Allogeneic human macrophages for cell therapy

Abstract

The present invention relates to allogeneic myeloid cells, e.g., macrophages, for use in therapy, e.g., cancer therapy, in human patients, wherein the allogeneic myeloid cells, e.g., allogeneic macrophages, contain at least one MHC-II complex identical to one of the MHC-II complexes of the human patient. This has the advantage of being able to trigger an antigen-specific response of the human patient's CD4+ T cells, e.g., against tumor neoantigens. The present invention also relates to allogeneic myeloid cells, e.g., allogeneic macrophages or allogeneic monocytes, for use in cancer therapy in human patients, wherein the allogeneic macrophages or allogeneic monocytes contain at least one protein complex selected from the group consisting of HLA-DR, HLA-DP, and HLA-DQ, which protein complex is identical to one of the corresponding HLA complexes of the human patient. Preferably, the allogeneic macrophages contain two protein complexes selected from this group, or the allogeneic macrophages contain as many as three complexes selected from this group.

Description

[Background technology]

[0001] BACKGROUND OF THE INVENTION Macrophages are plastic cells, and their phenotype depends on the environmental stimuli they encounter. When macrophages are exposed to bacterial components or inflammatory cytokines such as IFNγ, they enhance their bactericidal and tumoricidal capabilities and produce high levels of proinflammatory cytokines. This activation state is called M1 or classically activated macrophages (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 exhibit an anti-inflammatory phenotype, which typically includes the production of IL-10 and promotes tissue remodeling and repair, as well as angiogenesis (Murray and Wynn, 2011; Mantovani et al., 2002). This activation state is called M2 or alternatively activated macrophages.

[0002] Interestingly, macrophages are the most abundant cells in the tumor microenvironment. Clinical and experimental evidence has shown that tumor-associated macrophages (TAMs) promote tumor progression and malignant transformation (Mantovani et al., 1992; Condeelis and Pollard, 2006; Mantovani et al., 2006), and their presence is associated with poor survival rates (Qian and Pollard, 2010). Indeed, TAMs support cancer development by promoting tumor angiogenesis, tumor cell invasion, and metastasis, and by suppressing antitumor immune responses (Condeelis and Pollard, 2006; Pollard, 2004). Some cancers and their infiltrating cells have been reported to provide cytokine cocktails to the tumor environment, resulting in M2 macrophage polarization (Nevala et al., 2009). Thus, TAMs clearly resemble an M2 phenotype (Balkwill et al., 2005).

[0003] Bart et al. (2021) reviewed approaches to macrophage reprogramming in various disorders and discussed the potential implications and challenges of targeting macrophages in human disease. In particular, they discussed various attempts to reprogram tumor-associated macrophages to a non-M2-like phenotype using small molecules and cytokines, nanovectors, antibodies, nucleic acids, or viral vectors.

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

[0005] The present inventors have reported in patent application PCT / EP2022 / 056819 highly desirable human macrophages capable of resisting tumor-induced repolarization.

[0006] For the most effective medical use of M1-polarized myeloid cells, such as M1 macrophages, it is highly desirable to understand the mechanistic characteristics of the optimal activity of myeloid cells. Summary of the Invention

[0007] (Summary of the Invention) The present invention provides insight into the characteristics of M1-polarized myeloid cells that enable their optimal antitumor activity. In mice, lymphoid cells, particularly T cells and / or NK cells, were observed to be involved in the antitumor activity of myeloid cells, such as macrophages. Furthermore, dramatic upregulation of HLA class II genes was observed in myeloid cells that were resistant to tumor-induced repolarization and possessed antitumor activity. Based on these observations, but without being bound by any theory, it is suggested that optimal antitumor activity involves interactions between myeloid cells, such as macrophages, and T cells, such as CD4+ T cells, mediated by the interaction between the MHC II complex on myeloid cells, such as macrophages, and the T cell receptor on T cells. This has important implications for improving allogeneic anticancer cell therapies using macrophages, monocytes, or dendritic cells, and specifically macrophages.

[0008] Due to MHC restriction, T cells that may be specific for tumor-derived neoantigens should respond better to tumor-derived peptide antigens bound to matching MHC II complexes than to the same tumor-derived peptide antigens bound to mismatched MHC II complexes. Therefore, it is important to consider that allogeneic myeloid cells, e.g., allogeneic macrophages, possess at least one version of the HLA-DP, HLA-DQ, and / or HLA-DR complexes that are also present in the treated individual's CD4 T cells. In other words, there should be at least a partial match at the MHC-II complex level between the patient's lymphoid immune cells and the allogeneic macrophages, such that antigen-specific recognition of tumor-derived neoantigens by, for example, the neoantigen-specific patient's CD4 T cells is significantly improved when the neoantigen is presented on allogeneic antigen-presenting cells, e.g., macrophages.

[0009] Thus, the present invention relates to allogeneic myeloid cells, e.g., macrophages, for use in therapy, e.g., cancer therapy, in human patients, wherein the allogeneic myeloid cells, e.g., allogeneic macrophages, contain at least one MHC-II complex identical to any one of the MHC-II complexes of the human patient. This has the advantage of being able to trigger an antigen-specific response of the human patient's CD4+ T cells, e.g., against tumor neoantigens. The present invention also relates to allogeneic myeloid cells, e.g., allogeneic macrophages or allogeneic monocytes, for use in cancer therapy in human patients, wherein the allogeneic macrophages or allogeneic monocytes contain at least one protein complex selected from the group consisting of HLA-DR, HLA-DP, and HLA-DQ, which protein complex is identical to any one of the corresponding HLA complexes of the human patient. Preferably, the allogeneic macrophages contain two protein complexes selected from this group, or the allogeneic macrophages contain as many as three complexes selected from this group.

[0010] The partial, and preferably complete, MHC II match between allogeneic myeloid cells, e.g., macrophages, and the patient to be treated can also be explained at the genetic level. Thus, the present invention also relates to allogeneic myeloid cells, e.g., macrophages, for use in treating cancer in a patient, wherein the allogeneic myeloid cells and the patient to be treated share at least one pair of alleles selected from the group consisting of the following pairs: HLA-DRA / HLA-DRB1, HLA-DRA / HLA-DRB3, HLA-DRA / HLA-DRB4, HLA-DRA / HLA-DRB5, HLA-DPA1 / HLA-DPB1, and HLA-DQA1 / HLA-DQB1, and preferably the allogeneic myeloid cells and the patient to be treated share two, three, four, five, or even all six pairs of alleles. It should be noted that for heterozygous patients, this means overlap of one allele pair with one allele pair on the allogeneic myeloid cells, but it is not necessary that both allele pairs of the patient match both allele pairs on the allogeneic myeloid cells (although matching of both such allele pairs is of course included). [Brief explanation of the drawings]

[0011] (drawing) [Figure 1] Figure 1. Adoptive cell transfer of murine Maf-DKO macrophages inhibits in vivo tumor growth. (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 mice; WT: Mice treated with wild-type-derived bone marrow macrophages; BM-DKO: Mice treated with bone marrow-derived, MafB- and c-Maf-double-deficient macrophages. [Figure 2] Figure 2. Adoptive cell transfer of murine Maf-DKO macrophages reverses established in vivo tumor growth. (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-derived bone marrow macrophages; BM-DKO: Mice treated with MafB and c-Maf double-deficient bone marrow-derived macrophages. [Figure 3] Figure 3: Cellular content in peritoneal fluid. (a) Gating strategy for CD8 T cells and NK cells derived from peritoneal cells of tumor-bearing mice. (b) Histogram showing CD8α expression by peritoneal cells in the peritoneal cavities of untreated (dotted line, small circle), WT-BMDM (solid line, triangle), and Maf-DKO (hatched line, square) mice 27 days after tumor initiation. (c) Gating strategy for peritoneal macrophages. (d) MHC class II expression in peritoneal macrophages. (e) Mean fluorescence intensity of MHC II expression by peritoneal macrophages. (**: p ≤ 0.007). UN: untreated mice; WT: mice treated with wild-type-derived bone marrow macrophages; Maf-DKO: mice treated with MafB and c-Maf double-deficient macrophages. [Figure 4]Figure 4. Macrophages sorted from Maf-DKO-treated mice resemble M1-activated macrophages in vivo. Total mRNA was isolated from CD11b+ peritoneal cells of sorted 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 the mean of n=5. UN: untreated mice; WT: mice treated with wild-type-derived bone marrow macrophages; Maf-DKO: mice treated with MafB and c-Maf double-deficient (double double knockout) macrophages. [Figure 5] Figure 5: Murine Maf-DKO macrophages are more sensitive to M1 stimulation and more resistant to M2 stimulation than WT macrophages. Total mRNA was isolated for real-time PCR analysis of (a) IL-6, (b) IL-10, (c) C2TA, (d) arginase, and (e) NOS2 from cultured macrophages stimulated with compounds A, B, C, and D. Maf-DKO macrophages are shown as polka-dot bars, and WT macrophages are shown as striped bars. [Figure 6] Figure 6: Murine Maf-DKO macrophages are not re-educated by tumors in vitro. Cell supernatants were collected and analyzed for LPS-stimulated (a) IL-6 and (b) TNFα production in the absence (black bars) or presence (checkered bars) of ID8 tumor cell supernatant. SN is supernatant. [Figure 7] Figure 7. Murine Maf-DKO macrophages reverse established melanoma growth in vivo. (Experimental metastasis of B16 melanoma cells in C57B16 mice) (a) Representative scheme of the experimental procedure. Macrophages were injected 7 days after tumor initiation. (b) Lungs were excised and photographed. (c) The number of visible metastatic colonies on the lung surface was counted using a magnifying glass, and the median value was used for analysis.) Results were obtained from five mice per group. (*: p ≤ 0.04; **: p ≤ 0.007; ***: p ≤ 0.0005). UN: Untreated mice; WT: Mice treated with wild-type-derived bone marrow macrophages; Maf-DKO: Mice treated with MAFB and c-Maf double-deficient macrophages. [Figure 8] Figure 8: Mouse MAF DKO cells recruit other leukocytes in protecting against B16 melanoma. (Experimental metastasis of B16 melanoma cells in Rag2γc knockout mice.) (a, b) Representative scheme of the experimental procedure. In (a), mice were treated with macrophages immediately after tumor initiation (see results in 8c and 8e). In (b), mice were injected with macrophages after tumor establishment (see results in 8d and 8f). (c, d) Livers were excised and photographed, and bioluminescence was also recorded. (Photographs are of representative livers; white light is not the same as bioluminescence.) (e, f) The number of metastatic colonies visible on the surface of mice treated with (a) and (b) was counted using a magnifying glass, and the median value was used for analysis. Results were obtained from five mice per group. (*: p ≤ 0.04). B16: untreated mice; WT: mice treated with wild-type-derived bone marrow macrophages; Maf-DKO: mice treated with DKO macrophages. [Figure 9] Figure 9: Workflow for CRISPR / Cas9-mediated deletion of MAF and MAFB in doxycycline-inducible Cas9-expressing human iPSC lines. Step 1: Lipofectamine transfection with MAF-targeting sgRNA expression vector. Step 2: Cas9 induction by doxycycline (Dox) treatment. Step 3: Isolation of reporter-positive sgRNA-expressing cells by cell sorting and obtaining single-cell colonies. Step 4: Selection of MAF KO clones. Step 5: Repetition of steps 1-4 with MAFB-targeting sgRNA using the selected MAF KO iPSC clones and resulting MAFB knockout. [Figure 10]Figure 10: Gene structures of the human MAF and MAFB genes, showing the location of CRISPR / Cas9 target sites in the 5' and 3' UTRs and the resulting indels. The human MAF gene has short and long isoforms generated by alternative splicing; the significance of the long isoform is unknown. Exon 1 of the MAF gene was knocked out. Human MAFB is a single-exon gene. The first line of each sequence block indicates the wild-type locus, and subsequent lines indicate indels generated by CRISPR / Cas9 editing. Only one clone was isolated for MAF (indels in both alleles are shown), and three isolated clones for MAFB are shown (C1, C2, C3: clones 1, 2, and 3). In the sequence blocks, the start and stop codons are in bold, and the genomic target sequence is underlined. The protospacer position is indicated by a solid black line near the start or stop codon. [Figure 11] Figure 11: Volcano plot of differentially expressed genes in human wt and DKO macrophages. iPS cell-derived macrophages were cultured with M-CSF and GM-CSF for 7 days and then shifted to M-CSF alone for 2 hours. Differentially expressed genes identified by deep sequencing are shown. The X-axis indicates the magnitude of change, and the Y-axis indicates statistical significance.

[0012] (References) [Table 1] TIFF2025533449000002.tif216170TIFF2025533449000003.tif117170 DETAILED DESCRIPTION OF THE INVENTION

[0013] (Detailed Description of the Invention) (definition) As used herein, a "chromosome" is one of the 46 normal human chromosomes. A "chromosomally located gene" is a gene that is not extrachromosomal.

[0014] The term "gene" refers to a DNA sequence that encodes RNA or a specific sequence of amino acids that comprise all or part of one or more proteins or enzymes. This term may or may not include 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 capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. Some genes that are not structural genes may be transcribed from DNA into RNA but are 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 genomic sequence that encodes a protein, i.e., a sequence including regulatory elements, promoters, introns, and exon sequences.

[0015] In the context of the present invention, the terms "mutant" and "mutation" refer to a detectable change in genetic material, i.e., genomic DNA. Mutations include deletions, insertions, or substitutions of one or more nucleotides. Mutations can occur within the coding region (i.e., exons) of a gene, within introns, or within regulatory regions of a gene (e.g., enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, promoters). Mutations are generally identified in a subject by comparing the sequence of a nucleic acid or polypeptide expressed by the subject with the corresponding nucleic acid or polypeptide expressed in a control population. If the mutation is within a gene coding sequence, the mutation may be a "missense" mutation, which substitutes one amino acid for another in the gene product, or a "nonsense" mutation, which substitutes an amino acid codon for a stop codon. Mutations may also occur at splice sites where signals for exon-intron splicing are generated or disrupted, thereby resulting in a gene product of altered structure. A "mutation" in the context of the present invention is not silent, i.e., it results in at least a change in its nucleotide sequence (if the gene product is a functional RNA) or amino acid sequence (if the gene product is a protein) that renders the gene product non-functional or reduces the expression of the gene product at the RNA level by at least 80%. A preferred mutation is, for example, a deletion of the entire gene, thus abolishing gene expression.

[0016] As used herein, gene expression is "inhibited" when expression of the gene at the RNA level is reduced by at least 80% compared to the corresponding wild-type expression of the gene, as measured by quantitative RT-PCR. Preferably, expression of the gene at the RNA level is reduced by at least 90%, e.g., at least 95%.

[0017] As used herein, gene expression is "absent" when its expression is undetectable at the RNA level by q-PCR. When "expressed" mRNA is present at detectable levels in qPCR, it should (a) produce a sigmoidal fluorescence curve, (b) reach a plateau within at least 38 PCR cycles, preferably at least 36 PCR cycles, and (c) generate PCR products of the expected length, i.e., PCR products of lengths corresponding to those derived from mature mRNA and not from genomic DNA or unprocessed RNA intermediates. Preferably, mRNA expression can be confirmed by these three criteria in triplicate qPCR experiments.

[0018] "Mutagenesis," as used herein, is the experimental process by which the genetic information of an organism is intentionally altered to produce mutations. Preferred methods of mutagenesis herein are site-specific endonuclease-based methods.

[0019] As used herein, a "site-specific endonuclease" is an enzyme that cleaves phosphodiester bonds in a polynucleotide chain only at a very specific nucleotide sequence in the middle (end) of a double-stranded DNA molecule, and this sequence preferably occurs only once in the entire human genome, so as to enable specific genetic engineering of human target cells. Examples of site-specific endonucleases commonly used for genetic engineering in human target cells are zinc finger nucleases, TALENs, and CRISPR / Cas9 systems.

[0020] The term "expression," as used herein, can refer to gene expression of a polypeptide or protein, or gene expression of a polynucleotide, such as, for example, an miRNA or lncRNA, depending on the context. Expression of a polynucleotide can be determined, for example, by measuring the production of RNA transcript levels, using methods well known to those of skill in the art. Expression of a protein or polypeptide can be determined, for example, by immunoassay using an antibody or antibodies that specifically bind to the polypeptide, using methods well known to those of skill in the art.

[0021] "Expression of mRNA" as used herein relates to the transcription level of gene expression.

[0022] In the present invention, known methods can be used to detect gene expression, etc. Examples of methods for quantitatively detecting mRNA levels in a 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 new-generation sequencing. Representative 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 "Extinct Expression."

[0023] Those skilled in the art can detect mRNA or cDNA using the above-mentioned detection methods by preparing mRNA or cDNA and selecting a known method appropriate for the sample, taking into consideration the type and condition of the sample, etc. When comparing the gene expression level in the human macrophages of the present invention with the expression level of the same gene in wild-type macrophages, the comparison is made under otherwise identical conditions, i.e., both types of macrophages are cultured and treated in the same manner, to enable a scientifically meaningful comparison at the mRNA level.

[0024] As used herein, "allele" refers to one of two copies of the same human gene. The two copies of a gene, one on each of two homologous chromosomes, may be identical or may differ slightly in their individual sequences. Thus, the term "allele" is used somewhat differently herein because it also includes identical versions of the same human gene at the same corresponding locations on two homologous chromosomes. In a diploid cell, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

[0025] As used herein, an "allele" or "gene" is considered non-functional if, following a procedure that renders the allele or gene non-functional, no further expression of the protein encoded by that gene or allele is detected. That is, no new protein is expressed from the non-functional allele or gene. Eventually, the protein encoded by the non-functional allele or gene becomes undetectable in a cell population consisting of cells that only have the non-functional allele. The time it takes for the protein encoded by the gene or allele to become undetectable depends on the turnover kinetics of the protein and mRNA of that gene.

[0026] The term "deletion" as used herein means that a portion of a DNA sequence is missing compared to a wild-type reference sequence.

[0027] As used herein, an "exon" is any portion of a gene that encodes a portion of 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 in the RNA transcript. During RNA splicing, introns are removed and exons are covalently joined together to form the mature messenger RNA.

[0028] The term "chromosomal rearrangement" as used herein refers to a chromosomal abnormality involving a structural change in a native chromosome. Typically, chromosomal rearrangement occurs when the DNA double helix is ​​broken at two different locations, and then the broken ends rejoin to create a new chromosomal genetic makeup with a different gene order than the chromosome or chromosomes before the break. Such changes can include several different types of events, such as large deletions of more than 10,000 base pairs, gene duplications, gene inversions, and translocations within a chromosome or between two chromosomes.

[0029] As used herein, the term "karyotype analysis" refers to the analysis of chromosomes within cells from a sample. A skilled artisan will stain the chromosomes and then use a microscope to examine the size, shape, and number of chromosomes within the cells of a cell sample. Photographs of the stained sample are typically taken to show the arrangement of the chromosomes. A karyotype describes the number of chromosomes in an organism and how those chromosomes appear under a light microscope, including, among other things, the length of the chromosomes, the location of centromeres within those chromosomes, the banding pattern of the stained chromosomes, differences between sex chromosomes, and other physical characteristics.

[0030] The term "guide RNA" as used herein relates to "guide RNA" used in the context of the CRISPR / Cas9 DNA editing system. Guide RNA confers specificity to the CRISPR-Cas9 system for the target sequence. 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 through base pairing, where Cas9 acts as an endonuclease and cleaves the target DNA strand. Examples of guide RNAs are (a) synthetic trans-activating CRISPR RNA (tracrRNA) and synthetic CRISPR RNA (crRNA), where the crRNA is designed to specify a gene target site of interest, and (b) single guide RNA (sgRNA), which incorporates both crRNA and tracrRNA in a single construct.

