Methods for polarizing macrophages

Abstract

The present invention relates to chimeric cytokine receptors and their use for switching the polarization of macrophages or monocytes from M2 to M1. The present invention also relates to methods for the modification of monocytes to selectively increase phagocytic activity and modulate the polarization of progeny macrophages.

Description

[Technical Field]

[0001] This application claims priority to European Patent Application (EP) No. 22215498.1, filed December 21, 2022, and (EP) No. 23200945.6, filed September 29, 2023, both of which are incorporated herein by reference.

[0002] The present invention relates to chimeric cytokine receptors and their use for altering the polarization of macrophages or monocytes from M2 to M1. The present invention also relates to methods for modifying monocytes to selectively increase phagocytic activity and modulate the polarization of offspring macrophages. [Background technology]

[0003] Monocytes are white blood cells that circulate in the blood and can differentiate into macrophages, among other things, once they enter target tissues. One of the main properties of macrophages is the engulfment of dead or diseased cells, making them an important part of the immune system involved in many diseases.

[0004] Macrophages can adapt their immunological functions depending on the activation signals they are exposed to in target tissues. This process of macrophage polarization can give rise to M1- and M2-polarized macrophages. M1 macrophages, also known as classically activated macrophages, are important during acute infections, particularly those caused by intracellular bacteria and viruses, but are also important 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 macrophage activation, other mechanisms also regulate macrophage immune function. Macrophage engulfment of cells is regulated by the CD47-SIRPα axis. Ligation of the ubiquitously expressed cell surface protein CD47 with the cell surface molecule SIRPα on macrophages transmits a "do-not-eat-me" signal to macrophages. This mechanism prevents macrophages from engulfing healthy cells, but it also prevents macrophages from combating diseased cells, or at least reduces their effectiveness. Some diseased cells, such as tumor cells, are known to overexpress CD47, thereby protecting them from macrophage phagocytosis. Summary of the Invention [Problem to be solved by the invention]

[0006] Based on the above state of the art, the object of the present invention is to provide means and methods for enhancing macrophage activity against diseased cells. [Means for solving the problem]

[0007] This object is achieved by the subject matter of the independent claims herein, with further advantageous embodiments described in the dependent claims herein, the examples, the figures and the general description.

[0008] Summary of the Invention 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 chimeric cytokine receptor (ChCR) polypeptide and a second ChCR polypeptide; wherein the first ChCR polypeptide is: - the first extracellular domain, - the first type 1 transmembrane domain, - optionally a first flexible linker domain connecting said first extracellular domain and said first transmembrane domain, - a first intracellular domain, and the second ChCR polypeptide comprises: - a second extracellular domain, - a second type 1 transmembrane domain, - optionally a second flexible linker domain connecting said second extracellular domain and said second transmembrane domain, - a second intracellular domain, Including, where: - the first extracellular domain and the second extracellular domain are capable of inducing dimerization of said ChCR polypeptide heterodimer upon binding to a cytokine selected from IL-10 and TGFβ; and - the first and second intracellular domains, upon dimerization of the ChCR polypeptide heterodimer, are capable of activating IFNγ receptor / Jak1 / Jak2 / STAT1 signaling in monocytes and / or macrophages. Upon binding with IL-10 or TGFβ, the ChCR induces intracellular signaling (corresponding to interferon-γ stimulation).

[0009] A further aspect of the invention relates to an isolated monocyte or macrophage according to any one of the previous aspects for use in the treatment or prevention of cancer.

[0010] A further aspect of the present invention relates to a kit comprising: - an expression vector encoding a ChCR polypeptide as defined above; - an inhibitory nucleic acid molecule against SIRPα as defined above; and - an expression vector encoding a CAR polypeptide as described above.

[0011] A further aspect of the present invention relates to a method for modifying monocytes, said method comprising: i. providing monocytes obtained from a mammalian donor; ii. inserting into said monocytes a nucleic acid sequence encoding a ChCR polypeptide according to the first aspect; iii. Maintaining said monocytes under cell culture conditions.

[0012] Terms and Definitions For the purposes of interpreting this specification, the following definitions shall apply, and where appropriate, terms used in the singular shall also include the plural and vice versa. In the event that a definition set forth below conflicts with any document incorporated herein by reference, the definition set forth herein shall control.

[0013] As used herein, the terms "comprising," "having," "containing," "including," and other similar forms, and their grammatical equivalents, are intended to be equivalent in meaning and to be open-ended in that the listing of one or more items following any one of these words does not imply an exhaustive listing of such one or more items, or that it is limited to only the listed item or items. For example, an item "comprising" components A, B, and C can consist of components A, B, and C (i.e., contain only components A, B, and C), or it can include not only components A, B, and C, but also one or more other ingredients. Thus, "comprising" and its similar forms, and its grammatical equivalents, are intended and understood to include disclosure of "consisting essentially of" or "consisting of" embodiments.

[0014] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, and any other stated or intervening value within that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where one or both of the limits are included in the stated range, ranges excluding either or both of those included limits are also included in the disclosure.

[0015] As used herein, reference to "about" a value or parameter includes (and describes) a variation on the value or parameter itself. For example, a statement referring to "about X" also includes the statement "X."

[0016] As used in this specification, including the appended claims, the singular forms "a," "or," and "the" include plural references unless the context clearly dictates otherwise.

[0017] As used herein, "and / or" is considered to specifically describe each of the two specified features or components with or without the other features or components. 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 encompass each of the following aspects: A, B, and C; 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.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard procedures are used for molecular, genetic, and biochemical procedures (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.

[0019] Any patent documents cited herein are deemed to be incorporated herein by reference in their entirety.

[0020] array Sequences 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, sequence identity at the amino acid level can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater. Sequence identity at the nucleic acid level can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater. Alternatively, substantial identity exists when a nucleic acid segment hybridizes under selective hybridization conditions (e.g., under very high stringency hybridization conditions) to the complement of the strand. The nucleic acids can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.

[0021] In the present context, the terms "sequence identity" and "percentage of sequence identity" refer to a quantitative parameter that represents the results of sequence comparison, 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, Adv. Appl. Math. 2:482 (1981), the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988), or 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 through, for example, the National Center for Biotechnology Information (http: / / blast.ncbi.nlm.nih.gov / ).

[0022] An example of a comparison of amino acid sequences is the BLASTP algorithm using default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 11, Extension 1; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm using default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match / Mismatch Scores: 1.-2; Gap costs: Linear. Unless otherwise specified, sequence identity values ​​provided herein refer to values ​​obtained using the BLAST family of programs using the above-specified default parameters for protein and nucleic acid comparisons, respectively (Altschul et al., J. Mol. Biol. 215:403-410 (1990)).

[0023] Reference to identical sequences without specifying a percentage value includes the meaning of 100% identical sequences (ie, the same sequence).

[0024] General Biochemistry: Peptides, Amino Acid Sequences The term "polypeptide" in the context of this specification refers to a molecule consisting of 50 or more amino acids forming a linear chain, where the amino acids are connected by peptide bonds. The amino acid sequence of a polypeptide may refer to the amino acid sequence of an entire protein (as found physiologically) or a fragment thereof. The terms "polypeptide" and "protein" are used interchangeably herein and include proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences.

