Targeting pro-tumor bioactivity via macrophage transcriptional targeting for cancer therapy

Abstract

Described herein are methods of treating cancer in a subject in need thereof. The methods involved administering macrophages lacking a transcription factor that mediates one or more suppressive pathways in the macrophage; and administering a cancer therapy. Additional embodiments include administering a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based system to delete a DNA-binding region of a transcription factor that mediates a suppressive pathway in the macrophage; administering macrophages lacking a transcription factor that mediates one or more suppressive pathways in the macrophage; and administering a cancer therapy.

Description

TARGETING PRO-TUMOR BIOACTIVITY VIA MACROPHAGE TRANSCRIPTIONAL TARGETING FOR CANCER THERAPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application includes a claim of priority under 35 U.S.C. §119(e) to U.S. provisional patent application No. 63 / 541,463, filed September 29, 2023, the entirety of which is hereby incorporated by reference.REFERENCE TO SEQUENCE LISTING

[0002] This application contains a Sequence Listing submitted as a computer readable form named “065472-000928WOPT.xml”, having a size in bytes of 3,814 bytes, and created on September 26, 2024. The information contained in this computer readable form is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with Government support under CA220000 awarded by National Institutes of Health. The Government has certain rights in the invention.FIELD OF INVENTION

[0004] This invention relates to the treatment of cancer.BACKGROUND

[0005] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0006] Cancer remains one of the leading health problems in the modem world and despite advances in radiation, chemotherapy and immunotherapy many patients fail to respond. One reason is that tumors develop mechanisms to resist these therapies and our invention targets one of the main cells found in tumors that mediate this resistance, macrophages, and alters its programming to help make tumors more sensitive to other cancer therapies.

[0007] Radiation therapy (RT) is one of the most important therapies against cancer, but its efficacy is limited by the immune microenvironment of the tumors. Macrophages get programmed into a “pro-tumor” phenotype in tumors that limits the response to therapy; however, little is known about the molecular mechanisms by which macrophages maintain their “pro-tumor” behavior

[0008] As such, there remains a need in the art for additional and better therapeutic options for these patients.SUMMARY OF THE INVENTION

[0009] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0010] Various embodiments provide for a method of treating cancer in a subject in need thereof, comprising: administering macrophages lacking a transcription factor that mediates one or more suppressive pathways in the macrophage; and administering a cancer therapy.

[0011] Various embodiments provide for a method of treating cancer in a subject in need thereof, comprising: administering a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based system to delete a DNA-binding region of a transcription factor that mediates one or more suppressive pathways in the macrophage; administering macrophages lacking a transcription factor that mediates the one or more suppressive pathways in the macrophage; and administering a cancer therapy.

[0012] In various embodiments, the transcription factor can be GATA-3.

[0013] In various embodiments, the cancer therapy can be radiation, immunotherapy, chemotherapy or combinations thereof.

[0014] In various embodiments, the cancer therapy can be radiation and the radiation therapy can be given at a dosage of about 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-90, 91-95, or 96-100 Gy, and the doses are given in one or more fractions.

[0015] In various embodiments, the cancer therapy can be immunotherapy.

[0016] In various embodiments, the immunotherapy can comprise an immune checkpoint inhibitor. In various embodiments, the checkpoint inhibitor can be anti-CTLA4, anti-PDl, anti- PDL1, or combinations thereof. In various embodiments, the anti-CTLA4 can be ipilimumab (MDX-010) or tremelimumab (CP-675,206). In various embodiments, the anti-PDl can be selected from the group consisting of pembrolizumab, balstilimab, nivolumab, pidilizumab, AMP-224, AMP-514, spartalizumab, cemiplimab, penpulimab (AK105), prolgolimab (BCD- 100), ezabenlimab (BI 754091), toripalimab (JS001), lipustobart (LZM009), retifanlimab (MGA012), Sym021, dostarlimab (TSR-042), tebotelimab (MGD013), cadonilimab (AK104), vudalimab (XmAb20717), tislelizumab, PF-06801591, anti-PDl antibody expressing pluripotent killer T lymphocytes (PIK-PD-1), autologous anti-EGFRvIII 4SCAR-IgT cells, and combinations thereof. In various embodiments, the PD1 inhibitor can be pembrolizumab or dostarlimab. In various embodiments, the PDL1 inhibitor can be selected from the group consisting of garivulimab (BGB-A333), cosibelimab (CK-301), FAZ053, envafolimab (KN035), MDX-1105, betifisolimab (MSB2311), adebrelimab (SHR-1316), atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, M7824, and combinations thereof.

[0017] In various embodiments, the immunotherapy can comprise an adoptive cellular therapy, a cancer vaccine, monoclonal antibodies, or cytokine therapy. In various embodiments, the adoptive cellular therapy can be Chimeric antigen receptor (CAR) T cell therapy, Chimeric antigen receptor (CAR) natural killer (NK) cell therapy, Tumor infdtrating lymphocyte (TIL) therapy, Endogenous T cell (ETC) therapy, or combinations thereof.

[0018] In various embodiments, the cancer therapy can be chemotherapy.

[0019] In various embodiments, the chemotherapy can be selected from the group consisting of paclitaxel, cyclophosphamide, doxorubicin, irinotecan and combinations thereof. In various embodiments, the chemotherapy can be selected from the group consisting of dacarbazine, temozolomide, nab-paclitaxel, paclitaxel, cisplatin, carboplatin, and combinationsthereof. Tn various embodiments, the chemotherapy can be selected from the group consisting of Cisplatin, Carboplatin, Paclitaxel (Taxol), Albumin-bound paclitaxel (nab-paclitaxel, Abraxane), Docetaxel (Taxotere), Gemcitabine (Gemzar), Vinorelbine (Navelbine), Etoposide (VP- 16), Pemetrexed (Alimta), and combinations thereof.

[0020] In various embodiments, the cancer therapy can be an antibody-drug conjugate. In various embodiments, the antibody-drug conjugate can be selected from the group consisting of TDM-1, TdxD and sacituzumab govitecan, and combinations thereof.

[0021] In various embodiments, the macrophages can be differentiated from myeloid progenitor cells, induced pluripotent stem cells (iPSCs), or hematopoietic stem cells.

[0022] In various embodiments, the CRISPR-based system can comprise a vector encoding a guide RNA targeting Zn-finger DNA binding domain of GATA-3; and a vector encoding Cas9, Casl2a, or Casl3. In various embodiments, the guide RNA can comprise the sequence of SEQ ID NO:3.

[0023] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.BRIEF DESCRIPTION OF THE FIGURES

[0024] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0025] Figure 1 (panels A-C) shows tumors (~lcm) locally treated with indicated doses of RT, followed by harvesting at day 7 post RT. A) The graph represents number of CD8+ T cells in the TME of these mice treated with RT compared to control (untreated) tumors (>5 mice / group). B) Gzmb expression on CD8+ T cells, determined by flow cytometry (n=5 mice per group). C) Gzmb / Perforin+ cells within the CD8+ compartment is represented here (n=5 mice per group).

[0026] Figure 2 (panels A-b) shows locally radiated tumors (indicated doses of RT) were harvested at day 7 post RT. A) MHC-II expression among CDl lb+ F480 Hi macrophages wasdetermined (n=5 mice per group). B) Ly6G+ MDSCs / Neutrophils were determined within the CD1 lb + cell compartment by Flow Cytometry (n=5 mice per group).

[0027] Figure 3 shows tumor growth curves. Tumor bearing mice (>1.0cm) were treated with a localized gamma irradiation. Mean tumor burden post RT is depicted in the figure (n=5 mice / group).

[0028] Figure 4 shows UMAP: Uniform Manifold Approximation and Projection (UMAP) dimension 1 and 2 plots of tumor infiltrating CD45+ cells in E0771 tumors treated with fractionated (8Gyx 3) and its single dose equivalent 16.5Gy fraction with unirradiated control E0771 tumors in C57 / BL6 mice . Cell clusters were colored by cell populations. TAM: Tumor Associated Macrophage, eDC: Classical dendritic cells, mDC: Migratory dendritic cell, Gran: Granulocyte / Neutrophil, TC: T Cells.

[0029] Figure 5 shows DotPlot: Dot Plot heat map showing top 3 unique marker gene expression identified using the FindAllMarkers across all immune cell (CD45+) types. Darker red indicates higher levels of expression and orange-white signifies lower levels of expression. Dot size indicates the fraction of gene expressing cells in population.

[0030] Figure 6 shows Log2 (fold change) of both 16Gy for a single fraction and 8Gy for 3 fractions when compared to untreated control in all cell type clusters. Colors match designated cell cluster color presented in UMAP.

[0031] Figure 7 shows a heatmap: Gene expression of top 29 Unique genes based on differential expression in the myeloid population (including mDC and eDC) between with 8Gy 3 fractions, 16.5Gy single fraction, and non-treated tumors. Red signifies high levels of relative expression; whereas blue signifies low levels of relative expression.

[0032] Figure 8 (panels A-B) depicts Tumor-Associated Macrophage (TAM) exhibit phenotypic diversity that is dramatically altered post-RT. (A) tSNE plot of 47,560 individual TAMs (CD45+CD1 lb+) isolated from E0771 murine TNBC model after 16 Gy single-dose irradiation. (B) Quantification of TAM functional states from E0771 tumors at day 3, 7, 10 and 14 after radiation. RT, radiation.

[0033] Figure 9 (panels A-B) shows mice with GATA-3 deficient macrophages significantly enhance the response to RT. A) Tumor growth measured by caliper every 3 days following RT B) Kaplan-Meier survival curves (n=10 / group). WT = wild-type, RT = radiation(16 Gy, single dose), mG3K0 = Mice with GAT A-3 -deficient macrophages. One of three representative ** = p< 0.01, *** = p<0.001.

[0034] Figure 10 depicts an exemplary experimental diagram to investigate the effect of GATA-3-deficient macrophages on different doses of RT.

[0035] Figure 11 (panels A-C) shows Tumor Characterization in Irradiated Mice. Tumors were harvested from mice at various time points and analyzed by flow cytometry (FACS) (A), immunohistochemistry (IHC) (B) and scSeq (C). For FACS, tumors were enzymatically dissociated with and labeled with fluorescently tagged antibodies and then placed on a flow cytometer (A). For IHC, tumor fragments were fixed in formalin and embedded in paraffin. 5 micron sections were then cut and stained with various antibodies and developed with DAB prior to whole-slide scanning on the Aperio system (Leica Biosystems) (B). scSeq was performed on magnetically sorted immune cells (CD45+ leukocytes) and then subjected to scSeq using thelOX Genomics platform and analyzed in R with the Seurat software package (C).

