Dual CSF1-il-10 cytokine

Abstract

The invention relates to single-chain polypeptides having IL-10 and CSF1 activities and to the use thereof in therapy.

Description

[0001] DUAL CYTOKINE CSF1-IL-10 Field of the invention The invention relates to single chain polypeptides having both IL-10 and CSF1 activities and their use in therapy. Background of the invention Cytokines are proteins produced in the immune system and act as chemical messengers to communicate between cells of the immune system and also with other cells in the body. Cytokines control the full range of immune responses, including their initiation, type, strength, and duration. So much so that their unregulated production triggers a wide variety of inflammatory and autoimmune diseases, allergies, fibrosis, and can even cause cancer. For this reason, there is great medical interest in the development and use of cytokines as drugs to treat diseases. Interleukin 10 (IL-10) is an anti-inflammatory cytokine that plays a crucial role in regulating immune responses and maintaining immune homeostasis.IL-10 is a homodimeric cytokine produced as a monomer by various immune cells, including T cells, B cells, macrophages, and dendritic cells, and exerts its effects by binding to its receptor complex, which is composed of two subunits, IL-10R1 and IL-10R2. When IL-10 binds to its receptor, IL-10R1 first recognizes and binds to IL-10 with high affinity, forming a complex that is then stabilized by the binding of IL-10R2. Binding of IL-10 to its receptor complex leads to the activation of signaling pathways. When bound to its receptor on myeloid cells, IL-10 exerts potent anti-inflammatory activities, including inhibition of the production of proinflammatory mediators, the expression of MHCII, and costimulatory molecules. Simultaneously, the activation of myeloid cells by IL-10 leads to the expression of anti-inflammatory mediators and promotes the development of tolerogenic phenotypes, such assuch as anti-inflammatory M2 macrophages and tolerogenic dendritic cells. In contrast to its strong anti-inflammatory activity on myeloid cells, IL-10 is a growth factor for B cells and promotes plasma cell differentiation and antibody production. Similarly, IL-10 has stimulatory effects on CD8+ T cells, inducing their proliferation, IFNϒ production, and cytotoxicity. In humans, there is a strong genetic association between the IL-10 pathway and autoimmune diseases, such as inflammatory bowel disease. Loss-of-function mutations in IL-10, IL-10RA, or IL-10RB cause severe, early-onset, refractory enterocolitis. Restoring IL-10RA or IL-10 expression by hematopoietic stem cell transplantation rapidly alleviates clinical symptoms. In turn, mice deficient in IL-10 or IL-10R developcolitis spontaneously, while administration of IL-10 in different animal models of colitis has consistently been shown to be beneficial. Finally, mice in which all cells in the body respond to IL-10, except for specific subsets of myeloid cells including monocytes and macrophages, also develop spontaneous colitis, which is mediated by the production of the clinically validated target IL-23. Myeloid cells exclusively express the CSF1 receptor, i.e., CSF1R. Stimulation of CSF1R by its ligand, the cytokine CSF1, is essential for the differentiation, proliferation, and maintenance of anti-inflammatory M2 macrophages in the intestine, which play a key role in generating a tolerogenic environment through the production of IL-10. In autoimmune diseases, anti-inflammatory M2 macrophages in affected tissues, such as the mucosa of IBD patients, are replacedby inflammatory monocytes that drive disease progression. Although in preclinical studies IL-10 has shown promising results in improving inflammation and tissue damage in colitis models, clinical trials investigating the therapeutic potential of IL-10 in patients with colitis have not been successful. One possible explanation is that the delivery of IL-10 to the site of inflammation may be a limiting factor. However, some strategies aimed at improving IL-10 delivery to the site of inflammation did not show clinical benefits (AMT-101). Another possible explanation is that the anti-inflammatory effects of IL-10 on myeloid cells are counteracted by proinflammatory effects that damage tissue, including the activation, differentiation, and induction of antibodies by B cells and the induction of cytotoxic activities by CD8+ T cells. To address this limitation,IL-10-based therapeutic agents that can selectively activate myeloid cells while preventing the activation of other immune cell subsets. Several strategies have been proposed to develop IL-10-based therapeutic agents that selectively activate myeloid cells. These include fusing IL-10 with antibodies that bind to myeloid cell receptors or encapsulating IL-10 in nanocarriers (liposomes or nanoparticles) specifically targeting myeloid cells. The present invention seeks to overcome or at least alleviate one or more of the deficiencies of the prior art. Summary of the Invention The invention relates to a single chain polypeptide having IL-10 and CSF1 activities. The invention further relates to a pharmaceutical composition comprising the single chain polypeptide according to the invention. The invention further relates to the use of the single chain polypeptide according to the invention.as a medicament, in particular for use in the treatment of an inflammatory disease. Detailed description of the invention The inventors have designed single chain polypeptides that combine the activities of IL-10 and CSF1. Fusion proteins combining IL-10 and CSF1 monomers display either reduced IL-10 activity when the N-terminus of IL-10 is joined to the C-terminus of CSF1, or drastically reduced IL-10 activity when the C-terminus of IL-10 is joined to the N-terminus of CSF1. Unexpectedly, the design of fusion proteins combining single chain dimeric IL-10 and CSF1 monomer made it possible to obtain single chain polypeptides that display substantial IL-10 and CSF1 activities. On the one hand, the IL-10 monomers pair to form a "domain swapped" dimeric IL-10 protein. In a domain-swapped dimeric protein such as IL-10, the monomeric structure of the standard helix bundle ofthe cytokine IL-10 opens and embraces in an antiparallel manner another open IL-10 monomer generating two functional cytokine domains split together, defining two adjacent 3D domains. On the other hand, CSF1 monomers dimerize with the two face-to-face CSF1 monomeric molecules and are held together by a disulfide bond. Without wanting to be bound by theory, the inventors hypothesized that the fusion of IL-10 and CSF1 could result in the formation of concamers, since IL-10 is an exchanged dimer and CSF1 dimerizes, which would be prevented when the IL-10 monomer is replaced by a single-chain dimeric IL-10. Functional characterization of the single-chain dual polypeptide having both IL-10 and CSF1 activities has shown that the dual cytokine CSF1-IL-10 inhibits myeloid cell activation and induces the differentiation of monocyte-derived macrophages that have a regulatory phenotype (M2 phenotype). Furthermore, the dual cytokine CSF1-IL-10has specificity for monocytes and does not activate CD8 T cells or B cells. Functionally, in vivo, the dual cytokine CSF1-IL-10 was shown to alleviate the clinical symptoms of inflammatory disease, in a mouse model of TNBS-induced colitis. Single chain polypeptide with IL-10 and CSF1 activity Thus, there is provided a single chain polypeptide having both IL-10 and CSF1 activities. Herein, “IL-10 activity” or “IL-10 activities” indicates one or more biological activities mediated by IL-10 through binding to its receptor, the IL-10R. These comprise or consist of (i) binding to IL-10R on myeloid cells, in particular monocytes, or (ii) anti-inflammatory activities, or preferably both. Anti-inflammatory activities include (a) inhibition of the production of proinflammatory mediators (e.g., proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), IL-1, IL-12, IL-6, and factorgranulocyte-macrophage colony-stimulating enzyme, inflammatory enzymes, such as cyclooxygenase 2 and inducible nitric oxide synthase, chemokines, such as RANTES, membrane inflammatory protein 1α (MIP1α), IL-8, and eotaxin), and / or (b) the inhibition of the expression of MHCII and costimulatory molecules by myeloid cells, and / or (c) the induction of differentiation of monocytes into tolerogenic macrophages. In some embodiments, the anti-inflammatory activities comprise the inhibition of TNF-α and / or IL-6 production by activated monocytes. In some embodiments, the anti-inflammatory activities comprise the induction of differentiation of monocytes into tolerogenic macrophages. Tolerogenic macrophages have an M2-like phenotype and can be identified by their regulatory phenotype and high levels of CD206 and CD163 expression. In some embodiments, IL-10 activities comprise or consist of binding to the IL-10R on myeloid cells, inparticular monocytes, the inhibition of TNF-α and / or IL-6 production by activated monocytes, and the differentiation of monocytes into tolerogenic macrophages. In some embodiments, the single chain polypeptide having IL-10 and CSF1 activities has reduced T cell and B cell activation capabilities compared to IL-10. As used herein, "CSF1 activity" or "CSF1 activities" indicates one or more biological activities mediated by activation of the CSF1 receptor, CFS1-R. These comprise or consist of (i) activation of CFS1-R on the surface of monocytes or progenitors thereof, and / or (ii) differentiation of monocytes into macrophages, preferably both. In some embodiments, CSF1-R activation is triggered by CSF1 binding to CSF1-R. In some embodiments, the single chain polypeptide having IL-10 and CSF1 activities has a similar or higher affinity to CSF1R compared to CSF1.wild-type. In some embodiments, the single chain polypeptide having IL-10 and CSF1 activities has reduced affinity to the IL-10 receptor α (IL10RA) and the IL-10 receptor β (IL10RB) compared to wild-type IL-10. In some embodiments, the single chain polypeptide having IL-10 and CSF1 activities has similar or greater affinity to the CSF1R compared to wild-type CSF1 and has reduced affinity to the IL-10 receptor α (IL10RA) and the IL-10 receptor β (IL10RB) compared to wild-type IL-10. These properties are expected to favor selective binding of the single chain polypeptide having IL-10 and CSF1 activities to myeloid cells that exclusively express the CSF1 receptor, among the cells of the innate immune system, and subsequent activation thereof. By "similar affinity" it is meant herein that the affinity preferably does not vary (i.e., increases ordecreases) by a factor greater than 5-fold, preferably 4-fold, 3-fold, 2-fold, or 1.5-fold compared to the reference level of affinity of the wild-type cytokine (CSF1). By "higher" or "reduced" it is meant herein that the affinity preferably varies (i.e., increases or decreases) by at least a factor of 5-fold, preferably, 7-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold compared to the reference level of affinity of the wild-type cytokine (CSF1 or IL-10 as applicable). In a first aspect, there is thus provided a single chain polypeptide having IL-10 and CSF1 activities comprising an IL-10 monomer fused to a CSF1 monomer wherein the N-terminus of IL-10 is joined to the C-terminus of CSF1 through a linker. Indeed, in this orientation, the single chain polypeptide has a decreased IL-10 activity compared towild-type IL-10, but retains CSF1 activity similar to that of wild-type CSF1. According to this aspect, the single chain polypeptide having both IL-10 and CSF1 activities thus comprises, in the N-terminal to C-terminal direction, a CSF1 monomer, a peptide linker, and an IL-10 monomer. Preferably, the linker joining the C-terminal end of the CSF1 monomer to the N-terminal end of the IL-10 monomer is a (flexible) peptide sequence composed of Gly and Ser residues, in different ratios. Examples of suitable linkers comprise or consist of GGGSGGSGGSGGSGGSGGSGGGGG (25 amino acids in length, SEQ ID NO: 34), GGGGGSGGSGGSGGSGGSGGSGGSGGGG (30 amino acids in length, SEQ ID NO: 35), or GGGGGSGGGSGGSGGSGGSGGSGGSGGSGGSGGGGGG (35 amino acids in length, SEQ ID NO: 36). The CSF1 monomer may be as defined in the following section. The IL-10 monomer may be wild-type mature IL-10 as shown in SEQ IDNO:11, or a mutant thereof, or a polypeptide comprising the mature IL-10 sequence and at the N-terminus 1, 2, 3 or more additional amino acid residues consecutively present in the IL-10 signal peptide (i.e., A, RA, or VRA). In some embodiments, the single chain polypeptide