[0025] The term "peptide" in the present context relates to a molecule consisting of up to 50 amino acids, in particular 8 to 30 amino acids, more in particular 8 to 15 amino acids, which form a linear chain in which the amino acids are connected by peptide bonds.

[0026] The sequence of amino acid residues is written from the amino terminus to the carboxyl terminus. The capital letters at the sequence positions refer to the L-amino acid in single-letter code (Stryer, Biochemistry, Vol. 3, p. 21). The lowercase letters at the amino acid sequence positions refer to the corresponding D- or (2R)-amino acid. The sequence is written from left to right from the amino terminus to the carboxyl terminus. Following standard nomenclature, the sequence of amino acid residues is represented by either the three-letter or single-letter code 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).

[0027] As used herein, the term "dimer" refers to a unit consisting of two subunits.

[0028] As used herein, the term "homodimer" refers to a dimer composed of two subunits that are either identical or very similar members within the same class of subunits.

[0029] As used herein, the term "amino acid linker" refers to a polypeptide of variable length used to connect two polypeptides to generate a single polypeptide chain. Exemplary embodiments of linkers useful in practicing the invention defined herein are oligopeptide chains of 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 amino acids. A glycine-serine linker is composed of glycine and serine, while a glycine linker is composed of glycine subunits. A non-limiting example of an amino acid linker is the polypeptide GSGGGGSGGGGS (SEQ ID NO: 023), which links the extracellular antigen-binding domain and the transmembrane domain.

[0030] General molecular biology: nucleic acid sequence, expression The term "gene" refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. A polynucleotide sequence can be used to identify larger fragments or full-length coding sequences of the gene with which it is associated. Methods for isolating larger fragment sequences are known to those of skill in the art.

[0031] The term "transgene" as used herein refers to a gene or genetic material introduced from one organism into another. As used herein, the term may also refer to the transplantation of a naturally occurring or physiologically intact variant of a gene sequence into a patient's tissue that is deficient in that gene. Furthermore, it may refer to the transfer of a native coding sequence whose expression is driven by a promoter that is absent or silenced in the target tissue.

[0032] The term "recombinant" as used herein relates to a nucleic acid that is the product of one or more cloning, restriction, and / or ligation steps and that differs from naturally occurring nucleic acid. Recombinant viral particles contain recombinant nucleic acids.

[0033] The terms "gene expression" or "expression," or "gene product," can refer to either or both the process—and its product—of producing nucleic acids (RNA) or peptides or polypeptides, also called transcription and translation, respectively, or any of the intermediate processes that regulate the processing of genetic information to result in a polypeptide product. The term "gene expression" also applies to the transcription and processing of RNA gene products, such as regulatory RNAs or structural (e.g., ribosomal) RNAs. When the expressed polynucleotide is derived from genomic DNA, expression can include splicing of mRNA in eukaryotic cells. Expression can be assessed at both the level of transcription and translation, i.e., mRNA and / or protein product.

[0034] The term "knockdown" of an mRNA herein relates to a reduction in the amount of this particular mRNA.

[0035] The term "nucleotide" in the present context refers to a nucleic acid or nucleic acid analog building block, which oligomer can form selective hybrids with RNA or DNA oligomers based on base pairing. In this context, the term "nucleotide" includes the building blocks of classical ribonucleotides: adenosine, guanosine, uridine (and ribosylthymine), and cytidine; and the classical deoxyribonucleotides: deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Furthermore, it includes nucleic acid analogs such as phosphothioates, 2'O-methylphosphothioates, peptide nucleic acids (PNAs; N-(2-aminoethyl)-glycine units linked by peptide bonds, with the nucleobase attached to the alpha carbon of the glycine), or locked nucleic acids (LNAs; 2'O,4'C methylene-bridged RNA building blocks). When a "hybridizing sequence" is referred to herein, such a hybridizing sequence can be composed of any of the above nucleotides or a mixture thereof.

[0036] As used herein, the term "phosphothioate" is synonymous with the terms phosphorothioate and thiophosphate.

[0037] In the present context, the term "capable of forming hybrid or hybridizing sequences" relates to sequences that are capable of selectively binding to their target sequence under conditions present in the cytosol of a mammalian cell. Such hybridizing sequences may be continuously reverse-complementary to the target sequence or may contain gaps, mismatches or additional unmatched nucleotides. The minimum length of a hybridizable sequence depends on its composition (inclusion of C or G nucleotides, which contribute more to binding energy than A or T / U nucleotides) and backbone chemistry.

[0038] In the context of this specification, the term "hybridizing sequence" encompasses polynucleotide sequences comprising or consisting essentially of RNA (ribonucleotides), DNA (deoxyribonucleotides), phosphothioate deoxyribonucleotides, 2'-O-methyl modified phosphothioate ribonucleotides, LNA and / or PNA nucleotide analogs. In certain embodiments, hybridizing sequences according to the invention comprise 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, hybridizing sequences are at least 80% identical, more preferably 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reverse complements of SEQ ID NOs: 33 to 42. In certain embodiments, the hybridizing sequences comprise deoxynucleotides, phosphothioate deoxynucleotides, LNA and / or PNA nucleotides, or mixtures thereof.

[0039] As used herein, the term "inhibitory nucleic acid molecule" refers to a nucleic acid molecule that reduces the amount of functional mRNA of a target molecule in a cell. In particular, inhibitory nucleic acid molecules refer to oligonucleotides that have a sequence substantially complementary to and can hybridize with RNA. Antisense action against such RNA results in the modulation, particularly inhibition or suppression, of the biological action of the RNA. If the RNA is mRNA, the expression of the resulting gene product is inhibited or suppressed. Inhibitory nucleic acid molecules can consist of DNA, RNA, nucleotide analogs, and / or mixtures thereof. Those skilled in the art 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 both the nucleic acid base sequence and the backbone (ribonucleotide, deoxyribonucleotide, analog) composition. Many sources exist for the actual physical delivery of oligonucleotides.

[0040] In the present context, the term "antisense oligonucleotide" refers to an oligonucleotide that has a sequence substantially complementary to and can hybridize with RNA. Such antisense action on RNA results in the modulation, particularly inhibition or suppression, of the biological action of the RNA. If the RNA is mRNA, the expression of the resulting gene product is inhibited or suppressed. Antisense oligonucleotides can consist of DNA, RNA, nucleotide analogs, and / or mixtures thereof. Those skilled in the art are aware of various commercial and non-commercial sources for calculating the theoretically optimal antisense sequence for a given target. Optimization can be performed both in terms of the nucleic acid base sequence and the backbone (ribo, deoxyribo, analog) composition. There are many sources for the actual physical oligonucleotides, and they are generally synthesized by solid-state synthesis.