[0031] The term "MAF" refers to the human MAF transcription factor. MAF and other Maf family members form homodimers and heterodimers with each other and with Fos and Jun, 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- or 14-bp elements containing a core TRE (T-MARE) or CRE (C-MARE) palindrome, respectively. However, MAF can also bind to DNA sequences distant from these consensus sites, including composite AP-1 / MARE sites and MARE half-sites with AT-rich 5' extensions (Yoshida et al., 2005). MAF has been shown to stimulate transcription from some promoters and repress transcription from others. MAF has also been shown to induce T helper 2 (Th2) cell differentiation (Ho et al., 1996) due to its ability to activate tissue-specific transcription of interleukin 4 (IL-4) (Kim et al., 1999). Furthermore, overexpression of MAF in myeloid cell lines induces macrophage differentiation (Hegde et al., 1999). The human MAF gene is located on chromosome 16 at 16q23.2 and is fully described in the NCBI Gene database under gene ID 4094. Sequence and location information are from annotation release 109.20201120 (released as of December 9, 2020), the Genome Reference Consortium Human Build 38 Patch Release 13 reference sequence assembly GCF_000001405.39. This reference identifies the MAF gene on the complementary strand of 79,593,838 to 79,600,737.

[0032] 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, neurons, and hematopoietic cells, particularly macrophages) and encodes a protein containing a typical bZip motif in its carboxy-terminal region. Within the bZip domain, MAFB shares extensive homology not only with MAF but also with other Maf-related proteins. MAFB can form homodimers through its leucine repeat structure and specifically bind to Maf recognition element (MARE) palindromes, composite AP-1 / MARE sites, or MARE half-sites with AT-rich 5' extensions (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 (for "Kreisler Maf leucine zipper 1") because X-ray-induced chromosomal microinversions in kreisler mutant mice result in a tissue-specific loss of MAFB expression in the developing hindbrain, causing the kreisler phenotype (Cordes et al., 1994) (Eichmann et al., 1997). In the hematopoietic system, MAFB is selectively expressed in the myeloid lineage and is sequentially upregulated during myeloid cell differentiation from multipotent precursors to macrophages. Indeed, this induction reflects the essential role of MAFB in monocyte and macrophage differentiation. Thus, overexpression of MAFB in transformed chicken myeloblasts (Kelly et al., 2000; Bakri et al., 2005) and in human hematopoietic precursors (Gemelli et al., 2006) inhibits precursor proliferation (Tillmanns et al., 2007) and promotes macrophage formation (Kelly et al., 2000; Bakri et al., 2005; Gemelli et al., 2006), whereas a dominant-negative version of MAFB inhibits this process (Kelly et al., 2000).Combined deletion of MafB and Maf in mouse monocytes and macrophages allows for prolonged proliferation (Aziz 2009; Soucie 2016), which together demonstrate that MAF induction is a specific and critical determinant of the monocytic program in hematopoietic cells and is crucial for cell cycle arrest in differentiated monocytes and macrophages.

[0033] The human MAFB gene is located on chromosome 20q12 and is described in detail in the NCBI Gene database as gene ID 9935. The sequence and location information are from annotation release 109.20201120 (released as of December 9, 2020), the Genome Reference Consortium Human Build 38 Patch Release 13 reference sequence assembly GCF_000001405.39. This reference locates the MAFB gene on the complementary strand of 40685848–40689236.

[0034] IL-4, as used herein, 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 and has a molecular weight of approximately 20 kDa. It is a ligand for the interleukin 4 receptor. The interleukin 4 receptor also binds to IL13, which may account for the many overlapping functions of this cytokine and IL13. IL4 is a cytokine important in tissue repair and is thought to counteract the effects of proinflammatory type 1 cytokines.

[0035] The human IL-4 gene is located on chromosome 5 at 5q31.1 and is described in detail in the NCBI gene database as gene ID 3565. The sequence and location information are from annotation release 109.20210226. For cell culture experiments, recombinant human IL-4 (catalog no. 200-04) from Preprotech, a 15.1 kDa globular protein containing 130 AA, was used. Its specific activity in the human TF-1 cell proliferation assay was at least 5 × 106 U / mg.

[0036] IL-4 signaling occurs through interaction with its receptor. Interaction of IL-4 with its receptor results in receptor dimerization and activation. The activated receptor activates JAK1 and JAK3, which associate with the receptor subunit. Activated JAKs phosphorylate tyrosine residues, and the cytoplasmic tail of the receptor then serves as a binding site for many adaptors or signaling molecules, including STAT6. Activated STAT6 dimerizes, translocates into the nucleus, and transcriptionally activates genes that respond to IL-4.

[0037] IL-13 as used herein refers to human interleukin 13. The protein encoded by IL-13 is a cytokine produced by various immune cells, for example, primarily by activated Th2 cells, but also by CD4 cells, NKT cells, mast cells, basophils, and eosinophils. IL-13 is a central regulator of IgE synthesis, goblet cell hyperplasia, mucus hypersecretion, airway hyperresponsiveness, fibrosis, and chitinase upregulation.

[0038] The human IL-13 gene is located at 5q31.1 on chromosome 5 and is described in detail in the NCBI gene database as gene ID 3596. The sequence and location information are from annotation release 109.20210226.

[0039] As used herein, ARG-1 refers to human arginase 1. Arginase catalyzes the hydrolysis of arginine into ornithine and urea. The type I isoform encoded by this gene is a cytosolic enzyme, and under normal physiological conditions, it is expressed primarily in the liver as a component of the urea cycle. However, ARG1 is also an immunosuppressive signal found primarily in tumor-associated macrophages. Macrophages polarized to an immunosuppressive phenotype express ARG1.

[0040] The human ARG-1 gene is located on chromosome 6 at 6q23.2 and is described in detail in the NCBI gene database as gene ID 383. The sequence and location information are from annotation release 109.20210226.

[0041] As used herein, IL-10 refers to human interleukin-10. IL-10 is a cytokine produced primarily by monocytes and macrophages, and to a lesser extent by lymphocytes. This cytokine has pleiotropic effects in immune regulation and inflammation. It 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 toward an immunosuppressive phenotype express IL-10. IL-10 expression has been reported to be a predictor of advanced tumor stage and to be associated with poor overall survival.

[0042] The human IL-10 gene is located at 1q32.1 on chromosome 1 and is described in detail in the NCBI gene database as gene ID 3586. The sequence and location information are from annotation release 109.20210226.

[0043] IL-6 as used herein refers to human interleukin 6. The protein encoded by IL-6 is a cytokine that functions in inflammation and B cell maturation. Furthermore, this encoded protein has been shown to be an endogenous pyrogen that can induce fever in people with autoimmune or infectious diseases. This protein is primarily produced at sites of acute and chronic inflammation, where it is secreted into the serum and induces transcriptional inflammatory responses via interleukin 6 receptor α.

[0044] 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. The sequence and location information are from annotation release 109.20210226.

[0045] CXCL10, as used herein, refers to C-X-C motif chemokine ligand 10. The protein encoded by CXCL10 is a chemokine of the C-X-C subfamily and a ligand for the receptor CXCR3. Binding of this protein to CXCR3 produces pleiotropic effects, including stimulation of monocyte, natural killer, and T-cell migration, and modulation of adhesion molecule expression.

[0046] The human CXCL10 gene is located on chromosome 4 at 4q21.1 and is described in detail in the NCBI gene database as gene ID 3627. The sequence and location information are from annotation release 109.20210226.

[0047] As used herein, C2TA (CIITA) refers to class II major histocompatibility complex transactivator. C2TA is a protein with an acidic transcriptional activation domain, four leucine-rich repeats, and a 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.

[0048] 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. The sequence and location information are from annotation release 109.20210226.

[0049] TNF as used herein refers to tumor necrosis factor. TNF is a multifunctional proinflammatory cytokine that belongs 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 the regulation of a wide range of biological processes, including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation.

[0050] The human TNF gene is located on chromosome 6 at 6p21.33 and is described in detail in the NCBI gene database as gene ID 7124. The sequence and location information are from annotation release 109.20210226.

[0051] M-CSF, as used herein, is also referred to as CSF1 and relates to human colony-stimulating factor 1. M-CSF is a cytokine that regulates the generation, differentiation, proliferation, and function of macrophages. Various active isoforms of this protein have been identified as membrane-bound or extracellular disulfide-linked homodimers and are thought to be generated by proteolytic cleavage of a membrane-bound precursor.

[0052] The human M-CSF gene is located on chromosome 1p13.3 and is described in detail in the NCBI gene database as gene ID 1435. The sequence and location information are from annotation release 109.20210226. Recombinant human M-CSF (38 kDa homodimer produced in HEK293 cells, Gibco, catalog number PHC9501) was used in cell culture experiments. The ED50, determined by dose-dependent proliferation of M-NSF60 cells, was up to 5 ng / ml.

[0053] MHC II, as used herein, refers to class II of major histocompatibility complex (MHC) molecules, which are normally found only on professional antigen-presenting cells, such as mononuclear phagocytes, including dendritic cells and macrophages, and B cells. MHC II presents peptides to T cells, but it does so from extracellular proteins by endocytosis, not from cytosolic proteins as in the case of MHC class I.

[0054] In humans, the MHC class II protein complex is encoded by the human leukocyte antigen gene complex (HLA). The HLAs corresponding to MHC class II are HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.

[0055] HLA-A, or "human leukocyte antigen 1," refers to proteins belonging to the HLA class I heavy chain paralogs. These class I molecules are heterodimers consisting of a heavy chain and a light chain (β-2-microglobulin). The heavy chain is anchored within the membrane. Class I molecules play a central role in the immune system, presenting cytosolic peptides translocated into the endoplasmic reticulum lumen for recognition by cytotoxic T cells. Class I molecules are expressed in almost all cells. Their heavy chains are approximately 45 kDa, and the gene contains eight exons. Exon 1 encodes the leader peptide; exons 2 and 3 encode the α1 and α2 domains, both of which bind peptides; exon 4 encodes the α3 domain; exon 5 encodes the transmembrane region; and exons 6 and 7 encode the cytoplasmic tail. Polymorphisms in exons 2 and 3 determine the peptide-binding specificity of each class I molecule. Typing analysis of these polymorphisms is 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 fully described in the NCBI Gene database as gene ID 3105. Sequence and location information is from annotation release 109.20201120 (released as of December 9, 2020), the Genome Reference Consortium Human Build 38 Patch Release 13 reference sequence assembly GCF_000001405.39. This reference locates the HLA-A gene on the coding strand at positions 29942532–29945870.

[0056] HLA-B refers to proteins belonging to the HLA class I heavy chain paralogs. Several hundred 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 under gene ID 3106. Sequence and location information is from annotation release 109.20201120 (released as of December 9, 2020), the Genome Reference Consortium Human Build 38 Patch Release 13 reference sequence assembly GCF_000001405.39.

[0057] 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 anchored in the membrane. HLA-DR plays a central role in the immune system by presenting peptides derived from extracellular proteins. Class II molecules are expressed within antigen-presenting cells. Because the α chain is essentially constant, most of the variability in HLA-DR composition within individuals comes from the ability of the α chain to pair with β chains 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 polymorphisms that determine peptide binding specificity. In particular, hundreds of DRB1 alleles have been reported, and some alleles increase in frequency in association with specific diseases or pathologies. These alleles account for variability in HLA-DRB1 composition within populations. While some individuals may be homozygous for HLA-DRB1 (i.e., both the father and mother have identical HLA-DRB1 alleles), the majority of individuals are generally heterozygous for HLA-DRB1. The human HLA-DRB1 gene is located on chromosome 6 at 6p21.32 and is fully described in the NCBI Gene database under gene ID 31223. Sequence and location information is from annotation release 109.20201120 (released as of December 9, 2020), the Genome Reference Consortium Human Build 38 Patch Release 13 reference sequence assembly GCF_000001405.39.

[0058] 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 anchored in the membrane. HLA-DQ plays a central role in the immune system by presenting peptides derived from extracellular proteins. Class II molecules are expressed within antigen-presenting cells. Because both the α and β chains vary widely between individuals, multiple 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 (the DQA1 gene product) and β (the DQB1 gene product). These receptors can be generated from two different DQ haplotype α + β sets (one set derived from the maternal chromosome and one from the paternal chromosome). Individuals with the -AB- haplotype from one parent and the -ab- haplotype from the other parent produce two α isoforms (A and a) and two β isoforms (B and b). This results in four slightly different receptor heterodimers (or, more simply, DQ isoforms). Two isoforms are cis-haplotype pairings (AB and ab) and two isoforms are trans-haplotype pairings (Ab and aB). Such individuals are double heterozygous for these genes, the most common situation for DQ. While individuals with the -AB- and -Ab- haplotypes can only produce two DQs (AB and Ab), individuals with the AB- and -AB- haplotypes can only produce the DQ isoform AB and are called double homozygotes. The human HLA-DQA1 gene is located on chromosome 6, 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.

[0059] 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 anchored in the membrane. HLA-DP also plays a central role in the immune system by presenting peptides derived from extracellular proteins. The case of HLA-DP is similar to that described for HLA-DQ. Because both the α and β chains vary between individuals, several alleles have been reported for both HLA-DPA1 and HLA-DPB1. As described for HLA-DQ, HLA-DP can be generated from two different HLA-DP haplotype α + β sets (one set derived from the maternal chromosome and one from the paternal chromosome). Individuals can be double heterozygotes for HLA-DPA1 and HLA-DPB1, which is the most common scenario for DP, or single homozygotes, or double homozygotes for HLA-DP. The human HLA-DPA1 gene is located on chromosome 6 at 6p21.32 and is described in detail in the NCBI gene database as gene ID 3113. The human HLA-DPB1 gene is also located on chromosome 6 at 6p21.32 and is described in detail in the NCBI gene database as gene ID 3115.

[0060] CD2 as used herein, depending on the context, relates to the CD2 gene or to CD2 as a surface marker, which is the extracellular portion of the CD2 protein (also known as LFA-2). The CD2 gene is described in detail in the NCBI gene database under gene ID 914.

[0061] CD28 as used herein, depending on the context, relates to the CD28 gene or to the surface marker that is the extracellular portion of the CD28 protein. The CD28 gene is described in detail in the NCBI gene database under gene ID 940.

[0062] CD38 as used herein, depending on the context, relates to the CD38 gene or to the surface marker that is the extracellular portion of the CD38 protein (also known as ADPRC1). The CD38 gene is described in detail in the NCBI gene database under gene ID 952.

[0063] CD64 as used herein, depending on the context, relates to the CD64 gene or to the surface marker that is the extracellular part of FCGR1A (Fcγ receptor Ia), which is described in detail in the NCBI gene database under gene ID 2209.

[0064] CD74 as used herein, depending on the context, relates to the CD74 gene or to the surface marker that is the extracellular portion of the CD74 protein (also known as HLADG). The CD74 gene is described in detail in the NCBI gene database under gene ID 972.

[0065] C5AR1 as used herein, depending on the context, relates to the C5AR1 gene or to the surface marker that is the extracellular portion of the C5AR1 protein (also known as CD88). The C5AR1 gene is described in detail in the NCBI gene database under gene ID 728.

[0066] CD70 as used herein, depending on the context, relates to the CD70 gene or to the surface marker that is the extracellular portion of the CD70 protein. CD70 is described in detail in the NCBI gene database under gene ID 970.

[0067] CD206 as used herein, depending on the context, relates to the CD206 gene or to a surface marker that is the extracellular portion of the mannose receptor, type C1. CD206 is described in detail in the NCBI gene database as gene ID 4360.

[0068] CD163 as used herein relates to the CD163 gene or to the surface marker that is the extracellular part of the CD163 protein, depending on the context. CD163 is described in detail in the NCBI gene database under gene ID 9332.

[0069] IL15 as used herein, depending on the context, relates to the IL15 gene or to the secreted form of the IL15 protein, which is the activating cytokine interleukin 15. IL15 is described in detail in the NCBI gene database under gene ID 3600.

[0070] IL18 as used herein, depending on the context, relates to the IL18 gene or to the secreted form of the IL18 protein, which is the activating cytokine interleukin 18. IL18 is described in detail in the NCBI gene database under gene ID 3606.

[0071] IL23A as used herein, depending on the context, relates to the IL23A gene or to IL23A as a subunit of the heterodimeric cytokine IL23. In this context, IL23A is detectable as a secreted form of the activating cytokine interleukin 23. IL23A is described in detail in the NCBI gene database under gene ID 51561.

[0072] RXFP2 as used herein, depending on the context, relates to the RXFP2 gene or to the surface marker that is the extracellular portion of the RXFP2 protein (relaxin family peptide receptor 2). RXFP2 is described in detail in the NCBI gene database under gene ID 122042.

[0073] LYVE1 as used herein, depending on the context, relates to the LYVE1 gene or to the surface marker that is the extracellular portion of the LYVE1 protein (lymphatic hyaluronan receptor 1). LYVE1 is described in detail in the NCBI gene database under gene ID 10894.

[0074] STAB1 as used herein, depending on the context, relates to the STAB1 gene or to the surface marker that is the extracellular portion of the STAB1 protein, stabilin 1 (also called SCARH2 because it can act as a scavenger receptor). STAB1 is described in detail in the NCBI gene database under gene ID 23166.

[0075] LILRB5 as used herein, depending on the context, relates to the LILRB5 gene or to the surface marker that is the extracellular portion of the LILRB5 protein (leukocyte immunoglobulin-like receptor B5). LILRB5 is described in detail in the NCBI gene database under gene ID 10990.

[0076] RNASE1 as used herein relates to the RNASE1 gene or to the secreted form of the RNASE1 protein (ribonuclease A family member 1), depending on the context. RNASE1 is described in detail in the NCBI gene database under gene ID 6035.

[0077] F13A1 as used herein relates to the F13A1 gene or to the secreted form of the F13A1 protein (A subunit of coagulation factor XIII), depending on the context. F13A1 is described in detail in the NCBI gene database under gene ID 2162.

[0078] QPCT as used herein relates to the QPCT gene or to the secreted form of the QPCT protein (glutaminyl cyclase), depending on the context. QPCT is described in detail in the NCBI gene database under gene ID 25797.

[0079] CCL7 as used herein relates to the CCL7 gene or to the secreted form of the CCL7 protein (chemokine CCL7), depending on the context. CCL7 is described in detail in the NCBI gene database under gene ID 6354.

[0080] RNF128 as used herein relates to the RNF128 gene or the RNF128 protein (also known as E3 ubiquitin ligase, GRAIL), depending on the context. RNF128 is described in detail in the NCBI gene database under gene ID 79589.

[0081] STAT6 is described in detail in the NCBI gene database as gene ID 6778.

[0082] IRF4 is described in detail in the NCBI gene database as gene ID 3662.

[0083] PPARγ is described in detail in the NCBI gene database as gene ID 5468.

[0084] KLF4 is described in detail in the NCBI gene database as gene ID 9314.

[0085] C / EPBβ (CEBPB) is described in detail in the NCBI gene database as gene ID 1051.

[0086] GATA3 is described in detail in the NCBI gene database as gene ID 2625.

[0087] JMJD3 (KDM6B) is described in detail in the NCBI gene database as gene ID 23135.

[0088] SOCS2 is described in detail in the NCBI gene database under gene ID 8835.

[0089] SOCS1 is described in detail in the NCBI gene database under gene ID 8651.

[0090] AKT1 is described in detail in the NCBI gene database as gene ID 207.

[0091] FCGBP is described in detail in the NCBI gene database under gene ID 8857.

[0092] References to genes in the context of experiments conducted in mice refer to the mouse gene corresponding to the indicated human gene name (e.g., references to "IL-6" in the context of the mouse experiments described in Examples 1-8 refer to mouse IL-6).

[0093] As used herein, a negative regulator is a gene product, particularly a polypeptide, that interferes with the binding of RNA polymerase to a promoter region, inhibits the activity of an enhancer, or inhibits the activity of an activating transcription factor, resulting in decreased transcription of a target gene, e.g., C2TA.

