Proinflammatory immature myeloid cells and their use in treatment of cancer

Abstract

The present disclosure provides methods for making bone marrow hematopoietic progenitors lacking NF-κB p50 protein subunit (p50) and STAT6, or bone marrow hematopoietic progenitors lacking NF-κB p50 protein subunit and NF-κB p52 protein subunit. The progenitor cells are expanded, exposed to a myeloid cytokine, and provided intravenously to treat various malignancies. The infused cells have the potential to generate mature granulocytes, monocytes, macrophages, and dendritic cells. Methods for the genetically manipulation of a subject's hematopoietic progenitors during the expansion phase to reduce or eliminate expression of p50, p52, and / or STAT6 are also contemplated, and these progenitor cells may be combined with other therapeutic agents to maximize efficacy.

Description

[0001] 088933.0149

[0002] PATENT

[0003] PROINFLAMMATORY IMMATURE MYELOID CELLS AND THEIR USE IN TREATMENT OF CANCER

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims priority to U.S. Provisional Application No. 63 / 735,556 filed December 18, 2024, the contents of which is incorporated by reference in its entirety, and to which priority is claimed.

[0006] BACKGROUND OF THE INVENTION

[0007] In the U.S. population, mortality associated with the 15 most common cancer types alone has been estimated to approach 170 deaths annually In the U.S. population, mortality associated with the 15 most common cancer types alone has been estimated to approach 170 deaths annually per 100,000 individuals. Currently, there are an estimated 1,437,180 new cases of cancer and 565,650 deaths each year. The economic burden of cancer has been estimated to exceed $96B in 1990 dollars.

[0008] The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB) transcription factor activates inflammatory pathways in myeloid cells in response to extracellular signals. The canonical NF-KB subunits are p65 and p50; both contain the Rel Homology Domain that mediates homo- or hetero-dimerization and DNA-binding, with p65 also having a trans-activation domain. NF-KB p50 (p50) is an inhibitory subunit; in the basal state p65 is held in the cytoplasm by IKB, whereas p50:p50 homo-dimers enter the nucleus, bind DNA, and repress gene expression. Absence of p50 leads to activation of pro- inflammatory pathways. NF-KB p52 (p52) is a homolog of p50. The STAT6 transcription factor represses proinflammatory gene expression, while activating pathways that favor tumor growth.

[0009] T cell checkpoint inhibition has emerged as a novel cancer therapeutic approach effective in a subset of cancer patients. Effectiveness of T cell checkpoint inhibition is often limited by T cell-suppressive tumor myeloid cells. There is need to improve cancer immunotherapy through the development of novel approaches that augment T cell checkpoint therapy, or that augment other treatments designed to increase anti-tumor T cell immunity. 088933.0149

[0010] PATENT

[0011] SUMMARY OF THE INVENTION

[0012] The presently disclosed subject matter provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a synthetic hematopoietic progenitor cell or population of such cells, wherein expression of NF-KB p50 protein subunit and STAT6 protein in said cell or population of such cells are reduced when compared to wild-type cells; and wherein said disease is a cancer or a non-cancerous aberrant cellular proliferation.

[0013] The presently disclosed subject matter further provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a synthetic hematopoietic progenitor cell or population of such cells, wherein expression of NF-KB p50 protein subunit and NF-KB p52 protein subunit in said cell or population of such cells are reduced when compared to wild-type cells; and wherein said disease is a cancer or a non-cancerous aberrant cellular proliferation.

[0014] The presently disclosed subject matter provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a synthetic hematopoietic progenitor cell or population of such cells, wherein expression of NF-KB p52 protein subunit in said cell or population of such cells is reduced when compared to wild-type cells; and wherein said disease is a cancer or a non-cancerous aberrant cellular proliferation.

[0015] The presently disclosed subject matter provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a synthetic hematopoietic progenitor cell or population of such cells, wherein expression of STAT6 protein in said cell or population of such cells is reduced when compared to wild-type cells; and wherein said disease is a cancer or a non-cancerous aberrant cellular proliferation.

[0016] The presently disclosed subject matter provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a synthetic hematopoietic progenitor cell or population of such cells, wherein expression of NF-KB p50 protein subunit, NF-KB p52 protein subunit, and STAT6 protein in said cell or population of such cells are reduced when compared to wild-type cells; and wherein said disease is a cancer or a non-cancerous aberrant cellular proliferation. 088933.0149

[0017] PATENT

[0018] The presently disclosed subject matter provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a lipid nanoparticle or other carrier to allow in vivo gene editing of the genes encoding NF-KB p50, NF-KB p52, and STAT6, either individually or in pairwise (p50 + p52, p50 + STAT6) or triple (p50 + p52 + STAT6) combination.

[0019] In some embodiments, the subject is first treated with 15-150 mg / kg 5 -fluorouracil for 1-5 days and then the subject is administered 1 x 105to 5 x 109synthetic hematopoietic progenitor cells every 2 to 10 days later. In some embodiments, the subject is first treated with a chemotherapy agent other than 5-fluoruracil for 1-5 days and then the subject is administered 1 x 105to 5 x 109synthetic hematopoietic progenitor cells every 2 to 10 days later. In some embodiments, the subject also receives a T cell checkpoint inhibitor every 2-4 weeks targeting PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, or TIM-3 beginning prior to, simultaneous to, and / or subsequent to 1 x 105to 5 x 109cells of synthetic hematopoietic progenitor cells every 2 to 10 days. In some embodiments, the subj ect also receives a DNA methyltransferase inhibitor and / or a histone deacetylase inhibitor beginning prior to, simultaneous to, and / or subsequent to 1 x 105to 5 x 109cells of synthetic hematopoietic progenitor cells every 2 to 10 days.

[0020] In some embodiments, the gene for SIRPa was genetically deleted through the use of: a CRISPR / Cas9 gene editing construct, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).

[0021] In some embodiments, the cancer is melanoma, sarcoma, colon carcinoma, pancreatic ductal carcinoma, glioblastoma, prostate carcinoma or neuroblastoma. In some embodiments, the non-cancerous aberrant cellular proliferation is polycythemia vera.

[0022] The presently disclosed subject matter further provides a synthetic hematopoietic progenitor cell or population of such cells, wherein expression of NF-KB p50 protein subunit and STAT6 protein in said cell or population of such cells are reduced when compared to wildtype cells. In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal genetically modified to: (a) lack at least one copy of the STAT6 gene, or (b) to have reduced levels or activity of the mRNA for the STAT6 protein. In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the gene for the STAT6 protein was genetically deleted through the use of: a CRISPR / Cas9 gene editing construct, a zinc finger nuclease (ZFN), or a transcription 088933.0149

[0023] PATENT activator-like effector nuclease (TALEN). In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the level or activity of the mRNA for the STAT6 protein is genetically reduced through the use of an shRNA, antisense RNA, or anti-sense DNA construct. In some embodiments, said cell or population of such cells are obtained from iPSC genetically modified to lack at least one copy of the STAT6 gene or to have reduced levels or activity of the STAT6 mRNA for the STAT6 protein. In some embodiments, the iPSC cell or population of such cells was genetically modified to lack both copies of the STAT6 gene. In some embodiments, said cell or population of such cells are obtained by genetically modifying hematopoietic cells derived from iPSC to lack at least one copy of the STAT6 gene or to have reduced levels or activity of the STAT6 mRNA for the STAT6 protein. In some embodiments, the population of such cells was genetically modified to lack both copies of the STAT6 gene. In some embodiments, expression of NF-KB p52 protein subunit in said cell or population of such cells is reduced when compared to wild-type cells.

[0024] The presently disclosed subject matter further provides a synthetic hematopoietic progenitor cell or population of such cells, wherein expression of NF-KB p50 protein subunit and NF-KB p52 protein subunit in said cell or population of such cells are reduced when compared to wild-type cells. In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal genetically modified to: (a) lack at least one copy of the NF-KB p52 protein subunit gene, or (b) to have reduced levels or activity of the mRNA for the NF-KB p52 protein subunit. In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the gene for the NF-KB p52 protein subunit was genetically deleted through the use of: a CRISPR / Cas9 gene editing construct, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN). In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the level or activity of the mRNA for the NF-KB p52 protein subunit is genetically reduced through the use of an shRNA, anti-sense RNA, or anti-sense DNA construct. In some embodiments, said cell or population of such cells are obtained from iPSC genetically modified to lack at least one copy of the p52 gene or to have reduced levels or activity of the p52 mRNA for the NF-KB p52 protein subunit. In some embodiments, the iPSC cell or population of such cells was genetically modified to lack both copies of the p52 gene. In some embodiments, said cell or population of such cells are obtained 088933.0149

[0025] PATENT by genetically modifying hematopoietic cells derived from iPSC to lack at least one copy of the p52 gene or to have reduced levels or activity of the p52 mRNA for the NF-KB p52 protein subunit. In some embodiments, the population of such cells was genetically modified to lack both copies of the p52 gene.

[0026] In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal genetically modified to: (a) lack at least one copy of the NF-KB p50 protein subunit gene, or (b) to have reduced levels or activity of the mRNA for the NF-KB p50 protein subunit. In some embodiments, said cell or cells express one or more cell surface markers selected from the group consisting of: CDl lb, CD115 / MCSFR, CD14, CD64, CD16, HLA-DR, CD209, FLT3, CDl lc, CDlc, CD141, CD303, CD304, CDla, CD15, CD13, and CD33. In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the gene for the NF-KB p50 protein subunit was genetically deleted through the use of: a CRISPR / Cas9 gene editing construct, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN). In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the level or activity of the mRNA for the NF-KB p50 protein subunit is genetically reduced through the use of an shRNA, anti-sense RNA, or anti-sense DNA construct. In some embodiments, the mammal is a human. In some embodiments, said cell or population of such cells are obtained from iPSC genetically modified to lack at least one copy of the p50 gene or to have reduced levels or activity of the p50 mRNA for the NF-KB p50 protein subunit. In some embodiments, the iPSC cell or population of such cells was genetically modified to lack both copies of the p50 gene. In some embodiments, said cell or population of such cells are obtained by genetically modifying hematopoietic cells derived from iPSC to lack at least one copy of the p50 gene or to have reduced levels or activity of the p50 mRNA for the NF-KB p50 protein subunit. In some embodiments, the population of such cells was genetically modified to lack both copies of the p50 gene.

[0027] The presently disclosed subject matter further provides a synthetic hematopoietic progenitor cell or population of such cells, wherein expression of NF-KB p52 protein subunit in said cell or population of such cells is reduced when compared to wild-type cells. In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal genetically modified to: (a) lack at least one copy of the NF-KB p52 protein 088933.0149

[0028] PATENT subunit gene, or (b) to have reduced levels or activity of the mRNA for the NF-KB p52 protein subunit. In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the gene for the NF-KB p52 protein subunit was genetically deleted through the use of: a CRISPR / Cas9 gene editing construct, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN). In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the level or activity of the mRNA for the NF-KB p52 protein subunit is genetically reduced through the use of an shRNA, anti-sense RNA, or anti-sense DNA construct. In some embodiments, said cell or population of such cells are obtained from iPSC genetically modified to lack at least one copy of the p52 gene or to have reduced levels or activity of the p52 mRNA for the NF-KB p52 protein subunit. In some embodiments, the iPSC cell or population of such cells was genetically modified to lack both copies of the p52 gene. In some embodiments, said cell or population of such cells are obtained by genetically modifying hematopoietic cells derived from iPSC to lack at least one copy of the p52 gene or to have reduced levels or activity of the p52 mRNA for the NF-KB p52 protein subunit. In some embodiments, the population of such cells was genetically modified to lack both copies of the p52 gene.

[0029] The presently disclosed subject matter further provides a synthetic hematopoietic progenitor cell or population of such cells, wherein expression of STAT6 protein in said cell or population of such cells is reduced when compared to wild-type cells. In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal genetically modified to: (a) lack at least one copy of the STAT6 gene, or (b) to have reduced levels or activity of the mRNA for the STAT6 protein. In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the gene for the STAT6 protein was genetically deleted through the use of: a CRISPR / Cas9 gene editing construct, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN). In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the level or activity of the mRNA for the STAT6 protein is genetically reduced through the use of an shRNA, anti-sense RNA, or antisense DNA construct. In some embodiments, said cell or population of such cells are obtained from iPSC genetically modified to lack at least one copy of the STAT6 gene or to have reduced 088933.0149

[0030] PATENT levels or activity of the STAT6 mRNA for the STAT6 protein. In some embodiments, the iPSC cell or population of such cells was genetically modified to lack both copies of the STAT6 gene. In some embodiments, said cell or population of such cells are obtained by genetically modifying hematopoietic cells derived from iPSC to lack at least one copy of the STAT6 gene or to have reduced levels or activity of the STAT6 mRNA for the STAT6 protein. In some embodiments, the population of such cells was genetically modified to lack both copies of the STAT6 gene. In some embodiments, expression of NF-KB p52 protein subunit in said cell or population of such cells is reduced when compared to wild-type cells.