[0036] Figure 12 depicts an exemplary experimental diagram to investigate the effect of GATA-3-deficient macrophages on treatment-induced systemic anti-tumor immunity.

[0037] Figure 13 shows immune profiling and blockade experiments. Tumors from WT (LysM-Cre) or mGATA-3K0 mice that had been treated with RT cocktails were analyzed by multicolor flow cytometry. Analysis of the cell populations were completed with the FlowJo software package (TreeStar). n = 5 / group, *p< 0.05, **p < 0.01

[0038] Figure 14 shows exemplary diagram of experiments to study innate immune responses and adaptive immune responses.

[0039] Figure 15 (panels A-C) shows that macrophages can be adoptively transferred and transfer of mGATA-3K0 macrophages enhances response to RT compared to transfer of WT macrophages. (A) 5 x 106 BMDM (Mo) from either LysM-Cre or mGATA3-K0 mice were first labeled with the cell permeant dye CFDA and then given retro-orbitally to tumor bearing-mice tumors were then dissociated and analyzed by flow cytometry looking for CFDA+ cells. (n=4). WT tumor bearing mice were adoptively transferred 5 x 106BMDM from either LysM-Cre (WT) or mGATA3-K0 (mG3K0) mice 24 hours prior to RT and then irradiated with 16 Gy to the tumor. Mice were then followed for tumor growth (B) and survival (C). (n=4-6 mice / group). ** p <0.01.

[0040] Figure 16 (panels A-B) shows deletion of GATA-3 Zn Finger Binding Domain in iPSC-derived macrophages. (A) Partial gene map of the human GATA-3 gene (chromosome 10, pl 4) showing the first five exon. Schematic representation of guide RNA location for the lentiviral construct and the sequencing primers used for validation of the deletion. (B) iPSCs obtained from the Cedars-Sinai iPSC core were cultured with lentiviral vectors containing the guide RNA and Cas9 expression vector and then differentiated into myeloid cells using an M- CSF based procedure described in Zhang et al. The resulting macrophages were then lysed and genomic DNA isolated and subjected to PCR. The resulting gene product showed the 225 bp deletion in the GATA-3 gene which was confirmed by sequencing.

[0041] Figure 17 shows an exemplary diagram of experiments to determine parameters for adoptive transfer of modified macrophages in combination with different therapies.

[0042] Figure 18 shows generation of a macrophage-specific GATA3 KO Mice.

[0043] Figure 19 shows syngeneic orthotopic transplant model.

[0044] Figure 20 shows GATA3 ablation in macrophages dramatically enhances the antitumor potential of RT.

[0045] Figure 21 shows GATA3-KO Macrophages are More Inflammatory and Resistant to Polarization.

[0046] Figure 22 shows transferred GATA3-KO macrophages home to irradiated tumors.

[0047] Figure 23 shows transfer of GATA3-KO in macrophages improves response to RT.

[0048] Figure 24 shows transferred GATA-3KO macrophages enhance the response to immunotherapy.

[0049] Figure 25 shows human macrophages with GATA3-KO generated from iPSCs exhibit similar behavior as murine KO.

[0050] Figure 26 shows that IL4 signals through IL4-R and STAT-6 leading to activation of GATA-3.

[0051] Figure 27 shows that macrophages have different functional phenotypes depending on the cytokine mileu.

[0052] Figure 28 shows an example of method for the studies.

[0053] Figure 29 shows that IL-4 blockade improves the effectiveness of RT. Orthotopic PyMT derived tumors were grown to a median diameter of 1.0 cm and treated with localized gamma irradiation (5Gy). IL4 neutralizing antibody was administered 2 days prior to RT and then every 5 days for the duration of the experiment. Data is expressed as mean tumor burden ± SEM (5 mice per group).

[0054] Figure 30 shows IL4R Expression in tumors of WT C57BL / 6 mice. Mice were orthotopically injected with E0771 cells, and radiated when tumors were about 1.0 cm size. IL4R expression in irradiated vs unirradiated mice was determined by flow cytometery (n=4 mice per group)

[0055] Figure 31 shows tumor growth curve of WT-CRE and M<[) GATA-3 KO mice post radiation. Tumor bearing mice (-l.Ocrn) were treated with a single dose localized gamma radiation (16 Gy) and tumor sizes were monitored every two days until endpoint. Mean tumor burden is depicted in the figure (n=5 mice / group); one way annova was used to calculate statistical significance.

[0056] Figure 32 shows Survival Curve of WT-CRE and Mc[ GATA-3 KO mice post single dose of radiation. Tumor bearing mice (-l.Ocrn) were treated with a single dose localized gamma radiation (16 Gy) and tumor sizes were monitored every two days until endpoint. The figure represents the survival of the animals post radiation; statistical significance was determined by using one way annova.

[0057] Figures 33A-33B show immune profiling of WT-CRE and M<[) GATA-3 KO mice at Day 5 post radiation. Orthotopic PyMT-derived tumors were grown to a median diameter of 1.0 cm and then the tumor-bearing mice were subjected to a single dose of 16Gy focal radiation. Tumors were harvested on day 5 after radiation, and CD45+ cells were harvested; immune profiling was done using multicolor flow cytometry (n=5 mice per group).

[0058] Figure 34 shows tumor growth curve of WT-CRE and M<|) GATA-3 KO mice after T-Cell Depletion. Tumor bearing mice (-l.Ocrn) were treated with a single dose localized gamma irradiation (16 Gy) and tumor sizes were monitored every two days until endpoint. Anti- CD4 and anti-CD8 antibodies were administered via I.P injections starting two days prior to RT and continued twice a week till endpoint. (n=5 mice / group)

[0059] Figure 35 shows tumor growth curve and survival curve of anti-CTLA-4 in E0771 Breast Cancer.

[0060] Figure 36 (panels A-F) shows Macrophage specific deletion of GATA-3 improves response to radiation. A). Schematic representation of deleting GATA-3 in macrophages (mG3K0). B). Schematic overview of orthotopic injections of tumors cells followed by radiation. Orthotopic E0771 mammary tumors (C-D) were locally radiated once with 16 Gy (RT). Individual tumor growth were assessed every 2 days until endpoint and mean tumor burden ± SEM (C) and survival (D) was calculated for the indicated treatments. Mean tumor burden / animal ± SEM and survival were also assessed in subcutaneous B16 melanomas (E, F), treated with localized irradiation (16 Gy). Significance was determined by two-way ANOVA with post-hoc testing for tumor growth, Log-Rank test for the Kaplan-Meier survival curves. Numbers (n) for each experiment are listed on the figure and are pooled data from at least two independent experiments. For all figures, significance is shown as: n.s < non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

[0061] Figure 37 (panels A-G) shows Single Cell Sequence reveals significant remodeling of myeloid cells: UMAP: Uniform Manifold Approximation and Projection (UMAP) of tumor infiltrating CD45+ cells in E0771 tumors treated with 16 Gy radiation and non-treated E0771 tumors in control (CTRL) and mG3K0 mice. (A) Cell clusters were colored by cell populations; MDSC / neutrophils: Granulocytes and neutrophils, DC : dendritic cells, TC: T Cells, BC : B cells, Macrophages, Monocytes and Prolif: Proliferating Cells. B): Pie-chart representing changes in the CD45+ immune cells between control (CTRL) and mG3K0 mice with (+RT) and without radiation. C). UMAPs of myeloid cells split into varying treatment groups. D): Pie-chart representing changes in the myeloid compartment of tumor immune milieu of control (CTRL) and mG3K0 mice with (+RT) and without radiation. E). Lipid associated (LA-TAM signature) and regulatory gene signature (Reg-TAM signature) of CTRL and mG3K0 mice. F). Dot plot representation of relevant gene subset from CTRL and mG3K0 mice ± radiation. G). Violin plots representing expression of indicated genes in the CTRL versus mG3K0 mice ± radiation.

[0062] Figure 38 (panels A-E) shows CRISPR-Cas9 mediated GATA-3 KO in human stem cell derived macrophages shapes enhanced inflammatory potential of these cells: Development of a CRISPR / Cas9 platform for knockout of GATA-3 in primary human stemcells: A). Schematic representation of pLentiCRISPR-mCherry plasmid, designed for knockout of GATA-3 gene. The plasmid expresses Streptococcus pyogenes Cas9 nuclease fused with mCherry under the control of EF-la core promoter. B). The 20 nt target sequence along with the sgRNA scaffold is depicted. Blue rectangles indicate exon regions. The sequence of the singleguide RNAs (sgRNA) targeting exon 4 of GATA-3 is indicated in red. C). Western Blot analysis showing GATA-3 expression in CTRL vs CRSPR-CAS9 treated human stem cells. D). Schematic overview of isolation, culture and transduction of human stem cells (CD34+) and differentiation into macrophages. E). Human TNF- a levels in CTRL versus Gata-3 KO (G3K0) human stem cell derived macrophages primed with inflammatory stimuli (LPS + IFNy, labelled Ml) and immunosuppressive signal (IL4, labelled M2) before treating with LPS.DESCRIPTION OF THE INVENTION

[0063] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0064] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0065] As used herein the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 5% of that referenced numeric indication, unless otherwise specifically provided for herein. For example, the language “about 50%” covers the range of 45% to 55%. In various embodiments, the term “about” when used in connection with a referenced numeric indication can mean the referenced numeric indication plus or minus up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of that referenced numeric indication, if specifically provided for in the claims.

[0066] Treatment” and “treating,” as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, slow down and / or lessen the disease even if the treatment is ultimately unsuccessful.

[0067] As used herein, a “subject” means a human or non-human animal. Usually the non-human animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, and canine species, e.g., dog, fox, wolf. The terms, “patient”, “individual” and “subject” are used interchangeably herein. In an embodiment, the subject is mammal. The mammal may be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. In some embodiments, the subject is a human. In some embodiments, the subject has a cancer. In some embodiments, the subject has a tumor.

[0068] “Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.

[0069] A subject may be one who has been previously diagnosed with or identified as suffering from or having a disease, disorder or condition in need of treatment or one or more complications related to the disease, disorder, or condition, and optionally, have already undergone treatment for the disease, disorder, or condition or the one or more complications related to the disease, disorder, or condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a disease, disorder, or condition or one or more complications related to the disease, disorder, or condition. For example, a subject may be one who exhibits one or more risk factors for a disease, disorder, or condition or one or more complications related to the disease, disorder, or condition or a subject who does not exhibit risk factors. A “subject in need” of treatment for a particular disease, disorder, or condition may be a subject suspected of having that disease, disorder, or condition, diagnosed as having that disease, disorder, or condition, already treated or being treated for that disease, disorder, or condition, not treated for that disease, disorder, or condition, or at risk of developing that disease, disorder, or condition.

