Methods for macrophage polarization

Abstract

The invention relates to a chimeric cytokine receptor and application of the chimeric cytokine receptor in switching polarization of macrophages or mononuclear cells from M2 to M1. The invention also relates to a method for monocyte modification to selectively increase phagocytic activity and modulate polarization of progeny macrophages.

Description

[0001] This application claims the benefit of priority of European patent application EP22215498.1 filed on December 21, 2022, and European patent application EP23200945.6 filed on September 29, 2023, both of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to a chimeric cytokine receptor and its use in changing the polarization of macrophages or monocytes from M2 to M1. The present invention also relates to a method for modifying monocytes to selectively increase phagocytic activity and regulate the polarization of progeny macrophages. Background Art

[0003] Monocytes are white blood cells that circulate in the bloodstream and, upon entering their target tissues, can differentiate into, among other things, macrophages. One of the main properties of macrophages is the ingestion of dead or diseased cells, making them a crucial part of the immune system involved in many diseases.

[0004] Macrophages can adjust their immune function based on the activation signals they are exposed to in target tissues. This process of macrophage polarization can produce M1-polarized macrophages and M2-polarized macrophages. M1 macrophages, also known as classically activated macrophages, are important in acute infectious diseases, particularly those caused by intracellular bacteria and viruses, and are also crucial for phagocytosis of tumor cells. M2 macrophages, also known as alternatively activated macrophages, are important for parasite defense and tissue remodeling.

[0005] In addition to the activation of macrophages, there are other mechanisms that control their immune function. The phagocytosis of cells by macrophages is controlled by the CD47-SIRPα axis. The binding of the widely expressed cell surface protein CD47 to the cell surface molecule SIRPα on macrophages transmits the "don't eat me signal" to the macrophages. This mechanism is intended to prevent macrophages from engulfing healthy cells, but it also prevents macrophages from fighting diseased cells or at least reduces the effectiveness of macrophages against diseased cells. It has been found that some diseased cells (such as tumor cells) overexpress CD47, thereby protecting themselves from the effects of macrophage phagocytosis.

[0006] Based on the above-mentioned prior art, the object of the present invention is to provide means and methods for improving the activity of macrophages against diseased cells. This object is achieved by the subject matter of the independent claims of the present specification, and further advantageous embodiments are set forth in the dependent claims, examples, drawings and general description of the present specification. Summary of the Invention

[0007] A first aspect of the present invention relates to an isolated monocyte or macrophage comprising a chimeric cytokine receptor (ChCR) polypeptide heterodimer comprising or consisting of a first ChCR polypeptide and a second ChCR polypeptide;

[0008] Wherein the first ChCR polypeptide comprises:

[0009] - a first extracellular domain,

[0010] - the first type 1 transmembrane domain,

[0011] -optionally, a first flexible linker domain, which connects the first extracellular domain and the first transmembrane domain,

[0012] - a first intracellular domain, and

[0013] Wherein the second ChCR polypeptide comprises:

[0014] - a second extracellular domain,

[0015] - a second type 1 transmembrane domain,

[0016] -optionally, a second flexible linker domain, which connects the second extracellular domain and the second transmembrane domain,

[0017] - the second intracellular domain,

[0018] in

[0019] - the first extracellular domain and the second extracellular domain are capable of inducing dimerization of the ChCR polypeptide heterodimer upon binding to a cytokine selected from IL-10 and TGF-β; and

[0020] - the first intracellular domain and the second intracellular domain are capable of activating IFNγ receptor / Jak1 / Jak2 / STAT1 signaling in monocytes and / or macrophages upon dimerization of the ChCR polypeptide heterodimer. The ChCR induces intracellular signaling (equivalent to interferon γ stimulation) upon binding to IL-10 or TGF-β.

[0021] Another aspect of the present invention relates to the isolated monocytes or macrophages according to any one of the preceding aspects, for use in treating or preventing cancer.

[0022] Another aspect of the present invention relates to a kit comprising:

[0023] - an expression vector encoding a ChCR polypeptide as described above;

[0024] - an inhibitory nucleic acid molecule against SIRPα as described above; and

[0025] - an expression vector encoding a CAR polypeptide as described above.

[0026] Another aspect of the present invention relates to a method for modifying monocytes, comprising:

[0027] i. providing mononuclear cells obtained from a mammalian donor,

[0028] ii. inserting into the monocytes a nucleic acid sequence encoding the ChCR polypeptide according to the first aspect;

[0029] iii. maintaining the monocytes under cell culture conditions.

[0030] Terms and Definitions

[0031] For the purpose of interpreting this specification, the following definitions shall apply and, where appropriate, terms used in the singular shall include the plural and vice versa. In the event of a conflict between any definition set forth below and any document incorporated herein by reference, the set forth definition shall control.

[0032] As used herein, the terms "comprising," "having," "containing," and "including," and other similar forms and grammatical equivalents thereof, are intended to be equivalent in meaning and to be open-ended, in that one or more items following any of these words is not intended to be an exhaustive list of such one or more items or to be limited to only the listed one or more items. For example, an article "comprising" components A, B, and C can consist of components A, B, and C (i.e., contain only components A, B, and C), or can contain not only components A, B, and C, but also one or more additional components. Thus, it is intended and understood that "comprising" and similar forms, and grammatical equivalents thereof, include disclosure of embodiments that "consist essentially of" or "consist of."

[0033] Where a range of values ​​is provided, it is understood that unless the context clearly dictates otherwise, every intervening value (to the tenth of the unit of the lower limit) between the upper and lower limits of the stated range and any other stated or intervening value in the stated range is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the upper and lower limits, ranges excluding either or both of those included upper and lower limits are also encompassed within the disclosure.

[0034] Reference herein to "about" a value or parameter includes (and describes) variations directed to that value or parameter itself. For example, description referring to "about x" includes description of "X."

[0035] As used herein, including in the appended claims, the singular forms "a," "an," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0036] "And / or" when used in this document is considered to be a specific recitation of each of the two specified features or components with or without the other. Thus, the term "and / or" used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to cover each of the following: A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used for molecular, genetic and biochemical methods (see generally Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and Ausubel et al., Short Protocols in Molecular Biology (2002), 5th ed., John Wiley & Sons, Inc.) and chemical procedures.

[0038] Any patent documents cited herein should be deemed to be incorporated by reference in their entirety.

[0039] sequence

[0040] Sequences that are similar or homologous (e.g., at least about 70% sequence identity) to the sequences disclosed herein are also part of the present invention. In some embodiments, the sequence identity at the amino acid level can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. At the nucleic acid level, the sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. Alternatively, substantial identity exists when the nucleic acid segment will hybridize to the complementary sequence of the chain under selective hybridization conditions (e.g., very high stringency hybridization conditions). The nucleic acid can be present in whole cells, in cell lysates, or in partially purified or substantially pure form.

[0041] In the context of this specification, the terms sequence identity and sequence identity percentage refer to a single quantitative parameter representing a sequence comparison result determined by comparing two aligned sequences position by position. Methods for aligning sequences for comparison are well known in the art. Sequence alignment for comparison can be performed by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482 (1981), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by the similarity search method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85: 2444 (1988), or by computerized implementations of these algorithms, including but not limited to CLUSTAL, GAP, BESTFIT, BLAST, FASTA, and TFASTA. Software for performing BLAST analyses is publicly available, for example, through the National Center for Biotechnology Information (http: / / blast.ncbi.nlm.nih.gov / ).