[0031] In some embodiments, the endogenous gene(s) encoding NF-KB p50, NF-KB p52, and / or STAT6 are in vivo gene-edited or knocked down in bone marrow myeloid progenitors and / or tumor myeloid cells e.g., via systemic injection of lipid nanoparticles containing Cas9 mRNA and specific sgRNAs, or specific siRNAs.

[0032] The presently disclosed subject matter further provides a pharmaceutical composition comprising the hematopoietic progenitor cell or population of such cells disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises at least one additional therapeutic agent. In some embodiments, the composition is in the form of a graft.

[0033] The presently disclosed subject matter further provides use of the hematopoietic progenitor cell or population of such cells disclosed herein, or the pharmaceutical composition disclosed herein, for treatment of cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of said cells or said pharmaceutical compositions. In some embodiments, the cancer is pancreatic ductal carcinoma, glioblastoma, neuroblastoma, or prostate cancer. In some embodiments, the cancer is melanoma, sarcoma, or colon carcinoma.

[0034] The presently disclosed subject matter further provides use of the hematopoietic progenitor cell or population of such cells disclosed herein, or the pharmaceutical composition disclosed herein, for treatment of aberrant cellular proliferation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of said cells or said pharmaceutical compositions. In some embodiments, the aberrant cellular proliferation is a benign tumor, hemangiomas, a myeloproliferative disorder, or polycythemia vera. 088933.0149

[0035] PATENT

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIGS. 1A-1B show efficient gene-editing of p50, STAT6, or both in human CD34+hematopoietic stem cells. FIG. 1 A shows DNA Knockout Score (KO) obtained using STAT6 sgRNA pairs, a p50 sgRNA pair, or combining STAT6 and p50 sgRNAs, nucleofected as complexes with Cas9 into human CD34+marrow cells, compared with non-edited control DNA. DNA from each sample was isolated, subjected to PCR using primer pairs spanning the expected deletions, followed by DNA sequencing and comparison of sequences obtained using ICE software (Synthego) to obtain the KO score. FIG. IB shows protein extracts from nontargeted cells (NTC) and those edited using p50 sgAC, STAT6sg9AC, or both were subjected to Western blotting for p50, STAT6, and P-actin, confirming marked reduction of both p50 and STAT6 protein with single or dual CRISPR / Cas9 gene-editing.

[0038] FIGS. 2A-2B show efficient gene-editing of p50, p52, or both in human CD34+hematopoietic stem cells. FIG. 2A shows DNA Knockout Score obtained using a p50 sgRNA pair, a p52 sgRNA pair, or combining p50 and p52 sgRNAs, nucleofected as complexes with Cas9 into human CD34+marrow cells, compared with non-edited control DNA. DNA from each sample was isolated, subjected to PCR using primer pairs spanning the expected deletions, followed by DNA sequencing and comparison of sequences obtained using ICE software (Synthego) to obtain the KO score. FIG. 2B shows protein extracts from non-targeted cells (NTC) and those edited using p50 sgAC, p52sg6AB, or both were subjected to Western blotting for p50, p52, and P-actin, confirming marked reduction of both p50 and p52 with single or dual CRISPR / Cas9 gene-editing.

[0039] FIGS. 3A-3B show increased expression of proinflammatory cytokines in p50 / STAT6 double knockout murine macrophages compared with p50 knockout macrophages. FIG. 3A shows macrophages derived from WT, p50KO, STAT6KO, or p50 / STAT6 double KO (DKO) mice cultured in IL-4 or IL-10 / TGFp for 24 hrs (cytokines present in the immune-suppressive tumor microenvironment). RNAs were then analyzed for indicated RNAs by qRT-PCR, relative to cyclophilin A. Expression relative to that in WT macrophages is shown (n=3, mean and SE). B) The ratio of expression in p50 / STAT6 DKO vs p50KO macrophages is shown (n=3, mean and SE). *p<0.05, ** p<0.01, *** p<0.001. 088933.0149

[0040] PATENT

[0041] FIGS. 4A-4B show increased expression of proinflammatory cytokines in p50 / STAT6 double knockout human macrophages compared with p50 knockout macrophages. FIG. 4A shows human macrophages derived from CD34+cells gene-edited with a non-targeting (NT) sgRNA, or sgRNAs targeting p50, STAT6, or both cultured in IL-4 or IL-10 / TGFP for 24 hrs. RNAs were then analyzed for indicated RNAs by qRT-PCR, relative to P-actin. Expression relative to that obtained with NT sgRNA is shown (n=2, mean and SE). FIG. 4B shows the ratio of expression in p50 / STAT6 DKO vs p50KO macrophages is shown (n=2, mean and SE).

[0042] FIGS. 5A-5B show increased expression of proinflammatory cytokines in p50 / p52 double knockout murine macrophages compared with p50 knockout macrophages. FIG. 5A shows TIDE analysis for murine p52 after p52(sg7):Cas9 RNP nucleofection of Lin' marrow cells from p50'z' mice. FIG. 5B shows Lin' marrow cells from p50'z' mice were subjected to gene-editing using a non-targeting (NT) sgRNA or p52(sg7), followed by expansion, culture in M-CSF for 7 days to generate macrophages and then in IL-4 for 24 hours to favor M2 gene expression. RNAs were then analyzed by qRT-PCR, normalized to cyclophilin A. Foldincrease of four Ml RNAs and fold-decrease of three M2 RNAs in p50 / p52 double knockout compared to p50'z' M2 macrophages is shown (n=2, mean and SE).

[0043] FIG. 6 shows increased expression of proinflammatory cytokines in p50 / p52 double knockout human macrophages compared with p50 knockout macrophages. Human CD34+cells were gene-edited using p50 or p50 and p52 sgRNAs, followed by expansion, culture in M-CSF for 5 days and then in human serum for 7 days to generate macrophages, and then in IL-4 for 24 hours to favor M2 gene expression. RNAs were then analyzed by qRT-PCR, normalized to P-actin. Fold-increase in Ml RNAs in M2 -polarized human CD34+-cell-derived macrophages with p52 / p50 versus p50 KO (n=2, mean and SE).

[0044] FIGS. 7A-7D show reduced tumor growth and increased total and activated CD4+ T cells in glioblastoma tumors after p50 / STAT6-IMC immunotherapy. FIG. 7A shows C57BL / 6 mice inoculated intra-cranially with syngeneic GL261 -luciferase glioblastoma cells (5E3). Mice received 5FU (150 mg / kg i.p.) on day 5, followed either by no therapy, p50-IMC (1E7), or p50 / STAT6-IMC (1E7) on days 10 and 12. Tumor size was evaluated on day 14 by determining tumor bioluminescence using IVIS imaging (n=5 / group; mean, SE). IMC - immature myeloid cell; *p<0.05 vs 5FU. FIGS. 7B-7D show tumors isolated on day 15, dissociated into single cells, and evaluated for CD4+and CD8+T cell populations (FIG. 7A), 088933.0149

[0045] PATENT

[0046] IFNy+CD4+and IFNy+CD8+T cell populations (FIG. 7B), and TNFa+CD4+and TNFa CD8+T cell populations (FIG. 7C) by flow cytometry. Total numbers of each T cell population per tumor volume, estimated as relative bioluminescence units (RBU) are shown (n= 5 / group; mean, SE).

[0047] FIGS. 8A-8B show that p50 / STAT6-IMC are more effective against murine pancreatic cancer and lung cancer tumors than p50-IMC. C57BL / 6 mice were inoculated subcutaneously with 2E6 syngeneic Panc02 pancreatic ductal carcinoma (FIG. 8A) or 1E6 Lewis Lung Cancer (LLC) cells (FIG. 8B). Mice received 5 -fluorouracil (5FU, 150 mg / kg intra-peritoneally) on day 15 followed either by no therapy, p50-IMC (1E7 cells / dose), or p50 / STAT6-IMC (1E7 cell / dose) intra-venously on days 20 and 22. Tumor volumes were monitored using caliper measurements. Tumor growth curves were compared using an exponential model. P-values for 5FU or 5FU+p50-IMC versus 5FU+p50 / STAT6-IMC are shown, demonstrating increased efficacy of p50 / STAT6-IMC against both of these cancers.

[0048] FIGS. 9A-9B show gene expression analysis of human CD34+ cell-derived macrophages cultured in the immune-suppressive IL-4 cytokine for 24 hours. FIG. 9A shows macrophages obtained after non-targeting (NT) or STAT6 gene-editing of CD34+ cells. FIG. 9B shows macrophages obtained after non-targeting (NT) or NFKB1+STAT6 gene-editing of CD34+ cells. The NFKB1 gene encodes NF-KB p50 (p50). Heatmaps of the top 100 differentially expressed genes between NT and transcription factor-edited macrophages from human donors D1-D3 are shown, along with the top Hallmark pathways activated or repressed in the absence of STAT6 or p50+STAT6. NES, normalized enrichment score. Significantly altered pro-inflammatory pathways are bolded. Both STAT6 and p50+STAT6 gene-editing significantly activate several pro-inflammatory Hallmark pathways including “Interferon y Response,” Interferon a Response,” Inflammatory Response,” TNFa Signaling via NF-KB,” and “IL3 JAK STAT3 Signaling.”

[0049] FIGS. 10 A- 10C show protein and RNA expression relating to pro-inflammatory or immune-suppression pathways. FIG. 10A shows secreted pro-inflammatory protein expression by human CD34+ cell -derived macrophages cultured in IL-4, obtained after non-targeting (NT), STAT6, or NFKB1+STAT6 gene-editing. Data shown are for three human donors 48 hours (for CXCL10) or 72 hours (for TNFa or IL-6) after IL-4 addition. p50, STAT6, or p50+STAT6 gene-editing leads to increased TNFa production compared to NT cells. STAT6 088933.0149

[0050] PATENT editing increases and p50+STAT6 editing further increases IL-6 production. STAT6 or p50+STAT6 editing markedly increases CXCL10 production. FIGS. 10B-10C show expression of proinflammatory Ml (FIG. 1 OB) and immune-suppressive M2 (FIG. 10C) RNAs by human CD34+ cell-derived macrophages cultured in IL-4 for 24 hours. STAT6 or combined p50+STAT6 gene-editing increases TNFa, IL-6, CXCL10, and ILip RNA levels, and reduces ALOX15, CCL22, CD206, CCL18, and CCL13 RNA levels.

[0051] DETAILED DESCRIPTION OF THE INVENTION

[0052] In accordance with certain embodiments, the present disclosure provides a synthetic hematopoietic progenitor cell or populations of such cells, wherein the expression of the NF- KB p50 protein subunit and STAT6 of said cell or cells are absent or reduced when compared to wild-type. Additionally or alternatively, in some embodiments, the present disclosure provides a synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p50 protein subunit and the NF-KB p52 protein subunit of said cell or cells are absent or reduced when compared to wild-type. Additionally or alternatively, in some embodiments, the present disclosure provides a synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p52 protein subunit of said cell or cells is absent or reduced when compared to wild-type. Additionally or alternatively, in some embodiments, the present disclosure provides a synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of STAT6 of said cell or cells is absent or reduced when compared to wild-type. Additionally or alternatively, in some embodiments, the present disclosure provides a synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p50 protein subunit, the NF-KB p52 protein subunit, and STAT6 of said cell or cells are absent or reduced when compared to wild-type.

[0053] In some embodiments, the present disclosure provides compositions, methods, and systems for generating synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p50 protein subunit and STAT6 of said cell or cells are absent or reduced when compared to wild-type. Additionally or alternatively, in some embodiments, the present disclosure provides compositions, methods, and systems for generating synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p50 protein subunit and the NF-KB p52 protein subunit of said cell or 088933.0149

[0054] PATENT cells are absent or reduced when compared to wild-type. Additionally or alternatively, in some embodiments, the present disclosure provides compositions, methods, and systems for generating synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p52 protein subunit of said cell or cells is absent or reduced when compared to wild-type. Additionally or alternatively, in some embodiments, the present disclosure provides compositions, methods, and systems for generating synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of STAT6 of said cell or cells is absent or reduced when compared to wild-type. Additionally or alternatively, in some embodiments, the present disclosure provides compositions, methods, and systems for generating synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p50 protein subunit, the NF-KB p52 protein subunit, and STAT6 of said cell or cells are absent or reduced when compared to wild-type.

[0055] In some embodiments, the present disclosure provides compositions, systems, and methods for administering synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p50 protein subunit and STAT6 of said cell or cells are absent or reduced when compared to wild-type to a subject (e.g., to a subject with cancer or a non-cancerous aberrant cellular proliferation in an adoptive transfer type of procedure). Additionally or alternatively, in some embodiments, the present disclosure provides compositions, systems, and methods for administering synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p50 protein subunit and the NF-KB p52 protein subunit of said cell or cells are absent or reduced when compared to wildtype to a subject (e.g., to a subject with cancer or a non-cancerous aberrant cellular proliferation in an adoptive transfer type of procedure). Additionally or alternatively, in some embodiments, the present disclosure provides compositions, systems, and methods for administering synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p52 protein subunit of said cell or cells is absent or reduced when compared to wild-type to a subject (e.g., to a subject with cancer or a non-cancerous aberrant cellular proliferation in an adoptive transfer type of procedure). Additionally or alternatively, in some embodiments, the present disclosure provides compositions, systems, and methods for administering synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of STAT6 of said cell or cells is absent or reduced when compared to wild-type to a subject (e.g., to a subject 088933.0149

[0056] PATENT with cancer or a non-cancerous aberrant cellular proliferation in an adoptive transfer type of procedure). Additionally or alternatively, in some embodiments, the present disclosure provides compositions, systems, and methods for administering synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p50 protein subunit, the NF-KB p52 protein subunit, and STAT6 of said cell or cells are absent or reduced when compared to wild-type to a subject (e.g., to a subject with cancer or a non-cancerous aberrant cellular proliferation in an adoptive transfer type of procedure).