[0041] In the context of this specification, the term siRNA (small / short interfering RNA) refers to an RNA molecule that can interfere with (i.e., suppress or block) the expression of a gene containing a nucleic acid sequence complementary to or hybridizing to the siRNA sequence, in a process called RNA interference. The term siRNA is intended to encompass both single-stranded and double-stranded siRNAs. siRNAs are typically characterized by a length of 17 to 24 nucleotides. Double-stranded siRNAs can be derived from longer double-stranded RNA molecules (dsRNA). The leading theory is that this longer double-stranded RNA is cleaved by an endoribonuclease (called Dicer) to form double-stranded siRNAs. A nucleoprotein complex (called RISC) unwinds the double-stranded siRNA to form single-stranded siRNAs. RNA interference often functions by binding of siRNA molecules to mRNA molecules with complementary sequences, resulting in mRNA degradation. RNA interference can also occur when siRNA molecules bind to intronic sequences in pre-mRNA (immature, unspliced ​​mRNA) in the cell nucleus, causing the pre-mRNA to be degraded.

[0042] The term "shRNA (small hairpin RNA)" in the context of this specification relates to an artificial RNA molecule with a tight hairpin turn that can be used to silence the expression of a target gene via RNA interference (RNAi).

[0043] The term "sgRNA (single guide RNA)" in the present context relates to an RNA molecule capable of sequence-specifically silencing gene expression via the CRISPR (clustered regularly interspaced short palindromic repeat) mechanism.

[0044] The term "miRNA (microRNA)" in the present context relates to small non-coding RNAs (containing approximately 22 nucleotides) that function in RNA silencing and post-transcriptional regulation of gene expression.

[0045] In the context of this specification, the term "nucleic acid expression vector" refers to an artificial nucleic acid molecule used as a vehicle to deliver a foreign nucleic acid molecule to another cell. A "nucleic acid expression vector" refers to a plasmid, viral genome, or RNA that is used to transfect (in the case of a plasmid or RNA) or transduce (in the case of a viral genome) a target cell with a specific gene of interest, or, in the case of an RNA construct, to translate the corresponding protein of interest from the transformed mRNA. In vectors that operate at the level of transcription and subsequent translation, the gene of interest is under the control of a promoter sequence that is operable in the target cell so that the gene of interest is transcribed constitutively, in response to a stimulus, or depending on the state of the cell. In certain embodiments, a viral genome is packaged in a capsid, resulting in a viral vector that can transduce a target cell.

[0046] The term "promoter" in the context of this specification relates to a nucleic acid sequence that initiates transcription of a specific nucleic acid sequence in macrophages. Various promoters are well known in the art and are widely used in genetics as part of vectors that contain the nucleic acid sequence to be transcribed. Certain types of promoters are only active under specific conditions, either in the presence or absence of a specific molecule (inducible promoters), or in a specific cellular environment, such as cell type specific promoters.

[0047] The term "chimeric antigen receptor" in the context of this specification relates to an artificially engineered receptor comprising a portion of an antigen receptor.

[0048] Immunoreceptor tyrosine-based activation motifs (ITAMs) are important components of the intracellular signaling machinery of cell surface proteins of the immune system. They are located in the cytoplasmic tails of cell surface proteins, and their tyrosine residues become phosphorylated after the cell surface protein interacts with its respective ligand. The phosphorylated tyrosine residues form docking sites for other downstream components of the signaling machinery. An ITAM motif contains a tyrosine separated from a leucine or isoleucine by any two other amino acids.

[0049] The term "chimeric cytokine receptor" in the context of this specification relates to an artificially engineered receptor comprising a portion of a cytokine receptor.

[0050] "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 lack the WSXWS sequence characteristic of type I receptors. Typical type II cytokine receptors are heterodimers or multimers with high-affinity and low-affinity components.

[0051] "Type I cytokine receptors" are transmembrane receptors expressed on the cell surface that recognize and respond to cytokines with four alpha-helical chains. These receptors, also known as hemopoietin receptors, 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 interaction and others that are involved in signal transduction.

[0052] A "type I transmembrane domain" is derived from a type I membrane protein.

[0053] The term "type I membrane protein" in the context of this specification relates to a single-pass membrane protein that is anchored to the lipid membrane by its N-terminal domain, which targets the lumen of the endoplasmic reticulum (ER) during synthesis and the extracellular space in its mature form.

[0054] In the context of this specification, the term "IFNγ receptor / Jak1 / Jak2 / STAT1 signaling in monocytes and / or macrophages" refers to the pro-inflammatory activation of monocytes / macrophages via the interferon-γ receptor signaling cascade. Interferon-γ receptor-induced Jak1 / Jak2 / STAT1 signaling switches monocyte differentiation from dendritic cells to macrophages, induces the production of pro-inflammatory cytokines in macrophages, and converts macrophages into pro-inflammatory "M1" macrophages. Interferon-γ receptor signaling can be initiated by the ChCRs of the present invention. M1 activation of macrophages is positively associated with prolonged survival and the best clinical outcomes in many cancers.

[0055] (Cancer) Immunotherapy In the context of this specification, the terms "cancer immunotherapy," "biological therapy," or "immunomodulatory therapy" are meant to encompass types of cancer treatments that utilize the immune system to fight cancer.

[0056] As used herein, the term "pharmaceutical composition" refers to a compound of the present invention or a pharmaceutically acceptable salt thereof, together with 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.

[0057] As used herein, the term "pharmaceutically acceptable carrier" includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, as known to those of skill in the art (see, e.g., Remington: The Science and Practice of Pharmacy, ISBN 0857110624).

[0058] The term "cancer" as used in the context of this specification relates to malignant neoplastic diseases, and the terms "cancer" and "malignant neoplastic diseases" are used interchangeably herein. This includes, inter alia, carcinomas (cancers of epithelial origin), sarcomas (cancers of connective tissue origin), lymphomas and leukemias, germ cell-derived tumors and germinomas. Certain alternatives of any of the aspects and embodiments disclosed herein are directed to the use of the compounds and compositions of the present invention in the treatment of solid tumors. Other alternatives of any of the aspects and embodiments disclosed herein are directed to the use of the combinations of the present invention in the treatment of liquid cancers, such as myeloid or granulocytic leukemias, particularly AML, lymphocytic, lymphocytic, or lymphoblastic leukemias and lymphomas, polycythemia vera, or erythropenia.

[0059] As used herein, the term "treating" or "treatment" of any disease or disorder (e.g., cancer) refers, in one embodiment, to alleviating the disease or disorder (e.g., delaying, preventing, or reducing the onset 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 may not be discernible to the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. Methods for assessing the treatment and / or prevention of disease are generally known in the art, unless otherwise described herein below. DETAILED DESCRIPTION OF THE INVENTION

[0060] Detailed Description of the Invention A first aspect of the present invention relates to an isolated monocyte or macrophage cell. The isolated monocyte or macrophage cell 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.

[0061] The first ChCR polypeptide comprises, inter alia, from N-terminus to C-terminus: - the first extracellular domain, - the first type 1 transmembrane domain, - optionally a first flexible linker domain connecting said first extracellular domain and said first transmembrane domain, - The first intracellular domain.

[0062] The second ChCR polypeptide comprises, inter alia, from N-terminus to C-terminus: - a second extracellular domain, - a second type 1 transmembrane domain, - optionally a second flexible linker domain connecting said second extracellular domain and said second transmembrane domain, - A second intracellular domain.

[0063] 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 TGFβ.

