Human macrophages resistant to tumor-induced repolarization

Abstract

The present invention relates to human macrophages for use in cancer therapy, said macrophages comprising at least one mutation in both alleles of a chromosomal gene and resistant to tumor-induced reprogramming and / or exhibiting antitumor activity. The human macrophages of the invention demonstrate the presence of typical markers of M1 macrophages, such as MHC class II proteins, even after being cultured in an environment that promotes M2 polarization, such as in the presence of M-CSF and / or IL4 and / or IL13. The present invention also relates to a collection of human macrophages of the invention, their use in medicine, in particular in cancer therapy, such as, preferably, in the treatment of solid tumors.

Description

[Background technology]

[0001] (Background of the invention) Macrophages are plastic cells, and their phenotype depends on the stimuli of the environment they encounter. When macrophages are exposed to bacterial components or inflammatory cytokines such as IFNγ, they enhance their bactericidal and tumor-killing properties and produce high levels of pro-inflammatory cytokines. This activated state is called M1 or classical activated macrophage (O'Shea and Murray, 2008; Gordon, 2003). In contrast, when macrophages are stimulated with immunosuppressive cytokines such as IL-13, IL-4, or M-CSF, they typically exhibit an anti-inflammatory phenotype that promotes tissue remodeling and repair, as well as angiogenesis, including the production of IL-10 (Murray and Wynn, 2011; Mantovani et al., 2002). This activated state is called M2 or alternative activated macrophage.

[0002] Interestingly, macrophages are the most abundant cells in the tumor microenvironment. Clinical and experimental evidence suggests that tumor-associated macrophages (TAMs) promote tumor progression and malignancy (Mantovani et al., 1992; Condeelis and Pollard, 2006; Mantovani et al., 2006), and their presence correlates with low survival rates (Qian and Pollard, 2010). In fact, TAMs support cancer development by promoting tumor angiogenesis, tumor cell invasion and metastasis, and suppressing anti-tumor immune responses (Condeelis and Pollard, 2006; Pollard, 2004). Several cancers and their cell invasions have been reported to contribute a cytokine cocktail to their environment, leading to M2 macrophage polarization (Nevala et al., 2009). Therefore, TAMs are clearly similar to the M2 phenotype (Balkwill et al., 2005).

[0003] In their 2021 paper, Bart et al. re-examined approaches to macrophage reprogramming in various disorders and discussed the potential implications and challenges of macrophage-targeted approaches in human diseases. In particular, they discussed various attempts to reprogram tumor-associated macrophages to be non-M2-like phenotypes using small molecules, cytokines, nanovectors, antibodies, nucleic acids, or viral vectors.

[0004] Macrophages that have been polarized to the M1 phenotype before administration have been tested for the treatment of diseases such as cancer. However, the M1 phenotype is not stable, and administered macrophages may be repolarized to the M2-like phenotype by the tumor microenvironment.

[0005] Therefore, human macrophages that are resistant to tumor-induced repolarization are highly desirable for cell therapy.

[0006] Klichinsky et al. (2020) induced phagocytic activity of human macrophages against tumors using genetically modified human macrophages with CAR (chimeric antigen receptor). They observed that introducing an adenovirus vector expressing the chimeric antigen receptor resulted in a pro-inflammatory (M1) phenotype in human macrophages. The CAR-modified macrophages were reported to exhibit antitumor activity. It remains unclear to what extent this activity was mediated by the chimeric antigen receptor or the pro-inflammatory phenotype of the genetically modified macrophages. [Overview of the project] [Problems that the invention aims to solve]

[0007] Adenovirus DNA is not integrated into the genome of transduced cells and is not replicated during cell division. Since tissue macrophages are known to be able to self-replicate in vivo under certain conditions, the pro-inflammatory phenotype of human macrophages carrying adenovirus vectors is expected to be lost in their offspring and therefore lost over time. Furthermore, the presence of viral vector sequences in transduced cells poses complex control and safety problems when those cells are used in cell therapy.

[0008] This invention solves the problems of the prior art by providing macrophages that are resistant to repolarization to the M2 phenotype caused by specific mutations in their genomic DNA. [Means for solving the problem]

[0009] (Summary of the invention) The present invention relates to human macrophages comprising at least one mutation in both alleles of a chromosomal gene, which are resistant to tumor-induced reprogramming and / or exhibit antitumor activity. The human macrophages of the present invention demonstrate the presence of typical markers of M1 macrophages, such as MHC class II proteins, even after being cultured in an environment that promotes M2 polarization, for example, in the presence of M-CSF and / or IL4 and / or IL13.

[0010] Preferred mutations are (partial) deletions of genes involved in M-CSF signaling, such as genes encoding the transcription factor(s) MAF and / or MAFB.

[0011] The inventors have surprisingly found that a collection of macrophages containing at least one mutation in both alleles of a chromosomal gene can induce established tumor regression in vivo, even in the absence of tumor cell-targeting molecules such as CARs. The phenotypic feature exhibited is the unresponsiveness of these macrophages to tumor-induced repolarization. The human macrophages of the present invention can exhibit one or more markers indicating M1 polarization, such as the (increased) presence of MHC class II proteins on the macrophage surface, and / or the (elevated) expression of MHC class II genes, and / or the (elevated) expression of RXFP genes, and / or the (elevated) expression of GBP1 genes, and / or the (increased) secretion of IL18, and / or the (increased) secretion of IL23, and / or the (increased) secretion of IL15, and / or the absence of M2 surface markers such as CD28 and / or LYVE1, and / or the absence of RNF128 gene expression, even in the presence of an environment that promotes M2 polarization.

[0012] Accordingly, the present invention also relates to the collection of human macrophages of the present invention, their use in pharmaceuticals, and more particularly to their use in cancer therapy, preferably in the treatment of solid tumors.

[0013] Identifying human macrophages that are resistant to conversion to tumor-associated macrophages by the tumor microenvironment due to specific loss-of-function mutations in genes located on normal human chromosomes would be significant for all macrophage-based cell therapies, not limited to solid tumor therapies, where macrophages that do not convert to tumor-promoting tumor-associated macrophages are desired. [Brief explanation of the drawing]

[0014] (drawing) [Figure 1]Figure 1 shows that adoptive cell transfer of Maf-DKO macrophages suppresses tumor growth in vivo. (a) Representative scheme of the experimental procedure. (b) Quantification of bioluminescence (luciferase activity) from primary tumors obtained on day 27. (c) Quantification of intraperitoneal ID8 tumor cells. (**: p≦0.007). UN: Untreated mouse; WT: Mouse treated with wild-type bone marrow macrophages; BM-DKO: Mouse treated with bone marrow-derived MafB and c-Maf double-deficient macrophages. [Figure 2] Figure 2 shows that adoptive cell transfer of Maf-DKO macrophages reversed established tumor growth in vivo. (a) Representative scheme of the experimental procedure. (b) Quantification of bioluminescence (luciferase activity) from primary tumors obtained on day 27. (c) Gating strategy and quantification of ID8 tumor cells. (*: p≦0.04). UN: Untreated mice; WT-BMDM: Mice treated with wild-type bone marrow macrophages; BM-DKO: Mice treated with bone marrow-derived MafB and c-Maf double-deficient macrophages. [Figure 3] Figure 3 shows the cellular contents of ascites fluid. (a) Gating strategies for peritoneal cell-derived CD8 T cells and NK cells in tumor-bearing mice. (b) Columnar diagram showing CD8α expression by peritoneal cells in the peritoneal cavity of untreated mice (dotted line, small circles), WT-BMDM-treated mice (solid line, triangles), and Maf-DKO-treated mice (dashed line, squares) 27 days after carcinogenic initiation. (c) Gating strategies of peritoneal macrophages. (d) MHC class II expression in peritoneal macrophages. (e) Mean fluorescence intensity of MHCII expression by peritoneal macrophages. (**: p≦0.007). UN: Untreated mouse; WT: Mouse treated with wild-type bone marrow macrophages; Maf-DKO: Mouse treated with MafB and c-Maf double-deficient macrophages. [Figure 4]Figure 4 shows that macrophages sorted from Maf-DKO treated mice resemble M1-activated macrophages in vivo. Total mRNA was isolated from sorted CD11b+ peritoneal cells of tumor-bearing mice for real-time PCR analysis of (a) NOS2, (b) C2TA, (c) IL-6, (d) IL-12, and (e) IL-10. (*: p ≤ 0.04). Data are shown as mean values ​​for n=5. UN: Untreated mice; WT: Mice treated with wild-type bone marrow macrophages; Maf-DKO: Mice treated with MafB and c-Maf double knockout macrophages. [Figure 5] Figure 5 shows that Maf-DKO macrophages are more sensitive to M1 stimulation and more resistant to M2 stimulation than WT macrophages. Total mRNA was isolated from cultured macrophages and stimulated with compounds A, B, C, and D for real-time PCR analysis of (a) IL-6, (b) IL-10, (c) C2TA, (d) arginase, and (e) NOS2. Maf-DKO macrophages are shown as vertical bars with polka dots, and WT macrophages are shown as vertical bars with horizontal stripes. [Figure 6] Figure 6 shows that Maf-DKO macrophages are not re-educated by tumors in vitro. Cell supernatant was collected, and the production of (a) IL-6 and (b) TNFα in response to LPS stimulation was analyzed in the absence (black bars) or presence (grid bars) of ID8 tumor cell supernatant. SN represents the supernatant. [Figure 7]Figure 7 shows that Maf-DKO macrophages reversed established melanoma proliferation in vivo. (Metastasis experiment of B16 melanoma cells in C57Bl6 mice) (a) Representative scheme of the experimental procedure. Macrophages were injected 7 days after tumor development. (b) Lungs were removed and photographed. (c) The number of metastatic colonies visible on the lung surface was counted with a magnifying glass, and the median was used for analysis. Results were obtained from 5 mice in each group. (*: p≦0.04; **: p≦0.007; ***: p≦0.0005). UN: Untreated mouse; WT: Mouse treated with wild-type bone marrow macrophages; Maf-DKO: Mouse treated with MAFB and c-Maf double-deficient macrophages. [Figure 8] Figure 8 shows that MAF DKO cells also recruit other leukocytes in protection against B16 melanoma. (B16 melanoma cell metastasis experiment in Rag2γc knockout mice) (a, b) Representative scheme of the experimental procedure. In (a), mice were treated with macrophages immediately after tumor initiation (see 8c and 8e for results), and in (b), mice were injected with macrophages after tumor establishment (see 8d and 8f for results). (c, d) Livers were removed and photographed (photographs were taken from representative livers and under white light distinct from bioluminescence), and bioluminescence was also recorded. (e, f) The number of metastatic colonies visible under a magnifying glass on the surface of mice treated in (a) and (b) was counted, and the median was used for analysis. Results were obtained from 5 mice in each group. (*: p≦0.04). B16: Untreated mice; W: Mice treated with wild-type bone marrow macrophages; Maf-DKO: Mice treated with DKO macrophages. [Figure 9]Figure 9 shows the workflow for deleting MAF and MAFB via CRISPR / Cas9 in a human iPS cell line that expresses Cas9 under doxycycline induction. Step 1: Perform Lipofectamine transfection with a vector expressing sgRNA targeting MAF. Step 2: Induce Cas9 via doxycycline (Dox) treatment. Step 3: Isolate reporter-positive sgRNA-expressing cells by cell sorting to obtain single-cell colonies. Step 4: Select MAF KO clones. Step 5: Using the selected MAF KO iPS cell clone, repeat Steps 1 to 4 with sgRNA targeting MAFB to subsequently knock out MAFB.

[0015] [Figure 10] Figure 10 shows the gene structures of human MAF and MAFB, indicating the positions of CRISPR / Cas9 target sites in the 5' and 3' UTRs of both genes, as well as the resulting indels. The human MAF gene has short and long isoforms generated by alternative splicing, and the significance of the long isoform is unknown. We knocked out exon 1 of the MAF gene. Human MAFB is a single-exon gene. The first line of each sequence block refers to the wild-type locus, and the subsequent lines refer to the indels generated by CRISPR / Cas9 editing. Only one clone was isolated for MAF (showing indels in both alleles), and for MAFB, indels from three isolated clones are shown (C1, C2, C3: Clone 1, Clone 2, Clone 3). In the sequence blocks, the start codon and stop codon are represented in bold, and the genomic target sequences are underlined. The protospacer positions are shown as solid black lines near the start codon or stop codon.

[0016] [Figure 11] Figure 11(a) shows the karyotype analysis of human MAF / MAFB DKO iPS cells. (b) shows the karyotype analysis of human MAF / MAFB DKO iPS cell-derived macrophages. DKO cells at both developmental stages exhibit a normal karyotype. [Figure 12]Figure 12 shows that human MAF-DKO macrophages are functional macrophages similar to wild-type macrophages. After wild-type macrophages and MAF-DKO macrophages were cultured with M-CSF and GM-CSF for 7 days, various functional assays were performed: phagocytosis of latex beads, staining for reactive oxygen species (ROS) production after overnight stimulation with 100 ng / ml LPS, lysosomal staining with acridine orange, and cathepsin B activity using the Magic Red kit. [Figure 13] Figure 13 shows that human wild-type macrophages and MAF-DKO macrophages express major macrophage markers after being cultured with M-CSF and GM-CSF for 7 days. Cells were pre-gated for cells that are DAPI-negative and CD45+ / CD11b+. The dotted line is the FMO (fluorescence minus one) control, and the black solid line indicates stained cells. Wild-type macrophages and MAF-DKO macrophages express CD14 (LPS binding), CD33 (myeloid-specific sialoadhesin molecule), and CD64 (high-affinity Fc receptor binding IgG type antibody) at comparable levels. MAF-DKO expressed CD206 (mannose receptor) at low levels. In contrast to wild-type iPS cell-derived macrophages, HLA-DR is clearly detectable on MAF-DKO macrophages. [Figure 14] Figure 14 shows that human MAF-DKO macrophages do not express MAF or MAFB. (Left panel of 14) Shows RT-qPCR analysis of RNA extracted from three clones of wild-type macrophages, MAF single KO macrophages, or MAF-DKO macrophages. The y-axis shows the relative expression of the indicated genes (MAF in black, MAFB in white), normalized to wild-type (WT) which is the control condition. (Right panel of 14) Shows Western blot analysis of protein lysates extracted from three clones of wild-type macrophages, MAF single KO macrophages, or MAF-DKO macrophages. WT stands for "wild-type", n.d. stands for "not detected", and GRB2 stands for "loaded control". [Figure 15]Figure 15 shows the in vivo transplantation scheme for human wild-type macrophages and MAF-DKO macrophages. The same number of wild-type or MAF-DKO macrophages (2 × 10⁶ to 4 × 10⁶ per dose) were intratracheally transplanted once a week for 4 weeks with 1 μg M-CSF per animal. Animals were analyzed 4 weeks after the final transplant. Each point on the timeline represents one transplant, with a 1-week interval between transplants. [Figure 16] Figure 16 shows that human MAF-DKO macrophages showed improved rescue of the PAP phenotype compared to wild-type macrophages. (a) Protein concentration in BAL solution as measured by BCA assay. (b) Mouse surfactant protein D (mSPD) concentration in BAL solution as measured by ELISA. (c) Human GM-CSF concentration in BAL solution as measured by ELISA. [Figure 17] Figure 17 shows a volcanic plot of differentially expressed genes between WT macrophages and DKO macrophages. iPS cell-derived macrophages were cultured in M-CSF and GM-CSF for 7 days, then shifted to M-CSF for only 2 hours. Differentially expressed genes identified by deep sequencing are shown. The X-axis indicates the degree of change, and the Y-axis indicates statistical significance. [Prior art documents] [Non-patent literature]

[0017] (References) [Table 1] TIFF0007943862000002.tif248170TIFF0007943862000003.tif19170 [Modes for carrying out the invention]

[0018] (Detailed description of the invention) (definition) As used herein, "chromosome" refers to one of the 46 normal human chromosomes. "Genes located on a chromosome" refers to genes that are not located outside of a chromosome.

[0019] The term "gene" means a DNA sequence that codes for a specific sequence of amino acids, including, but not limited to, regulatory DNA sequences, such as promoter sequences that determine the conditions under which a gene is expressed. A "promoter" or "promoter sequence" is a DNA regulatory region that can bind to RNA polymerase within a cell and initiate the transcription of a downstream (3' direction) coding sequence. Some non-structural genes may be transcribed from DNA to RNA but not translated into amino acid sequences. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term "gene" may refer to a protein-coding genomic sequence, i.e., a sequence containing regulatory elements, promoters, introns, and exons.

[0020] In the context of this invention, the terms “mutant” and “transformation” mean a detectable change in genetic material, i.e., genomic DNA. A mutation includes the deletion, insertion, or substitution of one or more nucleotides. Mutations can occur within the coding region (i.e., exons), introns, or regulatory regions of a gene (e.g., enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, promoters). Generally, mutations are identified by comparing the sequence of a nucleic acid or polypeptide expressed by a subject with the corresponding nucleic acid or polypeptide expressed in a control population. If a mutation is within a coding sequence, it may be a “missense” mutation that replaces one amino acid in the gene product with another, or a “nonsense” mutation that replaces an amino acid codon with a stop codon. Mutations may also occur at splicing sites where signals for exon-intron splicing are generated or disrupted, resulting in a structurally altered gene product. In the context of the present invention, mutations are not silent; that is, mutations result in changes to at least the nucleotide sequence (if the gene product is functional RNA) or the amino acid sequence (if the gene product is a protein), rendering the gene product non-functional or reducing its expression at the RNA level by at least 80%. Preferred mutations include, for example, the deletion of an entire gene, which eliminates gene expression.

[0021] As used herein, gene expression is considered "inhibited" if its expression at the RNA level is reduced by at least 80% compared to the gene expression in the corresponding wild type, as measured by quantitative RT-PCR. Preferably, gene expression at the RNA level is reduced by at least 90%, for example, at least 95%.

[0022] As used herein, gene expression is considered "absent" if its expression is undetectable at the RNA level by q-PCR. If mRNA "expressed" in qPCR is present at a detectable level, it will (a) produce an S-shaped fluorescence curve, the curve will (b) reach a flat region within at least 38 PCR cycles, preferably at least 36 PCR cycles, and (c) produce a PCR product of predicted length, i.e., a PCR product of a length corresponding to a PCR product derived from mature mRNA, neither from genomic DNA nor from an untreated RNA intermediate. Preferably, mRNA expression can be confirmed by repeating the qPCR experiment three times according to these three criteria.

[0023] As used herein, "mutaogenesis" refers to an experimental process that intentionally alters the genetic information of an organism to produce a mutation. The preferred method of mutaogenesis as used herein is a site-specific endonuclease-based method.

[0024] As used herein, “site-specific endonuclease” is an enzyme that cleaves phosphate diester bonds within a polynucleotide chain only at a highly specific nucleotide sequence in the middle (end) portion of a double-stranded DNA molecule, which preferably occurs at only one location within the entire human genome to enable specific genetic manipulation of human target cells. Examples of site-specific endonucleases commonly used for genetic manipulation in human target cells include zinc finger nucleases, TALENs, and the CRISPR / Cas9 system.

[0025] As used herein, the term “expression” may, depending on the context, refer to the gene expression of a polypeptide or protein, or the gene expression of a polynucleotide, such as miRNA or lncRNA. Polynucleotide expression can be determined, for example, by measuring the production at the RNA transcript level using methods well known to those skilled in the art. Protein or polypeptide expression can be determined, for example, by immunoassay using one or more antibodies that specifically bind to the polypeptide, using methods well known to those skilled in the art.

[0026] As used herein, "mRNA expression" refers to the transcriptional level of gene expression.

[0027] In this invention, known methods can be used to detect gene expression and the like. Examples of methods for quantitatively detecting mRNA levels within a single cell or a collection of cells include, for example, PCR-based methods (real-time PCR, quantitative PCR) and DNA microarray analysis. Furthermore, mRNA levels can be quantitatively detected by counting the number of reads using a method called next-generation sequencing. Typical methods, conditions, and materials used to determine mRNA expression levels are described in the experimental section of this disclosure. A preferred method for determining mRNA expression is qPCR, as described above in "Abstract Expression".

[0028] Those skilled in the art can prepare mRNA or nucleic acid cDNA for detection by the aforementioned detection method by selecting a known method suitable for the type and condition of the sample and other factors. When comparing the gene expression level in human macrophages of the present invention with the expression level of the same gene in wild-type macrophages, in order to enable a scientifically meaningful comparison at the mRNA level, both types of macrophages are cultured and treated in the same manner and compared under otherwise identical conditions.

[0029] As used herein, the term "allele" refers to one of two copies of the same human gene. The two copies of a gene reside on two homologous chromosomes, one on each, and may be identical or have slightly different individual sequences. Therefore, the term "allele" is used herein somewhat differently from its usual usage, as it typically also includes identical versions of the same human gene located at the same corresponding site on two homologous chromosomes. In diploid cells, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

[0030] As used herein, an "allele" or "gene" is considered non-functional if, after a procedure to de-functionalize the allele or gene, no further expression of the protein encoded by that gene or allele is detected. In other words, no new protein is expressed from the non-functionalized allele or gene. Ultimately, the protein encoded by the non-functionalized allele or gene becomes undetectable within a cell population consisting solely of cells comprising non-functionalized alleles. The time it takes for the protein encoded by the gene or allele to become undetectable depends on the turnover dynamics of the gene's protein and mRNA.

[0031] As used herein, the term “deletion” means that a portion of a DNA sequence is missing compared to a wild-type reference sequence.

[0032] As used herein, “exon” refers to any part of a gene that codes for the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term “exon” refers to both the DNA sequence within a gene and the corresponding sequence within an RNA transcript. In RNA splicing, introns are removed and exons covalently bond to each other to form part of the mature messenger RNA.

[0033] As used herein, the term “chromosome rearrangement” refers to a chromosomal abnormality involving a structural change in the native chromosome. Typically, chromosome rearrangement occurs when the DNA double helix is ​​cut at two different locations, and then the cut ends rejoin, resulting in a new chromosomal gene configuration with a different gene order than that of the chromosome (or chromosome) before the cut. Such changes can include several different types of events, such as large deletions exceeding 10,000 base pairs within one chromosome or between two chromosomes, gene duplication, gene inversions, and translocations.

[0034] As used herein, the term "karyotype analysis" refers to the analysis of chromosomes within cells derived from a sample. Those skilled in the art stain chromosomes and examine the size, shape, and number of chromosomes within cells of a cell sample using a microscope. Typically, a photograph of the stained sample is taken to show the arrangement of chromosomes. The karyotype indicates the number of chromosomes in an organism, as well as how those chromosomes appear under a light microscope, in particular, the length of the chromosomes, the position of the centromeres within those chromosomes, the banding pattern of the stained chromosomes, the differences between sex chromosomes, and other physical characteristics.

[0035] As used herein, the term “guide RNA” refers to “guide RNA” as used in the context of the CRISPR / Cas9 DNA editing system. Guide RNA confers specificity to the target sequence for the CRISPR-Cas9 system. Guide RNA is a short non-coding RNA sequence that first binds to the Cas9 enzyme, and then the guide RNA sequence guides the complex to a specific location on the DNA via base pairing, where Cas9 acts as an endonuclease to cleave the target DNA strand. Examples of guide RNA include (a) synthetic transactivating CRISPR RNA (tracrRNA) and synthetic CRISPR RNA (crRNA) (crRNA is designed to identify the target gene site of interest), and (b) single guide RNA (sgRNA) which incorporates both crRNA and tracrRNA into a single construct.

[0036] The term "MAF" refers to the human MAF transcription factor. MAF and other Maf family members form homodimers and heterodimers with each other, as well as with Fos and Jun, which is consistent with the known ability of AP-1 proteins to pair with each other (Kerppola and Curran (1994); Kataoka, K. et al. (1994)). The DNA target sequences to which MAF homodimers bind are called MAF response elements (MAREs), which are 13 bp or 14 bp elements containing a core TRE (T-MARE) or CRE (C-MARE) palindrome, respectively. However, MAF can also bind to DNA sequences branched from these consensus sites, including sequences containing a composite AP-1 / MARE site and a MARE half site with an AT-rich 5' extension (Yoshida et al., 2005). MAF has been shown to stimulate transcription from several promoters and repress transcription from others. MAF has also been shown to induce differentiation of T helper 2 (Th2) cells due to its ability to activate tissue-specific transcription of interleukin 4 (IL-4) (Kim et al., 1999) (Ho et al., 1996). Furthermore, overexpression of MAF in myeloid cell lines induces macrophage differentiation (Hegde et al., 1999). The human MAF gene is located at 16q23.2 on chromosome 16 and is described in detail in the NCBI gene database as gene ID 4094. Sequence and positional information is from the reference sequence assembly GCF_000001405.39 of Genome Reference Consortium Human Build 38 Patch Release 13, annotation release 109.20201120 (as of December 9, 2020). This reference identifies the MAF gene on complementary strands 79,593,838 to 79,600,737.