[0042] An example of comparing amino acid sequences is the BLASTP algorithm using the default settings: expect threshold: 10; word length: 3; maximum number of matches within the query region: 0; matrix: BLOSUM62; gap cost: presence 11, extension 1; composition adjustment: conditional composition score matrix adjustment. An example of comparing nucleic acid sequences is the BLASTN algorithm using the default settings: expect threshold: 10; word length: 28; maximum number of matches within the query region: 0; match / mismatch score: 1.-2; gap cost: linear. Unless otherwise indicated, sequence identity values ​​provided herein refer to the values ​​obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol., 215:403-410 (1990)) using the default parameters defined above for protein and nucleic acid comparisons, respectively.

[0043] Reference to identical sequences without specifying a percentage value means 100% identical sequences (ie, identical sequences).

[0044] General biochemistry: peptides, amino acid sequences

[0045] In the context of this specification, the term polypeptide refers to a molecule consisting of 50 or more amino acids forming a linear chain in which the amino acids are linked by peptide bonds. The amino acid sequence of a polypeptide can represent the amino acid sequence of an entire (physiologically found) protein or a fragment thereof. The terms "polypeptide" and "protein" are used interchangeably herein and include proteins and fragments thereof. Polypeptides are disclosed herein as sequences of amino acid residues.

[0046] In the context of the present specification, the term peptide relates to a molecule consisting of up to 50 amino acids, particularly 8 to 30 amino acids, more particularly 8 to 15 amino acids, which form a linear chain wherein the amino acids are linked by peptide bonds.

[0047] The amino acid residue sequence is given from the amino terminus to the carboxyl terminus. Capital letters at the sequence position refer to the L-amino acid in the one-letter code (Stryer, Biochemistry, 3rd ed., p. 21). Lowercase letters at the amino acid sequence position refer to the corresponding D-amino acid or (2R)-amino acid. The sequence is written from left to right in the direction from the amino terminus to the carboxyl terminus. According to standard nomenclature, amino acid residue sequences are designated by three-letter or one-letter codes as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0048] In the context of this specification, the term dimer refers to a unit consisting of two subunits.

[0049] In the context of this specification, the term homodimer refers to a dimer comprising two subunits that are identical members or highly similar members of the same class of subunits.

[0050] In the context of this specification, the term amino acid linker refers to a polypeptide of variable length for connecting two polypeptides to generate single-chain polypeptides. Exemplary embodiments of the linker of the present invention that can be used for implementing the present invention specified herein are oligopeptide chains consisting of 1, 2, 3, 4, 5, 10, 20, 30, 40 or 50 amino acids. Glycine-serine linkers are composed of glycine and serine, while glycine linkers are composed of glycine subunits. The limiting example of an amino acid linker is the polypeptide GSGGGGSGGGGS (SEQ ID NO 023), which connects the extracellular antigen-binding domain to the membrane-spanning domain.

[0051] General molecular biology: nucleic acid sequences, expression

[0052] The term gene refers to a polynucleotide containing at least one open reading frame (ORF) that, when transcribed and translated, encodes a specific polypeptide or protein. Polynucleotide sequences can be used to identify larger fragments or full-length coding sequences of related genes. Methods for isolating larger fragment sequences are known to those skilled in the art.

[0053] In the context of this specification, the term transgenic refers to a gene or genetic material that has been transferred from one organism to another. In the context of the present invention, the term may also refer to the transfer of a natural or physiologically intact variant of a genetic sequence into the tissue of a patient lacking that variant. It may further refer to the transfer of a natural coding sequence whose expression is driven by a promoter that is absent or silenced in the target tissue.

[0054] In the context of this specification, the term recombinant relates to a nucleic acid that is the product of one or several steps of cloning, restriction and / or ligation and that differs from a naturally occurring nucleic acid. A recombinant viral particle comprises a recombinant nucleic acid.

[0055] The term gene expression or expression, or alternatively the term gene product, can refer to any one or both of the processes and products thereof that generate nucleic acids (RNA) or generate peptides or polypeptides (also referred to as transcription and translation, respectively), or any intermediate process that regulates the processing of genetic information to produce a polypeptide product. The term gene expression can also be applied to RNA gene products, such as the transcription and processing of regulatory RNA or structural (e.g., ribosomal) RNA. If the expressed polynucleotide is derived from genomic DNA, expression can include the splicing of mRNA in eukaryotic cells. Expression can be measured at the transcription and translation (in other words, mRNA and / or protein product) levels.

[0056] In the context of this specification, the term knockdown of mRNA relates to a reduction in the amount of that specific mRNA.

[0057] In the context of this specification, the term nucleotide refers to a nucleic acid or nucleic acid analog building block, an oligomer of which is capable of forming selective hybrids with RNA or DNA oligomers on the basis of base pairing. The term nucleotide herein includes the classical ribonucleotide building blocks adenosine, guanosine, uridine (and ribosylthymidine), cytidine, the classical deoxyribonucleotides deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. It further includes analogs of nucleic acids such as phosphorothioates, 2'O-methyl phosphorothioates, peptide nucleic acids (PNA; N-(2-aminoethyl)-glycine units linked by peptide bonds, wherein the nucleobase is attached to the α-carbon of glycine) or locked nucleic acids (LNA; 2'O, 4'C methylene-bridged RNA building blocks). Wherever a hybridization sequence is mentioned herein, such a hybridization sequence may be composed of any of the above nucleotides or a mixture thereof.

[0058] The term phosphothioate as used herein is synonymous with the terms phosphorothioate and thiophosphate.

[0059] In the context of this specification, the term capable of forming a hybrid or hybridizing sequence relates to a sequence that is capable of selectively binding to its target sequence under conditions present in the cytosol of a mammalian cell. Such hybridizing sequences can be continuous reverse complements to the target sequence, or can contain gaps, mispairings, or additional non-matching nucleotides. The minimum length of a sequence capable of forming a hybrid depends on its composition, with C or G nucleotides contributing more to the binding energy than A or T / U nucleotides, and on the backbone chemistry.

[0060] In the context of this specification, the term hybridizing sequence encompasses polynucleotide sequences comprising or essentially consisting of RNA (ribonucleotides), DNA (deoxyribonucleotides), phosphorothioate deoxyribonucleotides, 2'-O-methyl modified phosphorothioate ribonucleotides, LNA and / or PNA nucleotide analogs. In certain embodiments, the hybridizing sequence according to the present invention comprises 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In certain embodiments, the hybridizing sequence is at least 80% identical to the reverse complement of SEQ ID 33 to SEQ ID 42, more preferably 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical. In certain embodiments, the hybridizing sequence comprises deoxynucleotides, phosphorothioate deoxynucleotides, LNA and / or PNA nucleotides, or mixtures thereof.

[0061] In the context of this specification, the term inhibitory nucleic acid molecule relates to a nucleic acid molecule that reduces the amount of the functional mRNA of the target molecule inside the cell. In particular, inhibitory nucleic acid molecule relates to an oligonucleotide having a sequence that is substantially complementary to RNA and can hybridize with RNA. The antisense effect to this RNA will cause the biological effect of RNA to be regulated, particularly to be suppressed or suppressed. If RNA is mRNA, the expression of the resulting gene product is suppressed or suppressed. Inhibitory nucleic acid molecule can be made up of DNA, RNA, nucleotide analogs and / or their mixture. Technicians are aware of various commercial and non-commercial sources for calculating the theoretically optimal antisense sequence for a given target. Optimization can be performed in terms of core base sequence and backbone (ribose, deoxyribose, analogue) composition. There are many sources for delivering actual physical oligonucleotides.

