiments, the engineered macrophages or monocytes further comprise a transgene that encodes the inhibitory nucleic acid. The transgene may be operably linked to an ubiquitous promoter, a constitutive promoter, a macrophage-specific promoter, a monocyte-specific promoter, or an inducible promoter. In certain embodiments, the engineered macrophage or monocyte comprise a deletion, insertion, inversion, or frameshift mutation in a SIRPA, CLEC4a, or Siglec-E gene. In certain embodiments, the deletion, insertion, inversion, or frameshift mutation in a SIRPA, CLEC4a, or Siglec-E gene is generated using at least one sgRNA and at least one endonuclease (e.g., Cas9 endonuclease). Additionally or alternatively, in some embodiments, the engineered macrophage or monocyte is derived from an autologous donor or an allogeneic donor. In some embodiments, macrophages or monocytes can be transduced with a vector comprising inhibitory nucleic acids that target SIRPA, CLEC4a, or Siglec-E. The inhibitory nucleic acid may be an antisense oligonucleotide, a siRNA, a sgRNA or a shRNA.

[0235] In another aspect, the present disclosure provides methods for engineered macrophages or monocytes comprising: isolating macrophages or monocytes from a donor subject; transducing the macrophages or monocytes with (a) a vector comprising a heterologous ID3 nucleic acid and (b) an inhibitory nucleic acid that specifically targets and inhibits the expression of a SIRPA, CLEC4a, or Siglec-E nucleic acid. The inhibitory nucleic acid is an antisense oligonucleotide, a siRNA, a sgRNA or a shRNA.

[0236] Disclosed herein is a method for making engineered macrophages or monocytes comprising: introducing at least one single guide RNA (sgRNA) and at least one endonuclease into a macrophage or monocyte under conditions to produce a deletion, an insertion, an inversion, or a frameshift mutation in a SIRPA, CLEC4a, or Siglec-E gene, wherein the macrophage or monocyte comprises an endogenous genome and wherein the sgRNA comprises at least one sequence that is complementary to a SIRPA, CLEC4a, or Siglec-E nucleic acid sequence in the endogenous genome of the macrophage or monocyte. The at least one endonuclease may be Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1, c2c1, c2c3, Cas9HiFi, homologues thereof, or modified versions thereof. -74- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 In some embodiments, the engineered macrophages or monocytes lack detectable expression or activity of wild-type SIRPA, CLEC4a, or Siglec-E. Formulations

[0237] Engineered monocytes or engineered monocyte-derived macrophages that over- express the ID3 gene, alone or in combination with (a) overexpression of dectin-1, dectin-2, dectin-3, or mincle, (b) expression of a receptor disclosed herein (e.g., CAR) and / or (c) an inhibitory nucleic acid that specifically targets SIRPA, CLEC4a, or Siglec-E, and compositions comprising the same can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.

[0238] Sterile injectable solutions can be prepared by incorporating the compositions of the presently disclosed subject matter, e.g., a composition comprising engineered monocytes or engineered monocyte-derived macrophages, in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON' S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation. -75- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0239] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the presently disclosed subject matter, however, any vehicle, diluent, or additive used would have to be compatible with the engineered monocytes or engineered monocyte-derived macrophages of the presently disclosed subject matter.

[0240] The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions of the presently disclosed subject matter may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is suitable particularly for buffers containing sodium ions.

[0241] Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose can be used because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The concentration of the thickener can depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form).

[0242] Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert and will not affect the viability or efficacy of the engineered monocytes or engineered monocyte-derived macrophages as described in the presently disclosed subject matter. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein. -76- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0243] One consideration concerning the therapeutic use of the engineered monocytes or engineered monocyte-derived macrophages of the presently disclosed subject matter is the quantity of cells necessary to achieve an optimal effect. The quantity of cells to be administered will vary for the subject being treated. In certain embodiments, from about 102to about 1012, from about 103to about 1011, from about 104to about 1010, from about 105to about 109, or from about 106to about 108engineered monocytes or engineered monocyte-derived macrophages of the presently disclosed subject matter are administered to a subject. More effective cells may be administered in even smaller numbers. In some embodiments, at least about 1 × 108, about 2 × 108, about 3 × 108, about 4 × 108, about 5 × 108, about 1 × 109, about 5 × 109, about 1 × 1010, about 5 × 1010, about 1 × 1011, about 5 × 1011, about 1 × 1012or more engineered monocytes or engineered monocyte-derived macrophages of the presently disclosed subject matter are administered to a human subject. The precise determination of what would be considered an effective dose may be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art. Generally, engineered monocytes or engineered monocyte-derived macrophages are administered at doses that are nontoxic or tolerable to the patient.

[0244] The skilled artisan can readily determine the amount of cells and optional additives, vehicles, and / or carrier in compositions to be administered in methods of the presently disclosed subject matter. Typically, any additives (in addition to the active cell(s) and / or agent(s)) are present in an amount of from about 0.001% to about 50% by weight) solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as from about 0.0001 wt % to about 5 wt %, from about 0.0001 wt% to about 1 wt %, from about 0.0001 wt% to about 0.05 wt%, from about 0.001 wt% to about 20 wt %, from about 0.01 wt% to about 10 wt %, or from about 0.05 wt% to about 5 wt %. For any composition to be administered to an animal or human, and for any particular method of administration, toxicity should be determined, such as by determining the lethal dose (LD) and LD50 in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure -77- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 and the documents cited herein. And, the time for sequential administrations can be ascertained without undue experimentation. Administration

[0245] Engineered monocytes or engineered monocyte-derived macrophages that over- express the ID3 gene, alone or in combination with (a) overexpression of dectin-1, dectin-2, dectin-3, or mincle, (b) expression of a receptor disclosed herein (e.g., CAR) and / or (c) an inhibitory nucleic acid that specifically targets SIRPA, CLEC4a, or Siglec-E, can be provided systemically or directly to a subject for treating cancer.

[0246] In certain embodiments, engineered monocytes or engineered monocyte-derived macrophages are directly injected into an organ of interest. Additionally or alternatively, the engineered monocytes or engineered monocyte-derived macrophages are provided indirectly to the organ of interest, for example, by administration into the circulatory system (e.g., the tumor vasculature) or into the tissue of interest (e.g., solid tumor). Expansion and differentiation agents can be provided prior to, during or after administration of cells and compositions to increase production of the engineered monocytes or engineered monocyte- derived macrophages either in vitro or in vivo.

[0247] Engineered monocytes or engineered monocyte-derived macrophages of the presently disclosed subject matter can be administered in any physiologically acceptable vehicle, systemically or regionally, normally intravascularly, intraperitoneally, intrathecally, or intrapleurally, although they may also be introduced into bone or other convenient site where the cells may find an appropriate site for regeneration and differentiation (e.g., thymus). In certain embodiments, at least 1 × 105cells can be administered, eventually reaching 1 × 1010or more. In certain embodiments, at least 1 × 106cells can be administered. A cell population comprising engineered monocytes or engineered monocyte-derived macrophages can comprise a purified population of cells. Those skilled in the art can readily determine the percentage of engineered monocytes or engineered monocyte-derived macrophages in a cell population using various well-known methods, such as fluorescence activated cell sorting (FACS). The ranges of purity in cell populations comprising engineered monocytes or engineered monocyte-derived macrophages can be from about 50% to about 55%, from about 55% to about 60%, about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%; from about 85% to about 90%, from about 90% to -78- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 about 95%, or from about 95 to about 100%. Dosages can be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage). The engineered monocytes or engineered monocyte-derived macrophages can be introduced by injection, catheter, or the like. If desired, factors can also be included, including, but not limited to, interleukins, e.g., IL-2, IL-3, IL 6, IL-11, IL-7, IL-12, IL-15, IL-21, as well as the other interleukins, the colony stimulating factors, such as G-, M- and GM-CSF, interferons, e.g., γ- interferon.

[0248] In certain embodiments, compositions of the presently disclosed subject matter comprise pharmaceutical compositions comprising engineered monocytes or engineered monocyte-derived macrophages that over-express the ID3 gene, alone or in combination with (a) overexpression of dectin-1, dectin-2, dectin-3, or mincle, (b) expression of a receptor disclosed herein (e.g., CAR) and / or (c) an inhibitory nucleic acid that specifically targets SIRPA, CLEC4a, or Siglec-E, with a pharmaceutically acceptable carrier. Administration can be autologous or non-autologous.

[0249] For example, engineered monocytes or engineered monocyte-derived macrophages that over-express the ID3 gene, alone or in combination with (a) overexpression of dectin-1, dectin-2, dectin-3, or mincle, (b) expression of a receptor disclosed herein (e.g., CAR) and / or (c) an inhibitory nucleic acid that specifically targets SIRPA, CLEC4a, or Siglec-E, and compositions comprising the same can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived macrophages or monocytes of the presently disclosed subject matter or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a pharmaceutical composition of the presently disclosed subject matter (e.g., a pharmaceutical composition comprising engineered monocytes or engineered monocyte-derived macrophages that over- express the ID3 gene, alone or in combination with (a) overexpression of dectin-1, dectin-2, dectin-3, or mincle, (b) expression of a receptor disclosed herein (e.g., CAR) and / or (c) an inhibitory nucleic acid that specifically targets SIRPA, CLEC4a, or Siglec-E)), it can be formulated in a unit dosage injectable form (solution, suspension, emulsion). -79- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 Therapeutic Uses of the Engineered Monocytes or Engineered Monocyte-Derived Macrophages of the Present Technology

[0250] For treatment, the amount of the engineered monocytes or engineered monocyte- derived macrophages provided herein administered is an amount effective in producing the desired effect, for example, treatment of cancer, or one or more symptoms thereof. An effective amount can be provided in one or a series of administrations of the engineered monocytes or engineered monocyte-derived macrophages provided herein. An effective amount can be provided in a bolus or by continuous perfusion. For adoptive immunotherapy using CAR-expressing monocytes or macrophages, cell doses in the range of about 106to about 1010are typically infused. Lower doses of the engineered monocytes or engineered monocyte-derived macrophages may be administered, e.g., about 104to about 108.

[0251] The engineered monocytes or engineered monocyte-derived macrophages of the presently disclosed subject matter can be administered by any methods known in the art, including, but not limited to, pleural administration, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intrathecal administration, intrapleural administration, intraperitoneal administration, and direct administration to the thymus. In certain embodiments, the engineered monocytes or engineered monocyte-derived macrophages and the compositions comprising thereof are intravenously administered to the subject in need. Methods for administering cells for adoptive cell therapies, including, for example, donor lymphocyte infusion and engineered immune cell therapies, and regimens for administration are known in the art and can be employed for administration of the engineered monocytes or engineered monocyte-derived macrophages provided herein.

[0252] In one aspect, the present disclosure provides a method for treating cancer in a subject in need thereof comprising administering to the subject an effective amount of any and all embodiments of the ID3-overexpressing engineered monocytes or engineered monocyte-derived macrophages of the present technology. In some embodiments, the ID3- overexpressing engineered monocytes or engineered monocyte-derived macrophages further comprise a chimeric antigen receptor (CAR) that specifically binds to a tumor antigen. Additionally or alternatively, the ID3-overexpressing engineered monocytes or engineered monocyte-derived macrophages overexpress one or more of dectin-1, dectin-2, dectin-3, or -80- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 mincle, and / or (c) an inhibitory nucleic acid that specifically targets one or more of SIRPA, CLEC4a, or Siglec-E.

[0253] In another aspect, the present disclosure provides a method for treating cancer or inhibiting tumor growth in a subject in need thereof comprising administering to the subject an effective amount of nanoparticles comprising ID3 nucleic acid molecules (e.g., mRNA) or ID3 polypeptides, wherein the nanoparticles are configured to target macrophages or monocytes in the subject. In some embodiments, the nanoparticles target at least one of BTK, Siglec-1, TLR, TLR7, mannose receptor, TLR3, VEGF, SHP2, PIGF, CSF-1R or CCR2.

[0254] In any and all embodiments of the methods disclosed herein, the cancer is selected from the group consisting of adrenal cancers, bladder cancers, blood cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's disease, intestinal cancers, kidney cancers, larynx cancers, acute and chronic leukemias, liver cancers, lymph node cancers, lymphomas, lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, non-Hodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, and metastases thereof. In some embodiments, the cancer is a relapsed or refractory cancer. In some embodiments, the cancer is resistant to one or more cancer therapies, e.g., one or more chemotherapeutic drugs. Combination Therapy

[0255] In some embodiments, the compositions of the present technology may be employed in conjunction with other therapeutic agents useful in the treatment of cancer. For example, the ID3 over-expressing engineered monocytes or engineered monocyte- derived macrophages of the present technology may be separately, sequentially or simultaneously administered with at least one additional anti-cancer therapy.