[0037] The term "MAFB" refers to the human MAFB transcription factor. This gene is expressed in various cell types (including lens epithelial cells, pancreatic endocrine cells, epidermal cells, chondrocytes, nerve cells, and hematopoietic cells, particularly macrophages) and encodes a protein containing a typical bZip motif in its carboxyl-terminal region. Within the bZip domain, MAFB shares broad homology not only with MAF but also with other Maf-related proteins. MAFB forms homodimers through its leucine repeat structure and can specifically bind to the Maf recognition element (MARE) palindrom, composite AP-1 / MARE site, or MARE half site with AT-rich 5' extension (Yoshida et al., 2005). Furthermore, MAFB can form heterodimers with Maf or Fos through its zipper structure, but not with Jun or other Maf family members (Kataoka et al., 1994). MAFB is also known as kreisler, kr, or KrmI1 (of "Kreisler Maf leucine zipper 1"), because X-ray-induced microinversions of chromosomes in kreisler mutant mice cause tissue-specific loss of MAFB expression in hindbrain development, leading to the kreisler phenotype (Cordes et al., 1994) (Eichmann et al., 1997). In the hematopoietic system, MAFB is selectively expressed in myeloid lineages and is sequentially upregulated during myeloid cell differentiation from pluripotent progenitor cells to macrophages. In fact, this induction reflects the important role of MAFB in monocyte and macrophage differentiation. Therefore, overexpression of MAFB in transformed chicken myeloblasts (Kelly et al., 2000; Bakri et al., 2005) and in human hematopoietic progenitor cells (Gemelli et al., 2006) inhibits progenitor cell proliferation (Tillmanns et al., 2007) and promotes macrophage formation (Kelly et al., 2000; Bakri et al., 2005; Gemelli et al., 2006), while a dominant negative version of MAFB inhibits this process (Kelly et al., 2000).Deficiency of the MafB-Maf combination in mouse monocytes and macrophages prolongs proliferation (Aziz, 2009; Soucie, 2016). This suggests that MAFB induction is a specific and crucial determinant of the monocyte program in hematopoietic cells and is important for cell cycle arrest in differentiated monocytes and macrophages.

[0038] The human MAFB gene is located on chromosome 20 at 20q12 and is described in detail in the NCBI gene database as gene ID 9935. Sequence and positional information is based on the reference sequence assembly GCF_000001405.39 from the Genome Reference Consortium Human Build 38 Patch Release 13, annotation release 109.20201120 (as of December 9, 2020). This reference identifies the MAFB gene on the complementary strand from 40685848 to 40689236.

[0039] As used herein, IL-4 refers to human interleukin-4. The protein encoded by IL-4 is a pleiotropic cytokine produced by activated T cells. IL-4 is a glycoprotein consisting of 129 amino acids with a molecular weight of approximately 20 kDa. IL-4 is a ligand for the interleukin-4 receptor. The interleukin-4 receptor also binds to IL-13, which likely contributes to the many overlapping functions of this cytokine and IL-13. IL-4 is an important cytokine for tissue repair and is thought to counteract the effects of pro-inflammatory type 1 cytokines.

[0040] The human IL-4 gene is located at 5q31.1 on chromosome 5 and is described in detail as gene ID 3565 in the NCBI gene database. Sequence and positional information is based on annotation release 109.20210226. For cell culture experiments, recombinant human IL-4 (catalog number 200-04) from Preprotech, containing 130 AAs and a 15.1 kDa globular protein, was used. The specific activity in the human TF-1 cell proliferation assay was at least 5 × 10⁶.6 It was U / mg.

[0041] IL-4 signaling occurs through its interaction with its receptor. This interaction results in receptor dimerization and activation. The activated receptor activates JAK1 and JAK3, which associate with the receptor subunit. Activated JAK phosphorylates tyrosine residues, and the cytoplasmic end of the receptor then acts as a binding site for many adapters or signaling molecules, including STAT6. Activated STAT6 dimerizes, translocates to the nucleus, and transcribes and activates genes that respond to IL-4.

[0042] As used herein, IL-13 refers to human interleukin-13. The protein encoded by IL-13 is a cytokine produced by various immune cells, primarily activated Th2 cells, but also CD4 cells, NKT cells, mast cells, basophils, and eosinophils. IL-13 is a central regulator in IgE synthesis, goblet cell hyperplasia, mucus overgrowth, airway hyperresponsiveness, fibrosis, and chitinase upregulation.

[0043] The human IL-13 gene is located at 5q31.1 on chromosome 5 and is described in detail as gene ID 3596 in the NCBI gene database. Sequence and positional information is based on annotation release 109.20210226.

[0044] As used herein, ARG-1 refers to human arginase 1. Arginase catalyzes the hydrolysis of arginine to ornithine and urea. The type I isoform encoded by this gene is a cytosolic enzyme and, under normal physiological conditions, is expressed primarily in the liver as a component of the urea cycle. However, ARG1 is also an immunosuppressive signal primarily found in tumor-associated macrophages. Macrophages polarized into an immunosuppressive phenotype express ARG1.

[0045] The human ARG-1 gene is located at 6q23.2 on chromosome 6 and is described in detail as gene ID 383 in the NCBI gene database. Sequence and positional information is based on annotation release 109.20210226.

[0046] As used herein, IL-10 refers to human interleukin-10. IL-10 is a cytokine primarily produced by monocytes and macrophages, and less frequently produced by lymphocytes. This cytokine has pleiotropic effects in immunoregulation and inflammation. IL-10 downregulates the expression of Th1 cytokines, MHC class II, and costimulatory molecules in macrophages. This cytokine can inhibit NF-κB activity and participate in the regulation of the JAK-STAT signaling pathway. High expression of IL-10 is observed in tumor-associated macrophages. Macrophages polarized to an immunosuppressive phenotype express IL-10. IL-10 expression has been reported to be associated with worsening overall survival as a predictor of advanced tumor stage.

[0047] The human IL-10 gene is located at 1q32.1 on chromosome 1 and is described in detail as gene ID 3586 in the NCBI gene database. Sequence and positional information is based on annotation release 109.20210226.

[0048] As used herein, IL-6 refers to human interleukin-6. The protein encoded by IL-6 is a cytokine that functions in inflammation and B cell maturation. Furthermore, the encoded protein has been shown to be an endogenous pyrogen that can induce fever in people with autoimmune diseases or infections. This protein is primarily produced at acute and chronic inflammation sites, where it is secreted into the serum and induces a transcriptional inflammatory response via the interleukin-6 receptor α.

[0049] The human IL-6 gene is located on chromosome 7 at 7p15.3 and is described in detail in the NCBI gene database as gene ID 3569. Sequence and positional information is based on annotation release 109.20210226.

[0050] As used herein, CXCL10 refers to a CXC motif chemokine ligand 10. The protein encoded by CXCL10 is a chemokine of the CXC subfamily and a ligand for the receptor CXCR3. Binding of this protein to CXCR3 produces pleomorphic effects, including stimulation of monocyte, natural killer cell, and T cell migration, as well as regulation of adhesion molecule expression.

[0051] The human CXCL10 gene is located at 4q21.1 on chromosome 4 and is described in detail in the NCBI gene database as gene ID 3627. Sequence and positional information is based on annotation release 109.20210226.

[0052] As used herein, C2TA (CIITA) refers to the Class II major histocompatibility complex transactivator. C2TA is a protein comprising one acidic transcription activation domain, four leucine-rich repeats, and one GTP-binding domain. This protein is located in the nucleus and acts as a positive regulator of Class II major histocompatibility complex gene transcription, and is referred to as the “master regulator” of the expression of these genes.

[0053] The human C2TA gene is located on chromosome 16 at 16p13.13 and is described in detail in the NCBI gene database as gene ID 4261. Sequence and positional information is based on annotation release 109.20210226.

[0054] As used herein, TNF refers to tumor necrosis factor. TNF is a multifunctional pro-inflammatory cytokine belonging to the tumor necrosis factor (TNF) superfamily. This cytokine is primarily secreted by macrophages. TNF can function by binding to its receptors, TNFRSF1A / TNFR1 and TNFRSF1B / TNFBR. This cytokine is involved in regulating a wide range of biological processes, including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation.

[0055] The human TNF gene is located on chromosome 6 at 6p21.33 and is described in detail as gene ID 7124 in the NCBI gene database. Sequence and positional information is based on annotation release 109.20210226.

[0056] As used herein, M-CSF is also referred to as CSF1 and relates to human colony-stimulating factor 1. M-CSF is a cytokine that regulates macrophage production, differentiation, proliferation, and function. Various active isoforms of this protein have been identified as membrane-bound homodimers or extracellular disulfide-bonded homodimers, and are thought to be produced by proteolytic cleavage of the membrane-bound precursor.

[0057] The human M-CSF gene is located on chromosome 1 at 1p13.3 and is described in detail as gene ID 1435 in the NCBI gene database. Sequence and positional information is based on annotation release 109.20210226. For cell culture experiments, 38 kDa homodimers produced in HEK293 cells and recombinant human M-CSF from Gibco (catalog number PHC9501) were used. The maximum ED50, determined by dose-dependent proliferation of M-NSF60 cells, was 5 ng / ml.

[0058] As used herein, MHCII refers to class II of major histocompatibility complex (MHC) molecules, which are typically found only on mononuclear phagocytic cells, including dendritic cells and macrophages, as well as professional antigen-presenting cells such as B cells. MHC II presents peptides to T cells that are derived from extracellular proteins via endocytosis, rather than from cytosolic proteins as in the case of MHC class I.

[0059] In humans, MHC class II protein complexes are encoded by human leukocyte antigen gene complexes (HLA). The HLAs corresponding to MHC class II are HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.

[0060] HLA-A, or "human leukocyte antigen 1," refers to a protein belonging to the HLA class I heavy chain paralog. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (β-2-microglobulin). The heavy chain is fixed within the membrane. Class I molecules play a central role in the immune system, presenting cytosolic peptides that have been translocated into the endoplasmic reticulum lumen so that they can be recognized by cytotoxic T cells. HLA-A is expressed in almost all cells. Its heavy chain is approximately 45 kDa, and its gene contains eight exons. Exon 1 encodes the leader peptide, exons 2 and 3 both encode the α1 and α2 domains that bind to the peptide, exon 4 encodes the α3 domain, exon 5 encodes the transmembrane region, and exons 6 and 7 encode the cytoplasmic end. Polymorphisms in exons 2 and 3 are responsible for the peptide binding specificity of each class I molecule. These polymorphism analyses are routinely performed for bone marrow and kidney transplantation. Over 6,000 HLA-A alleles have been reported. The human HLA-A gene is located on chromosome 6 at 6p22.1 and is described in detail in the NCBI gene database as gene ID 3105. Sequence and positional information is based on the reference sequence assembly GCF_000001405.39 from the Genome Reference Consortium Human Build 38 Patch Release 13, annotation release 109.20201120 (as of December 9, 2020). This reference identifies the HLA-A gene on the coding strand between 29942532 and 29945870.

[0061] HLA-B refers to proteins belonging to the HLA class I heavy chain paralog. Hundreds of HLA-B alleles have been reported. The human HLA-B gene is located on chromosome 6 at 6p21.33 and is described in detail in the NCBI gene database as gene ID 3106. Sequence and positional information is from the reference sequence assembly GCF_000001405.39 of the Genome Reference Consortium Human Build 38 Patch Release 13, annotation release 109.20201120 (as of December 9, 2020).

[0062] HLA-DR is a dimeric protein belonging to HLA class II. This HLA class II molecule is a heterodimer consisting of an α chain (e.g., HLA-DRA) and a β chain (e.g., HLA-DRB1, HLA-DRB3, HLA-DRB4, or HLA-DRB5), both fixed within the membrane. HLA-DR plays a central role in the immune system by presenting peptides derived from extracellular proteins. This class II molecule is expressed in antigen-presenting cells. Since the α chain is substantially immutable, the variability of HLA-DR composition within an individual stems from the ability of the α chain to pair with the β chain derived from three different DRβ loci (HLA-DRB1 and two of the DRB3, DRB4, or DRB5 alleles). The β chain within the DR molecule contains essentially all the polymorphisms that determine the specificity of peptide bonds. In particular, hundreds of DRB1 alleles have been reported, and some alleles increase in frequency in association with specific diseases or conditions. These alleles are responsible for the variability in HLA-DR composition within a population. While some individuals may be homozygous for HLA-DRB1 (i.e., both parents have the same HLA-DRB1 allele), the vast majority of individuals are heterozygous for HLA-DRB1. The human HLA-DRB1 gene is located on chromosome 6 at 6p21.32 and is described in detail in the NCBI gene database as gene ID 31223. Sequence and positional information is based on the reference sequence assembly GCF_000001405.39 from Genome Reference Consortium Human Build 38 Patch Release 13, annotation release 109.20201120 (as of December 9, 2020).

[0063] HLA-DQ is a dimeric protein belonging to HLA class II. This HLA class II molecule is a heterodimer consisting of an α chain (e.g., HLA-DQA1) and a β chain (e.g., HLA-DQB1), both of which are fixed within the cell membrane. HLA-DQ plays a central role in the immune system by presenting peptides derived from extracellular proteins. This class II molecule is expressed in antigen-presenting cells. Both the α and β chains vary greatly between individuals, so numerous alleles have been reported for both HLA-DQA1 and HLA-DQB1. As an MHC class II antigen-presenting receptor, DQ functions as a DQ heterodimer, a dimer containing two protein subunits, α (DQA1 gene product) and β (DQB1 gene product). These receptors are generated from α+β sets (one set consisting of maternal and paternal chromosomes) of two different DQ haplotypes. When a person has the -AB- haplotype from one parent and the -ab- haplotype from the other parent, that person produces two α isoforms (A and a) and two β isoforms (B and b). In this case, four slightly different receptor heterodimers (more simply, DQ isoforms) can be produced. Two isoforms are cis-haplotype pairs (AB and ab), and two isoforms are trans-haplotype pairs (Ab and aB). Such a person is a double heterozygote with respect to these genes, which is the most common situation for DQ. In the case of a person with the -AB- and -Ab- haplotypes, only two DQ (AB and Ab) can be produced. However, a person with the AB- and -AB- haplotypes can only produce the DQ isoform AB and is called a double homozygote. The human HLA-DQA1 gene is located on chromosome 6 at 6p21.32 and is described in detail in the NCBI gene database as gene ID 3117. The human HLA-DQB1 gene is also located on chromosome 6 at 6p21.32 and is described in detail in the NCBI gene database as gene ID 3119.

[0064] HLA-DP is also a dimeric protein belonging to HLA class II. This HLA class II molecule is a heterodimer consisting of an α chain (e.g., HLA-DPA1) and a β chain (e.g., HLA-DPB1), both of which are fixed within the membrane. HLA-DP also plays a central role in the immune system by presenting peptides derived from extracellular proteins. The explanation for HLA-DP is similar to that for HLA-DQ. Since both the α and β chains vary between individuals, several alleles have been reported for both HLA-DPA1 and HLA-DPB1. As explained for HLA-DQ, HLA-DP is generated from an α+β set (one set consisting of maternal and paternal chromosomes) of two different HLA-DP haplotypes. Humans can be double heterozygotes of HLA-DPA1 and HLA-DPB1, which is the most common situation for DP, single homozygotes, or double homozygotes of HLA-DP. The human HLA-DPA1 gene is located at 6p21.32 on chromosome 6 and is described in detail as gene ID 3113 in the NCBI gene database. The human HLA-DPB1 gene is also located at 6p21.32 on chromosome 6 and is described in detail as gene ID 3115 in the NCBI gene database.

[0065] As used herein, CD2 refers, depending on the context, to the CD2 gene or to CD2 as a surface marker, which is the extracellular component of the CD2 protein (also known as LFA-2). The CD2 gene is described in detail in the NCBI gene database as gene ID 914.

[0066] As used herein, CD28 refers, depending on the context, to the CD28 gene or the surface marker which is the extracellular component of the CD28 protein. The CD28 gene is described in detail in the NCBI gene database as gene ID 940.

[0067] As used herein, CD38 refers, depending on the context, to the CD38 gene or the surface marker, which is the extracellular component of the CD38 protein (also known as ADPRC1). The CD38 gene is described in detail in the NCBI gene database as gene ID 952.

[0068] As used herein, CD64 refers, depending on the context, to the CD64 gene or to the surface marker which is the extracellular component of FCGR1A (Fc gamma receptor Ia). FCGR1A is described in detail in the NCBI gene database as gene ID 2209.

[0069] As used herein, CD74 refers, depending on the context, to the CD74 gene or the extracellular component (also known as HLADG) of the CD74 protein, which is a surface marker. The CD74 gene is described in detail in the NCBI gene database as gene ID 972.

[0070] As used herein, C5AR1 refers, depending on the context, to the C5AR1 gene or to the surface marker, which is the extracellular component (also known as CD88) of the C5AR1 protein. The C5AR1 gene is described in detail in the NCBI gene database as gene ID 728.

[0071] As used herein, CD70 refers, depending on the context, to the CD70 gene or the surface marker which is the extracellular component of the CD70 protein. CD70 is described in detail in the NCBI gene database as gene ID 970.

[0072] As used herein, CD206 refers, depending on the context, to the CD206 gene or to a surface marker which is the extracellular component of the mannose receptor C1 type. CD206 is described in detail in the NCBI gene database as gene ID 4360.

[0073] As used herein, CD163 refers, depending on the context, to the CD163 gene or the surface marker which is the extracellular component of the CD163 protein. CD163 is described in detail in the NCBI gene database as gene ID 9332.

[0074] As used herein, IL15 refers, depending on the context, to the IL15 gene, the secreted form of the IL15 protein, or the activating cytokine interleukin 15. IL15 is described in detail in the NCBI gene database as gene ID 3600.

[0075] As used herein, IL18 refers, depending on the context, to the IL18 gene, the secreted form of the IL18 protein, or the activating cytokine interleukin 18. IL18 is described in detail in the NCBI gene database as gene ID 3606.

[0076] As used herein, IL23A refers, depending on the context, to the IL23A gene or to IL23A as a subunit of the heterodimeric cytokine IL23. In this context, IL23A is detectable as the secreted activated cytokine interleukin 23. IL23A is described in detail in the NCBI gene database as gene ID 51561.

[0077] As used herein, RXFP2 refers, depending on the context, to the RXFP2 gene, or to the extracellular portion of the RXFP2 protein, or to the surface marker relaxin family peptide receptor 2. RXFP2 is described in detail in the NCBI gene database as gene ID 122042.

[0078] As used herein, LYVE1 refers, depending on the context, to the LYVE1 gene, or to the extracellular portion of the LYVE1 protein, or to the surface marker lymphatic hyaluronan receptor 1. LYVE1 is described in detail in the NCBI gene database as gene ID 10894.

[0079] As used herein, STAB1 refers, depending on the context, to the STAB1 gene, or to the extracellular portion of the STAB1 protein, or to the surface marker stabilin 1 (also known as SCARH2, as it can function as a scavenger receptor). STAB1 is described in detail in the NCBI gene database as gene ID 23166.

[0080] As used herein, LILRB5 refers, depending on the context, to the LILRB5 gene, or to the extracellular component of the LILRB5 protein, or to the surface marker leukocyte immunoglobulin-like receptor B5. LILRB5 is described in detail in the NCBI gene database as gene ID 10990.

[0081] As used herein, RNASE1 refers, depending on the context, to the RNASE1 gene or the secreted form of the RNASE1 protein, ribonuclease A family member 1. RNASE1 is described in detail in the NCBI gene database as gene ID 6035.

[0082] As used herein, F13A1 refers, depending on the context, to the F13A1 gene or the secreted form of the F13A1 protein, a subunit of coagulation factor XIII. F13A1 is described in detail in the NCBI gene database as gene ID 2162.

[0083] As used herein, QPCT refers, depending on the context, to the QPCT gene or the secreted form of the QPCT protein, glutaminyl cyclase. QPCT is described in detail in the NCBI gene database as gene ID 25797.

[0084] As used herein, CCL7 refers, depending on the context, to the CCL7 gene or the secreted form of the CCL7 protein, the chemokine CCL7. CCL7 is described in detail in the NCBI gene database as gene ID 6354.

[0085] As used herein, RNF128 refers, depending on the context, to the RNF128 gene, or the RNF128 protein, an E3 ubiquitin ligase also known as GRAIL. RNF128 is described in detail in the NCBI gene database as gene ID 79589.

[0086] STAT6 is described in detail in the NCBI gene database as gene ID 6778.

[0087] IRF4 is described in detail in the NCBI gene database as gene ID 3662.

[0088] PPARγ is described in detail in the NCBI gene database as gene ID 5468.

[0089] KLF4 is described in detail in the NCBI gene database as gene ID 9314.

[0090] C / EPBβ (CEBPB) is described in detail in the NCBI gene database as gene ID 1051.

[0091] GATA3 is described in detail in the NCBI gene database as gene ID 2625.

[0092] JMJD3(KDM6B) is described in detail in the NCBI gene database as gene ID 23135.

[0093] SOCS2 is described in detail in the NCBI gene database as gene ID 8835.

[0094] SOCS1 is described in detail in the NCBI gene database as gene ID 8651.

[0095] AKT1 is described in detail in the NCBI gene database as gene ID 207.

[0096] FCGBP is described in detail in the NCBI gene database as gene ID 8857.

[0097] References to genes in the context of experiments conducted on mice refer to the mouse genes corresponding to the human gene names shown (for example, references to "IL-6" in the context of the mouse experiments described in Examples 1-8 refer to mouse IL-6).

[0098] As used herein, negative regulators are gene products, particularly polypeptides, that interfere with the binding of RNA polymerase to the promoter region, inhibit the activity of enhancers, or inhibit the activity of activating transcription factors, resulting in a decrease in the transcription of a target gene, such as C2TA.

[0099] CD8+ T cells (also known as cytotoxic T lymphocytes or CTLs) develop in the thymus and express T cell receptors. They typically express the dimeric coreceptor CD8, which consists of one CD8α chain and one CD8β chain. CD8+ T cells recognize peptides presented by MHC class I molecules present on all nucleated cells. CD8+ T cells are crucial for immune defense against intracellular pathogens, including viruses and bacteria, as well as for tumor monitoring.

[0100] NK cells, also known as natural killer cells or large granular lymphocytes (LGLs), are a type of cytotoxic lymphocyte that plays a crucial role in the innate immune system. NK cells can be identified by the presence or absence of CD56 and CD3 (CD56+, CD3-). NK cells are innate immune cells that function similarly to cytotoxic T cells in adaptive immune responses. NK cells act on virus-infected cells approximately three days after infection, providing a rapid response and responding to tumorigenesis. NK cells have the ability to recognize and kill cells that are stressed and lack antibodies and MHC. This role is important because harmful cells that have lost the MHC I marker are not detected or destroyed by other immune cells, such as T lymphocytes.

[0101] The "mobilization" of cells, such as CD8+ T cells and / or NK cells, to a tumor can be examined in a mouse model, such as a humanized mouse model, by extracting the tumor mass, separating the cells, and analyzing the number of CD8+ T cells and / or NK cells associated with the tumor mass by FACS, for example, after appropriate staining. A procedure that can mobilize such cells to the tumor (such as the injection of macrophages according to the present invention) results in a state over time in which such cells constitute a higher proportion of the cell count in the tumor mass specimen compared to an appropriate control without such a procedure.

[0102] As used herein, the term “proliferative cell” refers to a cell capable of cell division. A cell is a proliferative cell if, after 8 days under appropriate culture conditions, a population of at least 1,000 “proliferative cells” increases in number by at least fourfold, i.e., n(192 hours) / n(0 hours) (where n is the total number of cells in the cell population at the indicated time) is at least 4.00.

[0103] As used herein, the term "differentiated human cell" refers to a cell whose cell type does not change, and which even during cell division produces two cells of the same cell type. This is in contrast to "pluripotent cells," which can differentiate into all adult cell types, and "oligopotent cells," which can differentiate into several closely related cell types.

[0104] As used herein, “myeloid cells” means cells of hematopoietic origin that are not lymphoid, nor erythrocyte-megakaryocyte, nor multilineage progenitor cells that may be of a non-myeloid lineage.

[0105] As used herein, iPS cells, or "induced pluripotent stem cells," refer to pluripotent cells obtained by reprogramming somatic cells. Several groups, including Professor Shinya Yamanaka's group at Kyoto University, Rudolf Jaenisch's group at MIT, James Thomson's group at the University of Wisconsin, and Konrad Hochedlinger's group at Harvard University, have succeeded in creating such induced pluripotent stem cells. Induced pluripotent stem cells have attracted considerable attention as ideal pluripotent cells that do not involve immunological rejection or ethical issues. For example, international publication WO2007 / 069666 reports somatic cell nuclear reprogramming factors containing gene products of Oct family genes, Klf family genes, and Myc family genes. This document also describes a method for creating induced pluripotent stem cells by nuclear reprogramming of somatic cells, a method that includes the step of contacting somatic cells with the aforementioned nuclear reprogramming factors.

[0106] Monocytes are mononuclear phagocytic cells found in peripheral blood. Monocytes are quite diverse in size, ranging from 10 to 30 μm in diameter. The ratio of the nucleus to the cytoplasm is in the range of 2:1 to 1:1. The nucleus is often band-shaped (horseshoe-shaped) or kidney-shaped (kidney-shaped). Monocytes may also fold themselves, exhibiting a gyral-like rotation. Nucleoli are not visible. The chromatin pattern is fine and arranged in a skein-like pattern. The cytoplasm is abundant and appears as a bluish-gray color with numerous fine azurophilic granules in Giemsa staining, exhibiting a ground-glass appearance. Vacuoles may also be present. More preferably, the expression of specific surface antigens is used to determine whether a cell is a monocyte. Phenotypic markers for human monocyte cells include CD11b, CD11c, CD33, CD45, and CD115. Generally, human monocyte cells are classified as CD9, CD11b, CD11c, CDw12, CD13, CD15, CDw17, CD31, CD32, CD33, CD35, CD36, CD38, CD43, CD45, CD49b, CD49e, CD49f, CD63, CD64, CD65s, CD68, CD84, CD85, CD86, CD87, CD89, CD91, CDw92, CD93, CD98, CD101, CD102, CD111, CD112, CD115, CD116, CD119, CDw121b, CDw123, CD127, CDw128, CDw131, CD147, CD They express 155, CD156a, CD157, CD162, CD163, CD164, CD168, CD171, CD172a, CD180, CD131a1, CD213a2, CDw210, CD226, CD281, CD282, CD284, and CD286, and optionally CD4, CD14, CD16, CD40, CD45RO, CD45RA, CD45RB, CD62L, CD74, CD141, CD142, CD169, CD170, CD181, CD182, CD184, CD191, CD192, CD194, CD195, CD197, CD206, and CX3CR1. Unless otherwise specified, the term "monocyte" as used herein includes the term "macrophage".