[0094] CD8+ T cells (also called cytotoxic T lymphocytes, or CTLs) develop in the thymus and express the T cell receptor, CD8, which is a dimeric coreceptor typically consisting of one CD8α chain and one CD8β chain. CD8+ T cells recognize peptides presented by MHC class I molecules, which are found on all nucleated cells. CD8+ T cells are crucial for immune defense against pathogens, including intracellular viruses and bacteria, as well as tumor surveillance.

[0095] NK cells, also known as natural killer cells or large granular lymphocytes (LGLs), are a type of cytotoxic lymphocyte important to the innate immune system. NK cells can be identified by the presence of CD56 and the absence of CD3 (CD56+, CD3-). NK cells are innate immune cells with functions similar to those of cytotoxic T cells in the adaptive immune response. NK cells act on virus-infected cells approximately three days after infection to provide a rapid response and respond to tumor formation. NK cells have the ability to recognize and kill stressed cells in the absence of antibodies and MHC. This role is important because harmful cells that lack the MHC I marker are not detected and destroyed by other immune cells, such as T lymphocytes.

[0096] The "recruitment" of cells, e.g., CD8+ T cells and / or NK cells, to tumors can be examined in mouse models, e.g., humanized mouse models, by removing the tumor mass, separating the cells, and analyzing the number of CD8+ T cells and / or NK cells associated with the tumor mass, e.g., by FACS after appropriate staining. A treatment capable of recruiting such cells to tumors (e.g., injection of macrophages of the invention) will over time result in these cells representing a higher proportion of the cell population in the tumor mass analyzed, when compared to an appropriate control without such treatment.

[0097] As used herein, the term "proliferative cells" refers to cells capable of cell division. A cell is a proliferative cell if a population of at least 1,000 "proliferative cells" increases in cell number by at least fourfold after 8 days under appropriate culture conditions, i.e., n(192 hours) / n(0 hours), where n is the total number of cells in the cell population at the indicated time point, is at least 4.00.

[0098] As used herein, the term "differentiated human cells" refers to cells that do not change cell type and, even upon cell division, give rise to two cells of the same cell type. This contrasts with "pluripotent cells," which can differentiate into all adult cell types, and "oligopotent cells," which can differentiate into several closely related cell types.

[0099] As used herein, "myeloid cells" refers to cells of hematopoietic origin that are not lymphoid, not erythroid-megakaryocyte, and not multilineage precursors with potential beyond the myeloid lineage.

[0100] As used herein, iPS cells or "induced pluripotent stem cells" refer to cells with pluripotency obtained by reprogramming somatic cells. Several groups, including the group of Professor Shinya Yamanaka et al. at Kyoto University, the group of Rudolf Jaenisch et al. at Massachusetts Institute of Technology, the group of James Thomson et al. at the University of Wisconsin, and the group of Konrad Hochedlinger et al. at Harvard University, have successfully created such induced pluripotent stem cells. Induced pluripotent stem cells have attracted considerable attention as ideal pluripotent cells that are free from immune rejection and ethical issues. For example, International Publication WO 2007 / 069666 reports somatic nuclear reprogramming factors, including gene products of Oct family genes, Klf family genes, and Myc family genes, as well as somatic nuclear reprogramming factors, including gene products of Oct family genes, Klf family genes, Sox family genes, and Myc family genes. This publication also describes a method for generating induced pluripotent stem cells by nuclear reprogramming of somatic cells, which includes contacting the aforementioned nuclear reprogramming factors with somatic cells.

[0101] "Monocytes" are mononuclear phagocytes found in peripheral blood. Monocytes vary considerably in size, ranging from 10 to 30 μm in diameter. The nucleus-to-cytoplasm ratio ranges from 2:1 to 1:1. The nucleus is often zonular (horseshoe-shaped) or reniform (kidney-shaped). Monocytes may also fold onto themselves and exhibit gyrus-like convolutions. Nucleoli are not visible. The chromatin pattern is delicate, consisting of skein-like filaments. The cytoplasm is abundant and appears blue-gray with numerous fine azurophilic granules on Giemsa staining, giving it a ground-glass appearance. Vacuoles may be present. More preferably, the expression of specific surface antigens is used to determine whether a cell is a monocytic cell. Phenotypic markers for human monocytic cells include CD11b, CD11c, CD33, CD45, and CD115. Generally, human monocytic cells are characterized by the following markers: 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 155, CD156a, CD157, CD162, CD163, CD164, CD168, CD171, CD172a, CD180, CD131a1, CD213a2, CDw210, CD226, CD281, CD282, CD284, CD286, and optionally express CD4, CD14, CD16, CD40, CD45RO, CD45RA, CD45RB, CD62L, CD74, CD141, CD142, CD169, CD170, CD181, CD182, CD184, CD191, CD192, CD194, CD195, CD197, CD206, CX3CR1. Unless specifically excluded, "monocytes" are included in the term "macrophage" as used herein.

[0102] Dendritic cells (DCs) are antigen-presenting cells that may exist in vivo, in vitro, ex vivo, or within a host or subject, or may be derived from hematopoietic stem cells, hematopoietic precursors, 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, with thin sheets (lamellipodia) extending in multiple directions from the dendritic cell body. DCs constitutively express both MHC class I and MHC class II molecules, which present peptide antigens to CD8+ and CD4+ T cells, respectively, and can activate naive T cells. Furthermore, human skin and mucosal DCs also express the CD1 gene family, MHC class I-related molecules, which present microbial lipid or glycolipid antigens. DC membranes are also rich in molecules that enable T cell adhesion (e.g., intercellular adhesion molecule-1 or CD54) or costimulate 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, CD286, and subsets of CD206, CD207, CD208, and CD209. Unless specifically excluded, "dendritic cells" are included in the term "macrophage" as used herein.

[0103] As used herein, "macrophage" includes macrophages, monocytes, and dendritic cells. However, preferably, "macrophage" as used herein refers to macrophages in the narrow sense. Macrophages are cells that exhibit phagocytic activity. Macrophage morphology varies between different tissues and between normal and pathological states, and not all macrophages can be identified by morphology alone. However, most macrophages are large cells with a round or cleaved nucleus, a well-developed Golgi apparatus, abundant endocytic vacuoles, lysosomes, and phagolysosomes, and a plasma membrane surrounded by folds or microvilli. The primary functions of macrophages in innate and adaptive immunity are the phagocytosis and subsequent degradation of senescent or apoptotic cells, microorganisms, and neoplastic cells, the secretion of cytokines, chemokines, and other soluble mediators, and the presentation of foreign antigens (peptides) on their surface to T lymphocytes. Macrophages originate from multipotent progenitors, common myeloid progenitors, and granulocyte-monocyte progenitors in the bone marrow of mammals. They eventually develop through additional precursor stages into monocytes 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 lifespan, some monocytes adhere to and migrate through capillary endothelium to all organs, where some can differentiate into tissue-resident macrophages or dendritic cells (see below). In addition to monocyte origin, tissue-resident macrophages can also arise from early primitive macrophage precursors in the yolk sac before the establishment of definitive hematopoiesis, from erythroid macrophage precursors (EMPs) in various hematopoietic sites in the embryo, or from fetal monocytes derived from embryonic hematopoietic stem cells. These embryonic-derived macrophages persist into adulthood and are maintained long-term, independent of input from adult hematopoietic stem cells and monocytes. Although macrophages are particularly abundant in lymphoid tissues, such as lymph nodes and the spleen, tissue-resident macrophages are present in essentially every organ in the body. In some organs, macrophages have special names, as summarized in Table 1.

[0104] (Table 1: Examples of tissue macrophages) [Table 2]

[0105] Further examples of macrophages are peritubular and testicular interstitial macrophages, cardiac macrophages from the heart, adipose tissue macrophages from adipose tissue, large intestinal and small intestinal macrophages from the intestine, skeletal muscle macrophages, synovial macrophages from the joints, arterial adventitial macrophages, arterial intimal macrophages, vascular-associated macrophages, pancreatic resident macrophages, meningeal macrophages, pleural macrophages, and omental macrophages.

[0106] In the context of the present invention, the macrophage may be selected from any of the macrophages described above. Preferably, the macrophage may be selected from the group consisting of microglia, histiocytes, Hofbauer cells, mesangial cells, Kupffer cells, peritoneal macrophages, alveolar macrophages, epithelial or dermal macrophages, marginal zone macrophages, metallophil macrophages, splenic red pulp macrophages, splenic white pulp macrophages, and osteoclasts. The macrophages may also be derived from human iPS cells following an in vitro differentiation protocol, as described elsewhere herein.

[0107] Macrophages are an important source of cytokines. Functionally, these numerous products can be divided into several groups: (1) cytokines that mediate proinflammatory responses, i.e., help recruit additional inflammatory cells (e.g., IL-1, IL-6, TNF, CC chemokines, and CXC chemokines, such as IL-8 and monocyte chemoattractant protein 1); (2) cytokines that mediate activation of T cells and natural killer (NK) cells (e.g., IL-1, IL-12, IL-15, IL-18); (3) cytokines that exert feedback effects on the macrophage itself (e.g., IL-1, IL-12, IL-15, IL-18). (4) cytokines that downregulate macrophages and / or help end inflammation (e.g., IL-10, TGFβ); (5) cytokines important for wound healing or for supporting tissue stem cells (e.g., EGF, PDGF, bFGF, TGFβ), or for supporting blood vessel growth (e.g., VEGF), or for supporting nerve cells (e.g., neurotrophic factors, kinins). Cytokine production by macrophages can be triggered by microbial products such as LPS, by interactions 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, CD286, and subsets of CD163, CD169, CD170, MARCO, FOLR2, and LYVE1. Activated macrophages can also express CD23, CD25, CD69, CD105, and HLA-DR, HLA-DP, and HLA-DQ.

[0108] As used herein, a macrophage is resistant to M2 polarization by M-CSF (or a cytokine combination of M-CSF and IL-4 and / or IL-13) if long-term culture of the macrophage(s) does not result in the loss of all characteristics characteristic of M1-polarized macrophages, and in particular does not result in the loss of all characteristics typical of M1-polarized macrophages. A characteristic of M1-polarized macrophages as used herein can be any one of upregulated expression of HLA class II genes, e.g., 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 can also be characterized by the surface markers they express or do not express and / or by the pattern of surface markers they express and / or do not express. Further examples of typical characteristics of M1 macrophages as used herein can be MHC II positive, CD74 positive, CD2 positive, LYVE1 negative, CD28 negative, STAB1 negative, and / or LILRB5 negative.

[0109] A "surface marker" is a molecule, typically a protein or carbohydrate structure, that is present and accessible on the outside plasma membrane of a cell and is specific to one particular cell type or a limited number of cell types, and is therefore a "marker" for those cell types. Examples of surface markers on human macrophages are 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.

[0110] A cell is considered "positive" for a surface marker if it produces a specific fluorescent signal when stained with a surface marker-specific antibody in a FACS assay. The principles of FACS are explained in detail in "Practical Flow Cytometry," 4th Edition, by Howard M. Shapiro. In a FACS assay, a collection of cells is typically stained with multiple fluorescent antibodies, each of which selectively binds to a different surface marker and has a different fluorescent dye. This allows the selection of specific cell types within a heterogeneous collection of cells by using an appropriate gating strategy in a FACS assay. The specific fluorescent signal from the surface marker-specific antibody is then typically verified in a one-dimensional histogram plot by comparing the histogram of staining with all antibodies with the histogram of staining with a mixed antibody excluding only the surface marker-specific antibody (the so-called "FMO" or "fluorescence minus one" signal). If the two histograms are different, and staining with the total antibody mix produces stronger fluorescence than the FMO control, then the tested collection of cells is positive for the tested cell surface marker. Visually, this means that the fluorescence peak of staining with the total antibody mix is ​​shifted to higher fluorescence values ​​compared to the FMO control. Preferably, the two histograms (one total antibody, one FMO) overlap by at most 70% area (area under the curve), e.g., at most 50% area, e.g., at most 25% area.

[0111] "Secretion" of cytokines, e.g., IL18 and / or IL15, can be determined by qualitatively and / or quantitatively measuring the appearance of the cytokine in the supernatant of a cell culture, e.g., by biochemical immunoassays, e.g., ELISA-based methods, or bead-based multiplex assays such as Luminex technology, to name just two examples. Briefly, the medium used for cell growth is analyzed for the presence of the specific cytokine being tested before adding the cytokine to a collection of cells and after culturing the cells whose cytokine secretion is being tested therein. A cytokine is "secreted" by cells cultured in the medium if its concentration in the supernatant increases over the culture period and this increase in cytokine concentration is confirmed by three separate, consecutive measurements. IL-6 can be detected at concentrations of 0.1 pg / ml or less, e.g., by the Meso Scale discovery immunoassay V-PLEX human IL-6 kit (Meso Scale Diagnostics). CXCL10 can be detected at concentrations of 5 pg / ml or even less using, for example, the LANCE Ultra Human CXCL10 Detection Kit (Perkin Elmer).

[0112] The terms "phagocyte" and "phagocyte" are used interchangeably herein to refer to cells capable of engulfment. There are different major categories of professional phagocytes: macrophages in the narrow sense, monocytes, and dendritic cells, as well as mononuclear phagocytes, including polymorphonuclear leukocytes (neutrophils). However, "non-professional" phagocytes are also known to participate in efferocytosis, the process by which professional and non-professional phagocytes rapidly and efficiently dispose of apoptotic cells.

[0113] The term "progenitor cell," as used herein, refers to a cell that is the progeny of a stem cell and can further differentiate to generate 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 other progenitor cells have the potential to differentiate into multiple cell types. Thus, progenitor cells are intermediate cell types with respect to the generation of mature cells within human tissues and organs, blood, and the central nervous system. Hematopoietic progenitor cells are intermediate cell types in the generation of blood cells. Hematopoietic progenitor cells are immature cells that arise from hematopoietic stem cells and ultimately differentiate into one of more than 10 different types of mature blood cells.

[0114] The term "CD34+ multipotent progenitors" as used herein refers to a stem cell-enriched hematopoietic progenitor population that expresses the CD34 surface antigen and is not a macrophage, monocyte, or dendritic cell.

[0115] The term "monoblast" as used herein refers to committed progenitor cells in the bone marrow that differentiate from myeloid progenitor cells in the hematopoietic process. They can mature into monocytes, which can then develop into macrophages.

[0116] The term "collection of cells" as used herein relates to at least 10,000 cells that are viable cells.

[0117] As used herein, the term "expanding" cells is the process of culturing cells under appropriate experimental conditions to increase the number of viable cells by mitosis of the cultured cells.

[0118] The term "after exposure" as used herein means that the cells are cultured in the continued presence of a particular agent, e.g., a cytokine, for the indicated time period and then immediately tested.

[0119] The term "genetically modified" cell as used herein refers to a cell whose DNA has been modified using biotechnology methods. For example, a cell whose DNA 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.

[0120] As used herein, the term "under appropriate culture conditions" refers to conditions under which the phagocytes of the present invention can grow. 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 (RPMI, named after Roswell Park Memorial Institute, is a medium often used for culturing human lymphocytes) supplemented with 10% FBS (PAA-GE Healthcare, A15-101), 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), 50 ng / ml M-CSF (Thermo Fisher, No. PHC9504), and, where indicated, 50 ng / ml GM-CSF (PeproTech, No. 300-03), as described in Example 3. Cultures are typically performed at 37°C in 5% CO2 and 21% O2.

[0121] The term "reactive oxygen species" as used herein refers to unstable molecules that contain oxygen and 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 insults.

[0122] The term "activation," as used herein, refers to the phenomenon in which an external stimulus induces a change in a cell, thereby activating the cell. For example, macrophages can be activated by cytokines such as interferon-γ (IFN-γ) and bacterial endotoxins such as lipopolysaccharide (LPS). Activated macrophages undergo numerous changes that enable them to kill invading bacteria or infected cells. They release toxic chemicals and proteins that have toxic effects on other cells. Activated macrophages increase in size, have an increased metabolism, increased levels of lysosomal proteins, and have an increased ability to phagocytose and kill microorganisms. 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 result in the type of tissue destruction that is characteristic of inflammation.

[0123] The term "adherent" as used herein refers to the ability of adherent cells to attach to a solid substrate, such as the bottom of a tissue culture flask. In contrast, suspension cells are suspended in culture medium and grow in suspension, without the need for mechanical or chemical removal.

[0124] As used herein, a "lamellipodia" is a protrusion at the leading edge of a cell. It comprises a quasi-two-dimensional actin network. Filopodia are finger-like structures within the lamellipodia that extend beyond and extend from the tip of the lamellipodia.

[0125] "Purified" and "isolated," when referring to a polypeptide or nucleotide sequence, mean that the indicated molecule is present in the substantial absence of other biological macromolecules. When referring to a "cell or cell population," the terms mean that the cell or cell population is present in the substantial absence of other cells or cell populations. As used herein, the term "purified" means that the same type of biological macromolecules or cells are present, preferably 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. An "isolated" nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the subject polypeptide, although the molecule may contain some additional bases or components that do not adversely affect the essential characteristics of the composition.

[0126] As used herein, the terms "tumor," "tumor cell," "cancer," and "cancer cell" refer to cells that exhibit relatively autonomous growth and exhibit an aberrant growth phenotype characterized by a significant loss of control over cell proliferation (i.e., deregulated cell division) and / or destruction / disregard of normal tissue organization and architecture. Tumor cells can be malignant or benign, while cancer cells are malignant. "Metastatic cells or tissue" means that the cells are capable of invading, colonizing, and destroying proximal and distal body structures.

[0127] As used herein, a "solid tumor" is a non-blood tumor. Solid tumors can be benign (not "cancerous") or malignant ("cancerous"). Different types of solid tumors are named for the cell type that forms them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas, although leukemias (blood cancers) do not generally form solid tumors.

[0128] The term "subject" as used herein refers to a human.

[0129] The term "treat" or "treatment" in the context of the present invention, as used herein, means to reverse, alleviate, inhibit the progression of, or prevent the disease or condition to which the term applies, or one or more symptoms of that disease or condition.

[0130] The term "chimeric antigen receptor" as used herein is 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 an activation-related response within the immune cell, such as the release of cytolytic molecules to induce tumor cell death, etc.

[0131] The term "ex vivo" as used herein means outside the living body.

[0132] As used herein, the term "in vitro" means outside a living organism and within a laboratory environment. For example, cells cultured "in vitro" are grown in a controlled, often artificial, medium.

[0133] "Autologous," as used herein, refers to cells that are derived from an individual's own cells. For example, in an autologous transfusion, a patient's own blood is withdrawn and reinfused into the body.

[0134] "Allogeneic," as used herein, is a term that refers to human cells that are not derived from the individual's own cells. For example, an allogeneic stem cell transplant differs from an autologous stem cell transplant, which uses stem cells from the patient's own body.

[0135] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 0.1. It is understood, although not always expressly stated, that all numerical designations are preceded by the word "about." It is also understood, although not always expressly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0136] It will be understood that the present invention is not limited to the particular materials and methods described herein. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to limit the scope of the present invention, which will be defined solely by the appended claims. As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide one of skill in the art with general definitions of many of the terms used in the present invention and are ranked in ascending order of preference unless otherwise specified herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (3rd ed., 2006), The Glossary of Genomics Terms, JAMA. 2013;309(14):1533-1535, Janeway's Immunobiology, 9th ed., and Practical Flow Cytometry, 4th ed., by H.M. Shapiro.