having IL-10 and CSF1 activities comprises or consists of the sequence SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical thereto and retaining IL-10 and CSF1 activities. In a second aspect, there is provided a single chain polypeptide having IL-10 and CSF1 activities comprising a single chain dimeric IL-10 fused to a CSF1 monomer. In some embodiments, the single chain polypeptide having IL-10 and CSF1 activities comprises a single chain dimeric IL-10 (SC)-fusion protein. In some embodiments, the polypeptidesingle chain having IL-10 and CSF1 activities comprises a single chain dimeric IL-10-CSF1 monomer fusion protein. In some embodiments, the single chain dimeric IL-10 and the CSF1 monomer are joined through a linker comprising 10 to 45 amino acid residues, preferably 12 to 40 amino acids, still preferably 15 to 40 amino acids, still preferably 20 to 35 amino acids, even more preferably 25 to 35 amino acids. Preferably, the linker joining the C-terminus of the SC dimeric IL-10 to the N-terminus of CSF1, or joining the N-terminus of the SC dimeric IL-10 to the C-terminus of CSF1, is a (flexible) peptide sequence composed of Gly and Ser residues, in different ratios. Examples of suitable linkers comprise or consist of GGSGGSGGSGGSGGG (15 amino acids in length, SEQ ID NO: 33), GGGSGGSGGSGGSGGSGGSGGG (25 amino acids in length, SEQ ID NO: 34),GGGGGSGGSGGSGGSGGSGGSGGSGGG (30 amino acids in length, SEQ ID NO: 35), or GGGGGSGGGGSGGSGGSGGSGGSGGSGGSGGSGGG (35 amino acids in length, SEQ ID NO: 36). Preferably, when the single chain polypeptide having IL-10 and CSF1 activities comprises a single chain dimeric CSF1 monomer-IL-10 fusion protein, the linker joining the C-terminus of CSF1 to the N-terminus of SC dimeric IL-10 comprises more than 15 amino acids, preferably at least 20 or 25 amino acids. CSF1 Monomer In said first and second aspects of the invention, the CSF1 monomer incorporated into the single chain polypeptide having IL-10 and CSF1 activities may be a wild type CSF1 monomer, or mutant thereof, or a circular permutant CSF1 monomer. A wild-type human CSF1 polypeptide sequence is shown in SEQ ID NO: 12. In some embodiments, the CSF1 monomer is a mutant CSF1 or a circular permutant CSF1 monomer having asimilar or greater affinity to CSF1R compared to wild-type CSF1. In some embodiments, the CSF1 monomer is a CSF1 monomer mutant comprising the sequence SEQ ID NO:12 modified by at least one substitution or substitutions: a) Q17R; b) V78W; c) T124I; d) V120I; e) Q17R, T124I and V120I; f) V78W, T124I and V120I; or g) Q17R, V78W, T124I and V120I. The Q17R or V78W substitution in the CSF1 monomer mutant was shown to improve EC-50 values ​​in HEK293 cells transfected with a CSF1R reporter kit (hence the affinity for CSF1R) compared to wild-type CSF1. The T124I or V120I substitutions in the CSF1 monomer mutant are designed to increase the stability of the CSF1 monomer in the CSF1R receptor-bound conformation and enhance the maximal response of HEK cells transformed with the CSFR1 reporter kit. A circular permutation in the structure of a protein is a rearrangement of the amino acid sequence, such thatthat the original N- and C-termini of the polypeptide appear to be joined, and new ones are created elsewhere. Circular permutations are therefore usually prepared by connecting the ends of the native protein, or terminal regions, by a covalent linker and introducing new N- and C-termini by cleaving an existing peptide bond. In some embodiments, the CSF1 monomer is a circular permutant of CSF1 generated by joining one amino acid in each of the N- and C-terminal regions of CSF-1 and deleting the peptide bond consisting of residues at positions 95 to 99 of SEQ ID NO: 12. As used herein, one amino acid in each of the N- and C-terminal regions of CSF-1 denotes amino acids located at the N-terminus of CSF1 (i.e., residue at position 1 of SEQ ID NO:12) and at the C-terminus of CSF1 (i.e., residue at position 150 of SEQ ID NO:12), or at aamino acid position located at most 2, 3, 4, 5, 6, 7 or 8 residues away from the N- or C-terminus of CSF1. Preferably, the CSF1 monomer is a circular permutant of CSF1 comprising or consisting of the sequence SEQ ID NO: 9 (referred to as ORKMCSF_013), or SEQ ID NO: 10 (referred to as ORKMCSF_014), or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and which retains CSF1 activity. Both circular permutants were shown to activate the downstream CSF1 signaling cascade with similar and slightly better EC-50 values ​​than the wild-type CSF1 monomer. Single Chain Dimeric IL-10 The SC dimeric IL-10 incorporated into the single chain polypeptide having IL-10 and CSF1 activities may be a fusion protein comprising a first IL-10 monomer fragment comprising at least α-helices A to F of IL-10, a peptide linker, and a secondIL-10 monomer fragment comprising at least α-helices A to F of IL-10, or a circular permutant thereof. The folding of the dimeric IL-10 of SC is such that a "continuous IL-10 3D domain" (left domain in Figure 2), and a split IL-10 3D domain (right domain in Figure 2) are formed. A wild type human IL-10 polypeptide sequence is shown in SEQ ID NO: 11: SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQ hhh hhhhhhhhhhh hhhhhh hhhhhhhhhhhhhhhhhhh HA HB AENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN hhh hhhhhhhhhhhhhhhhhhh hhhhhhhhhhhh hhhhhhhhhhhhh HC HD HE HF The positions of the helices are represented by strings of 'h' in the sequence above. For α-helix F, depending on the structural assignments made by different computer programs on different crystal structures of the same molecule, the helix extendssystematically up to M154, and may extend to M156 or I158 (underlined italic characters). Accordingly, the α-helix F is considered herein to be fully incorporated provided that the IL-10 monomer fragments comprise amino acid residues extending at least to M154. According to one embodiment, the SC dimeric IL-10 comprises or consists of the sequence MTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQ hhh hhhhhhhhhhh hhhhhh hhhhhhhhhhhhhhhhhhh HA HB AENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINY hhh hhhhhhhhhhhhhhhhhhh hhhhhhhhhhhh hhhhhhh hhhhhh HC HD HE HF IEAYX(NtCt)ZNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEV hhhh hhhhhhhhhhhh hhhhhh hhhhhhhhhhhhhhhh HF HA HB MPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIF hhhhhh hhhhhhhhhhhhhhhhhhh hhhhhhhhhhhh hhhhhhh hhh HC HD HE INYIEAYMTMKIRN hhhhhhhhhhhh HF (sequence SEQ ID NO: 65-X-(NtCt)-Z-SEQ ID NO:66), wherein (NtCt) is a peptide linker, wherein X is absent or present, and when present consists of one or more amino acids of the original IL-10 sequence in continuity with preceding amino acids on its N-terminal side, optionally with a mutation to accommodate the NtCt linker, wherein Z is absent or present, and when present consists of one or more amino acids of the original IL-10 sequence in continuity with preceding amino acids on its C-terminal side, optionally with a mutation to accommodate the NtCt linker, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and retaining at least the same stability, and / or at least the same level of interaction with the IL-10 receptor. For example, X may represent M, MT, MTM, MTMN (SEQ ID NO: 67) that are present in the original IL-10 sequence in continuity with the preceding amino acids on its N-side.terminal. X may also represent L or K, i.e. a mutation with respect to the original IL-10 sequence to accommodate the NtCt linker. Accordingly, X is preferably selected from the group consisting of M, MT, MTM, MTMN (SEQ ID NO: 67), L or K. For example, Z may represent LPs that are present in the original IL-10 sequence in continuity with the following amino acids on its C-terminal side. Z may also represent PEFA (SEQ ID NO: 68) or ELA. Accordingly, Y is preferably selected from the group consisting of LP, PEFA (SEQ ID NO: 68) or ELA. The sequence of the NtCt linker joining the C-terminus of one IL-10 monomer fragment and the N-terminus of the other IL-10 monomer fragment may have any suitable sequence. Preferably, it comprises a sequence defining a conformationally constrained structure, i.e. a "structured" linker. In this way, the designed linker helps to restrictconformationally the 3D domain structures in a suitable / desired structural orientation. These linkers may have from about 3 to about 20 amino acid residues, from about 3 to about 16 amino acid residues, from about 4 to about 12 amino acid residues, from about 4 to about 8 amino acid residues, from about 3 to 8 amino acid residues, or from about 3 to 6 amino acid residues. Advantageously, such linkers do not contain a plurality of Gly and / or Ser residues adjacent to each other; for example, advantageously the linker peptide contains 5 or fewer adjacent Gly and / or Ser residues; conveniently 3 or fewer adjacent Gly and / or Ser residues; 2 or fewer Gly and / or Ser residues; contains only isolated Gly and / or Ser residues; or in some embodiments does not contain any Gly and / or Ser residues. In one embodiment, the NtCt linker comprises from 3 to 20 amino acid residues and no more than 2adjacent Gly and / or Ser residues. According to some embodiments, the NtCt linker comprises the sequence NGGLDY (SEQ ID NO: 30), FGGLDY (SEQ ID NO: 48), YKTIT (SEQ ID NO: 31), or DKDIRDGD (SEQ ID NO: 32). The NtCt linker may be preceded, at its N-terminus, by X, i.e. amino acid residue(s) that are naturally and consecutively present at position(s) 152 to 157 of IL-10, as shown in SEQ ID NO: 11. Alternatively, or additionally, the NtCt linker may be followed, at its C-terminus, by Z, i.e. amino acid residue(s) that are naturally and consecutively present at position(s) 16 to 20 of IL-10, as shown in SEQ ID NO: 11. Thus, according to some embodiments, the NtCt linker and the sequence X or Z at its N-terminal or C-terminal side (sequences X and Z in bold have the same structure as in IL10 and correspond to the WT sequence of IL10 or could havepoint mutations to accommodate the linker) comprises or consists of the sequence NNGGLDYL (SEQ ID NO: 71), NFGGLDYL (SEQ ID NO: 45), LYKTITPEFA (SEQ ID NO: 46), KDKDIRDGDELA (SEQ ID NO: 47). In some embodiments, the single chain dimeric IL-10 comprises or consists of SEQ ID NO: 2 (ORK10-002), SEQ ID NO: 3, or SEQ ID NO: 49 (ORK10-003; with or without the addition of a HiBit tag sequence for quantification at the C-terminus of SEQ ID NO:3), or SEQ ID NO: 4 (ORK10-005), or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical thereto and retaining IL-10 activity. In some embodiments, the single chain dimeric IL-10 comprises a first IL-10 monomer fragment that includes a D84E substitution at the naturally occurring residue at position 84 of SEQ ID NO:11. The single chain dimeric IL-10 may then comprise or consist of SEQ ID NO: 1 or SEQ ID NO: 20 (Foldikine10 with orwithout the addition of a HiBit tag sequence for quantification at the C-terminus of SEQ ID NO:1). According to some embodiments, the SC dimeric IL-10 consists of a circular permutant of one of the above SC dimeric IL-10s. It is believed that the use of a circular permutant could be advantageous in minimizing a possible masking effect due to the way in which the fused CSF1 monomer is connected to the SC dimeric IL-10 (Figure 19). In some embodiments, the circular permutant comprises a first IL-10 monomer fragment comprising IL-10 α-helices E to F, a first dimeric SC IL-10 peptide linker, and a second IL-10 monomer fragment comprising at least IL-10 α-helices A to F, a second peptide linker, and a third IL-10 monomer fragment comprising at least IL-10 α-helices A to D. In some embodiments, the circular permutant comprises the sequence SEQ ID NO: 5 (Foldikine10_cut116, alsoreferred to herein as Foldikine10_cp116), or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and which retains IL-10 activity. Resulting single chain polypeptide comprising a single chain dimeric IL-10 fused to a CSF1 monomer and having both IL-10 and CSF1 activities According to some embodiments, the single chain polypeptide having IL-10 and CSF1 activities comprises or consists of the sequence SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:1, SEQ ID NO:20, SEQ ID NO: 21, SEQ ID NO: 22, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical thereto and retaining IL-10 and CSF1 activities. Pharmaceutical Composition The invention further relates to a pharmaceutical composition comprising the single chain polypeptide having IL-10 and CSF1 activities and apharmaceutically acceptable carrier. A suitable pharmaceutically acceptable "carrier" (such as diluents, adjuvants, excipients, or vehicles) is described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The