[0070] Cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0071] Although radiation therapy (RT) remains a cornerstone in the treatment of breast cancer, many trials combining RT with immune checkpoint blockade (ICB) have failed to demonstrate benefit in solid tumors including breast cancer. Maximal efficacy of RT relies on the generation of antitumor immunity following treatment which largely consists of cytotoxic T cells and macrophages. Broad depletion of macrophages modestly enhances tumor responses to RT suggesting that they can shape RT -induced antitumor immunity. Although IL4 signaling through GATA-3 is known to drive macrophages into an immunosuppressive Th2 phenotype, such central drivers of macrophage polarization are not well established. Given that macrophages abundantly express IL4 receptor, and while not wishing to be bound by any particular theory, we believe that GATA-3 may direct the transition of macrophages to M2 / alternative phase and that genetic ablation of GATA-3 in macrophages can enhance antitumor immunity by arresting the transition to an M2-like pro-tumor state.

[0072] Our data shows that GATA-3 is a central regulator of macrophage polarization in response to RT. Further, directed ablation of GATA-3 appears to drive macrophages towards an Ml -like phenotype, which enhances T cell recruitment to irradiated tumors. These data suggest that the antitumor efficacy of RT can be prolonged by targeting GATA-3 -dependent signaling within myeloid cells.

[0073] Macrophages have tremendous immunosuppressive capacity and in many tumors they are co-opted to limit anti-tumor immunity. We observed that the IL-4-pathway was one of the key suppressive pathway in tumors and that the major source of IL-4 receptor in tumors was macrophages. Blockade of IL-4 significantly enhanced the response to radiation and to understand whether macrophages were the reason we found a macrophage in which the suppressive programming was eliminated (via deletion of the transcription factor GATA-3 specifically in macrophages). Surprisingly, in this mouse when we repeated our radiation experiments with murine breast tumors we observed one of the strongest synergies with radiation that we have ever seen. We have confirmed this observation and also that it is immune-mediated. Thus, described herein include 1) targeting transcription factors that mediate suppressive pathways in macrophages can enhance the response to therapy, and 2) specifically targeting onepathway, GATA-3, in macrophages creates a macrophages that significantly enhances the response to other cancer therapies.

[0074] Other groups have sought to engineer macrophages, but to date they have put in new signals (such as CAR-MACs, chimeric antigen receptor macrophages) to attempt to activate macrophages, whereas described herein we eliminate immunosuppressive programs in macrophages.

[0075] Various embodiments provide for a cellular therapy targeting transcription factors that drive pro-tumor bioactivity. We identified GATA-3 as one of the key transcription factors downstream of IL-4 and when macrophages are genetically ablated of GATA-3 they synergize with radiation, immunotherapy and chemotherapy to generate better responses.

[0076] Described herein are data showing that in mice where GATA-3 has been eliminated in myeloid cells specifically that this synergizes with radiation and immunotherapy. We have additional data showing that macrophages generated ex vivo with GATA-3KO can be transferred and confer similar synergy as their genetic counterparts. Finally, we have generated CRISPR-based GATA-3 targeting constructs for both mouse and human where we can delete the DNA-binding region of GATA-3 and that this construct generates macrophages that behave similarly to the genetically-ablated macrophages.

[0077] Further shown herein, we can adoptively transfer macrophages grown ex vivo that lack GATA-3 that they 1) home to the tumor appropriately and in large numbers and 2) enhance the anti-tumor effect of RT. In addition to the mechanistic work, we are currently refining CRISPR-based targeting to delete GATA-3 in human macrophages by starting with myeloid progenitor cells and iPSCs that we modify and then differentiate into GATA-3 deficient macrophages.

[0078] Described herein we find that IL4 blockade along with RT improves the efficacy of RT by enhancing the cytotoxic immune responses against tumor cells. Further, GATA-3 is a critical transcription factor regulating cellular responses to IL-4 and macrophages are the largest expressers of IL4R in the tumor microenvironment. We show herein that deleting GATA-3 in macrophages dramatically enhances the response to RT.

[0079] Findings described herein include but are not limited to: (1) Macrophage (M$) specific GATA-3 ablation significantly improves the efficacy of RT; (2) TargetingGATA-3enhances anti-tumor immune responses elicited by RT and other anti-cancer therapies; (3) Immune profiling reveals significant enrichment of activated CD8+ T cells within the tumor microenvironment.

[0080] Various embodiments of the invention are based, at least in part, on these finding.

[0081] Various embodiments provide for a method of treating cancer in a subject in need thereof, comprising: administering macrophages lacking a transcription factor that mediates one or more suppressive pathways in the macrophage; and administering a cancer therapy.

[0082] In various embodiments, the transcription factor is GATA-3. Thus, macrophages lacking GATA-3 are administered.

[0083] In various embodiments, the macrophages are differentiated from myeloid progenitor cells, induced pluripotent stem cells (iPSCs), or hematopoietic stem cells.

[0084] In various embodiments, the cancer therapy is radiation, immunotherapy, chemotherapy or combinations thereof.

[0085] In various embodiments, the cancer therapy is radiation. Fraction sizes can be about 2-34 Gy given in 1-3 fractions. In particular embodiments, the fraction sizes can be about 2 Gy, 16, Gy or 34 Gy, given as one fraction. In particular embodiments, the fraction sizes can be about 2 Gy, 5 Gy or 9 Gy, given as three fractions.

[0086] In various embodiments, the fraction sizes range from 2-34 Gy in 1-35 doses. In various embodiments, the fraction sizes range from 2-10 Gy in 1-35 doses. In various embodiments, the fraction sizes range from 2-10 Gy in 1-20 doses, 21-25 doses, 26-30 doses, or 31-35 doses.

[0087] In various embodiments, the fraction sizes range from 11-20 Gy in 1-35 doses. In various embodiments, the fraction sizes range from 11-20 Gy in 1-20 doses or in 21-35 doses.

[0088] In various embodiments, the fraction sizes range from 21-30 Gy in 1-35 doses. In various embodiments, the fraction sizes range from 21-30 Gy in 1-10 doses, 11-20 doses, or 21- 35 doses.

[0089] In various embodiments, the fraction sizes range from 31-34 Gy in 1-35 doses. In various embodiments, the fraction sizes range from 31-34 Gy in 1-5 doses, 6-10 doses, 11-15 doses, 16-20 doses, or 21-35 doses.

[0090] In various embodiments, the fraction sizes can be 2 Gy x 25-30 doses, 6 Gy x 5 doses, 8 Gy x 1 dose, 8 Gy x 3 doses, 9 Gy x 3 doses, 16 Gy x 1 dose, 20 Gy x 1 dose, 24 Gy x 1 dose or 34 Gy x 1 dose.

[0091] In various embodiments, the fraction sizes is any one or more of about 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, 71-75, 76- 80, 81-85, 86-90, 91-95, or 96-100 Gy. These doses may be given in one or more fractions; for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 fractions.

[0092] In various embodiments, the cancer therapy is immunotherapy. Examples of immunotherapy include but are not limited to the immune checkpoint inhibitors such as anti- CTLA4 and anti-PDl / PDLl. Additional examples include but are not limited to adoptive cellular therapy (e.g., Chimeric antigen receptor (CAR) T cell therapy, Chimeric antigen receptor (CAR) natural killer (NK) cell therapy, Tumor infiltrating lymphocyte (TIL) therapy, Endogenous T cell (ETC) therapy), Cancer vaccines, Monoclonal antibodies, and Cytokine therapy.

[0093] Examples of anti-CTLA4 include but are not limited to ipilimumab (MDX-010) and tremelimumab (CP -675, 206) which are fully human antibodies against CTLA-4.

[0094] In various embodiments, the PD1 inhibitor can be selected from the group consisting of pembrolizumab, balstilimab, nivolumab, pidilizumab, AMP-224, AMP-514, spartalizumab, cemiplimab, penpulimab (AK105), prolgolimab (BCD- 100), ezabenlimab (BI 754091), toripalimab (JS001), lipustobart (LZM009), retifanlimab (MGA012), Sym021, dostarlimab (TSR-042), tebotelimab (MGD013), cadonilimab (AK104), vudalimab (XmAb20717), tislelizumab, PF-06801591, anti-PDl antibody expressing pluripotent killer T lymphocytes (PIK-PD-1), autologous anti-EGFRvIII 4SCAR-IgT cells, and combinations thereof. In particular embodiments, the PD1 inhibitor is pembrolizumab or dostarlimab.

[0095] In various embodiments, the PDL1 inhibitor can be selected from the group consisting of garivulimab (BGB-A333), cosibelimab (CK-301), FAZ053, envafolimab (KN035), MDX-1105, betifisolimab (MSB2311), adebrelimab (SHR-1316), atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, M7824, and combinations thereof.

[0096] In various embodiments, the cancer therapy is chemotherapy.

[0097] In various embodiments, the chemotherapy is paclitaxel, cyclophosphamide, or both. Additional examples include but are not limited to Ariamycin (doxorubicin), cyclophosphamide, antibody-drug conjugates (e.g., TDM-1, TdxD and sacituzumab govitecan), and irinotecan. These may be beneficial for breast cancer.

[0098] In various embodiments, the chemotherapy is selected from dacarbazine, temozolomide, nab-paclitaxel, paclitaxel, cisplatin, carboplatin, and combinations thereof. These may be beneficial for melanoma.

[0099] In various embodiments, the chemotherapy is selected from Cisplatin, Carboplatin, Paclitaxel (Taxol), Albumin-bound paclitaxel (nab-paclitaxel, Abraxane), Docetaxel (Taxotere), Gemcitabine (Gemzar), Vinorelbine (Navelbine), Etoposide (VP-16), Pemetrexed (Alimta), and combinations thereof. These may be beneficial for lung cancer.