[0107] Dendritic cells (DCs) are antigen-presenting cells that exist in vivo, in vitro, or ex vivo, or within a host or subject, or may be derived from hematopoietic stem cells, hematopoietic progenitor cells, or monocytes. Dendritic cells and their precursors can be isolated from various lymphoid organs, such as the spleen and lymph nodes, as well as from bone marrow and peripheral blood. DCs have a characteristic morphology, possessing thin sheets (membranous pseudopods) that extend in multiple directions from the dendritic cell body. DCs constitutively express both MHC class I and MHC class II molecules, which can present peptide antigens to CD8+ T cells and CD4+ T cells, respectively, and can activate naive T cells. Furthermore, DCs in human skin and mucous membranes also express the CD1 gene family and MHC class I-related molecules, which present microbial lipid or glycolipid antigens. The DC membrane is also rich in molecules that enable T cell adhesion (e.g., cell adhesion molecule-1 or CD54), or molecules that co-stimulate T cell activation, such as B7-1 and B7-2 (also known as CD80 and CD86, respectively). Generally, DCs express CD85, CD180, CD187, CD205, CD281, CD282, CD284, and CD286, and express CD206, CD207, CD208, and CD209 as subsets. Unless otherwise specified, the term “dendritic cell” as used herein is included in the term “macrophage.”

[0108] As used herein, “macrophage” includes macrophages, monocytes, and dendritic cells. However, preferably, as used herein, “macrophage” refers to macrophages in the narrow sense. Macrophages are phagocytic cells. The morphology of macrophages varies between different tissues and between normal and pathological states, and not all macrophages can be identified solely by morphology. However, most macrophages are large cells with a round or notched nucleus, a well-developed Golgi apparatus, abundant endocytic vacuoles, lysosomes, and phagolysosomes, and a plasma membrane covered with folds or microvilli. The primary functions of macrophages in innate and adaptive immunity are phagocytosis and subsequent degradation of senescent or apoptotic cells, microorganisms, and neoplastic cells, secretion of cytokines, chemokines, and other soluble mediators, and presentation of foreign antigens (peptides) on their surface to T lymphocytes. Macrophages originate from pluripotent progenitor cells, myeloid common progenitor cells, and granulocyte-monocyte progenitor cells in the bone marrow of mammalian organisms, ultimately developing into monocytes through further progenitor cell stages before entering the peripheral bloodstream. Unlike neutrophils, which have a multilobed nucleus, monocytes have a kidney-shaped nucleus and exhibit a large cell body during further differentiation and activation. Throughout their life cycle, some monocytes attach to the capillary endothelium, migrate from the endothelium to all organs, and some can differentiate into tissue-resident macrophages or dendritic cells in those organs (see below). In addition to monocyte-derived macrophages, tissue-resident macrophages can also arise from early primitive macrophage progenitor cells in the yolk sac before final hematopoietic synthesis is established, from erythroid macrophage progenitor cells (EMPs) in various hematopoietic sites of the embryo, or from embryonic monocytes derived from embryonic hematopoietic stem cells. These embryonic macrophages remain present until adulthood and are maintained for extended periods, independent of the introduction of adult hematopoietic stem cells and monocytes. While macrophages are particularly abundant in lymphatic tissues such as lymph nodes and the spleen, tissue-resident macrophages are essentially present in all organs of the body. Within some organs, macrophages have specific names, as summarized in Table 1.

[0109] (Table 1: Examples of tissue-resident macrophages) [Table 2]

[0110] Further examples of macrophages include peritubular macrophages and testicular interstitial macrophages, cardiac macrophages derived from the heart, adipose tissue macrophages derived from adipose tissue, colonic and small intestinal macrophages derived from the intestine, skeletal muscle macrophages, synovial macrophages derived from joints, adventitious macrophages, intima-arterial macrophages, vascular-associated macrophages, pancreatic resident macrophages, meningeal macrophages, pleural macrophages, and omental macrophages.

[0111] In the context of the present invention, macrophages may be selected from any of the macrophages described above. Preferably, macrophages may be selected from the group consisting of microglia, histiocytes, Hofbauer cells, mesangial cells, Kupffer cells, peritoneal macrophages, alveolar macrophages, epithelial or cutaneous macrophages, marginal zone lymphoma macrophages, metalloaffin macrophages, red pulp macrophages, white pulp macrophages, and osteoclasts. Macrophages may also be derived from human iPS cells in accordance with an in vitro differentiation protocol as described herein.

[0112] Macrophages are an important source of cytokines. Functionally, the numerous products can be divided into several groups: (1) cytokines that mediate the pro-inflammatory response, i.e., those that help recruit further inflammatory cells (e.g., IL-1, II-6, TNF, CC chemokines and CXC chemokines, e.g., IL-8, and monocyte chemotactic protein 1); (2) cytokines that mediate the activation of T cells and natural killer (NK) cells (e.g., IL-1, IL-12, IL-15, IL-18); (3) cytokines in which macrophages themselves exert a feedback effect (e.g., IL-1, T (4) Cytokines that downregulate macrophages and / or help to stop inflammation (e.g., IL-10, TGFβ); (5) Cytokines important for wound healing or supporting tissue stem cells (e.g., EGF, PDGF, bFGF, TGFβ), or cytokines important for supporting angiogenesis (e.g., VEGF), or cytokines important for supporting nerve cells (e.g., neurotrophic factors, kinins). Cytokine production by macrophages can be activated by microbial products such as LPS, by interaction with type 1 helper T cells, or by soluble factors including prostaglandins, leukotrienes, and most importantly, other cytokines (e.g., IFNγ). Generally, human macrophages express CD11c, CD11b, CD14, CD18, CD26, CD31, CD32, CD36, CD45RO, CD45RB, CD63, CD68, CD71, CD74, CD87, CD88, CD101, CD115, CD119, CD121b, CD155, CD156a, CD204, CD206, CDw210, CD281, CD282, CD284, and CD286, and express CD163, CD169, CD170, MARCO, FOLR2, and LYVE1 as subsets. Activated macrophages can further express CD23, CD25, CD69, CD105, as well as HLA-DR, HLA-DP, and HLA-DQ.

[0113] As used herein, a macrophage is said to be resistant to M2 polarization by M-CSF (or a combination of M-CSF and IL-4 and / or IL-13 cytokines) if long-term culture of one or more macrophages does not result in the loss of all the characteristics of an M1-polarized macrophage, in particular, if it does not result in the loss of all the normal characteristics of an M1-polarized macrophage. As used herein, the characteristics of an M1 polarized macrophage may be any one of the following: upregulated expression of an HLA class II gene, such as HLA-DPA1, HLA-DPB1, HLA-DPB2, HLA-DRA, HLA-DRB5, HLA-DQA1, HLA-DQB1, and / or upregulated expression of RXFP2, CD74, CD38, CD2, IL18, and / or IL23A, and / or upregulated secretion of IL18, IL15, and / or IL23, and / or downregulated expression of RNASE1, PPBP, CD28, LYVE1, FCGBP, F13A1, QPCT, CCL7, and / or RNF128, and / or downregulated secretion of PPBP, CCL7, RNASE1, F13A1, QPCT, and / or FCGBP. M1 macrophages are also characterized by the presence or absence of surface markers, and / or by patterns of expressed and / or unexpressed surface markers. Further examples of typical features of M1 macrophages as used herein may include MHC II positivity, CD74 positivity, CD2 positivity, LYVE1 negativity, CD28 negativity, STAB1 negativity, and / or LILRB5 negativity.

[0114] "Surface markers" are molecules, typically proteins or carbohydrate structures, that are accessiblely located on the outside of the cell's plasma membrane and are specific to one particular cell type or a limited number of cell types, and therefore serve as "markers" for those cell types. Examples of surface markers on human macrophages include CD11c, CD11b, CD14, CD16, CD18, CD26, CD31, CD32, CD33, CD36, CD45RO, CD45RB, CD63, CD64, CD68, CD71, CD74, CD87, CD88, CD101, CD115, CD119, CD121b, CD155, CD156a, CD163, CD169, CD170, CD204, CD206, CDw210, CD281, CD282, CD284, CD286, MARCO, FOLR2, CX3CR1, and LYVE1.

[0115] In FACS testing, cells are considered "positive" for a surface marker if they produce a specific fluorescence signal when stained with an antibody specific to that marker. The principle of FACS is explained in detail in Howard M. Shapiro's "Practical Flow Cytometry," 4th edition. In FACS testing, a collection of cells is typically stained with multiple fluorescent antibodies, each antibody selectively binding to a different surface marker and possessing a different fluorescent dye. This allows for the selection of specific cell types within a heterogeneous cell collection using an appropriate gating strategy in FACS testing. Next, the specific fluorescence signal from the surface marker-specific antibody is verified using a one-dimensional bar graph plot. This usually involves comparing the bar graph of staining with all antibodies to the bar graph of staining with a mix of antibodies excluding only the surface marker-specific antibody (the so-called "FMO" or "fluorescence minus 1" signal). If these two bar graphs differ, for example, if staining with the all-antibody mix produces stronger fluorescence than the FMO control, then the tested cell collection is positive for the tested cell surface marker. In terms of the visual appearance of the bar graph, this means that the fluorescence peak of the staining with the total antibody mix is ​​shifted to a higher fluorescence value compared to the FMO control. Preferably, the two bar graphs (one with the total antibody and the other with FMO) overlap by up to 70 area % (area under the curve), for example, up to 50 area %, for example, up to 25 area %.

[0116] The "secretion" of cytokines, such as IL-18 and / or IL-15, can be determined by immunobiochemical methods, qualitatively and / or quantitatively measuring the appearance of cytokines in the supernatant of cell cultures. Two examples include ELISA-based methods or bead-based multiplex assays, such as the Luminex technique. Briefly, the culture medium used for cell proliferation is analyzed for the presence of the specific cytokine to be tested, both before adding it to the cell collection and after culturing the cells in which cytokine secretion is to be tested. Cytokines are "secreted" by cells cultured in that medium if the cytokine concentration in the supernatant increases during the culture period, and this increase in cytokine concentration is confirmed by three consecutive separate measurements. IL-6 can be detected at concentrations of 0.1 pg / ml or less using, for example, the Meso Scale discovery immunoassay "V-PLEX Human IL-6 Kit" from Meso Scale Diagnostics. CXCL10 can be detected at concentrations of 5 pg / ml or less using, for example, the LANCE Ultra Human CXCL10 Detection Kit from Perkin Elmer.

[0117] The terms “phagocytic cell” and “phagocyte” are used interchangeably herein and refer to cells capable of phagocytosis. There are different major categories of professional phagocytes, including macrophages in the narrow sense, monocytes, and dendritic cells, as well as mononuclear phagocytes, including polymorphonuclear leukocytes (neutrophils). However, it is also known that “non-professional” phagocytic cells participate in efferocytosis. Efferocytosis is the process by which professional and non-professional phagocytes dispose of apoptotic cells in a rapid and efficient manner.

[0118] As used herein, the term “progenitor cell” refers to a cell that is a descendant of a stem cell and can further differentiate to produce a specific cell type. Many types of progenitor cells exist throughout the human body. Each progenitor cell can only differentiate into cells belonging to the same tissue or organ. Some progenitor cells ultimately differentiate into only one type of target cell, while others have the potential to differentiate into multiple cell types. Thus, progenitor cells are intermediate cell types involved in the production of mature cells in human tissues and organs, blood, and the central nervous system. Hematopoietic progenitor cells are intermediate cell types in the production of blood cells. Hematopoietic progenitor cells are immature cells that arise from hematopoietic stem cells and eventually differentiate into one of more than ten different types of mature blood cells.

[0119] As used herein, the term “CD34+ pluripotent progenitor cells” refers to a population of stem cell-enriched hematopoietic progenitor cells expressing the CD34 surface antigen, which are not macrophages, monocytes, or dendritic cells.

[0120] As used herein, the term “monoblast” refers to committed progenitor cells in the bone marrow that differentiate from myeloid progenitor cells in the hematopoietic process. They can mature into monocytes and then develop into macrophages.

[0121] As used herein, the term “collection of cells” refers to a collection of cells that are at least 10,000 living cells.

[0122] As used herein, the term "proliferation" of cells refers to the process of culturing cells under appropriate experimental conditions and increasing the number of viable cells through mitosis of the cultured cells.

[0123] As used herein, the term "after exposure" means that cells are cultured for the indicated time in the continuous presence of a specific agent, such as a cytokine, and then immediately tested.

[0124] As used herein, the term “genetically modified” cells refer to cells whose DNA has been altered using biotechnology. For example, a cell in which the DNA of a cell has been manipulated using a CRISPR / Cas9 DNA editing system, resulting in a detectable change in the cell’s DNA, is a genetically modified cell.

[0125] As used herein, the term "appropriate culture conditions" refers to conditions under which the phagocytic cells of the present invention can proliferate. Culturing is typically carried out in a cell culture medium supplemented with appropriate growth factors, at an appropriate temperature, and in an appropriately controlled atmosphere. A suitable culture medium is RPMI medium (named after Roswell Park Memorial Institute, a medium often used for culturing human lymphocytes) supplemented with 10% FBS (PAA-GE Healthcare, A15-101), and then supplemented with 100 units / ml penicillin, 100 ug / ml streptomycin (Thermo Fisher, no. 15140122), 2 mM GlutaMAX (Thermo Fisher, no. 35050038), 1 mM sodium pyruvate (Thermo Fisher, no. 11360-039), 50 ng / ml M-CSF (Thermo Fisher, no. PHC9504), and, if indicated, 50 ng / ml GM-CSF (Peprotech, no. 300-03), as described in Example 3. Culturing is usually carried out at 37 °C and under 5% CO2 and 21% O2.

[0126] As used herein, the term “reactive oxygen species” refers to unstable molecules containing oxygen that readily react with other molecules within cells. Typically, reactive oxygen species are free radicals. Reactive oxygen species are known as components of the killing response of immune cells to microbial invasion.

[0127] As used herein, the term "activation" refers to the phenomenon in which an external stimulus induces a change in a cell, thereby activating that cell. For example, macrophages can be activated by cytokines such as interferon-γ (IFN-γ) and bacterial endotoxins, such as lipopolysaccharides (LPS). Activated macrophages undergo many changes, thereby enabling them to kill invading bacteria or infected cells. They release toxic chemicals and proteins that have toxic effects on other cells. Activated macrophages become larger, their metabolism increases, their lysosomal protein levels rise, and their ability to phagocytose and kill microorganisms increases. Activated macrophages also release proteases, neutrophil chemotactic factors; reactive oxygen species such as nitric oxide and superoxide; cytokines such as tumor necrosis factor α (TNF-α), interleukin-1 and interleukin-8 (IL-1 and IL-8), eicosanoids, and growth factors. The products of these activated macrophages can lead to certain types of tissue damage, which are characteristic of inflammation.

[0128] As used herein, the term “adherence” refers to the property of adherent cells to a solid substrate, such as the bottom of a tissue culture flask. In contrast, suspension cells are suspended in a culture medium and grow while suspended, so they do not need to be removed mechanically or chemically.

[0129] As used herein, “membranous pseudopod” refers to a projection at the anterior margin of a cell. It contains a quasi-two-dimensional actin network. Filiform pseudopods are finger-like structures within the membranous pseudopod and extend beyond the tip of the membranous pseudopod.

[0130] When referring to polypeptides or nucleotide sequences, “purified” and “isolated” mean the presence of the molecule in the substantial absence of other biological macromolecules. When referring to “cells or populations of cells,” this term means the presence of those cells or populations in the substantial absence of other cells or populations. As used herein, the term “purified” means the presence of at least 75% by weight or number, more preferably at least 85% by weight or number, even more preferably at least 95% by weight or number, and most preferably at least 98% by weight or number of the same type of biological macromolecule or cells. An “isolated” nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that substantially does not contain other nucleic acid molecules that do not encode the polypeptide of interest, however this molecule may contain several additional bases or moieties that do not adversely affect the essential characteristics of the composition.

[0131] As used herein, the terms “tumor,” “tumor cell,” “cancer,” and “cancer cell” refer to cells that exhibit relatively autonomous growth, characterized by a marked loss of control over cell proliferation (i.e., deregulation of cell division and / or destruction / neglect of normal tissue organization and structure). Tumor cells may be malignant or benign, but cancer cells are malignant. “Metastatic cells or tissue” means that the cells are capable of invading, establishing, and destroying proximal and distal body structures.

[0132] As used herein, “solid tumor” refers to a non-hematological tumor. A solid tumor may be benign (not “cancerous”) or malignant (cancerous). Different types of solid tumors are named after the cell type that forms them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas, although leukemia (a blood cancer) does not generally form solid tumors.

[0133] As used herein, the term "subject" refers to a human being.

[0134] In the context of the present invention, the terms “to treat” or “to cure” as used herein mean to reverse, alleviate, inhibit the progression of, or prevent the disease or condition to which the term applies, or one or more symptoms of such disease or condition.

[0135] As used herein, the term "chimeric antigen receptor" refers to a fusion of an extracellular recognition domain (e.g., an antigen-specific targeting region), a transmembrane domain, and one or more intracellular signaling domains. Upon antigen engagement by the extracellular recognition domain, the intracellular signaling portion of the CAR can initiate activation-related responses within immune cells, such as the release of cytolytic molecules, thereby inducing tumor cell death.

[0136] As used herein, the term "exvivo" means the outside of a living organism.

[0137] As used herein, the term “in vitro” means outside of a living organism, within a laboratory environment. For example, cells cultured “in vitro” are controlled and often cultured in artificial media.

[0138] As used herein, "autologous" refers to cells from the individual themselves. For example, in autologous blood transfusion, the patient's own blood is drawn and reinjected into their body.

[0139] As used herein, "allogeneic" refers to human cells that are not the cells of the individual themselves. For example, allogeneic stem cell transplantation is different from autologous stem cell transplantation, which uses stem cells derived from the patient's own body.

[0140] All numerical values, such as pH, temperature, time, concentration, and molecular weight, including ranges, are (+) or (-) approximations that vary in increments of 0.1. While not always explicitly stated, please understand that all numerical values ​​are preceded by the term "approximately." Similarly, while not always explicitly stated, please understand that the reagents described herein are merely illustrative, and equivalents of such reagents are known in the art.

[0141] It should be understood that the present invention is not limited to the specific materials and methods described herein. Furthermore, it should be understood that the terms used herein are for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which will be limited only by the appended claims. Where used herein, the singular forms "a," "an," and "the" also include plural references unless the context clearly indicates otherwise. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The following references provide to those skilled in the art general definitions of many terms used in this invention and are ranked in ascending order of priority unless otherwise specified herein: Singleton et al., “Dictionary of Microbiology and Molecular Biology” (3rd edition, 2006), “The Glossary of Genomics Terms” JAMA. 2013;309(14):1533-1535, “Janeway's Immunobiology” 9th edition, and “Practical Flow Cytometry” 4th edition, edited by H.M. Shapiro.

[0142] All references cited herein are listed for the purpose of describing and disclosing cell lines, protocols, reagents, and vectors that are described in their respective publications and may be used in connection with the present invention. Nothing in this specification should be construed as implying that the present invention does not have prior rights over such disclosures in the prior art.

[0143] (Detailed explanation) The present invention relates to a human macrophage having at least one mutation in both alleles of a gene located on a chromosome, and which is resistant to M-CSF-induced M2 polarization. The macrophage of the present invention is antitumor-forming and is therefore useful in macrophage cell therapy, particularly in anticancer therapy.

[0144] In particular, the human macrophages of the present invention are resistant to M2 polarization by a combination of M-CSF and IL-4, and / or by a further combination of M-CSF, IL-4, and IL-13.

[0145] Resistance to M2 polarization means, for example, that the human macrophages of the present invention retain the normal characteristics of M1 macrophages even after exposure to M-CSF and / or IL-4 and / or IL-13 for 24 hours, particularly 48 hours, for example, 72 hours or more. For example, macrophages may still contain at least one normal characteristic of M1 macrophages even after exposure to 50 ng / ml M-CSF and / or 20 ng / ml IL-4 for 24 hours, or further, for example, 50 ng / ml M-CSF and / or 20 ng / ml IL-4 for 48 hours, for example, 72 hours. Preferred macrophages of the present invention exhibit at least one, for example, at least two, three, four, or even five, typical features of an M1 macrophage after 24 hours of exposure to a combination of 50 ng / ml M-CSF, 40 ng / ml IL-4, and 20 ng / ml IL-13, and especially after 48 hours, or even after a further 72 hours of exposure.

[0146] The human macrophages of the present invention can be characterized by their gene expression profile. A typical characteristic of M1 macrophages may be, for example, the expression of the HLA-DRA gene. In particular, the expression level of HLA-DRA mRNA in human macrophages containing at least one mutation in both alleles of a gene located on a chromosome is, for example, at least 4 times, for example at least 8 times, for example at least 16 times, or even more than 32 times, higher than the expression of HLA-DRA mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0147] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the expression of the HLA-DRB5 gene. In particular, the expression level of HLA-DRB5 mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is, for example, at least 4 times, for example at least 8 times, for example at least 16 times, or even more than 32 times, higher than the expression of HLA-DRB5 mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0148] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the expression of the HLA-DPA1 gene. In particular, the expression level of HLA-DPA1 mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is, for example, at least 4 times, for example at least 8 times, for example at least 16 times, or even more than 32 times, higher than the expression of HLA-DPA1 mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0149] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the expression of the HLA-DQA1 gene. In particular, the expression level of HLA-DQA1 mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is, for example, at least 4 times, for example at least 8 times, for example at least 16 times, or even more than 32 times, higher than the expression of HLA-DQA1 mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0150] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the expression of the RXFP2 gene. In particular, the expression level of RXFP2 mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is, for example, at least 4 times, for example at least 8 times, for example at least 16 times, or even more than 32 times, the expression of RXFP2 mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0151] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the expression of the CD74 gene. In particular, the expression level of CD74 mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is, for example, at least 4 times, for example, at least 8 times, or even more than 16 times, the expression of CD74 mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0152] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the expression of the CD70 gene. In particular, the expression level of CD70 mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is, for example, at least 4 times, for example, at least 8 times, or even more than 16 times, the expression of CD70 mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0153] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the expression of the CD38 gene. In particular, the expression level of CD38 mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is, for example, at least 4 times, for example, at least 8 times, or even more than 16 times, the expression of CD38 mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0154] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the expression of the IL15 gene. In particular, the expression level of IL15 mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is, for example, at least twice as high, and for example, at least 2.5 times higher, than the expression of IL15 mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0155] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the expression of the IL18 gene. In particular, the expression level of IL18 mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is, for example, at least four times higher and, for example, at least six times higher than the expression of IL18 mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0156] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the expression of the IL23A gene. In particular, the expression level of IL23A mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is, for example, at least four times higher and, for example, at least six times higher than the expression of IL23A mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0157] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the expression of the APOBEC3A gene. In particular, the expression level of APOBEC3A mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is, for example, at least four times higher and, for example, at least five times higher than the expression of APOBEC3A mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0158] Preferred and typical features of the human macrophages of the present invention are the expression of the HLA-DRA, HLA-DRB5, HLA-DPA1, HLA-DPB1, HLA-DQA1, and HLA-DQB1 genes. In particular, the expression levels of the respective mRNAs corresponding to the said genes in human macrophages containing at least one mutation in both alleles of the chromosomally located genes are, for example, at least 4 times, for example at least 8 times, for example at least 16 times, or even more than 32 times, higher than the expression of the said mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0159] Preferred and typical features of the human macrophages of the present invention include the expression of the HLA-DRA gene, HLA-DRB5 gene, HLA-DPA1 gene, HLA-DPB1 gene, HLA-DQA1 gene, HLA-DQB1 gene, RXFP2 gene, and CD74 gene. In particular, the expression levels of the respective mRNAs corresponding to the said genes in human macrophages containing at least one mutation in both alleles of the chromosomally located genes are, for example, at least 4 times, for example at least 8 times, and for example at least 16 times higher than the expression of said mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0160] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulated expression of the RNASE1 gene. In particular, the expression level of RNASE1 mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is, for example, at least 4 times lower, for example at least 8 times, for example at least 16 times, for example at least 32 times, or even at least 64 times lower than the expression of RNASE1 mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions. "64 times lower" means that the copy number of RNASE1 mRNA in the human macrophages of the present invention is only 1 / 64th the copy number of RNASE1 mRNA in the corresponding wild-type macrophages.