[0137] All references cited herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents, and vectors reported in those publications that may be used in connection with the present invention. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0138] (Detailed explanation) For example, in Example 8, it was observed that lymphoid cells, particularly T cells and / or NK cells, are involved in important aspects of the antitumor activity of macrophages. In lymphoid-depleted Rag2γc mice, administration of myeloid cells after the establishment of tumor metastases no longer resulted in regression of metastases, whereas wild-type mice with a physiological lymphoid compartment did. In Example 11, dramatic upregulation of specific HLA class II genes was observed in human MAF / MAFB DKO macrophages, which correspond to murine MAF / MAFB DKO macrophages with antitumor activity. Based on these observations, but without being bound by any theory, it is suggested that one aspect of the anti-tumor activity of myeloid cells, e.g., macrophages, involves interactions between myeloid cells, e.g., macrophages, and T cells, e.g., CD4+ T cells, via interactions between MHC II complexes on myeloid cells, particularly myeloid cells bearing at least one typical M1-marker, and T cell receptors on CD4+ cells. This has important implications for improving allogeneic anti-cancer cell therapies with myeloid cells, e.g., macrophages, monocytes, or dendritic cells, and particularly macrophages in the narrow sense.

[0139] Because of MHC restriction, T cells specific for tumor-derived neoantigens should respond better to tumor-derived peptide antigens bound to a given autologous MHC complex than to the same tumor-derived peptide antigen bound to an unmatched allogeneic MHC complex. Therefore, it is important to consider whether allogeneic myeloid cells, e.g., allogeneic macrophages, possess at least one version of the HLA-DP, HLA-DQ, and / or HLA-DR complexes that are also present in the individual being treated. In other words, there should be at least a partial match at the MHC-II complex level between the patient's lymphoid immune cells and the allogeneic myeloid cells, e.g., allogeneic macrophages; for example, antigen-specific recognition of tumor-derived neoantigens by the patient's neoantigen-specific CD4+ T cells is significantly improved when the neoantigens are presented on allogeneic myeloid antigen-presenting cells, e.g., macrophages.

[0140] Thus, in a further aspect, the present invention relates to allogeneic myeloid cells, e.g., allogeneic macrophages, for use in therapy, e.g., cancer therapy, in a human patient, wherein the allogeneic myeloid cells, e.g., allogeneic macrophages, comprise at least one MHC-II complex identical to any one of the MHC-II complexes of the human patient. This has the advantage of being able to trigger an antigen-specific response of the human patient's CD4+ T cells. The present invention also relates to allogeneic myeloid cells, e.g., allogeneic macrophages, for use in cancer therapy in a human patient, wherein the allogeneic macrophages comprise at least one protein complex selected from the group consisting of HLA-DR, HLA-DP, and HLA-DQ, wherein the protein complex is identical to any one of the corresponding HLA complexes of the human patient, and preferably the allogeneic myeloid cells, e.g., allogeneic macrophages, comprise two protein complexes selected from the group, or the allogeneic myeloid cells, e.g., allogeneic macrophages, comprise three complexes selected from the group. The present invention also relates to allogeneic myeloid cells, e.g., allogeneic macrophages, for use in treating cancer in human patients, wherein the allogeneic myeloid cells, e.g., allogeneic macrophages, comprise at least one HLA-DR protein complex identical to any of the corresponding HLA-DR complexes in the human patient. The present invention also relates to allogeneic myeloid cells, e.g., allogeneic macrophages, for use in treating cancer in human patients, wherein the allogeneic myeloid cells, e.g., allogeneic macrophages, comprise at least one HLA-DP protein complex identical to any of the corresponding HLA-DP complexes in the human patient. The present invention also relates to allogeneic myeloid cells, e.g., allogeneic macrophages, for use in treating cancer in human patients, wherein the allogeneic myeloid cells, e.g., allogeneic macrophages, comprise at least one HLA-DQ protein complex identical to any of the corresponding HLA-DQ complexes in the human patient.

[0141] A (partial) MHC II match between allogeneic myeloid cells, e.g., allogeneic macrophages, and the patient to be treated can also be described at the genetic level. Thus, the present invention also relates to allogeneic myeloid cells, e.g., allogeneic macrophages, for use in treating cancer in a patient, wherein the allogeneic myeloid cells, e.g., allogeneic macrophages, and the patient to be treated share at least one pair of alleles selected from the group consisting of the following pairs: HLA-DRA / HLA-DRB1, HLA-DRA / HLA-DRB3, HLA-DRA / HLA-DRB4, HLA-DRA / HLA-DRB5, HLA-DPA1 / HLA-DPB1, and HLA-DQA1 / HLA-DQB1, and preferably the allogeneic myeloid cells, e.g., allogeneic macrophages, and the patient to be treated share 2, 3, 4, 5, or even all six pairs of alleles. It should be noted that for heterozygous patients, this is the overlap of one allele pair with one allele pair in the allogeneic myeloid cells, e.g., allogeneic macrophages, and it is not necessary that both allele pairs in the patient match both allele pairs in the allogeneic myeloid cells, e.g., allogeneic macrophages (although matches of both such allele pairs are certainly included).

[0142] The principle of at least partial MHC-II overlap as described above applies to all myeloid cells, e.g., macrophages, intended for cancer therapy, including, for example, allogeneic macrophages polarized ex vivo to an M1 phenotype prior to administration, as discussed in the introduction, or human macrophages genetically modified with a chimeric antigen receptor (CAR) (e.g., as reported in Klichinsky et al., 2020). However, preferably, the allogeneic human myeloid cells contain at least one mutation in both alleles of a single gene located on a single chromosome, and the human macrophages are resistant to M-CSF-induced M2 polarization.

[0143] Accordingly, the present invention also relates to allogeneic human myeloid cells, e.g., allogeneic macrophages, for use in treating cancer in a human patient, wherein the allogeneic myeloid cells, e.g., allogeneic macrophages, comprise at least one MHC-II complex capable of triggering an antigen-specific response of CD4+ T cells in the human patient, and / or wherein the allogeneic myeloid cells and the patient to be treated share at least one pair of alleles selected from the group consisting of the following pairs: HLA-DRA / HLA-DRB1, HLA-DRA / HLA-DRB3, HLA-DRA / HLA-DRB4, HLA-DRA / HLA-DRB5, HLA-DPA1 / HLA-DPB1, and HLA-DQA1 / HLA-DQB1.

[0144] Furthermore, human myeloid cells, e.g., human macrophages, are resistant to M-CSF-induced M2 polarization, and in particular, after 48 hours of exposure to 50 ng / ml M-CSF, the human myeloid cells, e.g., human macrophages, acquire typical characteristics of M1 macrophages. Human myeloid cells, e.g., human macrophages, may be genetically modified by the introduction of a gene encoding a surface protein such as a chimeric antigen receptor, but human myeloid cells, e.g., human macrophages, may also be derived from non-genetically modified myeloid cells, e.g., human macrophages, e.g., iPS cells, and may be polarized to the M1 phenotype by, for example, LPS or interferon-γ.

[0145] The present invention also relates to a method of treating a human subject afflicted with cancer, the method comprising administering to the human subject allogeneic human myeloid cells, e.g., human macrophages, or a collection of allogeneic human myeloid cells, e.g., a collection of human macrophages, in an amount sufficient to at least inhibit further growth of the cancer, wherein the allogeneic myeloid cells, e.g., human macrophages, comprise at least one MHC-II complex capable of triggering a CD4+ T cell response in the human patient, and / or wherein the allogeneic myeloid cells, e.g., human macrophages, and the treated patient share at least one pair of alleles selected from the group consisting of the following pairs: HLA-DRA / HLA-DRB1, HLA-DRA / HLA-DRB3, HLA-DRA / HLA-DRB4, HLA-DRA / HLA-DRB5, HLA-DPA1 / HLA-DPB1, and HLA-DQA1 / HLA-DQB1.

[0146] Preferably, the human myeloid cells for therapy are myeloid cells containing at least one mutation in both alleles of a gene located on chromosome 1, and the macrophages are resistant to M-CSF-induced M2 polarization. The macrophages of the present invention are anti-tumorigenic and therefore useful for macrophage cell therapy, particularly anti-cancer therapy.

[0147] In particular, the human myeloid cells for the therapy of the present invention are resistant to M2 polarization by the combination of M-CSF and IL-4, and / or even by the combination of M-CSF, IL-4, and IL-13.

[0148] Resistance to M2 polarization means, for example, that allogeneic human myeloid cells, e.g., allogeneic human macrophages, for therapies of the invention, retain typical characteristics of M1 macrophages even after exposure to M-CSF and / or IL-4 and / or IL-13 for 24 hours, particularly 48 hours, e.g., 72 hours or more. For example, allogeneic human myeloid cells, e.g., allogeneic human macrophages, may be exposed to 50 ng / ml M-CSF and / or 20 ng / ml IL-4 for 24 hours, or even, e.g., 50 ng / ml M-CSF and / or 20 ng / ml IL-4 for 48 hours, e.g., 72 hours, and still retain at least one typical characteristic of M1 macrophages. Preferred allogeneic human myeloid cells, e.g., allogeneic human macrophages, for therapies of the present invention exhibit at least one, e.g., at least two, three, four, or even five, typical characteristic(s) of M1 macrophages after 24 hours of exposure, and particularly even after 48 hours or 72 hours of exposure, to a combination of 50 ng / ml M-CSF, 40 ng / ml IL-4, and 20 ng / ml IL-13.

[0149] Allogeneic human myeloid cells, e.g., allogeneic human macrophages, for use in the therapeutic methods of the present invention can be characterized by their gene expression profile. A typical characteristic of M1 macrophages can be, for example, expression of the HLA-DRA gene. In particular, the expression level of HLA-DRA mRNA in human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on a single chromosome is higher, e.g., at least 4-fold, e.g., at least 8-fold, e.g., at least 16-fold, or even at least 32-fold higher, than the expression of HLA-DRA mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0150] Alternatively or additionally, a typical characteristic of M1 macrophages may be, for example, expression of the HLA-DRB5 gene. In particular, the expression level of HLA-DRB5 mRNA in human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is higher, e.g., at least 4-fold, e.g., at least 8-fold, at least 16-fold, or even at least 32-fold higher, than the expression of HLA-DRB5 mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0151] Alternatively or additionally, a typical characteristic of M1 macrophages may be, for example, expression of the HLA-DPA1 gene. In particular, the expression level of HLA-DPA1 mRNA in human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is higher, e.g., at least 4-fold, e.g., at least 8-fold, at least 16-fold, or even at least 32-fold higher, than the expression of HLA-DPA1 mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0152] Alternatively or additionally, a typical characteristic of M1 macrophages may be, for example, expression of the HLA-DQA1 gene. In particular, the expression level of HLA-DQA1 mRNA in human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is higher, e.g., at least 4-fold, e.g., at least 8-fold, at least 16-fold, or even at least 32-fold higher, than the expression of HLA-DQA1 mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0153] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, expression of the RXFP2 gene. In particular, the expression level of RXFP2 mRNA in human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is higher, e.g., at least 4-fold, e.g., at least 8-fold, at least 16-fold, or even at least 32-fold higher, than the expression of RXFP2 mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0154] Alternatively or additionally, a typical characteristic of M1 macrophages may be, for example, expression of the CD74 gene. In particular, the level of CD74 mRNA expression in human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is higher, e.g., at least 4-fold, e.g., at least 8-fold, or even at least 16-fold higher, than CD74 mRNA expression in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0155] Alternatively or additionally, a typical characteristic of M1 macrophages may be, for example, expression of the CD70 gene. In particular, the level of expression of CD70 mRNA in human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is higher, e.g., at least 4-fold, e.g., at least 8-fold, or even at least 16-fold higher, than the expression of CD70 mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0156] Alternatively or additionally, a typical characteristic of M1 macrophages may be, for example, expression of the CD38 gene. In particular, the level of CD38 mRNA expression in human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is higher, e.g., at least 4-fold, e.g., at least 8-fold, or even at least 16-fold higher, than CD38 mRNA expression in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0157] Alternatively or additionally, a typical characteristic of M1 macrophages may be, for example, expression of the IL15 gene. In particular, the level of expression of IL15 mRNA in human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is higher, e.g., at least 2-fold, e.g., at least 2.5-fold higher, than the expression of IL15 mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0158] Alternatively or additionally, a typical characteristic of M1 macrophages may be, for example, expression of the IL18 gene. In particular, the level of expression of IL18 mRNA in human macrophages containing at least one mutation in both alleles of a gene located on one chromosome is higher, e.g., at least four-fold, e.g., at least six-fold higher, than the expression of IL18 mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0159] Alternatively or additionally, a typical characteristic of M1 macrophages may be, for example, expression of the IL23A gene. In particular, the expression level of IL23A mRNA in human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is higher, e.g., at least four-fold, e.g., at least six-fold higher, than the expression of IL23A mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0160] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, expression of the APOBEC3A gene. In particular, the level of APOBEC3A mRNA expression in human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is higher, e.g., at least four-fold, e.g., at least five-fold higher, than APOBEC3A mRNA expression in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0161] A preferred exemplary characteristic of the human macrophages of the present invention is 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 particular, the expression level of each mRNA corresponding to a gene in human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is higher, e.g., at least 4-fold, e.g., at least 8-fold, at least 16-fold, or even at least 32-fold higher, than the expression of the mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0162] A preferred exemplary characteristic of the human macrophages of the present invention is the expression of the HLA-DRA gene, the HLA-DRB5 gene, the HLA-DPA1 gene, the HLA-DPB1 gene, the HLA-DQA1 gene, the HLA-DQB1 gene, the RXFP2 gene, and the CD74 gene. In particular, the expression level of each mRNA corresponding to a gene in human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is higher, e.g., at least 4-fold, e.g., at least 8-fold, or even at least 16-fold higher, than the expression of the mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0163] 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 myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on a single chromosome is lower, e.g., at least 4-fold, e.g., at least 8-fold, e.g., at least 16-fold, at least 32-fold, or even at least 64-fold lower, than the expression of RNASE1 mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions. "64-fold lower" means that the copy number of RNASE1 mRNA in the human myeloid cells, e.g., human macrophages, of the invention is only 1 / 64 of the copy number of RNASE1 mRNA in the corresponding wild-type (wt) myeloid cells, e.g., human macrophages.

[0164] 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 myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is lower, e.g., at least 4-fold, e.g., at least 8-fold, e.g., at least 16-fold, at least 32-fold, or even at least 64-fold lower, than the expression of CD28 mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0165] 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 myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is lower, e.g., at least 4-fold, e.g., at least 8-fold, e.g., at least 16-fold, at least 32-fold, or even at least 64-fold lower, than the expression of LYVE1 mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0166] 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 myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is lower, e.g., at least 4-fold, e.g., at least 8-fold, e.g., at least 16-fold, or even at least 25-fold lower, than the expression of C5AR1 mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0167] 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 myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is lower, e.g., at least 4-fold, e.g., at least 8-fold, e.g., at least 16-fold, at least 32-fold, or even at least 64-fold lower, than the expression of FCGBP mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0168] 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 myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is lower, e.g., at least 4-fold, e.g., at least 8-fold, e.g., at least 16-fold, at least 32-fold, or even at least 64-fold lower, than the expression of F13A1 mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0169] 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 myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is lower, e.g., at least 4-fold, e.g., at least 8-fold, e.g., at least 16-fold, at least 32-fold, or even at least 64-fold lower, than the expression of QPCT mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0170] 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 myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is lower, e.g., at least four-fold, e.g., at least six-fold lower, than the expression of DUSP6 mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0171] 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 myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is lower, e.g., at least 4-fold, e.g., at least 8-fold, e.g., at least 16-fold, or even at least 32-fold lower, than the expression of CCL7 mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0172] 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 myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is lower, e.g., at least 4-fold, e.g., at least 8-fold, e.g., at least 16-fold, at least 32-fold, or even at least 64-fold lower, than the expression of RNF128 mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0173] 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 myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is lower, e.g., at least 4-fold, e.g., at least 8-fold, e.g., at least 16-fold, at least 32-fold, or even at least 64-fold lower, than the expression of IL10 mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0174] A preferred exemplary characteristic of the human macrophages of the present invention is downregulated expression of the RNASE1 gene, the CD28 gene, and the LYVE1 gene. In particular, the expression level of each mRNA corresponding to a gene in human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is lower, e.g., at least 4-fold, e.g., at least 8-fold, e.g., at least 16-fold, at least 32-fold, or even at least 64-fold lower, than the expression of the mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0175] Another preferred exemplary characteristic of the human macrophages of the present invention is the upregulated 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 on the one hand, and the downregulated expression of the RNASE1 gene, the CD28 gene, and the LYVE1 gene on the other hand. In particular, the expression level of each mRNA corresponding to the upregulated genes in human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is at least 4-fold higher, for example at least 8-fold, for example at least 16-fold, or even at least 32-fold higher than the expression of the mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions. On the other hand, the expression level of each one of the mRNAs corresponding to the downregulated genes in human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is at least 4-fold lower, such as at least 8-fold, e.g., at least 16-fold, at least 32-fold, or even at least 64-fold lower than the expression of that mRNA in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0176] Another preferred exemplary characteristic of human myeloid cells, e.g., human macrophages, for the therapy of the present invention is at least a 10-fold upregulated expression of the following gene combinations (compared to the expression of the respective mRNAs in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions): combinations (A) HLA-DRA and RXFP2, (B) HLA-DRA and CD74, (C) HLA-DOA and RXFP2, (D) HLA-DOA and CD74, (E) HLA-DPA1 and RXFP2, (F) HLA-DPA1 and CD74, (G) HLA-DRA and RXFP2 and CD74, (H) HLA-DOA and RXFP2 and CD74, and combination (I) HLA-DPA1 and RXFP2 and CD74.

[0177] Another preferred exemplary characteristic of human myeloid cells, e.g., human macrophages, for the therapy of the present invention is at least 50-fold downregulated expression of the following gene combinations: (m) RNASE1 and LYVE1, (n) RNASE1 and FCGBP, (o) RNASE1 and CD28, (p) RNASE1 and F13A1, (q) RNASE1 and RNF128, (r) LYVE1 and CD28, (s) LYVE1 and FCGBP, (t) LYVE1 and F13A1, (u) LYVE1 and RNF128, (v) CD28 and F13A1, and combination (w) CD28 and RNF128 (compared to the expression of the respective mRNAs in otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions).

[0178] Other preferred exemplary characteristics of human myeloid cells, e.g., human macrophages, for the therapy of the present invention are upregulated expression of one gene combination and downregulated expression of another gene combination, such as in the combinations described below: Am (A and m as described immediately above, i.e., a combination of 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, Au, 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, Eq, 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.

[0179] Human myeloid cells, e.g., human macrophages, for use in the therapy of the present invention can also be characterized by their secretion profile of cytokines and / or enzymes into the culture medium. Alternatively, or in addition, a typical characteristic of M1 macrophages may be, for example, the secretion of IL23A. In particular, the secretion of IL23A by human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on a single chromosome is higher, e.g., at least three-fold, e.g., at least four-fold, e.g., at least five-fold, six-fold, or even eight-fold higher, than the secretion of IL23A by otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0180] Alternatively or additionally, a typical characteristic of M1 macrophages may be, for example, the secretion of IL18. In particular, the secretion of IL18 by human myeloid cells, e.g., human macrophages, comprising at least one mutation in both alleles of a gene located on one chromosome is higher, e.g., at least 3-fold, e.g., at least 4-fold, e.g., at least 5-fold, 6-fold, or even 8-fold higher, than the secretion of IL18 by otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0181] Alternatively or additionally, a typical characteristic of M1 macrophages may be, for example, secretion of IL15. In particular, the secretion of IL15 by human myeloid cells, e.g., human macrophages, comprising at least one mutation in both alleles of a gene located on one chromosome is higher, e.g., at least two-fold, e.g., at least three-fold, e.g., at least four-fold higher, than the secretion of IL15 by otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0182] Alternatively or additionally, a typical characteristic of M1 macrophages may be, for example, downregulated secretion of RNASE 1. In particular, secretion of RNASE 1 by human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is lower, e.g., at least 3-fold, e.g., at least 5-fold, e.g., at least 7-fold, 10-fold, or even 30-fold lower, than secretion of RNASE 1 by otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0183] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulated secretion of FCGBP. In particular, secretion of FCGBP by human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is lower, e.g., at least 3-fold, e.g., at least 5-fold, e.g., at least 7-fold, 10-fold, or even 30-fold lower, than secretion of FCGBP by otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0184] Alternatively or additionally, a typical characteristic of M1 macrophages may be, for example, downregulated secretion of CCL7. In particular, the secretion of CCL7 by human myeloid cells, e.g., human macrophages, comprising at least one mutation in both alleles of a gene located on one chromosome is lower, e.g., at least 3-fold, e.g., at least 5-fold, e.g., at least 7-fold, 10-fold, or even 20-fold lower, than the secretion of CCL7 by otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0185] Alternatively or additionally, a typical characteristic of M1 macrophages may be, for example, downregulated secretion of IL10. In particular, the secretion of IL10 by human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is lower, e.g., at least 3-fold, e.g., at least 5-fold, e.g., at least 7-fold, 10-fold, or even 30-fold lower, than the secretion of IL10 by otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture conditions.