[0256] In some embodiments, the additional anti-cancer therapy is selected from among a chemotherapy, adoptive cell therapy, a radiation therapy, an immunotherapy, a monoclonal antibody, an anti-cancer nucleic acid, an anti-cancer protein, an anti-cancer -81- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 virus or microorganism, a cytokine, or any combination thereof. Radiation therapy includes, but is not limited to, exposure to radiation, e.g., ionizing radiation, UV radiation, as known in the art. Exemplary dosages include, but are not limited to, a dose of ionizing radiation at a range from at least about 2 Gy to not more than about 10 Gy or a dose of ultraviolet radiation at a range from at least about 5 J / m2to not more than about 50 J / m2, usually about 10 J / m2. In some embodiments, the methods further comprise sequentially, separately, or simultaneously administering an immunotherapy to the subject. In some embodiments, the immunotherapy regulates immune checkpoints. In further embodiments, the immunotherapy comprises, or consists essentially of, or yet further consists of an immune checkpoint inhibitor, such as an Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4) inhibitor, or a Programmed Cell Death 1 (PD-1) inhibitor, or a Programmed Death Ligand 1 (PD-L1) inhibitor. In yet further embodiments, the immune checkpoint inhibitor comprises, or consists essentially of, or yet further consists of an antibody or an equivalent thereof recognizing and binding to an immune checkpoint protein, such as an antibody or an equivalent thereof recognizing and binding to CTLA4 (for example, Yervoy (ipilimumab), CP-675,206 (tremelimumab), AK104 (cadonilimab), or AGEN1884 (zalifrelimab)), or an antibody or an equivalent thereof recognizing and binding to PD-1 (for example, Keytruda (pembrolizumab), Opdivo (nivolumab), Libtayo (cemiplimab), Tyvyt (sintilimab), BGB-A317 (tislelizumab), JS001 (toripalimab), SHR1210 (camrelizumab), GB226 (geptanolimab), JS001 (toripalimab), AB122 (zimberelimab), AK105 (penpulimab), HLX10 (serplulimab), BCD-100 (prolgolimab), AGEN2034 (balstilimab), MGA012 (retifanlimab), AK104 (cadonilimab), HX008 (pucotenlimab), PF-06801591 (sasanlimab), JNJ-63723283 (cetrelimab), MGD013 (tebotelimab), CT-011 (pidilizumab), or Jemperli (dostarlimab)), or an antibody or an equivalent thereof recognizing and binding to PD-L1 (for example, Tecentriq (atezolizumab), Imfinzi (durvalumab), Bavencio (avelumab), CS1001 (sugemalimab), or KN035 (envafolimab)). In some embodiments, the methods further comprise sequentially, separately, or simultaneously administering a cytokine to the subject. In some embodiments, the cytokine is administered prior to, during, or subsequent to administration of the one or more engineered monocytes or engineered monocyte-derived macrophages. In some embodiments, the cytokine is selected from the group consisting of interferon α, interferon β, interferon γ, complement C5a, IL-2, TNFα, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, -82- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2. The methods may further comprise sequentially, separately, or simultaneously administering to the subject at least one chemotherapeutic agent, optionally selected from the group consisting of nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, gemcitabine, triazenes, folic acid analogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs, antibiotics, enzyme inhibitors, epipodophyllotoxins, platinum coordination complexes, vinca alkaloids, substituted ureas, methyl hydrazine derivatives, adrenocortical suppressants, hormone antagonists, endostatin, taxols, camptothecins, SN-38, doxorubicin, doxorubicin analogs, antimetabolites, alkylating agents, antimitotics, anti-angiogenic agents, tyrosine kinase inhibitors, mTOR inhibitors, heat shock protein (HSP90) inhibitors, proteosome inhibitors, HDAC inhibitors, pro- apoptotic agents, methotrexate and CPT-11.

[0257] Additionally or alternatively, in some embodiments, the adoptive cell therapy comprises administration of lymphoid effector cells, such as T cells, B cells, or NK cells. In some embodiments, the lymphoid effector cells express a native or non-native receptor (e.g., T cell receptor, B cell receptor, NK receptor) that binds to a tumor antigen. In certain embodiments, the lymphoid effector cells, such as T cells, B cells, or NK cells, express a CAR that binds to a tumor antigen. Additionally or alternatively, in some embodiments, the lymphoid effector cells, such as T cells, B cells, or NK cells, are administered intra- arterially. Kits of the Present Technology

[0258] The presently disclosed subject matter provides kits for the treatment of cancer. In certain embodiments, the kit comprises a therapeutic or prophylactic composition containing an effective amount of an engineered monocyte or engineered monocyte-derived macrophage comprising a vector that overexpresses ID3. Also provided herein are kits for use in the manufacture of an engineered monocyte or engineered monocyte-derived macrophage that overexpresses ID3. In certain embodiments, the kit comprises a vector comprising a heterologous ID3 nucleic acid and instructions for using the same to transduce monocytes or monocyte-derived macrophages. -83- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0259] Additionally or alternatively, in some embodiments, the kit further comprises a vector comprising an engineered chimeric antigen receptor (CAR) or other cell-surface ligand that binds to a target antigen, such as a tumor antigen. Additionally or alternatively, the kit further comprises a vector that overexpresses dectin-1, dectin-2, dectin-3, or mincle, and / or an inhibitory nucleic acid that specifically targets SIRPA, CLEC4a, or Siglec-E.

[0260] In some embodiments, the vector comprising the heterologous ID3, dectin-1, dectin-2, dectin-3, mincle and / or the inhibitory nucleic acid that specifically targets SIRPA, CLEC4a, or Siglec-E, and the vector comprising the engineered CAR or cell-surface ligand that binds to a target antigen are the same. In other embodiments, the vector comprising the heterologous ID3, dectin-1, dectin-2, dectin-3, mincle and / or the inhibitory nucleic acid that specifically targets SIRPA, CLEC4a, or Siglec-E, and the vector comprising the engineered CAR or cell-surface ligand that binds to a target antigen are distinct.

[0261] In some embodiments, the kit comprises a sterile container; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0262] If desired, the kits of the present technology can be provided together with instructions for administering the engineered monocyte or engineered monocyte-derived macrophage to a subject having cancer. The instructions will generally include information about the use of the composition for the treatment or prevention of the disease or condition. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of the disease or condition, or symptoms thereof; precautions; warnings; indications; counter- indications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. In some embodiments, the engineered monocytes or engineered monocyte-derived macrophages of the present technology may be provided in the form of a prefilled syringe or autoinjection pen containing a sterile, liquid formulation or lyophilized preparation (e.g., Kivitz et al., Clin. Ther.28:1619-29 (2006)). -84- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0263] A device capable of delivering the kit components through an administrative route may be included. Examples of such devices include syringes (for parenteral administration) or inhalation devices.

[0264] The kit components may be packaged together or separated into two or more containers. In some embodiments, the containers may be vials that contain sterile, lyophilized formulations of engineered monocyte or monocyte-derived macrophage composition that are suitable for reconstitution. A kit may also contain one or more buffers suitable for reconstitution and / or dilution of other reagents. Other containers that may be used include, but are not limited to, a pouch, tray, box, tube, or the like. Kit components may be packaged and maintained sterilely within the containers. EXAMPLES

[0265] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way. Example 1: Materials and Methods

[0266] Mice: Animal procedures were performed in adherence with the Institutional Review Board (IACUC 15-04-006) at Memorial Sloan Kettering Cancer Center (MSKCC).15, p53LSL-R172H 16, KrasLSL-G12D 17,CD45.1 mice and C57BL / 6J mice were purchased from Jackson lab. Mice were bred under SPF conditions, 12light / 12dark cycle, ~21-22°C with 30-70% humidity. A list of mouse strains and genotyping protocol is provided in Supplementary Table 2.-85- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124-86- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124-87- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124-88- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0267] Human tissue samples: All procedures performed in studies involving human participants were conducted according to the Declaration of Helsinki. Human tissues were obtained with patient-informed consent and used under approval by the Institutional Review Boards from Memorial Sloan Kettering Cancer Center (IRB protocols #15-021).

[0268] Reagents, Plasmids, antibodies, and Q-PCR Primers purchased and used in this study are listed in Supplementary Table 3.-89- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124-90- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124-91- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124-92- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124-93- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124-94- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124-95- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124-96- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124-97- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124-98- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124-99- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124-100- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0269] Mouse cell lines. KPC-1 and KPC-2 cell lines18were obtained from pancreas tissue from p48Cre; p53LSL-R172H; KRASLSL-G12Dmice19. PAN0220,21(DCTD Tumor Repository) was generously provided by prof David Lyden. Colon adenocarcinoma cell line MC3821,22and melanoma cell line B16F1023(ATCC #CRL-6475 were a generous gift from prof Jedd D. Wolchok. Lewis lung carcinoma line LLC1 cells24(ATCC #CRL-1642) were purchased from ATCC. KPC-1, KPC-2 cells, PANC-1 cells were cultured in DMEM(Gibco) supplemented with 10% fetal bovine serum, 100 U / mL penicillin and 100 -101- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 μg / mL streptomycin (Invitrogen). Panc02 cells, LLC1 cells, MC38 cells and B16F10 cells were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin (Invitrogen) and incubated at 37°C in 5% CO2.

[0270] Mouse primary cells. Mouse bone marrow- derived macrophages (BMDM) were obtained as follow. Femur, tibia and iliac bones from C57BL / 6J mice were flushed with PBS red blood cells were lysed using red blood cell lysis buffer (eBioscience), and bone marrow cells were seeded per 15cm non- tissue culture plates in DMEM (Thermo Fisher Scientific) with 10% FBS (Thermo Fisher Scientific), 20ng / ml M-CSF (315-02- 50ug, PeproTech), 100 U / ml penicillin-streptomycin (Thermo Fisher Scientific) for 7 days.

[0271] Mouse Splenic NK cells. Mouse splenic NK cells were obtained as follow. Splenic cells suspensions from C57BL / 6J mice were dissociated and passed through a 100 μm cell strainer (BD), red blood cells were lysed using red blood cell lysis buffer (eBioscience), and resuspended in 50μl of blocking buffer containing anti-mouse CD16 / 32 (1:100) for 15min at 4°C, followed by staining with PE-anti-Nkp46 antibodies for 30min at 4°C, and anti PE microbeads (Miltenyi Biotec) for 30min at 4°C respectively. NKp46+NK cells were isolated by passing stained samples through Miltenyi biotec Magnetic separation system using LS columns according to instructions.

[0272] Human cell lines. PANC-1 cells25(ATCC#CRL-1469) were purchased from ATCC. PANC-1 cells (ATCC#CRL-1469) were purchased from ATCC. Human macrophages were obtained from Human induced Pluripotent Stem Cell (hiPSC) lines derived from frozen peripheral blood mononuclear cells (PBMCs) of two independent healthy donors. Written informed consent was obtained according to the Helsinki convention. The study was approved by the Institutional Review Board of St Thomas’Hospital; Guy’s hospital; the King’s College London University and the Memorial Sloan Kettering Cancer Center. hiPSC were derived according to published protocols26using Sendai viral vectors (ThermoFisher Scientific; A16517). Newly derived iPSC clones were maintained in culture for 10 passages (2-3 months) to remove any traces of Sendai viral particles and ensure the cells remain stable during prolonged culturing period. Over 90% of iPSCs in the derived lines expressed high levels of the pluripotency markers NANOG and OCT4 by flow cytometry. Karyotyping analysis showed normal karyotype (46, XX). iPSCs lines tested negative for mycoplasma contamination using MycoAlert Plus kit (Lonza). iPSC clones that passed all quality controls were frozen down and used for -102- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 downstream experiments. hiPSCs were maintained on irradiated CF1 mouse embryonic fibroblasts (MEFs, ThermoFisher Scientific; A34181) in ESC medium supplemented with 10 ng / ml basic fibroblast growth factor (bFGF, Peprotech; 100-18B). Media is changed every other day. Passaging was performed every 7 days at 1 / 4-1 / 6 dilution ratio depending on colony size. During passaging, iPSCs were detached as clusters by a 13 min incubation at 37°C with collagenase type IV (250 UI / ml final concentration) (ThermoFisher Scientific; 17104019) and were pelleted at room temperature by centrifugation at 150G.