[0161] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulated expression of the CD28 gene. In particular, the expression level of CD28 mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is, for example, at least 4 times lower, for example at least 8 times, for example at least 16 times, for example at least 32 times, or even at least 64 times lower than the expression of CD28 mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0162] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulated expression of the LYVE1 gene. In particular, the expression level of LYVE1 mRNA in human macrophages containing at least one mutation in both alleles of the chromosomally located gene is, for example, at least 4 times, for example at least 8 times, for example at least 16 times, for example at least 32 times, or even at least 64 times lower than the expression of LYVE1 mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0163] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulated expression of the C5AR1 gene. In particular, the expression level of C5AR1 mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is, for example, at least 4 times lower, for example at least 8 times, for example at least 16 times, and even at least 25 times lower than the expression of C5AR1 mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0164] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulated expression of the FCGBP gene. In particular, the expression level of FCGBP mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is, for example, at least 4 times lower, for example at least 8 times, for example at least 16 times, for example at least 32 times, or even at least 64 times lower than the expression of FCGBP mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0165] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulated expression of the F13A1 gene. In particular, the expression level of F13A1 mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is, for example, at least 4 times lower, for example at least 8 times, for example at least 16 times, for example at least 32 times, or even at least 64 times lower than the expression of F13A1 mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0166] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulated expression of the QPCT gene. In particular, the expression level of QPCT mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is, for example, at least 4 times lower, for example at least 8 times, for example at least 16 times, for example at least 32 times, or even at least 64 times lower than the expression of QPCT mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0167] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulated expression of the DUSP6 gene. In particular, the expression level of DUSP6 mRNA in human macrophages containing at least one mutation in both alleles of the chromosomally located gene is, for example, at least four times lower and, for example, at least six times lower than the expression of DUSP6 mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0168] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulated expression of the CCL7 gene. In particular, the expression level of CCL7 mRNA in human macrophages containing at least one mutation in both alleles of the chromosomally located gene is, for example, at least 4 times, for example at least 8 times, for example at least 16 times, or even at least 32 times lower than the expression of CCL7 mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0169] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulated expression of the RNF128 gene. In particular, the expression level of RNF128 mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is, for example, at least 4 times lower, for example at least 8 times, for example at least 16 times, for example at least 32 times, or even at least 64 times lower than the expression of RNF128 mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0170] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulated expression of the IL10 gene. In particular, the expression level of IL10 mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is, for example, at least 4 times lower, for example at least 8 times, for example at least 16 times, for example at least 32 times, or even at least 64 times lower than the expression of IL10 mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0171] A preferred and typical feature of the human macrophages of the present invention is the downregulated expression of the RNASE1 gene, the CD28 gene, and the LYVE1 gene. In particular, the expression levels of the respective mRNAs corresponding to the genes in human macrophages containing at least one mutation in both alleles of the chromosomally located genes are, for example, at least 4 times, for example at least 8 times, for example at least 16 times, for example at least 32 times, or even at least 64 times lower than the expression of the mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0172] Another preferred conventional feature of the human macrophages of the present invention is the upregulated expression of the HLA-DRA, HLA-DRB5, HLA-DPA1, HLA-DPB1, HLA-DQA1, and HLA-DQB1 genes on one side, and the downregulated expression of the RNASE1, CD28, and LYVE1 genes on the other side. In particular, the expression levels of each mRNA corresponding to the upregulated gene in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome are at least four times higher, for example at least eight times, for example at least sixteen times, or even more at least 32 times higher, than the expression levels of the mRNA in otherwise identical wild-type macrophages tested under otherwise uniform culture conditions. On the other hand, the expression levels of each mRNA corresponding to the downregulated gene in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome are at least four times lower, for example at least eight times, for example at least sixteen times, at least 32 times, or even more at least 64 times lower, than the expression levels of the mRNA in otherwise identical wild-type macrophages tested under otherwise uniform culture conditions.

[0173] Other preferred conventional features of the human macrophages of the present invention include the expression of the following gene combinations, which are at least 10-fold upregulated (compared to the expression of each mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions): combination (A) HLA-DRA and RXFP2, combination (B) HLA-DRA and CD74, combination (C) HLA-DOA and RXFP2, combination (D) HLA-DOA and CD74, combination (E) HLA-DPA1 and RXFP2, combination (F) HLA-DPA1 and CD74, combination (G) HLA-DRA, RXFP2, and CD74, combination (H) HLA-DOA, RXFP2, and CD74, and combination (I) HLA-DPA1, RXFP2, and CD74.

[0174] Other preferred conventional features of the human macrophages of the present invention are the expression of the following gene combinations, which are at least 50-fold downregulated (compared to the expression of each mRNA in otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions): combination (m)RNASE1 and LYVE1, combination (n)RNASE1 and FCGBP, combination (o)RNASE1 and CD28, combination (p)RNASE1 and F13A1, combination (q)RNASE1 and RNF128, combination (r)LYVE1 and CD28, combination (s)LYVE1 and FCGBP, combination (t)LYVE1 and F13A1, combination (u)LYVE1 and RNF128, combination (v)CD28 and F13A1, and combination (w)CD28 and RNF128.

[0175] Other preferred conventional features of the human macrophages of the present invention are the upregulated expression of one gene combination and the downregulated expression of another gene combination, such as the combinations described below: Am (a combination of A and m described immediately above, i.e., at least 10-fold upregulated expression of both HLA-DRA and RXFP2 (A) and at least 50-fold downregulated expression of both RNASE1 and LYVE1 (m)), An, Ao, Ap, Aq, Ar, As, At, A u, Av, Aw, Bm, Bn, Bo, Bp, Br, Bq, Bs, Bt, Bu, Bv, Bw, Vm, Cn, Co, Cp, Cq, Cr, Cs, Ct, Cu, Cv, Cw, Dm, Dn, Do, Dp, Dq, Dr, Ds, Dt, Du, Dv, Dw, Em, En, Eo, Ep, E q, Er, Es, Et, Eu, Ev, Ew, Fm, Fn, Fo, Fp, Fq, Fr, Fs, Ft, Fu, Fv, Fw, Gm, Gn, Go, Gp, Gq, Hr, Hs, Ht, Hu, Hv, Hw, Im, In, Io, Ip, Iq, Ir, Is, It, Iu, Iv, and Iw.

[0176] The human macrophages of the present invention may be characterized by the profile of their secretion of cytokines and / or enzymes into the culture medium. Alternatively, or additionally, a typical feature of M1 macrophages may be the secretion of, for example, IL23A. In particular, the secretion of IL23A by human macrophages containing at least one mutation in both alleles of a gene located on a chromosome is, for example, at least 3 times, for example at least 4 times, for example at least 5 times, 6 times, or even 8 times higher than the secretion of IL23A by otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0177] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, IL-18 secretion. In particular, IL-18 secretion by human macrophages containing at least one mutation in both alleles of a gene located on the chromosome is, for example, at least 3 times, for example at least 4 times, for example at least 5 times, 6 times, or even 8 times higher than IL-18 secretion by otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0178] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, IL-15 secretion. In particular, IL-15 secretion by human macrophages containing at least one mutation in both alleles of a gene located on the chromosome is, for example, at least twice, for example, at least three times, for example, at least four times higher than IL-15 secretion by otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0179] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulated secretion of RNASE1. In particular, RNASE1 secretion by human macrophages containing at least one mutation in both alleles of a gene located on the chromosome is, for example, at least 3 times lower, for example at least 5 times, for example at least 7 times, 10 times, or even 30 times lower than RNASE1 secretion by otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0180] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulated secretion of FCGBP. In particular, secretion of FCGBP by human macrophages containing at least one mutation in both alleles of a gene located on the chromosome is, for example, at least 3 times lower, for example at least 5 times, for example at least 7 times, 10 times, or even 30 times lower than secretion of FCGBP by otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0181] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulated secretion of CCL7. In particular, CCL7 secretion by human macrophages containing at least one mutation in both alleles of a gene located on the chromosome is, for example, at least 3 times lower, for example at least 5 times, for example at least 7 times, 10 times, or even 20 times lower than CCL7 secretion by otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0182] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulated secretion of IL-10. In particular, IL-10 secretion by human macrophages containing at least one mutation in both alleles of a gene located on the chromosome is, for example, at least 3 times lower, for example at least 5 times, for example at least 7 times, 10 times, or even 30 times lower than IL-10 secretion by otherwise identical wild-type macrophages tested under otherwise homogeneous culture conditions.

[0183] Another preferred conventional feature of the human macrophages of the present invention is, therefore, the upregulated secretion of IL18 and / or IL23A on the one hand, and the downregulated secretion of RNASE1 and / or FCGBP and / or IL10 and / or CCL7 on the other hand.

[0184] The human macrophages of the present invention may also be characterized by their cell surface markers. Alternatively, or additionally, the usual features of M1 macrophages may be, for example, the presence of the cell surface marker MHC II, or more specifically, the presence of HLA class II proteins, such as proteins selected from the group consisting of HLA-DRA, HLA-DRB5, HLA-DPA1, HLA-DPB1, HLA-DQA1, and HLA-DQB1, and in particular the presence of HLA-DRA on the surface of the macrophages of the present invention. In particular, the number of antibody-accessible MHC II proteins, such as HLA-DRA, on the cell surface of human macrophages containing at least one mutation in both alleles of a gene located on a chromosome is, for example, at least 3 times, for example at least 4 times, for example at least 5 times, 8 times, or even 10 times more than the number of antibody-accessible MHC II proteins on the cell surface of otherwise identical wild-type macrophages tested under otherwise homogeneous culture and immunostaining conditions.

[0185] Alternatively, or additionally, a typical characteristic of M1 macrophages may be the presence of, for example, the cell surface marker CD74. In particular, the number of CD74s on the cell surface of human macrophages containing at least one mutation in both alleles of a gene located on the chromosome is, for example, at least 3 times, for example at least 4 times, for example at least 5 times, 6 times, or even 8 times more than the number of CD74s on the cell surface of otherwise identical wild-type macrophages tested under otherwise uniform culture and immunostaining conditions.

[0186] Alternatively, or additionally, a typical feature of M1 macrophages may be the presence of, for example, the cell surface marker CD70. In particular, the number of CD70s on the cell surface of human macrophages containing at least one mutation in both alleles of a gene located on the chromosome is, for example, at least 3 times, for example at least 4 times, for example at least 5 times, 6 times, or even 8 times more than the number of CD70s on the cell surface of otherwise identical wild-type macrophages tested under otherwise homogeneous culture and immunostaining conditions.

[0187] Alternatively, or additionally, a typical characteristic of M1 macrophages may be the presence of, for example, the cell surface marker CD2. In particular, the number of CD2s on the cell surface of human macrophages containing at least one mutation in both alleles of a gene located on the chromosome is, for example, at least 3 times, for example at least 4 times, for example at least 5 times, 6 times, or even 8 times more than the number of CD2s on the cell surface of otherwise identical wild-type macrophages tested under otherwise uniform culture and immunostaining conditions.

[0188] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the downregulation of the cell surface marker CD28. In particular, the number of CD28 on the cell surface of human macrophages containing at least one mutation in both alleles of a gene located on a chromosome is, for example, at least twice, for example at least four times, for example at least six times, eight times, or even ten times less than the number of CD28 on the cell surface of otherwise identical wild-type macrophages tested under otherwise homogeneous culture and immunostaining conditions.

[0189] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the downregulation of the cell surface marker LYVE1. In particular, the number of LYVE1 cells on the cell surface of human macrophages containing at least one mutation in both alleles of a gene located on a chromosome is, for example, at least twice, for example at least four times, for example at least six times, eight times, or even ten times less than the number of LYVE1 cells on the cell surface of otherwise identical wild-type macrophages tested under otherwise uniform culture and immunostaining conditions.

[0190] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the downregulation of the cell surface marker STAB1. In particular, the number of STAB1 on the cell surface of human macrophages containing at least one mutation in both alleles of a gene located on a chromosome is, for example, at least twice, for example at least four times, for example at least six times, eight times, or even ten times less than the number of STAB1 on the cell surface of otherwise identical wild-type macrophages tested under otherwise uniform culture and immunostaining conditions.

[0191] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the downregulation of the cell surface marker LILRB5. In particular, the number of LILRB5s on the cell surface of human macrophages containing at least one mutation in both alleles of a gene located on a chromosome is, for example, at least twice, for example at least four times, for example at least six times, eight times, or even ten times fewer than the number of LILRB5s on the cell surface of otherwise identical wild-type macrophages tested under otherwise homogeneous culture and immunostaining conditions. For the purposes of the present invention, signal intensity in immunostaining assays was used to represent the number of specific types of cell surface markers.

[0192] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the downregulation of the cell surface marker CD163. In particular, the number of CD163 on the cell surface of human macrophages containing at least one mutation in both alleles of a gene located on a chromosome is, for example, at least twice, for example at least four times, for example at least six times, eight times, or even ten times fewer than the number of CD163 on the cell surface of otherwise identical wild-type macrophages tested under otherwise homogeneous culture and immunostaining conditions. For the purposes of the present invention, signal intensity in immunostaining assays was used to represent the number of specific types of cell surface markers.

[0193] Particularly preferred human macrophages of the present invention are positive for at least one, preferably two or all three, surface markers MHC II, CD70, and CD74, but negative for at least one, preferably two, for example, three, four, or all five, surface markers CD28, LYVE1, STAB1, CD163, and LILRB5.

[0194] Preferred surface marker combinations are: positive for HLA-DR and CD74 but negative for LYVE1; positive for HLA-DR and CD70 but negative for LYVE1; positive for HLA-DR and CD74 but negative for CD28; positive for HLA-DR and CD70 but negative for CD28; positive for HLA-DR and CD74 but negative for CD163; positive for HLA-DR and CD70 but negative for CD163; positive for HLA-DR and CD70 but negative for STAB1; positive for HLA-DR and CD74 but negative for STAB1; positive for HLA-DR and CD70 but negative for LILRB5; and HLA-DR and The results are as follows: positive for CD74 but negative for LILRB5; negative for LYVE1 and STAB1 but positive for HLA-DR; negative for LYVE1 and STAB1 but positive for CD70; negative for LYVE1 and STAB1 but positive for CD74; negative for LYVE1 and CD163 but positive for HLA-DR; negative for LYVE1 and CD163 but positive for CD70; negative for LYVE1 and CD163 but positive for CD74; negative for LYVE1 and LILRB5 but positive for HLA-DR; negative for LYVE1 and LILRB5 but positive for CD74; negative for LYVE1 and LILRB5 but positive for CD70.

[0195] The human macrophages of the present invention, which include at least one mutation in both alleles of a gene located on a chromosome, may also be characterized by any combination of the gene expression markers, particularly any combination of the preferred gene expression markers, having the secretion profile, particularly the preferred secretion profile.

[0196] The human macrophages of the present invention, which include at least one mutation in both alleles of a gene located on a chromosome, may also be characterized by any combination of the gene expression markers, particularly any combination of the preferred gene expression markers, the combination having the surface markers, particularly the preferred surface marker profiles.

[0197] The human macrophages of the present invention, which include at least one mutation in both alleles of a gene located on a chromosome, may also be characterized by any combination of the secretory profiles, particularly any combination of the preferred secretory profiles, having the surface markers, particularly the preferred surface marker profiles.

[0198] The human macrophages of the present invention, which include at least one mutation in both alleles of a gene located on a chromosome, may also be characterized by any combination of the gene expression profiles, particularly any combination of the preferred gene expression profiles, the combination having the surface marker, particularly the preferred surface marker profile, and the secretion profile, particularly the preferred secretion profile.

[0199] The human macrophages of the present invention contain at least one mutation in both alleles of a gene located on a chromosome. The mutation can defunction the gene and / or inhibit gene expression, or even eliminate gene expression.

[0200] Mutations may be insertions or frameshifts. However, preferred mutations are deletions, such as deletions within promoters, exons, or splice sites. Even small deletions of only a few base pairs can have an effect on gene function, but preferably the deletion is at least 50 base pairs, for example, at least 100 base pairs, at least 200 base pairs, at least 500 base pairs, or even more specifically, at least 1,000 base pairs.

[0201] A preferred human macrophage of the present invention comprises at least two distinct mutations, preferably these two mutations being deletions in at least two different genes.

[0202] As described above, the present invention relates to human macrophages having at least one mutation in both alleles of a gene located on a chromosome, wherein the macrophages are resistant to M-CSF-induced M2 polarization. The present invention provides a novel selectable phenotype for cells having at least one mutation in both alleles of a gene located on a chromosome, which is therefore screenable, and as a result, makes it possible to identify an unknown gene whose mutation causes the macrophage to be resistant to M-CSF-induced M2 polarization. Accordingly, the nature of the gene is not particularly limited as long as the biallelic mutation of the gene produces the desired phenotype. However, preferred human macrophages of the present invention have biallelic mutations in genes involved in M-CSF, IL-4, IL-10, and / or TGFB1-mediated downregulation of C2TA, such as negative regulators, and in particular negative transcription regulators of C2TA. Preferably, the gene is selected from the group consisting of STAT6, IRF4, PPARg, MAFB, MAF, KLF4, C / EPBb, GATA3, JMJD3, SOCS2, SOCS1, TMEM106A, and AKT1, and in particular, from the group consisting of STAT6, IRF4, TMEM106A, MAFB, and MAF.

[0203] A preferred human macrophage of the present invention, which contains at least one mutation in both alleles of a gene located on a chromosome and is resistant to M-CSF-induced M2 polarization, is a macrophage in which the mutated gene is MAFB. MAFB is preferably the only transcription factor-coding gene containing a biallelelic deletion. Alternatively, MAFB is preferably the only protein-coding gene containing a biallelelic deletion. Alternatively, MAFB is preferably the only gene containing a biallelelic deletion. Further deletion of transcription factors other than MAF and / or MAFB may impair macrophage function or make differentiation of induced pluripotent stem cells into macrophages impossible. For example, Buchrieser et al. showed that induced pluripotent stem cells with deletions of the transcription factors RUNX1 or SPI1 could not differentiate into iPS cell-derived macrophages.

[0204] A preferred human macrophage of the present invention, in which the mutated gene is MAFB, can also be characterized by the nature and size of the deletions. For example, all deletions may be in the exon region of MAFB. For example, each deletion may be greater than 50 base pairs, for example, at least 100 base pairs each, or at least 250 base pairs each. For example, deletions in or within the MAFB gene may be between 100 and 10,000 base pairs, for example, between 500 and 3,000 base pairs each, and particularly between 700 and 2,000 base pairs each. A preferred human macrophage of the present invention, in which the mutated gene is MAFB, may exhibit one or more, for example, all, of these deletions.

[0205] Preferred human macrophages of the present invention, in which the mutated gene is MAFB, can also be characterized by a specific deletion site on a chromosome. For example, the MAFB deletion is preferably located in the region 40685000-40690000 on human chromosome 22, e.g., 40685200-40689800, e.g., 40685400-40689600, e.g., 40685600-40689400, and particularly in the region 40685700-40689300.

[0206] A preferred human macrophage of the present invention, which contains at least one mutation in both alleles of a gene located on a chromosome, and which is resistant to M-CSF-induced M2 polarization, is a macrophage in which the mutated gene is MAF. Preferably, MAF is the only transcription factor-coding gene containing a biallelelic deletion. Alternatively, MAF is the only protein-coding gene containing a biallelelic deletion. Alternatively, MAF is the only gene containing a biallelelic deletion.

[0207] A preferred human macrophage of the present invention, in which the mutated gene is MAF, can also be characterized by the nature and size of the deletions. For example, all deletions may be in the exon region of MAF. For example, each deletion may be greater than 50 base pairs, for example, at least 100 base pairs each, or at least 250 base pairs each. For example, deletions in or within the MAF gene may be between 100 and 10,000 base pairs, for example, between 500 and 3,000 base pairs each, and particularly between 700 and 2,000 base pairs each. A preferred human macrophage of the present invention, in which the mutated gene is MAF, may exhibit one or more, for example, all, of these deletions.

[0208] Preferred human macrophages of the present invention, in which the mutated gene is MAF, can also be characterized by a specific deletion site on a chromosome. For example, the MAF deletion is preferably located in the region 79593000-79602000 on human chromosome 16, for example 79593200-79601500, for example 79593400-79601100, and particularly in the region 79593600-79600900.

[0209] A preferred human macrophage of the present invention, comprising at least one mutation in both alleles of a chromosomally located gene, and resistant to M-CSF-induced M2 polarization, is a macrophage in which both alleles of MAFB and both alleles of MAF are preferably deactivated by deletions of at least 50 base pairs. We have demonstrated that inhibiting MAFB and MAF gene expression in human cells is surprisingly more difficult than in mouse cells, partly because a genetic approach to double knockout cells is not an option for human cells. While the use of one guide RNA per gene in CRISPR / Cas9-based approaches has provided many successful examples in the past for inactivating the gene expression of other genes (e.g., Chu et al. (2016)), several attempts to deactivate both MAFB and MAF genes by creating small deletions using one guide RNA for each of the MAFB and MAF genes in a CRISPR / Cas9 approach have not yielded cells that proliferate in vitro. Surprisingly, the use of two guide RNAs per gene successfully induced large deletions of at least 50 base pairs, providing proliferating phagocytic cells. These cells had the additional advantage of being non-tumor-forming. MAF and MAFB are preferably the only transcription factor-coding genes containing biallelic deletions. Alternatively, MAF and MAFB are preferably the only protein-coding genes containing biallelic deletions. Alternatively, MAF and MAFB are preferably the only genes containing biallelic deletions.

[0210] Preferred human macrophages of the present invention, in which both alleles of MAFB and both alleles of MAF are defunctionalized, can also be characterized by the nature and size of deletions. For example, all deletions may be in the exon regions of the MAFB and MAF genes. For example, the deletions may each exceed 50 base pairs, for example, at least 100 base pairs each, or at least 250 base pairs each. For example, deletions in or within the MAFB and MAF genes may each range from 100 to 10,000 base pairs, for example, from 500 to 3,000 base pairs each, and particularly from 700 to 2,000 base pairs. Human macrophages of the present invention having stable M1 polarization may exhibit one or more, for example, all, of these deletions.

[0211] Preferred human macrophages of the present invention having stable M1 polarization can also be characterized by a specific deletion site on a chromosome. For example, MAFB deletions are preferably located in the region 40685000-40690000 on human chromosome 22, e.g., 40685200-40689800, e.g., 40685400-40689600, e.g., 40685600-40689400, and particularly in the region 40685700-40689300. For example, MAF deletions are preferably located in the region 79593000-79602000 on human chromosome 16, e.g., 79593200-79601500, e.g., 79593400-79601100, and particularly in the region 79593600-79600900.

[0212] The human macrophages of the present invention, which are resistant to M-CSF-induced M2 polarization, may be created by cell manipulation. The cells from which the human macrophages of the present invention may be derived are cells that are progenitor cells that develop into human macrophages, for example, iPS cells; blood monocytes; monocytes, monoblasts, myeloid or CD34+ pluripotent progenitor cells derived from umbilical cord blood; monocytes, monoblasts, myeloid or CD34+ pluripotent progenitor cells derived from bone marrow; mobilized monoblasts, myeloid or CD34+ pluripotent progenitor cells derived from adult blood; and monocytes, monoblasts, myeloid or CD34+ progenitor cells derived from extramedullary hematopoiesis. The human macrophages of the present invention, which are resistant to M-CSF-induced M2 polarization, may also be derived from macrophages, particularly alveolar macrophages or macrophages derived from adipose tissue, which are relatively readily available macrophages. A preferred cell from which the human macrophages of the present invention may be derived is human iPS cells. Another preferred cell from which the human macrophages of the present invention may be derived is CMP (myeloid common progenitor cell). Another preferred cell from which the human macrophages of the present invention may be derived is GMP (granulocyte / macrophage progenitor cell).

[0213] The creation of human induced pluripotent stem cells is now well established in this field. For example, Isogai et al. (2018) describe the creation of iPS cells from human blood monocytes. Fusaki N. et al. (2009) describe the production of human iPS cell lines using modified Sendai virus. In particular, when using human induced pluripotent stem cells as a starting material for macrophage differentiation, it is preferable that MAF and / or MAFB are the only transcription factor-coding genes (multiple are possible) containing biallelelic deletions. Alternatively, it is preferable that MAF and / or MAFB are the only protein-coding genes containing biallelelic deletions. Alternatively, it is preferable that MAF and / or MAFB are the only genes containing biallelelic deletions. If genes are further deleted, macrophage function may be impaired, or differentiation of induced pluripotent stem cells into macrophages may become impossible.

[0214] Human mononuclear phagocytes for biallelic mutations of the gene can also be obtained from human primary blood monocytes, which can be obtained by leukocyte apheresis and ertriation, or from monocyte-derived macrophages under culture conditions well known in this field. Human macrophages can also be obtained from bronchoalveolar lavage, or by differentiating CD34+ hematopoietic stem cells and progenitor cells obtained from umbilical cord blood, or stem cells recruited from peripheral blood or bone marrow, using differentiation protocols well known in this field.

[0215] Cas9 and gRNA can be expressed using commercially available DNA expression plasmids well-known in this field (e.g., Santa Cruz Sc-418922, https: / / www.scbt.com / de / p / control-crispr-cas9-plasmid). These DNA expression plasmids can be introduced into human mononuclear phagocyte target cells by electroporation or by lipid-based transfection protocols well-known in this field. Cas9 and gRNA can also be introduced into target cells as ribonucleoprotein complexes by electroporation. Cas9 / gRNA-mediated gene editing has been demonstrated in mononuclear phagocytes using this method (Zhang et al. (2020); Wang et al. (2018); Freund et al. (2020)), and in human CD34+ hematopoietic stem cells and progenitor cells differentiating into macrophages (Scharenberg et al. (2020)). Target genes, such as the MAFB and MAF genes, can also be deleted using recombinases that target the site to be manipulated (Lansing et al. (2020); Karpinski et al. (2016)), which can be introduced by methods known in the field, such as electroporation of protein, coding mRNA, or DNA expression plasmids, and have been used for gene editing in mononuclear phagocytic cells (Shi et al. (2018)).