[0062] In the context of this specification, the term antisense oligonucleotide relates to an oligonucleotide having a sequence that is substantially complementary to RNA and can hybridize with RNA. The antisense effect to this RNA will cause the biological effect of RNA to be regulated, particularly suppressed or suppressed. If RNA is mRNA, the expression of the resulting gene product is suppressed or suppressed. Antisense oligonucleotide can be made up of DNA, RNA, nucleotide analogs and / or their mixture. Technicians know various commercial and non-commercial sources for calculating the theoretically best antisense sequence for a given target. Optimization can be performed in terms of core base sequence and backbone (ribose, deoxyribose, analogue) composition. There are many sources for delivering actual physical oligonucleotides, and this actual physical oligonucleotide is synthesized by solid-state synthesis conventionally.

[0063] In the context of this specification, the term siRNA (small / short interfering RNA) refers to an RNA molecule that can interfere with the expression of a gene (in other words, inhibit or prevent the expression of a gene) that contains a nucleic acid sequence that is complementary to or hybridizes with the sequence of the siRNA, a process known as RNA interference. The term siRNA is intended to encompass both single-stranded siRNA and double-stranded siRNA. siRNA is typically characterized by a length of 17 to 24 nucleotides. Double-stranded siRNA can be derived from longer double-stranded RNA molecules (dsRNA). According to mainstream theory, longer dsRNAs are cut by endoribonucleases (called Dicer) to form double-stranded siRNAs. In a nucleoprotein complex (called RISC), double-stranded siRNAs unwind to form single-stranded siRNAs. RNA interference typically works by binding siRNA molecules to mRNA molecules with complementary sequences, resulting in mRNA degradation. siRNA molecules can also bind to intron sequences of precursor mRNAs (immature non-spliced ​​mRNAs) in the nucleus, resulting in precursor mRNA degradation, thereby performing RNA interference.

[0064] In the context of the present specification, the term shRNA (small hairpin RNA) relates to artificial RNA molecules with tight hairpin bends, which can be used to silence target gene expression by RNA interference (RNAi).

[0065] In the context of this specification, the term sgRNA (single guide RNA) relates to an RNA molecule capable of sequence-specific inhibition of gene expression via the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) mechanism.

[0066] In the context of this specification, the term miRNA (microRNA) relates to small non-coding RNA molecules (containing approximately 22 nucleotides) that play a role in RNA silencing and post-transcriptional regulation of gene expression.

[0067] In the context of this specification, term nucleic acid expression vector relates to a kind of artificial nucleic acid molecule, and this artificial nucleic acid molecule is used as the vehicle that exogenous nucleic acid molecule is carried into another cell.Nucleic acid expression vector relates to plasmid, viral genome or RNA, and it is used for transfecting (in the case of plasmid or RNA) or transducing (in the case of viral genome) target cell with specific gene of interest, or when RNA construct is transfected, translates corresponding protein of interest according to the mRNA of transfection.For the carrier operating on the level of transcription and subsequent translation, gene of interest is controlled by promoter sequence, and promoter sequence is operable inside target cell, therefore, gene of interest is transcribed in response to stimulation or depends on the state of cell.In certain embodiments, viral genome is packaged into capsid to become viral vector, and this viral vector can transduce target cell.

[0068] In the context of this specification, the term promoter relates to a nucleic acid sequence that initiates transcription of a specific nucleic acid sequence in macrophages. Different promoters are well known in the art, and they are widely used in genetics as part of a vector containing a nucleic acid sequence to be transcribed. Specific types of promoters are only active under certain conditions, either in the presence or absence of certain molecules (inducible promoters), or in certain cellular environments (such as cell type-specific promoters).

[0069] In the context of this specification, the term chimeric antigen receptor relates to an artificially engineered receptor comprising a portion of an antigen receptor.

[0070] The activation motif (ITAM) based on immunoreceptor tyrosine is an important component of the intracellular signal transduction mechanism of the cell surface protein of the immune system. This ITAM is located in the cytoplasmic tail of the cell surface protein, and after the cell surface protein interacts with its corresponding ligand, the tyrosine residue of ITAM can be phosphorylated. The tyrosine residue of phosphorylation forms a docking site for other downstream components in the signal transduction mechanism. The ITAM motif comprises a tyrosine separated by any other two amino acids and leucine or isoleucine.

[0071] In the context of this specification, the term chimeric cytokine receptor relates to an artificially engineered receptor comprising a portion of a cytokine receptor.

[0072] Type II cytokine receptors are transmembrane proteins expressed on the surface of certain cells that bind to and respond to a select group of cytokines. These receptors are similar to type I cytokine receptors, except that they do not have the signature sequence WSXWS that is unique to type I receptors. Typically, type II cytokine receptors are heterodimers or multimers with high- and low-affinity components.

[0073] Type I cytokine receptors are transmembrane receptors expressed on the surface of cells that recognize and respond to cytokines with four α-helical chains. These receptors are also known as erythropoietin receptors and share a common amino acid motif (WSXWS) in the extracellular portion adjacent to the cell membrane. Members of the type I cytokine receptor family contain different chains, some of which are involved in ligand / cytokine interactions, while others are involved in signal transduction.

[0074] Type 1 transmembrane domains are derived from type I membrane proteins.

[0075] In the context of this specification, the term type 1 membrane protein relates to single-pass membrane proteins anchored to the lipid membrane, whose N-terminal domain is targeted to the ER lumen during synthesis and to the extracellular space in its mature form.

[0076] In the context of this specification, the term IFN in monocytes and / or macrophages γ Receptor / Jak1 / Jak2 / STAT1 signaling is involved in the pro-inflammatory activation of monocytes / macrophages via the interferon gamma receptor signaling cascade. Interferon gamma receptor-induced Jak1 / Jak2 / STAT1 signaling converts monocyte differentiation from dendritic cells to macrophages and induces the production of pro-inflammatory cytokines in macrophages, thereby converting them into pro-inflammatory "M1" macrophages. Interferon gamma receptor signaling can be initiated by the ChCR of the present invention. In many cancers, M1 activation of macrophages is positively correlated with longer survival time and most positive clinical outcomes.

[0077] (Cancer) immunotherapy

[0078] In the context of this specification, the terms cancer immunotherapy, biological or immunomodulatory therapy are intended to encompass various types of cancer treatment that help the immune system fight cancer.

[0079] As used herein, the term pharmaceutical composition refers to a compound of the present invention or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the present invention is provided in a form suitable for topical, parenteral or injectable administration.

[0080] As used herein, the term pharmaceutically acceptable carrier includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and the like, and combinations thereof, known to those skilled in the art (see, e.g., Remington: the Science and Practice of Pharmacy, ISBN 085711624).

[0081] The term cancer used in the context of this specification relates to malignant neoplastic diseases; the terms "cancer" and "malignant neoplastic diseases" are used synonymously herein. They specifically include carcinomas (epithelial-derived cancers), sarcomas (connective tissue-derived cancers), lymphomas and leukemias, germ cell-derived tumors and blastomas. Specific alternatives to any aspect and embodiment disclosed herein relate to the use of the compounds and compositions of the present invention in the treatment of solid tumors. Other alternatives to any aspect and embodiment disclosed herein relate to the use of the combination of the present invention in the treatment of liquid cancers (such as myeloid or granulocytic leukemias, in particular AML, lymphoid, lymphocytic or lymphoblastic leukemias and lymphomas, polycythemia vera or polycythemia).

[0082] As used herein, the terms treating / treatment of any disease or condition (e.g., cancer) refer, in one embodiment, to ameliorating the disease or condition (e.g., slowing or preventing or reducing the development of the disease or at least one of its clinical symptoms). In another embodiment, "treating" or "treatment" refers to alleviating or improving at least one physical parameter, including those that the patient may not be able to discern. In yet another embodiment, "treating" or "treatment" refers to regulating the disease or condition physically (e.g., stabilizing discernible symptoms), physiologically (e.g., stabilizing physical parameters), or both. Unless specifically described below, methods for evaluating the treatment and / or prevention of a disease are generally known in the art. DETAILED DESCRIPTION

[0083] A first aspect of the present invention relates to an isolated monocyte or macrophage. The isolated monocyte or macrophage comprises a chimeric cytokine receptor (ChCR) polypeptide heterodimer. The ChCR polypeptide heterodimer comprises or consists of a first ChCR polypeptide and a second ChCR polypeptide.