[0273] hiPSC-derived macrophages. iPSC clusters were resuspended in ESC medium supplemented with 10 ng / ml bFGF (Peprotech; 100-18B) and plated on NUNC plates containing 12,500 to 16,000 MEFs per cm2. HiPSC derived macrophages were obtained using a previously published protocol27modified as follows. At day 0 of the differentiation, expanded hiPSCs were detached as described above and transferred (from 150mm plate, to 4wells) for cultivation in 6 well low adhesion plates in ESC media supplemented with 10μM ROCK Inhibitor (Sigma; Y0503). The plates were kept on an orbital shaker at 100rpm for 6 days to allow for a spontaneous formation of embryoid bodies (EB) with hematopoietic potential. At day 6 of the differentiation, 200-500μm cystic EBs were picked under a dissecting microscope and transferred onto adherent tissue culture plates (∼2.5 EBs / cm2) for cultivation in HD medium. At day 18 of the differentiation, macrophages produced by EBs were collected from suspension and cultivated on tissue culture plates at a density of ∼10,000 cells / cm2in MC media for 6 days in before use for downstream experiments. All cells were cultured at 37°C, 5% CO2, in standard tissue culture incubators. hiPSC culture and differentiation media-103- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0274] Human primary cells. Human NK cells (IQ biosciences.IQB-Hu1-NK5) were cultured in NK MACS medium (Miltenyi Biotec) supplemented with 10% heat-inactivated pooled human AB serum(Sigma), 100 U / mL penicillin and 100 μg / mL streptomycin (Invitrogen), 20ng / ml hIL-2 incubated at 37°C in 5% CO2. Human CD8 T cells (IQ biosciences. IQB-Hu1-CD8T10) were cultured with RPMI supplemented with 10% heat- inactivated pooled human AB serum, 100 U / mL penicillin and 100 μg / mL streptomycin, 20ng / ml hIL-2 incubated at 37°C in 5% CO2.\

[0275] In vivo treatment with diphtheria toxin, Csf1r inhibitor, Phosphatidylserine blockade, SIRPA blocking antibodies, and NK, CD8 blocking antibodies. Diphtheria toxin (DT) mediated depletion of Kupffer cells: Clec4fCreRosa26LSL-DTRmice and Rosa26LSL-DTRmice were intraperitoneally injected with 100ng DT (D0564-1MG, sigma) as a single dose or weekly injections13, as indicated in figure legends for the corresponding experiments. Efficiency of KC depletion was determined in FIG.7J. Csf1r inhibitor treatment with PLX562228: C57BL / 6J mice were placed on an ad libitum PLX5622- impregnated chow (1200 mg / kg, provided by Plexxicon) or control chow 2 weeks before injection of tumor cells. SIRPA blockage assay: Clec4fCreId3f / fmice and Id3f / flittermates were intraperitoneally injected with control IgG (HRPN, BioXcell) or 250μg anti-SIRPA (P84, BioXcell) 2 days prior and every two days after tumor cell injection. Phosphatidylserine blockade29: C57BL / 6J mice were injected i.v. with PBS or 1μg D89E30(gift from Dr. Shigekazu Nagata), 6 hours before injection of tumor cells. NK cells and CD8 T cells depletion: 8-12 weeks old Clec4fCremice and Id3f / flittermates were intraperitoneally injected with control IgG (HRPN, BioXcell), or 200μg anti-NK1.1 -104- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 (BE0036, BioXcell) and 200μg anti-CD8 (BP0061, BioXcell) 1 days prior tumor injection and every four days afterwards.

[0276] Metastasis-initiating cell assays In vitro oncosphere formation.Sphere formation assays31were performed by sorting 1000 CD9+CD133+KPC-1 cells / well and 1000 CD9-CD133- KPC-1 cells / well and plating them in ultra-low attachment 96 well plates (Corning) in DMEM / F-12 medium supplemented with B-27 serum (1:50, Invitrogen), 20 ng / mL bFGF(R&D system), and 50 U / mL penicillin– streptomycin for a total of 7 days. Images were acquired using Leica DM IL inverted phase contrast microscope with Leica application suiteX software and quantified using ImageJ software.

[0277] In vivo metastasis assay. In vivo metastasis assay were performed by sorting 5×104or 2×105CD47brightCD9+CD133+tumor cells and CD47lowCD9lowCD133lowtumor cells from KPC-1-gfp tumor cells, followed by intra-portal injection into C57BL / 6J mice for 1 week. Metatatic potential were determined by bioluminescence imaging anlaysis. See below:” Short term liver metastasis model (intraportal injection of tumor cell lines).”

[0278] Transduction of mouse and human tumor cell lines. To generate luciferase- tdTomato(luci-tdT), luciferase-gfp(luci-gfp), membrane tdTomato(mtdT) expressing tumor cell lines, KPC-1, KPC-2, PAN02, MC38, B16F10, LLC1 cells were seeded at a density of 5×105cells per well in 6-well culture plates. Lentiviral supernatant carrying luciferase-gfp gene (Plasmid pFUGW-FerH-ffLuc2-eGFP, Addgene #71393), or luciferase-tdT gene (Plasmid pUltra-Chili-Luc, Addgene #48688) and 10 µg / mL polybrene (MilliporeSigma) were added to tumor cells culture medium for 12 hours. The medium was then replaced, and 72 hours later, GFP+or tdTomato+tumor cells were FACS sorted three times prior to in vivo injection in mice. To generate membrane tdTomato expressing tumor cells, KPC-1 and PANC-1 cells were seeded at a density of 5×105cells per well in 6-well culture plate. Lentiviral supernatant carrying retrovirus expressing MemtdT gene (Plasmid pQC membrane tdTomato IX, Addgene #37351) and 10 µg / mL polybrene (MilliporeSigma) were added to tumor cells culture medium for 12 hours. The medium was then replaced, and 72 hours later, tdT+tumor cells were FACS sorted three times prior to their use in experiments. To generate Cd47 knockout KPC tumor cells, SgRNAs targeting CD47 sequence, sgCD47 5’-CCTTGCATCGTCCGTAATG-3’32were cloned into pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene #62988) according to the Addgene cloning protocol. To establish CD47 knock out KPC cell line, electroporation of pSpCas9-sgCD47 plasmid into 1×106KPC-1- luci-tdT cells according to Neon™ Transfection System protocol. Cells were FACS sorted -105- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 based on loss of CD47 staining with anti-mouse CD47-AF647 antibody (1:200, Biolegend) for three rounds to get pure populations of CD47 knockout cells.

[0279] Transduction of macrophages.

[0280] lentiviral transduction of BMDM. BMDM were seeded at a density of 1×106cells per well of 6 well plate after 5 days culture of Bone marrow cells. Lentiviral supernatant carrying control, mouse Id3 gene, mouse sh-Myc, sh-Hes1, sh-E2A, sh-Elk1, Scramble (Santa Cruz) and 10 µg / mL polybrene (MilliporeSigma) were added to BMDM culture medium for 12 hours. The medium was then replaced, and 48 hours later, transduced cells were selected with 2 μg / mL puromycin for 4 days. The transduced BMDM were analyzed by FACS, qRT-PCR, time-lapse imaging or in vivo rescue experiments. Lentiviral transduction of KCs. Kupffer cells were seeded at a density of 8×105cells per well of 6 well plates in the presence of 20ng / ml M-CSF and transduced with lentiviral supernatant carrying control, mouse sh-E2A, sh-Elk1, VPX supernatant (pSIV3-VPX plasmids, a generous gift by Mickaël Ménager) and 10 µg / mL polybrene for 12 hours. The medium was then replaced, and 48 hours later, transduced cells were selected with 1 μg / mL puromycin for 3 days. The transduced KCs were analyzed by FACS. Lentiviral transduction of Human iPSC derived macrophages. Human iPSC-derived macrophages were seeded at a density of 5 x 105cells per well of 6 well plate in the presence of 20ng / ml M-CSF and transduced with lentiviral supernatant carrying control, human Id3 gene, VPX supernatant and 10 µg / mL polybrene for 12 hours. The medium was then replaced, and 48 hours later, transduced cells were selected with 1 μg / mL puromycin for 3-4 days. The transduced human iPSC-derived macrophages were analyzed by qRT-PCR, time-lapse imaging.

[0281] Flow cytometry, cell sorting and cell counting.

[0282] Blood and BM cells preparation. Mice blood cells were obtained as follow. Mice were anesthetized by intraperitoneal injection of Ketamine / xylazine / Acepromazine anesthesia cocktail. Blood were collected by performing cardiac puncture approach, in brief, mice were placed on its back and insert needle of 1ml syringes, pre-treated with 1ml 100mM EDTA buffer, under the rib cage, Gently pull the plunger to collect blood. Red blood cells were lysed using red blood cell lysis buffer (eBioscience). Mouse bone marrow cells were obtained as follow. Femur, tibia and iliac bones from mice were flushed with PBS, red blood cells were lysed using red blood cell lysis buffer. -106- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0283] Tissue cells suspension preparation. Mice were perfused with 10ml PBS under terminal anesthesia, tissue samples were minced into small pieces and incubated in digestion buffer contain, 1X PBS, Collagenase D (1mg / ml, , Sigma), Dispase (2.4mg / ml, Thermo Fisher) , DNAse (0.2mg / ml, Sigma) and 3% heat-inactivated fetal bovie serum (FBS, Invitrogen) for 30min at 37°C. Cells suspensions were dissociated and passed through a 100 μm cell strainer (BD) and resuspended in 50μl of blocking buffer containing 1X PBS, 0.5%BSA, 2mM EDTA, anti-mouse CD16 / 32 (1:100), 5% normal rat, 5% normal mouse and 5% normal rabbit serum (Jackson ImmunoResearch) for 15min at 4°C. Samples were stained with indicated antibodies (listed in Supplementary Table 3, 1:200) for 30min at 4°C. Flow cytometry was performed using a BD Biosciences LSR Fortessa flow cytometer with Diva software. All data were analyzed using FlowJo 10.6 (Tree Star Inc.).

[0284] Staining and gating strategies. Mouse liver macrophages / myeloid cells panels were as follow. Pop1 macrophages, Hoechst-CD45+CD3-CD19-Nkp46-Ly6G-F4 / 80+Tim4+, Pop2 macrophages, Hoechst-CD45+CD3-CD19-Nkp46-Ly6G-F4 / 80+Tim4-MHCII+, Pop3 myeloid cells, Hoechst-CD45+CD3-CD19-Nkp46-Ly6G-F4 / 80+Tim4-MHCII-. KC subsets: KC subset1, Hoechst-CD45+CD3-CD19-Nkp46-Ly6G-F4 / 80+Tim4+CD206+, KC subset2, Hoechst-CD45+CD3-CD19-Nkp46-Ly6G-F4 / 80+Tim4+CD206hi. Other mouse macrophages panels were as follow. Kidney macrophages, Hoechst-CD45+CD3-CD19-Nkp46- CD11blowF4 / 80bright. Brain macrophages, Hoechst-CD45+CD3-CD19-Nkp46-CD11b+F4 / 80+. Lung alveolar macrophages, Hoechst-CD45+CD11b-CD11c+CD64+SiglecF+. Lung interstitial macrophages, Hoechst-CD45+CD11b+Ly6G-CD64+. Skin macrophages, Hoechst- CD45+CD3-CD19-Nkp46-CD11b+F4 / 80+.Spleen RPM, Hoechst-CD45+CD3-CD19-Nkp46- CD11blowF4 / 80+. Pancreas macrophages, Hoechst-CD45+CD3-CD19-Nkp46-F4 / 80+. Mouse myeloid cells panels were as follow. cDC1, Hoechst-CD45+F4 / 80-Ly6G-CD11b- CD11c+MHCII+. cDC2, Hoechst-CD45+F4 / 80-Ly6G-CD11b+CD11c+MHCII+.Ly6C+monocytes(blood), Hoechst- CD3-CD19-Nkp46-CD11b+CD115+Ly6C+. Ly6C+monocytes (spleen, liver), Hoechst-CD45+CD3-CD19-Nkp46-F4 / 80-Ly6G-CD11b+Ly6Chi. Ly6G+granulocytes(blood), Hoechst-CD3-CD19-Nkp46-Ly6G+. Ly6G+granulocytes (spleen, liver), Hoechst- CD45+CD3-CD19-F4 / 80-Nkp46- F4 / 80-Ly6G+. Mouse lymphocytes panels were as follow. NKT cells(liver), Hoechst-CD45+F4 / 80-TCRβ+CD1dTetramers+, γδT cells (liver), Hoechst-CD45+F4 / 80-CD3+TCRβ-TCRγδ+, CD3+T cells (spleen, liver), Hoechst- CD45+F4 / 80-CD3+. CD8+T cells (spleen, liver), Hoechst-CD45+F4 / 80-CD3+CD8+, CD4+T cells (spleen, liver), Hoechst-CD45+F4 / 80- CD3+CD4+, CD19+cells(spleen, liver), Hoechst- -107- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 CD45+F4 / 80-CD3-CD19+, Nkp46+cells(spleen, liver), Hoechst-CD45+F4 / 80- CD3- Nkp46+. CD3+T cells(blood), Hoechst-Ly6G-Nkp46-CD19-CD3+. CD8+T cells(blood), Hoechst- Ly6G-Nkp46-CD19-CD3+CD8+, CD4+T cells(blood), Hoechst-Ly6G-Nkp46-CD19- CD3+CD4+, CD19+cells(blood), Hoechst-Ly6G-Nkp46-CD19+, Nkp46+cells(blood), Hoechst-Ly6G-CD19-CD3-Nkp46+. Mouse bone marrow panels were as follow. long term HSCs (LT-HSCs), Hoechst-CD3-CD19-Nkp46-Ly6G-Kit+Sca1+CD150+CD48-), short term HSCs(ST-HSCs), Hoechst-CD3-CD19-Nkp46-Ly6G-Kit+Sca1+CD150-CD48-, multipotent progenitor(MPP), Hoechst-CD3-CD19-Nkp46-Ly6G-Kit+Sca1+CD150-CD48+. Human lymphocytes intracellular staining panels were as follows. CD8+T cells, Hoechst-CD8+IFN-γ+TNF+. CD56+NK cells, Hoechst-CD56+IFN-γ+.

[0285] Cell counting. Cells number was assessed using a cell counter (GUAVA easyCyte HT). Cell sorting was performed using an Aria III BD cell sorter. Single live cells were gated on DAPI- and using forward scatter width (FSC-W) and FSC-A to exclude doublets.

[0286] Cytokines intracellular staining.

[0287] IFN-γ and TNF intracellular staining in CD8+T cells. Mouse liver cells suspension, and human CD8+T cells were treated with cocktail of phorbol 12-myristate 13- acetate (PMA), ionomycin, brefeldin A and monensin (Thermofisher scientific) for 4-12h, Staining for IFN-γ and TNF was performed using the “eBioscience Transcription Factor Staining kit” (Thermofisher scientific) according to instruction. IFN-γ and TNF production were analyzed by Flow cytometry. IFN-γ intracellular staining of mouse splenic NK cells and human CD56+NK cells were performed using the “eBioscience Transcription Factor Staining kit” according to instruction. IFN-γ production was analyzed by Flow cytometry.