[0057] In some embodiments, cells of the present disclosure where the expression of the NF- KB p50 protein subunit and STAT6 of said cell or cells is absent or reduced when compared to wild-type are termed immature myeloid cells (IMC) or “p50 / STAT6-IMC”. In some embodiments, cells of the present disclosure where the expression of the NF-KB p50 protein subunit and the NF-KB p52 protein subunit of said cell or cells is absent or reduced when compared to wild-type are termed immature myeloid cells (IMC) or “p50 / p52-IMC”. In some embodiments, cells of the present disclosure where the expression of the NF-KB p52 protein subunit of said cell or cells is absent or reduced when compared to wild-type are termed immature myeloid cells (IMC) or “p52-IMC”. In some embodiments, cells of the present disclosure where the expression of STAT6 of said cell or cells is absent or reduced when compared to wild-type are termed immature myeloid cells (IMC) or “STAT6-IMC”. In some embodiments, cells of the present disclosure where the expression of the NF-KB p50 protein subunit, the NF-KB p52 protein subunit, and STAT6 of said cell or cells is absent or reduced when compared to wild-type are termed immature myeloid cells (IMC) or “p50 / p52 / STAT6- IMC”.

[0058] In some embodiments, IMC described herein (p50 / STAT6-IMC, p50 / p52-IMC, p52- IMC, STAT6-IMC, or p50 / p52 / STAT6-IMC) are generated from mammals such as mice lacking both copies of the gene encoding p50, p52, and / or STAT6. In some embodiments, IMC are generated from mammals such as mice lacking both copies of the gene encoding p50. In some embodiments, IMC are generated from mammals such as mice lacking both copies of the gene encoding STAT6. In some embodiments, IMC are generated from mammals such as mice lacking both copies of the gene encoding p52. In some embodiments, IMC are generated from mammals such as mice lacking both copies of the genes encoding p50 and STAT6. In some embodiments, IMC are generated from mammals such as mice lacking both copies of the genes 088933.0149

[0059] PATENT encoding p50 and p52. In some embodiments, IMC are generated from mammals such as mice lacking both copies of the genes encoding p50, p52, and STAT6.

[0060] In some embodiments, IMC described herein (p50 / STAT6-IMC, p50 / p52-IMC, p52- IMC, STAT6-IMC, or p50 / p52 / STAT6-IMC) are generated from mammals genetically modified to a) lack at least one copy of the genes encoding p50, p52, and / or STAT6, or b) to have reduced levels or activity of the mRNA for the p50, p52, and / or STAT6 proteins. In certain embodiments, the IMC described herein are generated from cells or a population of such cells that are obtained from the bone marrow or blood of a mammal genetically modified to a) lack at least one copy of the genes encoding p50, p52, and / or STAT6, or b) to have reduced levels or activity of the mRNA for the p50, p52, and / or STAT6 proteins.

[0061] The presently disclosed subject matter further provides a synthetic hematopoietic progenitor cell or population of such cells, wherein expression of NF-KB p50 protein subunit and STAT6 protein in said cell or population of such cells are reduced when compared to wildtype cells. In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal genetically modified to: (a) lack at least one copy of the STAT6 gene, or (b) to have reduced levels or activity of the mRNA for the STAT6 protein. In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the gene for the STAT6 protein was genetically deleted through the use of: a gene editing system comprising an RNA-guided nuclease and gRNA, e.g., a CRISPR / Cas9 gene editing construct; a zinc finger nuclease (ZFN); or a transcription activatorlike effector nuclease (TALEN). A “gene editing system” refers to any system having RNA- guided DNA editing activity. For example, but not by way of limitation, genome editing systems of the present disclosure can include at least two components adapted from naturally occurring CRISPR systems: a guide RNA (gRNA) and an RNA-guided nuclease (e.g., a Cas9 nuclease or a Casl2a nuclease). These two components form a complex that is capable of associating with a specific nucleic acid sequence and editing the DNA in or around that nucleic acid sequence, for instance by making one or more of a single-strand break (an SSB or nick), a double-strand break (a DSB) and / or a point mutation.

[0062] In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the level or activity of the mRNA for the STAT6 protein is genetically reduced through the use of an shRNA, anti-sense RNA, or anti-sense DNA 088933.0149

[0063] PATENT construct. In some embodiments, said cell or population of such cells are obtained from iPSC genetically modified to lack at least one copy of the STAT6 gene or to have reduced levels or activity of the STAT6 mRNA for the STAT6 protein. In some embodiments, the iPSC cell or population of such cells was genetically modified to lack both copies of the STAT6 gene. In some embodiments, said cell or population of such cells are obtained by genetically modifying hematopoietic cells derived from iPSC to lack at least one copy of the STAT6 gene or to have reduced levels or activity of the STAT6 mRNA for the STAT6 protein. In some embodiments, the population of such cells was genetically modified to lack both copies of the STAT6 gene. In some embodiments, expression of NF-KB p52 protein subunit in said cell or population of such cells is reduced when compared to wild-type cells.

[0064] The presently disclosed subject matter further provides a synthetic hematopoietic progenitor cell or population of such cells, wherein expression of NF-KB p50 protein subunit and NF-KB p52 protein subunit in said cell or population of such cells are reduced when compared to wild-type cells. In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal genetically modified to: (a) lack at least one copy of the NF-KB p52 protein subunit gene, or (b) to have reduced levels or activity of the mRNA for the NF-KB p52 protein subunit. In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the gene for the NF-KB p52 protein subunit was genetically deleted through the use of: a CRISPR / Cas9 gene editing construct, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN). In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the level or activity of the mRNA for the NF-KB p52 protein subunit is genetically reduced through the use of an shRNA, anti-sense RNA, or anti-sense DNA construct. In some embodiments, said cell or population of such cells are obtained from iPSC genetically modified to lack at least one copy of the p52 gene or to have reduced levels or activity of the p52 mRNA for the NF-KB p52 protein subunit. In some embodiments, the iPSC cell or population of such cells was genetically modified to lack both copies of the p52 gene. In some embodiments, said cell or population of such cells are obtained by genetically modifying hematopoietic cells derived from iPSC to lack at least one copy of the p52 gene or to have reduced levels or activity of the p52 mRNA for the NF-KB p52 protein 088933.0149

[0065] PATENT subunit. In some embodiments, the population of such cells was genetically modified to lack both copies of the p52 gene.

[0066] In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal genetically modified to: (a) lack at least one copy of the NF-KB p50 protein subunit gene, or (b) to have reduced levels or activity of the mRNA for the NF-KB p50 protein subunit. In some embodiments, said cell or cells express one or more cell surface markers selected from the group consisting of: CDl lb, CD115 / MCSFR, CD14, CD64, CD16, HLA-DR, CD209, FLT3, CDl lc, CDlc, CD141, CD303, CD304, CDla, CD15, CD13, and CD33. In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the gene for the NF-KB p50 protein subunit was genetically deleted through the use of: a CRISPR / Cas9 gene editing construct, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN). In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the level or activity of the mRNA for the NF-KB p50 protein subunit is genetically reduced through the use of an shRNA, anti-sense RNA, or anti-sense DNA construct. In some embodiments, the mammal is a human. In some embodiments, said cell or population of such cells are obtained from iPSC genetically modified to lack at least one copy of the p50 gene or to have reduced levels or activity of the p50 mRNA for the NF-KB p50 protein subunit. In some embodiments, the iPSC cell or population of such cells was genetically modified to lack both copies of the p50 gene. In some embodiments, said cell or population of such cells are obtained by genetically modifying hematopoietic cells derived from iPSC to lack at least one copy of the p50 gene or to have reduced levels or activity of the p50 mRNA for the NF-KB p50 protein subunit. In some embodiments, the population of such cells was genetically modified to lack both copies of the p50 gene.

[0067] The presently disclosed subject matter further provides a synthetic hematopoietic progenitor cell or population of such cells, wherein expression of NF-KB p52 protein subunit in said cell or population of such cells is reduced when compared to wild-type cells. In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal genetically modified to: (a) lack at least one copy of the NF-KB p52 protein subunit gene, or (b) to have reduced levels or activity of the mRNA for the NF-KB p52 protein subunit. In some embodiments, said cell or population of such cells are obtained from the bone 088933.0149

[0068] PATENT marrow or blood of a mammal where the gene for the NF-KB p52 protein subunit was genetically deleted through the use of: a CRISPR / Cas9 gene editing construct, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN). In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the level or activity of the mRNA for the NF-KB p52 protein subunit is genetically reduced through the use of an shRNA, anti-sense RNA, or anti-sense DNA construct. In some embodiments, said cell or population of such cells are obtained from iPSC genetically modified to lack at least one copy of the p52 gene or to have reduced levels or activity of the p52 mRNA for the NF-KB p52 protein subunit. In some embodiments, the iPSC cell or population of such cells was genetically modified to lack both copies of the p52 gene. In some embodiments, said cell or population of such cells are obtained by genetically modifying hematopoietic cells derived from iPSC to lack at least one copy of the p52 gene or to have reduced levels or activity of the p52 mRNA for the NF-KB p52 protein subunit. In some embodiments, the population of such cells was genetically modified to lack both copies of the p52 gene.

[0069] The presently disclosed subject matter further provides a synthetic hematopoietic progenitor cell or population of such cells, wherein expression of STAT6 protein in said cell or population of such cells is reduced when compared to wild-type cells. In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal genetically modified to: (a) lack at least one copy of the STAT6 gene, or (b) to have reduced levels or activity of the mRNA for the STAT6 protein. In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the gene for the STAT6 protein was genetically deleted through the use of: a CRISPR / Cas9 gene editing construct, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN). In some embodiments, said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the level or activity of the mRNA for the STAT6 protein is genetically reduced through the use of an shRNA, anti-sense RNA, or antisense DNA construct. In some embodiments, said cell or population of such cells are obtained from iPSC genetically modified to lack at least one copy of the STAT6 gene or to have reduced levels or activity of the STAT6 mRNA for the STAT6 protein. In some embodiments, the iPSC cell or population of such cells was genetically modified to lack both copies of the STAT6 088933.0149

[0070] PATENT gene. In some embodiments, said cell or population of such cells are obtained by genetically modifying hematopoietic cells derived from iPSC to lack at least one copy of the STAT6 gene or to have reduced levels or activity of the STAT6 mRNA for the STAT6 protein. In some embodiments, the population of such cells was genetically modified to lack both copies of the STAT6 gene. In some embodiments, expression of NF-KB p52 protein subunit in said cell or population of such cells is reduced when compared to wild-type cells.

[0071] In some embodiments, the endogenous gene(s) encoding NF-KB p50, NF-KB p52, and / or STAT6 are in vivo gene-edited or knocked down in bone marrow myeloid progenitors and / or tumor myeloid cells e.g., via systemic injection of lipid nanoparticles containing Cas9 mRNA and specific sgRNAs, or specific siRNAs. Editing systems and other strategies for gene knockout or knock down can be implemented (e.g. administered or delivered to a cell or a subject) in a variety of ways, and different implementations may be suitable for distinct applications. As described in detail herein, an editing system can, in certain embodiments, be implemented as a protein / RNA complex (a ribonucleoprotein, or RNP), which can be included in a pharmaceutical composition that optionally includes a pharmaceutically acceptable carrier and / or an encapsulating agent, such as, without limitation, a lipid or polymer micro- or nanoparticle, micelle, or liposome. Similar strategies can be implemented for the delivery of gene knock out or knock down approaches, e.g., delivery of siRNA. In certain embodiments, editing systems can be implemented via the delivery of one or more nucleic acids encoding the RNA- guided nuclease and guide RNA components described herein. For example, but not by way of limitation, editing systems can be implemented via the delivery of one or more vectors comprising such nucleic acids, e.g., viral vector such as an adeno-associated virus. Again, such delivery strategies are also appliable to the delivery of gene knock out and knock down approaches, e.g., siRNA-based approached. Additional or modified implementations operating according to the principles set forth herein will be apparent to the skilled artisan and are within the scope of this disclosure.