[0084] The first ChCR polypeptide comprises, in particular from N- to C-terminus:

[0085] - a first extracellular domain,

[0086] - the first type 1 transmembrane domain,

[0087] -optionally, a first flexible linker domain, which connects the first extracellular domain and the first transmembrane domain,

[0088] - first intracellular domain.

[0089] The second ChCR polypeptide comprises, in particular from N- to C-terminus:

[0090] - a second extracellular domain,

[0091] - a second type 1 transmembrane domain,

[0092] -optionally, a second flexible linker domain, which connects the second extracellular domain and the second transmembrane domain,

[0093] - second intracellular domain.

[0094] The first extracellular domain and the second extracellular domain are capable of inducing dimerization of the ChCR polypeptide heterodimer upon binding to a cytokine selected from IL-10 and TGF-β.

[0095] The first intracellular domain and the second intracellular domain are capable of activating IFNγ receptor / Jak1 / Jak2 / STAT1 signaling in monocytes and / or macrophages upon dimerization of the ChCR polypeptide heterodimer.

[0096] In certain embodiments,

[0097] - the first extracellular domain is or comprises the extracellular domain of IL-10 receptor α (CDW210A);

[0098] - the second extracellular domain is or comprises the extracellular domain of IL-10 receptor beta (CDW210B);

[0099] - the first intracellular domain is or comprises the IFNγ receptor 1 (CD119) intracellular domain; and

[0100] - The second intracellular domain is or comprises the IFNγ receptor 2 intracellular domain.

[0101] In certain embodiments, the ChCR polypeptide heterodimer comprises:

[0102] - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 21,

[0103] - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28;

[0104] - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 22,

[0105] - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29;

[0106] In particular, the ChCR polypeptide heterodimer has ≥85%, particularly ≥90%, ≥95% of the biological activity of the ChCR heterodimer of SEQ ID NO 53 and SEQ ID NO 54.

[0107] In certain embodiments,

[0108] - the first extracellular domain is or comprises the extracellular domain of IL-10 receptor α (CDW210A);

[0109] - the second extracellular domain is or comprises the extracellular domain of IL-10 receptor beta (CDW210B);

[0110] - the first intracellular domain is or comprises an IFNγ receptor 2 intracellular domain; and

[0111] - The second intracellular domain is or comprises the IFNγ receptor 1 (CD119) intracellular domain.

[0112] In certain embodiments, the ChCR polypeptide heterodimer comprises:

[0113] - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 21,

[0114] - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and

[0115] - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29;

[0116] - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 22,

[0117] - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and

[0118] - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28;

[0119] In particular, the ChCR polypeptide heterodimer has ≥85%, particularly ≥90%, ≥95% of the biological activity of the ChCR heterodimer of SEQ ID NO 59 and SEQ ID NO 60.

[0120] In certain embodiments,

[0121] - the first extracellular domain is or comprises a TGF-β receptor type I extracellular domain;

[0122] - the second extracellular domain is or comprises a TGF-β receptor type II extracellular domain;

[0123] - the first intracellular domain is or comprises the IFNγ receptor 1 (CD 119) intracellular domain; and

[0124] - The second intracellular domain is or comprises the IFNγ receptor 2 intracellular domain.

[0125] In certain embodiments, the ChCR polypeptide heterodimer comprises:

[0126] - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 23,

[0127] - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and

[0128] - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28;

[0129] - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 24,

[0130] - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and

[0131] - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29;

[0132] In particular, the ChCR polypeptide heterodimer has ≥85%, particularly ≥90%, ≥95% of the biological activity of the ChCR heterodimer of SEQ ID NO 55 and SEQ ID NO 56.

[0133] In certain embodiments,

[0134] - the first extracellular domain is or comprises a TGF-β receptor type I extracellular domain;

[0135] - the second extracellular domain is or comprises a TGF-β receptor type II extracellular domain;

[0136] - the first intracellular domain is or comprises an IFNγ receptor 2 intracellular domain; and

[0137] - The second intracellular domain is or comprises the IFNγ receptor 1 (CD119) intracellular domain.

[0138] In certain embodiments, the ChCR polypeptide heterodimer comprises:

[0139] - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 23,

[0140] - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and

[0141] - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29;

[0142] - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 24,

[0143] - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and

[0144] - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28;

[0145] In particular, the ChCR polypeptide heterodimer has ≥85%, particularly ≥90%, ≥95% of the biological activity of the ChCR heterodimer of SEQ ID NO 61 and SEQ ID NO 62.

[0146] The biological activity of chimeric cytokine receptors can be measured in reporter cells, which are HEK-Blue TM IFN-γ cells (InvivoGen). Briefly, ChCR was introduced into HEK-Blue TM In the IFN-γ cell line, which contains an IFN-γ-driven STAT-1-inducible secreted alkaline phosphatase (SEAP) reporter gene but lacks any IL-10 receptor or TGF-β receptor, respectively, the amount of SEAP can be easily assessed in a colorimetric assay (QuantiBlue), and the SEAP signal is proportional to the biological activity.

[0147] Another aspect of the present invention relates to an isolated monocyte or macrophage comprising a nucleic acid molecule encoding the ChCR polypeptide of the first aspect. The nucleic acid molecule encodes the ChCR together with a signal peptide. The signal peptide affects the transport of the ChCR to the cell surface.

[0148] In certain embodiments, the nucleic acid molecule is contained in an expression vector, wherein the ChCR polypeptide heterodimer is under the control of a promoter sequence operable in mammalian monocytes or macrophages. In certain embodiments, the expression vector is selected from the group consisting of a viral vector, a plasmid, a DNA molecule, or an RNA molecule. In certain embodiments, the expression vector is a lentiviral vector.

[0149] In certain embodiments, the monocytes or macrophages additionally comprise an inhibitory nucleic acid molecule against SIRPα.

[0150] In certain embodiments, the inhibitory nucleic acid molecule against SIRPα comprises a sequence selected from the group consisting of: SEQ ID NO 33 to SEQ ID NO 52.

[0151] In certain embodiments, the monocyte or macrophage additionally comprises a chimeric antigen receptor (CAR) polypeptide comprising:

[0152] - extracellular antigen binding domain,

[0153] - transmembrane domain, and

[0154] - an intracellular domain comprising at least one ITAM motif.

[0155] In certain embodiments, the CAR comprises a linker domain connecting the extracellular antigen binding domain and the transmembrane domain.

[0156] In certain embodiments, the monocyte or macrophage additionally comprises a chimeric antigen receptor (CAR) polypeptide comprising:

[0157] - an extracellular antigen-binding domain comprising a Fab fragment,

[0158] - a transmembrane domain comprising the CD8 transmembrane domain, and

[0159] - an intracellular domain comprising the intracellular domain of the FC receptor.

[0160] In certain embodiments, the intracellular domain of CAR comprises the intracellular domain of an Fc gamma receptor. In certain embodiments, the intracellular domain of CAR comprises the intracellular domain of CD32a.

[0161] In certain embodiments, the CAR polypeptide comprises a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% identical to any one of SEQ ID NO 13 to SEQ ID NO 18.

[0162] Another aspect of the present invention relates to the isolated monocytes or macrophages according to the fourth aspect for use in medicine.