[0216] Gonzalez F. et al. (2014) describe an iCRISPR platform for rapid, multiplexable, and inducible genome editing of human pluripotent stem cells based on the introduction of a doxycycline-inducible Cas9 expression cassette into the AAVS1 locus. This strategy can be used to introduce the inducible Cas9 expression cassette into a wide variety of self-generated or publicly available human iPS cell lines. These engineered cell lines can then be used to target selected genes, such as MAF and / or MAFB.

[0217] The human macrophages of this invention, in which both MAF and MAFB were defunctionalized, were found to be non-tumor-forming. Karyotype analysis showed that they contained 46 chromosomes, none of which showed chromosomal rearrangement. Therefore, these cells are useful for macrophage-based cell therapy.

[0218] The human macrophages of the present invention preferably maintain an M1-like phenotype even when cultured in the presence of the above-mentioned M2-inducing factors, such as M-CSF and / or IL-4. Preferably, the human macrophages maintain at least two, for example, at least four, at least six, at least eight, at least ten, at least fifteen, or all of the 25 M1 phenotypic markers listed below. Preferred M1 phenotypic markers are, respectively, at least four times higher expression of the HLA-DRA gene, at least four times higher expression of the HLA-DRB5 gene, at least four times higher expression of the HLA-DPA1 gene, at least four times higher expression of the HLA-DQA1 gene, at least four times higher expression of the HLA-DQB1 gene, at least four times higher expression of the RXFP2 gene, at least four times higher expression of the CD74 gene, at least four times higher expression of the CD38 gene, at least four times higher expression of the CD2 gene, at least four times higher expression of the IL18 gene, and at least four times higher expression of the IL23A gene compared to the expression levels of the corresponding genes in otherwise identical wild-type macrophages; and at least four times lower expression of the RNASE1 gene and at least four times lower expression of the CD28 gene compared to the expression levels of the corresponding genes in otherwise identical wild-type macrophages. All other genes exhibited four-fold lower expression, at least four-fold lower expression of the LYVE1 gene, at least four-fold lower expression of the FCGBP gene, at least four-fold lower expression of the STAB1 gene, at least four-fold lower expression of the LILRB5 gene, at least four-fold lower expression of the F13A1 gene, at least four-fold lower expression of the QPCT gene, at least four-fold lower expression of the RNF128 gene, and at least four-fold lower expression of the CCL7 gene; respectively, IL-18 secretion was at least three times higher and IL-23 secretion was at least three times higher compared to the secretion levels of the corresponding proteins in otherwise identical wild-type macrophages; respectively, RNASE1 secretion was at least three times lower, FCGBP secretion was at least three times lower, F13A1 secretion was at least three times lower and CCL7 secretion was at least three times lower compared to the secretion levels of the corresponding proteins in otherwise identical wild-type macrophages;The group can be selected from the following: the presence of cell surface markers HLA-DRA, HLA-DRB5, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, CD64, CD70, CD2, CD74, RXFP2, absence of cell surface marker CD28, absence of cell surface marker LYVE1, and absence of cell surface marker CD163.

[0219] These markers can be maintained even in the presence of in vitro tumor cells.

[0220] The present invention also relates to a collection of human macrophages having at least one mutation in both alleles of a gene located on a chromosome, wherein the human macrophages are resistant to M-CSF-induced M2 polarization. Such a collection of cells may be referred to below as "the human macrophage collection of the present invention." The human macrophage collection of the present invention is preferably a collection of a large number of cells, for example, at least 100,000, at least 1,000,000, for example at least 10,000,000, for example at least 100,000,000.

[0221] The human macrophage collection of the present invention can be described as being able to recruit CD8 T cells and / or NK cells against tumors in vivo. Surprisingly, the human macrophage collection of the present invention can reduce already established tumors in vivo, for example, tumor masses established in the peritoneum, lungs, or liver. Preferably, the human macrophage collection of the present invention induces regression of established tumors.

[0222] The human macrophage collection of the present invention can also be characterized by the functional characteristics of the human macrophages. For example, the macrophages included in the collection can be characterized by their phagocytic ability. For example, the human macrophages included in the collection can be characterized by their ability to produce reactive oxygen species when activated. For example, the human macrophages included in the collection can be characterized by their protease activity detectable within their lysosomes. For example, the human macrophages included in the collection can be characterized by their ability to take up lipids.

[0223] The human macrophages included in the collection of this invention may exhibit one or more, for example, all of these functional characteristics.

[0224] The human macrophage collection of the present invention may be characterized by including morphologically characterizable human macrophages. For example, human macrophages included in the collection may be characterized by appearing as large vacuolated cells under a microscope. For example, human macrophages included in the collection may be characterized by appearing as cells having an adhesive morphology. For example, human macrophages included in the collection may be characterized by appearing as cells characterized by membranous pseudopods and / or filopodia. Human macrophages included in the collection can be characterized by one or more, for example, all of these morphological features.

[0225] The human macrophages included in the collection of human macrophages of the present invention are intended for medical use as cell therapy drugs. Therefore, the human macrophages can be characterized by being non-tumor-forming. For example, the collection of human macrophages of the present invention can be characterized by consisting of cells having only 46 chromosomes. For example, the collection of human macrophages of the present invention can be characterized by the absence of cells with chromosomally rearranged chromosomes. These characteristics can be determined, for example, by karyotype analysis of a sufficient number of cells included in the collection, for example, at least 30 cells, at least 40 cells, or at least 50 cells. In tests to evaluate the tumorigenicity of the human macrophages of the present invention, at least 8 × 10⁶ cells are injected into the lungs of immunosuppressed huPAP mice. 6 Intratracheal injection of cells was observed not to result in the formation of visible tumors in the lungs of the animals. Therefore, the human macrophage collection of the present invention can be characterized by the absence of tumor formation when transplanted into the lungs of immunocompromised huPAP mice and examined four weeks later.

[0226] The human macrophage collection of the present invention can also be grown under appropriate conditions until a desired number of cells is achieved in the collection. After the depletion growth phase, the resulting collection of human phagocytic cells can be obtained through such growth and used in pharmaceuticals, for example, for the treatment of the aforementioned diseases.

[0227] The present invention also relates to the human macrophages and / or collections of the human macrophages of the present invention for use in cancer treatment, particularly in the treatment of cancer which is a solid tumor. Surprisingly, the inventors have found that macrophages resistant to M-CSF-induced M2 polarization can be created by introducing at least one mutation in both alleles of the chromosomal genes involved in M2 polarization, e.g., MAFB and / or MAF, and that the macrophages of the present invention have direct and indirect antitumor activity.

[0228] When human macrophages and / or collections of human macrophages are used as cell therapy, the macrophages may be autologous with respect to the patient being treated. In the autologous setting, the macrophages are preferably derived from myeloid common progenitor cells or granulocyte / macrophage progenitor cells. However, preferably, the human macrophages and / or collections of human macrophages are allogeneic with respect to the patient being treated. In the allogeneic setting, the macrophages are preferably derived from induced pluripotent stem cell lines. Stem cell lines that are homozygous for HLA-A, HLA-B, and HLA-DRB1 are particularly useful because they have higher immunocompatibility with recipient patients. This is because it is easier to find HLA-compatible recipients with HLA-homozygous cell lines than with HLA-heterozygous cell lines. Linkage disequilibrium occurs because the combinations of HLA-A, HLA-B, and HLA-DRB1 genes are not random due to genetic recombination and external pressures from environmental factors. According to Taylor et al. (2012), referring to the 2010 WHO HLA Nomenclature Report, there are 21 HLA-A, 44 HLA-B, and 15 HLA-DRB1 split specificities, resulting in a total of 13,860 HLA combinations. These HLA combinations are not evenly distributed among the populations of any particular country, and some HLA combinations in homozygous donors result in HLA matching with a larger number of potential recipients than in the populations of other countries. For example, the two homozygous HLA combinations that find the most potential recipients among the population of the UK are HLA-A1, HLA-B8, HLA-DRB1-17(3) and HLA-A2, HLA-B44(12), HLA-DRB1-4. Therefore, an induced pluripotent stem cell line is preferred if it is homozygous for HLA-A, HLA-B, and HLA-DRB1, and the HLA-A, HLA-B, and HLA-DRB1 combination is one of the 10 most frequent combinations in a population selected from groups consisting of residents of the European Union, the United States, China, and Japan.

[0229] The present invention also relates to a method for treating a human subject with cancer, comprising administering the human macrophages and / or a collection of human macrophages of the present invention to the human subject with cancer.

[0230] Depending on the specific medical use intended, the human macrophages of the present invention and / or the human macrophages included in the collection of human macrophages of the present invention may be further genetically modified.

[0231] The present invention also provides a pharmaceutical composition comprising a collection of human macrophages of the present invention. Pharmacopoecitable delivery methods and formulations for cell therapy are described in the art. The cells can be suspended in a pharmacopoecitable carrier, such as a buffer, e.g., PBS or PBS / EDTA supplemented with about 20% human serum albumin, or citrate plasma, or Plasmalyte-A pH 7.4 (Baxter, supplemented with about 2% HSA). The pharmacopoecitable carrier for macrophages of the present invention is compatible with cell viability. The carrier may contain physiological concentrations of NaCl.

[0232] The present invention also provides a method for generating macrophages that are resistant to M-CSF-induced M2 polarization, (a) A step in which at least one mutation is introduced into both alleles of a selected target gene located on a chromosome in human iPS cells; (b) The process of isolating colonies derived from single cells; (c) The process of differentiating a single-cell colony into a macrophage; (d) The method comprises the step of identifying colonies of macrophages that are resistant to M-CSF-induced M2 polarization.

[0233] As mentioned above, preferred mutations are deletions of more than 50 base pairs. In such cases, double-strand breaks are usually caused by site-specific endonucleases or recombinases, such as Cas-9 combined with at least two guide RNAs.

[0234] Preferably, the target cells for step (a) are iPS cells, monocytes, or macrophages, and in particular, the target cells are selected from the group consisting of iPS cells; blood monocytes; umbilical cord blood-derived monocytes, monoblasts, myeloid or CD34+ pluripotent progenitor cells; bone marrow-derived monocytes, monoblasts, myeloid or CD34+ pluripotent progenitor cells; mobilized adult blood-derived monoblasts, myeloid or CD34+ pluripotent progenitor cells, e.g., myeloid common progenitor cells, granulocyte / macrophage progenitor cells, macrophage / dendritic cell progenitor cells, or common monocyte progenitor cells; extramedullary hematopoietic-derived monocytes, monoblasts, myeloid or CD34+ progenitor cells; alveolar macrophages; and adipose tissue macrophages. One preferred example for the target cells of step (a) is human induced pluripotent stem cells. Another preferred example for the target cells of step (a) is myeloid common progenitor cells or granulocyte / macrophage progenitor cells.

[0235] After selecting cells that have biallelic mutations in the selected target gene, such as deletions of at least 50 base pairs in both chromosomal copies of the target gene, such as iPS cells (e.g., iPS cells with deletions in both alleles of MAFB and both alleles of MAF), they are usually cultured in a suitable medium, for example, in the presence of M-CSF (in the case of iPS cells differentiated into macrophages as detailed in Example 11). Examples of culture conditions for iPS cell-derived macrophages are described in Example 11.

[0236] Next, macrophage colonies can be tested for the presence of one or more markers typical of M1 macrophages. Preferably, macrophage colonies resistant to M-CSF-induced M2 polarization are identified by the presence and / or absence of surface markers, for example, by at least 4, at least 6, or at least 8 features selected from the list consisting of the presence of cell surface marker HLA-DRA, cell surface marker HLA-DRB5, cell surface marker HLA-DPA1, cell surface marker HLA-DPB1, cell surface marker HLA-DQA1, cell surface marker HLA-DQB1, cell surface marker CD64, cell surface marker CD74, cell surface marker CD70, absence of cell surface marker CD28, absence of cell surface marker LYVE1, and absence of cell surface marker CD163.

[0237] Alternatively, or additionally, colonies of macrophages resistant to M-CSF-induced M2 polarization can be identified by the secretion and / or absence of secretion of proteins, for example, by at least 4, at least 6, or at least 8 features selected from the list consisting of secretion of IL18, secretion of IL23, absence of RNASE1 secretion, absence of FCGBP secretion, absence of F13A1 secretion, absence of CCL7 secretion, and absence of PPBP secretion.

[0238] The present invention also provides a screening method for identifying genes that make macrophages resistant to repolarization by tumor cells, (a) A step of creating a collection of iPS cells, each iPS cell having at least one mutation in both alleles of a gene located on the chromosome of the iPS cell; (b) The process of isolating colonies derived from single cells; (c) The process of differentiating a single-cell colony into a macrophage; (d) A step to identify macrophage colonies, including the typical characteristics of M1 macrophages, after 48 hours of exposure to a combination of 40 ng / ml IL-4 and 50 ng / ml M-CSF; (e) The process of identifying mutated genes, The method includes the foregoing.

[0239] Preferred starting cells for screening are human iPS cells, and preferred means for identifying the colony of interest in step (d) is testing for the presence and / or absence of a surface marker, as described above with respect to the macrophage production method of the present invention.

[0240] The present invention also relates to human induced pluripotent stem cells, or human embryonic stem cells, or human myeloid common progenitor cells, or human granulocyte / macrophage progenitor cells, particularly human induced pluripotent stem cells, in which both alleles of a gene located on a chromosome contain at least one mutation, particularly at least one deletion, wherein the gene is selected from the group consisting of STAT6, IRF4, PPARg, MAFB, MAF, KLF4, C / EPBb, GATA3, JMJD3, SOCS2, SOCS1, TMEM106A, and AKT1, particularly selected from the group consisting of STAT6, IRF4, TMEM106A, MAFB, and MAF, and most preferably selected from the group consisting of MAF and MAFB. Examples include human induced pluripotent stem cells, human embryonic stem cells, human myeloid common progenitor cells, or human granulocyte / macrophage progenitor cells, particularly human induced pluripotent stem cells in which both alleles of MAFB and / or both alleles of MAF are deactivated by deletions of, for example, at least 50 base pairs. Surprisingly, the inventors have found that stem cells deactivated by deletions of both alleles of MAFB and both alleles of MAF are useful intermediates in the creation of professional phagocytic cells, and in particular in the creation of macrophages, because they are able to differentiate into human professional phagocytic cells. Preferably, all deletions in the human induced pluripotent stem cells, or human embryonic stem cells, or human myeloid common progenitor cells, or human granulocyte / macrophage progenitor cells of the present invention include exon DNA.

[0241] In particular, each deletion is at least 100 base pairs, for example, at least 250 base pairs each.

[0242] For example, the deletions are 100 to 10,000 base pairs, for example, 500 to 3,000 base pairs, or 600 to 1,500 base pairs. The preferred locations of MAFB deletions and / or MAF deletions in the human induced pluripotent stem cells, or human embryonic stem cells, or human myeloid common progenitor cells, or human granulocyte / macrophage progenitor cells of the present invention, particularly in human induced pluripotent stem cells, are as described above for the human phagocytic cells of the present invention. Preferably, the gene selected from the group consisting of STAT6, IRF4, PPARg, MAFB, MAF, KLF4, C / EPBb, GATA3, JMJD3, SOCS2, SOCS1, TMEM106A, and AKT1 is the only protein-coding gene containing a biallelelic deletion in the induced pluripotent stem cell, or embryonic stem cell, or human myeloid common progenitor cell, or human granulocyte / macrophage progenitor cell, particularly in human induced pluripotent stem cells.

[0243] MAF and MAFB are preferably the only transcription factor-coding genes containing both alleles. Alternatively, MAF and MAFB are the only protein-coding genes containing both alleles. Alternatively, MAF and MAFB are the only genes containing both alleles.

[0244] The present invention also relates to the use of human induced pluripotent stem cells, or human embryonic stem cells, or human myeloid common progenitor cells, or human granulocyte / macrophage progenitor cells, particularly the use of human induced pluripotent stem cells, for the generation of human phagocytic cells, and especially for the creation of human macrophages.

[0245] The present invention also relates to the use of human induced pluripotent stem cells, or human embryonic stem cells, or human myeloid common progenitor cells, or human granulocyte / macrophage progenitor cells, particularly the use of human induced pluripotent stem cells, for the preparation of compositions comprising human macrophages of the present invention.