pharmaceutical compositions of the invention are formulated to conform to regulatory standards and may be administered orally, intravenously, topically, or by other standard routes. As used herein, the term "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption-delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active ingredients is well known in the art. Except to the extent that any conventional media or agent is incompatible with the active ingredient, their use in the compositions is contemplated.Therapeutic. Supplementary active ingredients may also be incorporated into the compositions. The phrase "pharmaceutically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a human or non-human mammal. Acceptable pharmaceutical carriers may be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Pharmaceutical carriers may be saline, acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary agents, stabilizers, thickeners, lubricants, and colorants may be used. When administered to a subject, pharmaceutically acceptable carriers are preferably sterile. Water is a suitable vehicle.especially when the compound of the invention is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, limestone, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene glycol, water, ethanol, and the like. If desired, the present compositions may also contain small amounts of wetting or emulsifying agents, or buffering agents. The medicaments and pharmaceutical compositions of the invention may take the form of liquids, solutions, suspensions, lotions, gels, tablets, pills, granules, powders, modified release formulations (such as slow or sustained release), suppositories,emulsions, aerosols, sprays, capsules (e.g., capsules containing liquids or powders), liposomes, microparticles, or any other suitable formulation known in the art. Other examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th ed., 1995, see, e.g., pp. 1447-1676. In some embodiments, the therapeutic compositions or medicaments of the invention are formulated according to routine procedures as a pharmaceutical composition adapted for oral administration (most suitably for humans). Compositions for oral administration may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Thus, in various embodiments, the pharmaceutically acceptable carrier may be a capsule, a caplet, or a pill.Orally administered compositions may contain one or more agents, for example, sweeteners such as fructose, aspartame, or saccharin; flavorings such as peppermint, oil of wintergreen, or cherry; colorants; and preservatives, to provide a pharmaceutically palatable preparation. When the composition is presented in tablet or pill form, the compositions may be coated to delay disintegration and absorption in the gastrointestinal tract to provide sustained release of the active agent over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compositions. In these dosage forms, fluid from the environment surrounding the capsule is absorbed by the driving compound, which swells to displace the agent or agent composition through an opening. These dosage formsDosage forms can provide an essentially zero-order delivery profile, as opposed to the peaked profiles of immediate release formulations. A time-delay material such as glycerol monostearate or glycerol stearate may also be used. Oral compositions may include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such carriers are preferably of pharmaceutical grade. For oral formulations, the release site may be the stomach, the small intestine (duodenum, jejunum, or ileum), or the large intestine. One of skill in the art may prepare formulations that do not dissolve in the stomach, but release the material in the duodenum or elsewhere in the intestine. Suitably, the release will avoid the deleterious effects of the stomach environment, either by protecting the peptide (orderivative) or by release of the peptide (or derivative) beyond the stomach environment, such as in the intestine. To ensure full gastric resistance, an impermeable coating to a pH of at least 5.0 would be essential. Examples of the most common inert ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S and Shellac, which can be used as mixed films. To facilitate dissolution of the therapeutic agent(s) in the aqueous medium, a surfactant may be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate. Cationic detergents may be used and may include benzalkonium chloride orbenzethomium chloride. Possible non-ionic detergents that could be included in the formulation as surfactants include: lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene 10, 50 and 60 hydrogenated castor oil, glycerol monostearate, polysorbate 20, 40, 60, 65 and 80, sucrose fatty acid ester, methylcellulose and carboxymethylcellulose. These surfactants, when used, may be present in the formulation of the peptide or nucleic acid or derivative, alone or mixed in different proportions. Typically, compositions for intravenous administration comprise a sterile isotonic aqueous buffer. If necessary, the compositions may also include a solubilizing agent. Mixtures of the polypeptides described herein may be prepared in water conveniently mixed with one or more excipients, carriers or diluents. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, as well asin oils. Under normal conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Suitable pharmaceutical forms for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form may be sterile and sufficiently fluid to permit injection with a suitable syringe. It is desirable that the composition be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Adequate fluidity may be maintained, for example, byuse of a coating, such as lecithin, by maintaining the required particle size in the case of a dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be achieved by the use of absorption-delaying agents in the compositions, for example, aluminum monostearate and gelatin. For parenteral administration in an aqueous solution, for example, the solution can be suitably buffered if necessary and the liquid diluent is first made isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for the administrationintravenous, intramuscular, subcutaneous, intratumoral, and intraperitoneal. In this regard, those skilled in the art know the sterile aqueous media that can be employed. Another suitable route of administration for the pharmaceutical compositions of the invention is pulmonary or nasal administration. Additives can be included to improve cellular uptake of a therapeutic agent of the invention, such as fatty acids, oleic acid, linoleic acid, and linolenic acid. Therapeutic Applications The single chain polypeptide having IL-10 and CSF1 activities of the invention, or the pharmaceutical composition comprising same, is for use as a medicament. Accordingly, the invention further relates to a method of treatment comprising administering the single chain polypeptide having IL-10 and CSF1 activities of the invention to a subject in need thereof. The subject can be a human or non-human mammal, such as a rodent (mouse or rat), a primate (e.g.,example, a monkey), a pig, etc. The subject in need thereof is, in particular, a subject suffering from an inflammatory disease. The single chain polypeptide having IL-10 and CSF1 activities is designed to preferentially activate myeloid cells (monocytes, macrophages, dendritic cells) that co-express CSF1R and IL-10R, and that induce anti-inflammatory responses, over CD8 T cells and B cells expressing IL-10R that could induce cytotoxic responses and autoantibodies when activated. Accordingly, the invention further relates to a single chain polypeptide according to the invention for use in the treatment of an inflammatory disease. Also provided is a method of treating an inflammatory disease comprising administering the single chain polypeptide having IL-10 and CSF1 activities of the invention to a subject in need thereof. In some embodiments, the inflammatory disease is an inflammatory bowel disease, such asInflammatory bowel disease, including Crohn's disease and colitis, Behçet's disease, and / or other autoimmune diseases including but not limited to rheumatoid arthritis or type 1 diabetes mellius, systemic lupus erythematosus, among others. Treatment regimens may vary and often depend on the type and / or location of the disease, the stage / progression of the disease, and / or the health and age of the patient. The skilled physician will be able to determine an appropriate therapeutic treatment regimen in each circumstance. The invention will be illustrated in more detail in the following figures and examples. FIGURES Figure 1. Uncoupling of the pro- and anti-inflammatory functions of IL-10. The figure shows an engineered single molecule that brings together the activities of IL-10 and CSF-1, where the binding affinity of the IL-10 receptor is weakened and, as a result, only cells expressing the CSF-1 receptor will be activated by IL-10. Figure 2. IllustrationSchematic of the formation of a foldikine based on a domain-swapped dimeric class II cytokine (e.g., IL-10): (i) Wild-type domain-swapped dimer showing α-helices D, E, and F of each IL-10 monomeric unit aligned with α-helices A, B, and C of the opposite IL-10 monomer, to form adjacent 3D domains of IL-10 comprising α-helices A, B, and C of a first IL-10 monomer and α-helices D, E, and F of a second IL-10 monomer; (ii) single-chain foldikine-10 based on fused wild-type IL-10 monomers in which the monomeric sequences are joined to form a single-chain polypeptide by joining the C-terminus of one IL-10 monomer sequence to the N-terminus of a second monomer sequence via a peptide linker (dashed line). Wild-type linkers are shown in solid lines. Boxes indicate the α-helices that form each of the domains.Three-dimensional left and right images of IL-10 (continuous domain of IL-10 and split domain of IL-10). Figure 3. Analysis of Foldikine-10 versions. The different versions are label-free and quantified by ELISA (IL-10). The activity of the designed molecules was evaluated in commercial HEK-Blue10 cells in a dose-response experiment and the data were fitted to a sigmoidal dose-response to determine the EC-50 (see Methods). We set the reference line at 0.27 and the Hill slope at -1.6. IL-10WT (SEQ ID NO:11), Foldikine10 (SEQ ID NO: 1), ORK10-002 (SEQ ID NO: 2), ORK10-003 (SEQ ID NO: 3) and ORK10-005 (SEQ ID NO: 5). Figure 4. Circular permutants of foldikine-10. Schematic illustration of the circular permutations of foldikine-10. Wild-type linkers are shown in solid lines. Boxes indicate the α-helices that form each of the left and right three-dimensional domains of IL-10.(continuous domain of IL-10 and split domain of IL-10). The dashed lines represent the manipulated NtCt linkers. The new Nt and Ct resulting from the loop opening are shown as Nter and Cter. SEQ ID NO: 5 Foldikine_cut116, SEQ ID NO: 6 Foldikine_cut133, SEQ ID NO: 7 Foldikine_cut209, SEQ ID NO: 8 Foldikine_cut237. Figure 5. Dimeric structure of CSF1. CSF1 dimeric complex with the two subunits in different shades of gray. The position of the disulfide bridge is shown in black. Figure 6. Circular permutants of CSF1. Wild-type Nt-Ct of CSF1 and the two residues where we opened the loop (Y95, K100). Two circular permutants (ORKMCSF_013 and ORKMCSF_014) are generated with wild-type Nt-Ct linked with two different linkers. Figure 7. CSF1 circular permutants evaluated in HEK293 cells transfected with a reporter cell kit to measure the downstream MAPK / ERK signaling cascade upon CSF1 / CSF1R activation. CSF1(1-190) HiBit corresponds toa SEQ ID NO: 51. ORKMCSF_013 is SEQ ID NO: 9 and ORKMCSF_014 is SEQ ID NO: 10. Figure 8. Dose-response analysis of IL-10 variants with engineered affinity towards IL10RA in HEK-Blue10 cells. All mutations were introduced only in one half (continuous domain) of the foldikine ORK10-003b structure (SEQ ID NO: 49). The data have been fit to a sigmoidal dose-response to determine the EC-50 (see Methods). Reference and saturation values ​​have been set. Foldikine10 (ORK10-003REF is ORK10-003-HiBit of SEQ ID NO: 49). Mutant numbering is based on SEQ ID NO: 50. R24 is R163, K34 is 173, Q38 is 177, D44 is 183, E50 is 189 in ORK10-003b. We set the reference line at 0.27 and the Hill slope at -1.6. Figure 9. Dose-response analysis of IL-10 variants with engineered affinity toward IL10RB in HEK-Blue10 cells. All mutations were introduced into only one half (continuous domain) of the ORK10-003b foldikine structure (SEQ ID NO:49). The data were fit to a sigmoidal dose-response to