[0163] Another aspect of the present invention relates to the isolated monocytes or macrophages according to any one of the preceding aspects, for use in treating or preventing cancer.

[0164] Another aspect of the present invention relates to a kit comprising:

[0165] - an expression vector encoding a ChCR polypeptide as described above;

[0166] - an inhibitory nucleic acid molecule against SIRPα as described above; and

[0167] - an expression vector encoding a CAR polypeptide as described above.

[0168] In certain embodiments, the components of the kit are encoded on a single vector (e.g., on a lentiviral vector). In certain embodiments, the components of the kit are encoded on two or three vectors.

[0169] Another aspect of the present invention relates to a method for modifying monocytes, comprising:

[0170] i. providing mononuclear cells obtained from a mammalian donor,

[0171] ii. inserting a nucleic acid sequence encoding the ChCR polypeptide as described above into the monocytes;

[0172] iii. maintaining the monocytes under cell culture conditions.

[0173] In certain embodiments of the method, additionally,

[0174] - an inhibitory nucleic acid molecule against SIRPα as described above; and / or

[0175] - a CAR polypeptide as described above;

[0176] is inserted into the monocytes.

[0177] Another aspect of the present invention relates to a ChCR polypeptide heterodimer comprising:

[0178] - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 21,

[0179] - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and

[0180] - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28;

[0181] - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 22,

[0182] - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and

[0183] - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29.

[0184] Another aspect of the present invention relates to a ChCR polypeptide heterodimer comprising:

[0185] - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 21,

[0186] - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and

[0187] - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29;

[0188] - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 22,

[0189] - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and

[0190] - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28.

[0191] Another aspect of the present invention relates to a ChCR polypeptide heterodimer comprising:

[0192] - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 23,

[0193] - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and

[0194] - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28;

[0195] - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 24,

[0196] - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and

[0197] - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29.

[0198] Another aspect of the present invention relates to a ChCR polypeptide heterodimer comprising:

[0199] - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 23,

[0200] - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and

[0201] - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29;

[0202] - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 24,

[0203] - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and

[0204] - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28.

[0205] Medical treatment

[0206] Similarly, within the scope of the present invention is also a method for treating cancer in a patient in need thereof, comprising administering to the patient a monocyte or macrophage according to the above description.

[0207] Manufacturing method and treatment method according to the present invention

[0208] As an additional aspect, the present invention further encompasses the use of a monocyte or macrophage as identified herein in a method for the manufacture of a medicament for the treatment or prevention of cancer.

[0209] Similarly, the present invention encompasses methods of treating a patient who has been diagnosed with a disease associated with cancer. The methods entail administering to the patient an effective amount of monocytes or macrophages as identified herein.

[0210] When alternatives to a single separable feature (such as a monocyte or macrophage or receptor sequence or medical indication) are listed herein as "embodiments," it is understood that such alternatives can be freely combined to form separate embodiments of the invention disclosed herein. Thus, any alternative embodiment of a monocyte or macrophage can be combined with any alternative embodiment of a receptor sequence, and these combinations can be combined with any medical indication mentioned herein.

[0211] This specification further covers the following terms:

[0212] Terms:

[0213] 1. A CAR polypeptide comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to any one of SEQ ID NO 13 to SEQ ID NO 18.

[0214] 2. A ChCR polypeptide heterodimer comprising:

[0215] - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 21,

[0216] - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and

[0217] - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28;

[0218] - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 22,

[0219] - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and

[0220] - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29;

[0221] The ChCR polypeptide heterodimer has ≥85%, particularly ≥90%, ≥95% of the biological activity of the ChCR heterodimer of SEQ ID NO 53 and SEQ ID NO 54.

[0222] 3. A ChCR polypeptide heterodimer comprising:

[0223] - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 23,

[0224] - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and

[0225] - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28;

[0226] - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 24,

[0227] - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and

[0228] - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29;

[0229] The ChCR polypeptide heterodimer has ≥85%, particularly ≥90%, ≥95% of the biological activity of the ChCR heterodimer of SEQ ID NO 55 and SEQ ID NO 56.

[0230] 4. A ChCR polypeptide heterodimer comprising:

[0231] - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 21,

[0232] - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and

[0233] - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29;

[0234] - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 22,

[0235] - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and

[0236] - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28;

[0237] The ChCR polypeptide heterodimer has ≥85%, particularly ≥90%, ≥95% of the biological activity of the ChCR heterodimer of SEQ ID NO 59 and SEQ ID NO 60.

[0238] 5. A ChCR polypeptide heterodimer comprising:

[0239] - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 23,

[0240] - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and

[0241] - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29;

[0242] - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 24,

[0243] - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and

[0244] - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28;

[0245] The ChCR polypeptide heterodimer has ≥85%, particularly ≥90%, ≥95% of the biological activity of the ChCR heterodimer of SEQ ID NO 61 and SEQ ID NO 62.

[0246] The present invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be derived. These examples are intended to illustrate the present invention but are not intended to limit the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0247] Figure 1 Shown is A) knockdown of SIRPα in THP-1 cells using nine different siRNAs targeting SIRPα. Protein expression was normalized relative to nonspecific "non-targeting" siRNA (siRNA NT). B) The inventors further characterized the three most promising SIRPα-specific siRNAs (3, 6, and 9) as miRNAs in THP-1 and transduced them with lentiviral vectors expressing these miRNAs. C) The most promising miRNA 3 was then verified in primary human macrophages. As a negative control, the inventors used nonspecific "non-targeting" miRNA (miRNA NT). SIRPα expression was measured by flow cytometry, and the measured median fluorescence intensity (MFI) was normalized relative to miRNA NT.

[0248] Figure 2 Figure 2 shows the CD19-specific phagocytosis of tumor cells by macrophages expressing mCAR. A) Schematic diagram of mCAR design: the extracellular human CD19-specific scFv antibody fragment is connected to the intracellular signaling domain containing ITAM via the CD8a stem and transmembrane domain. B) and C) The present inventors evaluated the phagocytic activity of mCAR in transduced macrophages (which also express RFP) by FACS. CFSE-labeled Raji cells were used as targets. Macrophages that engulfed Raji cells were RFP and CFSE double positive. The assay was performed using macrophages from four different donors.

[0249] Figure 3 Shown is the EGFR-specific phagocytosis of tumor cells by macrophages expressing mCAR. A) Binding of human primary macrophages expressing cetuximab mCAR to EGFR. B) The present inventors evaluated the phagocytic activity of cetuximab mCAR in transduced macrophages (which also express RFP) by FACS. CFSE-labeled MDA-MB-231 cells (which express EGFR) were used as targets. Macrophages engulfing MDA-MB-231 cells were RFP and CFSE double positive. The assay was performed using macrophages from two different donors. Macrophages transduced with CD19-specific mCAR were used as controls for non-specific phagocytosis. In addition, both experimental groups were treated with cytochalasin D (CytD), which inhibits phagocytosis.

[0250] Figure 4 The generation of a functional chimeric cytokine receptor (ChCR) is shown. A) Design and mode of action of IL-10ChCR. The extracellular portion binds to IL-10, while the intracellular domain derived from the IFN-γ receptor induces the STAT1 signaling pathway, causing M1 polarization of macrophages. B) The inventors evaluated the function of ChCR IL-10-IFNGR in a cell line expressing the SEAP gene (driven by IFN-γ). Adding IL-10 to cells transduced with two IL-10ChCR subunits gave a positive signal. In C) transduced reporter cells and D) transduced THP-1, STAT1 phosphorylation after IL-10 stimulation of IL-10ChCR was verified by protein immunoblot (Western blot). Transduced THP-1 responded to IL-10 stimulation in a dose-dependent manner. E) STAT 1 phosphorylation was further verified by flow cytometry in primary macrophages that were not transduced, mock-transduced, or transduced with IL-10ChCR.