[0072] In some alternative embodiments, IMC described herein (p50 / STAT6-IMC, p50 / p52- IMC, p52-IMC, STAT6-IMC, or p50 / p52 / STAT6-IMC) are generated from hematopoietic cells, such as those obtained from a cancer patient or an allogeneic, HLA-matched or HLA- similar human donor, using gene editing tools such as CRISPR / Cas9 to knockout (KO) one or both gene alleles for p50, p52, and / or STAT6 in a subset of the cells. In some embodiments, 088933.0149

[0073] PATENT

[0074] IMC described herein (p50 / STAT6-IMC, p50 / p52-IMC, or p50 / p52 / STAT6-IMC) are generated from hematopoietic cells, such as those obtained from a cancer patient, using agents that knockdown (KD) expression of p50 mRNA, p52 mRNA, and / or STAT6 mRNA, such as shRNA, siRNA, anti-sense DNA, or anti-sense RNA.

[0075] In some embodiments, IMC express the monocyte markers CDl lb, MCSFR, CD14, CD64, and / or CD 16. In some embodiments, IMC express the dendritic cell markers HLA-DR, CD209, and / or FLT3. In other embodiments, IMC can also express CDl lc, CDlc, CD141, CD303, CD304, CDla, CD15, CD13, and / or CD33.

[0076] In accordance with certain embodiments, the present disclosure provides pharmaceutical compositions comprising a synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p50 protein subunit and STAT6 of said cell or cells are absent or reduced when compared to wild-type, and a pharmaceutically acceptable carrier. Additionally or alternatively, in some embodiments, the present disclosure provides a pharmaceutical composition comprising a synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p50 protein subunit and the NF-KB p52 protein subunit of said cell or cells is absent or reduced when compared to wildtype, and a pharmaceutically acceptable carrier. Additionally or alternatively, in some embodiments, the present disclosure provides a pharmaceutical composition comprising a synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p50 protein subunit, the NF-KB p52 protein subunit, and STAT6 of said cell or cells is absent or reduced when compared to wild-type, and a pharmaceutically acceptable carrier.

[0077] In accordance with an embodiment, the present disclosure provides a pharmaceutical composition comprising a synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p50 protein subunit and STAT6 of said cell or cells are absent or reduced when compared to wild-type, a pharmaceutically acceptable carrier, and at least one or more additional biologically active agents. Additionally or alternatively, in some embodiments, the present disclosure provides a pharmaceutical composition comprising a synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p50 protein subunit and the NF-KB p52 protein subunit of said cell or cells is absent or reduced when compared to wild-type, a pharmaceutically acceptable carrier, and at least one or more additional biologically active agents. Additionally or alternatively, in some 088933.0149

[0078] PATENT embodiments, the present disclosure provides a pharmaceutical composition comprising a synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p52 protein subunit of said cell or cells is absent or reduced when compared to wildtype, a pharmaceutically acceptable carrier, and at least one or more additional biologically active agents. Additionally or alternatively, in some embodiments, the present disclosure provides a pharmaceutical composition comprising a synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of STAT6 of said cell or cells is absent or reduced when compared to wild-type, a pharmaceutically acceptable carrier, and at least one or more additional biologically active agents. Additionally or alternatively, in some embodiments, the present disclosure provides a pharmaceutical composition comprising a synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p50 protein subunit, the NF-KB p52 protein subunit, and STAT6 of said cell or cells is absent or reduced when compared to wild-type, a pharmaceutically acceptable carrier, and at least one or more additional biologically active agents.

[0079] In accordance with an embodiment, the present disclosure provides a method of treating cancer or a non-cancerous aberrant cellular proliferation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of said cells or said pharmaceutical compositions described herein.

[0080] In accordance with certain embodiments, the present disclosure provides a method for making a synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p50 protein subunit and STAT6 of said cell or cells are absent or reduced when compared to wild-type. Additionally or alternatively, in some embodiments, the present disclosure provides a method for making a synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p50 protein subunit and the NF- KB p52 protein subunit of said cell or cells are absent or reduced when compared to wild-type. Additionally or alternatively, in some embodiments, the present disclosure provides a method for making a synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p52 protein subunit of said cell or cells is absent or reduced when compared to wild-type. Additionally or alternatively, in some embodiments, the present disclosure provides a method for making a synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of STAT6 of said cell or cells is absent or 088933.0149

[0081] PATENT reduced when compared to wild-type. Additionally or alternatively, in some embodiments, the present disclosure provides a method for making a synthetic hematopoietic progenitor cell or population of such cells, wherein the expression of the NF-KB p50 protein subunit, the NF-KB p52 protein subunit, and STAT6 of said cell or cells are absent or reduced when compared to wild-type.

[0082] In some embodiments, the synthetic hematopoietic progenitor cell or population of such cells comprise blood cells developed from mammalian induced pluripotent stem cells (iPSC). In this embodiment, reagents to knockout (KO) or knockdown (KD) mRNAs encoding p50, p52, and / or STAT6 are introduced into iPSC before culturing under conditions optimized to generate blood cells22, followed by isolating a population that includes hematopoietic stem and progenitor cells (e.g., isolation of lineage-negative or CD34+ cells). These cells can then be expanded in vitro, in the presence of Flt3 ligand (FL), thrombopoietin (TPO), and stem cell factor (SCF), and potentially additional or alternative cytokine combinations, or other biologically active agents that maintain cell immaturity. The cells can be further cultured with M-CSF and / or other myeloid cytokines that can include GM-CSF, IL-4, and FL, to generate IMC (e.g., p50 / STAT6-IMC, p50 / p52-IMC, p52-IMC, STAT6-IMC, or p50 / p52 / STAT6- IMC).

[0083] As an additional embodiment, hematopoietic stem and progenitor cells would be isolated from iPSC after culture under conditions optimized to generate blood cells, with KO and / or KD as these cells are expanded in conditions that maintain their immaturity, prior to transfer to myeloid cytokines to generate IMC (e.g., p50 / STAT6-IMC, p50 / p52-IMC, p52- IMC, STAT6-IMC, or p50 / p52 / STAT6-IMC).

[0084] As used herein, the term “wherein the expression of the NF-KB p50 protein subunit and STAT6 of said cell or cells are absent or reduced when compared to wild-type” means that the p50 protein subunit and STAT6 are not expressed in the cell or population of such cells at detectable levels, or the level of expression of the p50 protein subunit and STAT6 in the cell or population of such cells are less than the level of expression of a control or a wild-type cell or population of such cells. In addition, the term “wherein the expression of the NF-KB p50 protein subunit and the NF-KB p52 protein subunit of said cell or cells are absent or reduced when compared to wild-type” means that the p50 protein subunit and the p52 protein subunit are not expressed in the cell or population of such cells at detectable levels, or the level of 088933.0149

[0085] PATENT expression of the p50 protein subunit and the p52 protein subunit in the cell or population of such cells are less than the level of expression of a control or a wild-type cell or population of such cells. In addition, the term “wherein the expression of the NF-KB p52 protein subunit of said cell or cells is absent or reduced when compared to wild-type” means that the p52 protein subunit is not expressed in the cell or population of such cells at detectable levels, or the level of expression of the p52 protein subunit in the cell or population of such cells is less than the level of expression of a control or a wild-type cell or population of such cells. In addition, the term “wherein the expression of STAT6 of said cell or cells is absent or reduced when compared to wild-type” means that STAT6 is not expressed in the cell or population of such cells at detectable levels, or the level of expression of STAT6 in the cell or population of such cells is less than the level of expression of a control or a wild-type cell or population of such cells. In addition, the term “wherein the expression of the NF-KB p50 protein subunit, the NF- KB p52 protein subunit, and STAT6 of said cell or cells are absent or reduced when compared to wild-type” means that the p50 protein subunit, the p52 protein subunit, and STAT6 are not expressed in the cell or population of such cells at detectable levels, or the level of expression of the p50 protein subunit, the p52 protein subunit, and STAT6 in the cell or population of such cells are less than the level of expression of a control or a wild-type cell or population of such cells.

[0086] As used herein, the term “regression” refers to the return of a diseased subject, cell, tissue, or organ to a non-pathological, or less pathological state as compared to basal nonpathogenic exemplary subject, cell, tissue, or organ. For example, regression of a tumor includes a reduction of tumor mass as well as complete disappearance of a tumor or tumors.

[0087] As used herein the term, “zzz vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell cultures. The term “zzz vivo" refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment.

[0088] As used herein, the term “cell culture” refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro. 088933.0149

[0089] PATENT

[0090] In some embodiments, the IMC can be further expanded in vitro by exposure to IL-3, IL-6, Notch ligands or aryl hydrocarbon antagonists, and / or LSD1 inhibitors / degraders.

[0091] In some embodiments, the inventive methods include when a patient receives immunotherapy with one or more checkpoint inhibitors, prior to, at the same time, and / or after receiving the IMC (e.g., p50 / STAT6-IMC, p50 / p52-IMC, p52-IMC, STAT6-IMC, or p50 / p52 / STAT6-IMC) by adoptive transfer IV, or prior to, at the same time, and / or after direct administration of IMC to the patient's tumor. In various embodiments, the checkpoint inhibitor(s) target one or more of CTLA-4 or PD-1 / PD-L1, and / or other checkpoint inhibitors such as LAG-3 or TIM-3, which may include antibodies against such targets, such as monoclonal antibodies, or portions thereof, or humanized or fully human versions thereof. In some embodiments, the checkpoint inhibitor therapy comprises Yervoy (ipilimumab) or Keytruda (pembrolizumab).

[0092] In some embodiments, the inventive methods include when the patient receives a tumor or dendritic cell vaccine prior to, at the same time, and / or after receiving the IMC (e.g., p50 / STAT6-IMC, p50 / p52-IMC, p52-IMC, STAT6-IMC, or p50 / p52 / STAT6-IMC) by adoptive transfer IV, or prior to, at the same time, and / or after direct administration of IMC to the patient's tumor. In various embodiments, the tumor vaccine might be autologous tumor cells expressing GM-CSF or tumor cells mixed with other cells expressing GM-CSF. In some embodiments, the dendritic cell vaccine may be patient dendritic cells primed with a tumor antigen.

[0093] In some embodiments, the inventive methods include when the patient receives radiation therapy to the tumor prior to or subsequent to receiving IMC (e.g., p50 / STAT6-IMC, p50 / p52-IMC, p52-IMC, STAT6-IMC, or p50 / p52 / STAT6-IMC) by adoptive transfer IV or prior to or subsequent to direct administration of IMC to the patient’s tumor.

[0094] In some embodiments, the inventive methods include when the patient receives about 1 to 5 rounds of adoptive immunotherapy (e.g., one, two, three, four or five rounds) with the IMC (e.g., p50 / STAT6-IMC, p50 / p52-IMC, p52-IMC, STAT6-IMC, or p50 / p52 / STAT6- IMC). In some embodiments, each administration of adoptive immunotherapy is conducted prior to (e.g., from about 1 day to about 1 week prior to), simultaneously with, or after (e.g., from about 1 day to about 1 week after), a round of checkpoint inhibitor therapy. 088933.0149

[0095] PATENT

[0096] In particular embodiments, the inventive methods further comprise administering the synthetic hematopoietic progenitor cell or population of such cells, the IMC (e.g., p50 / STAT6- IMC, p50 / p52-IMC, p52-IMC, STAT6-IMC, or p50 / p52 / STAT6-IMC), to a subject (e.g. a patient). In some embodiments, the subject has a tumor and the contacting reduces the size (or eliminates) the tumor.

[0097] The subject referred to in the inventive methods can be any host. In certain embodiments, the host is a mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Lagomorpha, such as rabbits. In certain embodiments, the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). In certain embodiments, the mammals are from the order Artiodactyla, including Bovine (cows) and Swine (pigs) or of the order Perssodactyla, including Equine (horses). In certain embodiments, the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). In certain embodiments, the mammal is human.

[0098] In some embodiments, the inventive methods further comprise administering to the subject cytokines active on myeloid or T cells that might include M-CSF, G-CSF, GM-CSF, FL, IL-4, IL-2, and / or IL- 15, or chemical mimetics mimicking the action of one or more of these cytokines. In certain embodiments, after a subject has been treated with the adoptive transfer methods and compositions of the present disclosure, diagnostic procedures are employed to determine efficacy. In certain embodiments, tumor regression is analyzed. For example, clinical and radiographic responses (e.g. MRI and CT) can be used for monitoring the effector tumor-reactive IMC (e.g., p50 / STAT6-IMC, p50 / p52-IMC, p52-IMC, STAT6- IMC, or p50 / p52 / STAT6-IMC) on tumor growth. Certain procedures include clinical, histological and bioluminescent in vivo imaging for monitoring tumor growth. In some embodiments, the persistence of functional IMC is monitored by isolation of myeloid cells from tumor or draining lymph node followed by analysis for p50 mRNA or protein expression or p50 gene deletion, p52 mRNA or protein expression or p52 gene deletion, STAT6 mRNA or protein expression, or by staining tumor or lymph node tissue for proteins or RNAs expressed in activated myeloid cells, including in macrophages and dendritic cells. In other embodiments, the ability of IMC to induce an anti-tumor T cell response is monitored by staining tumor or lymph node tissue for total and activated CD4 and CD8 T cells and for 088933.0149

[0099] PATENT regulatory T cells or by isolation of tumor or lymph node T cell followed by flow cytometry or RNA isolation and mRNA analysis.

[0100] Administration and Dosing Regimes.