determine the EC-50 (see Methods). We fixed the reference and saturation values. The mutant numbering is based on SEQ ID NO: 50. N92 is 231 in ORK10-003b. We fixed the reference values ​​at 0.12 and -1.9 for the last two rows. Figure 10. Dose-response analysis in HEK reporter cells for CSF1 (SEQ ID NO: 12) predicted enhanced affinity mutants. The titration curve is shown and at the bottom of the table with the EC-50 values. The mutant numbering is based on SEQ ID NO: 12. The data were fit to a sigmoidal dose-response to determine the EC-50 (see Methods). We fixed the reference values ​​and the Hill slope. Figure 11. Dose-response analysis of CSF1 variants (SEQ ID NO: 12) engineered to enhance CSF1 stability. The analysis was performed in HEK293 reporter cells and a curve fit was derived. The numbering of themutants is based on SEQ ID NO: 12. Data were fitted to a sigmoidal dose-response to determine the EC-50 (see Methods). We fixed the reference values ​​and the Hill slope. The Hill slope was set at -1.7 and the reference line at 0.3. Figure 12. Diagram showing possible fusion proteins of IL-10 and CSF1 (A), as well as Foldikine10 and CSF1 (B). (C): Visualization of how the WT IL-10 monomer fused to CSF1 could aggregate or generate concatemers. Figure 13. EC-50 determination for IL10 or Foldikine10 fusion to CSF1 in orientation A. Titration curves of HEKblue cells of IL10R (upper section) and CSF1R reporter cells (lower section) with increasing concentrations of the candidate variants while maintaining orientation A (IL-10 variant-linker-CSF variant). The linker length is the same as in Figure 18 (30 aa). CSF1 (REF) is SEQ ID NO:12. Data have been fitted to a sigmoidal dose-response fordetermine the EC-50 (see Methods). For the top panel, we set the reference line at 0.08 and the Hill slope at -1.38. For the bottom panel, we set the reference line at 0.08 and the Hill slope at -1.38. For the bottom panel, we set the reference line at 0.3 and the Hill slope at -1.7. IL10 is IL10-HiBit (SEQ ID NO: 50). IL-10-CSF1 is IL-10-CSF1-HiBit (SEQ ID NO: 52), Foldikine10-CSF1 is Foldikine10-CSF1-HiBit (SEQ ID NO: 53). Figure 14. EC-50 determination for IL10 or Foldikine10 fusion to CSF1 in orientation B. Titration curves of IL10R reporter HEKblue cells (left section) and CSF1R reporter cells (right section) with increasing concentrations of the candidate variants while maintaining orientation B (CSF variant-linker-IL10 variant). The linker length is the same as in Figure 17 (30 aa). CSF1 (REF) is SEQ ID NO: 12. Data have been fitted to a sigmoidal dose-response to determine theEC-50 (see Methods). For the top panel, we set the reference line at 0.08 and the Hill slope at -1.38. For the bottom panel, we set the reference line at 0.08 and the Hill slope at -1.38. In the bottom panel, we set the reference line at 0.3 and the Hill slope at -1. IL10 is IL10-HiBit (SEQ ID NO: 50). CSF1-IL10 is CSF1-IL10-HiBit (SEQ ID NO: 54), CSF1-Foldikine10 is CSF1-Foldikine10-HiBit (SEQ ID NO: 55). Figure 15. EC-50 determination for the fusion of Foldikine10_cut116 to CSF1. Titration curves for HEKBlue IL10R reporter cells of Foldikine10_cut116 fused to CSF1. Data were fitted to a sigmoidal dose-response to determine the EC-50 (see Methods). We set the reference and Hill slope values. CSF1- Foldikine10_cut116 is CSF1-Foldikine10_cut116-HiBit (SEQ ID NO: 57), Foldikine10_cut116- CSF1 is Foldikine10_cut116-CSF1-HiBit (SEQ ID NO: 56), CSF1-Foldikine10 is CSF1- Foldikine10-HiBit (SEQEnglish: ID NO: 55), foldikine10_cut116 is SEQ ID NO: 5. Figure 16. EC-50 determination for the fusion of Foldikine10_cut116 to CSF1. HEK dose-response curve transformed with the CSF1R reporter kit. The data have been fitted to a sigmoidal dose-response to determine the EC-50 (see Methods). Reference and Hill slope values ​​have been set. CSF1-Foldikine10_cut116 is CSF1- Foldikine10_cut116-HiBit (SEQ ID NO: 57), Foldikine10_cut116-CSF1 is Foldikine10_cut116- CSF1-HiBit (SEQ ID NO: 56), CSF1-Foldikine10 is CSF1-Foldikine10-HiBit (SEQ ID NO: 55). Figure 17. Linker length and protein functionality for targeting B. Data for IL-10 R reporter HEKBlue cells. Data have been fitted to a sigmoidal dose response to determine the EC-50 (see Methods). We have set the reference and Hill slope values. CSF1-25aa-Foldikine10 is SEQ ID NO: 58, CSF1-30aa-Foldikine10 is SEQ ID NO: 55, CSF1-35aa-Foldikine10 is SEQ ID NO: 59,CSF1-15aa- Foldikine10 is SEQ ID NO: 60, CSF1-15aa-IL10 is SEQ ID NO: 61, CSF1-25aa-IL10 is SEQ ID NO: 62, CSF1-30aa-IL10 is SEQ ID NO: 63, CSF1-35aa-IL10 is SEQ ID NO: 64. The Hill slope was set at -1.5% for CSF1-35aa-IL10. The Hill slope was set at -1.2 and the reference line at 0.40. Figure 18. Linker length and protein functionality for targeting B. CSF1R HEK reporter cell data. The protein concentration for the 15 and 25 amino acid (aa) linker IL-10 variants was below the dynamic range of CSF1R HEK reporter cells. CSF1-25aa-foldikine10 is SEQ ID NO: 58, CSF1-30aa- foldikine10 is SEQ ID NO: 55, CSF1-35aa-foldikine10 is SEQ ID NO: 59, CSF1-15aa- foldikine10 is SEQ ID NO: 60, CSF1-15aa-IL10 is SEQ ID NO: 61, CSF1-25aa-IL10 is SEQ ID NO: 62, CSF1-30aa-IL10 is SEQ ID NO: 63, CSF1-35aa-IL10 is SEQ ID NO: 64. Protein expression for two mutants (CSF1-15AA-IL10 and CSF1-25AA-IL10 was below thedynamic range of CSFF1R HEK reporter cells). The Hill slope was set to -1.06 and the reference line to 1.2. Figure 19. Schematic diagram showing the possible masking effect due to the way the fused CSF1 protein binds to Foldikine 10 or to the circular permutations of Foldikine 10. A) Schematic diagram showing the binding of Foldikine 10 to its two receptors where the natural Nt and Ct target the cell membrane. B) Schematic diagram showing the binding of the fusion of the natural Nt of Foldikine 10 to the Ct of CSF1 (The same will happen if it is fused to the Ct of Foldikine10). In this case the presence of CSF1 fused to the Nt will affect the binding of the left receptor causing a masking effect and decreasing the binding affinity of Foldikine10. C) Schematic diagram showing fusion junction creating a new Nter by opening a loop pointing away from the membrane to a permutantcircular Foldikine 10 to the Ct of CSF1 (The same will occur if fused to the Ct of the circular permutant). In this case the presence of CSF1 fused to the Nt will not affect the binding of the left receptor. Figure 20. FLDK-1 and CSF1-IL10, similarly to IL-10, inhibit monocyte activation. (A) Peripheral blood mononuclear cells (PBMCs) total PBMCs from healthy donors were stimulated with LPS in the presence or absence of different concentrations of IL-10, FLDK-1 or CSF1-IL10. (B) TNFα levels in supernatants of PBMCs treated with rIL10 or FLDK1 were determined by ELISA 24 hours after stimulation. (C) TNFα levels in supernatants of PBMCs treated with CSF1-IL10 were determined by ELISA 17 hours after stimulation. Figure 21. FLDK1, similarly to CSF1, induces differentiation of monocyte-derived macrophages. (A) PBMCs were isolated from human buffy coats and CD14+ cells were purified.by positive selection with microspheres. Monocytes were cultured for 7 days in the presence or absence of: rIL-10, rCSF1, FLDK1 or untreated. Cytokines were added every 2 days (BC). On day 7, live cells were counted (B) and expression levels of macrophage markers (CD14, MHCII, CD206, CD163 and CD86) were determined by FACS (flow cytometry), only CD163 and CD86 were represented (C). Data shown as a group of 4-8 different experiments as mean + SEM. Figure 22. FLDK1-induced macrophages have a regulatory phenotype. (A) PBMCs were isolated from human buffy coats and CD14+ cells were purified by positive selection. Monocytes were cultured for 7 days in the presence or absence of: rCSF1 or FLDK1. Cytokines were added every 2 days. On day 7, cells were harvested and stimulated with LPS±IFNγ. (B) Cytokine levels in the supernatants were determined by ELISA 17 hours afterstimulation. Data shown as a group of 3-4 different experiments as mean + SEM. Figure 23. FLDK1-induced macrophages have a regulatory phenotype. (A) Monocytes were isolated from human buffy coat samples by selecting CD14-positive cells. Monocytes were cultured for 7 days in the presence or absence of either rCSF1 or FLDK1. Cytokines were added every 2 days. On day 7, cells were removed from the plates and counted. The same number of macrophages were replated and stimulated with LPS for 4 hours. CD3+ cells were purified from a different donor, counted, and added to CSF1- or FLDK1-differentiated macrophages. Cytostim (Miltenyi) was also added to the cocultures to enhance the interaction between both cell types. At 48 hours, supernatants were collected, and cells were counted after 5 days in coculture. (B) Data shown as a group of 4–6 different experiments as mean + SEM.Figure 24. FLDK-1 displays selectivity toward myeloid cells compared to lymphoid cells. (A) PBMC from healthy donors were seeded in 96-well plates and stimulated the next day with rIL10 or FLDK1 for 20 min at 37°C. Cells were stained with CD14 Alexa647 and CD3 APC / Cy7 and fixed with 4% PFA, then permeabilized for intracellular staining with 80% MetOH. Cells were stained with AlexaFluor488 pSTAT3 (pY705). (BC) Samples were analyzed using the LSRII cytometer, and MFI (“Mean Fluorescence Intensity”) values ​​obtained from STAT3 activation were plotted using GraphPad Prism software. A representative result with two technical replicates is shown as the mean, with an error bar representing the SEM. Each biological replicate was normalized by assigning the highest IL10 MFI value of the maximum concentration as 100% and the lowest MFI value of an untreated control as 0%. The MFI of the samplestreated with FLDK1 was normalized accordingly. Figure 25. FLDK-1 and CSF1-IL10 are less potent than IL-10 in activating CD8 T cells. (A) CD8 T cells were isolated from human PBMCs by positive selection with beads (Miltenyi). Lymphocytes were stimulated with anti-CD3 and anti-CD28 for 72 hours. Cells were then split in two and treated with (B) IL10 or FLDK1 or (C) IL10 or CSF1-IL10 with IL-2 for 48 hours. Cell supernatants were obtained, and Gzmb and IFNγ were detected by ELISA. Pooled normalized data from four independent experiments are shown in the graph. IL-10 vs. FLDK1 t-test, p-value < 0.05. Figure 26. FLDK-1 and CSF1-IL10 are less potent than IL-10 in B cell activation. (A) B cells were isolated from human PBMCs by positive selection with beads. Isolated lymphocytes were stimulated with CpG (InvivoGen) and (B) IL10 or FLDK1, or (C) CSF1-IL10 or IL10, on days 0 and 2. After 7 days, the cells ...C) CSF1-IL10 or IL10, on days 0 and 2.days, IgG levels in the supernatants were determined by ELISA. The graph shows the pooled data from 4 independent experiments. IL-10 vs. FLDK1 t-test, p-value < 0.05. Figure 27. FLDK1 protects mice from TNBS-induced chronic colitis. Evaluation of the therapeutic efficacy of FLDK1, compared to IL-10, (at three doses 1 µg per mouse, 3 µg per mouse or 10 µg per mouse) in an experimental model of TNBS-induced chronic IBD. Mice treated with FLDK1 showed less body weight loss compared to those treated with IL-10 or vehicle after 30 days of treatment. Figure 28. Structure-activity relationship. (A) PBMC from healthy donors were seeded in 96-well plates and stimulated the next day with rIL10, FLDK1, or FLDK1-E82A. (B) Samples were analyzed with the LSRII cytometer, and MFI values ​​obtained from STAT3 activation were graphed using GraphPad Prism software.Figure 29. Structure-activity relationship. Structure-activity relationship. (A) PBMC from healthy donors were seeded in 96-well plates and stimulated the next day with rIL10, FLDK1, or saturating concentrations of CSF1 + FLDK1. (B) Samples were analyzed using the LSRII cytometer, and the MFI values ​​obtained from STAT3 activation were graphed using GraphPad Prism software. EXAMPLES Example 1: Production of Combined CSF1-IL10 Bioactivity in a Single-Chain Protein Our goal is to create a dual molecule that can activate IL-10 signaling exclusively in CSF1R-expressing cells, which are predominantly myeloid cells and not other cells such as CD8+ T cells. To achieve this, we designed IL-10 and / or Foldikine-10 variants with reduced affinity for IL10RA and IL10RB linked to a CSF1 monomer. The CSF1 component acts as a targeting tool for circulating monocytes, facilitating bindingIL-10 preferentially to these cells (see Figure 1). Design of the Foldikine10 chassis The proposed product(s), Folkidine TM -1 involves IL-10 and CSF1 variants linked at a specific distance and orientation. To design the IL-10 variant, we designed Nter-Cter closing linkers (intralinkers) in the