[0186] Another preferred exemplary characteristic 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.

[0187] Human myeloid cells, e.g., human macrophages, for use in the therapy of the present invention may also be characterized by their cell surface markers. Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the presence of the cell surface marker MHC II, or more specifically, the presence of an HLA class II protein, e.g., a protein 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 myeloid cells, e.g., human macrophages, of the present invention. In particular, the number of antibody-accessible MHC II proteins, e.g., HLA-DRA, on the cell surface of human macrophages containing at least one mutation in both alleles of a gene located on one chromosome is greater, e.g., at least three-fold, e.g., at least four-fold, e.g., at least five-fold, eight-fold, or even ten-fold greater, than the number of antibody-accessible MHC II proteins on the cell surface of otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture and immunostaining conditions.

[0188] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the presence of the cell surface marker CD74. In particular, the number of CD74 markers on the cell surface of human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on a single chromosome is greater, e.g., at least three-fold, e.g., at least four-fold, e.g., at least five-fold, six-fold, or even eight-fold greater, than the number of CD74 markers on the cell surface of otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture and immunostaining conditions.

[0189] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the presence of the cell surface marker CD70. In particular, the number of CD70 on the cell surface of human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on a single chromosome is greater, e.g., at least three-fold, e.g., at least four-fold, e.g., at least five-fold, six-fold, or even eight-fold greater, than the number of CD70 on the cell surface of otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture and immunostaining conditions.

[0190] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, the presence of the cell surface marker CD2. In particular, the number of CD2 on the cell surface of human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on a chromosome is greater, e.g., at least three-fold, e.g., at least four-fold, e.g., at least five-fold, six-fold, or even eight-fold greater, than the number of CD2 on the cell surface of otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture and immunostaining conditions.

[0191] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulation of the cell surface marker CD28. In particular, the number of CD28 on the cell surface of human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on a single chromosome is less, e.g., at least 2-fold, e.g., at least 4-fold, e.g., at least 6-fold, 8-fold, or even 10-fold less, than the number of CD28 on the cell surface of otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture and immunostaining conditions.

[0192] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulation of the cell surface marker LYVE1. In particular, the number of LYVE1 on the cell surface of human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on a single chromosome is less, e.g., at least 2-fold, e.g., at least 4-fold, e.g., at least 6-fold, 8-fold, or even 10-fold less, than the number of LYVE1 on the cell surface of otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture and immunostaining conditions.

[0193] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulation of the cell surface marker STAB1. In particular, the number of STAB1 on the cell surface of human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome is less, e.g., at least 2-fold, e.g., at least 4-fold, e.g., at least 6-fold, 8-fold, or even 10-fold less, than the number of STAB1 on the cell surface of otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture and immunostaining conditions.

[0194] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulation of the cell surface marker LILRB5. In particular, the number of LILRB5 on the cell surface of human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on a single chromosome is less, e.g., at least 2-fold, e.g., at least 4-fold, e.g., at least 6-fold, 8-fold, or even 10-fold less, than the number of LILRB5 on the cell surface of otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture and immunostaining conditions. For purposes of the present invention, the signal intensity in the immunostaining assay was taken to represent the number of cell surface markers of a particular species.

[0195] Alternatively, or additionally, a typical characteristic of M1 macrophages may be, for example, downregulation of the cell surface marker CD163. In particular, the number of CD163 on the cell surface of human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on a single chromosome is less, e.g., at least 2-fold, e.g., at least 4-fold, e.g., at least 6-fold, 8-fold, or even 10-fold less, than the number of CD163 on the cell surface of otherwise identical wild-type myeloid cells, e.g., human macrophages, tested under otherwise equivalent culture and immunostaining conditions. For purposes of the present invention, signal intensity in an immunostaining assay is taken to represent the number of cell surface markers of a particular species.

[0196] Particularly preferred human macrophages for the therapy of the present invention are positive for at least one, preferably two or all three of the surface markers MHC II, CD70, and CD74, but negative for at least one, preferably two, e.g., three, four, or all five, of the surface markers CD28, LYVE1, STAB1, CD163, and LILRB5.

[0197] 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; 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.

[0198] Human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on a single chromosome for use in the therapy of the present invention can also be characterized by a combination of gene expression markers, particularly any of the preferred gene expression markers, and a secretion profile, particularly a preferred secretion profile.

[0199] For the purposes of the present invention, human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome can also be characterized by a combination of gene expression markers, particularly any of the preferred gene expression markers, and surface markers, particularly the preferred surface marker profile.

[0200] For the purposes of the present invention, human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on a single chromosome can also be characterized by a secretion profile, particularly any of the preferred secretion profiles, in combination with surface markers, particularly preferred surface marker profiles.

[0201] Human myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on a single chromosome for use in the therapy of the present invention can also be characterized by a combination of a gene expression profile, particularly any of the preferred gene expression profiles, with surface markers, particularly preferred surface marker profiles, and a secretion profile, particularly preferred secretion profiles.

[0202] Human myeloid cells, e.g., human macrophages, for therapy of the present invention contain at least one mutation in both alleles of a gene located on a single chromosome. The mutation can render the gene non-functional and / or inhibit or even abolish gene expression.

[0203] The mutation may be an insertion or a frameshift. However, preferred mutations are deletions, such as deletions within promoters, exons, or splice sites. Although small deletions of only a few base pairs may have an effect on gene function, preferably the deletion is at least 50 base pairs, e.g., at least 100 base pairs, at least 200 base pairs, at least 500 base pairs, or even at least 1000 base pairs.

[0204] Preferred human myeloid cells for therapy of the present invention, such as human macrophages, contain at least two different mutations, preferably the two mutations are deletions in at least two different genes.

[0205] As described above, the present invention preferably uses myeloid cells, e.g., human macrophages, containing at least one mutation in both alleles of a gene located on one chromosome for the therapy of the present invention, and the macrophages are resistant to M-CSF-induced M2 polarization. The present invention provides a new selectable phenotype for cells containing at least one mutation in both alleles of a gene located on one chromosome, which can be screened, thereby making it possible to identify previously unknown genes whose mutations render myeloid cells, e.g., human macrophages, resistant to M-CSF-induced M2 polarization. Therefore, the nature of the gene is not particularly limited, so long as mutations in both alleles of the gene produce the desired phenotype. However, preferred human myeloid cells, e.g., human macrophages, for the therapy of the present invention have mutations in both alleles in genes involved in M-CSF-, IL-4-, IL-10-, and / or TGFB1-mediated downregulation of C2TA, e.g., negative regulators, and particularly negative transcriptional 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 may be selected from the group consisting of STAT6, IRF4, TMEM106A, MAFB, and MAF.

[0206] A preferred human myeloid cell, e.g., a human macrophage, containing at least one mutation in both alleles of a gene located on one chromosome, wherein the myeloid cell, e.g., a human macrophage, is resistant to M-CSF-induced M2 polarization, is a myeloid cell, e.g., a human macrophage, in which the mutated gene is MAFB. Preferably, MAFB is the only transcription factor-encoding gene containing deletions in both alleles. Alternatively, MAFB is the only protein-encoding gene containing deletions in both alleles. Alternatively, MAFB is the only gene containing deletions in both alleles. Deletion of additional transcription factors other than MAF and / or MAFB may impair myeloid cell function, e.g., human macrophage function, or may render induced pluripotent stem cells unable to differentiate into macrophages. For example, Buchrieser et al. showed that induced pluripotent stem cells lacking the transcription factors RUNX1 or SPI1 cannot differentiate into iPS cell-derived macrophages.

[0207] Preferred human myeloid cells, e.g., human macrophages, for use in the therapeutic method 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 of the deletions may be in exonic portions of MAFB. For example, the deletions may each be greater than 50 base pairs, e.g., at least 100 base pairs, or at least 250 base pairs. For example, deletions in or within the MAFB gene may each be 100 to 10,000 base pairs, e.g., 500 to 3,000 base pairs, particularly 700 to 2,000 base pairs. Preferred human myeloid cells, e.g., human macrophages, for use in the therapeutic method of the present invention, in which the mutated gene is MAFB, may exhibit one or more, e.g., all, of these deletions.

[0208] Preferred human myeloid cells, e.g., human macrophages, for use in the present invention, in which the mutated gene is MAFB, can also be characterized by the location of the deletion in a particular chromosome. For example, the MAFB deletion is preferably within the region on human chromosome 22 between 40685000 and 40690000, e.g., 40685200 and 40689800, e.g., 40685400 and 40689600, e.g., 40685600 and 40689400, and particularly 40685700 and 40689300.

[0209] A preferred human myeloid cell, e.g., a human macrophage, containing at least one mutation in both alleles of a gene located on one chromosome, wherein the myeloid cell, e.g., human macrophage, is resistant to M-CSF-induced M2 polarization, is a myeloid cell, e.g., a human macrophage, in which the mutated gene is MAF. Preferably, MAF is the only transcription factor-encoding gene containing biallelic deletions. Alternatively, MAF is the only protein-encoding gene containing biallelic deletions. Alternatively, MAF is the only gene containing biallelic deletions.

[0210] Preferred human myeloid cells, e.g., human macrophages, for use in 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 exonic portions of MAF. For example, the deletions may each be greater than 50 base pairs, e.g., at least 100 base pairs, or at least 250 base pairs. For example, deletions in or within the MAF gene may each be 100 to 10,000 base pairs, e.g., 500 to 3,000 base pairs, particularly 700 to 2,000 base pairs. Preferred human myeloid cells, e.g., human macrophages, for use in the present invention, in which the mutated gene is MAF, can exhibit one or more, e.g., all, of these deletions.

[0211] Preferred human myeloid cells, e.g., human macrophages, for use in the present invention, in which the mutated gene is MAF, can also be characterized by the location of the deletion in a particular chromosome. For example, the MAF deletion is preferably within the region 79593000-79602000, e.g., 79593200-79601500, e.g., 79593400-79601100, and particularly 79593600-79600900, on human chromosome 16.

[0212] A preferred human myeloid cell, e.g., a human macrophage, containing at least one mutation in both alleles of a gene located on one chromosome, wherein the myeloid cell, e.g., human macrophage, is resistant to M-CSF-induced M2 polarization, is a myeloid cell, e.g., a human macrophage, in which both alleles of MAFB and both alleles of MAF have been rendered nonfunctional, preferably by a deletion of at least 50 base pairs. Inhibition of MAFB and MAF gene expression in human cells has proven surprisingly more difficult than in mouse cells, in part because genetic approaches to double-deficient cells cannot be selected for in human cells. While there have been many successful examples in the past where the use of one guide RNA per gene in a CRISPR / Cas9-based approach has inactivated gene expression of another gene (e.g., Chu et al., 2016), several attempts to disable both the MAFB and MAF genes by creating small deletions using one guide RNA each in a CRISPR / Cas9 approach failed to produce cells that proliferated in vitro. Surprisingly, the use of two guide RNAs per gene successfully generated large deletions of at least 50 base pairs and produced proliferative phagocytes. These cells had the additional advantage of being non-tumorigenic. Preferably, MAF and MAFB are the only transcription factor-encoding genes containing biallelic deletions. Alternatively, preferably, MAF and MAFB are the only protein-encoding genes containing biallelic deletions. Alternatively, preferably, MAF and MAFB are the only genes containing biallelic deletions.

[0213] Preferred human myeloid cells, e.g., human macrophages, of the present invention, in which both MAFB and MAF alleles are non-functional, can also be characterized by the nature and size of the deletions. For example, all deletions may be in exonic portions of the MAFB and MAF genes. For example, the deletions may be greater than 50 base pairs each, e.g., 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 be 100 to 10,000 base pairs each, e.g., 500 to 3,000 base pairs each, particularly 700 to 2,000 base pairs each. Human macrophages of the present invention with stable M1 polarization may exhibit one or more, e.g., all, of these deletions.

[0214] Preferred human myeloid cells, e.g., human macrophages, with stable M1 polarization of the present invention can also be characterized by the location of a deletion in a specific chromosome. For example, the MAFB deletion is preferably within the region of human chromosome 22 between 40685000 and 40690000, e.g., between 40685200 and 40689800, e.g., between 40685400 and 40689600, e.g., between 40685600 and 40689400, and particularly between 40685700 and 40689300. For example, the MAF deletion is preferably within the region of human chromosome 16 between 79593000 and 79602000, e.g., between 79593200 and 79601500, e.g., between 79593400 and 79601100, and particularly between 79593600 and 79600900.

[0215] The human myeloid cells of the present invention, e.g., human macrophages, which are resistant to M-CSF-induced M2 polarization, may be generated by cell engineering. Cells from which the human myeloid cells of the present invention, e.g., human macrophages, can be derived may be cells selected from the group consisting of cells that are precursors for the development of human myeloid cells, e.g., human macrophages, such as iPS cells; blood monocytes; monocytes, monoblasts, myeloid, or CD34+ pluripotent precursors from umbilical cord blood; monocytes, monoblasts, myeloid, or CD34+ pluripotent precursors from bone marrow; mobilized monoblasts, myeloid, or CD34+ pluripotent precursors from adult blood; and monocytes, monoblasts, myeloid, or CD34+ precursors from extramedullary hematopoiesis. The human myeloid cells of the present invention, e.g., human macrophages, which are resistant to M-CSF-induced M2 polarization, may also be derived from macrophages, particularly relatively easily obtainable macrophages, such as alveolar macrophages or adipose tissue-derived macrophages. A preferred cell from which the human macrophages of the present invention may be derived is a human iPS cell. Another preferred cell from which the human myeloid cells of the present invention, e.g., human macrophages, may be derived is a CMP (common myeloid precursor). Another preferred cell from which the human myeloid cells of the present invention, e.g., human macrophages, may be derived is a GMP (granulocyte / macrophage precursor).

[0216] The generation of human induced pluripotent stem cells is now well established in the art. For example, Isogai et al. (2018) reported the generation of iPS cells from human blood monocytes. Fusaki N. et al. (2009) reported the generation of human iPS cell lines using modified Sendai virus. In particular, when human induced pluripotent stem cells are used as starting materials for myeloid cell differentiation, such as human macrophage differentiation, it is preferred that MAF and / or MAFB be the only transcription factor-encoding gene(s) containing biallelic deletions. Alternatively, it is preferred that MAF and / or MAFB be the only protein-encoding gene containing biallelic deletions. Alternatively, it is preferred that MAF and / or MAFB be the only gene containing biallelic deletions. If additional genes are deleted, macrophage function may be impaired or differentiation of induced pluripotent stem cells into macrophages may be impossible.

[0217] Human mononuclear phagocytes for biallelic mutations of the gene can also be obtained from human primary blood monocytes, which can be obtained by leukapheresis and elutriation, or from monocyte-derived macrophages, the culture conditions of which are well known in the art. Human macrophages can also be obtained from bronchoalveolar lavage or by differentiation of CD34+ hematopoietic stem and progenitor cells obtained from umbilical cord blood, stem cells mobilized from peripheral blood, or bone marrow, using differentiation protocols well known in the art.

[0218] Cas9 and gRNA can be expressed using publicly available DNA expression plasmids (e.g., Santa Cruz Sc-418922, https: / / www.scbt.com / de / p / control-crispr-cas9-plasmid) that are well known in the art. The DNA expression plasmid can be introduced into human mononuclear phagocyte target cells by electroporation or lipid-based transfection protocols that are well known in the art. Cas9 and gRNA can also be introduced into target cells by electroporation as a ribonucleoprotein complex. Cas9 / gRNA-mediated gene editing has been demonstrated using this method in mononuclear phagocytes (Zhang et al., 2020; Wang et al., 2018; Freund et al., 2020) and in human CD34+ hematopoietic stem and progenitor cells that differentiate into macrophages (Scharenberg et al., 2020). Target genes, such as the MAFB and MAF genes, can also be deleted using recombinases that target the engineered site (Lansing et al., 2020; Karpinski et al., 2016), which can be introduced by methods known in the art, such as electroporation of protein-coding mRNA or DNA expression plasmids, and have been used for gene editing in mononuclear phagocytes (Shi et al., 2018).

[0219] Gonzalez F. et al. (2014) describe an iCRISPR platform for rapid, multiplexable, and inducible genome editing of human pluripotent stem cells, which is 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 genetically engineered cell lines can then be used to target selected genes, such as MAF and / or MAFB.

[0220] Human macrophages in which both MAF and MAFB were defunctionalized were found to be nontumorigenic. Karyotyping analysis showed that they contained 46 chromosomes, none of which showed chromosomal rearrangements. Therefore, these cells are useful for macrophage-based cell therapy.

[0221] These markers can be maintained in vitro even in the presence of tumor cells.

[0222] Stem cells are useful intermediates and / or starting materials for the preparation of allogeneic, MHC II-matched myeloid cells for therapeutic use in the present invention.

[0223] Accordingly, the present invention also relates to allogeneic human induced pluripotent stem cells, or allogeneic human embryonic stem cells, or allogeneic hematopoietic stem cells, or allogeneic pluripotent progenitor cells, or allogeneic human common myeloid progenitors, or allogeneic human granulocyte / macrophage progenitors - and in particular human induced pluripotent stem cells - wherein said allogeneic cells, such as allogeneic induced pluripotent stem cells, contain at least one MHC-II complex identical to one of the MHC-II complexes of the human patient to be treated, which has the advantage of being able to trigger an antigen-specific response of CD4+ T cells of said human patient. The present invention also relates to allogeneic stem or progenitor cells, e.g., allogeneic induced pluripotent stem cells, for use in the preparation of MHC II-matched allogeneic myeloid cells, comprising at least one protein complex selected from the group consisting of HLA-DR, HLA-DP, and HLA-DQ, wherein the protein complex is identical to any of the corresponding HLA complexes of the human patient, and preferably the allogeneic myeloid cells, e.g., allogeneic macrophages, comprise two protein complexes selected from said group, or the allogeneic myeloid cells, e.g., allogeneic macrophages, comprise even three complexes selected from said group. The present invention also relates to allogeneic stem or progenitor cells, e.g., allogeneic induced pluripotent stem cells, for use in the preparation of MHC II-matched allogeneic myeloid cells, e.g., allogeneic macrophages, wherein the allogeneic myeloid cells are for use in the treatment of cancer in a human patient, and the allogeneic myeloid cells, e.g., allogeneic macrophages, comprise at least one HLA-DR protein complex that is identical to any of the corresponding HLA-DR complexes in the human patient.

[0224] The present invention also relates to the use of the human induced pluripotent stem cells, or human embryonic stem cells, or human common myeloid progenitors, or human granulocyte / macrophage progenitors, of the present invention, in particular the use of said human induced pluripotent stem cells, for the generation of human MHC II-matched antigen-presenting cells, in particular for the generation of human MHC II-matched myeloid antigen-presenting cells, such as macrophages or dendritic cells.