[0101] One skilled in the art will appreciate that administration and dosing of cells for adoptive transfer may need to be customized to the patient for highest efficacy and tolerance. In human patients, this translates to a dose of about 3><108IMC cells (e.g., p50 / STAT6-IMC, p50 / p52- IMC, p52-IMC, STAT6-IMC, or p50 / p52 / STAT6-IMC), although higher and lower amounts of cells (e.g. one or more orders of magnitude different) may be employed. It is noted that, in certain embodiments, the number of IMC cells that is needed for therapeutic treatment using the methods and compositions of the present disclosure is generally less than disclosed in the prior art. For example, in some embodiments, the amount of the IMC applied to the patient can be e.g., 3*107to IMO8cells. It is further noted that while repeated transplantation can improve the efficacy of IMC-mediated anti-tumor activity, embodiments of the present disclosure may employ one or more administrations of the IMC. Such therapy may be sufficient for therapeutic treatment and may be further augmented by repeated checkpoint inhibitor, tumor or dendritic cell vaccine, and cytokine therapy.

[0102] In certain embodiments, the amount of the IMC administered to the patient can be from about IMO5IMC cells to about 5 MO9IMC cells, from about IMO5IMC cells to about IMO9IMC cells, from about 1 x 105IMC cells to about 1 x 108IMC cells, from about 1 x 105IMC cells to about 1X107IMC cells, from about 1X105IMC cells to about 1X106IMC cells, from about 1X106IMC cells to about 5xlO9IMC cells, from about 1X106IMC cells to about 1X109IMC cells, from about 1X106IMC cells to about 1X108IMC cells, from about 1X106IMC cells to about 1X107IMC cells, from about 1X107IMC cells to about 5xlO9IMC cells, from about 1X107IMC cells to about 1X109IMC cells, from about 1X107IMC cells to about 1X108IMC cells, from about 1X108IMC cells to about 5xlO9IMC cells, from about 1X108IMC cells to about IxlO9IMC cells, or from about IxlO9IMC cells to about 5xl09IMC cells. In certain embodiments, the amount of the IMC administered to the patient can be at least about IxlO5IMC cells, at least about IxlO6IMC cells, at least about IxlO7IMC cells, at least about IxlO8IMC cells, at least about IxlO9IMC cells, or at least about 5xl09IMC cells. In certain embodiments, the amount of the IMC administered to the patient can be up to about IxlO5IMC cells, up to about IxlO6IMC cells, up to about IxlO7IMC cells, up to about IxlO8IMC cells, 088933.0149

[0103] PATENT up to about l *109IMC cells, or up to about 5* 109IMC cells. In certain embodiments, the amount of the IMC administered to the patient can be about 1 x 105IMC cells, about 1 x 106IMC cells, about 1 X 107IMC cells, about 1 X 108IMC cells, about 1 X 109IMC cells, or about 5x l09IMC cells.

[0104] In certain embodiments, the IMC are administered to the patient about every 1 to 10 days, about every 1 to 5 days, about every 1 to 2 days, about every 2 to 10 days, about every 2 to 5 days, or about every 5 to 10 days. In certain embodiments, the IMC are administered to the patient about every day, about every 2 days, about every 3 days, about every 4 days, about every 5 days, about every 6 days, about every 7 days, about every 8 days, about every 9 days, or about every 10 days.

[0105] Types of Cancer.

[0106] Methods of some embodiments of the present disclosure find use in the treatment of cancer and are not limited by the type of cancer. In some embodiments, methods may be directed towards treatment of solid tumors. Examples of solid tumors include sarcomas and carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Additional types of malignancies and related disorders include but are not limited to leukemia (acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia), polycythemia vera, 088933.0149

[0107] PATENT lymphoma (Hodgkin's disease, non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, melanoma, sarcoma, and heavy chain disease.

[0108] It will be understood by those of ordinary skill in the art that the term “tumor” as used herein means a neoplastic growth which may, or may not be malignant. Additionally, the compositions and methods provided herein are not only useful in the treatment of tumors, but in their micrometastses and their macrometastses. Typically, micrometastasis is a form of metastasis (the spread of a cancer from its original location to other sites in the body) in which the newly formed tumors are identified only by histologic examination; micrometastases are detectable by neither physical exam nor imaging techniques. In contrast, macrometastases are usually large secondary tumors.

[0109] Co-administration with chemotherapeutic agents.

[0110] Chemotherapy and the adoptive IMC (e.g., p50 / STAT6-IMC, p50 / p52-IMC, p52-IMC, STAT6-IMC, or p50 / p52 / STAT6-IMC) cell transfer of the present disclosure may be performed sequentially or simultaneously. For example, myeloid depleting chemotherapy may be conducted prior to adoptive cell transfer. The present disclosure is not limited by type of anti-cancer agent co-administered. Indeed, a variety of anti-cancer agents are contemplated to be useful in the present disclosure including, but not limited to, Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Adriamycin; Aldesleukin; Alitretinoin; Allopurinol Sodium; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Annonaceous Acetogenins; Anthramycin; Asimicin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bexarotene; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Bullatacin; Busulfan; Cabergoline; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Celecoxib; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; DACA (N-[2-(Dimethyl- amino)ethyl]acridine-4-carboxamide); Dactinomycin; Daunorubicin Hydrochloride; Daunomycin; Decitabine; Denileukin Diftitox; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; 088933.0149

[0111] PATENT

[0112] Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized Oil I 131; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; 5- FdUMP; Fluorocitabine; Fosquidone; Fostriecin Sodium; FK-317; FK-973; FR-66979; FR- 900482; Gemcitabine; Geimcitabine Hydrochloride; Gemtuzumab Ozogamicin; Gold Au 198; Goserelin Acetate; Guanacone; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-nl; Interferon Alfa-n3; Interferon Beta- la; Interferon Gamma- lb; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Methoxsalen; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mytomycin C; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Oprelvekin; Ormaplatin; Oxisuran; Paclitaxel; Pamidronate Disodium; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rituximab; Rogletimide; Rolliniastatin; Safingol; Safingol Hydrochloride; Samarium / Lexidronam; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Squamocin; Squamotacin; Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Thymitaq; Tiazofurin; Tirapazamine; Tomudex; TOP-53; Topotecan Hydrochloride; Toremifene Citrate; Trastuzumab; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Valrubicin; Vapreotide; Verteporfin; Vinblastine; Vinblastine Sulfate; Vincristine; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride; 2-Chlorodeoxyadenosine; 2'-Deoxyformycin; 9- 088933.0149

[0113] PATENT aminocamptothecin; raltitrexed; N-propargyl-5,8-dideazafolic acid; 2-chloro-2'-arabino- fluoro-2 '-deoxy adenosine; 2-chloro-2'-deoxyadenosine; anisomycin; trichostatin A; hPRL- G129R; CEP-751; linomide; sulfur mustard; nitrogen mustard (mechlorethamine); cyclophosphamide; melphalan; chlorambucil; ifosfamide; busulfan; N-methyl-N-nitrosourea (MNU); N,N'-Bis(2-chloroethyl)-N-nitrosourea (BCNU); N-(2-chloroethyl)-N'-cyclohex-yl- N-nitrosourea (CCNU); N-(2-chloroethyl)-N'-(trans-4-methylcyclohexyl-N-nitrosourea (MeCCNU); N-(2-chloroethyl)-N'-(diethyl)ethylphosphonate-N-nit-rosourea (fotemustine); streptozotocin; diacarbazine (DTIC); mitozolomide; temozolomide; thiotepa; mitomycin C; AZQ; adozelesin; Cisplatin; Carboplatin; Ormaplatin; Oxaliplatin; Cl-973; DWA 2114R; JM216; JM335; Bis (platinum); tomudex; azacitidine; cytarabine; gemcitabine; 6- Mercaptopurine; 6-Thioguanine; Hypoxanthine; teniposide; 9-amino camptothecin; Topotecan; CPT-11; Doxorubicin; Daunomycin; Epirubicin; darubicin; mitoxantrone; losoxantrone; Dactinomycin (Actinomycin D); amsacrine; pyrazoloacridine; all-trans retinol; 14-hydroxy-retro-retinol; all-trans retinoic acid; N-(4-Hydroxyphenyl) retinamide; 13-cis retinoic acid; 3-Methyl TTNEB; 9-cis retinoic acid; fludarabine (2-F-ara-AMP); and 2- chlorodeoxyadenosine (2-Cda).

[0114] With respect to the pharmaceutical compositions used in combination with the IMC described herein (e.g., p50 / STAT6-IMC, p50 / p52-IMC, or p50 / p52 / STAT6-IMC), the carrier can be any of those conventionally used for cell therapy, and is limited only by considerations such as cell viability and by the route of administration. The carriers described herein are well- known to those skilled in the art and are readily available to the public. In certain embodiments, the carrier be one which is chemically inert to the active agent(s), and one which has little or no detrimental side effects or toxicity under the conditions of use. Examples of the carriers include tissue culture media or buffered saline, and these carriers may include cytokines used to generate IMC.

[0115] The choice of carrier will be determined, in part, by the particular pharmaceutical composition, as well as by the particular method used to administer the composition. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the present disclosure.

[0116] It will be understood to those of skill in the art that the term “chemotherapeutic agent” is any agent capable of affecting the structure or function of the body of a subject or is an agent 088933.0149

[0117] PATENT useful for the treatment or modulation of a disease or condition in a subj ect suffering therefrom. Examples of therapeutic agents can include any drugs known in the art for treatment of disease indications, including, for example, cancer.

[0118] An active agent and a biologically active agent are used interchangeably herein to refer to a chemical or biological compound, including cells that induce a desired pharmacological and / or physiological effect, wherein the effect may be prophylactic or therapeutic.

[0119] The dose of the chemotherapeutic agents used in conjunction with the IMC of the present disclosure (e.g., p50 / STAT6-IMC, p50 / p52-IMC, p52-IMC, STAT6-IMC, or p50 / p52 / STAT6-IMC) also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular composition. Typically, an attending physician will decide the dosage of the pharmaceutical composition with which to treat each individual subject, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, compound to be administered, route of administration, and the severity of the condition being treated.

[0120] By way of example, and not intending to limit the disclosure, the dose of one or more chemotherapeutic agents used in conjunction with IMC can be about 0.001 to about 1000 mg / kg body weight of the subject being treated, from about 0.01 to about 100 mg / kg body weight, from about 0.1 mg / kg to about 10 mg / kg, and from about 0.5 mg to about 5 mg / kg body weight. In certain embodiments, the dose of one or more chemotherapeutic agents used in conjunction with IMC can be from about 1 to about 1000 mg / kg, from about 1 to about 750 mg / kg, from about 1 mg / kg to about 500 mg / kg, from about 1 to about 250 mg / kg, from about 1 to about 150 mg / kg, from about 1 to about 100 mg / kg, from about 1 to about 50 mg / kg, from about 1 to about 25 mg / kg, from about 1 to about 15 mg / kg, from about 15 to about 1000 mg / kg, from about 15 to about 750 mg / kg, from about 15 mg / kg to about 500 mg / kg, from about 15 to about 250 mg / kg, from about 15 to about 150 mg / kg, from about 15 to about 100 mg / kg, from about 15 to about 50 mg / kg, from about 15 to about 25 mg / kg, from about 25 to about 1000 mg / kg, from about 25 to about 750 mg / kg, from about 25 mg / kg to about 500 mg / kg, from about 25 to about 250 mg / kg, from about 25 to about 150 mg / kg, from about 25 to about 100 mg / kg, from about 25 to about 50 mg / kg, from about 50 to about 1000 mg / kg, from about 50 to about 750 mg / kg, from about 50 mg / kg to about 500 mg / kg, from about 50 to about 250 mg / kg, from about 50 to about 150 mg / kg, from about 50 to about 100 mg / kg, from 088933.0149

[0121] PATENT about 100 to about 1000 mg / kg, from about 100 to about 750 mg / kg, from about 100 mg / kg to about 500 mg / kg, from about 100 to about 250 mg / kg, from about 100 to about 150 mg / kg, from about 150 to about 1000 mg / kg, from about 150 to about 750 mg / kg, from about 150 mg / kg to about 500 mg / kg, from about 150 to about 250 mg / kg, from about 250 to about 1000 mg / kg, from about 250 to about 750 mg / kg, from about 250 mg / kg to about 500 mg / kg, from about 500 to about 1000 mg / kg, from about 500 to about 750 mg / kg, or from about 750 to about 1000 mg / kg body weight of the subject being treated. In certain embodiments, the dose of one or more chemotherapeutic agents used in conjunction with IMC can be at least about 1 mg / kg, at least about 15 mg / kg, at least about 25 mg / kg, at least about 50 mg / kg, at least about 100 mg / kg, at least about 150 mg / kg, at least about 250 mg / kg, at least about 500 mg / kg, at least about 750 mg / kg, or at least about 1000 mg / kg body weight of the subject being treated. In certain embodiments, the dose of one or more chemotherapeutic agents used in conjunction with IMC can be up to about 1 mg / kg, up to about 15 mg / kg, up to about 25 mg / kg, up to about 50 mg / kg, up to about 100 mg / kg, up to about 150 mg / kg, up to about 250 mg / kg, up to about 500 mg / kg, up to about 750 mg / kg, or up to about 1000 mg / kg body weight of the subject being treated. In certain embodiments, the dose of one or more chemotherapeutic agents used in conjunction with IMC can be about 1 mg / kg, about 15 mg / kg, about 25 mg / kg, about 50 mg / kg, about 100 mg / kg, about 150 mg / kg, about 250 mg / kg, about 500 mg / kg, about 750 mg / kg, or about 1000 mg / kg body weight of the subject being treated.