[0288] Lineage tracing of bone marrow derived cells using genetic labelling. Bone marrow derived cells were labeled in Cxcr4gfp / +, Cx3cr1gfp / +mice, and by a single injection of 4-OH TAM (37.5 mg / kg body weight) supplemented with progesterone (18.75 mg / kg body weight) in 6 weeks-old Cxcr4CreERT2; Rosa26LSL-tdT 12(FIGs.8K and 8O). Cxcr4gfp / +and Cx3cr1gfp / +mice, and Cxcr4CreERT2; Rosa26LSL-tdTmice 2 weeks after 4-OH TAM injection, were injected with 1×106KPC-1 cells through the portal vein and sacrificed 2 weeks later for the analysis of tdT+cells, YFP+cells or GFP+cells among liver macrophage subsets. -108- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0289] Parabiosis. Generation of CD45.2parabionts. Female 6 to 8 weeks old CD45.2 were generated with age and weight matched female CD45.1 mice through parabiosis surgery described before33. Mice were maintained on trimethoprim / sulfamethoxazole diet after surgery to minimize infection. After 8 weeks, parabiotic mice were separated and perfused with phosphate buffered saline (PBS). Partner derived CD45.1+cells in Tim4+Kupffer cells were determined by flow cytometry analysis. Generation of CD45.2 / CD45.1 parabionts. Female 6-8 weeks old congenic CD45.1 and CD45.2 mice were connected through parabiosis surgery. After 8 weeks, CD45.2 parabiont were injected with 1×106KPC-1-luci- tdT cells through the portal vein to induce liver metastasis. Liver samples were harvested 2 weeks post tumor injection. Flow cytometry and immunofluorescence imaging were performed to analyze liver macrophage exchange ratios.

[0290] Quantitative RT-PCR. Mice Kupffer cells, BMDM and human iPSC- macrophages were lysed directly on tissue culture plates and RNA was extracted using the quick-RNA Microprep kit (Zymo research; R1050) in accordance with manufacturer’s instructions. cDNA preparation was performed with Quantitect Reverse transcription kit (Qiagen; 205313) using the manufacturer’s protocol. qRT-PCR were performed on a Quant Studio 6 Flex System with 10ng cDNA per reaction using probes (Table S6) and TaqMan Fast Advance Mastermix (ThermoFisher Scientific; 4444557), or PowerUp SYBR Green Master Mix (ThermoFisher Scientific; A25742), in line with manufacturer’s instructions. Expression values for each tested gene relative to a GAPDH endogenous control were calculated using the formula: 2^-(Cttest_gene-CtGAPDH).

[0291] Cytology. Cytospin preparation were carried using Cytospin 3 (Thermo Shandon) and Cytofunnels (Fisher Scientific, BMP-CYTO-DB25) by centrifuging sorted cells onto Super-frost slides (Thermo Scientific, 12-550-15) at 800r.p.m for 10min (medium acceleration) Slides were air-dried for at least 30 min and fixed for 10 mins in 100% methanol (Fisher Scientific, A412SK-4). Methanol fixed cells were stained in 50% May- Grünwald solution (Sigma-Aldrich, MG500-500mL) for 5 min, 5% Giemsa (Sigma- Aldrich, 48900-500mL-F) for 15 min, washed with Sorensons buffered distilled water (pH 6.8) three times for 2 mins after each staining. Slides were mounted with Entellan New (Millipore, 1079610100) after air-drying, representative pictures were taken using an Axio Lab.A1 microscope (Zeiss) under a N-Achroplan 100x / 01.25 objective. -109- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0292] Bioluminescence imaging. Depending on experiments bioluminescence imaging was conducted either on isolated organs (ex-vivo) in long term orthotopic pancreatic tumor experiments, or in anesthetized mice (in vivo) in shorter term models (below) on the In ViVo Imaging System spectrum (Perkin Elmer). Quantification of bioluminescent images was performed using LivingImage 2.60.1 software (Perkin Elmer). Photoradiance was measurement of photons / s / cm2 / sr.

[0293] Immunofluorescence and wholemount imaging. Immunofluorescence imaging of mouse liver. Mice were perfused with 10ml PBS, liver samples were dissected and fixed overnight at 4°C with PLP fixative in phosphate buffer33. Following PBS wash, livers were dehydrated in 30% sucrose in PBS and embedded in OCT. Cryoblocks were cut at a thickness of 16 μm and blocked with PBS containing 5% normal goat serum (Jackson ImmunoResearch), 1% BSA and 0.3% Triton X-100(Sigma) for 1 hour at room temperature. Samples were incubated with anti-mouse-F4 / 80- eF450 / AF647 / AF488 / eF570(1:200, BM8, eBioscience), anti-mouse Tim4-AF647 / PE(1:200, RMT4-54,Biolegend), anti-mouse CD45.1-AF488(1:200, A20, Biolegend), Chicken-anti- GFP (1:500, A10262,Invitrogen, recognize YFP), Rabbit-anti-RFP (1:200, 600-401-379, Rockland), Goat-anti mouse Clec4f (1:200, AF2784,R&D system), anti-mouse CCL3(1:200, 50-7532-82,Thermo Fisher Scientific), anti-mouse CCL4(1:200, AF-451- NA,Thermo Fisher Scientific), anti-mouse CCL5(1:200, 701030,Thermo Fisher Scientific), anti-mouse IL12p70(1:200, MM121B,Thermo Fisher Scientific), anti-mouse IL15(1:200, AF447-SP, Thermo Fisher Scientific), anti-mouse IL18(1:200, PA5-79481,Thermo Fisher Scientific) antibodies for 2 hour at room temperature. Secondary antibody staining using anti-chicken Alexa Fluor 488 (1:500; a11039,Thermo Fisher Scientific), anti-rabbit Alexa Fluor 555 (1:500, A32794 ,Thermo Fisher Scientific), anti-rabbit Alexa Fluor 647(1:500, A32795, Thermo Fisher Scientific), anti-goat Alexa Fluor 555 (1:500，a32816, Thermo Fisher Scientific), anti-goat Alexa Fluor 647(1:500，a21447, Thermo Fisher Scientific), anti-goat Alexa Fluor 488 (1:500，a32814,Thermo Fisher Scientific), anti-sheep Alexa Fluor 568(1:500, A21099, Thermo Fisher Scientific), Streptavidin Alexa Fluor 647 (1:500，405237, BioLegend) were performed for 1 hour at room temperature. Nuclei were counterstained with DAPI (Invitrogen). Sections were mounted with Fluoromount-G (eBiosciences). Images were acquired on a Zeiss LSM880 confocal microscope using oil immersion 40x 1.4 N.A. objective. Immunofluorescence imaging of human PDAC patients’ metastatic liver. Human PDAC patient metastatic liver samples were embedded in OCT. -110- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 Cryoblocks were cut at a thickness of 16 μm, fixed with 4%PFA for 30min, blocked with PBS containing 5% normal goat serum (Jackson ImmunoResearch), 1% BSA and 0.3% Triton X-100(Sigma) for 1 hour. Samples were incubated with anti-human CD14- AF488(1:200, BD), sheep-anti-human CK19(1:200, AF3506, R&D system), rabbit-anti- human Tim4(1:200, PA5-53346, Thermo Fisher Scientific), Goat-anti-human IL12(1:200, AF-219-NA, R&D system), Goat-anti-human IL18(1:200, AF2548, R&D system), Mouse- anti-human IL15(1:200, MAB2471, R&D system), Goat-anti-human CCL3(1:200, AF-270- NA, R&D system), Goat-anti-human CCL4(1:200, AF-271-NA, R&D system), Goat-anti- human CCL5(1:200, AF-278-NA, R&D system) for 2 hours at room temperature. Followed by incubation with anti-rabbit- Alexa Fluor 647 / Alexa Fluor 555, anti-sheep- Alexa Fluor 568(1:500, A21099, Thermo Fisher Scientific), anti-goat- Alexa Fluor 647(1:500，a21447, Thermo Fisher Scientific), anti-mouse- Alexa Fluor 555(1:500, A-31570, Thermo Fisher Scientific) for 1 hour. Nuclei were stained with DAPI for 10min. Sections were mounted with Fluoromount-G (eBiosciences). Images were acquired on a Zeiss LSM880 confocal microscope using oil immersion 40x 1.4 N.A. objective. Whole mount immunofluorescence imaging of mouse liver. Liver pieces were fixed in 4% paraformaldehyde (PFA) diluted in PBS for 30 min at room temperature with agitation. Samples were permeabilized with 1X PBS containing 0.3% TritonX-100, 4% BSA for 1 hour at room temperature and incubated with, anti-F4 / 80-eF450(1:100, eBioscience), anti-Tim4-AF647(1:100, Biolegend) antibodies mix for 2 hours at room temperature. Data were acquired using LSM880 Zeiss microscope. Imaris (Bitplane) was used to analyze the acquired images.

[0294] Quantification of chemokines / cytokines and tumor materials relative MFI in KCs. Quantification of tdTomato relative MFI in tdT+KCs lysosomes. Immunofluorescence staining for F4 / 80, Tim4, Lamp1+and tdT on frozen liver sections from C57BL / 6j mice 2 weeks after intra-portal injection of 1×106KPC-1-tdT cells. Images were acquired on a Zeiss LSM880 confocal microscope. Imaris (Bitplane) was used to reconstruct 3D surface of Lamp1+lysosome or 97.5 μm2non-Lamp1- region in tdT+KCs, tdT relative mean fluorescence intensity (MFI) in Lamp1+lysosome was determined by normalized to non-Lamp1- region MFI of 1. Quantification of chemokines / cytokines relative MFI in mouse KCs. Immunofluorescence staining for CCL3, CCL4, CCL5, IL12p70, IL15, IL18, GFP, F4 / 80 and Tim4 on frozen liver sections from C57BL / 6j mice, Clec4fCreId3f / fmice and Id3f / flittermates 2 weeks after intra-portal injection of 1×106KPC-1- gfp cells. Images were acquired on a Zeiss LSM880 confocal microscope. Imaris (Bitplane) -111- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 was used to reconstruct 3D surface of F4 / 80+Tim4+KCs. Chemokines / cytokines relative mean fluorescence intensity (MFI) in F4 / 80+Tim4+KCs was determined by normalized to tumor free region (distance from tumor>50 μm) in C57BL / 6j mice or Id3f / fmice MFI of 1. Quantification of chemokines / cytokines, CK19+tumor materials relative MFI in human KCs. Immunofluorescence staining for CCL3, CCL4, CCL5, IL12, IL15, IL18, CK19, CD14 and Tim4 on frozen liver sections from human PDAC liver metastatic patients. Images were acquired on a Zeiss LSM880 confocal microscope. Imaris (Bitplane) was used to reconstruct 3D surface of CD14+Tim4+KCs. Chemokines / cytokines, CK19+tumor material relative mean fluorescence intensity (MFI) in CD14+Tim4+KCs was determined by normalized to background MFI of 1.

[0295] Tumor growth, metastasis, and rescue models

[0296] Endogenous KPC model. KPC mice heterozygous forand KrasLSL-G12Dalleles19were generated by crossing p48Cre15, p53LSL-R172H 16, KrasLSL-G12D17mice under SPF conditions. KPC mice were monitored on a regular basis to check for symptoms of abdominal distension, moribund animals were euthanized by CO2 asphyxiation according to IACUC guidelines. Mice were sacrificed at 6 month and livers were harvested and fixed as described above. Quantification of CK19+tumor materials / chemokines / cytokines relative MFI were performed as follows. Frozen liver sections from KPC mice and control (KrasLSL-G12Dp53LSL-R172H) mice were performed immunofluorescence staining for CCL3, CCL4, CCL5, IL12p70, IL15, IL18, CK19, F4 / 80 and Tim4. Images were acquired on a Zeiss LSM880 confocal microscope. Imaris (Bitplane) was used to reconstruct 3D surface of F4 / 80+Tim4+KCs. CK19+tumor material, chemokines / cytokines relative mean fluorescence intensity (MFI) in F4 / 80+Tim4+KCs were determined by normalized to KrasLSL-G12Dp53LSL-R172Hmice MFI of 1.

[0297] Long term orthotopic pancreatic tumor model. Orthotopic injection of pancreatic cell lines in the pancreas was performed according to a published protocol34. Mice were anesthetized under isoflurane gas, sterile sharp scissors were used to cut a single incision off the abdominal skin and muscle above the pancreas, the pancreas was gently positioned to allow slow injection in pancreas of 2 ×105KPC-2-luci-tdT or 2 ×105KPC-2- luci-gfp cells per mice, resuspended in 50 μl of PBS and Matrigel (354234, corning) at 2 / 1 ratio using 31G insulin syringes (BD). The pancreas was gently placed back into the abdominal cavity. The muscle layer was closed using sterile absorbable vicryl suture (J463G, Ethicf on). Skin edges were closed with sterile 9mm wound clips (Braintree -112- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 Scientific). Eight weeks after pancreatic orthotopic injection, mice received retro-orbital injection of 1 mg D-Luciferin (Goldbio Technology) in 100 μl sterile water, and liver, spleen, lung and pancreas were dissected for ex-vivo analysis by bioluminescent imaging. Livers were harvested and fixed as described above, %tdT+Tim4+in Tim4+KCs were analyzed by whole mount imaging as described in” Whole mount immunofluorescence imaging of mouse liver”. (FIG.9F). Conditional depletion of KC by Diphteria toxin - In indicated experiments Clec4fCreRosa26LSL-DTRand Rosa26LSL-DTRlittermates received weekly intra-peritoneal injection of 100ng DT, starting 1 week afted pancreatic orthotopic injection of tumor cells.