swapped domain of IL-10 to terminate a single chain (Foldikine structure). TM, Figure 2) (Foldikine10, based on the so-called MutSC1 construct of patent application PCT / EP2023 / 052202 with D>E substitution at position 79 of MutSC1; Montero-Blay et al., Molecular Systems Biology (2023)19:e11037), and the molecules ORK10-002, ORK10-003, ORK10-005). Foldikine10 (SEQ ID NO: 1) comprises amino acids at positions 5 to 157 of IL-10 (SEQ ID NO: 11) with the substitution G5M, D84E, and K157N (SEQ ID NO: 28), an Nt-Ct linker peptide consisting of NGGLDY (SEQ ID NO: 30), and amino acids at positions 19 to 160 of IL-10 (i.e., SEQ ID NO: 29). SEQ ID NO: 20 (foldikine10-HiBit) is the same as SEQ ID NO: 1 except for the addition at the C-terminus of a HiBit tag for protein quantification.ORK10-002 (SEQ ID NO: 2) differs from Foldikine10 (SEQ ID NO: 1) by the N154F substitution and the E80D reverse substitution (i.e., ORK10-002 includes the Asp (D) residue present at position 84 of IL-10 as shown in SEQ ID NO:11). ORK10-002 comprises amino acids at positions 5 to 152 of IL-10 with the G5M substitution (SEQ ID NO: 39), an Nt-Ct linker peptide consisting of FGGLDY (SEQ ID NO: 46), and amino acids at positions 19 to 156 of IL-10 (from SEQ ID NO: 40). ORK10-003 and ORK10-003-HiBit (SEQ ID NO: 3 and SEQ ID NO: 49) and ORK10-005 (SEQ ID NO: 4) also comprise the E80D reverse substitution compared to Foldikine10 (SEQ ID NO: 1) and further differ by the design of the Nt-Ct bond and surrounding residues of the IL-10 monomers.ORK10-003 (SEQ ID NO: 3) comprises amino acids at positions 5 to 154 of IL-10 (SEQ ID NO: 11) with the substitutions G5M (SEQ ID NO: 39) and M154 mutated to L (SEQ ID NO: 41), an Nt-Ct linker peptide consisting of YKTIT (SEQ ID NO: 31), and amino acids at positions 17 to 156 of IL-10 (SEQ ID NO: 424) where residues GNLP are mutated to PEFA. SEQ ID NO: 49 (ORK10-003-HiBit) is the same as SEQ ID NO: 3 except for the addition at the C-terminus of a HiBit tag for protein quantification. ORK10-005 (SEQ ID NO: 4) comprises amino acids at positions 5 to 153 of IL-10 (SEQ ID NO: 43), an Nt-Ct linker peptide consisting of DKDIRDGD (SEQ ID NO: 32), and amino acids at positions 18 to 156 of IL-10 (SEQ ID NO: 44).Comparison of the EC-50 for Foldikine10 (SEQ ID NO: 1), and ORK10-002 (SEQ ID NO: 2), ORK10-003 (SEQ ID NO: 3), and ORK10-005 (SEQ ID NO: 4) shows that the ORK10-003 molecule has an improved EC-50 compared to Foldikine-10 (Figure 3, Table 1). Table 1. EC-50 values ​​and their associated errors for the different foldikine IL-10 variants EC-50 error EC-50 IL-10 8.91E-10 4.24E-11 Foldikine10 6.01E-10 4.22E-11 ORK10-002 3.87E-10 2.70E-11 ORK10-003 2.30E-10 2.95E-11 ORK10-005 6.25E-10 2.69E-10 Reference line set at 0.27 and Hill slope set at -1.6 for all variants except ORK10-005 which has a clearly different slope. Circular permutants of Foldikine 10. The natural Nt and Ct of Foldikine10 and the various ORK-10 target the cell membrane when cytokine molecules bind to the IL10RA and IL10RB receptors (Figure 4).This could lead to a masking effect when fused to another cytokine such as CSF1 due to the steric hindrance of the fused molecule with the cell membrane or receptors. To overcome this effect, we produced circular permutations of Foldikine10 by exploring the opening of the structure over all flexible regions connecting α-helices (thus creating unnatural Nt and Ct) while ligating the free Nt and Ct with the same linker used to design Foldikine10 (Sequences Foldikine10_cut116 (SEQ ID NO: 5), Foldikine10_cut133 (SEQ ID NO: 6), Foldikine10_cut209 (SEQ ID NO: 7), Foldikine10_cut237 (SEQ ID NO: 8)). We first expressed the different versions and quantified them by ELISA. Only for Foldikine10_cut116 did we have enough protein to test in HEK reporter cells.This molecule (Foldikine10_cut116) was active (cp116) but decreased the EC-50 (the HEK EC50 of IL-10 is 6.04E-10 for Foldikine10, and 1.67E-09 for Foldikine10_cut116). This molecule involves the opening of one of the regions connecting the two 3D domains of IL-10 at position 116 (see Figure 4). The cut at this point should allow the connection to other molecules without causing a masking effect (see next section), since the new Nt and Ct point opposite the membrane and away from the receptors (see Figure 3). Structure of CSF1 and circular permutants of CSF1 CSF1 naturally dimerizes with the two monomeric molecules facing each other and they are held together by a disulfide bond (see Figure 4).Circular permutants of CSF1 were generated, in which native Nt-Cts are connected by an engineered linker and novel Nt- and Cts are generated by disconnecting the peptide bond in a region of the protein that does not disrupt the interaction with the receptor (between residues 95 and 100, Figure 6). Both novel molecules (ORKMCSF_013 (SEQ ID NO: 9) and ORKMCSF_014 (SEQ ID NO: 10); Figure 7) were able to activate the downstream CSF1 signaling cascade (Figure 8) with similar and slightly better EC-50 values ​​than monomeric CSF1 molecules (Table 2). English: Table 2. EC-50 values ​​and their associated errors for the different CSF1 variants EC-50 error EC-50 CSF1 (1-190) HiBiT 1.45E-09 3.00E-10 ORKMCSF_013 7.37E-10 9.49E-11 ORKMCSF_014 7.71E-10 1.74E-10 Manipulation of IL-10 affinity We manipulated a set of mutants based on the active structure ORK10-003-HiBit foldikine10 (SEQ ID NO: 49) to decrease the affinity for IL10RA.We screened 11 mutants using FoldX and sequence conservation analysis, through which we identified several mutations that did not compromise protein expression and reduced the EC-50 (R24A, E50A, D44N, K34A, Q38A, D44A, numbering based on >SEQ ID NO: 11b IL10b, see Methods). R24 is R163, K34 is 173, Q38 is 177, D44 is 183, E50 is 189 in ORK10-003-HiBit. These mutations are point mutations integrated into the continuous domain of Foldikine10. These mutants showed varying degrees of activation of HEKblue cell reporters with EC-50 (see Figure 8, Table 3). A mutation that weakened IL10RB interaction (N92E; numbering based on >SEQ ID NO: 11 IL10, N92 is 231, in ORK10-003-HiBit) was also identified (see Figure 9, Table 3). Table 3.EC-50 values ​​and their associated errors EC-50 error EC-50 Foldikine10 (ORK10-003 REF) 9.44E-11 4.12E-12 R24A 1.51E-10 1.16E-11 E50A 5.43E-11 3.40E-11 D44N 4.37E-10 1.84-E11 D44A 2.01E-9 2.57E-11 K34A 9.22E-10 5.51E-12 Q38A 2.07E-10 6.38E-11 Foldikine10 (ORK10-003 REF)9.53E-11 4.14E-12 N92E 1.19E-10 3.84E-12 We set the reference line fit at -0.27 and the Hill slope at -1.6 for the first 7 rows and at 0.12 and -1.9 for the last two rows. Mutants to Increase the EC-50 of CSF1 with the CSF1 Receptor For CSF1, we engineered a set of one- or two-point mutations to increase the affinity for CSF1R (Table 4).We selected a set of conserved and non-conserved residues based on changes in the interaction energy of the complex with the receptor and the stability of the mutated CSF1 predicted by FoldX, and on visual inspection of the complex interface (bold for conserved residues (allowing for one mismatch) between mouse, hamster, human, pig, cow, sheep, alpaca, camel, fox, dog, mink, walrus, otter, polar bear, cat, puma, dolphin, bat, raccoon, blue whale, and horse CSF1 sequences; asterisk for residues mutated to improve binding, and x for residues mutated to increase CSF1 stability). EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFR x* xx * DNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFS * xx xx * * * * NO:12). Table 4.Selected CSF1 mutants including predicted FoldX energies modeled on the CSF1 / CSF1R complex (note that FoldX has an error of about 0.8 kcal / mol in its predictions). Mutations Δ Complex Δ Stability Δ VdW Shocks Interaction energy (kcal / mol) (kcal / mol) (kcal / mol). To increase affinity, we selected the mutations H9F, Q17R, S18Q, V78W, Q79L, Q81F, Q81Y, E82K. In a second round based on sequence conservation, we selected V78F, S18F-R21Q, Q17V / L, S18F, V78F / L / Y, Q79T / K, K93E, T93E, T34L, and M10L. In Figure 10, we show the titration curve of different CSF1 mutants and the EC-50 values ​​and their associated errors. These variants were evaluated in HEK293 cells transfected with a CSF1R reporter kit (see Methods). Of all mutants, two had EC-50 values ​​similar to WT CSF1 (Q17R and V78W), being better in one of the two biological replicates. Table 5.EC-50 values ​​and their associated errors CSF1 H9F Q17R S18Q V78W Q79L Q81F E82K EC50 Replicate 1.88E-09 2.66E-9 2.70E-9 2.51E-9 6.23E-9 6.76E-9 1.08E-8 1.27E-8 biological 1 Error 8.63E-11 9.5E-10 5.31E-10 9.61E-10 3.57E-10 9.27E-10 1.45E-9 3.71E-9 Hill slope set at -1.7 and reference line at 0.3 EC50 Replicate 5.58E-09 1.18E-08 7.94E-09 1.99E-08 2.10E-08 1.28E-08 6.77E-08 2.49E-08 Biological 2 Error 1.09E-09 1.04E-09 9.38E-10 1.85E-09 3.96E-09 1.17E-09 5.34E-09 3.45E-09 Hill slope set at -1.7 and reference line at 0.45 In parallel, as discussed above, we designed different variants to increase the stability of the CSF1 conformation in the CSF1R-bound state. These variants were evaluated in HEK293 cells transfected with a CSF1R reporter kit (see Methods) (see Table 6).The mutants were selected to include residues that do not directly interact with the CSFR1 receptor and are partially or completely buried within the structure. Among the mutants, we observed a slight increase in binding affinity for the V117L mutant in one of the biological replicates and similar values ​​to the WT for T124I in both biological replicates. More interestingly, we found an increase in the maximum activation value in HEK293 cells transfected with a CSF1R reporter kit in both replicates, but only significant in the second replicate.

[0002] Table 6. The table shows the EC_50 and the maximum response value determined in HEK cells transformed with the CSFR1 reporter kit. EC-50 CSF1 V117L V117 V120I T124I A76I, V117L Biological replicate 1 EC-50 4.52E-10 1.04E-09 1.00E-09 8.80E-10 5.74E-10 1.18E-09 Error 1.55E-10 1.36E-10 1.52E-10 2.48E-10 6.87E-11 1.91E-10 Maximum activation value 8.49 8.66 7.64 8.86 8.73 8.55 Error 0.59 0.26 0.26 0.55 0.22 0.31 Hill slope set at -1.2 and reference line at 0.33 EC-50 CSF1 V117L V117I V120I T124I A76I, V117L Biological replicate 2 EC-50 3.37E-10 3.07E-10 4.37E-10 4.43E-10 4.21E-10 5.97E-10 Error 3.14E-11 5.36E-11 7.68E-11 1.39E-11 3.79E-10 9.27E-11 Maximum activation value 1.14 1.56 1.28 1.37 1.40 1.14 Error 0.017 0.05 0.04 0.08 0.02 0.04 Hill slope set at -1.33 and reference line at 0.12.Based on this analysis, we propose combinations of mutants that within the error could improve the binding affinity (Q78R, V78W, V120I) and / or the signaling range (V117L, V120I, T124I) of CSF1 for its receptor. i) Q17R, T124I and V120I ii) V78W, T124I and V120I iii) Q17R, V78W, T124I and V120I iv) V117L, T124I. Example 2: Generation of a dual CSF1-IL10 molecule Determination of the optimal orientation of the CSF1-IL10 product Design of the chimeric Foldikine-10 with CSF1 or Foldikine-CSF1. To assess whether by fusing Foldikine-10 with CSF1, we still maintained the activity of both cytokines, we first tested IL10 activity using the HEKBlue 10 reporter and CSF1 activity using HEK293 transfected with the CSF1R reporter kit.There are two possibilities to make the fusion protein, we join the Ct of IL-10 or Foldikine10 to the Nt of CSF1, or we fuse the Nt of IL-10 or Foldikine10 to the Ct of CSF1 (and the same with the circular permutations of Foldikine10 and CSF1) (See Figure 12). The fusion of IL-10 with CSF1 could lead to the formation of concatemers since IL-10 is an exchanged dimer and CSF1 dimerizes (See Figure 12). This will not be the case for the other fusions. For a CMC reason, for the protein fusion we decided to continue with foldikine-10 instead of ORK10-003. Orientation A - IL10-CSF1 In this orientation, we fuse the Ct of IL-10, or Foldikine10, to the Nt of CSF1, resulting in IL-10-CSF1 (SEQ ID NO:52), or Foldikine10-CSF1 (SEQ ID NO:53). The EC50 of IL-10 WT is affected when fused to CSF1 at the level of functionality. The EC-50 of Foldikine-10 is also affected, but to a lesser extent than IL-10 WT.Regarding the functionality of CSF1, it loses activity when fused to the C-terminus of IL-10 and less when fused to Foldikine-10 (see Figure 13 and Tables 7 and 8). Orientation B - CSF1-IL10 In this orientation, we fuse the Ct of CSF1 to the NT of IL-10, or Foldikine10, resulting in CSF1-IL-10 (SEQ ID NO: 54), or CSF1-Foldikine10 (SEQ ID NO: 55). The EC50 of IL-10 WT and Foldikine10 is affected when fused to CSF1. Regarding the functionality of CSF1, it loses activity when fused to IL-10 but not to Foldikine10 as in the case of orientation A (see Figure 14 and Tables 7 and 8).