[0100] Additional examples of chemotherapeutic agents include cytotoxic agents (e.g., 5- fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin (Adriamycin®), vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucine phosphate sodium, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon alfa-2a recombinant, paclitaxel, teniposide, and streptozoci), cytotoxic akylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylesulfonic acid), alkylating agents (e.g., asaley, AZQ, BCNU, busulfan, bisulphan, carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cis-platinum, clomesone, cyanomorpholinodoxorubicin, cyclodisone, cyclophosphamide, dianhydrogalactitol, fluorodopan, hepsulfam, hycanthone, iphosphamide, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, streptozotocin, teroxirone, tetraplatin, thiotepa, triethylenemelamine, uracil nitrogen mustard, and Yoshi-864), antimitotic agents (e.g., allocolchicine, Halichondrin M, colchicine, colchicine derivatives, dolastatin 10, maytansine, rhizoxin, paclitaxel derivatives, paclitaxel, thiocolchicine, trityl cysteine, vinblastine sulfate, and vincristine sulfate), plant alkaloids (e.g., actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mitramycin, mitomycin, daunorubicin, VP-16-213, VM-26, navelbine and taxotere), biologicals (e.g., alpha interferon, BCG, G-CSF, GM-CSF, and interleukin-2),topoisomerase I inhibitors (c.g, camptothecin, camptothecin derivatives, and morpholinodoxorubicin), topoisomerase II inhibitors (e.g., mitoxantron, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene HCL, daunorubicin, deoxydoxorubicin, menogaril, N,N-dibenzyl daunomycin, oxanthrazole, rubidazone, VM-26 and VP-16), and synthetics (e.g., hydroxyurea, procarbazine, o,p'-DDD, dacarbazine, CCNU, BCNU, cis- diamminedichloroplatimun, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, gliadel and porfimer sodium).

[0101] In various embodiments, the cancer is breast cancer. Examples of breast cancer include but are not limited to breast cancer such as a ductal carcinoma in duct tissue in a mammary gland, medullary carcinomas, colloid carcinomas, tubular carcinomas, and inflammatory breast cancer. In other embodiments, the cancer is melanoma or lung cancer. Examples of lung cancer include but are not limited to as non-small cell lung cancer (NSCLC), which is divided into squamous cell carcinomas, adenocarcinomas, and large cell undifferentiated carcinomas, and small cell lung cancer.

[0102] Various embodiments provide for a method of treating cancer in a subject in need thereof, comprising: administering a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based system to delete a DNA-binding region of a transcription factor that mediates one or more suppressive pathways in the macrophage; administering macrophages lacking a transcription factor that mediates one or more suppressive pathways in the macrophage; and administering a cancer therapy.

[0103] In various embodiments, the transcription factor is GATA-3. As such, macrophages lacking GATA-3 are administered.

[0104] In various embodiments, the macrophages are differentiated from myeloid progenitor cells, induced pluripotent stem cells (iPSCs), or hematopoietic stem cells.

[0105] In various embodiments, the cancer therapy is radiation, immunotherapy, chemotherapy or combinations thereof.

[0106] In various embodiments, the cancer therapy is radiation. Fraction sizes can be about 2-34 Gy given in 1-3 fractions. In particular embodiments, the fraction sizes can be about2 Gy, 16, Gy or 34 Gy, given as one fraction. In particular embodiments, the fraction sizes can be about 2 Gy, 5 Gy or 9 Gy, given as three fractions.

[0107] In various embodiments, the fraction sizes range from 2-34 Gy in 1-35 doses. In various embodiments, the fraction sizes range from 2-10 Gy in 1-35 doses. In various embodiments, the fraction sizes range from 2-10 Gy in 1-20 doses, 21-25 doses, 26-30 doses, or 31-35 doses.

[0108] In various embodiments, the fraction sizes range from 11-20 Gy in 1-35 doses. In various embodiments, the fraction sizes range from 11-20 Gy in 1-20 doses or in 21-35 doses.

[0109] In various embodiments, the fraction sizes range from 21-30 Gy in 1-35 doses. In various embodiments, the fraction sizes range from 21-30 Gy in 1-10 doses, 11-20 doses, or 21- 35 doses.

[0110] In various embodiments, the fraction sizes range from 31-34 Gy in 1-35 doses. In various embodiments, the fraction sizes range from 31-34 Gy in 1-5 doses, 6-10 doses, 11-15 doses, 16-20 doses, or 21-35 doses.[0U1] In various embodiments, the fraction sizes can be 2 Gy x 25-30 doses, 6 Gy x 5 doses, 8 Gy x 1 dose, 8 Gy x 3 doses, 9 Gy x 3 doses, 16 Gy x 1 dose, 20 Gy x 1 dose, 24 Gy x 1 dose or 34 Gy x 1 dose.

[0112] In various embodiments, the fraction sizes is any one or more of about 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, 71-75, 76- 80, 81-85, 86-90, 91-95, or 96-100 Gy. These doses may be given in one or more fractions; for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 fractions.

[0113] In various embodiments, the cancer therapy is immunotherapy. Examples of immunotherapy include but are not limited to the immune checkpoint inhibitors such as anti- CTLA4 and anti-PDl / PDLl. Additional examples include but are not limited to adoptive cellular therapy (e.g., Chimeric antigen receptor (CAR) T cell therapy, Chimeric antigen receptor (CAR) natural killer (NK) cell therapy, Tumor infdtrating lymphocyte (TIL) therapy, Endogenous T cell (ETC) therapy), Cancer vaccines, Monoclonal antibodies, and Cytokine therapy.

[0114] Examples of anti-CTLA4 include but are not limited to ipilimumab (MDX-010) and tremelimumab (CP-675,206) which are fully human antibodies against CTLA-4.

[0115] In various embodiments, the PD1 inhibitor can be selected from the group consisting of pembrolizumab, balstilimab, nivolumab, pidilizumab, AMP -224, AMP-514, spartalizumab, cemiplimab, penpulimab (AK105), prolgolimab (BCD- 100), ezabenlimab (BI 754091), toripalimab (JS001), lipustobart (LZM009), retifanlimab (MGA012), Sym021, dostarlimab (TSR-042), tebotelimab (MGD013), cadonilimab (AK104), vudalimab (XmAb20717), tislelizumab, PF-06801591, anti-PDl antibody expressing pluripotent killer T lymphocytes (PIK-PD-1), autologous anti-EGFRvIII 4SCAR-IgT cells, and combinations thereof. In particular embodiments, the PD1 inhibitor is pembrolizumab or dostarlimab.

[0116] In various embodiments, the PDL1 inhibitor can be selected from the group consisting of garivulimab (BGB-A333), cosibelimab (CK-301), FAZ053, envafolimab (KN035), MDX-1105, betifisolimab (MSB2311), adebrelimab (SHR-1316), atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, M7824, and combinations thereof.

[0117] In various embodiments, the cancer therapy is chemotherapy.

[0118] In various embodiments, the chemotherapy is paclitaxel, cyclophosphamide, or both. Additional examples include but are not limited to Ariamycin (doxorubicin), cyclophosphamide, antibody-drug conjugates (e.g., TDM-1, TdxD and sacituzumab govitecan), and irinotecan. These may be beneficial for breast cancer.

[0119] In various embodiments, the chemotherapy is selected from dacarbazine, temozolomide, nab-paclitaxel, paclitaxel, cisplatin, carboplatin, and combinations thereof. These may be beneficial for melanoma.

[0120] In various embodiments, the chemotherapy is selected from Cisplatin, Carboplatin, Paclitaxel (Taxol), Albumin-bound paclitaxel (nab-paclitaxel, Abraxane), Docetaxel (Taxotere), Gemcitabine (Gemzar), Vinorelbine (Navelbine), Etoposide (VP- 16), Pemetrexed (Alimta), and combinations thereof. These may be beneficial for lung cancer.

[0121] Additional examples of chemotherapeutic agents include cytotoxic agents (e.g., 5- fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin (Adriamycin®), vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucine phosphate sodium, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon alfa-2a recombinant, paclitaxel, teniposide, and streptozoci), cytotoxic akylatingagents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylesulfonic acid), alkylating agents (e.g., asaley, AZQ, BCNU, busulfan, bisulphan, carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cis-platinum, clomesone, cyanomorpholinodoxorubicin, cyclodisone, cyclophosphamide, di anhydrogal actitol, fluorodopan, hepsulfam, hycanthone, iphosphamide, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, streptozotocin, teroxirone, tetraplatin, thiotepa, triethylenemelamine, uracil nitrogen mustard, and Yoshi-864), antimitotic agents (e.g., allocolchicine, Halichondrin M, colchicine, colchicine derivatives, dolastatin 10, maytansine, rhizoxin, paclitaxel derivatives, paclitaxel, thiocolchicine, trityl cysteine, vinblastine sulfate, and vincristine sulfate), plant alkaloids (e.g., actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mitramycin, mitomycin, daunorubicin, VP-16-213, VM-26, navelbine and taxotere), biologicals (e.g., alpha interferon, BCG, G-CSF, GM-CSF, and interleukin-2), topoisomerase I inhibitors (e.g., camptothecin, camptothecin derivatives, and morpholinodoxorubicin), topoisomerase II inhibitors (e.g., mitoxantron, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene HCL, daunorubicin, deoxydoxorubicin, menogaril, N,N-dibenzyl daunomycin, oxanthrazole, rubidazone, VM-26 and VP-16), and synthetics (e.g., hydroxyurea, procarbazine, o,p'-DDD, dacarbazine, CCNU, BCNU, cis- diamminedichloroplatimun, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, gliadel and porfimer sodium).

[0122] In various embodiments, the CRISPR-based system comprises a vector encoding a guide RNA targeting Zn-finger DNA binding domain of GATA-3; and a vector encoding Cas9, Casl2a, or Casl3.

[0123] In various embodiments, the cancer is breast cancer. Examples of breast cancer include but are not limited to breast cancer such as a ductal carcinoma in duct tissue in a mammary gland, medullary carcinomas, colloid carcinomas, tubular carcinomas, and inflammatory breast cancer. In other embodiments, the cancer is melanoma or lung cancer. Examples of lung cancer include but are not limited to as non-small cell lung cancer (NSCLC), which is divided into squamous cell carcinomas, adenocarcinomas, and large cell undifferentiated carcinomas, and small cell lung cancer.

[0124] In various embodiments, the present invention provides pharmaceutical compositions including a pharmaceutically acceptable excipient along with a therapeutically effective amount of macrophages lacking a transcription factor that mediates one or more suppressive pathways in the macrophages. “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, nontoxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0125] In certain embodiments, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term “pharmaceutically acceptable salts, esters, amides, and prodrugs” as used herein refers to those carboxylate salts, amino acid addition salts, esters, amides, and prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use of the compounds of the invention. The term “salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. These may include cations based on the alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylanunonium, tetraethyl ammonium, methyl amine, dimethyl amine, trimethylamine, triethylamine, ethylamine, and the like (see, e.g., Berge S. M., et al. (1977) J. Pharm. Sci. 66, 1, which is incorporated herein by reference).