[0298] Short term liver metastasis model (intraportal injection of tumor cell lines). 1×106KPC-1-luci-gfp cells, 5×105B16F10-luci- gfp cells, 1×106LLC1-luci- gfp cells, 1×106MC38-luci-gfp cells or 1×106KPC-1-luci-tdT cells, 5×105B16F10-luci-tdT cells, 1×106LLC1-luci-tdT cells or 1×106Pan02-luci-tdT cells, were resuspended in 50 μl PBS. Mice were anesthetized with isoflurane gas and sterile sharp scissors were used to cut a single incision off the abdominal skin and muscle. While holding the median side of the incision aside with forceps, including skin and peritoneal lining, a sterile cotton swab was used to carefully pull the large and small intestines out until the portal vein is visualized. After covering intestines with the sterile gauze soaked in sterile PBS, a 31G needle (BD) loaded with tumor cells was inserted into the portal vein below the liver and the full volume (50µl) was slowly injected. The needle was then removed while simultaneously placing a sterile cotton tip applicator on the vein with pressure, for 5 min, to keep the injection site intact. The internal organs were gently placed back into the abdominal cavity. The muscle layer was closed using sterile absorbable vicryl suture (J463G, Ethicon). Skin edges were closed with sterile 9mm wound clips (Braintree Scientific). Tumor bearing mice were analyzed as follows. Conditional depletion of KC by Diphteria toxin - In indicated experiments Clec4fCreRosa26LSL-DTRand Rosa26LSL-DTRlittermates received intra-peritoneal injection of 100ng DT before and / or after the tumor cell grafts, see above. Survival experiments. In the indicated experiments, cohort of tumor bearing mice were examined by a veterinarian twice a week for 5 weeks. Moribund animals were determined by the veterinarian and euthanized by CO2asphyxiation according to IACUC guidelines. Comparison of survival curves were determined by Log-rank (Mantel-Cox) test (FIGs.1G, 3J, and 6G). Liver tumor burden at 24 hours. In the indicated experiments, CD45-GFP+or CD45-tdT+liver tumor cells numbers in tumor bearing mice were analyzed by Flow -113- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 cytometry. %GFP+Tim4+or %tdT+Tim4+in KCs was analyzed by immunofluorescence staining. Liver tumor burden at 2 weeks. In the indicated experiments, liver tumor burden at 2 weeks was assessed by in-vivo bioluminescence imaging. Chemokines and cytokines production by KC was analyzed by qRT-PCR and immunofluorescence staining, %GFP+Tim4+or %tdT+Tim4+in KCs was analyzed by immunofluorescence staining. Numbers of immune cells was analyzed by flow cytometry and Immunofluorescence staining. Production of IFN-g and TNF by NK cells or CD8 T cells was analyzed by flow cytometry.

[0299] Rescue of KPC liver metastasis in Clec4fCreId3f / fmice by BMDMs. 6-12 weeks-old Clec4fCreId3f / fmice received 1x106KPC-1-luci-gfp cells by intra-portal injection, followed after one week (day7 post tumor injection) by intra-portal injection of either 1x106BMDM expressing lenti-control or lenti-mId3 cells. Tumor burden was performed 14 days after tumor injection by bioluminescent images described above.

[0300] Rescue of LLC liver metastasis in wt mice by BMDMs. 6-8 weeks-old C57BL / 6J mice received 1x106LLC1-luciferase cells by intra-portal injection, followed after one week (day7 post tumor injection) by intra-portal injection of either 1x106BMDM expressing lenti-control, lenti-mId3 cells or not. Tumor burden was performed 14 days after tumor injection by bioluminescent images described above. Survival was analyzed described above. Comparison of survival curves were determined by Log-rank (Mantel- Cox) test.

[0301] B16F10 melanoma subcutaneous tumors and rescue by BMDM. 6-12 weeks- old C57 / BL6J mice received subcutaneously injection of 1×106B16F10-luci-gfp cells, into left and right flank, followed by intra-tumor injection of 5×105BMDM expressing lenti- control, lenti-mId3 cells at day7 post tumor injection.7 days later, tumor burden was assessed by in-vivo bioluminescent imaging described as above. Numbers of immune cells and production of IFN-g and TNF by NK cells or CD8 T cells was analyzed by flow cytometry.

[0302] Intravital imaging of liver Kupffer cells and KPC-1- MemtdT tumor cells in vivo. 6-12 weeks-old C57BL / 6J mice were injected with 1×106KPC-1-MemtdT cells via intra-portal injection as described above.2 weeks after tumor cell injection. Mouse was anesthetized under isoflurane gas and anesthesia was maintained via continuous inhalation of Isoflurane (0.5L / min) in oxygen via a nose cone. Mice were then injected retro-orbitally -114- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 with 5μL CellEvent™ Caspase-3 / 7 Green reagent (Invitrogen), a four amino acid peptide (DEVD) caspase3 / 7 cleavage reporter conjugated to a nucleic acid binding dye that become fluorescent when bound to DNA (Cas-Green) to monitor tumor cell apoptosis and death35, and 10 μL of anti-TIM4-AF647 antibodies (Biolegend) in 50 μl PBS to label apoptotic / dead cells and Kupffer cells respectively. Sterile eye lubricant was applied to both eyes to prevent corneal drying during the experiment. Cut a 1.5-cm horizontal incision off the skin and muscle above the liver, extrude liver left lobe gently. The mouse was then inverted and positioned on a custom-made aluminum tray stage inserted with circular 2.5 cm diameter hole, covered with a glass coverslip attached with silicone grease. PBS-soaked sheets of paper were pre-positioned on the cover slip to surround the area of the exposed liver, then mouse was ready to perform intravital imaging. During the whole imaging period, PBS was gently added every 20 min on both sides of the mouse to keep the area moist. A thermostat controlled heated chamber keeps the whole microscope, mice, tray, and microscope objectives at 32°C to prevent hypothermia during the experiment. Imaging was performed using Zeiss LSM880 confocal laser scanning microscope. Acquisition of CellEvent™ Caspase 3 / 7 Green, tdTomato and AF647 fluorescent signals was performed in line in a single channel. The power used for each laser lines; 1% 488nm, 1% 568nm and 5% 647nm were the lowest required to obtain a sufficient signal for each fluorescent probe and chosen to minimize phototoxicity. Seven consecutive stacks with 2.5µm interval were captured using Zeiss Plan-Apochromat” 20x / 0.75 objective, with digital zoom set to 1, every 1 min per position for up to 8 hours. Time-lapse videos and 3D surface reconstructions were generated using Imaris (Bitplane).

[0303] In vitro mouse coculture assays.

[0304] Engulfment assay: Ex-vivo 3D co-culture and time-lapse imaging of Kupffer cells and KPC-1- MemtdT tumor cells. F4 / 80+Tim4+Kupffer cells were sorted from freshly isolated liver of C57BL / 6J ormice using digestion buffer and antibody panel described in Flow cytometry and cell sorting section.1x104Kupffer cells were mixed with 2x103KPC-1-MemtdT cells (5:1 ratio), embedded in growth factor reduced Matrigel (356231, Corning) and cultured overnight in 24 wells µ-plate (Ibidi, USA) with DMEM medium in the presence of 20ng / ml M-CSF and when indicated with 1µg D89E, 60mm Latrunculin A, 50µg / ml anti-SIRPA (P84, BioXcell), or 20 μg / ml anti-Dectin1(R1-8g7, InvivoGen). Prior to imaging, the co-cultures were stained with 2μM CellEvent™ Caspase- 3 / 7 Green reagent (Invitrogen), anti-F4 / 80-AF647 antibodies (1:200, Biolegend) for 30min. -115- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 Imaging was performed using Zeiss LSM880 confocal laser scanning microscope equipped with an imaging chamber maintained at 37°C, 5% CO2, 20% O2 and 90% relative humidity. Five consecutive stacks with 2.5µm interval were captured using Zeiss LD”C-Apochromat” 40x / 1.1 water immersion objective (x=212.55 µm, y=212.55 µm, z=15 µm) every 5 min per position for 20 hours. The data were analyzed using Imaris (Bitplane). For each sample, % engulfing KCs values were determined by averaging the percentage of F4 / 80+cells engulfing live tumor cells (CellEvent, clevage caspase3 / 7 negative tumor cells) from at least 3 simultaneously imaged field of views. Time to engulfment values was determined by time from stable interaction between macrophages and tumor cells to tumor cells engulfment. Time to Cas cleavage was determined by time from stable interaction between macrophages and tumor cells to the detection of Casp.3 / 7 cleavage. The total n numbers of macrophages tracked per each sample were indicated.

[0305] Engulfment assay: Ex-vivo 3D co-culture and time-lapse imaging of BMDM and KPC-1 MemtdT tumor cells. BMDM / KPC-1-MemtdT tumor cells time-lapse imaging. 1x104BMDM expressing lenti-control cells or lenti-mouse Id3 cells were mixed with 2x103KPC-1-MemtdT cells (5:1 ratio), embedded in growth factor reduced Matrigel (356231, Corning) and cultured overnight in 24 wells µ-plate (Ibidi, USA) with DMEM medium in the presence of 20ng / ml M-CSF and when indicated with 50µg / ml anti-SIRPA (P84, BioXcell). Prior to imaging, the co-cultures were stained with 2μM CellEvent™ Caspase- 3 / 7 Green reagent (Invitrogen), anti-F4 / 80-AF647 antibodies (1:200, Biolegend) for 30min. Imaging was performed using Zeiss LSM880 confocal laser scanning microscope and analyzed as descripted in “Ex-vivo 3D co-culture and time-lapse imaging of Kupffer cells and KPC-1- MemtdT tumor cells”. Production of chemokines, cytokines by Kupffer cells in coculture assay with tumor cells.3x 105KCs from Id3f / for Clec4fCreId3f / fmice were seeded in a 12 well plate, with 1.5x105KPC tumor cells or not.48 hour later, supernatants from coculture were harvested for the following study. Production of chemokines and cytokines by KCs was analyzed by qRT-PCR described as above. Role of supernatants in lymphoid cell activation. Mouse NK cells / supernatant coculture assay. Mouse splenic NK cells from c57bl6j mice were seeded in a 96 well round bottom plate at 3x 104cells / well in 100 μl NK culture medium (RPMI supplemented with 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin, 20ng / ml mIL-2) in the presence of 100 μl above mentioned supernatant.3 days later, IFN-γ production by NK cells were analyzed by Flow cytometry.

[0306] In vitro Human coculture assays. -116- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0307] Engulfment assay: Ex-vivo 3D co-culture and time-lapse imaging of human iPSC derived macrophages and PANC-1- MemtdT tumor cells. Human iPSC derived macrophages / PANC-1- MemtdT tumor cells time-lapse imaging.1x104Human iPSC derived macrophages expressing lenti-control cells or lenti-human Id3 cells were mixed with 2x103PANC-1-MemtdT cells (5:1 ratio), embedded in growth factor reduced Matrigel (356231, Corning) and cultured overnight in 24 wells µ-plate (Ibidi, USA) with RPMI1640 medium in the presence of 100ng / ml M-CSF. Prior to imaging, the co-cultures were stained with 2μM CellEvent™ Caspase-3 / 7 Green reagent (Invitrogen), anti-CD14-AF647 antibodies (1:200, Biolegend) for 30min. Imaging was performed using Zeiss LSM880 confocal laser scanning microscope and analyzed as descripted in “Ex-vivo 3D co-culture and time-lapse imaging of Kupffer cells and KPC-1- MemtdT tumor cells”. Production of chemokines, cytokines by human macrophage in coculture assay with tumor cells. 105human iPSC-derived macrophages expressing lenti-control or lenti-human Id3 were seeded in a 12 well plate, treated with 5x104Panc-1 tumor cells or not.48 hour later. Coculture supernatants were harvested for the following study. Production of chemokines and cytokines by human iPSC-derived macrophages was analyzed by qRT-PCR described as above. Role of supernatants in lymphoid cell activation. Human NK cells / supernatant coculture assay. Human NK cells were seeded in a 96 well round bottom plate at 104cells / well in 100 μl NK culture medium (see above) in the presence of 100 μl above mentioned supernatant.3 days later, IFN-γ production were analyzed by Flow cytometry. Human CD8 T cells / supernatant coculture assay. Human CD8 T cells, stained with CFSE, were seeded in a 96 well round bottom plate at 104cells / well in 100 μl CD8 culture medium (see above) in the presence of 100 μl above mentioned supernatant, PBS washed anti- hCD3 / hCD28 activation beads were added to the medium.3 days later, treated with cocktail of phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A and monensin for 6h. CFSE proliferation, TNF and IFN-γ production by CD8 T cells were determined by Flow cytometry.