[0003] Table 7. IL-10 EC-50 values ​​as well as the error of this EC-50. IL-10 EC-50 error EC-50 IL-10 8.00E-10 5.42E-11 Foldikine10 2.14E-10 4.87E-12 LCR-IL10 1.16E-9 2.31e-10 CSF1-Foldikine10 1.06e-9 1.04e-10 IL-10-CSF1 5.9 E-9 6.06E-10 Foldikine10-CSF1 1.36E-9 1.02E-10 Reference line set at 0.08, and Hill slope at -1.38. *The concentration of IL10 of the active dimeric form estimated by Hibit is twice that of Foldikine10, so we divided the EC50 by two Table 8. EC-50 values ​​of CSF1 as well as the error of this EC-50.CSF1 EC-50 error EC-50 CSF1 7.34E-10 1.06E-10 CSF-1-IL10 7.81e-10 3.18e-10 CSF1-Foldikine10 9.53e-10 7.37e-11 IL10-CSF1 4.64E-09 5.04E-10 Foldikine10-CSF1 3.18E-09 4.06E-10 Baseline set at 0.3 and Hill slope at -1.7 Testing the activity of CSF1 fused to a circular permutant of Foldikine10 As described above, fusing Foldikine10 to CSF1 at either its N- or C-terminus increases the EC-50 of Foldikine10. This could be due, as explained above, to the fact that the natural Nt and Ct of Foldikine10 point towards the cell membrane in complex with the IL10RA and IL10RB receptors and therefore there could be a steric hindrance effect. To address this, we designed different circular permutations of Foldikine10 and found as reported above one (Foldikine10_cut116 (SEQ ID NO: 5)) that was expressed at WT levels and had around three times worse EC-50 (See Figure 4).In this circular permutant, the Nt and Ct do not point to the membrane and are far from the receptors, so we expect no steric hindrance when fused to CSF1. To test this, we fused Foldikine10_cut116 to CSF1 in both orientations (N-terminal and C-terminal: Foldikine10_cut116-CSF1-HiBit (SEQ ID NO: 56); CSF1-Foldikine10_cut116-HiBit (SEQ ID NO: 57)). Fusing the circular permutant compared to the CSF1-Foldikine10 structure resulted in similar EC-50 values ​​of CSF1 in both orientations (see Figures 15-16). We observed that the EC-50 for IL10 of the circular permutation of Foldikine alone (Foldikine_cut116) or in combination with CSF1 have similar EC-50 values, in contrast to the behavior of Foldikine10 which loses activity when fused to CSF1, supporting the hypothesis of the steric hindrance effect mentioned above (see Table 9). Table 9. EC-50 values ​​and associated error for CSF1 and IL10 activity.Circular permutant CSF-1 IL-10 EC-50 error EC-50 EC-50 error EC-50 CSF1-foldikine10_cut116 4.14E-09 3.38E-10 3.06E-10 1.73E-11 foldikine10_cut116-CSF1 2.69E-09 2.13E-10 4.95E-10 2.01E-11 CSF1-Foldikine10 3.62E-09 2.60E-10 1.67E-10 3.29E-11 Foldikine10 cut_116 2.41E-10 2.08E-11 Impact of linker length on protein functionality We tested orientation B (CSF-linker-IL10) to determine if longer linkers short or longer ones could interfere with the protein functionality. While CSF1-Foldikine10 showed IL-10 signaling with linker lengths ranging from 15 to 35 amino acids (aa) (CSF1-25AA-Foldikine10-HiBit (SEQ ID NO: 58); CSF1-30AA-Foldikine10-HiBit (SEQ ID NO: 55); CSF1-35AA-Foldikine10-HiBit (SEQ ID NO: 59); CSF1-15AA-Foldikine10-HiBit (SEQ ID NO: 60)), CSF1-IL-10 with a short linker (i.e., 15 aa: CSF1-15AA-IL10-HiBit (SEQ ID NO: 61)) was not active.CSF1-IL-10 with linker lengths ranging from 25 to 35 amino acids showed IL-10 signaling (CSF1-25AA-IL10-HiBit (SEQ ID NO: 62), CSF1-30AA-IL10-HiBit (SEQ ID NO: 63), CSF1-35AA-IL10-HiBit (SEQ ID NO: 64)). In terms of CSF1 activity, protein expression for two mutants (CSF1-15AA-IL10-HiBit (SEQ ID NO: 61) and CSF1-25AA-IL10-HiBit (SEQ ID NO: 62)) was below the dynamic range of CSF1R HEK reporter cells. The CSF1-Foldikine10 fusion supports different linker lengths, whereas IL-10 loses activity or expression with short linkers. See Figures 17-18 and Tables 10 and 11. Table 10. EC-50 values ​​and associated error for IL10 activity fused to linkers of different lengths.CSF1- CSF1- CSF1- CSF1- CSF1- CSF1- CSF1- 15AA- 25AA- 30AA- 35AA- 15AA- 25AA- 30AA- CSF1-35AA- IL10 IL10 IL10 IL10 Foldikine10 Foldikine10 Foldikine10 Foldikine10 EC-50 4,10E-10 2.35E-10 1.9E-10 2.60E-10 3.61E-10 3.32E-10 3.18E-10 Error 2.11E-11 1.57E-11 1.23E-11 6.61E-12 2.81E-12 6.74E-12 2.35E-12 ______________________________________________________________________________ The slope of Hill was set at -1.2 and the reference line at 0.40. *The concentration of IL-10 in the active dimeric form estimated by Hibit is twice that of Foldikine10, so we divided the EC50 by two. Table 11. EC50 values ​​and associated error for CSF1 activity fused to linkers of different lengths.CSF1- CSF1- CSF1-15AA- CSF1-25AA- CSF1-30AA- CSF1-35AA- 30AA-IL10 35AA-IL10 Foldikine10 Foldikine10 Foldikine10 Foldikine10 CE- 50 5.50E-10 6.50E-10 1.19E-09 1.64E-09 1.97E-09 1.83E-09 Error 2.79E-10 2.50E-10 3.21E-10 4.84E-10 4.76E-10 4.39E-10 The Hill slope was set to -1.06 and the reference line to 1.2. Example 2: Methods Generating circular permutant molecules with FoldX-ModelX software Circular permutants are generated in two steps: first, by bridging naturally occurring Nt-Ct molecules, a cyclic structural model with no beginning or end is generated; second, by disconnecting a peptide bond in a flexible region of the model. The linkers between Nt-Ct molecules are designed using ModelX Bridging. FoldX’s AlaScan assists in the selection of nicking residues by predicting those that are less important for protein stability.Generation of single-chain (SC) molecules using ModelX software SC-IL10 variants were generated by re-connectivizing the IL-10 dimeric structure in X-ray (PDBs: 1y6k, 2ilk). The new connectivities were designed using the ModelX tools suite. The ModelX Bridging command (cross-link mode), which connects a selected residue pair as anchors with all geometrically compatible peptide fragments from a custom-built protein fragment library (PepXDB), was used. The PepXDB 120k structures were extracted from PDB and digested into peptides of different lengths (3–45 aa) and included the geometric descriptors required for the Bridging algorithm.The Bridging command allows the user to select different peptide lengths; the result is a set of bridged models with different conformations, sequences, and lengths, thus discarding linkers / connections with phi and psi dihedrals prohibited in the Ramachandran representation. Once the stitching models are created, extensive linker screening is performed by running them through the Bridging algorithm with exhaustive combinations of anchor points in an overlapping sliding window around the flanks of the regions to be joined. Different peptide lengths (6–20 aa) were queried in each window. The linkers in the bridged models contain the same side-chain rotamers as they did in the PDB structure from which they were digested; thus, adaptation to the new SC-IL10 context is performed using the FoldX RepairPDB command (Montero-Blay et al. 2023, Molecular Systems Biology, 19:e11037).RepairPDB identifies residues with poor torsion angles, VanderWaals clashes, or total energy, and mutates them and their neighbors to themselves, exploring different rotamer combinations to find new energy minima. The resulting models are sorted by global energy (FoldX Stability command). When peptides are longer than the numerical positions between the anchors, Bridging renumbers the excess residues with RES codes that are not recognized by FoldX in subsequent modeling steps. For this reason, the design of SC-IL10 required a numerical rearrangement of the monomeric protein residues into the dimer, allowing the 18-aa-long numerical gaps between regions to be bridged. This is very important for the extreme case where the anchor residues flank the numerical gap.To address this situation, renumbering was performed to create gaps of 18 numerical positions around the regions to be connected, which was sufficient to accept fragments ranging in length from 6 to 20 residues without using residue codes. The difference between 18 and 20 is that the anchor points are required for the algorithm to position the bridges, but they will be replaced by the terminal residues of the peptides found. Bridging also replaces all terminal residues between the anchors with those of the peptide. For clarity, the templates generated before using Bridging are referred to as "stitch patterns." Since the numerical gap is not a spatial gap, Bridging can also accept smaller peptide fragments, creating discontinuous numbering.Generation of linkers to link Foldikine10 variants to CSF1 To generate the linker between Foldikine10 variants and CSF1, we first looked at the 3D structure of the complexes between IL-10 and IL-10 RA and RB (PDB 6x93; 3.5 Å) and of CSF1 with its receptor (PDB 4wrm; 6.5 Å) by placing the receptors in the orientation we would expect them to have in the membrane and determined the distance between the wild-type Nt or CT of IL10 to the corresponding Ct or NT of CSF1. Using this distance as an approximation, we estimated that we needed a random linker between aa Cys146 of CSF1 and Cys12 of IL10 composed of GGSGG repeat units (SEQ ID NO: 27) of around 30 amino acids. Generation of IL-10 and CSF1 affinity mutants using FoldX software A) IL-10. To generate IL-10 versions with decreased affinity toward R1, we used hIL-10 molecules crystallized in complex with IL-10RA at the best crystal resolution (PDB 1y6k; 2.5Å).Next, we performed an in silico mutagenesis scan of the complex interface residues using the BuildModel FoldX command, which individually mutates all IL-10 residues to the other 19 naturally occurring amino acids (Schymkowitz et al, 2005), and the AnalyseComplex FoldX command to estimate the change in interaction energy for each mutation. Mutant selection was based on the complex interaction energy ( ^ ^G) and stability predicted by FoldX, as well as visual inspection of the complex interface. From these, we selected R24A, Q34A, M39A, D44N, D44A, L46A, E50A, E50Y, E151R, D144Q. B) CSF1. To generate CSF1 mutants with higher affinity for its receptor, we used CSF1 crystallized with CSF1 (PDB 4wrl; 2.8 Å).We then performed in-silico mutagenesis screening of the complex interface residues using the BuildModel FoldX command, which individually mutates all CSF1 residues to the other 19 naturally occurring amino acids (Schymkowitz et al, 2005, PNAS, 102(29), 10147-10152), and the AnalyseComplex FoldX command to estimate the change in interaction energy for each mutation. We selected a set of conserved and non-conserved residues based on the complex interaction energy ( ^ ^G) and stability predicted by FoldX, as well as visual inspection of the complex interface. From them we selected H9F, Q17R, S18Q, V78W, Q79L, Q81F, Q81Y, E82K for a first round of mutants and V78F, S18F-R21Q, Q17V / L, S18F, V78F / L / Y, Q79T / K, K93E, T93E, T34L and M10L for a second round.Generation of CSF1 Stability Mutants Using FoldX Software We used CSF1-crystallized CSF1 (PDB 4wrl; 2.8 Å) to generate CSF1 mutants with increased CSF1 stability toward CSF1R in the bound state. We then performed in-silico mutagenesis screening of the interface residues of the complex using the BuildModel FoldX command, which individually mutates all CSF1 residues to the other 19 naturally occurring amino acids (Schymkowitz et al, 2005, PNAS, 102(29), 10147-10152). Based on the stability of the complex predicted by FoldX and a visual inspection, we selected A76I, V117I, V117L, V120I, T124I.Protein Quantification Since we have generated different point mutations to improve or decrease the affinity to the receptors, as well as broken loops in some of the constructs or constructed new linkers, there is a danger that the resulting molecules are not recognized by the antibody used in commercial ELISA kits. To avoid this problem, we have used the HIBIT method to quantify all our constructs (https: / / www.promega.es / en / resources / technologies / hibit-protein-tagging-system / ). Essentially, this involves fusing a peptide tag spaced by a random linker to the Nt or Ct of. The Nano-Glo® HiBiT extracellular detection system is a method that can detect HiBiT-tagged proteins secreted into the extracellular medium. Detection is performed by adding a non-lytic detection reagent containing a substrate and Large BiT (LgBiT).HiBiT binds strongly to LgBiT, promoting complex formation and generating a bright, luminescent enzyme. The amount of luminescence generated is proportional to the amount of HiBiT-tagged protein present, and the system can detect protein quantities over seven orders of magnitude. The detection process is straightforward, requiring only a add-mix-read assay protocol. Having a reference peptide of known concentrations makes it extremely easy to quantify the expression of any protein. Plasmid Generation All plasmids generated in this work were assembled using the Gibson method (Gibson DG et al. Nat Methods. 