[0126] The term “pharmaceutically acceptable esters” refers to the relatively nontoxic, esterified products of the compounds of the present invention. These esters can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting thepurified compound in its free acid form or hydroxyl with a suitable esterifying agent. Carboxylic acids can be converted into esters via treatment with an alcohol in the presence of a catalyst. The term is further intended to include lower hydrocarbon groups capable of being solvated under physiological conditions, e.g., alkyl esters, methyl, ethyl and propyl esters.

[0127] As used herein, “pharmaceutically acceptable salts or prodrugs” are salts or prodrugs that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subject without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use.

[0128] The term “prodrug” refers to compounds that are rapidly transformed in vivo to yield the functionally active one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof. A thorough discussion is provided in T. Higachi and V. Stella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 of the A. C. S. Symposium Series, and in Bioreversible Carriers in: Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are hereby incorporated by reference. As used herein, a prodrug is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound. A prodrug of the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof can be designed to alter the metabolic stability or the transport characteristics of one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof, to mask side effects or toxicity, to improve the flavor of a compound or to alter other characteristics or properties of a compound. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, once a pharmaceutically active form of the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof, those of skill in the pharmaceutical art generally can design prodrugs of the compound (see, e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, N. Y., pages 388-392). Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in “Design of Prodrugs,” ed. H. Bundgaard, Elsevier, 1985. Suitable examples of prodrugs include methyl, ethyl and glycerol esters of the corresponding acid.

[0129] In various embodiments, the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration. “Route ofadministration” may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal or parenteral.

[0130] “Transdermal” administration may be accomplished using a topical cream or ointment or by means of a transdermal patch. “Parenteral” refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders. Via the enteral route, the pharmaceutical compositions can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection. Via the topical route, the pharmaceutical compositions based on compounds according to the invention may be formulated for treating the skin and mucous membranes and are in the form of ointments, creams, milks, salves, powders, impregnated pads, solutions, gels, sprays, lotions or suspensions. They can also be in the form of microspheres or nanospheres or lipid vesicles or polymer vesicles or polymer patches and hydrogels allowing controlled release. These topical-route compositions can be either in anhydrous form or in aqueous form depending on the clinical indication. Via the ocular route, they may be in the form of eye drops.

[0131] The pharmaceutical compositions according to the invention can also contain any pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier” as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity,irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

[0132] The pharmaceutical compositions according to the invention can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

[0133] The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or nonaqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.

[0134] The pharmaceutical compositions according to the invention may be delivered in a therapeutically effective amount. The precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for instance, by monitoring a subject’s response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

[0135] Typical dosages of an effective macrophages lacking a transcription factor that mediates one or more suppressive pathways in the macrophages can be as indicated to the skilled artisan by the in vitro responses or responses in animal models. Such dosages typically can be reduced by up to about one order of magnitude in concentration or amount without losing the relevant biological activity. Thus, the actual dosage will depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsiveness of the relevant primary cultured cells or histocultured tissue sample, such as biopsied malignant tumors, or the responses observed in the appropriate animal models, as previously described.Kits

[0136] The present invention is also directed to a kit to treat cancer. The kit is useful for practicing the inventive method of treating cancer The kit is an assemblage of materials or components, including at least one of the inventive compositions. Thus, in some embodiments the kit contains a composition including macrophages lacking a transcription factor that mediates one or more suppressive pathways in the macrophages, as described above.

[0137] The exact nature of the components configured in the inventive kit depends on its intended purpose. For example, some embodiments are configured for the purpose of treating cancer. In one embodiment, the kit is configured particularly for the purpose of treating mammalian subjects. In another embodiment, the kit is configured particularly for the purpose of treating human subjects. In further embodiments, the kit is configured for veterinary applications, treating subjects such as, but not limited to, farm animals, domestic animals, and laboratory animals.

[0138] Instructions for use may be included in the kit. “Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit to affect a desired outcome, such as to treat cancer. Optionally, the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandaging materials or other useful paraphernalia as will be readily recognized by those of skill in the art.

[0139] The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. The packaging materials employed in the kit are those customarily utilized in treating cancer As used herein, the term “package” refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. Thus, for example, a package can be a glass vial used to contain suitable quantities of an inventive composition containing macrophages lacking a transcription factor that mediates one or more suppressive pathways in the macrophages. The packaging material generally has an external label which indicates the contents and / or purpose of the kit and / or its components.EXAMPLES

[0140] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.Example 1

[0141] We generated a macrophage specific GATA-3 KO mouse model (mG3K0) driven by the LysM-Cre promoter. Using a syngeneic orthotopic murine model of breast cancer (EO771), we evaluated the differential effect of RT (16Gy x 1, X-RAD SmART) in WT and mG3K0 mice. Multiparametric flow cytometry was performed to investigate the immune changes within the tumor microenvironment on day 3, day 5 and day 10 after RT. T celldepletion was performed using antibodies to CD4 and CD8 by intraperitoneal injections to understand the role of adaptive immunity in the response to RT in WT and mG3K0 mice.

[0142] We found that mG3K0 mice bearing advanced EO771 tumors demonstrated significantly improved tumor regression compared to WT mice (p<0.001), which translated to increased overall survival. In vitro characterization of bone-marrow derived macrophages from mG3K0 and WT mice suggest that macrophages with ablated GATA-3 expressed increased levels of iNOS and decreased levels of Arginase (Arg-1), consistent with an Ml-like phenotype. Immune profiling of the tumors also revealed that mGATA-3 KO animals have significant enrichment of CD8+ T cells in the tumor milieu post RT and these CD8+ T cells express higher amounts of interferon gamma (p<0.001) and Granzyme B (p<0.0015) than their WT counterparts. Using neutralizing antibodies to deplete CD8+ T cells, we show that anti-tumor effects in the mG3K0 mice were abolished, suggesting that mG3K0 macrophages impact survival, at least, in part by reinvigorating cytotoxic CD8+T cells.Example 2E0771 Model and macrophages in breast cancer

[0143] We have demonstrated using the E0771 mouse model of breast cancer that the efficacy of RT depends in part on the development of an anti-tumor immune response. This model of breast cancer is a model where the original mammary cancer cells arose in a C57BL / 6 mouse and genetically mimics the human triple-negative subtype of breast cancer. Moreover, the profile of leukocytes that infiltrate mammary adenocarcinomas in E0771 mice mirrors that found in human breast cancers as evaluated histologically or by polychromatic flow cytometry. Studies in E0771 mice revealed that macrophages regulate the response to RT through modulation of their pro-tumor bioactivity. Thus, the behavior of tumors in this model is subject to immune regulation leading us to believe that targeting the macrophage pro-tumor bioactivity following RT by ablating the master downstream transcription factor GATA-3 would enhance anti-tumor activity elicited by RT. We created a GATA-3 fl / fl mouse that targets the DNA-binding domain of the transcription factor them crossed it to a LysM-Cre mouse which expressed the Cre recombinase in all myeloid cells.

[0144] In order to evaluate the efficacy of RT in our models, we developed an orthotopic transplantation strategy. Tumor fragments from E0771 tumor bearing mice are harvested and transplanted into the mammary fat pad on the lower abdomen of naive syngeneic mice and tumor development monitored until palpable tumors appear and achieve a size of 0.5 cm. Tumorbearing mice are then randomized and enrolled into treatment groups where primary tumor growth is monitored with and without genetically-modified macrophages and with and without RT prospectively to the study end-point.Macrophages deficient in GATA-3 dramatically enhances the efficacy of RT

[0145] To address the belief that suppressing the pro-tumor bioactivity of macrophages would enhance the response to RT, we used the orthotopic syngeneic E0771 transplantation in mice and irradiated them with a single-fraction of 16 Gy which we had determined to be a dose that modestly delays tumor growth, but does not inhibit entirely. Surprisingly, we observed in several experiments that in tumor-bearing mice whose macrophages lack GATA-3, a single-dose of RT dramatically slowed the tumor growth whereas the same dose in the wild-type control showed a modest delay comparable to unirradiated mice (Fig. 9A). This elimination led to longterm survival for the mice whose tumors had been eliminated (Fig. 9B). These data indicate that reprogramming immune microenvironments by targeting macrophage pro-tumor bioactivity may alter the survival of breast cancer patients by enhancing sensitivity to cytotoxic therapy.Example 3Effect of GATA-3-deficient macrophages on different doses of RT.

[0146] In our studies, we have used a single, experimentally-determined dose of RT (16 Gy, single-fraction) that we observed would delay tumor growth when delivered alone. Clinically, RT is typically delivered over multiple daily fractions (fractionated RT) ranging from 1 to 7 weeks, though advances in RT delivery have allowed for increasingly larger doses to be delivered safely, and clinical trials with these larger doses have demonstrated improved local efficacy.

[0147] Studies in a humanized mouse model of melanoma suggest that low dose RT (2 Gy) can prime the immune system, whereas other groups have shown in mouse models of breast cancer that multiple, larger doses (8-20 Gy) produce the strongest anti-tumor immune response.Given the overall paucity of data regarding the most immunostimulatory dose of RT, we will explore the effect of targeted disruption of GATA-3 in macrophages in conjunction with several different doses and fraction numbers to characterize the role the pro-tumor macrophage bioactivity plays in shaping the RT-induced anti-tumor immune responses.

[0148] To better model the doses and type of RT that is delivered clinically, we utilize an X-RAD Small Animal Image Guided Irradiation System (SmART) developed by Precision X- Ray. This device combines 3D volumetric imaging (computerized tomography) and optical imaging with accurate specimen positioning, allowing for precise, conformal image- guided radiation therapy to specific targets in mice. This approach allows for delivering highly focused and well-quantified doses of radiation to tumors in mice in a manner that is essentially identical to that used in the clinic for breast cancer patients. It is far superior to using a cesium-source irradiator or modified shielding techniques as is commonly employed in mouse studies.Table 1. Experimental Radiation Doses and Fractionation Schedules

[0149] Mice bearing either the LysM-Cre (mCre) or GATA-3 fl / fl x LysM-Cre (mGATA-3K0) will be implanted in their lower mammary fat pads with syngeneic E0771 mammary tumor cells as described above. Transplanted mice will be treated at 2 weeks when tumors are approximately 0.5 cm, tumor- bearing mice will then be divided into the appropriate experimental cohorts (10 mice / group) with treatment groups receiving RT with or without antibacterial or antifungal drug cocktails as described above, versus the control group receiving water or antibiotics alone with no RT. RT will consist of a single low dose (2 Gy), medium dose (16 Gy) or high dose (34 Gy). We will also test the effect of multiple daily doses of low dose (2 Gy, 5 Gy) or high dose (9 Gy) (Table 1 & Fig. 10). RT will be administered to a local field centered on the transplanted mammary tumor using the XRAD SmART, delivering X-rays from 50 - 225 kV using different lead applicators to deliver beam sizes ranging from 0.5 - 2.0 cm.