[0251] Figure 5 The results of genetically engineered macrophages expressing ChCR stimulated with IL-10 are shown. Monocyte-derived macrophages (MDMs) expressing IL-10ChCR undergo M1 polarization after stimulation with IL-10. Monocytes from healthy donors were either not transduced, simulated transduced, or transduced with a lentivirus expressing IL-10ChCR. After differentiation into MDMs, they were polarized for 2 days with 5ng / ml IFN-γ or 100ng / ml IL-10. The expression of polarization markers was assessed by flow cytometry. N=3-4, p values ​​were calculated using paired t-tests (for CD38+% and CD163+%) and ratio paired t-tests (for MFI).

[0252] Figure 6 TGF-β ChCR induces pSTAT1. Flow cytometric analysis of STAT 1 phosphorylation in THP-1 cells and macrophages that were mock-transduced or transduced with TGF-β ChCR was performed. Cells were stimulated as indicated and pSTAT 1 was measured by flow cytometry.

[0253] Figure 7 The results of genetically engineered macrophages expressing TGF-β ChCR with TGF-β stimulation are shown. The monocyte-derived macrophages (MDMs) expressing ChCR undergo M1 polarization after stimulation with TGF-β. Monocytes from healthy donors are either simulated transduced or transduced with a lentivirus expressing TGF-β ChCR. After differentiation into MDMs, they are polarized for 2 days as indicated. The expression of surface markers is assessed by flow cytometry. N=3, p values ​​are calculated using paired t-test (for CD38+) and ratio paired t-test (for MFI).

[0254] Figure 8 Shown is the secretion of IP-10 by monocyte-derived macrophages (MDMs) expressing ChCR upon stimulation with TGF-β or IL-10. Monocytes from healthy donors were either not transduced, mock-transduced, or transduced with a lentivirus expressing ChCR. After differentiation into MDMs, they were polarized for 2 days with the indicated concentrations of IFN-γ, IL-10, or TGF-β. IP-10 secretion in the supernatant was analyzed by ELISA. N=4.

[0255] Figure 9 Figure 20 illustrates the tumoricidal effect of the conditioned medium of the genetically engineered macrophages expressing ChCR on the TNBC cell line.Monocytes from healthy donors were either not transduced, or simulated transduction was performed, or transduced with a lentivirus expressing IL-10ChCR (A) or TGF-βChCR (B).After differentiation into MDM, they were polarized for 2 days as indicated.MDA-MB-231 or BT-549 cell lines were processed using the conditioned medium from the polarized macrophages for 3 days.As a control, MDA-MB-231 and BT-549 were directly processed using the cytokines for stimulating macrophages.WST1 was used to determine viability.Results using the conditioned medium from 1 to 4 donors were shown.

[0256] Figure 10Shown are A) Transduction efficacy of human primary macrophages transduced with an empty lentiviral vector ("mock"), lentiviral vectors expressing IL-10 ChCR, and TGF-β ChCR. N=14-33. B) Surface expression of the two subunits of IL-10 ChCR, IL-10Ra and IL-10Rb, on human primary macrophages, shown as MFI and % IL-10Ra and IL-10Rb+, N=4. C) Surface expression of TGF-β ChCR on human primary macrophages, shown as MFI and % TGFbR2+ cells, N=5.

[0257] Example

[0258] Example 1: SIRPα miRNA: Enhances the phagocytic activity of macrophages

[0259] Signal regulatory protein (SIRP)-α on MΦ binds to the cell surface molecule CD47, which is ubiquitous on almost all cells. SIRPα's recognition of CD47 transmits an inhibitory "don't eat me signal" to MΦ. Therefore, the SIRPα-CD47 axis serves as a major checkpoint for phagocytosis and is involved in the cell renewal of senescent cells. Notably, senescent cells lose their CD47 expression and are therefore susceptible to phagocytosis. By virtue of the potential to exploit phagocytosis, this "don't eat me signal" has become the focus of new cancer therapies. The inventors will promote the phagocytic activity of MΦ by downregulating SIRPα expression using specific microRNAs (miRNAs). miRNAs are small non-coding RNA molecules that regulate post-transcriptional gene expression. The inventors identified a macrophage-like cell line, THP-1, which expresses SIRPα at high levels and is therefore optimal for screening miRNAs against SIRPα. In THP-1, the inventors screened a total of 10 siRNA candidates ( Figure 1 A), and three of them were validated as miRNAs with different knockdown abilities ( Figure 1 B). The inventors then transduced human monocytes isolated from peripheral blood mononuclear cells (PBMCs) with a lentiviral vector encoding one of the most promising (miRNA3). The inventors then validated SIRPα knockdown in human primary macrophages ( Figure 1 C). Non-targeting (NT) miRNA was used as a negative control. Indeed, miRNA 3 downregulated SIRPα expression in human primary macrophages to a level of approximately 0.5, compared to a NT miRNA level of 1.0.

[0260] Example 2: Generation of chimeric antigen receptors (mCARs) that are expressed on macrophages and induce tumor cell Antigen-specific phagocytosis

[0261] In the first case, the inventors were investigating which cytoplasmic tail region was most effective in activating genetically modified macrophages. Therefore, the inventors used the same external portion of the CAR in all of the constructs they designed, i.e., they designed a macrophage chimeric antigen receptor (mCAR) with a single-chain variable fragment (scFv) mouse antibody (i.e., FMC63), which binds to human CD19 (huCD19) and has been successfully used in CAR T cell receptors ( Figure 2 A). Importantly, the amino acid sequence of the anti-CFD19 scFv is publicly available (ADM64594.1), suitable reagents are commercially available to evaluate mCAR, and CD19-expressing Raji cells (which serve as the target for CAR-expressing cells) are already available.

[0262] The cytoplasmic tail region that the present inventors are exploring is the FcγR chain and FcγRIIa (CD32A) ( Figure 2 A variant 1 and 2). For reference, the present inventors include a CAR published by CARISMA, which contains a CD3ζ signaling domain. CD3ζ is usually part of the T cell receptor complex, but it appears that it can also induce Syk signaling in macrophages, thereby promoting phagocytosis. However, the present inventors are convinced that the Fcγ chain and FcγRIIa chain are better choices for generating mCARs because they are inherently expressed by macrophages and play a major role in antibody-dependent cellular phagocytosis (ADCP). The common feature of these FcγR chains is the immunoreceptor tyrosine-based activation motif (ITAM), and activation of these motifs by phosphorylation leads to strong Syk signaling in macrophages and promotes antigen-specific cellular phagocytosis.

[0263] The inventors found that all three mCARs showed similar cell surface expression levels and antigen binding abilities (data not shown). More interestingly, the inventors verified the antigen-specific phagocytosis of human primary macrophages after recognizing target cells (Raji cells) expressing CD 19. In brief, the inventors transduced macrophages with viral vectors encoding different receptors and then co-cultured them with fluorescently labeled Raji cells as target cells. The inventors also transduced macrophages with a lentiviral vector encoding only a reporter gene but lacking mCAR ("mock" control). The inventors found that macrophages with all different mCARs engulfed substantially more Raji cells ( Figure 2 B)-There is no difference between the various types of cytoplasmic tails ( Figure 2 C).