[0246] The present invention further relates to the following embodiments. 1. A human macrophage having at least one mutation in both alleles of a gene located on a chromosome, and being resistant to M-CSF-induced M2 polarization, preferably the human macrophage is not a dendritic cell, and more preferably the human macrophage is neither a dendritic cell nor a monocyte. 2. Human macrophages as described in Clause 1, which are resistant to M2 polarization by a combination of M-CSF and IL-4. 3. Human macrophages as described in Clause 1 or 2, which are resistant to M2 polarization by a combination of M-CSF, IL-4, and IL-13. 4. Human macrophages as described in any one of clauses 1 to 3, having the usual characteristics of M1 macrophages after 24 hours of exposure to 50 ng / ml M-CSF. 5. Human macrophages as described in Clause 4, having the usual characteristics of M1 macrophages after 48 hours of exposure to 50 ng / ml M-CSF. 6. Human macrophages described in any one of clauses 1 to 5, having the usual characteristics of M1 macrophages after 24 hours of exposure to 20 ng / ml IL-4. 7. Human macrophages as described in Clause 6, having the usual characteristics of M1 macrophages after 48 hours of exposure to 20 ng / ml IL-4. 8. Human macrophages as described in any one of clauses 1 to 7, having the usual characteristics of M1 macrophages after 24 hours of exposure to a combination of 40 ng / ml IL-4 and 50 ng / ml M-CSF. 9. Human macrophages as described in Clause 8, having the normal characteristics of M1 macrophages after 48 hours of exposure to a combination of 40 ng / ml IL-4 and 50 ng / ml M-CSF. 10. A human macrophage as described in any one of clauses 1 to 9, having the usual characteristics of an M1 macrophage in which GM-CSF is absent for the last two hours. 11. A human macrophage as described in Clause 10, having the usual characteristics of an M1 macrophage in which GM-CSF is absent for the last 6 hours. 12. A typical characteristic of an M1 macrophage is that the expression of at least one mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least four times higher than the expression of said mRNA in otherwise identical wild-type macrophages, wherein the at least one mRNA is selected from the list consisting of HLA-DRA, HLA-DRB5, HLA-DPA1, HLA-DQA1, RXFP2, CD74, CD38, CD2, IL18, and IL23A, as described in any one of clauses 4 to 11. 13. A human macrophage as described in Clause 12, wherein the expression of at least one mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least six times higher than the expression of the same mRNA in an otherwise identical wild-type macrophage. 14. A human macrophage according to any one of Clauses 4 to 11, characterized in that the expression of at least three mRNAs in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least four times higher than the expression of the same at least three mRNAs in otherwise identical wild-type macrophages, wherein the at least three mRNAs are selected from the list consisting of HLA-DRA, HLA-DRB5, HLA-DPA1, HLA-DQA1, RXFP2, CD74, CD38, CD2, IL18, and IL23A. 15. A human macrophage as described in Clause 14, wherein the expression of at least three mRNAs in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least six times higher than the expression of the same at least three mRNAs in otherwise identical wild-type macrophages. 16. A human macrophage according to any one of Clauses 4 to 11, wherein the usual characteristics of an M1 macrophage are that the expression of at least six mRNAs in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least four times higher than the expression of the same at least six mRNAs in otherwise identical wild-type macrophages, wherein the at least six mRNAs are selected from the list consisting of HLA-DRA, HLA-DRB5, HLA-DPA1, HLA-DQA1, RXFP2, CD74, CD38, CD2, IL18, and IL23A. 17. A human macrophage as described in Clause 16, wherein the expression of at least six mRNAs in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least six times higher than the expression of the same at least six mRNAs in otherwise identical wild-type macrophages. 18. A human macrophage according to any one of clauses 12 to 17, wherein the expression of HLA-DRB5 mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least 4 times, for example, at least 8 times, at least 16 times, or even more, at least 32 times higher than the expression of HLA-DRB5 mRNA in otherwise identical wild-type macrophages. 19. A human macrophage according to any one of clauses 12 to 18, wherein the expression of HLA-DPA1 mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least 4 times, for example, at least 8 times, at least 16 times, or even more, at least 32 times higher than the expression of HLA-DPA1 mRNA in otherwise identical wild-type macrophages. 20. A human macrophage according to any one of clauses 12 to 19, wherein the expression of HLA-DQA1 mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least 4 times, for example, at least 8 times, at least 16 times, or even more, at least 32 times higher than the expression of HLA-DQA1 mRNA in otherwise identical wild-type macrophages. 21. A human macrophage according to any one of clauses 12 to 20, wherein the expression of HLA-DRA mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least 4 times, for example, at least 8 times, at least 16 times, or even more, at least 32 times higher than the expression of HLA-DRA mRNA in otherwise identical wild-type macrophages. 22. A human macrophage according to any one of clauses 4 to 11, wherein the usual characteristics of an M1 macrophage are the expression of the HLA-DRA gene, the HLA-DRB5 gene, the HLA-DPA1 gene, the HLA-DPB1 gene, the HLA-DQA1 gene, and the HLA-DQB1 gene in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome, wherein the expression of the HLA-DRA gene, the HLA-DRB5 gene, the HLA-DPA1 gene, the HLA-DPB1 gene, the HLA-DQA1 gene, and the HLA-DQB1 gene is at least 4 times, for example, at least 8 times, at least 16 times, or even more, at least 32 times higher than the expression of the mRNA in otherwise identical wild-type macrophages. 23. A human macrophage according to any one of clauses 4 to 22, wherein the usual characteristic of an M1 macrophage is the expression of the RXFP2 gene, and the expression of RXFP2 mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least four times, for example, at least eight times, or even more than at least sixteen times, higher than the expression of RXFP2 mRNA in otherwise identical wild-type macrophages. 24. A human macrophage according to any one of clauses 4 to 23, wherein the usual characteristic of an M1 macrophage is the expression of the CD74 gene, and the expression of CD74 mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least four times, for example, at least eight times, or even more, at least sixteen times higher than the expression of CD74 mRNA in otherwise identical wild-type macrophages. 25. A human macrophage according to any one of clauses 4 to 24, wherein the usual characteristic of an M1 macrophage is the expression of the CD38 gene, and the expression of CD38 mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least four times, for example, at least eight times, or even more, at least sixteen times higher than the expression of CD38 mRNA in otherwise identical wild-type macrophages. 26. A human macrophage according to any one of clauses 4 to 25, wherein the usual characteristic of an M1 macrophage is the expression of the CD2 gene, and the expression of CD2 mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least four times, for example, at least eight times, or even more at least sixteen times higher than the expression of CD2 mRNA in otherwise identical wild-type macrophages. 27. A human macrophage according to any one of clauses 4 to 26, wherein the usual characteristic of an M1 macrophage is the expression of the IL18 gene, and in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome, the expression of IL18 mRNA is at least four times higher, and for example, at least six times higher, compared to the expression of IL18 mRNA in otherwise identical wild-type macrophages. 28. A human macrophage according to any one of clauses 4 to 27, wherein the usual characteristic of an M1 macrophage is the expression of the IL23A gene, and in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome, the expression of IL23A mRNA is at least four times higher, and for example, at least six times higher, compared to the expression of IL23A mRNA in otherwise identical wild-type macrophages. 29. A typical characteristic of an M1 macrophage is that the expression of at least one mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least 10 times lower than the expression of said mRNA in an otherwise identical wild-type macrophage, wherein the at least one mRNA is selected from the list consisting of RNASE1, CD28, LYVE1, FCGBP, F13A1, QPCT, CCL7, and RNF128, as described in any one of Clauses 4 to 28. 30. A human macrophage according to clause 30, wherein the expression of at least one mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least 50 times lower than the expression of the same mRNA in an otherwise identical wild-type macrophage. 31. The human macrophage described in any one of Clauses 4 to 28, wherein the usual characteristics of the M1 macrophage are that the expression of at least three mRNAs in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least 10 times lower than the expression of the same at least three mRNAs in otherwise identical wild-type macrophages, wherein the at least three mRNAs are selected from the list consisting of RNASE1, CD28, LYVE1, FCGBP, F13A1, QPCT, CCL7, and RNF128. 32. A human macrophage as described in Clause 31, wherein the expression of at least three mRNAs in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least 50 times lower than the expression of the same at least three mRNAs in otherwise identical wild-type macrophages. 33. A typical characteristic of an M1 macrophage is that the expression of at least six mRNAs in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least 10 times lower than the expression of the same at least six mRNAs in otherwise identical wild-type macrophages, wherein the at least six mRNAs are selected from the list consisting of RNASE1, CD28, LYVE1, FCGBP, F13A1, QPCT, CCL7, and RNF128, as described in any one of Clauses 4 to 28. 34. A human macrophage as described in Clause 33, wherein the expression of at least six mRNAs in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least 50 times lower than the expression of the same at least six mRNAs in otherwise identical wild-type macrophages. 35. A human macrophage according to any one of clauses 4 to 34, wherein the usual characteristic of an M1 macrophage is reduced expression of the RNASE1 gene, and the expression of RNASE1 mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least four times lower than the expression of RNASE1 mRNA in otherwise identical wild-type macrophages, for example, at least eight times, at least sixteen times, at least 32 times, or even more at least 64 times lower. 36. A human macrophage according to any one of clauses 4 to 35, wherein the usual characteristic of an M1 macrophage is reduced expression of the CD28 gene, and the expression of CD28 mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least four times lower than the expression of CD28 mRNA in otherwise identical wild-type macrophages, for example, at least eight times, at least sixteen times, at least 32 times, or even more at least 64 times lower. 37. A human macrophage according to any one of Clauses 4 to 36, wherein the usual characteristic of M1 macrophages is reduced expression of the LYVE1 gene, and the expression of LYVE1 mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is at least four times lower than the expression of LYVE1 mRNA in otherwise identical wild-type macrophages, for example, at least eight times, at least sixteen times, at least 32 times, or even more at least 64 times lower. 38. A human macrophage according to any one of Clauses 4 to 37, wherein the usual characteristic of an M1 macrophage is reduced expression of the FCGBP gene, and the expression of FCGBP mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least four times lower than the expression of FCGBP mRNA in otherwise identical wild-type macrophages, for example, at least eight times, at least sixteen times, at least 32 times, or even more at least 64 times lower. 39. A human macrophage according to any one of clauses 4 to 38, wherein the usual characteristic of an M1 macrophage is reduced expression of the F13A1 gene, and the expression of F13A1 mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least four times lower than the expression of F13A1 mRNA in otherwise identical wild-type macrophages, for example, at least eight times, at least sixteen times, at least 32 times, or even more at least 64 times lower. 40. A human macrophage according to any one of clauses 4 to 39, wherein the usual characteristic of M1 macrophages is reduced expression of the QPCT gene, and the expression of QPCT mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is at least 4 times lower than the expression of QPCT mRNA in otherwise identical wild-type macrophages, for example, at least 8 times, at least 16 times, at least 32 times, or even more at least 64 times lower. 41. A human macrophage according to any one of clauses 4 to 40, wherein the usual characteristic of an M1 macrophage is reduced expression of the CCL7 gene, and the expression of CCL7 mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least 4 times lower, for example, at least 8 times, at least 16 times, or even more than 32 times lower, compared to the expression of CCL7 mRNA in otherwise identical wild-type macrophages. 42. A human macrophage according to any one of clauses 4 to 41, wherein the usual characteristic of an M1 macrophage is reduced expression of the C5AR1 gene, and the expression of C5AR1 mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least four times lower, for example, at least eight times, at least sixteen times, or even at least 25 times lower, compared to the expression of C5AR1 mRNA in otherwise identical wild-type macrophages. 43. A human macrophage according to any one of Clauses 4 to 42, wherein the usual characteristic of M1 macrophages is reduced expression of the RNF128 gene, and the expression of RNF128 mRNA in human macrophages containing at least one mutation in both alleles of the gene located on the chromosome is at least 4 times lower than the expression of RNF128 mRNA in otherwise identical wild-type macrophages, for example, at least 8 times, at least 16 times, at least 32 times, or even more at least 64 times lower. 44. A human macrophage described in any one of clauses 4-43, whose usual characteristic feature is the secretion of IL23A. 45. A typical characteristic of M1 macrophages is the secretion of IL-18, as described in any one of clauses 4-44. 46. ​​Human macrophages as described in any one of clauses 4 to 45, whose usual characteristics are the secretion of IL18 and IL23A. 47. Human macrophages as described in any one of clauses 4-46, whose usual characteristic feature is the downregulated secretion of RNASE1. 48. Human macrophages as described in any one of clauses 4-47, whose usual characteristic feature is the downregulated secretion of FCGBP. 49. Human macrophages as described in any one of clauses 4 to 48, whose usual characteristics are downregulated secretion of RNASE1 and FCGBP, and upregulated secretion of IL18 and IL23A. 50. A human macrophage as described in any one of clauses 1 to 49, which is positive for the surface marker HLA-DRA. 51. A human macrophage as described in any one of clauses 1 to 50, which is positive for the surface marker HLA-DPA1. 52. A human macrophage as described in any one of clauses 1 to 51, which is positive for the surface marker HLA-DQA1. 53. A human macrophage as described in any one of clauses 1 to 52, which is positive for the surface marker HLA-DRB5. 54. A human macrophage as described in any one of clauses 1 to 53, which is positive for the surface marker CD74. 55. A human macrophage as described in any one of clauses 1 to 54, which is positive for the surface marker CD2. 56. A human macrophage as described in any one of clauses 1 to 55, which is negative for the surface marker CD28. 57. A human macrophage as described in any one of clauses 1 to 56, which is negative for the surface marker LYVE1. 58. A human macrophage as described in any one of clauses 1 to 57, which is negative for the surface marker STAB1. 59. A human macrophage as described in any one of clauses 1 to 58, which is negative for the surface marker LILRB5. 60. A human macrophage as described in any one of clauses 1 to 59, which is positive for the surface marker HLA-DR but negative for the surface markers CD28 and LYVE1. 61. A human macrophage as described in any one of clauses 1 to 60, which is positive for the surface markers HLA-DR and CD74, but negative for the surface markers CD28, LYVE1, STAB1, and LILRB5. 62. The mutation in a human macrophage as described in any one of the clauses 1 to 61, which renders the gene nonfunctional. 63. The mutation inhibits gene expression in any one of the human macrophages described in any one of the clauses 1 to 61. 64. The mutation causes the elimination of gene expression in any one of the human macrophages described in any one of the clauses 1 to 61. 65. A human macrophage as described in any one of clauses 1 to 64, wherein the mutation is a deletion. 66. The human macrophage described in Clause 65, wherein the deletion is located in the promoter, exon, or splice site. 67. The human macrophage described in Clause 65 or 66, wherein the deletion is a deletion of at least 50 base pairs. 68. A human macrophage as described in any one of clauses 1 to 67, containing at least two different mutations. 69. The human macrophage described in Clause 68, wherein the mutation is a deletion in at least two different genes. 70. A human macrophage as described in any one of the clauses 1 to 64 or 68, wherein the mutation is an insertion or a frameshift. 71. A human macrophage as described in any one of clauses 1 to 70, wherein the gene is a gene involved in the downregulation of M-CSF, IL-4, IL-10, and / or TGFB1-mediated C2TA. 72. The human macrophage according to any one of clauses 1 to 71, wherein the gene is selected from the group consisting of STAT6, IRF4, PPARγ, MAFB, MAF, KLF4, C / EPBβ, GATA3, JMJD3, SOCS2, SOCS1, and AKT1. 73. The human macrophage according to any one of clauses 1 to 72, wherein the gene encodes a negative regulator of C2TA transcription. 74. The human macrophage according to any one of clauses 1 to 73, wherein the gene is MAFB. 75. The human macrophage according to clause 74, wherein the mutation is a mutation within the region from 40685000 to 40690000 on human chromosome 22. 76. The human macrophage according to clause 75, wherein the mutation is a mutation within the region from 40685200 to 40689800, such as 40685400 to 40689600, for example 40685600 to 40689400, and particularly 40685700 to 40689300 on human chromosome 22. 77. The human macrophage according to any one of clauses 74 to 76, wherein MAFB is rendered non-functional by a deletion of at least 50 base pairs. 78. The human macrophage according to clause 77, wherein the deletion is a deletion of at least 100 base pairs. 79. The human macrophage according to clause 77 or 78, wherein the deletion is a deletion of at least 250 base pairs. 80. The human macrophage according to any one of clauses 77 to 79, wherein the deletion is a deletion of 100 base pairs to 10,000 base pairs. 81. The human macrophage according to clause 80, wherein the deletion is a deletion of 500 base pairs to 3,000 base pairs, for example 600 base pairs to 1,500 base pairs. 82. The human macrophage according to any one of clauses 74 to 81, further comprising a mutation in the MAF gene. 83. The human macrophage according to clause 82, wherein the mutation in MAF is a deletion. 84. The human macrophage according to any one of clauses 1 to 73, wherein the gene is MAF. 85. The human macrophage described in Clause 84, wherein the mutation is located in the region 79593000–79602000 on human chromosome 16. 86. The human macrophage described in Clause 85, wherein the mutation is located in the region 79593200–79601500 on human chromosome 16, for example, 79593400–79601100, and in particular, 79593600–79600900. 87. MAF is a human macrophage as described in any one of clauses 84-86, which is defunctionalized by the deletion of at least 50 base pairs. 88. The human macrophage described in Clause 87, wherein the deletion is a deletion of at least 100 base pairs. 89. The human macrophage described in Clause 87 or 88, wherein the deletion is a deletion of at least 250 base pairs. 90. The human macrophage described in Clause 88 or 89, wherein the deletion is a deletion of 100 to 10,000 base pairs. 91. The human macrophage described in Clause 90, wherein the deletion is a deletion of 500 to 3,000 base pairs, for example, 600 to 1,500 base pairs. 92. Human macrophages as described in any one of clauses 84-91, further including mutations in the gene MAFB. 93. The human macrophage described in Clause 92, wherein the mutation in the MAFB is a deletion. 94. The two genes mentioned above are MAFB and MAF, as described in Clause 69, of the human macrophage. 95. A human macrophage as described in Clause 94, wherein both alleles of MAFB and both alleles of MAF are defunctionalized by deletions of at least 50 base pairs. 96. Human macrophages as described in Clause 95, in which all deletions include exon DNA. 97. A human macrophage as described in any one of clauses 94 to 96, wherein each deletion is at least 100 base pairs. 98. A human macrophage as described in any one of clauses 94 to 96, wherein each deletion is at least 250 base pairs. 99. A human macrophage as described in any one of clauses 94 to 98, wherein each deletion is between 100 and 10,000 base pairs. 100. A human macrophage as described in any one of clauses 94 to 99, wherein the deletion is between 500 and 3,000 base pairs, for example, between 600 and 1,500 base pairs. 101. The MAFB deletion is located in the region 40685000-40690000 on human chromosome 22, in a human macrophage as described in any one of clauses 94-100. 102. The human macrophage described in Clause 101, wherein the MAFB deletion is located in the region 40685200–40689800 on human chromosome 22, for example, 40685400–40689600, for example, 40685600–40689400, and in particular, 40685700–40689300. 103. The MAF deletion is located in the region 79593000-79602000 on human chromosome 16, in a human macrophage as described in any one of clauses 94-102. 104. The human macrophage described in Clause 103, wherein the MAF deletion is located on human chromosome 16 in the region 79593200–79601500, for example, 79593400–79601100, and in particular in the region 79593600–79600900. 105. A non-tumor-forming human macrophage as described in any one of clauses 1 to 104. 106. A human macrophage having 46 chromosomes, as described in any one of clauses 1 to 105. 107. Human macrophages as described in any one of clauses 1 to 106, which do not contain chromosomes with chromosomal rearrangements. 108. Human macrophages as described in any one of clauses 1 to 107, which maintain the expression of at least two, for example, at least four, M1 phenotypic markers in vitro in the presence of tumor cells. 109. A human macrophage as described in any one of clauses 1 to 108, wherein the gene located on a chromosome, including the biallelelic deletion, is the only gene containing the biallelelic deletion. 110. A human macrophage as described in any one of Clauses 1 to 109, wherein the gene located on a chromosome, including the biallelelic deletion, is the only protein-coding gene including the biallelelic deletion. 111. The human macrophage described in any one of the clauses 1 to 110, wherein the gene located on a chromosome, including a biallelelic deletion, is a transcription factor-coding gene including a biallelelic deletion. 112. A human macrophage as described in any one of clauses 1 to 111, wherein MAF and / or MAFB is the sole transcription factor-coding gene (or more) containing a biallelelic deletion. 113. A human macrophage as described in any one of Clauses 1 to 112, in which MAF and / or MAFB is the sole gene (or gene) encoding the aforementioned protein, containing both allelic deletions. 114. A human macrophage as described in any one of clauses 1 to 113, wherein MAF and / or MAFB is the sole such gene (or more) containing both allelic deletions. 115. A collection of human macrophages comprising at least one mutation in both alleles of a gene located on a chromosome, wherein the collection of human macrophages is capable of recruiting T cells and / or NK cells against a tumor in vivo. 116. The tumor is a collection of human macrophages as described in Clause 115, which is a tumor established within the peritoneum. 117. The tumor is a collection of human macrophages as described in Clause 115, which is an established tumor in the lung. 118. The tumor is a collection of human macrophages as described in Clause 115, which is an established tumor within the liver. 119. A collection of human macrophages containing at least one mutation in both alleles of a gene located on a chromosome, the collection of human macrophages that induces regression of an established tumor in vivo. 120. A collection of human macrophages as described in any one of clauses 1 through 114. 121. A collection of human macrophages as described in any one of clauses 115-120, wherein the number of human macrophages is at least 1,000,000. 122. The collection of human macrophages described in Clause 121, wherein the number of human macrophages is at least 10,000,000, for example, at least 100,000,000. 123. A collection of human macrophages according to any one of clauses 115 to 122, wherein the number of macrophages can be increased at least tenfold under appropriate culture conditions. 124. A collection of human macrophages according to any one of clauses 115-123, wherein the number of macrophages can be increased at least 20-fold under appropriate culture conditions. 125. Human macrophages as described in any one of Clauses 1 to 114, and / or collections of human macrophages as described in any one of Clauses 115 to 124, for use as a pharmaceutical product. 126. Human macrophages as described in any one of Clauses 1 to 114, and / or collections of human macrophages as described in any one of Clauses 115 to 124, for use in cancer treatment. 127. The said cancer is a solid tumor, and a collection of human macrophages as described in any one of Clauses 1 to 114, and / or as described in any one of Clauses 115 to 124, for use as described in Clause 126. 128. The macrophages are human macrophages and / or collections of such human macrophages, which are of autologous origin with respect to the patient being treated, for use as a medicinal agent as described in any one of Clauses 125 to 127. 129. The macrophages are allogeneic with respect to the patient being treated, human macrophages for use as a medicinal agent as described in any one of paragraphs 125 to 127, and / or a collection of such human macrophages. 130. A method for treating a human subject with cancer, comprising administering to a human subject with cancer a human macrophage as described in any one of clauses 1 to 114, and / or a collection of human macrophages as described in any one of clauses 115 to 124. 131. The treatment method described in Clause 130, wherein the macrophages are of autologous origin with respect to the patient being treated. 132. The treatment method described in Clause 130, wherein the macrophages are allogeneic with respect to the patient being treated. 133. The macrophages are derived from an induced pluripotent stem cell line, as described in any one of Clauses 125-129, or as described in any one of Clauses 130-132. 134. The therapeutic method according to Clause 133, wherein the induced pluripotent stem cell line is homozygous for HLA-A, HLA-B, and HLA-DRB1. 135. The medical use or treatment method described in Clause 132, wherein the combination of HLA-A, HLA-B, and HLA-DRB1 is one of the ten most frequent combinations among residents selected from a group consisting of residents of the European Union, residents of the United States, residents of China, and residents of Japan. 136. A method for generating human macrophages as described in any one of clauses 1 to 114, and / or a collection of human macrophages as described in any one of clauses 115 to 124, (a) A step in which at least one mutation is introduced into both alleles of a selected target gene located on a chromosome in human iPS cells; (b) The process of isolating colonies derived from single cells; (c) The process of differentiating a single-cell colony into a macrophage; (d) The method comprising the step of identifying colonies of macrophages resistant to M-CSF-induced M2 polarization. 137. The method according to Clause 136, wherein in step (d), the macrophage colonies are identified by having the usual characteristics of M1 macrophages after being exposed to 50 ng / ml M-CSF for 48 hours. 138. The method according to clause 136, wherein in step (d), the macrophage colonies are identified by comprising the normal characteristics of M1 macrophages after being exposed to the combination of 40 ng / ml IL-4 and 50 ng / ml M-CSF for 48 hours. 139. The method according to any one of clauses 136 to 138, wherein the normal characteristics of M1 macrophages are elevated expression of the HLA-DRA gene. 140. The method according to any one of clauses 136 to 139, wherein the normal characteristics of M1 macrophages are secretion of IL18 and IL23A. 141. The method according to any one of clauses 136 to 140, wherein the normal characteristics of M1 macrophages are that the macrophages are positive for the surface markers HLA-DRA and CD74, but negative for the surface markers CD28 and LYVE1. 142. A screening method for identifying a gene that renders macrophages resistant to repolarization by tumor cells, comprising: (a) preparing a collection of iPS cells, which comprises at least one mutation in both alleles of a gene located on the chromosome of the iPS cells; (b) isolating single cell-derived colonies; (c) differentiating the single cell-derived colonies into macrophages; (d) identifying macrophage colonies that comprise the normal characteristics of M1 macrophages after being exposed to the combination of 40 ng / ml IL-4 and 50 ng / ml M-CSF for 48 hours; (e) identifying the mutated gene, the method as described above. 143. The method according to clause 142, wherein in step (d), the macrophage colonies are identified by secreting IL18 and IL23A. 144. The method according to any one of clauses 142 to 143, wherein in step (d), the macrophage colonies are identified by being positive for the surface markers HLA-DRA and CD74, but negative for the surface markers CD28 and LYVE1. 145. Human induced pluripotent stem cells or human embryonic stem cells having at least one mutation in both alleles of a gene located on a chromosome, wherein the gene is selected from the group consisting of STAT6, IRF4, PPARg, MAFB, MAF, KLF4, C / EPBb, GATA3, JMJD3, SOCS2, SOCS1, TMEM106A, and AKT1, and the mutation is a deletion. 146. The gene is selected from the group consisting of STAT6, IRF4, TMEM106A, MAFB, and MAF, and is a human induced pluripotent stem cell or human embryonic stem cell as described in Clause 145. 147. The gene is selected from the group consisting of MAF and MAFB, a human induced pluripotent stem cell or human embryonic stem cell as described in Clause 145 or 146. 148. Human induced pluripotent stem cells or human embryonic stem cells as described in any one of clauses 145-147, wherein both alleles of MAFB and both alleles of MAF are rendered functional by deletions, particularly deletions of at least 50 base pairs. 149. Human induced pluripotent stem cells or human embryonic stem cells as described in any one of clauses 145-148, all of which contain exon DNA. 150. The deletions, each comprising at least 100 base pairs, are human induced pluripotent stem cells or human embryonic stem cells as described in any one of clauses 145 to 149. 151. The deletions, each comprising at least 250 base pairs, are human induced pluripotent stem cells or human embryonic stem cells as described in any one of clauses 145 to 150. 152. The deletions are each between 100 and 10,000 base pairs, and are human induced pluripotent stem cells or human embryonic stem cells as described in any one of clauses 145 to 151. 153. The deletion is 500 to 3,000 base pairs, for example, 600 to 1,500 base pairs, of human induced pluripotent stem cells or human embryonic stem cells as described in any one of clauses 145 to 152. 154. The gene is a single protein-coding gene containing both allelic deletions, as described in any one of clauses 145-153, for human induced pluripotent stem cells or human embryonic stem cells. 155. A human induced pluripotent stem cell or human embryonic stem cell as described in any one of clauses 145 to 154, wherein the gene is the only gene containing both allelic deletions. 156. A human induced pluripotent stem cell or human embryonic stem cell as described in any one of clauses 145 to 155, wherein the gene is MAFB, and the MAFB deletion is located in the region 40685000-40690000 on human chromosome 22. 157. The MAFB deletion is located in the region 40685200–40689800 on human chromosome 22, for example, 40685400–40689600, for example, 40685600–40689400, and in particular, 40685700–40689300, as described in Clause 156. 158. A human induced pluripotent stem cell or human embryonic stem cell as described in any one of clauses 145 to 155, wherein the gene is MAF, and the MAF deletion is located in the region 79593000-79602000 on human chromosome 16. 159. The MAF deletion is located on human chromosome 16 in the region 79593200–79601500, for example, 79593400–79601100, and in particular in the region 79593600–79600900, as described in Clause 158. 160. The genes are MAF and MAFB, and MAF and MAFB are the only transcription factor-coding genes containing both allelic deletions, as described in any one of clauses 156 to 159, for human induced pluripotent stem cells or human embryonic stem cells. 161. The genes are MAF and MAFB, and MAF and MAFB are the only protein-coding genes containing both allelic deletions, as described in any one of clauses 156 to 159, for human induced pluripotent stem cells or human embryonic stem cells. 162. The genes are MAF and MAFB, and MAF and MAFB are the only genes containing both allelic deletions, as described in any one of clauses 156 to 159, for human induced pluripotent stem cells or human embryonic stem cells. 163. Use of human induced pluripotent stem cells or human embryonic stem cells as described in any one of clauses 145 to 162 for the generation of human phagocytic cells. 164. The use described in Clause 163, wherein the human phagocytic cell is a human macrophage. 165. The use described in Clause 164, wherein the human macrophage is non-tumoric and can produce at least four grandchild cells under appropriate culture conditions ex vivo. 166. Use of human induced pluripotent stem cells or human embryonic stem cells as described in any one of clauses 145 to 165 for the creation of a collection of human macrophages as described in any one of clauses 115 to 124. 167. Common human myeloid progenitor cells or human granulocyte / macrophage progenitor cells having at least one mutation in both alleles of a gene located on a chromosome, wherein the gene is selected from the group consisting of STAT6, IRF4, PPARg, MAFB, MAF, KLF4, C / EPBb, GATA3, JMJD3, SOCS2, SOCS1, TMEM106A, and AKT1, and the mutation is a deletion. 168. The gene is selected from the group consisting of STAT6, IRF4, TMEM106A, MAFB, and MAF, and is a human myeloid common progenitor cell or human granulocyte / macrophage progenitor cell as described in Clause 167. 169. The gene is selected from the group consisting of MAF and MAFB, and is a human myeloid common progenitor cell or human granulocyte / macrophage progenitor cell as described in clause 167 or 168. 170. Human myeloid common progenitor cells or human granulocyte / macrophage progenitor cells as described in any one of clauses 167-169, wherein both alleles of MAFB and both alleles of MAF are rendered functional by deletions, particularly deletions of at least 50 base pairs. 171. Human myeloid common progenitor cells or human granulocyte / macrophage progenitor cells as described in any one of clauses 167-170, in which all deletions include exon DNA. 172. The deletions are each at least 100 base pairs, and are human myeloid common progenitor cells or human granulocyte / macrophage progenitor cells as described in any one of clauses 167 to 171. 173. The deletions are each at least 250 base pairs, and the cells are human myeloid common progenitor cells or human granulocyte / macrophage progenitor cells as described in any one of clauses 167-172. 174. Human myeloid common progenitor cells or human granulocyte / macrophage progenitor cells as described in any one of clauses 167 to 173, wherein the deletions are each between 100 and 10,000 base pairs. 175. The deletion is 500 to 3,000 base pairs, for example, 600 to 1,500 base pairs, in human myeloid common progenitor cells or human granulocyte / macrophage progenitor cells as described in any one of clauses 167 to 174. 176. The gene is the only protein-coding gene containing both allelic deletions in a human myeloid common progenitor cell or human granulocyte / macrophage progenitor cell as described in any one of clauses 167-175. 177. Human myeloid common progenitor cells or human granulocyte / macrophage progenitor cells as described in any one of clauses 167-176, wherein the gene is the only gene containing both allelic deletions. 178. A human myeloid common progenitor cell or human granulocyte / macrophage progenitor cell as described in any one of clauses 167 to 177, wherein the gene is MAFB, and the MAFB deletion is located in the region 40685000-40690000 on human chromosome 22. 179. The MAFB deletion is located in the region 40685200–40689800 on human chromosome 22, for example, 40685400–40689600, for example, 40685600–40689400, and in particular, in the region 40685700–40689300, in human myeloid common progenitor cells or human granulocyte / macrophage progenitor cells as described in Clause 178. 180. A human myeloid common progenitor cell or human granulocyte / macrophage progenitor cell as described in any one of clauses 167 to 179, wherein the gene is MAF, and the MAF deletion is located in the region 79593000-79602000 on human chromosome 16. 181. The MAF deletion is located in the region 79593200–79601500 on human chromosome 16, for example, 79593400–79601100, and in particular in the region 79593600–79600900, as described in Clause 180, in human myeloid common progenitor cells or human granulocyte / macrophage progenitor cells. 182. The genes are MAF and MAFB, and MAF and MAFB are the only transcription factor-coding genes containing both allelic deletions, as described in any one of clauses 178-181, for human myeloid common progenitor cells or human granulocyte / macrophage progenitor cells. 183. The genes are MAF and MAFB, and MAF and MAFB are the only protein-coding genes containing both allelic deletions, as described in any one of clauses 178-181, for human myeloid common progenitor cells or human granulocyte / macrophage progenitor cells. 184. The genes are MAF and MAFB, and MAF and MAFB are the only genes containing both allelic deletions, as described in any one of clauses 178-181, for human myeloid common progenitor cells or human granulocyte / macrophage progenitor cells. 185. Use of human myeloid common progenitor cells or human granulocyte / macrophage progenitor cells as described in any one of clauses 167-184 for the generation of human phagocytic cells. 186. The use described in Clause 185, wherein the human phagocytic cell is a human macrophage. 187. The use described in Clause 186, wherein the human macrophage is non-tumoric and can produce at least four grandchild cells under appropriate culture conditions ex vivo. 188. Use of a collection of human macrophages as described in any one of Clauses 115-124 for the creation of human myeloid common progenitor cells or human granulocyte / macrophage progenitor cells as described in any one of Clauses 167-184.