[0150] Since the study endpoint will be determined by tumor size, we will quantitatively measure tumor growth every 3-days throughout the studies using calipers. Tumors will be evaluated at three endpoints: 2, 4 and 10 days following RT. The leukocyte profile from treated and untreated tumors will be evaluated by scSeq, FACS and immunohistopathology as shown above (Fig. 8) and as we have previously published. T cell, macrophage and DC subsets will be evaluated for 1. activation and maturation status, 2. cytokine profile by intracellular FACS or quantitative PCR (qPCR) on FACS-isolated cells, and 3. functional phenotypes as recently described30 (Fig. 11). Together these data will reveal the activation and TH status of the individual leukocyte subtypes. The typical yield from a 1.0-cm tumor when dissociated is approximately 2-3xl06cells - approximately 2 x 104are needed for scSeq and 2.5xl05cells are required for FACS; thus, sufficient immune cells can be analyzed in distinct populations, and 10 mice / group will provide sufficient statistical power using the Student’s t-test to reveal differences between treatment groups. Tumor histopathology will be examined via immunohistochemical (IHC) and immunofluorescent (IF) approaches in paraffin-embedded and / or OCT-frozen tissue sections for other parameters regulating tumor progression including proliferation via BrdU, cell death via cleaved caspase-3, angiogenesis via CD31, and hypoxia via hypoxyprobe staining as previously published. Sections will be analyzed by digital scanning and enumeration using the Aperio digital pathology system and quantitatively evaluated using the Student’s t-test with significance at a p value < 0.05 between groups (Fig. 11). Together these parameters, in concert with leukocyte profiles, will reveal how different RT doses influence the angiogenic, proliferative and apoptotic properties of the tumor and will shed light on potential mechanism(s) underlying the effect of reprogramming macrophages on RT.Effect of GATA-3-deficient macrophages on treatment-induced systemic anti-tumor immunity

[0151] In addition to a local inflammatory response, several case reports and multiple mouse models have demonstrated that RT can produce a systemic anti-tumor immune response targeting tumors outside of the irradiated field (an “abscopal” response). This RT-mediated abscopal effect depends on DCs and generating a productive cytotoxic CD8+ T cells. Recentpublications from our lab and others have revealed that this systemic anti-tumor immune response can be highly potentiated by immunotherapy with immune checkpoint inhibitors, different immune agonists and combinations of these two agents. Further, that immunotherapy in combination with chemotherapy show tremendous synergy clinically. In addition, studies in several mouse models have revealed that depletion of macrophages affects the efficacy of chemotherapy the immune checkpoint inhibitors anti-CTLA4 and anti-PDl / PDLl mAbs. However, the impact of targeting the GAT A-3 -mediated programming in macrophages on the effect of immunotherapeutic agents remains unknown, and the impact of GAT A-3- mediated programming in macrophages on the RT-induced abscopal response has not yet been described.

[0152] To address this, we propose several studies to understand the effect of macrophages lacking GATA-3 on the development of systemic anti-tumor immunity following RT and / or immunotherapy in our mouse model of breast cancer. We will again utilize our orthotopic transplant model, however for these experiments mCre or mGATA-3KO mice will be implanted in two opposing mammary fat pads with syngeneic E0771 mammary tumor cells to mimic metastatic disease (Fig. 12). For RT, the tumor will be treated with either a single dose (16 Gy) or multiple doses (9 Gy x 3) of RT. Both doses of RT have been described to optimally prime a systemic immune response following RT in murine models though doses determined from the above example would also inform the RT doses for this sub-Aim and subsequent ones. We will similarly test anti-PD-1 and anti-CTLA-4 using doses and schedules determined previously. Tumor growth will be monitored for both tumors and altered tumor growth kinetics will be compared between the mCre and mGATA-3K0 groups following treatment. As the abscopal effect mediated by RT alone is difficult to elicit, we will also explore the role of the mGATA-3K0 in the RT-induced abscopal response in the presence of the checkpoint inhibitor anti-PDl, which has been described previously to enhance the RT -mediated systemic anti -tumor immune response. Tumor growth, immune profiles and parameters will be quantitatively assessed using the Aperio digital pathology system and quantitatively evaluated using the Student’s T-test with significance at a p value < 0.05 between groups.Expected outcomes and alternative approaches

[0153] These studies will establish the relationship between macrophage pro-tumor bioactivity and the efficacy of RT and immunotherapy in generating both a local and systemicanti-tumor immune responses. As we already have data herein showing its positive impact in RT, we anticipate that in the absence of one of the key pro-tumor pathways in macrophages that the TH1 immune microenvironment induced following treatment will have enhanced anti-tumor activity both in the primary tumor and systemically for immunotherapy as well. Our studies will reveal to what degree this response is mediated by the varied presence or altered activation / maturation of TH1 CD4+ T cells, CD8+ T cells, T regulatory (Treg) cells, different myeloid programs or other myeloid suppressive cells, accompanied by possibly more mature DCs (based on phenotypic markers such as MHC class II and cytokine expression, e.g., interferon IFNy, IL-12, Granzyme A and B). Alternatively, a distinct leukocyte subtype may not emerge as specifically important, but instead a shifted cytokine microenvironment may emerge (lower IFNy, IL- 17 and possibly elevated TGF0) indicative of a “stronger” THl-type anti-tumor state. Our experimental strategy will not discriminate but instead will monitor all possibilities. If the abscopal response is not optimal with RT and anti-PDl we will utilize anti-CTLA-4 mAb with RT or in combination with the anti-PD-1 Ab to elicit the RT -induced abscopal response. This detailed examination of therapy-induced immune responses will identify potential molecular / cellular pathways to target therapeutically.Example 4Innate Immune Responses

[0154] We will first study the effect of myeloid GATA-3 ablation in mice deficient in neutrophils or DCs to elucidate which specific innate leukocyte populations may be important. As above, mice will be implanted in their mammary fat pads with syngeneic E0771 mammary tumor cells, and primary tumors will be treated with either single-dose or fractionated RT in mGATA-3K0 mice or mCre controls. We will then use the anti-Ly6G antibody (1A8, Bio X Cell) to deplete neutrophils or the CDl lc-DTR transgenic crossed to the LysM-Cre x GATA-3 fl / fl mice in which a transgene for diphtheria toxin is under control of the CDl lc promoter combined with diphtheria toxin administration to deplete DCs which are CDl lc+ (Fig. 14). Additionally, key cells / cytokines identified above will also be evaluated for significance by comparing between mice that are specifically depleted of leukocyte subsets or cytokines as appropriate and following their effect on the growth parameters on the implanted tumors. Alteredtumor growth kinetics will be compared between immune- complete versus immune-modified groups following treatment with RT (16 Gy, 9 Gy x 3), or PD-l / CTLA-4 directed immunotherapy in mGATA-3Ko versus Cre. We will follow tumor growth with calipers; assess intratumoral immune profiles via scSeq and flow cytometry; and quantify changes in angiogenesis, proliferation and apoptosis using the Aperio digital pathology system. Resulting data will be evaluated using the Student’s t-test with significance at a p value < 0.05 between groups.Adaptive Immune Responses

[0155] As above, mice will be implanted in their mammary fat pads with syngeneic E0771 mammary tumor cells and primary tumors will be treated with either RT (16 Gy, 9 Gy x 3), or PD-l / CTLA-4 directed immunotherapy in mGATA-3KO versus mCre mice. We will first study the effect of mGATA-3KO and treatment in mice deficient in CD4+ T cells and CD8+ T cells. We will also test mice that have the key cytokines driving Thl and Th2 polarization, IL-4 and IFNy, using blocking antibodies (Fig. 14). This set of experiments will allow us to elucidate which specific leukocyte populations and polarizing cytokines mediate the effects we see on the efficacy of RT in the setting of macrophages without pro-tumor programming based on our hypothesis that the RT-mediated anti-tumor immune response depends on the generation of a productive THl-mediated response. Additionally, cells / cytokines identified above may also be evaluated for significance by comparing between mice that are specifically depleted of leukocyte subsets or cytokines as appropriate and following their effect on the growth parameters on the implanted tumors in mGATA-3KO and Cre controls. Altered tumor growth kinetics will be compared between immune-complete versus immune-modified groups following treatment with RT and immunotherapy in mGATA-3KO and Cre control mice. Tumor growth, immune profiles and parameters will be quantitatively assessed using the Aperio digital pathology system and quantitatively evaluated using the Student’s T-test with significance at a p value < 0.05 between groups.Macrophage Functional Analysis

[0156] To elucidate the cellular mechanisms driving the enhanced therapeutic efficacy in tumors bearing GATA-3- deficient macrophages, we will test the functional capacity of these macrophages in vitro for the key inflammation associated parameters: ability to polarize,cytokine production, generation of reactive oxygen species (ROS), phagocytosis and antigen presentation. In order to test these functions, we will generate bone-marrow derived macrophages from mGATA-3K0 and LysM- Cre controls. To identify whether mGATA-3K0 macrophages polarize differently in response to cytokines, we will treat mGATA-3K0 and LysM-Cre macrophages with classic polarizing cytokines lipopolysaccharide (LPS)+IFNy and IL-4 for 24-48 hours and look for markers of classic (iNOS, CD38, IL-12a) and alternative activation pathways (Arginase-1, Retnla, Chi313) as described in Jablonski et al. To test for cytokine production, we will treat mGATA-3K0 and LysM-Cre macrophages with different key inflammatory stimuli for 6-12 hours including LPS (TLR4 ligand), imiquimod (TLR7 ligand) and CpG (TLR9 ligand) and look for major inflammatory cytokines tumor necrosis factor (TNF)-a and IL-6 production by sandwich ELISA (Biolegend). We will also test STING agonist (cGAMP) in both macrophage types to look for Type I interferon production by ELISA. To look at ROS production we will prime BMDMs with from either mGATA-3K0 and LysM-Cre with IFNg / LPS for 24 hours and then feed them the fungal ligand zymosan using the CellROX colorimetric assay (ThermoFisher) as previously published. For phagocytosis, we will test the capacity of mGATA-3K0 and LysM-Cre macrophages to consume fluorescently-labeled polystrene beads (Vybrant Phagocytosis Assay Kit, Molecular Probes) to test for pure phagocytic capacity as well as fluorescently-labeled irradiated E0771 tumor cells. Briefly, we will plate mGATA-3K0 and LysM-Cre macrophages and incubate them with a set number of beads after 24 hours the unconsumed beads are quenched with trypan blue and the macrophages are then subjected to flow cytometry for analysis. Similarly, E0771 cells will be labeled with the cell permeant dye carboxyfluorescein di acetate succinimidyl ester (CFDA) then irradiated with 16 Gy, cells will then be transferred to pre-plated macrophages and incubated for 24 hours. The amount of cellular material consumed will then be assessed by flow cytometry. Finally, to assess for antigen presentation, we will employ an ovalbumin (OVA) culture where we will feed LPS- primed mGATA-3K0 and LysM-Cre macrophages OVA and then co-culture them with CFDA- labeled CD8+ T cells harvested from OT-I mice, a transgenic line where all CD8+ T cells express a high-affinity receptor for OVA. The co-culture will then be incubated for 5 days as described previously.Expected outcomes and alternative approaches