[0122] The dose of the compositions of the present disclosure also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular composition. Typically, an attending physician will decide the dosage of the pharmaceutical composition with which to treat each individual subject, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, compound to be administered, route of administration, and the severity of the condition being treated.

[0123] As used herein, the terms “effective amount” or “sufficient amount” are equivalent phrases which refer to the amount of a therapy (e.g., a prophylactic or therapeutic agent), which is sufficient to reduce the severity and / or duration of a disease, ameliorate one or more symptoms thereof, prevent the advancement of a disease or cause regression of a disease, or which is sufficient to result in the prevention of the development, recurrence, onset, or 088933.0149

[0124] PATENT progression of a disease or one or more symptoms thereof, or enhance or improve the prophylactic and / or therapeutic effect(s) of another therapy (e.g., another therapeutic agent) useful for treating a disease, such as a neoplastic disease or tumor.

[0125] As noted above, compositions comprising the IMC (e.g., p50 / STAT6-IMC, p50 / p52- IMC, p52-IMC, STAT6-IMC, or p50 / p52 / STAT6-IMC) can be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intratumor, intraperitoneal, intracranial, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants.

[0126] In certain embodiment, the dosage form of the IMC (e.g., p50 / STAT6-IMC, p50 / p52- IMC, p52-IMC, STAT6-IMC, or p50 / p52 / STAT6-IMC) is suitable for injection or intravenous administration.

[0127] In certain embodiments, chemotherapy (e.g., an anti-cancer agent) is administered prior to, simultaneous to, and / or subsequent to performing adoptive IMC (e.g., p50 / STAT6-IMC, p50 / p52-IMC, p52-IMC, STAT6-IMC, or p50 / p52 / STAT6-IMC) cell transfer. In certain embodiments, the chemotherapy is 5 -fluorouracil. In certain embodiments, the chemotherapy is a chemotherapy agent other than 5 -fluorouracil.

[0128] In certain embodiments, the chemotherapy is administered prior to performing adoptive IMC cell transfer. In certain embodiments, chemotherapy is administered between about 1 day and about 10 days, between about 1 day and about 5 days, between about 1 day and about 2 days, between about 2 days and about 10 days, between about 2 days and about 5 days, or between about 5 days and about 10 days prior to performing adoptive IMC cell transfer.

[0129] In certain embodiments, chemotherapy is administered for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks prior to performing adoptive IMC cell transfer. In certain embodiments, chemotherapy is administered for up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about 1 week, up to about 2 weeks, up to about 3 weeks, or up to about 4 weeks prior to performing adoptive IMC cell transfer. In certain embodiments, chemotherapy is administered for about 1 day, about 2 days, about 3 days, about 088933.0149

[0130] PATENT

[0131] 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks prior to performing adoptive IMC cell transfer.

[0132] In certain embodiments, a T cell checkpoint inhibitor is administered prior to, simultaneous to, and / or subsequent to performing adoptive IMC (e.g., p50 / STAT6-IMC, p50 / p52-IMC, p52-IMC, STAT6-IMC, or p50 / p52 / STAT6-IMC) cell transfer. In certain embodiments, the T cell checkpoint inhibitor targets PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, and / or TIM-3.

[0133] In certain embodiments, the T cell checkpoint inhibitor is administered for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks prior to performing adoptive IMC cell transfer. In certain embodiments, the T cell checkpoint inhibitor is administered for up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about 1 week, up to about 2 weeks, up to about 3 weeks, or up to about 4 weeks prior to performing adoptive IMC cell transfer. In certain embodiments, the T cell checkpoint inhibitor is administered for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks prior to performing adoptive IMC cell transfer.

[0134] In certain embodiments, a DNA methyltransferase inhibitor and / or a histone deacetylase inhibitor is administered prior to, simultaneous to, and / or subsequent to performing adoptive IMC (e.g., p50 / STAT6-IMC, p50 / p52-IMC, p52-IMC, STAT6-IMC, or p50 / p52 / STAT6-IMC) cell transfer.

[0135] In certain embodiments, the DNA methyltransferase inhibitor and / or a histone deacetylase inhibitor is administered for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks prior to performing adoptive IMC cell transfer. In certain embodiments, the DNA methyltransferase inhibitor and / or a histone deacetylase inhibitor is administered for up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about 1 week, up to about 2 weeks, up to about 3 weeks, or up to about 4 weeks prior to performing adoptive IMC cell transfer. In certain embodiments, the DNA methyltransferase inhibitor and / or a histone deacetylase inhibitor is administered for about 1 day, about 2 days, about 3 088933.0149

[0136] PATENT days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks prior to performing adoptive IMC cell transfer.

[0137] In certain embodiments, the chemotherapy, the T cell checkpoint inhibitor, or the DNA methyltransferase inhibitor and / or a histone deacetylase inhibitor is administered prior to performing adoptive IMC (e.g., p50 / STAT6-IMC, p50 / p52-IMC, p52-IMC, STAT6-IMC, or p50 / p52 / STAT6-IMC) cell transfer. In certain embodiments, the amount of the IMC administered to the patient can be from about 1 x 105IMC cells to about 5* 109IMC cells, from about IxlO5IMC cells to about l><109IMC cells, from about IxlO5IMC cells to about IxlO8IMC cells, from about IxlO5IMC cells to about IxlO7IMC cells, from about IxlO5IMC cells to about IxlO6IMC cells, from about IxlO6IMC cells to about 5xlO9IMC cells, from about IxlO6IMC cells to about IxlO9IMC cells, from about IxlO6IMC cells to about IxlO8IMC cells, from about IxlO6IMC cells to about IxlO7IMC cells, from about IxlO7IMC cells to about 5X109IMC cells, from about IxlO7IMC cells to about IxlO9IMC cells, from about IxlO7IMC cells to about IxlO8IMC cells, from about IxlO8IMC cells to about 5xlO9IMC cells, from about IxlO8IMC cells to about IxlO9IMC cells, or from about IxlO9IMC cells to about 5xl09IMC cells. In certain embodiments, the amount of the IMC administered to the patient can be at least about IxlO5IMC cells, at least about IxlO6IMC cells, at least about IxlO7IMC cells, at least about IxlO8IMC cells, at least about IxlO9IMC cells, or at least about 5xl09IMC cells. In certain embodiments, the amount of the IMC administered to the patient can be up to about IxlO5IMC cells, up to about IxlO6IMC cells, up to about IxlO7IMC cells, up to about IxlO8IMC cells, up to about IxlO9IMC cells, or up to about 5xl09IMC cells. In certain embodiments, the amount of the IMC administered to the patient can be about IxlO5IMC cells, about IxlO6IMC cells, about IxlO7IMC cells, about IxlO8IMC cells, about IxlO9IMC cells, or about 5xl09IMC cells. In certain embodiments, the IMC are administered to the patient about every 1 to 10 days, about every 1 to 5 days, about every 1 to 2 days, about every 2 to 10 days, about every 2 to 5 days, or about every 5 to 10 days. In certain embodiments, the IMC are administered to the patient about every day, about every 2 days, about every 3 days, about every 4 days, about every 5 days, about every 6 days, about every 7 days, about every 8 days, about every 9 days, or about every 10 days. 088933.0149

[0138] PATENT

[0139] Exemplary methods for making the IMC (e.g., p50 / STAT6-IMC, p50 / p52-IMC, p52- IMC, STAT6-IMC, or p50 / p52 / STAT6-IMC)of the present disclosure from human bone marrow.

[0140] For clinical application, CRISPR / Cas9 or adenine or cytosine base-editing can be used to knockout (KO) the p50 gene to generate human p50-IMC. In addition, p50 shRNA can be used to knockdown (KD) the p50 RNA to generate human p50-IMC. The same methods can be employed to KO or KD other genes, in particular STAT6 and / or the NF-KB p52 protein subunit, to generate human p50 / STAT6-IMC, p50 / p52-IMC, p52-IMC, STAT6-IMC, or p50 / p52 / STAT6-IMC. CD34+ hematopoietic stem and progenitor cells would be isolated from patient bone marrow or peripheral blood, for example using nanobead-conjugated CD34 antibody and immunomagnetic selection.17

[0141] These cells will then be expanded for 6-16 days (and potentially longer), e.g., in serum- free medium such as X-Vivo-20 with FL (30-100 ng / mL), TPO (10-100 ng / mL), and SCF (30- 100 ng / mL) cytokines, potentially under hypoxic (e.g. 5% oxygen) conditions, and potentially in the presence of additional biologic agents. Lentiviral vectors (LV) expressing either Cas9 / sgRNA (for KO) or shRNA (for KD) will be packaged in 293T cells using pMDLg / pRRE, pRSV-Rev, and pMD2.G, or related LV packaging plasmids, followed by concentration of cell supernatants. CD34+ marrow cells will then transduced for 2-3 days as they expand using purified LV with Retronectin-coated plates, via spinoculation, or in liquid culture, potentially in the presence of 4-8 pg / mL protamine sulfate.18

[0142] As a second method, Cas9 protein can be combined with HPLC-purified 100 bp sgRNA having 2’-O-methyl and phosphorothioate stabilizing modifications on three 5’ and 3’ nucleotides, e.g. in a 1 :2.5 molar ratio, to generate ribonucleoprotein complexes (RNPs), followed by nucleofection into CD34+ cells as they expand.19Additional methods for gene KO include co-nucleofection of chemically stabilized sgRNA and Cas9 mRNA or nucleofection of plasmids encoding Cas9 and sgRNAs.19,20Use of two sgRNAs targeting different segments of the p50, STAT6, and / or p52 gene might be used in each of these methods of gene KO. After gene KO or KD and cell expansion, the cells will be transferred to serum- free media with myeloid cytokines such as M-CSF (10-100 ng / mL), GM-CSF (10-100 ng / mL), or GM-CSF (10-100 ng / mL) with IL-4 (10-100 ng / mL) or FL (10-100 ng / mL) for 1-3 days prior to cell infusion. As an example, gene KO can use sgRNAs cloned into LentiCRISPRv2 088933.0149

[0143] PATENT

[0144] LV, and p50 mRNA KD, and using shRNAs in pLKO.l LV, in the Ml murine and human U937 myeloid cell lines and in murine marrow cells expanding in TPO / FL / SCF.

[0145] The following examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration, and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods.

[0146] EXAMPLES

[0147] Generation of murine WT-IMC, p50 / STAT6-IMC, p50 / p52-IMC, and p50 / p52 / STAT6-IMC

[0148] Progenitor cell products of the present disclosure can be generated as follows: Bone marrow from WT or gene KO mice is flushed from the extremity bones and subjected to red blood cell lysis using ammonium chloride. The gene KO mice were p50'A, STAT6 / _, p52' / _, p50’ "STAT6 / _, p50' / 'p52' / ‘, or p50’ / ’p52’ / ’STAT6’ / ’ mice. Lineage-negative cells are isolated using a magnetic column after staining with a cocktail of biotin-anti-Lineage antibodies (CD3, B220, CDl lb, Gr-1, and Teri 19) that bind mature blood cells, and anti-biotin microbeads. The obtained Lin- cells are then expanded for 6 days in IMDM media with 10% heat-inactivated fetal bovine serum (HI-FBS) and the 30 ng / mL FL, 10 ng / mL TPO, and 30 ng / mL SCF cytokines, with lx penicillin-streptomycin (P / S). The cells are then transferred to IMDM with 10% HI-FBS, P / S, and 10 ng / mL M-CSF for 1 day in ultra-low attachment plates. Cell morphology is assessed by Wright’ s-Giemsa staining of cell cytospins. Cell surface marker expression is assessed by flow cytometry (FC) using anti-CD45-BV650, anti-FLT3-BV421, anti-MCSFR-PE, and anti-CDl Ic-PE-Dazzle (Biolegend) and anti-CDl lb-PerCPCy5.5 (BD) antibodies.

[0149] Node, marrow, and spleen myeloid and T cell subset and activation.

[0150] Spleen and lymph node cells are dissociated by passage through 40 M cell strainers. All antibody staining is preceded by 15 min of 1 :50 FcyR block in FC buffer, on ice. Myeloid 088933.0149

[0151] PATENT subsets are stained with anti-CDl lb-FITC, anti-CD45-BV650, anti-Ly6C-AF700, anti-MR- PE-Cy7, anti-CDl Ic-PE / Dazzle594, anti-Ly6G-BV605 (BioLegend), anti-MHCII-eFluor450 (eBioscience), and anti-F4 / 80-APC (BioRad).

[0152] Western blot analyses.

[0153] Total cellular proteins prepared in Laemmli sample buffer are subjected to Western blotting using p50 (13586, Cell Signaling) and P-actin (AC-15, Sigma) antibodies.