[0064] The first intracellular domain and the second intracellular domain can activate IFNγ receptor / Jak1 / Jak2 / STAT1 signaling in monocytes and / or macrophages upon dimerization of the ChCR polypeptide heterodimer.

[0065] In certain embodiments, - the first extracellular domain is or comprises the extracellular domain of IL-10 receptor alpha (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.

[0066] In certain embodiments, the ChCR polypeptide heterodimer comprises: - a first extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 21; - a first transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 26, and - a first intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 28; - a second extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 22; - a second transmembrane domain comprising a sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 27, and - a second intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 29; In particular, the ChCR polypeptide heterodimer has 85% or more, in particular 90% or more, 95% or more of the biological activity of the ChCR heterodimers of SEQ ID NO:53 and SEQ ID NO:54.

[0067] In certain embodiments, - the first extracellular domain is or comprises the extracellular domain of IL-10 receptor alpha (CDW210A); - the second extracellular domain is or comprises the extracellular domain of IL-10 receptor beta (CDW210B); - the first intracellular domain is or comprises an IFNγ receptor 2 intracellular domain; and - the second intracellular domain is or comprises the IFNγ receptor 1 (CD119) intracellular domain.

[0068] In certain embodiments, the ChCR polypeptide heterodimer comprises: - a first extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 21; - a first transmembrane domain comprising a sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 27, and - a first intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 29; - a second extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 22; - a second transmembrane domain comprising a sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 26, and - a second intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 28; In particular, the ChCR polypeptide heterodimer has 85% or more, in particular 90% or more, 95% or more of the biological activity of the ChCR heterodimers of SEQ ID NO:59 and SEQ ID NO:60.

[0069] In certain embodiments, - the first extracellular domain is or comprises a TGFβ receptor type I extracellular domain; - the second extracellular domain is or comprises a TGFβ 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.

[0070] In certain embodiments, the ChCR polypeptide heterodimer comprises: - a first extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 23; - a first transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 26, and - a first intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 28; - a second extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 24; - a second transmembrane domain comprising a sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 27, and - a second intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 29; In particular, the ChCR polypeptide heterodimer has 85% or more, in particular 90% or more, 95% or more of the biological activity of the ChCR heterodimers of SEQ ID NO:55 and SEQ ID NO:56.

[0071] In certain embodiments, - the first extracellular domain is or comprises a TGFβ receptor type I extracellular domain; - the second extracellular domain is or comprises a TGFβ receptor type II extracellular domain; - the first intracellular domain is or comprises an IFNγ receptor 2 intracellular domain; and - the second intracellular domain is or comprises the IFNγ receptor 1 (CD119) intracellular domain.

[0072] In certain embodiments, the ChCR polypeptide heterodimer comprises: - a first extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 23; - a first transmembrane domain comprising a sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 27, and - a first intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 29; - a second extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 24; - a second transmembrane domain comprising a sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 26, and - a second intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 28; In particular, the ChCR polypeptide heterodimer has 85% or more, in particular 90% or more, 95% or more of the biological activity of the ChCR heterodimers of SEQ ID NO:61 and SEQ ID NO:62.

[0073] The biological activity of chimeric cytokine receptors can be measured in reporter cells, which are HEK-Blue™ IFN-γ cells (InvivoGen). Briefly, the ChCR is introduced into the HEK-Blue™ IFN-γ cell line, which contains an IFN-γ-driven, STAT-1-inducible secreted alkaline phosphatase (SEAP) reporter but does not contain either the IL-10 receptor or the TGFβ receptor, respectively. The amount of SEAP can be easily assessed with a colorimetric assay (QuantiBlue), and the SEAP signal is proportional to biological activity.

[0074] A further aspect of the present invention relates to an isolated monocyte or macrophage comprising a nucleic acid molecule encoding a ChCR polypeptide as described in the first aspect, wherein the nucleic acid molecule encodes the ChCR with a signal peptide that affects transport of the ChCR to the cell surface.

[0075] 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.

[0076] In certain embodiments, the monocytes or macrophages further comprise an inhibitory nucleic acid molecule against SIRPα.

[0077] In certain embodiments, the inhibitory nucleic acid molecule against SIRPα comprises a sequence selected from the group of SEQ ID NO:33 to SEQ ID NO:52.

[0078] In certain embodiments, the monocyte or macrophage further comprises a chimeric antigen receptor (CAR) polypeptide, comprising: - an extracellular antigen-binding domain, - a transmembrane domain, and - an intracellular domain containing at least one ITAM motif.

[0079] In certain embodiments, the CAR comprises a linker domain connecting the extracellular antigen-binding domain and the transmembrane domain.

[0080] In certain embodiments, the monocyte or macrophage further comprises a chimeric antigen receptor (CAR) polypeptide, comprising: - an extracellular antigen-binding domain comprising a Fab fragment, - a transmembrane domain, including the CD8 transmembrane domain, and - Intracellular domains, including the intracellular domain of the FC receptor.

[0081] In certain embodiments, the intracellular domain of the CAR comprises the intracellular domain of an Fc gamma receptor. In certain embodiments, the intracellular domain of the CAR comprises the intracellular domain of CD32a.

[0082] In certain embodiments, the CAR polypeptide comprises a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to any one of SEQ ID NOs: 13-18.

[0083] A further aspect of the invention relates to an isolated monocyte or macrophage according to the fourth aspect for use in medicine.

[0084] A further aspect of the invention relates to an isolated monocyte or macrophage according to any one of the previous aspects for use in the treatment or prevention of cancer.

[0085] A further aspect of the present invention relates to a kit comprising: - an expression vector encoding a ChCR polypeptide as defined above; - an inhibitory nucleic acid molecule against SIRPα as defined above; and - an expression vector encoding a CAR polypeptide as described above.

[0086] In certain embodiments, the components of the kit are encoded on a single vector, such as a lentiviral vector, hi certain embodiments, the components of the kit are encoded on two or three vectors.

[0087] A further aspect of the present invention relates to a method for modifying monocytes, said method comprising: i. providing monocytes obtained from a mammalian donor; ii. inserting into said monocytes a nucleic acid sequence encoding a ChCR polypeptide as described above; iii. Maintaining said monocytes under cell culture conditions.

[0088] In certain embodiments of the method, further comprising: an inhibitory nucleic acid molecule against SIRPα as defined above; and / or - a CAR polypeptide as defined above; is inserted into the monocyte.

[0089] A further aspect of the invention relates to a ChCR polypeptide heterodimer comprising: - a first extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 21; - a first transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 26, and - a first intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 28; - a second extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 22; - a second transmembrane domain comprising a sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 27, and - a second intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 29.

[0090] A further aspect of the invention relates to a ChCR polypeptide heterodimer comprising: - a first extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 21; - a first transmembrane domain comprising a sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 27, and - a first intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 29; - a second extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 22; - a second transmembrane domain comprising a sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 26, and - a second intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 28.

[0091] A further aspect of the invention relates to a ChCR polypeptide heterodimer comprising: - a first extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 23; - a first transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 26, and - a first intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 28; - a second extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 24; - a second transmembrane domain comprising a sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 27, and - a second intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 29.