[0225] The present invention further relates to the following embodiments: 1. Allogeneic human myeloid cells for therapeutic use in a human patient, said allogeneic human myeloid cells comprising at least one MHC-II complex that is identical to one of the MHC-II complexes of said human patient. 2. The allogeneic human myeloid cells for therapeutic use according to clause 1, wherein said therapeutic use is cancer treatment. 3. The allogeneic human myeloid cell for therapeutic use of any one of clauses 1 or 2, wherein said myeloid cell is a macrophage, dendritic cell, or monocyte. 4. The allogeneic human myeloid cells for use in treating cancer in a human patient of clause 2, wherein said allogeneic myeloid cells are allogeneic macrophages, said allogeneic macrophages comprising at least one MHC-II complex identical to any one of the MHC-II complexes of said human patient. 5. The allogeneic human myeloid cells for therapeutic use of any one of clauses 1 to 4, wherein said allogeneic myeloid cells comprise at least one protein complex selected from the group consisting of HLA-DR, HLA-DP, and HLA-DQ, and said protein complex is identical to any of the corresponding HLA complexes of said human patient. 6. The allogeneic human myeloid cells for therapeutic use of any one of clauses 1 to 5, wherein said allogeneic myeloid cells comprise two protein complexes selected from said group, and preferably said allogeneic macrophages comprise three complexes selected from said group. 7. The allogeneic human myeloid cells for therapeutic use of any one of clauses 1 to 6, wherein said allogeneic myeloid cells comprise at least one HLA-DR protein complex that is identical to one of the corresponding HLA-DR complexes of said human patient. 8. The allogeneic human myeloid cells for therapeutic use of any one of clauses 1 to 7, wherein said allogeneic myeloid cells comprise at least one HLA-DP protein complex that is identical to one of the corresponding HLA-DP complexes of said human patient. 9. The allogeneic human myeloid cells for therapeutic use of any one of clauses 1 to 8, wherein said allogeneic macrophages comprise at least one HLA-DQ protein complex that is identical to any of the corresponding HLA-DQ complexes of said human patient. 10. Allogeneic human myeloid cells for therapeutic use in a patient, wherein the allogeneic human myeloid cells and the patient to be treated share at least one pair of alleles selected from the group consisting of the following pairs: HLA-DRA / HLA-DRB1, HLA-DRA / HLA-DRB3, HLA-DRA / HLA-DRB4, HLA-DRA / HLA-DRB5, HLA-DPA1 / HLA-DPB1, and HLA-DQA1 / HLA-DQB1. 11. Allogeneic human myeloid cells for therapeutic use according to clause 10, wherein said therapeutic use is cancer treatment. 12. The allogeneic human myeloid cells for therapeutic use according to clause 11, wherein the cancer is a solid tumor. 13. Allogeneic human myeloid cells for therapeutic use according to any one of clauses 10 to 12, wherein said myeloid cells are macrophages, monocytes or dendritic cells, in particular macrophages. 14. The allogeneic human myeloid cells for therapeutic use of any one of clauses 10 to 13, wherein the allogeneic myeloid cells and the patient to be treated have 2, 3, 4, 5, or even all 6 pairs of alleles in common. 15. The allogeneic human myeloid cells for therapeutic use according to any one of clauses 1 to 14, wherein said human myeloid cells, particularly said human macrophages, are resistant to M-CSF-induced M2 polarization. 16. Allogeneic human myeloid cells for therapeutic use according to any one of clauses 1 to 15, wherein said human myeloid cells, in particular said human macrophages, have typical characteristics of M1 macrophages after exposure to 50 ng / ml M-CSF for 48 hours. 17. Allogeneic human myeloid cells for therapeutic use according to any one of clauses 1 to 16, wherein said human myeloid cells, and in particular said human macrophages, are genetically modified. 18. The allogeneic human myeloid cells for therapeutic use according to clause 17, wherein the genetic modification is the introduction of a gene encoding a surface protein, such as a chimeric antigen receptor. 19. The allogeneic human myeloid cells for therapeutic use of any one of clauses 1 to 16, wherein said human macrophages are non-genetically modified macrophages. 20. The allogeneic human myeloid cells for therapeutic use of any one of clauses 1 to 19, wherein said human myeloid cells are derived from iPS cells. 21. An allogeneic human myeloid cell, and in particular an allogeneic human macrophage, for therapeutic use according to any one of clauses 1 to 20, wherein said allogeneic human myeloid cell, such as said allogeneic human macrophage, is a myeloid cell according to any one of clauses 22 to 114. 22. A human myeloid cell, preferably a macrophage, comprising at least one mutation in both alleles of a gene located on a chromosome, wherein the macrophage is resistant to M-CSF-induced M2 polarization, preferably the human macrophage is not a dendritic cell, more preferably the human macrophage is not a dendritic cell and is not a monocyte. 23. The human myeloid cell, preferably a macrophage, of clause 22, wherein said myeloid cell, preferably a macrophage, is resistant to M2 polarization by a combination of M-CSF and IL-4. 24. The human myeloid cell, preferably a macrophage, of any one of clauses 22-23, wherein the macrophage is resistant to M2 polarization by a combination of M-CSF, IL-4, and IL-13. 25. A human myeloid cell, preferably a macrophage, according to any one of clauses 22 to 24, wherein the myeloid cell has typical characteristics of an M1 macrophage after exposure to 50 ng / ml M-CSF for 24 hours. 26. A human myeloid cell, preferably a macrophage, according to clause 25, wherein said myeloid cell has typical characteristics of an M1 macrophage after exposure to 50 ng / ml M-CSF for 48 hours. 27. A human myeloid cell, preferably a macrophage, according to any one of clauses 22 to 26, wherein the myeloid cell has typical characteristics of an M1 macrophage after exposure to 20 ng / ml IL-4 for 24 hours. 28. A human myeloid cell, preferably a macrophage, according to clause 27, wherein said myeloid cell has typical characteristics of an M1 macrophage after exposure to 20 ng / ml IL-4 for 48 hours. 29. The human myeloid cells, preferably macrophages, of any one of clauses 22 to 28, wherein the myeloid cells have typical characteristics of M1 macrophages after exposure to a combination of 40 ng / ml IL-4 and 50 ng / ml M-CSF for 24 hours. 30. A human myeloid cell, preferably a macrophage, according to clause 29, wherein the myeloid cell has typical characteristics of an M1 macrophage after being exposed to a combination of 40 ng / ml IL-4 and 50 ng / ml M-CSF for 48 hours. 31. The human myeloid cells, preferably macrophages, of any one of clauses 22 to 30, wherein the myeloid cells have typical characteristics of M1 macrophages and GM-CSF has not been present for the final 2 hours. 32. The human myeloid cells, preferably macrophages, of clause 31, wherein said myeloid cells have typical characteristics of M1 macrophages and GM-CSF has been absent for the final 6 hours. 33. The human myeloid cell, preferably macrophage, of any one of clauses 25 to 32, wherein a typical characteristic of M1 macrophages is that expression of at least one mRNA in said human myeloid cell comprises at least one mutation in both alleles of a gene located on one chromosome, and is at least four-fold increased compared to expression of said mRNA in an otherwise identical wild-type myeloid cell, wherein said 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. 34. The human myeloid cell of clause 33, wherein expression of said at least one mRNA in said human myeloid cell, comprising at least one mutation in both alleles of a gene located on one chromosome, is at least six-fold higher compared to expression of the same mRNA in an otherwise identical wild-type myeloid cell. 35. The human myeloid cell of any one of clauses 25 to 32, wherein a typical characteristic of M1 macrophages is that expression of at least three mRNAs in said human myeloid cell, comprising at least one mutation in both alleles of a gene located on one chromosome, is increased by at least four-fold compared to expression of the same at least three mRNAs in an otherwise identical wild-type myeloid cell, 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. 36. The human myeloid cell of clause 35, wherein expression of said at least three mRNAs in said human myeloid cell, comprising at least one mutation in both alleles of a gene located on one chromosome, is at least six-fold higher compared to expression of the same at least three mRNAs in an otherwise identical wild-type myeloid cell. 37. The human myeloid cell of any one of clauses 25 to 32, wherein a typical characteristic of M1 macrophages is at least a four-fold increase in expression of at least six mRNAs in said human myeloid cell, said human myeloid cell comprising at least one mutation in both alleles of a gene located on a single chromosome, compared to the expression of the same at least six mRNAs in an otherwise identical wild-type myeloid cell, said at least six mRNAs being selected from the list consisting of HLA-DRA, HLA-DRB5, HLA-DPA1, HLA-DQA1, RXFP2, CD74, CD38, CD2, IL18, and IL23A. 38. The human myeloid cell of clause 37, wherein expression of said at least six mRNAs in said human myeloid cell, comprising at least one mutation in both alleles of a gene located on one chromosome, is at least six times higher compared to expression of the same at least six mRNAs in an otherwise identical wild-type myeloid cell. 39. The human myeloid cell of any one of clauses 33 to 38, wherein the expression of HLA-DRB5 mRNA in said human myeloid cell, comprising at least one mutation in both alleles of a gene located on a single chromosome, is at least four-fold, such as at least eight-fold, at least 16-fold, or even at least 32-fold higher than the expression of HLA-DRB5 mRNA in an otherwise identical wild-type myeloid cell. 40. The human myeloid cell of any one of clauses 33 to 39, wherein the expression of HLA-DPA1 mRNA in said human myeloid cell, comprising at least one mutation in both alleles of a gene located on a single chromosome, is at least four-fold, such as at least eight-fold, at least 16-fold, or even at least 32-fold higher than the expression of HLA-DPA1 mRNA in an otherwise identical wild-type myeloid cell. 41. The human myeloid cell of any one of clauses 33 to 40, wherein the human myeloid cell contains at least one mutation in both alleles of a gene located on one chromosome, and wherein expression of HLA-DQA1 mRNA in said human myeloid cell is at least four-fold, such as at least eight-fold, at least 16-fold, or even at least 32-fold higher than expression of HLA-DQA1 mRNA in an otherwise identical wild-type myeloid cell. 42. The human myeloid cell of any one of clauses 33 to 41, wherein the expression of HLA-DRA mRNA in said human myeloid cell, comprising at least one mutation in both alleles of a gene located on a single chromosome, is at least four-fold, such as at least eight-fold, at least 16-fold, or even at least 32-fold higher than the expression of HLA-DRA mRNA in an otherwise identical wild-type myeloid cell. 43. The human myeloid cell according to any one of clauses 33 to 42, wherein a typical characteristic of an M1-myeloid cell is 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, each of which contains at least one mutation in both alleles of one gene located on one chromosome, and wherein 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 the human myeloid cell is at least four-fold, for example at least eight-fold, at least 16-fold, or even at least 32-fold higher than expression of the mRNA in an otherwise identical wild-type myeloid cell. 44. The human myeloid cell of any one of clauses 31 to 43, wherein a typical characteristic of M1 macrophages is that the expression of at least one mRNA in said human myeloid cell comprises at least one mutation in both alleles of a gene located on one chromosome, is reduced by at least 10-fold compared to the expression of said mRNA in an otherwise identical wild-type myeloid cell, wherein said at least one mRNA is selected from the list consisting of RNASE1, CD28, LYVE1, FCGBP, F13A1, QPCT, CCL7, and RNF128. 45. The human myeloid cell of clause 44, wherein the human myeloid cell contains at least one mutation in both alleles of a gene located on one chromosome, and wherein expression of said at least one mRNA in said human myeloid cell is at least 50-fold lower compared to expression of the same mRNA in an otherwise identical wild-type myeloid cell. 46. ​​The human myeloid cell of any one of clauses 31 to 45, wherein a typical characteristic of M1 macrophages is that the expression of at least three mRNAs in said human myeloid cell, comprising at least one mutation in both alleles of a gene located on one chromosome, is reduced by at least 10-fold compared to the expression of the same at least three mRNAs in an otherwise identical wild-type myeloid cell, wherein the at least three mRNAs are selected from the list consisting of RNASE1, CD28, LYVE1, FCGBP, F13A1, QPCT, CCL7, and RNF128. 47. The human myeloid cell of clause 46, wherein expression of said at least three mRNAs in said human myeloid cell, comprising at least one mutation in both alleles of a gene located on one chromosome, is at least 50-fold lower compared to expression of the same at least three mRNAs in an otherwise identical wild-type myeloid cell. 48. The human myeloid cell of any one of clauses 31 to 47, wherein a typical characteristic of M1 macrophages is that the expression of at least six mRNAs in said human myeloid cell, comprising at least one mutation in both alleles of a gene located on one chromosome, is reduced by at least 10-fold compared to the expression of the same at least six mRNAs in an otherwise identical wild-type myeloid cell, wherein the at least six mRNAs are selected from the list consisting of RNASE1, CD28, LYVE1, FCGBP, F13A1, QPCT, CCL7, and RNF128. 49. The human myeloid cell of any one of clauses 31 to 48, wherein the typical characteristic of M1 macrophages is downregulated secretion of RNASE1. 50. The human myeloid cell of any one of clauses 31 to 49, wherein the typical characteristic of M1 macrophages is downregulated secretion of FCGBP. 51. The human myeloid cell of any one of clauses 22 to 50, wherein said myeloid cell is positive for said surface marker HLA-DRA. 52. The human myeloid cell of any one of clauses 22 to 51, wherein said myeloid cell is positive for said surface marker HLA-DPA1. 53. The human myeloid cell of any one of clauses 22 to 52, wherein said myeloid cell is positive for said surface marker HLA-DQA1. 54. The human myeloid cell of any one of clauses 22 to 53, wherein said myeloid cell is positive for said surface marker HLA-DRB5. 55. The human myeloid cell of any one of clauses 22 to 54, wherein said myeloid cell is positive for said surface marker CD74. 56. The human myeloid cell of any one of clauses 22 to 55, wherein said myeloid cell is positive for said surface marker CD2. 57. The human myeloid cell of any one of clauses 22 to 56, wherein said myeloid cell is negative for said surface marker CD28. 58. The human myeloid cell of any one of clauses 22 to 57, wherein the myeloid cell is negative for the surface marker LYVE1. 59. The human myeloid cell of any one of clauses 22 to 58, wherein said myeloid cell is negative for said surface marker STAB1. 60. The human myeloid cell of any one of clauses 22 to 59, wherein said myeloid cell is negative for said surface marker LILRB5. 61. The human myeloid cell of any one of clauses 22 to 60, wherein said myeloid cell is positive for the surface marker HLA-DR but negative for the surface markers CD28 and LYVE1. 62. The human myeloid cell of any one of clauses 22 to 61, wherein said myeloid cell is positive for the surface markers HLA-DR and CD74, but negative for the surface markers CD28, LYVE1, STAB1, and LILRB5. 63. The human myeloid cell of any one of clauses 22-62, wherein said mutation renders said gene non-functional. 64. The human myeloid cell of any one of clauses 22-62, wherein said mutation inhibits gene expression. 65. The human myeloid cell of any one of clauses 22-62, wherein the mutation abolishes gene expression. 66. The human myeloid cell of any one of clauses 22-65, wherein the mutation is a deletion. 67. The human myeloid cell of clause 66, wherein the deletion is within a promoter, exon, or splice site. 68. The human myeloid cell of any one of clauses 66-67, wherein the deletion is a deletion of at least 50 base pairs. 69. The human myeloid cell of any one of clauses 22 to 68, comprising at least two different mutations. 70. The human myeloid cell of clause 69, wherein the two mutations are deletions in at least two different genes. 71. The human myeloid cell of any one of clauses 22 to 70, wherein the gene is a gene involved in M-CSF, IL-4, IL-10, and / or TGFB1-mediated downregulation of C2TA. 72. The human myeloid cell of any one of clauses 22 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 myeloid cell of any one of clauses 22 to 72, wherein the gene encodes a negative regulator of C2TA transcription. 74. The human myeloid cell of any one of clauses 1 to 73, wherein the gene is MAFB. 75. The human myeloid cell of claim 74, wherein the mutation is within the region of 40,685,000 to 40,690,000 on human chromosome 22. 76. The human myeloid cell of clause 75, wherein the mutation is within the region 40685200 to 40689800, such as 40685400 to 40689600, such as 40685600 to 40689400, and particularly 40685700 to 40689300, on human chromosome 22. 77. The human myeloid cell of any one of clauses 74 to 76, wherein the MAFB is non-functionalized by a deletion of at least 50 base pairs. 78. The human myeloid cell of any one of clause 77, wherein the deletion is a deletion of at least 100 base pairs. 79. The human myeloid cell of any one of clauses 77-78, wherein the deletion is a deletion of at least 250 base pairs. 80. The human myeloid cell of any one of clauses 77 to 79, wherein the deletion is a deletion of between 100 base pairs and 10,000 base pairs. 81. The human myeloid cell of clause 80, wherein the deletion is between 500 base pairs and 3000 base pairs, for example, between 600 base pairs and 1500 base pairs. 82. The human myeloid cell of any one of clauses 74 to 81, further comprising a mutation in the gene MAF. 83. The human myeloid cell of clause 82, wherein the mutation in said MAF is a deletion. 84. The human myeloid cell of any one of clauses 1 to 73, wherein the gene is MAF. 85. The human myeloid cell of claim 84, wherein the mutation is within the region 79593000 to 79602000 on human chromosome 16. 86. The human myeloid cell of clause 85, wherein the mutation is within the region 79593200 to 79601500, for example 79593400 to 79601100, and particularly 79593600 to 79600900, on human chromosome 16. 87. The human myeloid cell of any one of clauses 84 to 86, wherein the MAF is non-functionalized by a deletion of at least 50 base pairs. 88. The human myeloid cell of clause 87, wherein the deletion is a deletion of at least 100 base pairs. 89. The human myeloid cell of any one of clauses 87-88, wherein the deletion is a deletion of at least 250 base pairs. 90. The human myeloid cell of any one of clauses 88-89, wherein the deletion is a deletion of between 100 base pairs and 10,000 base pairs. 91. The human myeloid cell of clause 90, wherein the deletion is between 500 base pairs and 3000 base pairs, for example, between 600 base pairs and 1500 base pairs. 92. The human myeloid cell of any one of clauses 84 to 91, further comprising a mutation in the gene MAFB. 93. The human myeloid cell of clause 92, wherein the mutation in said MAFB is a deletion. 94. The human myeloid cell of clause 69, wherein the two genes are MAFB and MAF. 95. The human myeloid cell of clause 94, wherein both alleles of MAFB and both alleles of MAF are non-functional due to a deletion of at least 50 base pairs. 96. The human myeloid cell of clause 95, wherein all deletions involve exonic DNA. 97. The human myeloid cell of any one of clauses 94-96, wherein the deletions are at least 100 base pairs each. 98. The human myeloid cell of any one of clauses 94-96, wherein the deletions are each at least 250 base pairs. 99. The human myeloid cell of any one of clauses 94 to 98, wherein the deletions are each between 100 base pairs and 10,000 base pairs. 100. The human myeloid cell of any one of clauses 94 to 99, wherein the deletion is between 500 base pairs and 3000 base pairs, for example between 600 base pairs and 1500 base pairs. 101. The human myeloid cell of any one of clauses 94 to 100, wherein the MAFB deletion is within the region 40685000 to 40690000 on human chromosome 22. 102. The human myeloid cell of clause 101, wherein the MAFB deletion is within the region 40685200 to 40689800, such as 40685400 to 40689600, such as 40685600 to 40689400, and particularly 40685700 to 40689300, on human chromosome 22. 103. The human myeloid cell of any one of clauses 94 to 102, wherein the MAF deletion is within the region 79593000-79602000 on human chromosome 16. 104. The human myeloid cell of clause 103, wherein the MAF deletion is within the region 79593200-79601500, for example 79593400-79601100, and particularly 79593600-79600900, on human chromosome 16. 105. The human myeloid cell of any one of clauses 1 to 104, which is non-tumorigenic. 106. The human myeloid cell of any one of clauses 1 to 105, having 46 chromosomes. 107. The human myeloid cell of any one of clauses 1 to 106, which does not contain a chromosome with a chromosomal rearrangement. 108. The human myeloid cell of any one of clauses 1-107, which maintains expression of at least two, e.g., at least four, M1 phenotype markers in vitro in the presence of tumor cells. 109. The human myeloid cell of any one of clauses 1-108, wherein the gene located on the chromosome that contains a biallelic deletion is the only gene that contains a biallelic deletion. 110. The human myeloid cell of any one of clauses 1-109, wherein the gene located on the chromosome that contains a biallelic deletion is the only protein-coding gene that contains a biallelic deletion. 111. The human myeloid cell of any one of clauses 1 to 110, wherein the gene located on a chromosome that contains a biallelic deletion is a transcription factor-encoding gene that contains a biallelic deletion. 112. The human myeloid cell of any one of clauses 1-111, wherein MAF and / or MAFB are the only transcription factor-encoding gene(s) that contain biallelic deletions. 113. The human myeloid cell of any one of clauses 1-112, wherein MAF and / or MAFB are the only protein-coding gene(s) that contain biallelic deletions. 114. The human myeloid cell of any one of clauses 1 to 113, wherein MAF and / or MAFB are the only gene(s) that contain biallelic deletions.