[0157] Based on our pilot studies and data, we anticipate that the major cell type affected will be CD8+ T cells. These studies will reveal the degree to which the enhanced therapeutic outcome in mice with GATA-3 -deficient macrophages is reliant upon neutrophils, DCs and T cell responses. Cytokine depletion of IL-4 and fFNy, would allow us to evaluate if an altered cytokine milieu created by macrophages lacking pro-tumor programming instead regulates antitumor responses by CD8+ T cells. Regardless of the mechanism, our studies are designed to reveal dominant pathways for generation of anti-tumor responses following radiation and immune-based therapy that are regulated by the pro-tumor macrophage bioactivity. These can, in turn, be further manipulated in vivo to further explore augmentation of any responses revealed herein. Recognizing that many immune-based mechanisms regulated by interactions with the macrophages other than those we propose to examine exist and may play an important role in the development of therapy-mediated anti-tumor immunity, we would also plan to study alternative immune subsets and cytokines such as Treg cells, IL10, IL12 and IL17 as time and resources permit. Moreover, since macrophages have a diverse and multi-pronged interaction with immunity we will determine which cellular mechanisms of inflammation in macrophages contributing to the therapy-elicited anti-tumor immune response.Example 5Determine parameters for adoptive transfer of modified macrophages in combination with different therapies

[0158] We will test the effect of transferred GATA-3KO macrophages on the therapy- induced anti-tumor immune response. Our preliminary studies with RT and transferred GATA- 3KO macrophages demonstrated that transferred macrophages can similarly enhance the response to RT to the genetically ablated model. To understand whether this therapeutic enhancement could be further optimized, we will implant tumors in WT mice and transfer various numbers of macrophages deficient in GATA-3 with different timing (multiple doses) in combination with a single-dose of RT (16 Gy). These mice experiments will enable us to identify the optimal number and dosing schedule for transferred cells. Using this optimized protocol, we will then test it with multi- fraction RT (9 Gy x 3), chemotherapy (paclitaxel, cyclophosphamide)and immunotherapy to determine whether GATA-3K0 macrophages can act as therapeutic partner for different therapies (Fig. 17).

[0159] We will quantitatively measure tumor growth every 3-days throughout the studies using calipers. Tumors will be evaluated at three endpoints: 1, 4 and 10 days following. The leukocyte profile from untreated and treated tumors will be evaluated by scSeq, FACS and immunohistopathology as we have previously published and shown above in Figure 8. T cell, macrophage and DC subsets will be evaluated for 1. activation and maturation status, 2. cytokine profile by intracellular FACS or quantitative PCR (qPCR) on FACS-isolated cells, and 3. functional phenotypes as recently described30 (Fig. 11). Together these data will reveal the activation and TH status of the individual leukocyte subtypes from mice where only some of the macrophages are deficient in GATA-3 in comparison to the studies from above. Tumor histopathology will be examined via immunohistochemical (IHC) and immunofluorescent (IF) approaches in paraffin-embedded and / or OCT-frozen tissue sections for other parameters regulating tumor progression including proliferation via BrdU, cell death via cleaved caspase-3, angiogenesis via CD31, and hypoxia via hypoxyprobe staining as previously published and shown above (Fig. 11). Sections will be analyzed by digital scanning and enumeration using the Aperio digital pathology system and quantitatively evaluated using the Student’s t-test with significance at a p value < 0.05 between groups. Together these parameters, in concert with leukocyte profiles, will reveal how a GATA-3-deficeint macrophage cellular therapy alters the tumor immune microenvironment alone and in combination with other therapies and how a combination treatment would influence the angiogenic, proliferative and apoptotic properties of a tumor and will shed light on the potential of this treatment to enhance the efficacy of multiple cancer treatments in breast cancer.Generation of human macrophages deficient in GATA-3

[0160] We will characterize the behavior of human macrophages generated from iPSC cells that have been genetically-engineered with lentivirus to delete the DNA binding domain of GATA-3. Macrophages generated from iPSC behave similarly to differentiated macrophages- derived from the circulation in response to various stimuli 1 and thus given the more readily alterable starting iPSC cell, we elected to use these cells as the testing ground for the experiments to engineer human myeloid cells. To understand whether macrophages lackingGATA-3 generated from human iPSC cells behave similarly to their murine counterparts, we will examine them first in vitro. To that end we will examine the functional capacity of these macrophages in vitro for the key inflammation associated parameters: ability to polarize, cytokine production, generation of reactive oxygen species (ROS) and phagocytosis. The behavior of the human mGATA-3K0 macrophages will be compared to the results from above to ensure they mirror the behavior of their murine counterparts. We will then confirm the viability of our approach using additional iPSC cells lines to get a measure of the impact of the different baseline genetics of the different lines on the behavior of our engineered macrophages. Then, time and resources, permitting we will test one of our lines in a PDX model of breast cancer in collaboration with the Cedars-Sinai Rodent Genetics Core which maintains several breast PDX model animal lines.Expected outcomes and alternative approaches

[0161] Given our preliminary studies, we do not anticipate major problems doing adoptive transfer of macrophages nor difficulty generating GATA-3-deficeint human macrophages. As our pilot experiments found that transfer of mGATA-3K0 macrophages enhances the response to RT, we believe that it will behave similarly with other therapies though either a positive or negative result would be informative for future preclinical and clinical studies. For the human macrophage studies, we might find that genetically-modified macrophages from iPSCs do not behave like their murine counterparts in which case we will utilize hematopoietic stem cells or monocytes from PBMC as an alternative starting cell for modification.Example 6

[0162] All animal experiments will be performed with 10 animals per experimental group as we have been able to demonstrate statistically significant differences in the past with tumor growth curves (using mixed model regression) and overall survival (log-rank test). Analysis of FACS data will be performed with two-way ANOVA. All post hoc tests will be adjusted for multiple comparisons with Tukey’s method.

[0163] All testing will be two-tailed, and differences will be considered significant where p<0.05. As a general estimate of power for all animal experiments, using preliminary data fromtumor growth curves and SD=675mm2(see Fig 13 ), assuming a study design of two factors, (one with 4 levels and one with 2 levels) groups of 10 animals per group, with at least 15 repeated measures within each subject, in mixed model regression we will have at least 80% power to detect a minimum difference of 250mm2for interaction terms with time. (Power estimated with 100 simulations using PASS vl4 software.)

[0164] Taken together, the data from these experiments will reveal (1) how the macrophages that lack pro-tumor programming via GATA-3 affects the efficacy of RT, chemotherapy and immunotherapy on both on primary tumors and systemically; (2) the immunologic and cellular mechanism of the enhanced anti-tumor immune response following therapy when GATA-3 is eliminated from macrophages (3) whether targeting GATA-3 in macrophages can create an effective cellular therapy to use in combination with the current standard of care therapies in breast cancer. The data from these experiments will indicate how the anti -turn or immune response following different breast cancer treatments is regulated by the macrophage population and provide the basis for human trials to explore the benefit of manipulating macrophage programming to shape the therapeutically- mediated anti-tumor immune responses.Example 7 Macrophage DifferentiationCD34+ HSC isolation

[0165] Human CD34+stem cells were isolated from leukopaks obtained from the Cedars- Sinai Blood Bank. Leukopaks were removed from liquid nitrogen storage and thawed in a 37°C water bath. Immediately after thawing, approximately 30mL of blood was transferred from the leukopak into 3-4 50mL conical tubes. 15mL of dPBS was added to each tube and the tubes were centrifuged at 500rcf for lOmin. To remove large chunks and other debris in the blood, the pellet was resuspended in 15mL of dPBS and then filtered through a 70pm filter followed by a 40pm filter (both filters pre-wet with 2mL of PBS). The remaining cells were then centrifuged again at 800rcf for 5min, resuspended in 5mL of dPBS and then filtered through another pre-wet 40pm filter. At this point a cell count was performed on the Countess II FL, and lOOpL of FcRBlocking Reagent and CD34 Microbeads (Miltenyi, 130-046-702) were added per 1 x 108cells and incubated at 4°C for 30min. Following incubation, the samples were magnetically separated on the autoMACS NEO and then added to a 6-well plate for culturing in StemSpan SFEM II media (STEMCELL Technologies, 09655) supplemented with StemSpan CD34+ Expansion Supplement (STEMCELL Technologies, 02691) according to manufacturer protocol. After 4 days in culture, myeloid differentiation was initiated by adding StemSpan Myeloid Expansion Supplement II 100X (STEMCELL Technologies, 02694).GATA3 knockout and sorting

[0166] The GATA3 plasmid was created using a pLentiCRISPR-mCherry backbone (Addgene, 75161). The bacteria were streaked onto an LB agar plate with lOOpg / mL of ampicillin and incubated overnight. Colonies were picked the following morning and cultured on a shaker (37°C, 250rpm) in 5mL of LB broth with lOOpg / mL of ampicillin again overnight. 2mL of the turbid media was taken the next morning and added to 98mL of LB broth and cultured for an additional 4-6hrs on the shaker. The turbid media was transferred to two 50mL conical tubes and a midi prep (Omega Bio-Tek D6904-03) was performed to isolate the GATA3 plasmid. Lentiviral packaging was conducted in HEK293T cells that were above 80% confluency in a 10cm plate using the 3rdGeneration Packaging System (Applied Biological Materials Inc., LV053) and Lipofectamine 3000 (Invitrogen™, L3000008) according to manufacturer protocol. Viral supernatants were pooled together, and the lentivirus was concentrated using the Speedy Lentivirus Purification reagent (Applied Biological Materials Inc., LV999) according to manufacturer protocol.