[0092] A further aspect of the invention relates to a ChCR polypeptide heterodimer comprising: - a first extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 23; - a first transmembrane domain comprising a sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 27, and - a first intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 29; - a second extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 24; - a second transmembrane domain comprising a sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 26, and - a second intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 28.

[0093] medical treatment Similarly, within the scope of the present invention is a method or method for treating cancer in a patient in need thereof, comprising administering to the patient monocytes or macrophages as described above.

[0094] Manufacturing and treatment methods according to the present invention The present invention further encompasses, in a further aspect, the use of monocytes or macrophages as specified herein in a method for the manufacture of a medicament for the treatment or prevention of cancer.

[0095] Similarly, the present invention encompasses a method for treating a patient diagnosed with a cancer-related disease, the method comprising administering to the patient an effective amount of monocytes or macrophages as identified herein.

[0096] For example, when alternative forms of a single separable feature, such as a monocyte or macrophage, or a receptor sequence, or a medical indication, are described herein as "embodiments," it is to be understood that such alternative forms can be freely combined to form separate embodiments of the invention disclosed herein. Thus, any of the alternative embodiments of a monocyte or macrophage can be combined with any of the alternative embodiments of a receptor sequence, and these combinations can be combined with any of the medical indications described herein.

[0097] This specification further includes the following items:

[0098] item: Item 1. A CAR polypeptide comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to any one of SEQ ID NOs: 13 to 18.

[0099] Item 2. A ChCR polypeptide heterodimer, comprising: - a first extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 21; - a first transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 26, and - a first intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 28; - a second extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 22; - a second transmembrane domain comprising a sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 27, and - a second intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 29; Including, wherein the ChCR polypeptide heterodimer has 85% or more, particularly 90% or more, 95% or more of the biological activity of the ChCR heterodimers of SEQ ID NO: 53 and SEQ ID NO: 54; The ChCR polypeptide heterodimer.

[0100] Item 3. A ChCR polypeptide heterodimer, comprising: - a first extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 23; - a first transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 26, and - a first intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 28; - a second extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 24; - a second transmembrane domain comprising a sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 27, and - a second intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 29; Including, wherein the ChCR polypeptide heterodimer has 85% or more, particularly 90% or more, 95% or more of the biological activity of the ChCR heterodimers of SEQ ID NO: 55 and SEQ ID NO: 56; The ChCR polypeptide heterodimer.

[0101] Item 4. A ChCR polypeptide heterodimer, comprising: - a first extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 21; - a first transmembrane domain comprising a sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 27, and - a first intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 29; - a second extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 22; - a second transmembrane domain comprising a sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 26, and - a second intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 28; Including, wherein the ChCR polypeptide heterodimer has 85% or more, particularly 90% or more, 95% or more of the biological activity of the ChCR heterodimers of SEQ ID NO: 59 and SEQ ID NO: 60; The ChCR polypeptide heterodimer.

[0102] Item 5. A ChCR polypeptide heterodimer, comprising: - a first extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 23; - a first transmembrane domain comprising a sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 27, and - a first intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 29; - a second extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 24; - a second transmembrane domain comprising a sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 26, and - a second intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 28; Including, wherein the ChCR polypeptide heterodimer has 85% or more, particularly 90% or more, 95% or more of the biological activity of the ChCR heterodimers of SEQ ID NO: 61 and SEQ ID NO: 62; The ChCR polypeptide heterodimer.

[0103] The present invention is further explained by the following examples and figures, from which further embodiments and advantages can be derived, which are intended to illustrate the invention without limiting its scope. [Brief explanation of the drawings]