2009 May;6(5):343-5). When required, gene (gBlock double-stranded fragments) and oligonucleotide synthesis were performed by Integrated DNA Technologies (IDT) Corporation.Gene amplification was carried out using Phusion DNA polymerase (Thermo Fisher Scientific) and transformed into Escherichia coli DH5-alpha competent cells (NEB). For mammalian protein expression in ExpiCHO cells, the pcDNA3.1 vector (V790-20, Invitrogen) was used. All plasmids were verified by Sanger sequencing (Eurofins Genomics). Expression in CHO cells The ExpiCHO Protein Expression Kit cells were purchased from Thermofisher (A29133). For low-scale production (2.5 ml), 24-deep-well plates (AXYPDW10ML24CS, Merck) were used and covered with gas-permeable film (ThermoFisher). On day -1 of production, ExpiCHO cells were split to a final density of 3 x 10. 6 viable cells / ml. They were grown at 37°C, 8% CO2, and shaking at 110 rpm. On Day 0 of production, ExpiCHO cells were split to a final density of 3 x 10 6viable cells / ml per sample 2.5 ml aliquot of the cell suspension into a well of the plate. Per sample, 2 ug of DNA was added to 200 ul of cold OptiPRO SFM and 9.0 μl of ExpiFectamine CHO reagent. Then, 200 μl of the complexation mix was added to each corresponding well. The entire plate was covered with a gas permeable seal and placed at 37°C, 8% CO2 and 225 rpm. On day 1 after 18-22 hours post-transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed were added to each well. For a full 24-well plate, 400 μl of ExpiFectamine CHO Enhancer and 16 ml of ExpiCHO Feed were mixed in a conical tube. From this mixture, 600 μl was added to each well of the plate. The plate was placed at 37°C, 8% CO2, and 225 rpm. After 4 days, the samples were collected. The plates were centrifuged for 5 minutes at 4600 rpm at room temperature. The supernatants were aliquoted, rapidly frozen in liquid nitrogen, and stored at -80°C.Cell culture HEK-Blue cell lines. TM carrying a SEAP reporter construct were purchased from InvivoGen (InvivoGen, San Diego, CA, USA). In this work, we used human IL-10 reporter cells (hkb-il10). HEK293 cells were purchased directly from InvivoGen. Both the HEK-Blue cell lines TMHEK293 cells were cultured in DMEM (Lonza, BE12-604F) supplemented with 10% FBS, 2 mM L-glutamine, and the manufacturer-specified selection antibiotics for each reporter cell. Cells were passaged at 70% confluence following the manufacturer's recommendations. THP-1 cells (ATCC, lot: 70047949) were cultured in RPMI-1640 with 10% fetal bovine serum, 2-mercaptoethanol to a final concentration of 0.05 mM, and Pen Strep (Gibco / 15140122) at 37°C in a CO2 incubator. Cell concentrations were maintained between 0.2 and 1e6 cells / ml. Colorimetric analysis in HEK-BlueTM cells for Foldikine-10 activity After quantification of the supernatant, the samples were adjusted to fit the dynamic range of the HEK reporter cells using DMEM medium to make the different dilutions.The maximum concentration of each reporter cell was set to match saturation, and 8 serial dilutions (0.5X each) were made for each sample. HEK reporter cells were then prepared following the manufacturer’s instructions. Briefly, 180 µL of cells were seeded at 280,000 cells / ml per well in a 96-well plate (Nunc Microwell, ThermoFisher Scientific, #167008). Thereafter, 20 µL of each supernatant at the adjusted concentration was added, and cells were maintained for 20–24 h at 37°C and 5% CO2 (HEK-Blue™ induced). Next, 180 µl of QUANTI-Blue solution (alkaline phosphatase detection medium, #repqbs, InvivoGen) was mixed with 20 µl of induced HEK-BlueTM cells in a new 96-well plate. The cells were then incubated for 60 min at 37°C and the absorbance (630 nm) was measured on a Tecan i-control spectrophotometer, 2.0.10.0.HEK293 CSF1 Reporter Cells Colorimetric analysis in HEK-Blue cells. TMfor CSF1 activity. The CSF1R / SER reporter kit (MAPK / ERK Signaling Pathway, #79379) was purchased from BPS Bioscience (BPS Bioscience Inc, San Diego, CA, USA). The day before DNA transfection, HEK293T cells (3x104 cells / well) were seeded in a 96-well multiwell plate (Nunc Microwell, ThermoFisher Scientific, #167008) in 100 μl of DMEM (Lonza, BE12-604F) supplemented with 10% FBS, 2 mM L-glutamine and 1% penicillin / streptavidin and incubated for 20-24 h at 37°C and 5% CO2. The next day, we prepared 100 μl of SER luciferase reporter vector (inducible luciferase vector), 100 μl of CSF1R expression vector (plasmid C), and 1500 μl of Opti-MEM (GIBCO, #31985-070). In parallel, 30 μl of Lipofectamine 2000 (Invitrogen, #11668019) was diluted in 1500 μl of Opti-MEM (GIBCO, #31985-070). After 5 min of incubation, the diluted DNA was combined with the diluted Lipofectamine 2000, mixed gently, and incubated for 25 min.minutes at room temperature to form the complexes. After that time, 30 μl of the complexes were added to each well containing the cells. The cells were incubated at 37°C in a 5% CO2 incubator. After ~5 to 6 hours of transfection, the medium was changed to growth medium with 0.5% serum. The cells were incubated at 37°C in a 5% CO2 incubator overnight. The following day, the SRE reporter was induced by activating the CSFR1R signaling cascade with our CSF1-IL10 fusion variants. After 6 hours of treatment, the media was removed and 50 μl of 5X diluted passive lysis buffer (Promega, #E1941) was added. Firefly Luciferase Reagent Substrate (Component B) was diluted 100x in Firefly Luciferase Reagent Buffer (Component A), 20 μl of this mixture was added to 20 μl of its lysate placed in a white 96-well multi-well plate (PerkinElmer, #6005290) and the plate was incubated at room temperature.ambient for 15 minutes. The luminescence of the fireflies was then measured using a luminometer. 20 μl of 100x diluted Renilla Luciferase Reagent Substrate (component D) in Renilla Luciferase Reagent Buffer (component C) was then added to the well and the luminescence was immediately measured using a luminometer. To obtain the normalized luciferase activity for the SRE reporter, we calculated the ratio of firefly luminescence to renilla luminescence and inferred a linear dose-response fit (Hill slope), see below. Inference of EC-50 Data For inference of an EC-50, we measured a dose-response analysis by monitoring absorbance (HEKBlue10) or fluorescence (HEK293-CSF1 reporter). The changes in absorbance at 630 nm due to the different concentrations of IL-10 tested were fitted to a saturation binding model using the following equation: Y =Baseline + ( B Max - Baseline) / (1+(X / EC50)^ - Hill slope). In this equation, EC-50 is the apparent dissociation constant (since the number of active receptors per cell is unknown), h is the Hill slope, and B Max is the saturation signal. This equation assumes only specific binding; all nonspecific signals have been subtracted. For the EC-50 calculated in Tables 3 and 6, the equation used was log(agonist) vs. response - variable slope (Y = Bottom + (Top – Bottom) / (1+10^((LogEC50-X)*Hill slope)) from Prism 9 software. For the CSF1 analysis we measured the changes in the firefly / renilla luminescence ratio. Different experiments were analyzed independently and fitted using GraphPad Prism 9 software. For IL-10 and Foldikin10 variants in those experiments where the reference or final lines of some mutants were not present, we added the following equation:defined, we used the average values ​​of those mutants with the lowest EC-50 (final value at saturation) or high EC-50 (initial value without ligand). In the case of CSF1, as we cannot be sure that the transformation efficiency with the CSFR1 kit was the same in all cases, we set the initial value as indicated above and fixed the Hill slope to the average of the mutants with the lowest EC-50. In each Figure we indicate how the adjustment was performed. Analysis of CSF1-Foldikine activity in THP-1 cells Thp1 cells were prepared at a concentration of 1e6 / mL of live cells. Then, 100 μL of cells / well were plated in a U-shaped 96-well plate and incubated for 15 minutes. In parallel, supernatants were prepared at the target dilution (60 nM) in a 96-well plate prewarmed with RPMI medium. Then, 20 μl of the dilutions were added to 100 μl of cells and incubated for 20 minutes.Cells were then immediately fixed to maintain the phosphorylation state by adding an equal volume of prewarmed BD Cytofix Buffer to the cell suspension, resulting in a total volume of 240 μl. Cells were incubated at 37°C for 10 minutes and pelleted by centrifugation. Cells were subsequently permeabilized by adding 100 μl of BD™ Phosflow Perm Buffer III (for 1×106 cells) and incubating for 30 minutes on ice (protected from light). Note: Longer incubation times in this permeabilization buffer may decrease the signal intensity of the surface marker staining. Cells were then washed twice with 200 μl of wash buffer (1X PBS + 1% FBS + 0.09% sodium azide) and centrifuged to remove the supernatant. Next, 5 μl of the Alexa Fluor 488 pSTAT3 Y407-conjugated antibody was added to each well of the U-bottom 96-well plate. The plateincubated at room temperature for 60 minutes in the dark. Finally, cells were washed and resuspended in PBS for flow cytometry analysis. Example 3: In vitro functional assessment of the dual cytokine CSF1-IL10 For the experiments in Examples 3 and 4, FLDK1 (SEQ ID NO: 72, i.e., SEQ ID NO:16 or 55 with Tev-HisHiBit tag), and / or CSF1-IL-10 (SEQ ID NO: 74) was used as the exemplary CSF1-IL10 dual cytokine. PBMCs (150,000 cells / well) from healthy donors were stimulated with 10 ng / ml LPS (Sigma) in the presence or absence of different concentrations of rIL-10 or FLDK-1 (0.00001 to 1 nM) or of CSF1-IL-10 (Fig. 20 A). TNFα levels measured in the presence of rIL-10 or FLDK-1 show that FLDK-1 inhibits monocyte activation similarly to IL-10 (the dotted line indicates LPS levels alone) (Fig. 20 B). CSF1-IL-10 was also shown to inhibit monocyte activation (Fig. 20 C). Similar results were obtained with IL-6 and whenIsolated monocytes (CD14+ cells) were used (data not shown). In addition, PBMCs were isolated from human buffy coats and CD14+ cells were purified by positive bead selection (Miltenyi). Monocytes (0.5 million / ml) were cultured for 7 days in the presence or absence of: rIL-10, rCSF1, FLDK1 (10 ng / ml per treatment) or untreated. Cytokines were added every 2 days. On day 7, live cells were counted and characterized by FACS (CD163, CD206, MHCII, CD14, CD86) (Fig. 21 A). The results show that FLDK1, similarly to CSF1, induces the differentiation of monocyte-derived macrophages, as identified by the expression of CD163 (Fig. 21 B, C). Further characterization was performed to determine the phenotype of the induced monocyte-derived macrophages. PBMCs were isolated from human buffy coats, and CD14+ cells were purified by positive selection (Miltenyi). Monocytes (0.5 million / ml) were cultured for 7days in the presence or absence of: rCSF1 or FLDK1 (10 ng / ml). Cytokines were added every 2 days. On day 7, cells were harvested and stimulated with LPS, with or without IFNγ (Fig. 22 A). TNF-α levels in the supernatants were determined by ELISA, 17 hours after stimulation. CSF-1-differentiated macrophages produce TNF-α, which is further enhanced by the addition of IFNγ. In contrast, FLDK1-differentiated macrophages do not produce TNF-α, even when stimulated with LPS+IFNγ. These results show that FLDK1-induced macrophages have a regulatory phenotype (Fig. 22 B). Furthermore, monocytes were isolated from human buffy coat samples by selecting CD14-positive cells (Miltenyi). Monocytes (0.5 million / ml) were cultured for 7 days in the presence or absence of rCSF1 or FLDK1 (10 ng / ml per treatment). On day 7, cells were removed from the plates and counted. The same number of macrophages was replated.