[0308] Identification of candidate functional E2A and ELK1 motifs. The position weight matrix (PWM) of E2A / TCF3 and ELK1 motifs were downloaded from JASPAR database with motif IDs MA0522.1 and MA0028.2, respectively36. To find motif matches, a motif score or PWM score was first computed for all the putative regulatory elements of Kupffer cells at the Sirpα locus and filtered with a minimum PWM score cutoff that passed a false positive rate < 0.2%. Then the DeepLIFT scores were computed based on a deep -117- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 learning model (see below) at every regulatory element and overlaid these scores with motif matches for E2A and ELK1 to predict functional motifs. The final set of functional motifs all have a PWM score exceeding the score cutoff and at least 3 positions within top 20% based on DeepLIFT scores.

[0309] Training and interpretation of deep learning model. The deep learning model was trained and interpreted as described previously37. In brief, a strategy of AgentBind38was adapted and a pre-trained DeepSEA model39was fine-tuned using all active enhancers in Kupffer cells based on previously published ATAC-seq and H3K27ac ChIP-seq data under GEO accession GSE12833840. AgentBind model consists of (1) pre-training convolutional neural networks (CNNs), which infer important sequence context features and learn combinations and orientations of these features that were predictive of binding, using ChIP-sequencing and DNaseI-sequencing profiles collected from ENCODE18 and the Epigenomics Roadmap Project20 across dozens of cell types, and (2) fine-tuning an individual model for each TF to identify bound vs. unbound sequences, detailed in ref:38. DeepSEA model (deep learning–based sequence analyzer), is a fully sequence-based algorithmic framework for noncoding-variant effect prediction, detailed in ref:39. Softw were used for this methodology were Python 3, Keras 2.3.1, tensorflow 2.1.0, scikit-learn 0.21.3, deeplift 0.6.10.0, biopython 1.76. Training data were prepared as follows. Positive data labeled as 1 were 300-bp sequences of ATAC-seq peaks associated with strong levels of H3K27ac. The processed data file from GEO accession GSE128338 was obtained, which includes the reproducible ATAC-seq peaks merged from Kupffer cells of both healthy and NASH diet mice and their tag counts of H3K27ac ChIP-seq in the expanded 2000-bp regions40. The sex chromosomes were removed and peaks with a minimum cutoff of 32 tags of H3K27ac ChIP-seq were filtered. The positive sequences were balanced with the same number of 300-bp negative sequences, which were GC content-matched random genomic regions selected from the mm10 genome and were labeled as 0. During the training, sequences on chromosome 8 were left out for cross validation and those on chromosome 9 for testing. The final model had an area under the receiver operating characteristic curve (auROC) equal to 0.828 on the testing data. Next, DeepLIFT41was used to generate importance scores with single-nucleotide resolution using uniform nucleotide backgrounds. For each input sequence, two sets of scores wree generated, one for the original sequence and the other for its reverse complement. The final scores were the -118- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 absolute maximum at each aligned position. Predicted functional nucleotides were defined by the top 20% (i.e., top 60) positions within each input 300-bp sequence.

[0310] Experimental analysis of candidates E2A and ELK1 binding motifs by Cleavage Under Targets and Release Using Nuclease (CUT&RUN). CUT&RUN was performed with Epicypher (14-1048) kit according to the manufacturer’s protocol with modification.2x105Tim4+Kupffer cells were sorted from liver and resuspended in 1ml Nuclei isolation buffer (0.5mM Tris, pH8.0, 0.5mMEDTA, 5mM magnesium chloride,0.1M Sucrose, 0.05%Triton X-100, 1X EDTA-free protease inhibitor (11836170001, Sigma) and incubated on ice for 10 min. Nuclei were resuspended in 100μl wash buffer, DNA was purified with QIAamp DNA Micro Kit, using 5µl sample as Input. The rest of the sample was mixed with 10µl Concanavalin A beads and rotated at room temperature for 30 min. The supernatant was removed by placing beads and nuclei mixture on a magnetic stand. Nuclei were resuspended in 50µl antibody buffer mixed with 3µl rabbit-anti-E2A (gift from Kees Murre lab, made by David Wiest), or 4µl rabbit-anti-Elk1(Cell Signaling Technology,9182S) and incubated overnight at 4°C. The next morning, nuclei were washed in wash buffer twice, resuspend in 50µl cell permeabilization buffer contain 2.5µl pAG- MNase and incubated for 10min at RT. After 2 washes, 1µl of chromatin digest additive was added, and samples were incubated at 4°C for 2 hours with rotation. After addition of 33µl of stop buffer samples were incubated for 10min at 37°C. Tubes were then placed on a magnetic stand and the supernatant containing enriched DNA was transferred to 1.5ml tubes. DNA was purified using Chip DNA Clean&Concentrator kit (Zymo research). CUT&RUN enriched DNA and Input DNA was analyzed by qPCR on a QuantStudio (TM) 6 Flex System (Applied Biosystems) with Power SYBR Green PCR Master Mix (Thermo Scientific, A25742) and calculated as % of input.

[0311] Bulk RNAseq analysis.80,000 Kupffer cells per sample were FACS-sorted into 1.5ml Eppendorf tube with 800µl TRIzol™ LS Reagent (ThermoFisher catalog # 15596018) or in 1.5ml Eppendorf tube precoated with 10% BSA. RNA samples were submitted to the Integrated Genomics Operation (IGO) at MSKCC for quality and quantity analysis, library preparation and sequencing. Briefly, Phase separation in cells lysed in TRIzol Reagent was induced with chloroform. RNA was precipitated with isopropanol and linear acrylamide and washed with 75% ethanol. The samples were resuspended in RNase- free water. After RiboGreen quantification and quality control by Agilent BioAnalyzer, 2ng total RNA with RNA integrity numbers ranging from 9.4 to 10 underwent amplification -119- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 using the SMART-Seq v4 Ultra Low Input RNA Kit (Clonetech catalog # 63488), with 12 cycles of amplification. Subsequently, 10ng of amplified cDNA was used to prepare libraries with the KAPA Hyper Prep Kit (Kapa Biosystems KK8504) using 8 cycles of PCR. Samples were barcoded and run on a HiSeq 4000 in a PE100 run, using the HiSeq 3000 / 4000 SBS Kit (Illumina). RNA-Seq data processing and analysis. Sequenced reads from the RNAseq were aligned to the mouse reference genome GRCm39 or mm10 using STAR v2.7.10a42. The aligned reads were quantified as gene counts using HTSeq43with GENCODE release M3044. DESeq245was applied to the gene counts table to identify differentially expressed genes (DEGs). Adjust p-values were applied by DEseq2 using Benjamini and Hochberg method for multiple comparison with two-sided test. DEGs were ranked based on their log2 fold change and associated p-values (adjusted p value <0.05). Gene set enrichment analysis. Pathways enriched in the ranked DEGs were identified against the mouse Molecular Signatures Database (MSigDB)46pathway collection (p adj < 0.25) using the fgsea package in R, and the most biologically informative list were shown.

[0312] Single-cell RNA-seq analysis. The CRC dataset (GSE146409)47contained 3 colorectal liver metastasis patients and a non tumor individual. The PDAC dataset (GSE205013)48contained 3 PDAC liver metastasis patients, the nontumor dataset (GSE115469)49control contained 5 non tumor individuals. ScRNA-seq analysis was conducted using “Seurat” package (4.3.0) in R studio (4.2.0). For each dataset, quality control was performed by retaining cells with nFeature_RNA > 200 but < 10000, and mitochondrial content <10%. The PDAC and the nontumor datasets were integrated using SCTransform workflow. Firstly, the two Seurat objects were merged, normalized, and scaled with 2000 features. Subsequently, SCTransform() function was applied to the merged object with dataset source regressed out. Linear dimension reduction was performed to the “SCT” assay and the first 50 principal components. Harmony (0.1.1) was used to correct dataset and samples. The clustering analysis was based on the harmonized Seurat object. The first 40 principal components were used in RunTSNE() and FindNeighbors() functions, whereas the resolution parameter was set to 1.8 in FindCluster() function. For other parameters unspecified above, default values were used in the Seurat workflow. The CRC dataset was pre-integrated and the analysis was performed with the standard Seurat pipeline. Clusters were visualized in a two-dimensional t-distributed stochastic neighbor embedding (tSNE) and were annotated using differential expressed marker genes based on the human protein atlas (https: / / www.proteinatlas.org / ). The expression patterns of characteristic genes -120- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 were presented in the tSNE plot. Expression data of characteristic genes in KCs and TAMs clusters were extracted and presented in violin plots using ggplot2 package (3.4.1). Average expression level in each cluster was labeled on the violin plots. Adjusted p-values were obtained from Seurat FindMarkers() function using Wilcoxon test and Bonferroni correction based on the total number of genes in the dataset.

[0313] Statistics and Reproducibility. Analysis of bulk RNAseq and scRNAseq data is included in the corresponding sections. For other experiments, error bars in graphical data represent means ± SD. Statistical significance was determined using a two-tailed Student’s t- test and ANOVA for normally distributed data, or Mann-Whitney test, Kruskal-Wallis test when data were not normally distributed based on Shapiro-Wilk test or Anderson- Darling test (p<0.05). Comparison of survival curves were determined by Log-rank (Mantel-Cox) test. P < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism software. The n value represents biological replicates unless otherwise specified in the legend. Experiments were repeated to ensure reproducibility of the observations. The investigators were not blinded to allocation during experiments and outcome assessment. No statistical methods were used to predetermine sample size. For FIGs.1A-1E, 2G, 3H, 3E, and 6H. FIG.8C, 16A, and 16C results were obtained from at least 3 independent experiments. For FIGs.1H, 1I, and 1J results were obtained from 2 independent experiments: Example 2: Kupffer cells restrict tumor growth

[0314] Depletion of macrophages in C57BL / 6 mice with the CSF1R inhibitor PLX5622 increased liver engraftment of the pancreatic adenocarcinoma cell lines KPC1 (P48Cre, KRASG12D, p53R172Hand Pan02, the melanoma cell line B16F10, and Lewis lung carcinoma LLC1 in comparison to control untreated mice (FIGs.7A-7B), consistent with the proposed anti-tumor role of liver macrophages17,19,21,29. An analysis was performed in genetic models of macrophage-deficient mice of the roles of liver macrophage subsets in long-term syngeneic pancreatic adenocarcinoma models. After 8 weeks, littermate control mice developed large pancreatic and splenic tumors, and ~half of the mice developed detectable liver and lung metastasis (59%±3 and 52%±7) (FIGs.1A-1E, Supplementary Table 1). The same tumoral phenotype was observed in Flt3cre;Csf1rf / f(FIG.1A) and Ccr2- / -mice (FIG.1B), which carry normal number of TIM4+Kupffer cells (KCs) (FIGs.7C-7D) but are deficient in Csf1r-dependent bone marrow-derived macrophages and monocyte-derived macrophages, respectively30,31. -121- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124-122- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0315] In contrast, specific targeting of KCs (FIGs.7E-7G) in Clec4fCre;Csf1rf / f(FIGs. 1C and 7H), or Clec4fCre;Spi1f / fmice (FIGs.1D and 7I), or the inducible depletion of Kupffer cells upon diphtheria toxin (DT) administration to tumor bearing Clec4fCre;R26LSL-DTRmice25(FIGs.1E, 7J, and 7K) resulted in the development of larger liver, lung, and (less reproducibly) larger spleen metastasis in all mice (100% and 95%±7), while the size of pancreatic tumors was unchanged in comparison to cre-negative littermates controls (FIGs. 1C-1E, Supplementary Table 1). Because lung macrophages do not express Clec4f (FIG. 7F) and are not depleted in Clec4fCre; R26LSL-DTRmice treated with DT (FIG.7J) the increase in lung metastasis in KC-deficient mice is likely a consequence of the higher tumor burden in the liver, as the hepatic veins drain into the lung via the right ventricle of the heart (FIG.1F). In addition, survival experiments after intraportal injection of KPC cells, showed that Clec4fCreR26LSL-DTRmice treated with DT have a reduced survival in comparison to cre- negative littermates controls (FIG.1G). KC depletion in Clec4fCre;R26LSL-DTRmice also increased the number of tumor cells present in the liver 24 hr after portal vein injection of -123- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 KPC cells by >3 fold (FIG.1H). In addition, KC depletion before, as well as 3 days after, tumor injection increased the tumor burden in the liver after 2 weeks by >5 fold (FIGs.1I and 1J). Finally, flow cytometry analysis showed that a subset of tumor cells which coexpress CD47brightand markers previously associated with metastatic potential such as CD9 and CD13332,33(FIG.7L) and endowed with metastatic potential in vivo and in vitro (FIG.7M) is increased ~10 fold in the liver of KC-deficient Clec4fCre;R26LSL-DTRin comparison to littermates controls (FIG.7N). These data indicated that KC, in contrast to bone marrow derived macrophages, represent a potent barrier to the liver engraftment of tumor cells circulating in the portal vein, and exert a strong and long-lasting inhibitory effect on their subsequent growth in the liver and the lung. Example 3: KCs nucleate a peritumoral niche

[0316] Tim4+Clec4f+KCs were always located outside and around the liver tumor nodules in an endogenous tumor model with spontaneous metastasis (KPC mice) (FIG. 2A), as well as in the orthotopic graft model (FIG.8A), and in short-term models after intra-portal injection of 5 different carcinoma and melanoma cell lines (FIGs.8B-8G). Analysis of CD45.1 / CD45.2 parabionts where the CD45.2 partner received intraportal injection of KPC cells confirmed the location of Tim4+KCs around the metastatic nodules (FIG.2B). Although parabiosis experiments underestimate the contribution of blood circulating cells to tissues, our results also showed that the contribution of partner-derived cells to Tim4+KCs was below <0.5%, to be compared with ~25% contribution of partner- derived cells to CD45+Tim4- cells (FIGs.2B and 8H-8J), suggesting that up to 99% Tim4+KCs remain host-derived (CD45.2+) in the tumor bearing liver, whether they belong to the main KC CD206+subset or the smaller CD206brightsubset34-36(FIGs.8H-8J). In contrast partner-derived F4 / 80+Tim4- macrophages accumulated within the metastatic nodules (FIG.2B). In addition, genetic labeling of bone-marrow derived cells from tumor free and tumor bearing mice using 3 genetic models (Cx3cr1gfpmice, Cxcr4gfpmice, and Cxcr4CreERT2R26LSL-tdTmice pulsed with OH-TAM at 6 weeks of age), confirmed that most Tim4+cells (KCs) from both CD206+and CD206brightsubsets are not labeled (FIGs.8K- 8O).