[0104] [Figure 1AB] Figure 1 shows A) knockdown of SIRPα in THP-1 cells using nine siRNAs against SIRPα. Protein expression was normalized to a nonspecific, "non-targeting" siRNA (siRNA NT). B) We further characterized the three most promising SIRPα-specific siRNAs (3, 6, and 9) as miRNAs in THP-1 cells by transducing them with lentiviral vectors expressing these miRNAs. [Figure 1C] C) The most promising miRNA 3 was validated in primary human macrophages. As a negative control, we used a nonspecific "non-targeting" miRNA (miRNA NT). SIRPα expression was measured by flow cytometry, and the median fluorescence intensity (MFI) was normalized to miRNA NT. [Figure 2A] Figure 2 shows CD19-specific phagocytosis of tumor cells by mCAR-expressing macrophages. A) Diagram of mCAR design: An extracellular human CD19-specific scFv antibody fragment is linked to an ITAM-containing intracellular signaling domain via a CD8α stalk and transmembrane domain. [Figure 2B]B) and C) We evaluated the phagocytic activity of mCAR in transduced macrophages that also express RFP by FACS. CFSE-labeled Raji cells were used as targets. Macrophages that phagocytosed Raji cells were RFP and CFSE double-positive. This assay was performed using macrophages from four different donors. [Figure 2C] B) and C) We evaluated the phagocytic activity of mCAR in transduced macrophages that also express RFP by FACS. CFSE-labeled Raji cells were used as targets. Macrophages that phagocytosed Raji cells were RFP and CFSE double-positive. This assay was performed using macrophages from four different donors. [Figure 3] Figure 3 shows EGFR-specific phagocytosis of tumor cells by mCAR-expressing macrophages. A) EGFR binding in cetuximab mCAR-expressing human primary macrophages. B) We evaluated the phagocytic activity of cetuximab mCAR in transduced macrophages that also express RFP by FACS. CFSE-labeled MDA-MB-231 cells expressing EGFR were used as targets. Macrophages that phagocytosed MDA-MB-231 cells were RFP and CFSE double positive. This assay was performed using macrophages from two different donors. Macrophages transduced with CD19-specific mCAR served as a control for nonspecific phagocytosis. Additionally, both experimental groups were treated with cytochalasin D (CytD), which inhibits phagocytosis. [Figure 4ABC]Figure 4 shows the generation of functional chimeric cytokine receptors (ChCRs). A) Design and mechanism of action of the IL-10 ChCR. The extracellular portion binds IL-10, while the intracellular domain derived from the IFN-γ receptor induces the STAT1 signaling pathway, leading to M1 polarization of macrophages. B) We evaluated the function of the ChCR IL-10-IFNGR pair in a cell line expressing the SEAP gene, which is driven by IFN-γ. Addition of IL-10 to cells transduced with both IL-10 ChCR subunits resulted in a positive signal. STAT1 phosphorylation upon IL-10 stimulation of the IL-10 ChCR was confirmed by Western blot in C) transduced reporter cells and D) transduced THP-1 cells. Transduced THP-1 cells responded to IL-10 stimulation in a dose-dependent manner. E) STAT1 phosphorylation was further verified by flow cytometry in primary macrophages that were either untransduced, mock transduced, or transduced with IL-10 ChCR. [Figure 4DE] STAT1 phosphorylation upon IL-10 stimulation of IL-10 ChCR was confirmed by Western blot in C) transduced reporter cells and D) transduced THP-1. Transduced THP-1 responded to IL-10 stimulation in a dose-dependent manner. E) STAT1 phosphorylation was further verified by flow cytometry in primary macrophages that were either untransduced, mock-transduced, or transduced with IL-10 ChCR. [Figure 5]Figure 5 shows the results of IL-10 stimulation of genetically engineered macrophages expressing ChCR. Monocyte-derived macrophages (MDMs) expressing IL-10 ChCR undergo M1 polarization upon stimulation with IL-10. Monocytes from healthy donors were either untransduced, mock-transduced, or transduced with IL-10 ChCR-expressing lentivirus. After differentiation into MDMs, they were polarized with either 5 ng / ml IFN-γ or 100 ng / ml IL-10 for 2 days. Expression of polarization markers was assessed by flow cytometry. N = 3–4. p values ​​were calculated using paired t-tests (for the percentage of CD38 and CD163+ cells) and paired t-tests of ratios (for MFI). [Figure 6] Figure 6 shows that TGF-β ChCR induces pSTAT1. Flow cytometric analysis of STAT1 phosphorylation in THP-1 cells and macrophages either mock-transduced or transduced with TGF-β ChCR. Cells were stimulated as indicated, and pSTAT1 was measured by flow cytometry. [Figure 7] Figure 7 shows the results of TGF-β stimulation of genetically modified macrophages expressing the TGF-β ChCR. Monocyte-derived macrophages (MDMs) expressing the ChCR undergo M1 polarization upon stimulation with TGFβ. Monocytes from healthy donors were either mock-transduced or transduced with a lentivirus expressing the TGF-β ChCR. After differentiation into MDMs, they were allowed to polarize for 2 days as indicated. Surface marker expression was assessed by flow cytometry. N=3. p values ​​were calculated using a paired t-test (for CD38+) and a paired t-test of the ratios (for MFI). [Figure 8]Figure 8 shows IP-10 secretion from ChCR-expressing monocyte-derived macrophages (MDMs) upon stimulation with TGF-β or IL-10. Monocytes from healthy donors were either untransduced, mock-transduced, or transduced with ChCR-expressing lentivirus. After differentiation into MDMs, they were polarized with either IFN-γ, IL-10, or TGFβ at the indicated concentrations for 2 days. IP-10 secretion in the supernatants was analyzed by ELISA. N=4. [Figure 9A] Figure 9 shows the tumoricidal effect of conditioned medium from genetically engineered macrophages expressing ChCR on TNBC cell lines. Monocytes from healthy donors were either untransformed, mock-transduced, or transduced with lentiviruses expressing IL-10 ChCR (A) or TGFβ ChCR (B). After differentiation into MDMs, they were polarized for 2 days as indicated. Conditioned medium from polarized macrophages was used to treat MDA-MB-231 or BT-549 cell lines for 3 days. As controls, MDA-MB-231 and BT-549 were directly treated with the cytokines used to stimulate macrophages. Viability was assessed using the WST1 assay. Results using conditioned medium from 1 to 4 donors are shown. [Figure 9B] Figure 9 shows the tumoricidal effect of conditioned medium from genetically engineered macrophages expressing ChCR on TNBC cell lines. Monocytes from healthy donors were either untransformed, mock-transduced, or transduced with lentiviruses expressing IL-10 ChCR (A) or TGFβ ChCR (B). After differentiation into MDMs, they were polarized for 2 days as indicated. Conditioned medium from polarized macrophages was used to treat MDA-MB-231 or BT-549 cell lines for 3 days. As controls, MDA-MB-231 and BT-549 were directly treated with the cytokines used to stimulate macrophages. Viability was assessed using the WST1 assay. Results using conditioned medium from 1 to 4 donors are shown. [Figure 10]Figure 10 shows A) the efficacy of transduction of human primary macrophages with empty lentiviral vector ("mock") and lentiviral vectors expressing IL-10 ChCR and TGF-β ChCR. N=14-33. B) Surface expression of IL-10Ra and IL-10Rb, two subunits of IL-10 ChCR, on human primary macrophages, expressed as MFI and the percentage of IL-10Ra and IL-10Rb+ cells. N=4. C) Surface expression of TGF-β ChCR on human primary macrophages, expressed as MFI and the percentage of TGFbR2+ cells. N=5. [Example]

[0105] Example 1: SIRPα miRNA: Enhancement of phagocytic activity of macrophages Signal regulatory protein (SIRP)-α on macrophages (MΦ) binds to the cell surface molecule CD47, which is ubiquitously present on all cells. Recognition of CD47 by SIRPα transmits an inhibitory "do not eat me" signal to MΦ. Thus, the SIRPα-CD47 axis acts as a master checkpoint for phagocytosis and is involved in cell turnover in senescent cells. Senescent cells, in particular, lose CD47 expression, making them susceptible to phagocytosis. Given the potential for exploiting phagocytosis, this "do not eat me" signal has become a focus of novel cancer therapeutics. We have demonstrated that downregulating SIRPα expression using specific microRNAs (miRNAs) promotes the phagocytic activity of MΦs. miRNAs are small non-coding RNA molecules that regulate gene expression post-transcriptionally. We identified the macrophage-like cell line THP-1, which expresses SIRPα at high levels and is therefore ideal for screening miRNAs targeting SIRPα. In THP-1, we screened a total of 10 siRNA candidates (Figure 1A), and confirmed that three of them had distinct knockdown potentials (Figure 1B). We then transduced human monocytes isolated from peripheral blood mononuclear cells (PBMCs) with a lentiviral vector encoding the most promising miRNA, miRNA 3. We then verified SIRPα knockdown in primary human macrophages (Figure 1C). A non-targeting (NT) miRNA was used as a negative control. Indeed, miRNA 3 downregulated SIRPα expression in primary human macrophages to approximately 0.5 of the NT miRNA level (1.0).

[0106] Example 2: Generation of chimeric antigen receptors (mCARs) that are expressed on macrophages and induce antigen-specific phagocytosis of tumor cells First, we investigated which cytoplasmic tail best activates genetically modified macrophages. Specifically, we designed a macrophage chimeric antigen receptor (mCAR) with FMC63, a single-chain variable fragment (scFv) mouse antibody that binds to human CD19 (huCD19) and has been successfully used in CAR T cell receptors (CAR T cell receptors) (Figure 2A). Importantly, the amino acid sequence of the anti-CFD19 scFv was publicly available (ADM64594.1), suitable reagents for evaluating mCAR were commercially available, and CD19-expressing Raji cells, which serve as targets for CAR-expressing cells, were already available.

[0107] The cytoplasmic tails we explore are the FcγR chain and FcγRIIa (CD32A) (Variant 1 and 2 in Figure 2A). For reference, we included a CAR published by CARISMA, which contains a CD3ζ signaling domain. CD3ζ is normally part of the T cell receptor complex, but it also appears to be able to induce Syk signaling in macrophages and promote phagocytosis. Nevertheless, we believe that the Fcγ chain and FcyRIIa chain are better choices for creating mCARs because they are constitutively expressed by macrophages and play a key role in antibody-dependent cell phagocytosis (ADCP). A common feature of these FcγR chains is the immunoreceptor tyrosine-based activation motif (ITAM), whose activation via phosphorylation enhances Syk signaling in macrophages and promotes antigen-specific phagocytosis.