[0226] The practice of the present invention will employ, unless otherwise specified, conventional techniques of molecular biology (including recombinant DNA technology), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in detail in references such as "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 making and practicing the present invention.

[0227] Those skilled in the art will recognize that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all steps, features, compositions, and compounds referred to or shown herein, individually or collectively, as well as any and all combinations of such steps or features, or any two or more thereof. Accordingly, the present disclosure should be considered in all embodiments shown and is not intended to be limiting; the scope of the invention is indicated by the appended claims, and all changes that come within the meaning and range of equivalents are intended to be embraced therein.

[0228] Throughout this application various references are cited, each of which is incorporated herein by reference in its entirety.

[0229] The above description will be more fully understood by reference to the following examples, which are illustrative of methods of practicing the invention and are not intended to limit the scope of the invention. [Example]

[0230] (Example) Aziz et al., 2009, showed that combined MafB and c-Maf deficiency in mice did not adversely affect hematopoiesis, as all lineages, including monocytes and macrophages, were able to be generated (Aziz et al., 2009). Instead, the combined deficiency allowed for a prolonged proliferation period of mature mouse macrophages without loss of differentiated phenotype or function (Aziz et al., 2009).

[0231] We investigated the role of Maf-DKO macrophages in ID8 ovarian tumor models or B16 melanoma tumor models. In these studies, tumor-bearing mice were treated with Maf-DKO mouse macrophages and showed that mouse Maf-DKO macrophages inhibited early and established tumor growth. Furthermore, in vitro results demonstrated that Maf-DKO macrophages possessed an M1-like phenotype and were not repolarized to an M2-like phenotype by typical M2 stimuli. Importantly, tumor cells also failed to repolarize mouse Maf-DKO macrophages.

[0232] Next, we generated human MAF / MAFB DKO macrophages. In vitro experiments confirmed that, like mouse Maf-DKO macrophages, these human MAF / MAFB DKO macrophages possess a stable M1-like phenotype and are not repolarized to an M2-like phenotype by typical M2 stimuli or tumor cells. To our knowledge, MAF / MAFB DKO macrophages are the first example of human macrophages resistant to tumor-induced repolarization due to loss-of-function mutations in both alleles of a single chromosomal gene. More generally, this indicates that human macrophages in a stable antitumor polarization state, generated by loss-of-function mutations in both alleles of a single chromosomal gene required for M2 induction, may be useful for cell therapy, e.g., tumor cell therapy.

[0233] (Mouse MafB / c-Maf DKO macrophages) Materials and Methods Mice: Female mice (C57BL / 6 or Rag2γc knockout), 6–8 weeks old, were used for all experiments. Mice were housed under specific pathogen-free conditions and handled at the CIML (Centre d'Immunologie de Marseille Luminy) in accordance with French and European Union directives.

[0234] Cell lines: Mouse ovarian cancer cell line ID8 and B16 melanoma cell line were cultured in DMEM medium supplemented with 10% fetal calf serum (FCS), 1% penicillin, and streptomycin at 37°C under 5% CO2. Cells were passaged twice a week with trypsin.

[0235] Generation of mouse macrophages: Mouse macrophages were derived from the bone marrow of wild-type or Maf double knockout mice and cultured for 10–12 days in DMEM medium supplemented with 10% FBS and 10–50 ng / ml rMCSF or L929 cell-conditioned medium (20% M-CSF IMDM / 0.5FCS medium, LCM), 2 mM glutamine, 1% sodium pyruvate, and 50 μg / ml penicillin / streptomycin at 37°C in 5% CO2. The Maf-DKO primary cell line (sorted from blood (Aziz et al., 2009)) was passaged every 4 days with partial medium changes every 2 days.

[0236] Adoptive cell transfer of macrophages into tumor-bearing mice: 5 x 10 6 ID8-Luc cells were injected intraperitoneally (ip). Mice were either immediately treated with macrophages or tumors were allowed to establish for 14 days before macrophage transfer. 5 × 10 6 Mice were intraperitoneally transferred with WT or Maf-DKO macrophages. Transfers were repeated four times consecutively on days 0, 2, 4, and 6, or 14, 16, 18, and 20, respectively, with immediate or delayed treatment. To assess tumor progression, mice were dissected on day 27, and ID8 cells were counted by flow cytometry, and luciferase activity in cell lysates was measured.

[0237] 1 x 10 in 100 μl PBS 5 Mice were injected intravenously (iv) with 1 × 10 B16-F10 (or B16-Luc) cells into the eye. 6 The mice were treated with macrophages either immediately after tumor injection or after tumor development for 7 days. The macrophage treatment was repeated four times consecutively. 14 days after tumor injection, the mice were sacrificed, and the lungs and livers were removed. The number of metastatic colonies visible on the surface of the lungs and liver was counted.

[0238] Bioluminescence imaging: Bioluminescence imaging was performed by Berthold Night-Owl technologies. Mice were injected i.p. with luciferin solution (3 mg per mouse) and imaged until peak luminescence was achieved (10 min). Total luminous flux values ​​were obtained from anatomical regions of interest. Data were expressed as relative light units (RLU) of photon emission / s / mm 2 Images were taken of the dynamics of tumor development from day 0 to day 27 (in the ID8 model) or day 0 to day 14 (in the B16 model).

[0239] Luciferase assay: Luciferase assays were performed using the Promega luciferase assay system (TB281). Briefly, cells were washed with 1x PBS and lysed with 20 μl of the provided cell lysis buffer. Cells were then resuspended in 100 μl of luciferase assay reagent, and the light generated was measured using a luminometer.

[0240] Isolation of cells from the peritoneal cavity: After sacrifice, cells were obtained from the peritoneal exudate by lavaging the peritoneal cavity with 10 ml of PBS. Red blood cells were removed by incubation in red blood cell lysis buffer for 5 minutes at room temperature. The peritoneal cell content was assessed using multicolor flow cytometry. Gene expression of the total population was assessed by qPCR.

[0241] Flow cytometry analysis: Cell suspensions were resuspended in FACS buffer (PBS 1X 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. Absolute cell counts were achieved by adding 20 μl of a calibrated microbead suspension to each sample prior to acquisition. Flow cytometry analysis and cell sorting were performed on an LSRII and a FACSAria (Becton Dickinson).

[0242] M1 / M2 in vitro stimulation: 200,000 WT BMDM macrophages or Maf-DKO macrophages were incubated in DMEM medium containing 20 ng / ml murine recombinant M-CSF. Cells were then stimulated with 100 ng / ml LPS (8 hours) or 100 ng / ml IFNγ (8 hours), or 20 ng / ml IL-4 (24 hours). Macrophage stimulation was performed in the presence or absence of 50% supernatant from ID8 conditioned medium. ID8 conditioned medium was harvested after 72 hours of culture. At the indicated time points, supernatants were harvested and cytokine production was assessed by CBA, and cells were lysed by RLT and gene expression was assessed by QPCR. Conditions are summarized in the table below. [Table 3]

[0243] RNA extraction and reverse transcription: Total peritoneal cell populations, CD11b+CD45+Lin-selected macrophages, or in vitro stimulated macrophages were harvested at the time points described above. 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 content was measured using a nanodrop.

[0244] 500 ng of RNA from each sample was used for reverse transcription. 1 μl of oligodt (500 μg / ml) and 1 μl of dNTP mix (10 mM each) were added to nuclease-free sterile water to a final volume of 12 μl. The mixture was heated to 65°C to denature the RNA and rapidly cooled to maintain the denatured form. After a brief centrifugation, 4 μl of first-strand buffer (5x), 2 μl of 0.1 M DTT, and 1 μl of RNaseOut (40 units / μl) (Invitrogen) were added. The mixture was incubated at 42°C for 2 minutes, followed by the addition of 1 μl of Super-Script II reverse transcriptase (200 units, Invitrogen). The mixture was then incubated for 50 minutes at 42°C. The reverse transcriptase was heat-inactivated at 70°C for 15 minutes.

[0245] Quantitative PCR: cDNA was diluted 1:10 and quantitative PCR was performed using 2 μl of 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.

[0246] CBA: Cytokine Bead Array: Cytokines were analyzed using the BD Biosciences "Mouse Inflammatory CBA" kit, catalog number 552364, and Canto II according to the manufacturer's instructions. Sample supernatants were analyzed undiluted.

[0247] Statistical analysis: All experiments were performed in triplicate and representative data are shown. Results were tested for statistical significance using Student's T-test with GraphPad Prism software.

[0248] (result) Example 1 MafB and c-Maf double knockout macrophages inhibit early tumor growth. We previously demonstrated that MafB and c-Maf double-knockout (Maf-DKO) mouse macrophages can grow indefinitely ex vivo upon stimulation with M-CSF without loss of function or malignant transformation (Aziz et al., 2009). To assess the role of MafB and c-Maf in TAM biology, we examined the development of ID8 ovarian tumor cells in mice treated with Maf-DKO macrophages. We first investigated the role of macrophages in the early stages of tumor establishment.

[0249] The ID8 mouse ovarian surface epithelial cell line is frequently used as a syngeneic mouse model for human ovarian cancer. ID8 cancer cells expressing firefly luciferase were injected i.p. into wild-type C57BL6 mice on day 0. The 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 assessed in situ by bioluminescence imaging at the indicated days, from 0 to 27, after adoptive cell transfer (data not shown). Untreated tumor-bearing mice develop malignant ascites on days 4–27, as assessed by quantification of emitted photons. Similarly, tumor-bearing mice treated with WT bone marrow-derived macrophages (WT-BMDM) develop ID8 tumors over the course of the experiment. In contrast, mice treated with bone marrow-derived Maf-DKO macrophages (DKO-BMDM) showed a significant reduction in tumor burden 27 days after tumor induction. Next, peritoneal cells were lysed and luciferase assays were performed to quantify luciferase levels in the ascites (Figure 1b). Luciferase levels were significantly lower in the DKO-BMDM-treated group (squares) compared with the WT-treated or untreated groups (filled circles). Next, we investigated the absolute number of tumor cells in the peritoneum of tumor-bearing mice. To this end, flow cytometry analysis of peritoneal cells was performed, as shown in Figure 1c. ID8 cells were high in SSC and CD45-negative (CD45-). Quantification of tumor cell number showed that the number of ID8 cells was elevated in the peritoneum of untreated and WT-treated mice, but this number was significantly reduced when mice were treated with DKO-BMDM macrophages. Thus, Maf-DKO macrophages were able to reduce intraperitoneal tumor burden.

[0250] Example 2 MafB and c-Maf DKO macrophages induce regression of established tumors. To assess the role of Maf-DKO macrophages in tumor progression, ID8-luciferase tumor cells were injected intraperitoneally into C57BL6 mice on day 0 (Figure 2a). By day 14, these mice had malignant ascites, and tumors were established, visible by bioluminescence (data not shown). Next, WT-BMDM, DKO-BMDM, or blood DKO were adoptively transferred four times at 2-day intervals (Figure 2a). As previously described, tumor progression was assessed by in situ bioluminescence imaging on the indicated days between days 0 and 27. Bioluminescence quantification of emitted photons showed that untreated tumor-bearing mice had massive intraperitoneal malignant ascites on day 27 (data not shown). Similarly, mice treated with WT-BMDM developed robust peritoneal tumors on day 27 (data not shown). In contrast, DKO macrophage-treated mice showed significantly reduced bioluminescence signals on day 27, thus indicating significantly reduced tumor burden (data not shown). For further analysis, mice were sacrificed on day 27, and tumor burden was quantified by luciferase assay and flow cytometry. Luciferase levels were high in peritoneal cells from untreated mice and slightly higher in WT-BMDM-treated mice (Figure 2b, filled circles), reflecting a high level of intraperitoneal tumor burden. Conversely, DKO macrophage-treated mice showed reduced luciferase levels, reflecting a reduced tumor burden (Figure 2b, squares, right column). To confirm the reduction in tumor cell number, the absolute number of ID8 cells in the peritoneal cavity was analyzed by flow cytometry. As previously described, ID8 cells were identified as high-SSC CD45- cells (Figure 2c). While untreated and WT-BMDM-treated mice showed a high percentage of ID8 cells (Fig. 2c, left panel), Maf-DKO-treated mice showed a significantly reduced percentage of ID8 cells (Fig. 2c, right panel). These data indicate that Maf-DKO macrophages can combat and even reduce the burden of established tumors.

[0251] Example 3 (Cellular composition of ascites) Tumors are generally composed of various immune cells that contribute to the tumor stroma of growing malignancies (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 from tumor-bearing mice was performed on day 27. Peritoneal lavage fluid was collected and stained with a cocktail of antibodies against NK cells, CD8 T cells, granulocytes, and macrophages, which are the most representative cells within the tumor stroma (Kerkar and Restifo, 2012).

[0252] First, using this strategy on established tumors 21 days after tumor initiation (Fig. 2a), as shown in Fig. 3a, in addition to macrophages, NK cells and T cells, such as CD8 T cells, were detected in the peritoneal leukocyte fraction. These cell populations were not detected when mice were immediately treated with macrophages as in Example 1 (data not shown). Almost no CD8 T cell infiltration was observed in the peritoneum of untreated or WT-BMDM-treated mice (Fig. 3b, circles and triangles). However, mice treated with Maf-DKO macrophages showed a significant increase in the CD8 T cell population (Fig. 3b, squares). Furthermore, greater NK cell recruitment was observed in Maf-DKO-treated mice compared with untreated or WT-BMDM-treated mice. These results suggest that CD8 T cells and NK cells may be involved in the tumor burden reduction mediated by Maf-DKO macrophages.

[0253] Next, we examined the peritoneal macrophage population based on the CD11b and F4 / 80 markers (Figure 3c). Although the peritoneal cavities of untreated, WT-BMDM-treated, and Maf-DKO-treated mice were filled with macrophages, the proportion of macrophages did not differ among these three groups (data not shown). Therefore, based on a previously published study suggesting that MHCII levels vary within macrophage populations, being high in tumor-suppressing macrophages and low in tumor-promoting macrophages (Wang et al., 2011), we hypothesized that the activation state of macrophage populations may vary, which may explain our previous findings regarding reduced tumor burden in Maf-DKO-treated mice. To test this hypothesis, we stained peritoneal cells with different macrophage activation markers (CD80, CD86, CD69, and MHCII). We observed that all macrophages from Maf-DKO-treated mice were MHCII+ (Fig. 3d, squares), whereas only a small fraction of macrophages from untreated or WT-BMDM-treated mice were MHCII+ (Fig. 3d, circles and triangles). Furthermore, the mean fluorescence intensity of MHCII+ in macrophages from Maf-DKO macrophages-treated mice was higher than that of MHCII-low, MHCII+ macrophages from untreated or WT-BMDM-treated mice (Fig. 3e), suggesting that Maf-DKO macrophages are MHCII-rich. Our data indicate that MHCII-rich TAMs are associated with tumor suppression (in the case of Maf-DKO macrophages), whereas MHCII-low macrophages are associated with tumor promotion (in the case of WT macrophages).

[0254] Example 4 (Deficiency of MafB and c-Maf leads to classical macrophage activation in vivo.) The production of pro-inflammatory versus anti-inflammatory cytokines by tumor-infiltrating leukocytes plays an important role in regulating the immune response to tumors (Balkwill and Mantovani, 2001). Classically activated macrophages (M1) exhibit a cytotoxic pro-inflammatory phenotype (high IL-12, IL-6, IFNγ, and NOS2), whereas 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). The mRNA levels of NOS2, C2TA (MHC II transactivator), IL-6, IL-12, and IL-10 were measured by real-time RTPCR.

[0255] CD11b+ cells from Maf-DKO-treated mice (Fig. 4, striped pattern) showed significantly increased levels of NOS2, CIITA, and IL-6 mRNA, as well as IL-12 mRNA (Fig. 4a-4d), unlike those from WT-treated or untreated control mice (polka dots and solid patterns, respectively). In contrast, CD11b+ cells from WT-treated mice showed elevated IL-10 expression compared with CD11b+-sorted macrophages from Maf-DKO mice (Fig. 4e). Collectively, these results indicate that Maf-DKO macrophages are refractory to in vivo re-education to an M2 phenotype by tumor cells.

[0256] Example 5 Maf-DKO macrophages exhibit stronger classical M1 activation in vitro. The in vivo results for macrophage phenotype indicate that Maf-DKO macrophages most likely resemble M1 macrophages in tumor-bearing mice. To further characterize Maf-DKO macrophages, in vitro experiments were performed in which macrophages (WT or Maf-DKO) were stimulated with either known M1 stimuli (LPS, IFNγ) or M2 stimuli (IL-4). Cells were then lysed and gene expression was measured by real-time QPCR.

[0257] As expected, IL-6 expression was detected upon stimulation with LPS (Fig. 5a), and IL-6 mRNA levels were significantly higher in Maf-DKO macrophages than in WT macrophages (Fig. 5a; note that DKO macrophages are polka-dotted and WT macrophages are striped in Fig. 5). Expression of C2TA (a positive regulator of MHCII genes, an M1 marker) was increased in Maf-DKO macrophages compared with WT macrophages upon stimulation with IFNγ, LPS, and, to a lesser extent, IL-4 (Fig. 5b, polka-dotted vs. striped), another indication of the M1-like phenotype of Maf-DKO macrophages. Expression of NOS2 was also increased in Maf-DKO macrophages compared with WT macrophages upon stimulation with LPS or IFNγ (Fig. 5c). In contrast, monitoring the expression of M2 genes revealed that IL-10 (upon stimulation with IL-4 and LPS) (Fig. 5d) and arginase (upon stimulation with IL-4) (Fig. 5e) were highly expressed in WT macrophages but not in Maf-DKO macrophages. These results suggested that Maf-DKO macrophages were sensitive to M1 stimuli, similar to classically activated macrophages, but were much less sensitive, if at all, to M2 stimuli.

[0258] Example 6 Maf-DKO macrophages are refractory to re-education with tumor cells in vitro. Next, we investigated whether Maf-DKO macrophages could be educated by tumors in vitro. To do so, we created a mimicking in vivo environment by culturing either WT or Maf-DKO macrophages in medium containing ID8 tumor supernatant. We confirmed the production of proinflammatory cytokines by CBA (see Materials and Methods). IL-6 was produced by both WT and Maf-DKO macrophages upon stimulation with LPS (Fig. 6a, black bars). IL-6 production by WT macrophages was significantly reduced in the presence of ID8 tumor cell supernatant, whereas the level produced by Maf-DKO macrophages under the same conditions remained high (Fig. 6a, checkered bars). Similar to IL-6, TNFα was also produced by both WT and Maf-DKO macrophages in the absence of ID8-SN (Fig. 6b, black bars). However, in the presence of ID8-SN, Maf-DKO macrophages still produced large amounts of TNFα, whereas WT macrophages showed a significant reduction in TNFα production (Fig. 6b, checkered bars). These results suggest that Maf-DKO macrophages are M1-like macrophages that are refractory and resistant to reprogramming by the tumor microenvironment, whereas WT macrophages can be reprogrammed by tumors.