[0154] Retroviral transduction.

[0155] DNA oligonucleotides encoding sgRNAs targeting the murine or human p50, p52, and / or STAT6 gene were inserted into the LentiCRISPRv2 LV vector, which were then transfected into 293 T cells with the pCMV-AR8.91 and pMD.G(VSV.G) LV packaging plasmids using Lipofectamine 2000, followed by collection of cell supernatant 2d and 3d later to obtain LV particles. These were then filtered through 0.45 pM low protein-binding filters and used to transduce Ml or U937 myeloid cells in RPMI with 10% HI-FBS in the presence of 4 pg / mL Polybrene. Cells were then cultured in the presence of 2 pg / mL puromycin to select for transduced cells. pLKO. l LV vectors expressing shRNAs targeting p50, p52, and / or STAT6 mRNA were packaged similarly and used to transduce Lin- murine marrow isolated from WT mice in media containing IMDM, 10% HI-FBS, 10 ng / mL TPO, 30 ng / mL FL, and 30 ng / mL SCF in the presence of 4 pg / mL Polybrene. p50 gene knockout (KO) and mRNA knockdown (KD) in murine and human myeloid cell lines and in murine bone marrow cells. sgRNAs for murine and human p50, p52, and STAT6 were designed using a Broad Institute website21and the corresponding oligonucleotides were introduced into the lentiCRISPRv2 plasmid, which encodes the sgRNA, hSpCas9, and puromycin-resistance.

[0156] EXAMPLES 1 and 2

[0157] Generation of human p50‘ 'STAT6' ' and p50' / 'p52' / ‘ hematopoietic stem cells.

[0158] Human CD34+hematopoietic stem cells were isolated and gene-edited using a nontargeting (NT) sgRNA, or sgRNAs targeting p50, p52, and / or STAT6. DNA from the gene- edited hematopoietic stem cells was isolated, and subj ected to PCR using primer pairs spanning the expected deletions and DNA sequencing to confirm gene deletions (FIGS. 1A and 2A). 088933.0149

[0159] PATENT

[0160] Protein extracts from non-targeted cells (NTC) or gene-edited cells were subjected to Western blotting to confirm reduction in protein expression (FIGS. IB and 2B).

[0161] EXAMPLE 3

[0162] Expression of proinflammatory cytokines in macrophages generated from p50'A, STAT6 - and p50‘ 'STAT6' ' mice.

[0163] Macrophages derived from WT, p50'A, STAT6A, or pSO'^STATb ' double KO (DKO) mice were cultured in IL-4 or IL-10 / TGFP (cytokines present in the immune-suppressive tumor microenvironment) for 24 hrs. RNAs were then analyzed for expression of IL-12P, TNFa, IL- ip, and Nos2 by qRT-PCR (FIG. 3 A). After culturing with IL-4 or IL-10 / TGFP, expression of IL-12P, IL-ip, TNFa, and Nos2 were increased in macrophages derived from p50 / STAT6 DKO mice versus macrophages derived from p50 KO mice (FIG. 3B).

[0164] EXAMPLE 4

[0165] Expression of proinflammatory cytokines in human p50"STAT6" macrophages generated from CD34+cells.

[0166] Human macrophages derived from CD34+ cells were gene-edited using a non-targeting (NT) sgRNA, or sgRNAs targeting p50, STAT6, or both. The macrophages were cultured in IL-4 or IL-10 / TGFP for 24 hrs, and RNAs were analyzed for expression of IL-12P, TNFa, IL- ip, and Nos2 qRT-PCR (FIG. 4A). After culturing with IL-4 or IL-10 / TGFP, expression of IL- 12P, IL-ip, TNFa, and Nos2 were increased in pSCk'STATb ' macrophages versus p50' / _macrophages (FIG. 4B).

[0167] EXAMPLE 5

[0168] Expression of proinflammatory cytokines in murine p50' / 'p52' / ‘ macrophages.

[0169] Lin- marrow cells were isolated from p50' / _mice and gene edited using a non-targeting (NT) sgRNA, or sgRNAs targeting p52 (FIG. 5A). The cells were cultured in M-CSF for 7 days to generate macrophages and then in IL-4 for 24 hours to favor M2 gene expression.

[0170] RNAs from Ml and M2 macrophages were analyzed by qRT-PCR (FIG. 5B). Ml p50‘ / 'p52' / ' macrophages exhibited increased expression of IL-6, IL-12P, TNFa, and IL-ip versus 088933.0149

[0171] PATENT

[0172] Ml p50' / _macrophages. M2 p50' / 'p52' / " macrophages exhibited decreased expression of MR, Arg, and Fizz versus M2 p50‘ ' macrophages.

[0173] EXAMPLE 6

[0174] Expression of proinflammatory cytokines in human p50' / 'p52' / ' macrophages generated from CD34+cells.

[0175] Human CD34+ cells were gene-edited using a non-targeting (NT) sgRNA, or sgRNAs targeting p50, p52, or both. The cells were then cultured in M-CSF for 5 days and then in human serum for 7 days to generate macrophages, and then in IL-4 for 24 hours to favor M2 gene expression. Ml RNAs in the M2-polarized cells were analyzed for expression of IL-12P, TNFa, and IL-ip by qRT-PCR. The p50' / 'p52' / ‘ macrophages exhibited increased expression of IL-12P, IL-ip, and TNFa, and Nos2 versus p50' / _macrophages (FIG. 6).

[0176] EXAMPLE 7

[0177] Increased total and activated CD4+ T cells in glioblastoma tumors after p50 / STAT6- IMC immunotherapy.

[0178] C57BL / 6 mice were inoculated intra-cranially with syngeneic GL261 -luciferase glioblastoma cells (5E3). Mice received 5FU (150 mg / kg i.p.) on day 5, followed either by no therapy, p50-IMC (1E7), or p50 / STAT6-IMC (1E7) on days 10 and 12. Tumor size was evaluated on day 14. Tumors were isolated on day 15, dissociated into single cells, and evaluated for the indicated T cell populations by flow cytometry (FIGS. 7B-7D).

[0179] EXAMPLE 8 p50 / STAT6-IMC are more effective against murine pancreatic cancer and lung cancer tumors than p50-IMC.

[0180] C57BL / 6 mice were inoculated subcutaneously with 2E6 syngeneic Panc02 pancreatic ductal carcinoma or 1E6 Lewis Lung Cancer (LLC) cells. Mice received 5 -fluorouracil (5FU, 150 mg / kg intra-peritoneally) on day 15 followed either by no therapy, p50-IMC (1E7 cells / dose), or p50 / STAT6-IMC (1E7 cell / dose) intra-venously on days 20 and 22. p50 / STAT6-IMC exhibited increased efficacy against both cancers (FIGS. 8A-8B). 088933.0149

[0181] PATENT

[0182] EXAMPLE 9

[0183] Absence of STAT6 or p50 and STAT6 in human macrophages cultured in conditions that mimic the immune-suppressive tumor microenvironment leads to activation of multiple pro-inflammatory pathways.

[0184] Human bone marrow CD34+ myeloid progenitors from three human donors (D1-D3) were gene-edited by nucleofection with a non-targeting (NT), NFKB1, STAT6, or NFKB1+STAT6 sgRNAs and Cas9. The NFKB1 gene encodes NF-KB p50 (p50). The edited cells were differentiated into macrophages, which were then cultured with IL-4, an immune- suppressive cytokine, for 24 hours. RNAs were then prepared and subjected to RNA sequencing. Resulting RNA expression data was subjected to Gene Set Enrichment Analysis to identify Hallmark pathways activated by the absence of STAT6 or of both p50 and STAT6 (FIGS. 9A-9B).

[0185] EXAMPLE 10

[0186] Absence of STAT6 or p50 and STAT6 in human macrophages in conditions that mimic the immune-suppressive tumor microenvironment leads to increased pro-inflammatory protein and RNA expression and reduced immune-suppressive RNA expression.

[0187] Human bone marrow CD34+ myeloid progenitors from four human donors (D1-D4) were gene-edited by nucleofection with a non-targeting (NT), NFKB1, STAT6, or NFKB1+STAT6 sgRNAs and Cas9. The edited cells were differentiated into macrophages by culture with M-CSF and then human serum and then cultured with IL-4, an immune- suppressive cytokine. Cell culture supernatants from three donors, collected 48 hours (for CXCL10) or 72 hours (for TNFa or IL-6) after IL-4 addition, were analyzed for protein levels using the Luminex assay (FIG. 10A). RNAs prepared from Donors 1-4, collected 24 hours after IL-4 addition, were subjected to quantitative real time-PCR (qRT-PCR) analysis (FIGS. 10B- 10C). The student’s t test was used to compare protein or RNA levels between groups. Mean values and standard deviations are shown. TNFa protein levels were below the lower limit of the Luminex assay (4.7 pg / ml) for two of the NT and one of the p50 and STAT6 samples at 48 or 72 hours. CXCL10 protein levels were below the lower limit (2.1 pg / ml) for one of the NT protein samples and above the upper limit (2,995 pg / ml) for one of the STAT6 samples - therefore CXCL10 protein data was analyzed at 48 hours to allow statistical analysis. Results 088933.0149

[0188] PATENT for TNFa, IL-6, IL-ip, and IL-12P come from analysis of RNAs from four human donors; results for the other RNAs analyzed come from analysis of three human donors. *p,0.05; **p<0.01; ***p<0.001.

[0189] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0190] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the subject matter of the instant disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can 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 (e.g., “such as”) provided herein, is intended merely to better illuminate the subject matter of the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0191] Certain embodiments of the compositions and methods of the present disclosure are presented herein, including the best mode known to the inventors for carrying out such embodiments. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the subject matter of the disclosure to be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims 088933.0149

[0192] PATENT appended hereto as permitted by applicable law. Moreover, any combination of the abovedescribed elements in all possible variations thereof is encompassed by the subject matter of the present disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[0193] References

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[0197] 4. Lee S, Kivimae S, Dolor A, Szoka FC. Macrophage-based cell therapies: the long and winding road. J. Controlled Release 2016; 240:527-40.

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[0201] 8. Ellwood-Yen K, Graeber TG, Wongvipat J, Irueala- Arispe ML, Zhang J, Matusik R, Thomas GV, Sawyers CL. Myc-driven murine prostate cancer shares molecular features with human prostate tumors. Cancer Cell 2003; 4(3):223-38.

[0202] 9. Barakat DJ, Suresh R, Barberi T, Pienta K, Simons BW, Friedman AD. Absence of myeloid Klf4 reduces prostate cancer growth with pro-atherosclerotic activation of tumor myeloid cells and infiltration of CD8 T cells. PLoS One 2018; 13(l):e0191188. 088933.0149

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[0208] 14. Brinkman EK, Chen T, Amendola M, van Steensel B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucl. Acids. Res. 2014; 42(22):el68.

[0209] 15. Delaney C, Milano F, Othus M, Becker PS, Sandhu V, Nicoud I, Dahlberg A, Bernstein ID. Infusion of a non-HLA-matched ex-vivo expanded cord blood progenitor cell product after intensive acute myeloid leukemia chemotherapy: a phase 1 trial. Lancet Haematol. 2016; 3(7):e330-9.

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Claims

1. 088933.0149PATENTClaims:

1. A method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a synthetic hematopoietic progenitor cell or population of such cells, wherein expression of NF-KB p50 protein subunit and STAT6 protein in said cell or population of such cells are reduced when compared to wild-type cells; and wherein said disease is a cancer or a non-cancerous aberrant cellular proliferation.

2. A method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a synthetic hematopoietic progenitor cell or population of such cells, wherein expression of NF-KB p50 protein subunit and NF-KB p52 protein subunit in said cell or population of such cells are reduced when compared to wildtype cells; and wherein said disease is a cancer or a non-cancerous aberrant cellular proliferation.

3. A method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a synthetic hematopoietic progenitor cell or population of such cells, wherein expression of NF-KB p52 protein subunit in said cell or population of such cells is reduced when compared to wild-type cells; and wherein said disease is a cancer or a non-cancerous aberrant cellular proliferation.

4. A method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a synthetic hematopoietic progenitor cell or population of such cells, wherein expression of STAT6 protein in said cell or population of such cells is reduced when compared to wild-type cells; and wherein said disease is a cancer or a non-cancerous aberrant cellular proliferation.

5. A method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a synthetic hematopoietic progenitor cell or population of such cells, wherein expression of NF-KB p50 protein subunit, NF-KB p52 protein subunit, and STAT6 protein in said cell or population of such cells are reduced when compared to wild-type cells; and wherein said disease is a cancer or a non-cancerous aberrant cellular proliferation.088933.0149PATENT6. A method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of: a) a gene editing system comprising an RNA-guided nuclease and gRNA or nucleic acid encoding such a system; a zinc finger nuclease (ZFN) or nucleic acid encoding such a ZFN; or a transcription activator-like effector nuclease (TALEN) or a nucleic acid encoding such a TALEN, wherein the gene editing system, ZFN or TALEN is capable of in vivo gene editing at least one gene selected from the group consisting of NF-KB p50, NF-KB p52, STAT6, or combinations thereof, or b) an shRNA, siRNA, or antisense nucleic acid to reduce the level or activity of the mRNA encoding at least one gene selected from the group consisting of NF-KB p50, NF-KB p52, STAT6, or combinations thereof thereby treating said disease in the subject.