[0247] The implementation of this invention will utilize prior art in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, unless otherwise specified. Such techniques are detailed in literature such as, for example, "Molecular Cloning: A Laboratory Manual," 4th edition (Sambrook et al., 2012); "Handbook of Experimental Immunology" (Weir, 1997); "Short Protocols in Molecular Biology" (Ausubel, 2002); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting" (Babar, 2011); and "Current Protocols in Immunology" (Coligan, 2002). These techniques may be considered in the preparation and implementation of this invention.

[0248] Those skilled in the art will recognize that the invention described herein is subject to modifications and alterations other than those specifically described. It should be understood that the invention includes all such modifications and alterations without departing from its spirit or essential features. The invention also includes all of the processes, features, compositions, and compounds mentioned or shown herein individually or collectively, as well as any combination of any and all of such processes or features, or any two or more. Therefore, this disclosure should be considered in all aspects shown and is not restrictive; the scope of the invention is indicated by the appended claims, and all modifications falling within the meaning and scope of equivalents are intended to be encompassed therein.

[0249] Throughout this specification, various references are cited, each incorporated into this specification as a whole by reference.

[0250] The above description will be better understood by referring to the following examples. However, such examples are illustrative of ways of carrying out the present invention and are not intended to limit the scope of the invention. [Examples]

[0251] (Examples) Aziz et al. (2009) showed that deficiency of the MafB and c-Maf combination in mice does not adversely affect hematopoiesis, as all lineages, including monocytes and macrophages, can be produced (Aziz et al., 2009). Instead, the deficiency allowed for an extension of the proliferation period of mature mouse macrophages without differentiated phenotypes or functional defects (Aziz et al., 2009).

[0252] We investigated the role of Maf-DKO macrophages in ID8 ovarian tumor models and B16 melanoma tumor models. To this end, we treated tumor-bearing mice with Maf-DKO mouse macrophages and demonstrated that mouse Maf-DKO macrophages inhibited early tumor growth and tumor establishment. Furthermore, our in vitro results demonstrated that Maf-DKO macrophages possess an M1-like phenotype and are not repolarized to an M2-like phenotype by normal M2 stimulation. Importantly, tumor cells also do not repolarize mouse Maf-DKO macrophages.

[0253] Next, we created human MAF / MAFB DKO macrophages. In in vitro experiments, we confirmed that, similar to mouse Maf-DKO macrophages, human MAF / MAFB DKO macrophages possess a stable M1-like phenotype and do not repolarize to an M2-like phenotype by normal M2 stimulation or tumor cells. To our knowledge, MAF / MAFB DKO macrophages are the first examples of human macrophages that exhibit resistance to tumor-induced repolarization due to loss-of-function mutations in both alleles of the chromosomal gene. More generally, this suggests that human macrophages in a stable anti-tumor-polarized state due to loss-of-function mutations in both alleles of the chromosomal gene required for M2 induction are useful in cell therapy, such as tumor cell therapy.

[0254] (Mouse MafB / c-Maf DKO macrophages) (Materials and Methods) Mice: Female mice (C57BL / 6 or Rag2γc knockout), 6-8 weeks old, were used in all experiments. The mice were raised at CIML (Center for Immunology in Marseille Luminy) under specific pathogen-free conditions in accordance with French and European Union directives.

[0255] Cell lines: Mouse ovarian cancer cell line ID8 and B16 melanoma cell line were cultured in DMEM medium supplemented with 10% fetal bovine serum (FCS), 1% penicillin, and streptomycin at 37°C under 5% CO2. Cells were passaged twice weekly with trypsin.

[0256] Preparation of mouse macrophages: Mouse macrophages were derived from the bone marrow of wild-type mice or Maf double knockout mice and cultured in DMEM medium for 10-12 days, supplemented with 10% FBS, 10-50 ng / ml rMCSF, or 20% M-CSF. They were cultured in L929 cell-adapted IMDM / 0.5FCS medium (LCM), 2 mM glutamine, 1% sodium pyruvate, 50 ug / ml penicillin / streptomycin, under 5% CO2 at 37°C. Primary Maf-DKO cell lines (selected from blood (Aziz et al., 2009)) were subculturified every 4 days with partial medium changes every 2 days.

[0257] Macrophage adoptive cell transfer into tumor-bearing mice: 5 × 10¹⁶ cells into 6-8 week old C57BL / 6 female mice 6 ID8-Luc cells were injected intraperitoneally (ip). Mice were immediately treated with macrophages, or allowed to establish tumors for 14 days before macrophage transfer. 5 × 10 6 Individual WT macrophages or Maf-DKO macrophages were transferred to mice via ip (intracellular matrix) in four consecutive transfers, each performed on days 0, 2, 4, and 6, or days 14, 16, 18, and 20, with either immediate or delayed treatment. To evaluate tumor progression, mice were dissected on day 27, ID8 cells were counted by flow cytometry, and luciferase activity in cell lysates was measured.

[0258] 1 × 10⁶ in 100 μl PBS 5 1 × 10¹ B16-F10 (or B16-Luc) cells were injected intravenously (iv) into the eye. As described above, the mice were 1 × 10¹⁶ 6 Macrophage treatment was performed either immediately after tumor injection or after tumor development on day 7. Macrophage treatment was repeated four times consecutively. On day 14 after tumor injection, mice were sacrificed and their lungs and livers were removed. The number of metastatic colonies visible on the surface of the lungs and livers was counted.

[0259] Bioluminescence imaging: Bioluminescence imaging was performed using Berthold Technologies' Night-Owl. Luciferin solution (3 mg per mouse) was injected intravenously into mice, and imaging was performed until the emission peak was obtained (10 minutes). Total luminous flux values ​​were obtained from the target anatomical region. Data were expressed in relative light units (RLU) photon emission / s / mm². 2 The images were presented as follows: (for the ID8 model) images captured the dynamics of tumor development from day 0 to day 27, or (for the B16 model) from day 0 to day 14.

[0260] Luciferase Assay: The luciferase assay was performed using the Promega luciferase assay system (TB281). Briefly, the cells were washed with 1×PBS and lysed with 20 μl of the cell lysis buffer included in the system. Next, the cells were suspended in 100 μl of luciferase assay reagent, and the resulting light was measured with a luminometer.

[0261] Isolation of peritoneal cells: After sacrificing, cells were obtained from the peritoneal exudate by washing the peritoneal cavity with 10 ml of PBS. Red blood cells were removed by incubation in erythrocyte lysis buffer at room temperature for 5 minutes. Cell content in the peritoneal cavity was assessed using multicolor flow cytometry. Gene expression in the entire population was evaluated by qPCR.

[0262] Flow cytometry analysis: Cell suspensions were resuspended in FACS buffer (PBS1X containing 2 mM EDTA and 0.5% FCS). All samples were blocked with 1:100 CD16 / 32 (2.4G2, BD PharMingen) and then surface-stained on ice with antibodies against CD45, CD11b (M1 / 70), F4 / 80, CD3e, Ly6G (1A8), NK1.1, CD19, CD8α, and MHCII. The absolute number of cells could be measured by adding 20 μl of calibrated microbead suspension to each sample before obtaining the absolute number. Flow cytometry analysis and cell sorting were performed using LSRII and FACSAria (Becton Dickinson).

[0263] M1 / M2 in vitro stimulation: 200,000 WT BMDM macrophages or Maf-DKO macrophages were incubated in DMEM medium containing 20 ng / ml recombinant mouse M-CSF (rMCSF). The cells were then stimulated with 100 ng / ml LPS (8 hours), 100 ng / ml IFNγ (8 hours), or 20 ng / ml IL-4 for 24 hours. Macrophage stimulation was performed in or without 50% supernatant from ID8 conditioned medium. After 72 hours of incubation, the ID8 conditioned medium was collected. At the indicated time point, the supernatant was collected and cytokine production was evaluated by CBA, and the cells were lysed by RLT and gene expression was evaluated by QPCR. The conditions are summarized in the table below. [Table 3]

[0264] RNA extraction and reverse transcription: The entire peritoneal cell population, CD11b+CD45+Lin-selected macrophages, or in vitro stimulated macrophages were collected at the points described above. The cells were washed with PBS and lysed in RLT buffer containing β-ME. RNA was extracted using the Qiagen RNeasy mini-kit (Qiagen Biotech) according to the manufacturer's instructions. RNA levels were measured by NanoDrop.

[0265] 500 ng of RNA from each sample was used for reverse transcription. To nuclease-free sterile water, 1 μl of Oligodt (500 μg / ml) and 1 μl of dNTP mix (10 mM each) were added to a final volume of 12 μl. This mixture was heated to 65°C to denature the RNA, and then rapidly cooled to maintain the denatured form. After simple centrifugation, 4 μl of First Strand Buffer (5×), 2 μl of 0.1 M DTT, and 1 μl of RNaseOut (40 units / μl, Invitrogen) were added. This mixture was incubated at 42°C for 2 minutes, and then 1 μl of Super-Script II reverse transcriptase (200 units, Invitrogen) was added. The mixture was then incubated at 42°C for 50 minutes. The reverse transcriptase was inactivated by heating at 70°C for 15 minutes.

[0266] Quantitative PCR: cDNA was diluted 1:10, and quantitative PCR was performed using 2 μl of the diluted cDNA. QPCR was performed using SyberGreen (Applied Biosciences) 2X master mix according to the manufacturer's instructions. The primers used for QPCR are listed in the table below.

[0267] Cytokine Bead Array (CBA): Cytokines were analyzed using the BD Biosciences "Mouse Inflammatory CBA" kit, catalog number 552364, and Canto II, following the manufacturer's instructions. Sample supernatants were analyzed without dilution.

[0268] Statistical Analysis: All experiments were performed in triplicate, and representative data are shown. The results were tested for statistical significance using Student's t-test with GraphPad prism software.

[0269] (result) (Example 1) (MafB and c-Maf double knockout macrophages suppress the early growth of tumors.) We previously demonstrated that double knockout (Maf-DKO) mouse macrophages for MafB and c-Maf can proliferate permanently ex vivo without loss of function or malignant transformation upon M-CSF stimulation (Aziz et al., 2009). To evaluate the roles of MafB and c-Maf in the ecology of TAM, we examined the development of ID8 ovarian tumor cells treated with mouse Maf-DKO macrophages. First, we investigated the role of macrophages in the early stages of tumor establishment.

[0270] The ID8 mouse ovarian surface epithelial cell line is often used as a syngeneic mouse model for human ovarian cancer. ID8 cancer cells expressing firefly luciferase were injected via ip injection into wild-type C57BL6 mice on day 0. These mice were then treated with either wild-type bone marrow-derived macrophages (WT) or Maf-DKO bone marrow-derived macrophages (BM-DKO) on days 0, 1, 2, and 3, or left untreated (Figure 1a). Tumor progression was evaluated in situ by bioluminescence imaging at the indicated number of days from day 0 to day 27 after adoptive cell transfer (data not shown). Untreated tumor-bearing mice developed malignant ascites between days 4 and 27, as evaluated by quantification of emitted photons. Similarly, tumor-bearing mice treated with WT bone marrow-derived macrophages (WT-BMDM) developed ID8 tumors during the experimental period. In contrast, mice treated with bone marrow-derived Maf-DKO macrophages (DKO-BMDM) showed a significant reduction in tumor volume on day 27 after tumor induction. Next, peritoneal cells were lysed and a luciferase assay was performed to quantify luciferase levels in the ascites fluid (Figure 1b). The DKO-BMDM-treated group (squares) had significantly lower luciferase levels compared to the WT-treated group or the untreated group (black dots). Next, the absolute number of tumor cells in the peritoneum of tumor-bearing mice was investigated. To this end, flow cytometry analysis of peritoneal cells was performed as shown in Figure 1c. ID8 cells had high SSC values ​​and were CD45-negative (CD45-). Quantification of tumor cell count showed an increase in the number of ID8 cells in the peritoneum of untreated and WT-treated mice, but this number significantly decreased when the mice were treated with DKO-BMDM macrophages. Therefore, Maf-DKO macrophages were able to reduce peritoneal tumor volume.

[0271] (Example 2) (MafB and c-Maf DKO macrophages induce established tumor regression.) To evaluate the role of Maf-DKO macrophages in advanced tumors, ID8-luciferase tumor cells were intravenously injected into C57BL6 mice on day 0 (Figure 2a). By day 14, these mice showed malignant ascites and established tumors, which was visible by bioluminescence (data not shown). Next, adoptive cells were transferred with WT-BMDM, DKO-BMDM, or blood DKO four times at 2-day intervals (Figure 2a). As previously described, tumor progression was evaluated between days 0 and 27 by creating in-situ bioluminescence images on the indicated days. Bioluminescence quantification of emitted photons showed that untreated tumor-bearing mice had highly malignant ascites on day 27 (data not shown). Similarly, mice treated with WT-BMDM developed aggressive peritoneal tumors on day 27 (data not shown). In contrast, mice treated with DKO macrophages showed significantly reduced bioluminescence signaling and, consequently, significantly reduced tumor volume on day 27 (data not shown). For further analysis, mice were sacrificed on day 27, and tumor volume was quantified by luciferase assay and flow cytometry. Luciferase levels were high in peritoneal cells of untreated mice and slightly higher in WT-BMDM treated mice (Figure 2b, black dots), reflecting a high level of peritoneal tumor volume. On the other hand, mice treated with DKO macrophages showed decreased luciferase levels, reflecting a reduction in tumor volume (Figure 2b, squares, right column). To confirm the reduction in tumor cell count, the absolute number of ID8 cells in the peritoneal cavity was analyzed by flow cytometry. As before, ID8 cells were identified as SSChi CD45- cells (Figure 2c). Untreated mice (Figure 2c, left panel) and WT-BMDM-treated mice showed a high percentage of ID8 cells, while Maf-DKO-treated mice showed a significantly reduced percentage of ID8 cells (Figure 2c, right panel). These data indicate that Maf-DKO macrophages can fight tumors and even reduce established tumor masses.

[0272] (Example 3) (Cell composition of ascites fluid) Generally, tumors are composed of various immune system cells that contribute to the tumor stroma of a growing malignant tumor (Kerkar and Restifo, 2012). To determine whether leukocyte subsets (e.g., T cells, NK cells, etc.) were recruited during tumor development, flow cytometry analysis of ascites fluid from tumor-bearing mice was performed on day 27. Peritoneal lavage fluid was collected and stained with antibody cocktails targeting NK cells, CD8 T cells, granulocytes, and macrophages, which are the most representative cells in the tumor stroma (Kerkar and Restifo, 2012).

[0273] Initially, on day 21, after cancer initiation and using established tumor strategies (Figure 2a), NK cells and T cells, such as CD8 T cells, were detected in the peritoneal leukocyte fraction in addition to macrophages, as shown in Figure 3a. These cell populations were not detected when mice were immediately treated with macrophages, as in Example 1 (data not shown). Little CD8 T cell infiltration was observed in the peritoneum of untreated or WT-BMDM treated mice (Figure 3b, circles and triangles). However, mice treated with Maf-DKO macrophages showed a significant increase in the CD8 T cell population (Figure 3b, squares). Furthermore, greater NK cell recruitment was observed in Maf-DKO treated mice compared to untreated or WT-BMDM treated mice. These results suggest that CD8 T cells and NK cells may be involved in the reduction of tumor burden mediated by Maf-DKO macrophages.

[0274] Next, we examined the peritoneal macrophage population based on the CD11b and F4 / 80 markers (Figure 3c). The peritoneal cavities of untreated, WT-BMDM-treated, and Maf-DKO-treated mice were filled with macrophages, but there was no difference in the proportion of macrophages among these three groups (data not shown). Therefore, based on previous research reports (Wang et al., 2011) suggesting that MHCII levels within macrophage populations fluctuate, being higher in tumor-suppressing macrophages and lower in tumor-promoting macrophages, we hypothesized that the activation states of macrophage populations may differ, which may explain our aforementioned results regarding the reduction of tumor mass in Maf-DKO-treated mice. To test this hypothesis, we stained peritoneal cells with different macrophage activation markers (CD80, CD86, CD69, and MHCII). All macrophages from Maf-DKO treated mice were MHCII+ (Figure 3d, squares), while only a very small fraction of macrophages from untreated or WT-BMDM treated mice were MHCII+ (Figure 3d, circles and triangles). Furthermore, the average fluorescence intensity of MHCII+ in macrophages from Maf-DKO treated mice was higher than that of low-MHCII MHCII+ macrophages from untreated or WT-BMDM treated mice (Figure 3e), suggesting that Maf-DKO macrophages are high in MHCII. Our data indicate that high-MHCII TAMs are associated with tumor suppression (in the case of Maf-DKO macrophages), while low-MHCII macrophages are associated with tumor promotion (in the case of WT macrophages).

[0275] (Example 4) (MafB and c-Maf deficiencies lead to classical macrophage activation in vivo.) The production of pro-inflammatory cytokines versus anti-inflammatory cytokines by tumor-infiltrating leukocytes plays a crucial role in regulating the immune response against tumors (Balkwill and Mantovani, 2001). Classically, activated macrophages (M1) exhibit a cytotoxic pro-inflammatory phenotype (high IL-12, IL-6, IFNγ, NOS2), while alternatively, activated macrophages (M2) suppress immune and pro-inflammatory responses by increasing the production of IL-10 or arginase (O'Shea and Murray, 2008) (Gordon, 2003) (Murray and Wynn, 2011) (Mantovani et al., 2002). To analyze macrophage phenotypes in vivo, peritoneal macrophages (CD11b+Lin-) were selected from tumor-bearing mice (treated as described above). mRNA levels of NOS2, C2TA (MHC II transactivator), IL-6, IL-12, and IL-10 were measured using real-time RTPCR.

[0276] CD11b+ cells derived from Maf-DKO treated mice (Figure 4, striped) showed significantly elevated NOS2, CIITA (C2TA), and IL-6 mRNA levels, as well as IL-12 mRNA levels, unlike those derived from WT treated mice or untreated control mice (polka dots and black, respectively) (Figures 6a-6d). In contrast, CD11b+ cells from WT treated mice showed higher IL-10 expression compared to CD11b+-selected Maf-DKO macrophage cells (Figure 6e). These results suggest that Maf-DKO macrophages are refractory to in vivo retraining to the M2 phenotype by tumor cells.

[0277] (Example 5) (Maf-DKO macrophages show more potent classical M1 activation in vitro.) Our in vivo results regarding macrophage phenotypes indicate that Maf-DKO macrophages are most similar to M1 macrophages in tumor-bearing mice. To further characterize Maf-DKO macrophages, we performed in vitro experiments stimulating macrophages (WT or Maf-DKO) with either known M1 stimuli (LPS, IFNγ) or M2 stimuli (IL-4). The cells were then lysed, and gene expression was measured by real-time QPCR.

[0278] As predicted, IL-6 expression was detected upon LPS stimulation (Figure 5a), and IL-6 mRNA levels were significantly higher in Maf-DKO macrophages compared to WT macrophages (Figure 5a; note that DKO macrophages are represented by dots and WT macrophages by stripes in Figure 5). C2TA (a positive regulator of the MHCII gene and an M1 marker) expression was increased in Maf-DKO macrophages compared to WT macrophages (Figure 5b stripes) upon IFNγ, LPS stimulation, and to a lesser extent, IL-4 stimulation (Figure 5b dots), which is another indication of the M1-like phenotype in Maf-DKO macrophages. Similarly, NOS2 expression was enhanced in Maf-DKO macrophages compared to WT macrophages upon LPS or IFNγ stimulation (Figure 5c). In contrast, monitoring of M2 gene expression revealed that IL-10 (Figure 5d) and arginase (Figure 5e) (in response to IL-4 stimulation) were highly expressed in WT macrophages but not in Maf-DKO macrophages. These results suggest that Maf-DKO macrophages are sensitive to M1 stimulation, similar to classical macrophage activation, but their sensitivity to M2 stimulation is, if any, much lower.

[0279] (Example 6) (Maf-DKO macrophages are refractory to retraining by tumor cells in vitro.) Next, we investigated whether Maf-DKO macrophages could be educated by tumors in vitro. To this end, we created a pseudo-in vivo environment by culturing either WT macrophages or Maf-DKO macrophages in a medium containing ID8 supernatant. We confirmed the production of inflammatory cytokines by CBA (see section "Materials and Methods"). IL-6 was produced by both WT macrophages and Maf-DKO macrophages upon stimulation with LPS (black columns in Figure 6a). The level of IL-6 production by WT macrophages dramatically decreased in the presence of ID8 tumor cell supernatant, while the level produced by Maf-DKO macrophages remained high under the same conditions (grid-patterned columns in Figure 6a). Similar to IL-6, TNFα is produced by both WT macrophages and Maf-DKO macrophages in the absence of ID8-SN (Figure 6b, black columns). However, in the presence of ID8-SN, Maf-DKO macrophages still produce large amounts of TNFα, while WT macrophages show a significant decrease in TNFα production (Figure 6b, grid-patterned columns). These results suggest that Maf-DKO macrophages are M1-like macrophages that are refractory to and resistant to reprogramming by the tumor microenvironment, while WT macrophages can be reprogrammed by the tumor.

[0280] (Example 7) (Maf-DKO macrophages inhibit the development of melanoma.) To investigate the antitumor activity of Maf-DKO macrophages in a second tumor model, we examined the effect of these macrophages on B16 melanoma development. B16 was used as the experimental metastasis model. B16 melanoma is a mouse tumor cell line used in research as a model for metastasis and solid tumor formation studies. B16 tumor cells (1 × 10⁶) 5) was intravenously (i.v.) transferred into C57BL6 mice via retro-orbital injection, and tumors developed in the lungs. Next, mice were injected with Maf-DKO macrophages, WT macrophages, or left uninjected. Mice were treated with different types of macrophages only after tumor establishment (Figure 7a). When the mice were sacrificed on day 14, metastatic tumors appeared as black pigmented colonies with a diameter of 1 to 3 mm, and a tendency to fuse with each other was observed. As confirmed by quantification of tumor colonies (Figure 7c, left panel), B16 tumor cells formed a vast number of tumor nodules in the lungs of untreated mice (Figure 7b, left column). Maf-DKO macrophages (Figure 7b, right panel and 7c, right column) strongly reduced tumor formation compared to untreated control mice, and exhibited a stronger effect than WT-treated mice (Figure 7b, middle panel and 7c, middle column. Therefore, the absence of MafB and c-Maf in macrophages reduces lung metastasis.

[0281] Collectively, these data indicate that Maf-DKO macrophages can suppress tumor development in both prophylactic and therapeutic settings, regardless of the tumor model.

[0282] (Example 8) (T cells, NK cells, and B cells are also involved in defense against B16 melanoma.) To evaluate the potential contribution of other immune cells such as T cells, B cells, and NK cells to the anti-tumor mechanism, Rag2γc knockout mice deficient in T cells, B cells, and NK cells were used. B16 tumor cells expressing firefly luciferase were administered at 1×10 5The treatment was initiated by intravenous injection of Maf-DKO macrophages into mice. Immediately after injection (Figure 8a) or on day 7 after tumor establishment (Figure 8b), mice were treated with Maf-DKO macrophages four consecutive times, or left untreated. Tumor growth was monitored using bioluminescence imaging and quantification of tumor nodules. Tumors developed in all mice, as demonstrated by bioluminescence imaging on day 14. The tumors were localized in the lungs and livers, and an increased number of metastatic colonies was shown in both organs, compared to our results for B16 development in WT mice (Example 7) (data shown for liver only: Figures 8c-8f). Strong infiltration of B16 tumor cells was shown in both the lungs and livers of both untreated and WT-treated mice (liver data shown: Figure 8c). Importantly, mice immediately treated with Maf-DKO macrophages showed a significant reduction in liver metastases compared to WT-treated mice (left panel) or untreated mice (not shown) (Figure 8c, right panel). The tumor reduction mediated by Maf-DKO was confirmed by quantification of metastatic nodules (Figure 8e, polka-dot columns). However, in tumor-established mice, only slight tumor reduction was observed in the lungs after treatment with both Maf-DKO and WT macrophages. Furthermore, no reduction in liver metastasis was observed in this second group of mice (Figures 8d, 8f), suggesting that effector immune cells (NK cells, T cells, and B cells) are involved in the antitumor effect of Maf-DKO. These data indicate that Maf-DKO macrophages have both a direct effect of inhibiting tumor growth and metastasis in the early stages of tumorigenesis, and an indirect effect of sustained tumor suppression by cooperating with other immune cells, thereby achieving the therapeutic role described above.

[0283] (Consideration of mouse experiments) This study provided evidence for the role of Maf-DKO macrophages in preventing tumor growth. Transplantation of Maf-DKO macrophages, rather than WT macrophages, into tumor-bearing mice (ID8 ovarian carcinoma or B16 melanoma) resulted in a significant reduction in both initial and established tumor volume (Figures 1, 2, and 7), and increased mouse survival. Furthermore, the antitumor activity of Maf-DKO macrophages did not decrease with long-term maintenance with M-CSF (Maf-DKO cells grown from blood and maintained in culture for extended periods showed comparable antitumor activity to bone marrow-derived Maf-DKO cells).