[0259] Example 7 (Maf-DKO macrophages inhibit melanoma development.) To investigate the antitumor activity of Maf-DKO macrophages in a second independent tumor model, we examined the effect of these macrophages on B16 melanoma development. B16 was used as an experimental metastasis model. B16 melanoma is a murine tumor cell line used in research as a model for studying metastasis and solid tumor formation. B16 tumor cells (1 × 10 5) was intravenously transferred into C57BL6 mice via retroorbital injection, resulting in the development of tumors in the lungs. Mice were then injected with Maf-DKO macrophages, WT macrophages, or left uninjected. Mice were treated with the different types of macrophages only after tumor establishment (Figure 7a). When mice were sacrificed on day 14, metastatic tumors appeared as darkly pigmented colonies measuring 1–3 mm in diameter and tending to fuse with each other. B16 tumor cells formed numerous tumor nodules in the lungs of untreated mice (Figure 7b, left column), which was confirmed by quantification of tumor colonies (Figure 7c, left panel). Maf-DKO macrophages (Figures 7b and 7c, right column) strongly reduced tumor formation compared with untreated control mice and had a stronger effect than WT-treated mice (Figures 7b and 7c, center column). Thus, the absence of MafB and c-Maf in macrophages reduces lung metastasis.

[0260] Taken together, these data indicate that Maf-DKO macrophages can suppress tumor progression in preventative and therapeutic settings, regardless of tumor model.

[0261] 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 antitumor mechanism, we used Rag2γc knockout mice, which lack T cells, B cells, and NK cells. 1 × 10 firefly luciferase-expressing B16 tumor cells were injected into the Rag2γc knockout mice. 5Mice were challenged intravenously with Maf-DKO macrophages, WT macrophages, or left untreated. Immediately after challenge (Fig. 8a) or on day 7 after tumor establishment (Fig. 8b), mice were treated four consecutive times with Maf-DKO macrophages, WT macrophages, or left untreated. Tumor growth was monitored using bioluminescence imaging and quantitative analysis of tumor nodules. As evidenced by bioluminescence imaging on day 14, tumors developed in all mice. Compared with our results on B16 development in WT mice (Example 7), tumors were localized in the lungs and liver, indicating an increased number of metastatic colonies in both organs (Figs. 8c–8f, data show liver only). Both the lungs and liver of untreated and WT-treated mice showed strong infiltration of B16 tumor cells (c shows liver data). Importantly, mice immediately treated with Maf-DKO macrophages showed a significant reduction in liver metastases compared with WT-treated mice (left panel) or untreated mice (not shown) (Fig. 8c, right panel). This Maf-DKO-mediated tumor regression was confirmed by quantitative analysis of metastatic nodules (Figure 8e, polka-dot columns). However, in mice with established tumors, only slight tumor regression 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 and 8f), suggesting that effector immune cells (NK cells, T cells, and B cells) are involved in the Maf-DKO antitumor effect. These data indicate that Maf-DKO macrophages achieve the above-mentioned therapeutic role by both directly inhibiting tumor growth and metastasis at the early stage of tumor development and indirectly suppressing tumors sustainably by cooperating with other immune cells.

[0262] (Examination of mouse experiments) In this study, we provided evidence for the role of Maf-DKO macrophages in preventing tumor growth. Transfer of Maf-DKO macrophages, but not WT macrophages, into tumor-bearing mice (ID8 ovarian carcinoma or B16 melanoma) significantly reduced both the initial and established tumor burden (Figures 1, 2, and 7) and increased mouse survival. Furthermore, the antitumor activity of Maf-DKO macrophages was not impaired even after long-term maintenance with M-CSF (expanded Maf-DKO cells obtained from blood and maintained in culture for long periods showed antitumor activity comparable to that of bone marrow-derived Maf-DKO cells).

[0263] Without intending to be bound by any theory, the as yet unknown mechanism of Maf-DKO tumor inhibition is likely both direct and indirect. Results from Rag2γc knockout mice (which lack T cells, B cells, and NK cells) suggest that Maf-DKO macrophages, unlike WT macrophages, continuously cooperate with other immune cells to achieve tumor regression even in the presence of tumors in vivo. Furthermore, we demonstrated that T cells, such as CD8 T cells and, to a lesser extent, NK cells, were recruited to the peritoneal cavity of established tumor-bearing mice at early time points after Maf-DKO adoptive cell transfer (Figures 3a and 3b). Collectively, these results suggest that Maf-DKO macrophages may similarly inhibit tumor growth through the activation of T cells, such as CD4 T cells or CD8 T cells, or NK cells. Furthermore, we show that macrophages from Maf-DKO-treated mice are (high) MHC II compared with peritoneal macrophages from WT-treated mice. Importantly, a shift from (high) MHC II to (low) MHC II in TAMs has been demonstrated to mediate tumor progression in mice (Wang et al., 2011). Similarly, another study showed that CD169+ macrophages trigger antitumor immune responses through their ability to phagocytose and cross-present tumor cell peptides to cytotoxic T cells (Asano et al., 2011). Collectively, these data and our results suggest that Maf-DKO antitumor activity may be due in part to the activation of T cells via tumor peptide presentation, leading to their subsequent activation.

[0264] Preliminary in vitro results on coculture of ID8 tumor cells with Maf-DKO or WT macrophages showed that more lactate dehydrogenase (LDH) was released during coculture of tumor cells with Maf-DKO macrophages (data not shown), supporting the idea that more ID8 cell lysis occurs upon contact with Maf-DKO macrophages, indicating that Maf-DKO macrophages have a direct killing effect on tumor cells, at least in vitro.

[0265] Previous literature suggests that coculture with ID8 cells polarizes macrophages toward an M2-like phenotype by increasing IL-10 expression and decreasing IL-12 (Hagemann et al., 2006). Ex vivo monitoring of the profiles of TAMs from Maf-DKO-treated mice demonstrated that Maf-DKO macrophages, compared with WT macrophages, remained stable M1-like macrophages despite exposure to the tumor environment. Furthermore, the phenotype of Maf-DKO macrophages was analyzed in vitro by measuring the expression levels of IL-6, C2TA, NOS2, arginase, and Il-10 mRNA. Maf-DKO macrophages were shown to be more sensitive to M1 stimuli (LPS and IFNγ) through upregulation of NOS2, C2TA, and IL-6, and downregulation of arginase and IL-10, whereas WT macrophages were more sensitive to M2 stimuli (IL-4), exhibiting the opposite expression profile.

[0266] Furthermore, in vitro treatment of Maf-DKO macrophages with tumor cell-conditioned medium (ID8-SN) did not switch their phenotype from M1 to M2, further demonstrating that Maf-DKO macrophages resist repolarization by the tumor secretome.

[0267] These experiments provide evidence for the role of Maf-DKO macrophages in driving antitumor immune responses, opening new avenues for cell therapy of cancer.

[0268] (Human MAF / MAFB DKO macrophages) Methods for generating human induced pluripotent stem cells are well known in the art. Human iPS cell lines were generated from skin fibroblasts obtained from the foreskin of a healthy Caucasian newborn boy. Reprogramming was performed using a non-integrating Sendai virus containing POU5F1, SOX2, KLF4, and MYC (Fusaki N. et al., 2009). Viral screening for HIV1, HIV2, hepatitis B virus, and hepatitis C virus, as well as mycoplasma, was negative. Karyotyping revealed a normal 46(X,Y) karyotype. A doxycycline-inducible Cas9 expression cassette was introduced into this cell line by TALEN-mediated gene targeting at the AAVS1 locus, generating the cell line BIHi001-A-1, essentially as described in Gonzalez et al. (2014).

[0269] Example 9 (Creation of double knockout (DKO) cells from human iPS cells) (a) iPS cell culture iPS cells were cultured under feeder-free conditions in StemFlex medium (Thermo Fisher, No. A3349401) on vitronectin-coated (Thermo Fisher, No. A14700) tissue culture plates at 37°C under normoxic conditions with 5% CO2. At 60–80% confluency, cells were passaged in ReLeSR (Stem Cell Technologies, No. 05872) and maintained the day after passage in the presence of 10 μM ROCK inhibitor Y-27632 (biomol, No. Cay10005583). Single-cell suspensions of iPS cells were obtained by incubation with Accutase (Merck Millipore, No. SF006).

[0270] (b) Genome editing We used the CRISPR / Cas9 system to delete the coding sequences of MAF (gene ID: 4094, 373 amino acids) and MAFB (gene ID: 9935, 323 amino acids) in human cells. To this end, an iPS cell line derived from a healthy donor, carrying a doxycycline-inducible Cas9 expression cassette integrated into the AAVS1 locus (BIHi001-A-1, https: / / hpscreg.eu / cell-line / BIHi001-A-1; also known as iBCRT Cas9v1-3G-Kl.16), was transfected with an sgRNA expression plasmid targeting MAF and MAFB (pU6-(BbsI)sgRNA_CAG-venus-bpA, Addgene ID 86985, https: / / www.addgene.org / 86985 / ) according to published protocols (Yumlu, 2017).

[0271] To delete the complete CDS, two sgRNAs per gene were designed using the CrispRGold program for sequence design (https: / / crisprgold.mdc-berlin.de / Chu et al., 2016) to target sequences in 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. The two sgRNAs for each gene were co-expressed from the sgRNA expression vector pU6-(BbsI)sgRNA_CAG-venus-bpA, which also encodes a fluorescent reporter gene (Venus, YFP) for positive selection by FACS of transfected cells.

[0272] [Table 4] Table 1: sgRNA and protospacer sequences for CRISPR / Cas9-mediated deletion of 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 sequences of the sgRNAs expressed from pU6-(BbsI)sgRNA_CAG-venus-bpA with the indicated protospacer sequences are located in the UTRs, 5'UTRs, and 3'UTRs of MAF and MAFB, respectively.

[0273] [ka]

[0274] BIHi001-A-1 was transfected with the sgRNA expression vector pCAG-mTrex2-bpA (Addgene ID 86984, https: / / www.addgene.org / 86984 / ) using Lipofectamine 3000 (ThermoFisher, no. L3000001) to enhance indel formation. Control cells, designated "WT" or "wild-type" throughout, were treated identically except that pU6-(BbsI)sgRNA_CAG-venus-bpA was used without the protospacer sequence. Transfected BIHi001-A-1 cells were treated with 1 μg / ml doxycycline hyclate (Sigma-Aldrich, no. D9891) to induce Cas9 expression, and cells expressing the Venus reporter gene from the sgRNA-carrying vector were then FACS-isolated. Sorted cells were seeded at low density and single-cell derived colonies were isolated. In the first round of transfection, sgRNA targeting MAF was used, and colonies were screened for full-length deletion of the MAF CDS using PCR and Sanger sequencing analysis of the edited locus (Figure 9). Cells were cultured under iPS cell conditions as described above.

[0275] After a second transfection of MAF KO iPS cells with MAFB-targeting sgRNA, PCR and sequencing analysis of the resulting colonies were performed. 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 loci are shown in Figure 10.

[0276] Example 10 (Induced differentiation of iPS cells into macrophages) Directed differentiation of iPS cells into macrophages was performed according to 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 in ultra-low attachment U-bottom 96-well plates (Nunclon Sphera, Thermo Fisher Scientific, #174925) and resuspended in EB medium (StemFlex medium, Thermo Fisher Scientific, #A3349401) supplemented with 50 ng / ml BMP-4 (R&D Systems, #314-BP), 20 ng / ml SCF (R&D Systems, #255-SC), 50 ng / ml VEGF (PeproTech, #AF-100-20A), and 10 μM Y-27632 to generate embryoid bodies (EBs). The 96-well plate was centrifuged at 100 g for 3 minutes to collect the cells at the bottom, and then incubated at 37°C, 5% CO for 4 days. After 1 and 2 days, half of the EB medium was replaced with freshly prepared EB medium.

[0277] (Wild-type and MAF-DKO EBs were indistinguishable by size or morphology.) After 4 days, EBs were manually selected and resuspended in EB differentiation medium consisting of X-VIVO 15 (Lonza, #BE02-060F) supplemented with 2 mM GlutaMAX (Thermo Fisher, #35050038), 0.055 mM 2-mercaptoethanol (Sigma-Aldrich, #M6250), 100 ng / ml human M-CSF (Thermo Fisher, #PHC9504), and 25 ng / ml human IL-3 (R&D Systems, #203-IL). The EBs were seeded onto ultra-low attachment 6-well tissue culture plates (8 EBs / well) or 90 mm tissue culture dishes (24 EBs / dish; Nunclon Sphera, Thermo Fisher, #174932 and #174945). Two-thirds of the culture medium was replaced with fresh differentiation medium every 5 days. Monocyte / macrophage generation from EBs began approximately 15 days after EB seeding, and suspension cells were harvested, counted, and used for immunophenotyping by flow cytometry or EdU incorporation assay.

[0278] For further experiments, cells harvested from the supernatant of EB cultures were replated for terminal macrophage differentiation in RPMI supplemented with 10% FBS (PAA-GE Healthcare, A15-101), 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 incubated at 37°C in 5% CO2 in ultra-low attachment 12-well tissue culture plates (Nunclon Sphera, Thermo Fisher, no. 174931, no. 174932) and 50 ng / ml GM-CSF (PeproTech, no. 300-03). The cells were counted manually using a Neubauer hemocytometer or automatically using a CASY cell counter (OMNI Life Science).

[0279] Deletion of MAF and MAFB did not affect myeloid differentiation potential; similar to wild-type EBs, MAF-DKO EBs began releasing monocytes / macrophages into the EB differentiation medium approximately 15 days after EB plating. MAF-DKO suspension 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 beads, produced ROS, and exhibited a mature macrophage phenotype, staining positive for lysosomes and cathepsin activity.

[0280] Example 11 iPS cell-derived MAF-DKO and WT macrophages obtained from EB differentiation cultures essentially as described in Example 11 were harvested 20 days after EB seeding by collecting cells in suspension. 500,000 cells were replated per well in 6-well plates (3 ml medium per well) and maintained for 5 days for terminal differentiation in the presence of M-CSF and GM-CSF (partial medium changes with fresh medium every other day, essentially as described in Example 11). On day 5, cells were harvested and replated at 100,000 cells per well in Nunclon 12-well plates in complete medium (containing M-CSF and GM-CSF as described in Example 11) and maintained for an additional 2 days (2 ml medium per well). Seven days after harvesting the suspension cells, the medium was replaced with fresh complete medium (containing M-CSF and GM-CSF, both at 50 ng / ml). Cells were then harvested 2 hours after medium change, and 10,000 DAPI-negative, CD45-positive, C11b-positive cells were sorted directly into LRT plus lysis buffer, and RNA was extracted using the RNeasy Micro Kit (Quiagen, catalog no. 74034). Differential gene expression patterns were then analyzed by RNAseq, as described in detail in Picelli et al. (2013) Nature Methods 10:1096-1098. RNA isolated from 10,000 cells was analyzed at a sequencing depth of 38 million fragments per library. Alignment of fragments to the human reference (hg38) was performed with GSNAP (v2020-12-16; [Thomas D. et al. (2005) Bioinformatics 21:1859-1875], [Thomas D. et al. (2010) Bioinformatics 26:873-881]), and splice sites were detected using Ensembl gene annotation 98 ([Howe et al. (2021) NAR vol. 49(1):884-891]).Uniquely aligned fragments were counted using featureCounts (v2.0.1; [Liao et al., 2014] "featureCounts: an efficient general-purpose program for assigning sequence reads to genomic features"] with the same Ensembl annotation support. Meanwhile, the sequenced libraries were subjected to quality control with RNA-SeQC (v1.1.8; [DeLuca et al., 2012] Bioinformatics 28.11:1530-1532). This included the exon, intron, and intergenic distribution of reads and rRNA ratios within each library. Normalization of raw fragment counts based on library size and testing for differential expression between the two conditions (DKO for 2 hours and WT for 2 hours) were performed using DESeq2 (v1.24.0; [Love et al., 2014] Genome Biology, 15, 550]), and 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 the R package (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 differentially expressed.

[0281] The differences in gene expression patterns between MAF-DKO and wt macrophages are dramatic, as visualized in the volcano plot shown in Figure 11. As shown in the table below, compared to wt macrophages, the indicated M1 markers were strongly upregulated in DKO macrophages, whereas the indicated M2 markers were strongly downregulated, despite the presence of M-CSF at a concentration of 50 ng / ml during terminal differentiation. This indicates that human MAF-DKO macrophages have essentially the same polarized phenotype as murine MAF-DKO macrophages and are therefore predicted to have essentially the same anti-tumorigenic properties as murine MAF-DKO macrophages.

[0282] [Table 5]

Claims

1. Allogeneic human myeloid cells for therapeutic use in a human patient, comprising at least one MHC-II complex identical to any one of the MHC-II complexes of the human patient.

2. Allogeneic human myeloid cells according to claim 1, for use in cancer treatment.

3. Allogeneic human myeloid cells for use in cancer treatment according to claim 2, wherein the allogeneic myeloid cells comprise at least one protein complex selected from the group consisting of HLA-DR, HLA-DP, and HLA-DQ, and the protein complex is identical to any of the corresponding HLA complexes of the human patient.

4. Allogeneic human myeloid cells for use in cancer treatment according to claim 3, wherein the allogeneic myeloid cells comprise two protein complexes selected from the group, preferably comprising three complexes selected from the group.

5. Allogeneic human myeloid cells for use in cancer treatment according to claim 2 or 3, wherein the allogeneic myeloid cells comprise at least one HLA-DR protein complex identical to any of the corresponding HLA-DR complexes of the human patient, and / or at least one HLA-DP protein complex identical to any of the corresponding HLA-DP complexes of the human patient, and / or at least one HLA-DQ protein complex identical to any of the corresponding HLA-DQ complexes of the human patient.

6. Allogeneic human myeloid cells for use in cancer treatment according to claim 2, wherein the allogeneic myeloid cells and the patient being treated have in common at least one pair of alleles selected from the group consisting of: HLA-DRA / HLA-DRB1, HLA-DRA / HLA-DRB3, HLA-DRA / HLA-DRB4, HLA-DRA / HLA-DRB5, HLA-DPA1 / HLA-DPB1, and HLA-DQA1 / HLA-DQB1.

7. Allogeneic human myeloid cells for use in cancer treatment according to claim 6, wherein the allogeneic myeloid cells and the patient being treated also have in common all 2, 3, 4, 5, or 6 pairs of alleles.

8. Allogeneic human myeloid cells for use in cancer treatment according to claim 2, wherein the allogeneic human myeloid cells are resistant to M-CSF-induced M2 polarization.

9. Allogeneic human myeloid cells for use in cancer treatment according to claim 2, wherein the allogeneic human myeloid cells are genetically modified.

10. Allogeneic human myeloid cells for use in cancer treatment according to claim 9, wherein the gene recombination is the introduction of a gene encoding a surface protein, such as a chimeric antigen receptor.

11. Allogeneic human myeloid cells for use in cancer treatment according to claim 2, wherein the allogeneic human myeloid cells are derived from iPS cells.

12. A collection of allogeneic human myeloid cells for use in cancer treatment according to claim 2, wherein the collection of allogeneic human myeloid cells is capable of recruiting T cells and / or NK cells to a tumor in vivo.

13. The collection of allogeneic human myeloid cells for use in cancer treatment according to claim 12, wherein the number of allogeneic human myeloid cells is at least 10,000,000.

14. Allogeneic myeloid cells for use according to claim 2, wherein the allogeneic myeloid cells are macrophages.

15. The collection of allogeneic myeloid cells for use according to claim 13, wherein the allogeneic myeloid cells are macrophages.

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