[0108] We found that all three mCARs exhibited similar cell surface expression levels and antigen-binding capabilities (data not shown). More interestingly, we examined antigen-specific phagocytosis by primary human macrophages after recognizing CD19-expressing target cells (Raji cells). Briefly, we transduced macrophages with viral vectors encoding different receptors and cocultured them with fluorescently labeled Raji cells as target cells. We also transduced macrophages with a lentiviral vector encoding only a reporter gene but lacking mCAR (a "mock" control). We found that macrophages bearing all different mCARs phagocytosed significantly more Raji cells than "mock"-transduced macrophages (Figure 2B), and no differences were observed between the types of cytoplasmic tails (Figure 2C).

[0109] Next, we performed an initial characterization of the EGFR-specific cetuximab mCAR in primary human macrophages for its antigen-binding ability. We stained macrophages transduced with recombinant EGFR protein and confirmed the binding of the protein by staining the Avi tag of the recombinant EGFR (Figure 3A). We also evaluated the ability of cetuximab mCAR to induce antigen-specific phagocytosis of the EGFR-expressing tumor cell line MDA-MB-231. Because the MDA-MB-231 cell line does not express CD19, a CD19-specific mCAR should not induce phagocytosis. As an additional control, we added cytochalasin D, which inhibits phagocytosis, to demonstrate that the mCAR induces phagocytosis, not just binding to target cells. We were able to clearly demonstrate that cetuximab mCAR induces specific phagocytosis of MDA-MB-231 cells.

[0110] Example 3: Chimeric cytokine receptors on macrophages The TME (tumor microenvironment) is rich in anti-inflammatory cytokines such as IL-10 and TGF-β. It is true that we would benefit from the presence of IL-10 or TGF-β in the TME. For this reason, we will generate genetically modified macrophages that express chimeric cytokine receptors that bind, for example, IL-10 but induce pro-inflammatory signals via the cytoplasmic IFN-γ chain.

[0111] Example 4: IL-10-IFNγ Chimeric Cytokine Receptor (IL-10 ChCR) As a first prototype, we designed and generated a chimeric cytokine receptor pair containing the extracellular recognition domains of IL-10, i.e., IL-10Rα and Rβ, and the intracellular cytoplasmic domains of IFN-γR1 and R2 (Figure 4A).

[0112] Homodimerization of the prototypic chimeric cytokine receptor in response to IL-10 binding should trigger the IFN-γ signaling pathway through phosphorylation of signal transducer and activator of transcription 1 (STAT1) by the corresponding cytoplasmic domain. IFN-γ-triggered phosphorylated STAT1 signaling leads to the polarization of macrophages (MΦ) toward the classical "M1" phenotype rather than the alternative "M2" phenotype.

[0113] We verified the expression and functionality of the chimeric cytokine receptor in HEK-Blue™ IFN-γ cells (InvivoGen) (Figure 4B). Briefly, we transduced the IL-10 ChCR into the HEK-Blue™ IFN-γ cell line, which contains an IFN-γ-driven, STAT-1-inducible secreted alkaline phosphatase (SEAP) reporter but lacks either IL-10 receptor. The amount of SEAP can be easily assessed using a colorimetric assay (QuantiBlue). Coexpression of IL-10Rα-IFN-γR1 and IL-10Rβ-IFN-γR2 in response to IL-10 resulted in the highest overall SEAP activity. Furthermore, we observed elevated SEAP activity in IL-10-stimulated cells transduced with Rα alone, without Rβ. We concluded that high expression of the high-affinity IL-10Rα-IFN-γR1 subunit is sufficient to induce partial STAT1 signaling upon binding to IL-10. We confirmed the above data by demonstrating STAT1 phosphorylation upon ChCR stimulation with IL-10 via Western blot in HEK-Blue™ IFN-γ cells and the human monocytic cell line THP-1 (Figures 4C and 4D). In primary human macrophages, we also demonstrated STAT1 phosphorylation upon ChCR stimulation with IL-10 (Figure 4E). STAT1 is the first non-receptor kinase phosphorylated upon activation of specific cytokine receptors. Here, we induced the IFNγ signaling pathway by stimulating the chimeric cytokine with IL-10.

[0114] Given this data obtained with the IL-10ChCR, we set out to create a second variant of the ChCR (TGFβ chimeric cytokine receptor) consisting of the extracellular domain of the TGF-β receptor and the intracellular domain of the IFN-γ receptor.

[0115] As a next step, we transduced primary human monocytes with the IL-10 ChCR lentiviral vector. As controls, we used monocytes transduced with a control lentivirus ("mock") or untransduced monocytes. After transduction, we differentiated the monocytes into macrophages. The macrophages were stimulated with IFNγ for M1 activation, IL-10 for M2 activation, or left unstimulated. After stimulation, we harvested the cells and analyzed the expression of HLA-DR and CD38, a marker of M1 activation, and CD163, a marker of M2 activation (Figure 5).

[0116] Example 5: TGF-β-IFNγ Chimeric Cytokine Receptor (TGF-β ChCR) As mentioned above, after obtaining the first functional data for the prototype ChCR, we initiated the TGF-β-IFNγ ChCR combination. We have already collected initial data on this ChCR variant in THP-1 and primary human macrophages. TGF-β strongly induces pSTAT1 only in THP-1 cells and primary human macrophages expressing the TGF-β ChCR. THP-1 cells and primary human macrophages transduced with an irrelevant lentiviral vector ("mock") did not phosphorylate STAT1 (Figure 6).

[0117] Next, we transduced primary human monocytes with the TGF-β ChCR lentiviral vector and used monocytes transduced with a control lentivirus ("mock") or untransduced monocytes as controls. After transduction, we differentiated the monocytes into macrophages. These macrophages were stimulated with IFNγ and 1, 10, or 100 ng / mL TGF-β for M1 activation, or were left unstimulated. After stimulation, we harvested the cells and analyzed the expression of HLA-DR, CD38, and CD86 (as M1 markers) (Figure 7).

[0118] Example 6: Stimulation of IL-10 and TGF-β chimeric cytokine receptors We transduced primary human monocytes with ChCR (IL-10 or TGF-β) lentiviral vectors and used monocytes transduced with control lentivirus ("mock") or untransduced monocytes as controls. After transduction, we differentiated the monocytes into macrophages. After stimulation with 1, 10, and 100 ng / mL of TGF-β for TGF-β ChCR or 1, 10, 100, and 500 ng / mL of IL-10 for IL-10 ChCR, we measured the secretion of IP-10 by stimulated macrophages by ELISA. As controls, we used medium without cytokine ("0") or containing IFN-γ. Although IP-10 is normally secreted only upon IFN-γ stimulation, we show in Figure 8 that it is also secreted when macrophages express ChCR and are stimulated with the respective cytokine (e.g., IL-10 or TGF-β).