[0167] Stem cells that had undergone myeloid differentiation for four days were seeded at 1.5 x 106cells per well in a 12-well plate and infected with virus at a 1 :4 virus to media ratio. 300pL of StemSpan SFEM II was added to each well along with 0.5pL of 8mg / mL polybrene and incubated for 5min. 150pL of the concentrated viral supernatant was then added to each well and cultured at 37°C overnight. The media was removed the following day and fresh media with myeloid expansion supplements was added, and cells were cultured for an additional 2-3 days before FACS sorting for mCherry+ cells. Sorted cells were seeded in a clear 96-well plate at 20,000 cells per well in RPMI (supplemented with 10% FBS and penicillin / streptomycin) andallowed to continue differentiating for seven days in preparation for the activation / pol arization experiment.Activation and polarization experiment

[0168] Macrophages positive and negative for the GATA3 knockout were first primed for 24hrs by treating with either 150U / mL IFNy (Peprotech) or 20ng / mL IL-4 (Peprotech). After priming, media was removed, and treatments were applied to the cells for 24hrs: untreated, 0.5pg / mL LPS, IFNy + LPS, or IL-4 + LPS. IFNy + LPS treatment was applied only to macrophages that had been primed with IFNy, and IFNy was added together with the LPS at the same concentration as used during priming. IL-4 + LPS treatment was applied only to macrophages primed with IL-4, and IL -4 was added together with the LPS at the same concentration as used during priming. Following the 24hrs of treatment, the supernatants were collected from each well and analyzed for TNFc concentration using the ELISA MAX™ Deluxe Set Human TNF-a (Biolegend, 430204).

[0169] Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and / or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[0170] The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particularuse contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

[0171] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of’ or “consisting essentially of.”

[0172] Unless stated otherwise, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the application (especially in the context of claims) may be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with theterm “for example.” No language in the specification should be construed as indicating any nonclaimed element essential to the practice of the application.

[0173] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0174] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating cancer in a subject in need thereof, comprising: administering macrophages lacking a transcription factor that mediates one or more suppressive pathways in the macrophage; and administering a cancer therapy.

2. A method of treating cancer in a subject in need thereof, comprising: administering a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based system to delete a DNA-binding region of a transcription factor that mediates one or more suppressive pathways in the macrophage; administering macrophages lacking a transcription factor that mediates the one or more suppressive pathways in the macrophage; and administering a cancer therapy.

3. The method of claim 1 or claim 2, wherein the transcription factor is GATA-3.

4. The method of any one of claims 1-3, wherein the cancer therapy is radiation, immunotherapy, chemotherapy or combinations thereof.

5. The method of any one of claims 1-3, wherein the cancer therapy is radiation and the radiation therapy is given at a dosage of about 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31- 35, 36-40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-90, 91-95, or 96-100 Gy, and the doses are given in one or more fractions.

6. The method of any one of claims 1-3, wherein the cancer therapy is immunotherapy.

7. The method of claim 6, wherein the immunotherapy comprises an immune checkpoint inhibitor.

8. The method of claim 7, wherein the checkpoint inhibitor is anti-CTLA4, anti-PDl, anti- PDL1, or combinations thereof.

9. The method of claim 8, wherein the anti-CTLA4 is ipilimumab (MDX-010) or tremelimumab (CP-675,206).

10. The method of claim 8, wherein the anti-PDl is selected from the group consisting of pembrolizumab, balstilimab, nivolumab, pidilizumab, AMP-224, AMP-514, spartalizumab, cemiplimab, penpulimab (AK105), prolgolimab (BCD- 100), ezabenlimab(BI 754091), toripalimab (JS001), lipustobart (LZM009), retifanlimab (MGA012), Sym021, dostarlimab (TSR-042), tebotelimab (MGD013), cadonilimab (AK104), vudalimab (XmAb20717), tislelizumab, PF-06801591, anti-PDl antibody expressing pluripotent killer T lymphocytes (PIK-PD-1), autologous anti-EGFRvIII 4SCAR-IgT cells, and combinations thereof.

11. The method of claim 8, wherein the PD1 inhibitor is pembrolizumab or dostarlimab.

12. The method of claim 8, wherein the PDL1 inhibitor is selected from the group consisting of garivulimab (BGB-A333), cosibelimab (CK-301), FAZ053, envafolimab (KN035), MDX-1105, betifisolimab (MSB2311), adebrelimab (SHR-1316), atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, M7824, and combinations thereof.

13. The method of claim 6, wherein the immunotherapy comprises an adoptive cellular therapy, a cancer vaccine, monoclonal antibodies, or cytokine therapy.

14. The method of any one of claims 1-3, wherein the cancer therapy is chemotherapy.

15. The method of claim 14, wherein the chemotherapy is selected from the group consisting of paclitaxel, cyclophosphamide, doxorubicin, irinotecan and combinations thereof.

16. The method of claim 14, wherein the chemotherapy is selected from the group consisting of dacarbazine, temozolomide, nab-paclitaxel, paclitaxel, cisplatin, carboplatin, and combinations thereof.

17. The method of claim 14, wherein the chemotherapy is selected from the group consisting of Cisplatin, Carboplatin, Paclitaxel (Taxol), Albumin-bound paclitaxel (nab-paclitaxel, Abraxane), Docetaxel (Taxotere), Gemcitabine (Gemzar), Vinorelbine (Navelbine), Etoposide (VP- 16), Pemetrexed (Alimta), and combinations thereof.

18. The method of any one of claims 1-3, wherein the cancer therapy is an antibody-drug conjugate.

19. The method of claim 18, wherein the antibody-drug conjugate is selected from the group consisting of TDM-1, TdxD and sacituzumab govitecan, and combinations thereof.

20. The method of any one of claims 1-19, wherein the macrophages are differentiated from myeloid progenitor cells, induced pluripotent stem cells (iPSCs), or hematopoietic stem cells.

21. The method of any one of claims 2-19, wherein the CRISPR-based system comprisesa vector encoding a guide RNA targeting Zn-finger DNA binding domain of G ATA-3; and a vector encoding Cas9, Cast 2a, or Cast 3.

22. The method of claim 21, wherein the guide RNA comprises the sequence of SEQ ID NO:3.WHAT IS CLAIMED IS:

1. A method of treating cancer in a subject in need thereof, comprising: administering macrophages lacking a transcription factor that mediates one or more suppressive pathways in the macrophage; and administering a cancer therapy.

2. A method of treating cancer in a subject in need thereof, comprising: administering a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based system to delete a DNA-binding region of a transcription factor that mediates one or more suppressive pathways in the macrophage; administering macrophages lacking a transcription factor that mediates the one or more suppressive pathways in the macrophage; and administering a cancer therapy.

3. The method of claim 1 or claim 2, wherein the transcription factor is GATA-3.

4. The method of any one of claims 1-3, wherein the cancer therapy is radiation, immunotherapy, chemotherapy or combinations thereof.

5. The method of any one of claims 1-3, wherein the cancer therapy is radiation and the radiation therapy is given at a dosage of about 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31- 35, 36-40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-90, 91-95, or 96-100 Gy, and the doses are given in one or more fractions.

6. The method of any one of claims 1-3, wherein the cancer therapy is immunotherapy.

7. The method of claim 6, wherein the immunotherapy comprises an immune checkpoint inhibitor.

8. The method of claim 7, wherein the checkpoint inhibitor is anti-CTLA4, anti-PDl, anti- PDL1, or combinations thereof.

9. The method of claim 8, wherein the anti-CTLA4 is ipilimumab (MDX-010) or tremelimumab (CP-675,206).

10. The method of claim 8, wherein the anti-PDl is selected from the group consisting of pembrolizumab, balstilimab, nivolumab, pidilizumab, AMP-224, AMP-514, spartalizumab, cemiplimab, penpulimab (AK105), prolgolimab (BCD- 100), ezabenlimab(BI 754091), toripalimab (JS001), lipustobart (LZM009), retifanlimab (MGA012), Sym021, dostarlimab (TSR-042), tebotelimab (MGD013), cadonilimab (AK104), vudalimab (XmAb20717), tislelizumab, PF-06801591, anti-PDl antibody expressing pluripotent killer T lymphocytes (PIK-PD-1), autologous anti-EGFRvIII 4SCAR-IgT cells, and combinations thereof.

11. The method of claim 8, wherein the PD1 inhibitor is pembrolizumab or dostarlimab.

12. The method of claim 8, wherein the PDL1 inhibitor is selected from the group consisting of garivulimab (BGB-A333), cosibelimab (CK-301), FAZ053, envafolimab (KN035), MDX-1105, betifisolimab (MSB2311), adebrelimab (SHR-1316), atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, M7824, and combinations thereof.

13. The method of claim 6, wherein the immunotherapy comprises an adoptive cellular therapy, a cancer vaccine, monoclonal antibodies, or cytokine therapy.

14. The method of any one of claims 1-3, wherein the cancer therapy is chemotherapy.

15. The method of claim 14, wherein the chemotherapy is selected from the group consisting of paclitaxel, cyclophosphamide, doxorubicin, irinotecan and combinations thereof.

16. The method of claim 14, wherein the chemotherapy is selected from the group consisting of dacarbazine, temozolomide, nab-paclitaxel, paclitaxel, cisplatin, carboplatin, and combinations thereof.

17. The method of claim 14, wherein the chemotherapy is selected from the group consisting of Cisplatin, Carboplatin, Paclitaxel (Taxol), Albumin-bound paclitaxel (nab-paclitaxel, Abraxane), Docetaxel (Taxotere), Gemcitabine (Gemzar), Vinorelbine (Navelbine), Etoposide (VP- 16), Pemetrexed (Alimta), and combinations thereof.

18. The method of any one of claims 1-3, wherein the cancer therapy is an antibody-drug conjugate.

19. The method of claim 18, wherein the antibody-drug conjugate is selected from the group consisting of TDM-1, TdxD and sacituzumab govitecan, and combinations thereof.

20. The method of any one of claims 1-19, wherein the macrophages are differentiated from myeloid progenitor cells, induced pluripotent stem cells (iPSCs), or hematopoietic stem cells.

21. The method of any one of claims 2-19, wherein the CRISPR-based system comprisesa vector encoding a guide RNA targeting Zn-finger DNA binding domain of G ATA-3; and a vector encoding Cas9, Cast 2a, or Cast 3.

22. The method of claim 21, wherein the guide RNA comprises the sequence of SEQ ID NO:3.49

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