7. The method of claims 1-6, wherein the subject is first treated with 15-150 mg / kg 5- fluorouracil for 1-5 days and then the subject is administered 1 x 105to 5 x 109synthetic hematopoietic progenitor cells every 2 to 10 days later.

8. The method of claims 1-6, wherein the subject is first treated with a chemotherapy agent other than 5-fluoruracil for 1-5 days and then the subject is administered 1 x 105to 5 x 109synthetic hematopoietic progenitor cells every 2 to 10 days later.

9. The method of claims 1-6, wherein the subject also receives a T cell checkpoint inhibitor every 2-4 weeks targeting PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, or TIM-3 beginning prior to, simultaneous to, and / or subsequent to 1 x 105to 5 x 109cells of synthetic hematopoietic progenitor cells every 2 to 10 days.

10. The method of claims 1-6, wherein the subject also receives a DNA methyltransferase inhibitor and / or a histone deacetylase inhibitor beginning prior to, simultaneous to, and / or subsequent to 1 x 105to 5 x 109cells of synthetic hematopoietic progenitor cells every 2 to 10 days.088933.0149PATENT11. The method of any one of claims 1-10, wherein the gene for SIRPa was genetically deleted through the use of: a gene editing system comprising an RNA-guided nuclease and gRNA, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).

12. The method of any one of claims 1-11, wherein the cancer is melanoma, sarcoma, colon carcinoma, pancreatic ductal carcinoma, glioblastoma, prostate carcinoma or neuroblastoma.

13. The method of any one of claims 1-12, wherein the non-cancerous aberrant cellular proliferation is polycythemia vera.

14. A synthetic hematopoietic progenitor cell or population of such cells, wherein expression of NF-KB p50 protein subunit and STAT6 protein in said cell or population of such cells are reduced when compared to wild-type cells.

15. The hematopoietic progenitor cell or population of such cells of claim 14, wherein said cell or population of such cells are obtained from the bone marrow or blood of a mammal genetically modified to:(a) lack at least one copy of the STAT6 gene, or(b) to have reduced levels or activity of the mRNA for the STAT6 protein.

16. The hematopoietic progenitor cell or population of such cells of claim 14 or 15, wherein said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the gene for the STAT6 protein was genetically deleted through the use of: a gene editing system comprising an RNA-guided nuclease and gRNA, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).

17. The hematopoietic progenitor cell or population of such cells of any one of claims 14- 16, wherein said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the level or activity of the mRNA for the STAT6 protein is genetically reduced through the use of an shRNA, anti-sense RNA, or anti-sense DNA construct.088933.0149PATENT18. The hematopoietic progenitor cell or population of such cells of any one of claims 14- 17, wherein said cell or population of such cells are obtained from iPSC genetically modified to lack at least one copy of the STAT6 gene or to have reduced levels or activity of the STAT6 mRNA for the STAT6 protein.

19. The hematopoietic progenitor cell or population of such cells of claim 18, wherein the iPSC cell or population of such cells was genetically modified to lack both copies of the STAT6 gene.

20. The hematopoietic progenitor cell or population of such cells of any one of claims 14- 19, wherein said cell or population of such cells are obtained by genetically modifying hematopoietic cells derived from iPSC to lack at least one copy of the STAT6 gene or to have reduced levels or activity of the STAT6 mRNA for the STAT6 protein.

21. The hematopoietic progenitor cell or population of such cells of claim 20, wherein the population of such cells was genetically modified to lack both copies of the STAT6 gene.

22. The hematopoietic progenitor cell or population of such cells of any one of claims 14- 21, wherein expression of NF-KB p52 protein subunit in said cell or population of such cells is reduced when compared to wild-type cells.

23. A synthetic hematopoietic progenitor cell or population of such cells, wherein expression of NF-KB p50 protein subunit and NF-KB p52 protein subunit in said cell or population of such cells are reduced when compared to wild-type cells.

24. The hematopoietic progenitor cell or population of such cells of claim 23, wherein said cell or population of such cells are obtained from the bone marrow or blood of a mammal genetically modified to:(a) lack at least one copy of the NF-KB p52 protein subunit gene, or(b) to have reduced levels or activity of the mRNA for the NF-KB p52 protein subunit.

25. The hematopoietic progenitor cell or population of such cells of claim 23 or 24, wherein said cell or population of such cells are obtained from the bone marrow or blood of a088933.0149PATENT mammal where the gene for the NF-KB p52 protein subunit was genetically deleted through the use of: a gene editing system comprising an RNA-guided nuclease and gRNA, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).

26. The hematopoietic progenitor cell or population of such cells of any one of claims 23-25, wherein said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the level or activity of the mRNA for the NF-KB p52 protein subunit is genetically reduced through the use of an shRNA, anti-sense RNA, or anti-sense DNA construct.

27. The hematopoietic progenitor cell or population of such cells of any one of claims 23-26, wherein said cell or population of such cells are obtained from iPSC genetically modified to lack at least one copy of the p52 gene or to have reduced levels or activity of the p52 mRNA for the NF-KB p52 protein subunit.

28. The hematopoietic progenitor cell or population of such cells of claim 27, wherein the iPSC cell or population of such cells was genetically modified to lack both copies of the p52 gene.

29. The hematopoietic progenitor cell or population of such cells of any one of claims 23- 28, wherein said cell or population of such cells are obtained by genetically modifying hematopoietic cells derived from iPSC to lack at least one copy of the p52 gene or to have reduced levels or activity of the p52 mRNA for the NF-KB p52 protein subunit.

30. The hematopoietic progenitor cell or population of such cells of claim 29, wherein the population of such cells was genetically modified to lack both copies of the p52 gene.

31. The hematopoietic progenitor cell or population of such cells of any one of claims 14- 30, wherein said cell or population of such cells are obtained from the bone marrow or blood of a mammal genetically modified to:(a) lack at least one copy of the NF-KB p50 protein subunit gene, or(b) to have reduced levels or activity of the mRNA for the NF-KB p50 protein subunit.088933.0149PATENT32. The hematopoietic progenitor cell or population of such cells of any one of claims 14-31, wherein said cell or cells express one or more cell surface markers selected from the group consisting of: CDl lb, CD115 / MCSFR, CD14, CD64, CD16, HLA-DR, CD209, FLT3, CDl lc, CDlc, CD141, CD303, CD304, CDla, CD15, CD13, and CD33.

33. The hematopoietic progenitor cell or population of such cells of any one of claims 14-32, wherein said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the gene for the NF-KB p50 protein subunit was genetically deleted through the use of: a gene editing system comprising an RNA-guided nuclease and gRNA, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).

34. The hematopoietic progenitor cell or population of such cells of any one of claims 14-33, wherein said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the level or activity of the mRNA for the NF-KB p50 protein subunit is genetically reduced through the use of an shRNA, anti-sense RNA, or anti-sense DNA construct.

35. The hematopoietic progenitor cell or population of such cells of any one of claims 14-34, wherein the mammal is a human.

36. The hematopoietic progenitor cell or population of such cells of any one of claims 14-35, wherein said cell or population of such cells are obtained from iPSC genetically modified to lack at least one copy of the p50 gene or to have reduced levels or activity of the p50 mRNA for the NF-KB p50 protein subunit.

37. The hematopoietic progenitor cell or population of such cells of claim 36, wherein the iPSC cell or population of such cells was genetically modified to lack both copies of the p50 gene.

38. The hematopoietic progenitor cell or population of such cells of any one of claims 14- 37, wherein said cell or population of such cells are obtained by genetically modifying hematopoietic cells derived from iPSC to lack at least one copy of the p50 gene or to have reduced levels or activity of the p50 mRNA for the NF-KB p50 protein subunit.088933.0149PATENT39. The hematopoietic progenitor cell or population of such cells of claim 38, wherein the population of such cells was genetically modified to lack both copies of the p50 gene.

40. A synthetic hematopoietic progenitor cell or population of such cells, wherein expression of NF-KB p52 protein subunit in said cell or population of such cells is reduced when compared to wild-type cells.

41. The hematopoietic progenitor cell or population of such cells of claim 40, wherein said cell or population of such cells are obtained from the bone marrow or blood of a mammal genetically modified to:(a) lack at least one copy of the NF-KB p52 protein subunit gene, or(b) to have reduced levels or activity of the mRNA for the NF-KB p52 protein subunit.

42. The hematopoietic progenitor cell or population of such cells of claim 40 or 41, wherein said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the gene for the NF-KB p52 protein subunit was genetically deleted through the use of: a gene editing system comprising an RNA-guided nuclease and gRNA, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).

43. The hematopoietic progenitor cell or population of such cells of any one of claims 40-42, wherein said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the level or activity of the mRNA for the NF-KB p52 protein subunit is genetically reduced through the use of an shRNA, anti-sense RNA, or anti-sense DNA construct.

44. The hematopoietic progenitor cell or population of such cells of any one of claims 40-43, wherein said cell or population of such cells are obtained from iPSC genetically modified to lack at least one copy of the p52 gene or to have reduced levels or activity of the p52 mRNA for the NF-KB p52 protein subunit.088933.0149PATENT45. The hematopoietic progenitor cell or population of such cells of claim 44, wherein the iPSC cell or population of such cells was genetically modified to lack both copies of the p52 gene.

46. The hematopoietic progenitor cell or population of such cells of any one of claims 40- 45, wherein said cell or population of such cells are obtained by genetically modifying hematopoietic cells derived from iPSC to lack at least one copy of the p52 gene or to have reduced levels or activity of the p52 mRNA for the NF-KB p52 protein subunit.

47. The hematopoietic progenitor cell or population of such cells of claim 46, wherein the population of such cells was genetically modified to lack both copies of the p52 gene.

48. A synthetic hematopoietic progenitor cell or population of such cells, wherein expression of STAT6 protein in said cell or population of such cells is reduced when compared to wild-type cells.

49. The hematopoietic progenitor cell or population of such cells of claim 48, wherein said cell or population of such cells are obtained from the bone marrow or blood of a mammal genetically modified to:(a) lack at least one copy of the STAT6 gene, or(b) to have reduced levels or activity of the mRNA for the STAT6 protein.

50. The hematopoietic progenitor cell or population of such cells of claim 48 or 49, wherein said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the gene for the STAT6 protein was genetically deleted through the use of: a gene editing system comprising an RNA-guided nuclease and gRNA, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).

51. The hematopoietic progenitor cell or population of such cells of any one of claims 48- 50, wherein said cell or population of such cells are obtained from the bone marrow or blood of a mammal where the level or activity of the mRNA for the STAT6 protein is genetically reduced through the use of an shRNA, anti-sense RNA, or anti-sense DNA construct.088933.0149PATENT52. The hematopoietic progenitor cell or population of such cells of any one of claims 48- 51, wherein said cell or population of such cells are obtained from iPSC genetically modified to lack at least one copy of the STAT6 gene or to have reduced levels or activity of the STAT6 mRNA for the STAT6 protein.

53. The hematopoietic progenitor cell or population of such cells of claim 52, wherein the iPSC cell or population of such cells was genetically modified to lack both copies of the STAT6 gene.

54. The hematopoietic progenitor cell or population of such cells of any one of claims 48- 53, wherein said cell or population of such cells are obtained by genetically modifying hematopoietic cells derived from iPSC to lack at least one copy of the STAT6 gene or to have reduced levels or activity of the STAT6 mRNA for the STAT6 protein.

55. The hematopoietic progenitor cell or population of such cells of claim 54, wherein the population of such cells was genetically modified to lack both copies of the STAT6 gene.

56. The hematopoietic progenitor cell or population of such cells of any one of claims 48- 55, wherein expression of NF-KB p52 protein subunit in said cell or population of such cells is reduced when compared to wild-type cells.

57. A pharmaceutical composition comprising the hematopoietic progenitor cell or population of such cells of any one of claims 14-56 and a pharmaceutically acceptable carrier.

58. The pharmaceutical composition of claim 57, further comprising at least one additional therapeutic agent.

59. The pharmaceutical composition of claim 57 or 58, wherein the composition is in the form of a graft.

60. Use of the hematopoietic progenitor cell or population of such cells of any one of claims 14-56, or the pharmaceutical composition of any one of claims 57-59, for treatment of088933.0149PATENT cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of said cells or said pharmaceutical compositions.

61. The use of claim 60, wherein the cancer is pancreatic ductal carcinoma, glioblastoma, neuroblastoma, or prostate cancer.

62. The use of claim 61, wherein the cancer is melanoma, sarcoma, or colon carcinoma.

63. Use of the hematopoietic progenitor cell or population of such cells of any one of claims 14-56, or the pharmaceutical composition of any one of claims 57-59, for treatment of aberrant cellular proliferation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of said cells or said pharmaceutical compositions.

64. The use of claim 63, wherein the aberrant cellular proliferation is a benign tumor, hemangiomas, a myeloproliferative disorder, or polycythemia vera.

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