[0284] While we do not intend to be bound by any theory, the still-unknown mechanisms of Maf-DKO tumor inhibition are likely both direct and indirect. Results in Rag2γc knockout mice (lacking T cells, B cells, and NK cells) suggest that Maf-DKO macrophages, unlike WT macrophages, continuously cooperate with other immune cells to achieve tumor reduction even in the presence of tumors in vivo. Furthermore, it was shown that T cells, e.g., CD8 T cells, and to a lesser extent, NK cells, were recruited into the peritoneal cavity of established tumor-bearing mice at an early stage after Maf-DKO adoptive cell transfer (Figures 3a, 3b). Taken together, these results suggest that Maf-DKO macrophages may inhibit tumor growth through the activation of T cells, e.g., CD4 T cells or CD8 T cells, or through the activation of NK cells. Moreover, macrophages in Maf-DKO treated mice were shown to have "high" MHC II compared to peritoneal macrophages in WT treated mice. Importantly, recent studies have demonstrated that a shift from "high" MHC II to "low" MHC II in TAMs mediates tumor progression in mice (Wang et al., 2011). Another study showed that CD169+ macrophages induce an antitumor immune response via their ability to act on phagocytic cells, and cross-presentation of tumor cell peptides to cytotoxic T cells (Asano et al., 2011). In summary, these data and our results suggest that Maf-DKO antitumor activity may partially activate T cells through tumor peptide presentation.

[0285] Preliminary in vitro results on co-culturing Maf-DKO macrophages or WT macrophages with ID8 tumor cells have shown increased lactate dehydrogenase (LDH) release (data not shown), and it has been claimed that when tumor cells are co-culturified with Maf-DKO macrophages, more ID8 cell lysis occurs upon contact with the Maf-DKO macrophages. This suggests that, at least in vitro, Maf-DKO macrophages have a direct killing effect on tumor cells.

[0286] Previous literature suggests that co-culture with ID8 cells polarizes macrophages into an M2-like phenotype by increasing IL-10 expression and decreasing IL-12 (Hagemann et al., 2006). Monitoring the TAM profile in ex vivo Maf-DKO treated mice showed that Maf-DKO macrophages were more stable M1-like macrophages compared to WT macrophages. Furthermore, we analyzed the phenotype of Maf-DKO macrophages in vitro by measuring the mRNA expression levels of IL-6, C2TA, NOS2, arginase, and IL-10. Maf-DKO macrophages were shown to be more sensitive to M1 stimulation (LPS and IFNγ) based on upregulation of NOS2, C2TA, and IL-6, and downregulation of arginase and IL-10, while WT macrophages were shown to be more sensitive to M2 stimulation (IL-4), exhibiting the opposite expression profile.

[0287] Furthermore, even when Maf-DKO macrophages were in vitro treated with tumor cell conditioned medium (ID8-SN), their phenotype did not switch from M1 to M2.

[0288] These experiments provide evidence of the role of Maf-DKO macrophages in driving anti-tumor immune responses, opening new avenues for cancer cell therapy.

[0289] (Human MAF / MAFB DKO macrophages) Methods for generating human induced pluripotent stem cells are well known in this field. A human iPS cell line was created from dermal fibroblasts obtained from the penile foreskin of a healthy Caucasian neonatal boy. Reprogramming was performed using non-integrated Sendai viruses including POU5F1, SOX2, KLF4, and MYC (Fusaki N. et al., (2009)). Screening for HIV1, HIV2, hepatitis B virus, hepatitis C virus, and mycoplasma virus was negative. Karyotyping showed a normal 46(X,Y) karyotype. Basically as described in Gonzalez et al. (2014), a doxycycline-inducible Cas9 expression cassette was introduced into this cell line at the AAVS1 locus by TALEN-mediated gene targeting to prepare the cell line BIHi001-A-1.

[0290] (Example 9) (Creation of double knockout (DKO) cells from human iPS cells) (a) Culture of iPS cells iPS cells were cultured in StemFlex medium (Thermo Fisher, A3349401) on a vitronectin-coated tissue culture plate (Thermo Fisher, A14700) under feeder-free conditions at 37°C and normal oxygen pressure of 5% CO2. When the culture density reached 60-80%, the cells were passaged using ReLeSR (Stem Cell Technologies, 05872) and maintained the following day in the presence of 10 μM ROCK inhibitor Y-27632 (biomol, Cay10005583). Single-cell suspensions of iPS cells were obtained by incubation with Accutase (Merck Millipore, SF006).

[0291] (b) genome editing The coding sequences for MAF (gene ID: 4094, 373 amino acids) and MAFB (gene ID: 9935, 323 amino acids) were deleted from human cells using the CRISPR / Cas9 system. For this purpose, iPS cell lines from healthy donors, possessing a doxycycline-inducible Cas9 expression cassette (BIHi001-A-1, https: / / hpscreg.eu / cell-line / BIHi001-A-1; also known as iBCRT Cas9v1-3G-Kl.16) integrated into the AAVS1 locus, were transfected with an sgRNA expression plasmid (pU6-(BbsI)sgRNA_CAG-venus-bpA, Addgene ID 86985, https: / / www.addgene.org / 86985 / ) targeting MAF and MAFB, according to a published protocol (Yumlu's literature (2017)).

[0292] To completely remove the CDS, we used the CrispRGold program for sequence design (https: / / crisprgold.mdc-berlin.de / Chu et al. (2016)) to design two sgRNAs per gene, targeting the 5'UTR at least 20 nt upstream of the start codon and the 3'UTR at least 20 nt downstream of the stop codon for each gene. The two sgRNAs for each gene were co-expressed from the sgRNA expression vector pU6-(BbsI)sgRNA_CAG-venus-bpA, which also encodes the fluorescent reporter gene (Venus, YFP) used for positive selection of transfected cells by FACS.

[0293] [Table 4] (Table 1: sgRNA and protospacer sequences for CRISPR / Cas9-mediated deficiency in MAF and MAFB CDS) The location of the target-specific 20-nucleotide protospacer is marked with an "N" within the sgRNA sequence. The genomic target sequence of the sgRNA expressed from pU6-(BbsI)sgRNA_CAG-venus-bpA, which has the displayed protospacer sequence, is located at the 5'UTR and 3'UTR of the UTR (untranslated region) of the MAF and MAFB, respectively.

[0294] [ka]

[0295] BIHi001-A-1 cells were transfected with pCAG-mTrex2-bpA (Addgene, ID 86984, https: / / www.addgene.org / 86984 / ) in an sgRNA expression vector using lipofectamine 3000 (Thermo Fisher, No. L3000001) to enhance indel formation. Control cells described as "WT" or "wild-type" throughout this specification were treated in the same manner, except that pU6-(BbsI)sgRNA_CAG-venus-bpA was used without inserting a protospacer sequence. Transfected BIHi001-A-1 cells were treated with 1 μg / ml doxycycline hicrat (Sigma-Aldrich, No. D9891) to induce Cas9 expression, and cells expressing the Venus reporter gene were subsequently isolated from the sgRNA-containing vector by FACS. Selected cells were seeded at low density, and single-cell-derived colonies were isolated. In the first transfection, colonies were screened for full-length deletion of MAF CDS using sgRNA targeting MAF, PCR, and Sanger sequencing of edited loci (Figure 9). The cells were cultured under iPS cell conditions as described above.

[0296] After a second transfection of MAF KO iPS cells using MAFB-targeted sgRNA, the resulting colonies were subjected to PCR and sequencing. Three MAF / MAFB DKO (MAF-DKO) iPS cell clones were isolated (clones 1, 2, and 3). The genomic target regions and edited MAF and MAFB gene loci are shown in Figure 10.

[0297] (Example 10) (Characterization of MAF / MAFB DKO iPS cells) Three isolated MAF-DKO iPS cell clones were analyzed by karyotyping. Karyotyping was performed using conventional cytogenetic analysis (G-banding). Briefly, cells were blocked with 100 ng / ml colsemid for 2.5 hours at 37°C under 5% CO2, expanded with 0.075 M KCl, and fixed with 3:1 methanol:glacial acetic acid. Metaphase chromosomes were spread on coverslips and stained with Giemsa stain. Twenty metaphase spreads were analyzed per sample. All clones had a normal appearance and karyotype (Figure 11).

[0298] (Example 11) (Induction and differentiation of iPS cells into macrophages) Induction and differentiation of iPS cells into macrophages followed a previously published protocol (Buchrieser, 2017). Single-cell suspensions of wild-type or MAF-DKO iPS cells were seeded at a density of 12,500 cells / well into ultra-low adhesion U-bottom 96-well plates (Nunclon Sphera, Thermo Fisher, No. 174925) and resuspended in EB medium consisting of StemFlex medium (Thermo Fisher, No. A3349401) supplemented with 50 ng / ml BMP-4 (R&D Systems, No. 314-BP), 20 ng / ml SCF (R&D Systems, No. 255-SC), 50 ng / ml VEGF (Peprotech, AF-100-20A), and 10 μM Y-27632 to generate embryoid bodies (EBs). A 96-well plate was centrifuged with 100 g for 3 minutes, and cells from the bottom were collected and incubated at 37°C and 5% CO2 for 4 days. After 1 and 2 days, half of the EB medium was replaced with freshly prepared EB medium.

[0299] (Wild-type and MAF-DKO EB could not be distinguished by size or morphology.) After 4 days, EB cells were manually selected and resuspended in EB differentiation medium consisting of X-VIVO 15 (Lonza, BE02-060F) supplemented with 2 mM GlutaMAX (Thermo Fisher, No. 35050038), 0.055 mM 2-mercaptoethanol (Sigma-Aldrich, No. M6250), 100 ng / ml human M-CSF (Thermo Fisher, No. PHC9504), and 25 ng / ml human IL-3 (R&D Systems, No. 203-IL). The cells were then seeded onto ultra-low adhesion 6-well tissue culture plates (8 EB / well) or 90 mm tissue culture dishes (24 EB / dish; Nunclon Sphera, Thermo Fisher, Nos. 174932 and 174945). Two-thirds of the culture medium was replaced with fresh differentiation medium every 5 days. Monocyte / macrophage production from EB cells began approximately 15 days after EB seeding. Suspended cells were collected, counted, and used for immunophenotyping by flow cytometry or EdU uptake assays.

[0300] For further experiments, cells harvested from the supernatant of EB cultures were supplemented with 10% FBS (PAA-GE Healthcare, A15-101), and then re-plated for final macrophage differentiation in RPMI supplemented with 100 units / ml penicillin, 100 μg / ml streptomycin (Thermo Fisher, No. 15140122), 2 mM GlutaMAX (Thermo Fisher, No. 35050038), 1 mM sodium pyruvate (Thermo Fisher, No. 11360-039), and 50 ng / ml M-CSF (Thermo Fisher, No. PHC9504). The cells were then processed on ultra-low adhesion 12-well tissue culture plates (Nunclon Sphera, Thermo Fisher, No. 174931, No. 174932) and 50 ng / ml Cells were seeded on GM-CSF (Peprotech, No. 300-03) at 37°C and 5% CO2. Cells were counted manually using a Neubauer hemocytometer or automatically using a CASY cell counter (OMNI Life Science).

[0301] MAF and MAFB deletions did not affect myeloid differentiation ability, and, similar to wild-type EB, MAF-DKO EB cells began releasing monocytes / macrophages into EB differentiation medium approximately 15 days after EB seeding. MAF-DKO suspended cells were viable and, after plating in differentiation medium containing 50 ng / ml M-CSF and GM-CSF, both wild-type and MAF-DKO macrophages phagocytosed the beads, produced ROS, and exhibited a mature macrophage phenotype, staining positively for lysosome and cathepsin activity (Figure 12).

[0302] (Example 12) (Characterization of MAF / MAFB DKO cells after final macrophage differentiation) For flow cytometry, re-seeded cells obtained from the EB differentiated culture described in Example 11 and grown for 7 days under final macrophage differentiation conditions were blocked with FcR blocking reagent (Miltenyi, no. 130-059-901) at 4°C for 15 minutes, washed, and stained with the antibodies in Table 2 at 4°C for 30 minutes. The cells were recorded using an LSRFortessa (BD) hemocytometer and analyzed using FlowJo (BD).

[0303] [Table 5] (Table 2: Antibodies used for immunophenotyping)

[0304] No significant differences were observed between wild-type macrophages and MAF-DKO macrophages in terms of the morphology or immunophenotype of CD45, CD11b, CD33, CD14, and CD64, as evaluated by flow cytometry (Figure 13) or tissue staining. CD206 expression was lower in MAF-DKO macrophages. Surprisingly, wild-type iPS cell-derived macrophages lacked detectable HLA-DR on their surface, while MAF-DKO macrophages strongly expressed HLA-DR.

[0305] (MAF-DKO macrophages were tested for MAF and MAFB protein and mRNA expression.) RNA was extracted from wild-type and MAF-DKO cells using the RNeasy Mini Kit (Qiagen, No. 74104), and then cDNA was synthesized using the High Capacity Reverse Transcription Kit (Applied Biosystems, No. 4368814). qPCR was performed using TB Green Premix Ex Taq (Tli RNase plus, Takara, No. RR420L). qPCR products were spread onto a 2% agarose gel. By Western blotting, wild-type and MAF-DKO proteins were extracted by lysing cells with Laemmli buffer. Lysates were clarified and denatured with QIAshredder (Qiagen, No. 79656), loaded onto polyacrylamide gels for electrophoresis (SDS-PAGE), and then semi-dry blotting was performed. The blots were blocked with 5% milk TBST solution, incubated with anti-MAFB antibody (Sigma, no. HPA005653) or anti-MAF antibody (Sigma, no. HPA028289), then incubated with a secondary antibody, anti-rabbit IgG-HRP, and subsequently incubated with ECL® Prime Western Blot detection reagent (GE Healthcare Amersham®, no. RPN2232).

[0306] Importantly, wild-type iPS cell-derived macrophages expressed both MAF and MAFB mRNA and protein, respectively, while MAF-DKO macrophages were negative for both MAF and MAFB expression at the mRNA and protein levels (Figure 14).

[0307] (Example 13) (MAF-DKO macrophages function in a humanized mouse model and treat alveolar proteinosis.) To test the in vivo function of MAF-DKO macrophages, we selected a mouse strain called huPAP (nominal: C;129S4-Rag2tm1.1Flv Csf2 / Il3tm1.1(CSF2,IL3)Flv Il2rgtm1.1Flv / J;JAX # 014595), an animal model of alveolar proteinosis (PAP), a human lung disease. The huPAP strain is an immunodeficient mouse strain designed for human cell transplantation. Due to the lack of mouse GM-CSF expression, alveolar macrophages are absent in their lungs, and therefore the mice exhibit signs of alveolar proteinosis, such as high protein content in the fluid obtained by bronchoalveolar lavage (BAL) and the resulting high turbidity. Instead of mouse GM-CSF, huPAP expresses human GM-CSF (and IL-3), enabling not only the rearrangement of alveolar macrophages by transplanted human cells but also the rescue of the alveolar proteinosis phenotype. Therefore, this model is a useful tool for studying the in vivo function of human macrophages.

[0308] We use the same number (2 x 10 per transplant). 6 ~4×10 6 Wild-type macrophages or MAF-DKO macrophages (collected from EB cultures 15-25 days after resuspending in EB differentiation medium) were intratracheally transplanted into huPAP mice along with 1 μg M-CSF per animal (Figure 15), and both cell engraftment and PAP rescue were checked. Control mice were given the same volume of PBS (cell resuspension buffer).

[0309] HuPAP recipients with either wild-type or MAF-DKO macrophages recovered rapidly after transplantation and showed no abnormal behavior compared to PBS-treated or untreated animals (data not shown). All animals received four transplants of either wild-type or MAF-DKO macrophages, with one week intervals between each transplant. Animals were analyzed four weeks after the last transplant.

[0310] Macroscopic examination revealed no tumors in the lung tissue or other organs, which is consistent with the fact that MAF-DKO macrophages maintain a normal karyotype without clear chromosomal abnormalities (Figure 11).

[0311] We found that MAF-DKO macrophages showed better engraftment to huPAP mice than wild-type macrophages, and improved PAP phenotype rescue, as demonstrated by a slight improvement in bronchoalveolar lavage fluid turbidity, protein concentration, and SP-D levels compared to rescue by wild-type macrophages (Figure 16). Furthermore, wild-type and MAF-DKO macrophages isolated from transplanted mice 4 weeks after the final treatment phagocytosed beads, produced ROS, stained positively for lysosome and cathepsin activity, and were also capable of phagocytosing lipids.

[0312] (Example 14) MAF-DKO macrophages and WT macrophages derived from iPS cells obtained from EB differentiated cultures were collected on day 20 after EB seeding by collecting cells from a suspension, basically as described in Example 11. 500,000 cells per well were re-plated into a 6-well plate (3 ml of medium per well) and maintained for 5 days for terminal differentiation in the presence of M-CSF and GM-CSF (basically as described in Example 11, with a portion of the medium replaced with fresh medium every other day). On day 5, cells were collected and re-plated with 100,000 cells per well in complete medium (containing M-CSF and GM-CSF as described in Example 11) in a NuNCLON 12-well plate, and maintained for a further 2 days (2 ml of medium per well). On day 7 after suspension cell collection, the medium was replaced with fresh complete medium (containing 50 ng / ml of both M-CSF and GM-CSF). Next, cells were harvested two hours after the medium change, and 10,000 DAPI-negative, CD45-positive, and C11b-positive cells were selected and treated with LRT plus cell lysis buffer. RNA was extracted using the RNeasy microkit (Quiagen, catalog number 74034). Subsequently, as described in detail in Picelli et al.'s paper, (2013) Nature Methods 10:1096-1098, differences in gene expression patterns were analyzed by RNA sequencing, with RNA isolated from the 10,000 cells analyzed at a sequencing depth of 38 million fragments per library. Fragment alignment with the human reference (hg38) was performed using GSNAP (v2020-12-16; [Thomas D. et al. (2005) Bioinformatics 21:1859-1875], [Thomas D. et al. (2010) Bioinformatics 26:873-881]) and Ensembl gene annotation 98 ([Howe et al. (2021) NAR vol. 49(1):884-891]), and splice sites were detected.Uniquely aligned fragments were counted using featureCounts (2.0.1; [Liao et al., (2014) "featureCounts: an efficient general purpose program for assigning sequence reads to genomic features"]) and the same Ensembl annotation support. Simultaneously, the sequenced libraries underwent quality control for exon distribution, intron distribution, gene distribution, and rRNA ratio of read sequences within each library using RNA-SeQC (1.1.8; [DeLuca et al., (2012) Bioinformatics 28.11:1530-1532.]). Normalization of the number of raw fragments based on library size and differential expression testing between two conditions (2 hours in DKO and 2 hours in WT) were performed using DESeq2 (v1.24.0; [Love et al., (2014) Genome Biology, 15, The tests were performed using

[550] ), IHW (1.12.0; [Ignatiadis et al. (2016) Nature Methods. doi:10.1038 / nmeth.3885; and Ignatiadis N, Huber W (2017) "Covariate-powered weighted multiple testing with false discovery rate control" arXiv. doi:arXiv:1701.05179]), and R packages (3.6.3; [R Core Team (2020) "R: A language and environment for statistical computing" R Foundation for Statistical Computing, Vienna, Austria, URL https: / / www.R-project.org / ]).Genes with a corrected p-value (padj) < 0.05 and / or log2FoldChange > 0.58 or < -0.58 were considered to be differentially expressed.

[0313] The differences in gene expression patterns between MAF-DKO macrophages and WT macrophages are dramatic, as visualized in the volcanic plot shown in Figure 17. As shown in the table below, the M1 markers shown were strongly upregulated in DKO macrophages compared to WT macrophages, while the M2 markers shown were strongly downregulated despite the presence of M-CSF at a concentration of 50 ng / ml during the final differentiation stage. This indicates that human MAF-DKO macrophages have essentially the same polarization phenotype as mouse MAF-DKO macrophages, and therefore are expected to have essentially the same antitumor properties as mouse MAF-DKO macrophages.

[0314] [Table 6] This application provides the invention in the following embodiments. (Aspect 1) A human macrophage having at least one mutation in both alleles of a gene located on a chromosome, and being resistant to M-CSF-induced M2 polarization, for use in cancer treatment. (Aspect 2) Human macrophages for use according to Embodiment 1, having the normal characteristics of M1 macrophages after exposure to 50 ng / ml M-CSF for 48 hours. (Aspect 3) A typical characteristic of M1 macrophages is that the expression of at least one mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least four times higher than the expression of said mRNA in otherwise identical wild-type macrophages, wherein the at least one mRNA is selected from the list consisting of HLA-DRA, HLA-DRB5, HLA-DPA1, HLA-DQA1, RXFP2, CD74, CD38, CD2, IL18, and IL23A, as described in Embodiment 2 of human macrophages for use. (Aspect 4) A typical characteristic of M1 macrophages is that the expression of at least one mRNA in a human macrophage containing at least one mutation in both alleles of the gene located on the chromosome is at least 10 times lower than the expression of said mRNA in otherwise identical wild-type macrophages, wherein the at least one mRNA is selected from the list consisting of RNASE1, CD28, LYVE1, FCGBP, F13A1, QPCT, CCL7, and RNF128, as described in Embodiment 2 or 3 of the human macrophage for use. (Appendix 5) The macrophages are positive for the surface markers HLA-DRA and / or HLA-DPA1 and / or HLA-DQA1 and / or CD74 and / or CD2, and are human macrophages for use according to any one of embodiments 1 to 4. (Aspect 6) The macrophages are human macrophages for use according to any one of embodiments 1 to 5, wherein the macrophages are negative for the surface markers CD28 and / or LYVE1 and / or STAB1 and / or LILRB5. (Aspect 7) The mutation is a deletion, and the gene is a gene selected from the group consisting of STAT6, IRF4, PPARg, MAFB, MAF, KLF4, C / EPBb, GATA3, JMJD3, SOCS2, SOCS1, TMEM106A, and AKT1, a human macrophage for use according to any one of embodiments 1 to 6. (Pattern 8) A human macrophage for use according to any one of embodiments 1 to 7, wherein the gene located on the chromosome, which contains the biallelelic deletion, is the only gene containing the biallelelic deletion. (Aspect 9) A human macrophage for use according to any one of embodiments 1 to 7, wherein the gene located on a chromosome, including the biallelelic deletion, is the only protein-coding gene including the biallelelic deletion. (Aspect 10) A collection of human macrophages according to any one of embodiments 1 to 9, wherein the number of human macrophages is at least 1,000,000, for use in cancer treatment. (Aspect 11) A human induced pluripotent stem cell or human embryonic stem cell having at least one mutation in both alleles of a gene located on a chromosome, wherein the gene is selected from the group consisting of STAT6, IRF4, PPARg, MAFB, MAF, KLF4, C / EPBb, GATA3, JMJD3, SOCS2, SOCS1, TMEM106A, and AKT1, and the mutation is a deletion. (Aspect 12) Human induced pluripotent stem cells or human embryonic stem cells according to embodiment 11, wherein both alleles of MAFB and both alleles of MAF are defunctionalized by deletion of at least 50 base pairs. (Aspect 13) The human induced pluripotent stem cell or human embryonic stem cell according to embodiment 12, wherein MAF and MAFB are the only transcription factor-coding genes containing biallelic deletions.

Claims

1. A human macrophage for use in cancer treatment, comprising at least one mutation in each of both alleles of the gene MAFB and at least one mutation in each of both alleles of the gene MAF located on a chromosome, thereby defunctionalizing both alleles of MAFB and both alleles of MAF, wherein the mutation is a deletion of at least 50 base pairs including exon DNA, and the macrophage is resistant to M-CSF-induced M2 polarization.

2. The human macrophage according to claim 1, wherein, after exposure to 50 ng / ml M-CSF for 48 hours, the macrophage exhibits at least four times higher expression of at least one mRNA selected from the list consisting of HLA-DRA, HLA-DRB5, HLA-DPA1, HLA-DQA1, RXFP2, CD74, CD2, IL18, and IL23A compared to mRNA expression in the same wild-type macrophage.

3. The human macrophage according to claim 1 or 2, wherein, after exposure to 50 ng / ml M-CSF for 48 hours, the macrophage exhibits at least 10-fold lower expression of at least one mRNA selected from the list consisting of RNASE1, CD28, LYVE1, FCGBP, F13A1, QPCT, CCL7, and RNF128 compared to mRNA expression in the same wild-type macrophage.

4. The macrophage is positive for the surface markers HLA-DRA and / or HLA-DPA1 and / or HLA-DQA1 and / or CD74 and / or CD2, according to any one of claims 1 to 3.

5. The macrophage is a human macrophage according to any one of claims 1 to 4, wherein the macrophage is negative for the surface marker CD28 and / or LYVE1 and / or STAB1 and / or LILRB5.

6. The human macrophage according to any one of claims 1 to 5, wherein the MAFB and MAF are the only genes containing both allelic deletions.

7. The human macrophage according to any one of claims 1 to 6, wherein the MAFB and MAF are the only protein-coding genes containing both allelic deletions.

8. A collection of human macrophages for use in cancer treatment, wherein the macrophages are human macrophages according to any one of claims 1 to 7, and the number of human macrophages is at least 1,000,000.

9. A human induced pluripotent stem cell or human embryonic stem cell comprising at least one mutation in each of both alleles of the gene MAFB and at least one mutation in each of both alleles of the gene MAF located on a chromosome, thereby defunctionalizing both alleles of MAFB and both alleles of MAF, wherein the mutation is a deletion of at least 50 base pairs, the deletion comprises exon DNA, and macrophages derived from the stem cell are resistant to M-CSF-induced M2 polarization.

10. The human induced pluripotent stem cell or human embryonic stem cell according to claim 9, wherein MAF and MAFB are the only transcription factor-coding genes containing biallelic deletions.

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