[0317] RNAseq analysis of sorted KC from tumor-bearing liver in comparison to control showed macrophage activation, inflammatory response, and a cellular chemotaxis profile (FIG.9A). This profile included the increased expression of several receptors involved in macrophage activation and phagocytosis such as the activating receptors -124- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 Dectins that recognize carbohydrate antigens on tumor cells37, and C-C chemokines (CCL) 2, 3, 4, 5, 6 and 7, and interleukins (IL) 12, 15, 18 which are involved in the recruitment and activation of effector lymphoid cells at tumor sites38-42(FIGs.2C and 9B-9C). In addition, the most differentially expressed mRNA in KC from tumor-bearing mice consisted in epithelial mRNA such as Cytokeratins 8 and 19 (Krt19, Krt8) (FIG.9B), which is compatible with the phagocytosis of KPC tumor cells, although a contamination cannot be excluded. The transcriptional response to tumor cells of the main and minor KC were similar (FIG.3D). These data indicated that resident KCs surround the tumors and suggested several mechanisms for the KC-mediated restriction of tumor growth.

[0318] In favour of phagocytosis of tumor cells, KCs contained abundant tumor-derived material, in short-term (FIGs.2D and 9E) and long-term orthotopic models (FIG.9F) and the endogenous KPC tumor model with spontaneous metastasis (FIG.9G), as visualized by tdTomato (FIGs.2D and 9F) or Krt19 staining (FIGs.3E and 3G). The percentage of KCs containing tumor material increased over time from 40 to ~100% over 2 months in the orthotopic model (FIG.3F), and was ~100% in the endogenous model (FIG.9G). Spatially, in the short term models ~90% of KCs contained tumor material at the tumor margin, while only ~60% and ~30% did between 50 to 500µm and more than 500µm away from the tumor margin, respectively (FIG.2D), and tumor material (tdT) in KC was colocalized with Lamp1+phagolysosomes (FIG.2E). Twenty four hours after intraportal injection of KPC cells, ~35% of liver KC contained tumor material as assessed by flow cytometry, independently of phosphatidylserine blockade with MFG-E8 D89E (FIG.9H). Intravital microscopy in the liver of wt mice, using a caspase3 / 7 cleavage reporter (Cas- Green) to monitor tumor cell apoptosis and death, documented the engulfment of live KPC tumor cells by KC (FIGs.2F and 10A), however the phagocytic process spanned several hours which made quantification difficult. A 2-cell in vitro time lapse imaging assay was developed where KCs and KPC cells are cultivated together in Matrigel with the Cas-Green reporter, and with either PBS control, MFG-E8 D89E, or the inhibitor of actin polymerization Latrunculin-A (FIGs.2G and 10B). Results showed that ~50% of wild type KCs actively engulf ≥ 1 Cas-greennegKPC cells in the course of a 20h observation, independently of phosphatidylserine blockade, while Latrunculin-A blocked this process (FIG.2G). In addition, Caspase3 / 7 cleavage in tumor cells followed rather than preceded engulfment by KC (FIGs.2G and 10B). The time from contact between KC and tumor -125- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 cells to engulfment was ~4 hours in average while time from contact between KC and tumor cells to Caspase3 / 7 cleavage was ~6 hours in this assay (FIG.2G).

[0319] Immunofluorescence staining in tumor bearing liver from KPC mice and littermate controls confirmed that the CCR5 ligands CCL3, CCL4, and CCL5, and the cytokines IL12, IL15 and IL18 were produced by KC in peri-tumoral liver (FIG.11A). Similarly, immunofluorescence staining in tumoral liver 2 weeks after intra-portal injection of KPC cells in wt mice indicated that CCL3, 4, and 5 and IL-12,15, and 18, are most prominently produced by KCs present at the tumor margin (FIGs.2H and 11B). Consistently, quantification of activated NK cells and CD8 T cells in the metastatic liver showed that they were also preferentially enriched at the tumor margin, next to CCL3 / 4 / 5- IL12 / 15 / 18 expressing KCs (FIGs.2I and 2J). These data altogether suggested that resident KC that surround the tumor cells may exert their anti-tumor activity via sustained phagocytosis of live tumor cells and recruitment and activation of lymphoid effectors cells. Example 4: KC anti-tumor activity is Id3-dependent

[0320] Inhibitor of differentiation (ID) proteins43are early genes that regulate cell fate determination during development and cellular functions in differentiated cells44. ID3 was shown to be a KC lineage-determining nuclear factor because embryonic pre-macrophages lacking ID3 expression fail to differentiate into KCs during organogenesis, resulting in a selective KC deficiency24(FIG.11C). As expected for resident macrophages KCs are not replaced by WT bone marrow-derived cells in Id3-deficient parabiotic mice (FIG.11D). Mice with ID3 deficiency during embryogenesisdeveloped larger liver tumor and lung metastasis in comparison to littermates controls 2 weeks after intra-portal injection of KPC cells (FIG.3A), comparable to the phenotype of other KC-deficient mice (see FIGs.1 and 7). ID3 remains preferentially expressed at high levels in KC after birth (24and FIG. 3B), but its role for function of adult KCs is unknown. KCs acquire expression of Clec4f after birth (FIG.3C), a time when KC specification has been completed24, and the consequences of Id3 deletion was examined in adult KC in Clec4fCre;mice. Clec4fCre; Id3f / fmice presented with KC in normal numbers, morphology, and ability to uptake 2- micron latex beads after IV injection as compared to wt controls (FIGs.3D, 3E, and 11E). In addition, Id3-deficient KCs were normally located outside and around metastatic tumors, similar to controls (FIG.3F). However, Clec4fCre; Id3f / fmice still developed large liver and lung metastasis comparable to that of KC-deficient mice in short-term (FIG.3H) and orthotopic models (FIG.3H). Flow cytometry analysis confirmed that liver tumor cells as -126- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 well as the subset of phenotypic metastasis initiating cells were increased in comparison to control (FIGs.3I and 11F). Survival experiments after intraportal injection of KPC cells, showed that Clec4fCre; Id3f / fmice have a reduced survival in comparison to controls (FIG. 3J), comparable to that of Clec4fCreR26LSL-DTRmice treated with DT (see FIG.1G). Finally, Clec4fCre; Id3f / fmice also developed larger liver metastasis after intraportal injection of B16F10 melanoma, MC38 colon adenocarcinoma, and LLC1 lung carcinoma (FIG.3K). These data therefore suggested that in addition to being required during embryonic development for KC differentiation, expression of ID3 is also necessary in adult KCs for their anti-tumor activity. Example 5: Id3 controls the KC peritumoral niche

[0321] Differential gene expression analysis of RNAseq of KC from Clec4fCre; Id3f / fand control animals showed the downregulation of pathways associated with signaling receptor activity, leukocyte mediated cytotoxicity, leukocyte migration and T-cell mediated immunity in Id3-deficient KCs (FIG.4A). Id3 deficiency shifted the activatory / inhibitory receptor balance towards inhibitory receptors expression (FIG.9C). Notably the activating receptor Dectin-1 / Clec7a1,37was downregulated in Id3-deficient KCs from control and tumoral liver, while in contrast the macrophage inhibitory receptor Sirpα was overexpressed in the same KCs (FIGs.4B-4D and 12A). This analysis also identified Id3-independent receptors, including the activating receptor LRP1 which binds tumor-expressed calreticulin18and the inhibitory receptor SiglecG / 10 which binds CD24 on tumor cells7, which were expressed in Id3-deficient and control KCs (FIGs.9C and 12B). In addition, expression of the C-C chemokines CCL3, CCL4 and CCL5, and the cytokines Il12, IL15 and IL18, were also downregulated in Id3-deficient KCs from tumoral liver (FIGs.4B-4C).

[0322] Despite normal KC numbers and peritumoral location, the percentage of KCs that carry tumor material 2 weeks after intraportal injection of KPC cells was decreased by ~half in Id3-deficient mice in comparison to control (FIG.4E). The percentage of KCs carrying tumor material in the liver of Clec4fCre; Id3f / fmice 24hrs after portal injection of KPC cells was also decreased ~3-fold (FIG.4F), and the number of live tumor cells (CD45negtdT+) was increased 2.5-fold (FIG.4F). The percentage of Id3-deficient KCs that engulf one or more Caspase 3 / 7 green reporter (Cas-Green)negKPC cells in the course of the 20h in vitro time lapse imaging assay was decreased 5-fold (FIG.4G), although the average delay between contact, and engulfment or Caspase3 / 7 cleavage in Id3-deficient and control KCs were similar (FIG.4G). -127- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124

[0323] Immunofluorescence staining confirmed that expression of chemokines CCL3, CCL4, and CCL5 and the cytokines IL12, IL15 and IL18 by KC in the liver of Clec4fCre; Id3f / fmice was reduced or abolished at the tumor margin and in the tumor in comparison to littermates controls (FIGs.4H and 12C-12D). NK and CD8 T cell numbers were selectively reduced in the livers of Clec4fCre; Id3f / ftumor-bearing mice as shown by flow cytometry analysis (FIGs.4I and 12E-12G), and specifically from the peritumoral zone and the tumors as shown by immunofluorescence analysis (FIGs.4J and 12H). In addition, while NK and CD8+T cells recruited to liver tumors of wt mice produce IFNg and TNF (FIG.4K), the production of IFNg and TNF was reduced in the remaining NK and CD8+T cells present in the liver of tumor bearing Clec4fCre; Id3f / fmice (FIG.4K). Furthermore, in vitro co-culture of FACS-sorted KC from Clec4fCre; Id3f / fand Id3f / f(wt) littermates with or without KPC cells showed that tumor cells induced high expression by KC of CCL3, CCL4, and IL18 in an Id3- dependent manner and to a lesser extent of CCL5, IL15, and IL12 (FIG. 12I). Moreover, the supernatants from Id3f / f(wt) KC / KPC cocultures, but not from ID3- deficient KC / KPC cocultures, were sufficient to stimulate IFNg expression by NK cells (FIG 12J).

[0324] Depletion of CD8 and NK cells with antibodies increased tumor growth in wt mice, but not to the level of Clec4fCre; Id3f / fmice, and did not further increase tumor growth in Clec4fCre; Id3f / fmice (FIG.4L), suggesting that both phagocytosis and the recruitment and / or activation of effector lymphoid cells contribute to the anti-tumor activity of KC. Altogether, these data indicated that ID3 deficiency in KC impairs their activation by tumor cells, possibly via dysregulating expression by KC of macrophage inhibitory and activating receptors, resulting in impaired phagocytosis of tumor cells, and decreased recruitment and non-cognate activation of effector CD8+T cells and NK cells. Example 6: SIRPA blockade rescues Id3-deficient KC

[0325] Wild-type KCs express higher levels of Id3 (see FIG.3B), and lower levels of Sirpα than other macrophage subsets including microglia, alveolar macrophages, and bone marrow-derived macrophages (BMDM) (FIG.5A). SIRPA binding to its ligand CD47 inhibits macrophage activation and phagocytosis4,5. In contrast Dectin-1, which recognize tumor cell antigens, activates macrophages in tumors37, and can prime cytotoxic T-cell responses45,46. It was reasoned that ID3 may regulate the inhibitory / activating receptor balance in macrophages, and that the control of SIRPA and Dectin1 expression may underlie at least part of the anti-tumor activities of wt KCs. In vivo blockade of SIRPA with -128- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 antibodies rescued expression of Dectin1, CCL3, CCL4, CCL5, IL12, IL15 and IL18 by Id3-deficient KCs (FIGs.5B and 13A-13D). SIRPA blockade restricted the development of liver metastasis in Clec4fCre; Id3f / fmice (FIG.5C), and rescued the phagocytosis of tumor cells by ID3-deficient KCs to wild-type levels in vivo (FIG.5D), and in vitro (FIG.5E). Conversely, Dectin1 blocking antibodies abolished phagocytosis of tumor cells by wt KCs (FIG.5E), and also decreased their production of chemokines and cytokines (FIG.5F). SIRPA blockade also rescued the numbers of CD8+T-cells and NKp46+NK cells in the tumor bearing liver in Clec4fCre; Id3f / fmice (FIGs.5G and 13E), the formation of the peritumoral CD8+T-cells and NKp46+NK-rich zone (FIGs.5H, and 13F), and the production of IFNg and TNF by CD8+T-cells and NK cells (FIGs.5I-5J). In addition, genetic deletion of the SIRPA ligand CD47 on tumor cells also restricted tumor growth in Clec4fCre; Id3f / fmice (FIGs.13G-13I) and rescued CD8 T-cell and NK cell recruitment to wt levels (FIG.13J). Therefore, the regulation of SIRPA and Dectin1 expression by ID3 in KC underlies at least partly mediates the phagocytosis, inflammatory chemokines production, and the recruitment and activation of NK and CD8 T cells. Of note, these results suggest that signaling by SIRPA itself controlled in part expression of the activating receptor Dectin1 by KCs. Example 7: ID3 buffers SIRPA transactivation