[0264] We then characterized the antigen binding ability of the EGFR-specific cetuximab mCAR in primary human macrophages for the first time. The inventors stained the transduced macrophages with recombinant EGFR protein and verified the binding of the protein by staining the Avi tag of the recombinant EGFR ( Figure 3 A). The inventors also evaluated the ability of the cetuximab mCAR to induce antigen-specific phagocytosis of the EGFR-expressing tumor cell line MDA-MB-231. As a control, the CD19-specific mCAR was cut off, which should not induce phagocytosis because the MDA-MB-231 cell line does not express CD19. As a further control, the inventors added cytochalasin D, which inhibits phagocytosis, to show that the mCAR induces phagocytosis rather than simply binding to target cells. The inventors could clearly show that the cetuximab mCAR specifically induces phagocytosis of MDA-MB-231 cells.

[0265] Example 3: Macrophage Chimeric Cytokine Receptor

[0266] The TME is rich in anti-inflammatory cytokines such as IL-10 and TGF-β. In fact, the present inventors will benefit from the presence of IL-10 or TGF-β in the TME. To this end, the present inventors will generate genetically modified macrophages that express chimeric cytokine receptors that bind, for example, IL-10 but trigger pro-inflammatory signals via the cytosolic IFN-γ chain.

[0267] Example 4: IL-10-IFNγ Chimeric Cytokine Receptor (IL-10ChCR)

[0268] As a first prototype, the present inventors designed and generated a chimeric cytokine receptor pair that has the extracellular recognition domains of IL-10, IL-10Rα and Rβ, and the intracellular cytoplasmic domains of IFN-γR1 and -R2 ( Figure 4 A).

[0269] Homodimerization of the prototype chimeric cytokine receptor in response to IL-10 binding should trigger the IFN-γ signaling pathway through phosphorylation of the corresponding cytoplasmic domain via signal transducer and activator of transcription 1 (STAT 1). Phosphorylated STAT 1 signaling triggered by IFN-γ leads to polarization of MΦ toward the classic "M1" rather than the alternative "M2" phenotype.

[0270] The present inventors verified that the chimeric cytokine receptor was expressed in HEK-Blue TM Expression and function of IFN-γ in cells (InvivoGen) Figure 4 B) Briefly, the present inventors introduced IL-10ChCR into HEK-Blue TMIFN-γ cell line, which contains an IFN-γ driven STAT-1 inducible secretory alkaline phosphatase (SEAP) reporter gene, but lacks any IL-10 receptor. The amount of SEAP can be easily assessed in a colorimetric assay (QuantiBlue). IL-10Rα-IFN-γR1 together with IL-10Rβ-IFN-γR2 resulted in the highest overall SEAP activity in response to IL-10 expression. In addition, the inventors observed an increase in SEAP activity in cells transduced with only Rα but not with Rβ upon IL-10 stimulation. The inventors concluded that high expression of the high affinity IL-10Rα-IFN-γR1 subunit is sufficient to induce some STAT1 signaling upon binding to IL-10. The inventors investigated this by using a HEK-Blue TM The above data were confirmed by Western blotting in IFN-γ cells and the human monocytic cell line THP-1, demonstrating that STAT1 was phosphorylated after ChCR stimulation with IL-10 ( Figure 4 C and Figure 4 D). Also in human primary macrophages, the present inventors demonstrated that STAT1 was phosphorylated after ChCR stimulation with IL-10 ( Figure 4 E) STAT1 is the first non-receptor kinase to be phosphorylated upon activation of certain cytokine receptors. Here, the inventors induced the IFNγ signaling pathway by stimulating a chimeric cytokine with IL-10.

[0271] Having obtained this data with the IL-10 ChCR, the inventors started by generating a second variant of the ChCR consisting of the extracellular domain of the TGF-β receptor and the intracellular domain of the IFN-γ receptor (TGF-β chimeric cytokine receptor).

[0272] As a next step, the inventors transduced human primary monocytes with the IL-10ChCR lentiviral vector, and used monocytes transduced with a control lentivirus ("mock") as a control or were not transduced. After transduction, the inventors differentiated the monocytes into macrophages. These macrophages were stimulated with IFNγ to achieve M1 activation, with IL-10 to achieve M2 activation, or without stimulation. After stimulation, the inventors harvested these cells and analyzed the expression of HLA-DR and CD38 (as markers of M1 activation) and CD163 (as a marker of M2 activation). Figure 5 )

[0273] Example 5: TGF-β-IFNγ Chimeric Cytokine Receptor (TGF-βChCR)

[0274] As described above, after obtaining the first functional data on the prototype ChCR, the inventors started with the TGF-β-IFNγ ChCR combination. The inventors have collected the first data on this ChCR variant in THP-1 and human primary macrophages. TGF-β strongly induced pSTAT 1 only in THP-1 cells and human primary macrophages expressing TGF-β ChCR. THP-1 cells and human primary macrophages transduced with an irrelevant lentiviral vector ("mock") did not phosphorylate STAT1 ( Figure 6 ).

[0275] Next, the inventors transduced human primary monocytes with the TGF-β ChCR lentiviral vector, and used monocytes transduced with a control lentivirus ("mock") as a control or were not transduced. After transduction, the inventors differentiated the monocytes into macrophages. These macrophages were stimulated with IFNγ to achieve M1 activation, and stimulated with 1, 10, or 100 ng / mL TGF-β, or were not stimulated. After stimulation, the inventors harvested these cells and analyzed the expression of HLA-DR, CD38, and Cd86, which are M1 markers. ( Figure 7 )

[0276] Example 6: Stimulation of IL-10 and TGF-β Chimeric Cytokine Receptors

[0277] The inventors transduced human primary monocytes with ChCR (IL-10 or TGF-β) lentiviral vectors, and used monocytes transduced with control lentivirus ("mock") as a control or were not transduced. After transduction, the inventors differentiated the monocytes into macrophages. After stimulation with 1, 10 and 100 ng / mL TGF-β in the case of TGF-βChCR, and with 1, 10, 100 and 500 ng / mL IL-10 in the case of IL-10ChCR, IP-10 secretion of stimulated macrophages was measured by ELISA. As a control, culture medium without cytokines ("0") or IFN-γ was used. IP-10 is usually secreted only after IFN-γ stimulation, but the inventors Figure 8 demonstrated that when macrophages express ChCR and are stimulated with corresponding cytokines (such as IL-10 or TGF-β), IP-10 is also secreted.

[0278] Example 7: Tumoricidal effect of macrophage-conditioned medium

[0279] The present inventors used empty lentiviral vector ("mock"), lentiviral vector expressing IL-10ChCR ( Figure 9 A) or TGF-β ChCR ( Figure 9B) Human primary monocytes from healthy donors were transduced with or without lentivirus. After 7 days of differentiation into monocyte-derived macrophages (MDMs), they were stimulated for 2 days with medium containing no cytokines, IFN-γ, IL-10, or TGF-β. Figure 9 As shown. Conditioned medium from stimulated macrophages was used to treat MDA-MB-231 or BT-549 cell lines for 3 days. The viability of tumor cells was assessed using the WST1 assay. The present inventors demonstrated that conditioned medium from ChCR-expressing macrophages stimulated with IL-10 or TGF-β had a negative effect on tumor cell viability, similar to conditioned medium from IFNγ-stimulated macrophages ( Figure 9 To control for potential effects of recombinant IL-10 or TGF-β on MDA-MB-231 and BT-549 cells, the cell lines were directly treated with the corresponding cytokines. The results demonstrated that the effects on viability were indeed caused by factors secreted by ChCR-expressing macrophages, rather than by the recombinant cytokines.

[0280] Example 8:

[0281] The present inventors were able to transduce human primary macrophages with mock, IL-10 ChCR, and TGF-β ChCR lentiviruses with efficacies of 69.62% ± 16.97%, 72.3% ± 15.5%, and 69.3% ± 11.58% (mean ± SD), respectively ( Figure 10 A). They also validated the IL-10ChCR ( Figure 10 B) and TGF-β ChCR ( Figure 10 C) Expression on the surface of transduced cells.