[0119] Example 7: Tumoricidal effect of macrophage-conditioned medium We transduced primary human monocytes from healthy donors with an empty lentiviral vector ("mock"), lentiviruses expressing the IL-10 ChCR (Figure 9A), or TGFβ ChCR (Figure 9B), or left untransduced. After 7 days of differentiation into monocyte-derived macrophages (MDMs), they were stimulated for 2 days with cytokine-free, IFN-γ, IL-10, or TGF-β-containing medium, as shown in Figure 9. Conditioned medium from stimulated macrophages was used to treat MDA-MB-231 or BT-549 cell lines for 3 days. The viability of these tumor cells was assessed using a WST1 assay. We demonstrate that conditioned medium from ChCR-expressing macrophages stimulated with IL-10 or TGFβ negatively impacts tumor cell viability, similar to conditioned medium from macrophages stimulated with IFNγ (Figure 9). To control for potential effects of recombinant IL-10 or TGF-β on MDA-MB-231 and BT-549 cells, we treated the cell lines directly with the corresponding cytokines, demonstrating that the effects on viability were indeed caused by factors secreted by ChCR-expressing macrophages, rather than by the recombinant cytokines.

[0120] Example 8: We were able to transduce primary human macrophages with efficiencies of 69.62% ± 16.97%, 72.3% ± 15.5%, and 69.3% ± 11.58% (mean ± SD) using mock, IL-10, and TGF-β lentiviruses, respectively (Fig. 10A). We also confirmed the expression of IL-10 (Fig. 10B) and TGF-β (Fig. 10C) on the surface of transduced cells.

[0121] Example 9: Sequences Table 1 lists the sequences of the present invention that are also attached to the ST.26 sequence protocol. Repeated portions of the sequences are labeled with the corresponding common font.

[0122] [Table 1] JPEG2025542299000002.jpg214153JPEG2025542299000003.jpg216153JPEG2025542299000004.jpg166153JPEG2025542299000005.jpg200153

Claims

1. 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; The first ChCR polypeptide comprises: - a first extracellular domain, - the first type 1 transmembrane domain, - optionally a first flexible linker domain connecting said first extracellular domain and said first transmembrane domain, - a first intracellular domain, and the second ChCR polypeptide comprises: - a second extracellular domain, a second type 1 transmembrane domain, - optionally a second flexible linker domain connecting said second extracellular domain and said second transmembrane domain, - a second intracellular domain, where: - the first extracellular domain and the second extracellular domain are capable of inducing dimerization of said ChCR polypeptide heterodimer upon binding to a cytokine selected from IL-10 and TGFβ; and - 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 said ChCR polypeptide heterodimer, The isolated monocytes or macrophages.

2. - the first extracellular domain is or comprises the extracellular domain of IL-10 receptor alpha (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, The isolated monocyte or macrophage of claim 1.

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

4. - the first extracellular domain is or comprises the extracellular domain of IL-10 receptor alpha (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, The isolated monocyte or macrophage of claim 1.

5. The ChCR polypeptide heterodimer comprises: - a first extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 21; - a first transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 27, and - a first intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 29; - a second extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 22; - a second transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 26, and - a second intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 28; Including, wherein the ChCR polypeptide heterodimer has 85% or more, particularly 90% or more, 95% or more of the biological activity of the ChCR heterodimers of SEQ ID NO: 59 and SEQ ID NO: 60; 10. The isolated monocyte or macrophage of claim 1 or 4.

6. - the first extracellular domain is or comprises a TGFβ receptor type I extracellular domain; - the second extracellular domain is or comprises a TGFβ 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, The isolated monocyte or macrophage of claim 1.

7. The ChCR polypeptide heterodimer comprises: - a first extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 23; - a first transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 26, and - a first intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 28; - a second extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 24; - a second transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 27, and - a second intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 29; Including, wherein the ChCR polypeptide heterodimer has 85% or more, particularly 90% or more, 95% or more of the biological activity of the ChCR heterodimers of SEQ ID NO: 55 and SEQ ID NO: 56; 10. The isolated monocyte or macrophage of claim 1 or 6.

8. - the first extracellular domain is or comprises a TGFβ receptor type I extracellular domain; - the second extracellular domain is or comprises a TGFβ 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, The isolated monocyte or macrophage of claim 1.

9. The ChCR polypeptide heterodimer comprises: - a first extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 23; - a first transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 27, and - a first intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 29; - a second extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 24; - a second transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 26, and - a second intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 28; Including, wherein the ChCR polypeptide heterodimer has 85% or more, particularly 90% or more, 95% or more of the biological activity of the ChCR heterodimers of SEQ ID NO: 61 and SEQ ID NO: 62; 10. The isolated monocyte or macrophage of claim 1 or 8.

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

11. 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, the expression vector is selected from the group of a viral vector, a plasmid, a DNA molecule, or an RNA molecule, The isolated monocyte or macrophage of claim 10, more particularly wherein the expression vector is a lentiviral vector.

12. The isolated monocyte or macrophage of any one of claims 1 to 11, wherein the monocyte or macrophage further comprises an inhibitory nucleic acid molecule against SIRPα.

13. The isolated monocyte or macrophage of claim 12, wherein the inhibitory nucleic acid molecule against SIRPα comprises a sequence selected from the group of SEQ ID NO: 33 to SEQ ID NO:

52.

14. The monocyte or macrophage: - an extracellular antigen-binding domain, in particular a Fab fragment, a transmembrane domain, in particular the CD8 transmembrane domain, and an intracellular domain comprising at least one ITAM motif, in particular the intracellular domain of an Fc receptor, more in particular the intracellular domain of an Fc gamma receptor or the intracellular domain of CD32a, o optionally a linker domain connecting said extracellular antigen-binding domain and said transmembrane domain; 14. The isolated monocyte or macrophage of any one of claims 1 to 13, further comprising a chimeric antigen receptor (CAR) polypeptide comprising:

15. 15. The isolated monocyte or macrophage of claim 14, wherein the CAR polypeptide comprises a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to any one of SEQ ID NOs: 13 to 18.

16. 16. The isolated monocyte or macrophage of any one of claims 1 to 15 for use in the treatment or prevention of cancer.

17. - an expression vector encoding a ChCR polypeptide according to any one of claims 1 to 9; - an inhibitory nucleic acid molecule against SIRPα according to claim 12 or 13; and - an expression vector encoding the CAR polypeptide of claim 14 or 15. Kit including:

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

19. an inhibitory nucleic acid molecule against SIRPα according to claim 12 or 13; and / or - a CAR polypeptide according to claim 14 or 15; 19. The method of claim 18, wherein further inserted into said monocytes.

20. - a first extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 21; - a first transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 26, and - a first intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 28; - a second extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 22; - a second transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 27, and a second intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO:

29. A ChCR polypeptide heterodimer comprising:

21. - a first extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 21; - a first transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 27, and - a first intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 29; - a second extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 22; - a second transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 26, and a second intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO:

28. A ChCR polypeptide heterodimer comprising:

22. - a first extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 23; - a first transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 26, and - a first intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 28; - a second extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 24; - a second transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 27, and a second intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO:

29. A ChCR polypeptide heterodimer comprising:

23. - a first extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 23; - a first transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 27, and - a first intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 29; - a second extracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 24; - a second transmembrane domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO: 26, and a second intracellular domain comprising a sequence having at least 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to SEQ ID NO:

28. A ChCR polypeptide heterodimer comprising:

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