[0326] Experiments were performed to identify the molecular level mechanism by which ID3 may control Sirpα expression in KC, test its potential physiological significance, and investigate whether the same mechanism can endow other macrophages with anti-tumor activity. ID proteins exert their biological effects by blocking the DNA-binding activity of class I bHLH E-proteins, Pax, Ets, and Ets-domain transcription factors from the ternary complex factor (TCF) family43,47-49. Among these, the E-protein E2A encoded by the Tcfe2a gene, as well as the TCF factor Elk-1, are highly expressed in macrophages in general and KCs in particular (FIGs.14A-14B). ELK1 links gene transcription to RAS / MAPK / ERK signaling in response to cellular stress and environmental cues such as LPS. ELK1 DNA binding and transcriptional activity are stimulated by phosphorylation of its C‐terminal domain (C‐box) by ERK1 / 2. E2A is a transcriptional activator conserved from yeast to humans and its expression and DNA binding activity is also induced by LPS50. The liver drains blood from the gut via the portal circulation and is thus constantly and directly exposed to stress signals including bacterial products27. From a physiological perspective, it was hypothesized that the expression of ID3 by KC may allow -129- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 downregulation of Sirpα expression by limiting binding of E-proteins and ELK1 to Sirpα promoter / enhancer regions, in order to maintain KC phagocytic activity within an inflammatory environment (FIG.5K). A deep learning analysis identified 12 putative ELK1-binding sites and 6 putative E-Box binding sites at upstream enhancer regions, intronic enhancers, and the Sirpα promoter in mouse Kupffer cells and bone marrow derived macrophages (BMDM) (FIGs.14C-14D). These regions are all in proximity to or in connection with the Sirpα promoter according to the H3K4me3 HiChIP data of bone marrow derived macrophages (FIG.14C). ‘Cut and run’ analyses were performed of E2A and ELK1 binding to DNA at these sites in KCs from control and Clec4fCreId3f / flittermates. These experiments indicated that ID3 prevents binding of E2A and ELK1at the Sirpα promoter and the upstream and intronic Sirpα enhancer regions (FIG.5L). In addition, Sh- RNAs targeting E2A and ELK1 both reduced Sirpα expression in Id3-deficient KCs to wild type levels (FIG.5M), indicating that E2A and ELK1 are required for up-regulation of Sirpα expression in Id3 deficient KCs. BMDM express low levels of Id3 and high Sirpα (see FIGs.3B and 5A) but share active regulatory regions at the Sirpα locus with KCs (FIG.14C). Accordingly, it was found that overexpression of Id3, as well as sh-RNAs targeting E2A and ELK1, all reduce Sirpα expression in BMDM (FIG.5N). As expected, LPS (2 mg / kg) further increased ELK1 and E2A binding to Sirpα enhancer / promoter regions in Id3-deficient but not wt KCs (FIGs.14E-14F). Consistently, LPS increased Sirpα expression in Id3-deficient KCs and in wild type BMDM, but not in wt KC or BMDM overexpressing ID3 (FIGs.14G-14H). Finally, sh-RNA against E2A or ELK1 in BMDM were sufficient to abrogate the LPS-mediated increase in Sirpα expression in BMDM (FIG. 14H). Although the comparison of human and mice non-coding sequences is difficult, a simple mouse / human Blastn alignment for Sirpα regulatory elements identified conserved Elk1 binding motifs in Sirpα enhancer and promoter regions (FIG.15A), suggesting that the role of ID3 may be conserved in human macrophages. Accordingly, it was found that lentiviral-mediated expression of mouse Id3 in mouse BMDM and human Id3 in hiPSC- derived macrophages (hiPSC-Macs) resulted in the selective down-regulation of Sirpα expression as well as the upregulation of Dectin-1 in the two cell types (FIGs.6A-6B). Altogether, these results strongly suggest that ID3 represses Sirpα expression by preventing DNA binding of E2A and ELK1 to Sirpα enhancer / promoter regions, a property that characterizes wt KC but can also be transferred to other macrophages such as BMDM or human macrophages, via enforced expression of ID3 or knock-down of E2A or ELK1. -130- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 Example 8: Conserved features of Human KC

[0327] Human KCs express higher levels of Id3 and lower levels of Sirpα than other monocytes and macrophage subsets51,52(https: / / www.proteina...

Claims

Atty. Dkt. No.: 115872-3124 WHAT IS CLAIMED IS 1. An engineered monocyte or engineered monocyte-derived macrophage comprising a non-endogenous expression vector including a mammalian ID3 nucleic acid, optionally wherein the mammalian ID3 nucleic acid is operably linked to an expression control sequence.

2. The engineered monocyte or engineered monocyte-derived macrophage of claim 1, wherein the engineered monocyte or engineered monocyte-derived macrophage is derived from bone marrow or induced pluripotent stem cells.

3. The engineered monocyte or engineered monocyte-derived macrophage of claim 1 or 2, wherein the expression control sequence comprises an inducible promoter, a constitutive promoter, a native ID3 promoter, or a heterologous promoter.

4. The engineered monocyte or engineered monocyte-derived macrophage of any one of claims 1-3, wherein the non-endogenous expression vector is a plasmid, a cosmid, a bacmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, or a retroviral vector.

5. The engineered monocyte or engineered monocyte-derived macrophage of any one of claims 1-4, wherein the mammalian ID3 nucleic acid comprises the sequence of SEQ ID NO: 3 or wherein the mammalian ID3 nucleic acid encodes an Id3 polypeptide comprising the amino acid sequence of SEQ ID NO:

4.

6. The engineered monocyte or engineered monocyte-derived macrophage of any one of claims 1-5, wherein the engineered monocyte or engineered monocyte-derived macrophage is derived from an autologous donor or an allogenic donor.

7. The engineered monocyte or engineered monocyte-derived macrophage of any one of claims 1-6, wherein the non-endogenous vector including the mammalian ID3 nucleic acid further comprises at least one of a bioluminescent protein, a fluorescent protein, a chemiluminescent protein, an epitope tag, or a selectable marker.

8. The engineered monocyte or engineered monocyte-derived macrophage of any one of claims 1-7, further comprising a non-endogenous expression vector including a heterologous nucleic acid encoding one or more genes selected from the group consisting of dectin-1, dectin-2, dectin-3, or mincle, optionally wherein the non-endogenous expression vector including the mammalian ID3 nucleic acid and the non-endogenous expression -139- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 vector including the heterologous nucleic acid encoding the one or more genes is the same or are distinct.

9. The engineered monocyte or engineered monocyte-derived macrophage of any one of claims 1-8, wherein the engineered monocyte or engineered monocyte-derived macrophage lacks detectable expression or activity of SIRPA, CLEC4a, or Siglec-E.

10. The engineered monocyte or engineered monocyte-derived macrophage of any one of claims 1-9, wherein the engineered monocyte or engineered monocyte-derived macrophage expresses at least one inhibitory nucleic acid that specifically targets and inhibits expression of one or more of SIRPA, CLEC4a, or Siglec-E.

11. The engineered monocyte or engineered monocyte-derived macrophage of claim 10, wherein the at least one inhibitory nucleic acid is an antisense oligonucleotide, a siRNA, a sgRNA or a shRNA.

12. The engineered monocyte or engineered monocyte-derived macrophage of any one of claims 1-9, wherein the engineered monocyte or engineered monocyte-derived macrophage comprises a deletion, insertion, inversion, or frameshift mutation in one or more genes selected from among SIRPA, CLEC4a, or Siglec-E.

13. The engineered monocyte or engineered monocyte-derived macrophage of any one of claims 1-12, further comprising a chimeric antigen receptor (CAR) that specifically binds to a tumor antigen.

14. The engineered monocyte or engineered monocyte-derived macrophage of claim 13, wherein the CAR comprises an extracellular antigen-binding domain, a transmembrane domain and an intracellular domain.

15. The engineered monocyte or engineered monocyte-derived macrophage of claim 14, wherein the extracellular antigen binding domain comprises a single chain variable fragment (scFv) or a human scFv.

16. The engineered monocyte or engineered monocyte-derived macrophage of claim 14 or 15, wherein the extracellular antigen binding domain comprises a signal peptide that is covalently joined to the N-terminus of the extracellular antigen binding domain.

17. The engineered monocyte or engineered monocyte-derived macrophage of any one of claims 14-16, wherein the transmembrane domain comprises a CD8 transmembrane domain or a CD28 transmembrane domain. -140- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 18. The engineered monocyte or engineered monocyte-derived macrophage of any one of claims 14-17, wherein the intracellular domain comprises one or more costimulatory domains.

19. The engineered monocyte or engineered monocyte-derived macrophage of claim 18, wherein the one or more costimulatory domains are selected from among a CD28 costimulatory domain, a 4-1BB costimulatory domain, an OX40 costimulatory domain, an ICOS costimulatory domain, a DAP- 10 costimulatory domain, a PD-1 costimulatory domain, a CTLA-4 costimulatory domain, a LAG-3 costimulatory domain, a 2B4 costimulatory domain, a BTLA costimulatory domain, a CD3ζ-chain, or any combination thereof.

20. A composition comprising an effective amount of the engineered monocyte or engineered monocyte-derived macrophage of any one of claims 1-19, and a pharmaceutically acceptable carrier.

21. A method for treating cancer or inhibiting tumor growth in a subject in need thereof comprising administering to the subject an effective amount of the engineered monocyte or engineered monocyte-derived macrophage of any one of claims 1-19 or the composition of claim 20.

22. The method of claim 21, wherein the cancer or tumor is selected from the group consisting of adrenal cancers, bladder cancers, blood cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's disease, intestinal cancers, kidney cancers, larynx cancers, acute and chronic leukemias, liver cancers, lymph node cancers, lymphomas, lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, non-Hodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, and metastases thereof.

23. The method of claim 21 or 22, wherein the engineered monocyte or engineered monocyte-derived macrophage is administered pleurally, intravenously, subcutaneously, intranodally, intratumorally, intrathecally, intrapleurally or intraperitoneally. -141- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 24. The method of any one of claims 21-23, further comprising sequentially, separately, or simultaneously administering to the subject an additional anti-cancer therapy.

25. The method of claim 24, wherein the additional anti-cancer therapy is selected from among chemotherapeutic agents, adoptive cell therapy, immune checkpoint inhibitors, monoclonal antibodies that specifically target tumor antigens, immune activating agents (e.g., interferons, interleukins, cytokines), oncolytic virus therapy and cancer vaccines.

26. The method of claim 25, wherein the adoptive cell therapy comprises administering to the subject an effective amount of lymphoid effector cells selected from among T cells, B cells, or NK cells.

27. The method of claim 26, wherein the lymphoid effector cells express a native or non-native receptor (e.g., a CAR) that binds to a tumor antigen.

28. The method of claim 26 or 27, wherein the lymphoid effector cells are administered intra-arterially.

29. A method of preparing immune cells for adoptive cell therapy comprising: (a) isolating monocytes or monocyte-derived macrophages from a donor subject, and (b) transducing the isolated monocytes or monocyte-derived macrophages with a nucleic acid encoding ID3 or an expression vector comprising said nucleic acid, optionally wherein the nucleic acid encodes an Id3 polypeptide comprising the amino acid sequence of SEQ ID NO:

4.

30. The method of claim 29, further comprising transducing the isolated monocytes or monocyte-derived macrophages with at least one additional nucleic acid encoding one or more of dectin-1, dectin-2, dectin-3, mincle, or a CAR that binds a tumor antigen, or an expression vector comprising the at least one additional nucleic acid.

31. The method of claim 29 or 30, further comprising transducing the isolated monocytes or monocyte-derived macrophages with at least one inhibitory nucleic acid that specifically targets and inhibits the expression of one or more of SIRPA, CLEC4a, or Siglec-E.

32. The method of any one of claims 29-31, comprising administering the transduced monocytes or monocyte-derived macrophages to a recipient subject.

33. The method of claim 32, wherein the donor subject and the recipient subject are the same or different. -142- 4855-2652-7480.1Atty. Dkt. No.: 115872-3124 34. A method for treating cancer or inhibiting tumor growth in a subject in need thereof comprising administering to the subject an effective amount of nanoparticles comprising ID3 nucleic acid molecules (e.g., mRNA) or ID3 polypeptides, wherein the nanoparticles are configured to target macrophages or monocytes in the subject.

35. The method of claim 34, wherein the nanoparticles target at least one of BTK, Siglec-1, TLR, TLR7, mannose receptor, TLR3, VEGF, SHP2, PIGF, CSF-1R or CCR2. -143- 4855-2652-7480.1

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