[0282] Example 9: Sequence

[0283] Table 1 lists the sequences of the present invention, which are also attached to the ST.26 sequence protocol. The repeated parts of the sequence are marked with the corresponding consistent font.

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

Claims

1. An isolated monocyte or macrophage comprising a chimeric cytokine receptor (ChCR) polypeptide heterodimer, wherein the ChCR polypeptide heterodimer comprises or consists of a first ChCR polypeptide and a second ChCR polypeptide; wherein the first ChCR polypeptide comprises: - a first extracellular domain, - the first type 1 transmembrane domain, -optionally, a first flexible linker domain, wherein the first flexible linker domain connects the first extracellular domain and the first transmembrane domain, - a first intracellular domain, and wherein the second ChCR polypeptide comprises: - a second extracellular domain, - a second type 1 transmembrane domain, -optionally, a second flexible linker domain, wherein the second flexible linker domain connects the second extracellular domain and the second transmembrane domain, - the second intracellular domain, in - the first extracellular domain and the second extracellular domain are capable of inducing dimerization of the ChCR polypeptide heterodimer upon binding to a cytokine selected from IL-10 and TGF-β; and - the first intracellular domain and the second intracellular domain are capable of activating IFNγ receptor / Jak1 / Jak2 / STAT 1 signaling in monocytes and / or macrophages upon dimerization of the ChCR polypeptide heterodimer.

2. The isolated monocytes or macrophages according to claim 1, wherein - the first extracellular domain is or comprises the extracellular domain of IL-10 receptor α (CDW210A); - the second extracellular domain is or comprises the extracellular domain of IL-10 receptor beta (CDW210B); - the first intracellular domain is or comprises the IFNγ receptor 1 (CD119) intracellular domain; and - the second intracellular domain is or comprises the IFNγ receptor 2 intracellular domain.

3. The isolated monocyte or macrophage of claim 1 or 2, wherein the ChCR polypeptide heterodimer comprises: - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 21, - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28; - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 22, - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29; The ChCR polypeptide heterodimer has ≥85%, particularly ≥90%, ≥95% of the biological activity of the ChCR heterodimer of SEQ ID NO 53 and SEQ ID NO 54.

4. The isolated monocytes or macrophages according to claim 1, wherein - the first extracellular domain is or comprises the extracellular domain of IL-10 receptor α (CDW210A); - the second extracellular domain is or comprises the extracellular domain of IL-10 receptor beta (CDW210B); - the first intracellular domain is or comprises the IFNγ receptor 2 intracellular domain; and - the second intracellular domain is or comprises the IFNγ receptor 1 (CD119) intracellular domain.

5. The isolated monocyte or macrophage of claim 1 or 4, wherein the ChCR polypeptide heterodimer comprises: - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 21, - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29; - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 22, - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28; The ChCR polypeptide heterodimer has ≥85%, particularly ≥90%, ≥95% of the biological activity of the ChCR heterodimer of SEQ ID NO 59 and SEQ ID NO 60.

6. The isolated monocytes or macrophages according to claim 1, wherein - the first extracellular domain is or comprises a TGF-β receptor type I extracellular domain; - the second extracellular domain is or comprises a TGF-β receptor type II extracellular domain; - the first intracellular domain is or comprises the IFNγ receptor 1 (CD119) intracellular domain; and - the second intracellular domain is or comprises the IFNγ receptor 2 intracellular domain.

7. The isolated monocyte or macrophage of claim 1 or 6, wherein the ChCR polypeptide heterodimer comprises: - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 23, - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28; - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 24, - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29; The ChCR polypeptide heterodimer has ≥85%, particularly ≥90%, ≥95% of the biological activity of the ChCR heterodimer of SEQ ID NO 55 and SEQ ID NO 56.

8. The isolated monocytes or macrophages according to claim 1, wherein - the first extracellular domain is or comprises a TGF-β receptor type I extracellular domain; - the second extracellular domain is or comprises a TGF-β receptor type II extracellular domain; - the first intracellular domain is or comprises the IFNγ receptor 2 intracellular domain; and - the second intracellular domain is or comprises the IFNγ receptor 1 (CD119) intracellular domain.

9. The isolated monocyte or macrophage of claim 1 or 8, wherein the ChCR polypeptide heterodimer comprises: - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 23, - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29; - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 24, - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28; The ChCR polypeptide heterodimer has ≥85%, particularly ≥90%, ≥95% of the biological activity of the ChCR heterodimer of SEQ ID NO 61 and SEQ ID NO 62.

10. An isolated monocyte or macrophage comprising a nucleic acid molecule encoding the ChCR polypeptide of any one of claims 1 to 9.

11. The isolated monocyte or macrophage of claim 10, wherein the nucleic acid molecule is contained in an expression vector, wherein the ChCR polypeptide heterodimer is under the control of a promoter sequence operable in mammalian monocytes or macrophages, In particular, wherein the expression vector is selected from the group consisting of a viral vector, a plasmid, a DNA molecule or an RNA molecule, More particularly, the expression vector is a lentiviral vector.

12. The isolated monocyte or macrophage according to any one of the preceding claims, wherein the monocyte or macrophage additionally comprises an inhibitory nucleic acid molecule against SIRPα. 13 . The isolated monocyte or macrophage according to claim 12 , wherein the inhibitory nucleic acid molecule against SIRPα comprises a sequence selected from the group consisting of: SEQ ID NO 33 to SEQ ID NO 52.

14. The isolated monocyte or macrophage of any one of claims 1 to 4, wherein the monocyte or macrophage additionally comprises a chimeric antigen receptor (CAR) polypeptide comprising: - extracellular antigen-binding domains, in particular Fab fragments, - transmembrane domains, in particular the CD8 transmembrane domain, and an intracellular domain comprising at least one ITAM motif, in particular an intracellular domain of an Fc receptor, more particularly an intracellular domain of an Fcγ receptor or CD32a, o Optionally, a linker domain, which connects the extracellular antigen-binding domain and the transmembrane domain.

15. The isolated monocyte or macrophage of claim 14, wherein the CAR polypeptide comprises a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to any one of SEQ ID NO 13 to SEQ ID NO 18.

16. An isolated monocyte or macrophage according to any preceding claim for use in treating or preventing cancer.

17. A kit comprising: - an expression vector encoding the ChCR polypeptide according to any one of claims 1 to 9; - The inhibitory nucleic acid molecule against SIRPα according to any one of claims 12 or 13; and -An expression vector encoding the CAR polypeptide according to any one of claims 14 or 15.

18. A method for modifying monocytes, comprising: i. providing mononuclear cells obtained from a mammalian donor, ii. inserting into the monocyte a nucleic acid sequence encoding the ChCR polypeptide according to any one of claims 1 to 9; iii. maintaining the monocytes under cell culture conditions.

19. The method according to claim 18, wherein additionally, - The inhibitory nucleic acid molecule against SIRPα according to any one of claims 12 or 13; and / or -the CAR polypeptide of any one of claims 14 or 15; is inserted into the monocytes.

20. A ChCR polypeptide heterodimer comprising: - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 21, - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28; - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 22, - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29.

21. A ChCR polypeptide heterodimer comprising: - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 21, - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29; - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 22, - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28.

22. A ChCR polypeptide heterodimer comprising: - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 23, - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28; - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 24, - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29.

23. A ChCR polypeptide heterodimer comprising: - a first extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 23, - a first transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 27, and - a first intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 29; - a second extracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 24, - a second transmembrane domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 26, and - a second intracellular domain comprising a sequence that is at least ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identical to SEQ ID NO 28.

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