(40,000 cells) and stimulated with LPS (Sigma) (10 ng / ml) for 4 hours. CD3+ cells from a different donor were purified, counted (120,000 cells), and added to CSF1- or FLDK1-differentiated macrophages. Cytostim (Miltenyi) (2 µl / million cells) was also added to the cocultures to enhance the interaction between both cell types. Cytokines in the supernatants were analyzed by ELISA at 48 hours, and T cell proliferation was analyzed after 5 days in coculture (Fig. 23 A). FLDK1-differentiated macrophages inhibited T cell proliferation and TNF-α and IFNγ production in these cocultures, demonstrating their regulatory phenotype (Fig. 23 B). PBMCs (150,000 cells / well) from healthy donors were seeded in 96-well plates and stimulated the next day with rIL10 or FLDK1 (10 nM per treatment, 8 1 / 3 dilutions) for 20 min at 37°C. Cells were stained with CD14 Alexa647 (Biolegend) and CD3 APC / Cy7 (Biolegend), fixedwith 4% PFA and permeabilized for intracellular staining with 80% MetOH. Cells were stained with AlexaFluor488 pSTAT3 (pY705) (Becton Dickinson) for 1 h (Fig. 24 A). Samples were analyzed using the LSRII cytometer, and the obtained MFI values ​​of STAT3 activation were graphed using GraphPad Prism software. A representative result with two technical replicates is shown as mean with an error bar representing the SEM (Fig. 24 BC). Similar results were obtained in 10 different donors. Each biological replicate was normalized by assigning the highest IL10 MFI value of the maximum concentration as 100% and the lowest MFI value of an untreated control as 0%. The MFI of FLDK1-treated samples was normalized accordingly. These results indicate that both monocytes and T cells respond similarly to IL-10. In contrast, the dual cytokine CSF1-IL10 FLDK-1 preferentially activates monocytes.monocytes compared to T cells (Fig. 24 BC). These results show that FLDK1 displays selectivity towards myeloid cells compared to lymphoid cells, unlike IL-10. The respective potencies of FLDK-1 and IL-10, as well as CSF1-IL-10 and IL-10 in activating CD8 T cells and B cells, were further characterized. T lymphocytes were isolated from human PBMCs by positive selection with beads (Miltenyi). Lymphocytes (1.5 million / ml) were stimulated with anti-CD3 (ThermoFisher) (1 µg / ml) and anti-CD28 (ThermoFisher) (3 µg / ml) for 72 hours. Cells were then split in two and treated with IL-10, or FLDK1 or CSF1-IL10 (10 nM per treatment) with IL-2 (5 ng / ml) for 48 hours. Cell supernatants were obtained and granzyme B (Gzmb) and IFNγ were detected by ELISA (Fig. 25 A). Pooled normalized data from 4 independent experiments are shown in the graph for Gzmb. IL-10 vs FLDK1 t-test,p-value < 0.05. The results show that FLDK-1 (Fig. 25 B) or CSF1-IL-10 (Fig. 25 C) is less potent than IL-10 in activating CD8 T cells. B cells were isolated from human PBMCs by positive selection with microspheres (Miltenyi). Isolated lymphocytes (50,000 cells / well) were stimulated with CpG (InvivoGene) (1 µg / ml) and IL10, or FLDK1 or CSF1-IL10 (10 nM per treatment) on days 0 and 2. After 7 days, cell supernatants were obtained for determination of IgG levels by ELISA (Fig. 26 A). Similar results were obtained when stimulated with CD40L. Pooled data from 4 independent experiments are shown in the graph (Fig. 26 B). IL-10 versus FLDK1 t-test, p-value < 0.05. The results show that FLDK-1 (Fig. 26 B) or CSF1-IL-10 (Fig. 26 C) are less potent than IL-10 in B cell activation. Example 4: In vivo, the dual cytokine CSF1-IL10 alleviates clinical symptoms in a mouse model of colitis and other autoimmune diseases.inflammatory diseases. The therapeutic efficacy of FLDK1 (at three doses) was evaluated in an experimental model of chronic IBD induced by TNBS (trinitrobenzene sulfonic acid [TNBS]-induced colitis mouse model), with comparison with a reference molecule (recombinant IL-10). TNBS was infused intrarectally into male Balb / c mice at four increasing doses (0.7 mg / mouse on day 0; 0.8 mg / mouse on day 8; 1.0 mg / mouse on day 15; and 1.2 mg / mouse on day 22). Body weight measurements were taken daily. The experimental groups included 10 mice per group and were as follows: 1. 50% ethanol intrarectally 2. TNBS / 50% ethanol + vehicle treatment intrarectally 3. TNBS / 50% ethanol + FLDK1 treatment (1 µg dose per mouse) intrarectally 4. TNBS / 50% ethanol + FLDK1 treatment (3 µg dose per mouse) intrarectally 5. TNBS / 50% ethanol + FLDK1 treatment (10 µg doseper mouse) intrarectally 6. TNBS / 50% ethanol + rIL10 treatment (1 µg dose per mouse) intrarectally 7. TNBS / 50% ethanol + rIL10 treatment (3 µg dose per mouse) intrarectally 8. TNBS / 50% ethanol + rIL10 treatment (10 µg dose per mouse) intrarectally FLDK1 and rIL10 were administered intraperitoneally, at each dose, 3 times per week (every other day, starting on the day of each TNBS infusion). The results show that FLDK1-treated mice had less body weight loss compared to IL10- or vehicle-treated mice after 30 days of treatment, in a dose-dependent manner (Fig.27 AC). Example 5: Selectivity of the dual cytokine CSF1-IL10 for monocytes versus T cells is mediated by the CSF1 part of the cytokine. A structure-activity assessment of FLDK1 was performed as follows. PBMCs (150,000cells / well) from healthy donors in 96-well plates and stimulated the next day with FLDK1, IL-10, or FLDK1-E82A (SEQ ID NO: 73) (10 nM per treatment) (a mutated version of FLDK1 in which CSF1 cannot bind to CSF1-R due to the E82A mutation) for 20 min at 37°C (Fig. 28 A). Cells were stained with CD14 Alexa647 (Biolegend) and CD3 APC / Cy7 (Biolegend), fixed with 4% PFA, and permeabilized for intracellular staining with 80% MetOH. Cells were stained with AlexaFluor488 pSTAT3 (pY705) (Becton Dickinson) for 1 h. Samples were analyzed using the LSRII cytometer, and MFI values ​​obtained from STAT3 activation were plotted using GraphPad Prism software. A representative result with two technical replicates is shown as the mean with an error bar representing the SEM. Each condition was normalized by assigning the highest IL10 MFI value of the maximum concentration as 100% and the lowest MFI value of auntreated control as 0%. The MFI of the remaining treated samples was normalized accordingly. The results show that selective monocyte activation by FLDK1 is mediated by the CSF-1 portion of the cytokine, as selective monocyte versus T cell activation is greatly reduced by the FLDK1-E82A mutant (Fig. 28 B). PBMCs (150,000 cells / well) from healthy donors were also seeded in 96-well plates and stimulated the next day with either rIL10, FLDK1 (10 nM per treatment), or saturating concentrations of CSF1 (100 nM) (for 2 min) followed by FLDK1 (10 nM) for 20 min at 37°C (Fig. 29 A). Cells were stained with CD14 Alexa647 (Biolegend) and CD3 APC / Cy7 (Biolegend), fixed with 4% PFA, and permeabilized for intracellular staining with 80% MetOH. Cells were stained with AlexaFluor488 pSTAT3 (pY705) (Becton Dickinson) for 1 h. Samples were analyzed using the LSRII cytometer, and MFI values ​​were obtained from activation.of STAT3 were plotted using GraphPad Prism software. A representative result with two technical replicates is shown as the mean with an error bar representing the SEM. Each condition was normalized by assigning the highest IL10 MFI value from the maximum concentration as 100% and the lowest MFI value from an untreated control as 0%. The MFI of the remaining treated samples was normalized accordingly. The results show that selective monocyte activation by FLDK1 is mediated by the CSF-1 moiety of the cytokine, as saturating concentrations of CSF-1 prevented the selective activation of monocytes versus T cells by FLDK1 (Fig. 29 B). SEQUENCES NtCt linkers are underlined. Intermolecular bonds are underlined and italicized. Mutations are in bold. Tags such as HiBit or TevHisHibit for protein quantification are shown in bold and italics, as they are the Gly linkers between them.show the signal peptides used to secrete the proteins that are cleaved after secretion. For proteins beginning with IL10 and Foldikine10, we used the signal sequence: MHSSALLCCLVLLTGVRA (SEQ ID NO: 69). For proteins beginning with CSF1, we used MTAPGAAGRCPPTTWLGSLLVCLLASRSIT (SEQ ID NO: 70). >SEQ ID NO: 1 foldikine10: MTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKN AFNKLQEKGIYKAMSEFDIFINYIEAYMTMNNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQL DNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRL RRNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN >SEQ ID NO: 20 foldikine10-HiBit: MTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKN AFNKLQEKGIYKAMSEFDIFINYIEAYMTMNNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQL DNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGVSGWR LFKKIS >SEQ ID NO: 2 ORK10-002 MTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKN AFNKLQEKGIYKAMSEFDIFINYIEAYMTMNFGGLDYLPNMLRDLRDAFSRVKTFFQMKDQL DNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRL RRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTM >SEQ ID NO: 3 ORK10-003 MTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKN AFNKLQEKGIYKAMSEFDIFINYIEAYLYKTITPEFANMLRDLRDAFSRVKTFFQMKDQLDNLL LKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCH RFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTM >SEQ ID NO: 49 ORK10-003-HiBit MTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKN AFNKLQEKGIYKAMSEFDIFINYIEAYLYKTITPEFANMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCH RFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMGGVSGWRLFKKIS >SEQ ID NO: 4 ORK10-005 MTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKN AFNKLQEKGIYKAMSEFDIFINYIEAKDKDIRDGDELANMLRDLRDAFSRVKTFFQMKDQLD NLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLLR RCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYTM >SEQ ID NO: 5 Foldikine_cut116 NKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMNNGGLDYLPNMLRDALRDAFSR VKTFFQMKDQLDNLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSL GENLKTLRLRLRRNRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTM NNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLKESLLEDFKGYLGCQALSEMIQFY LEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNRFLPCE >SEQ ID NO: 6 Foldikine_cut133 EKGIYKAMSEFDIFINYIEAYMTMNNGGLDYLPNMLRDAFSRVKTFFQMKDQLDNLLK ESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNRHF LPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMNNGGLDYLPNMLRDALRDAFSRVKTFFQMKDQLDNLLKESLLEDFKYLGCQALSEMIQFYLEEVMPQAENQDPDIKAH VNSLGENLKTLRLRLRRNRFLPCENKSKAVEQVKNAFNKLQ >SEQ ID NO: 7 Foldikine_cut209 GYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMNNGGLDYLPNMLRDALDFSRVKTFFQMKDQLDNLLKESLLEDFKYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGE NLKTLRLRLRRNRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMNN GGLDYPNMLRDALDFSRVKTFFQMKDQLDNLLKESLLEDFK >SEQ ID NO: 8 Foldikine_cut237 DIKAHVNSLGENLKTLRLRLRRNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFIN YIEAYMTMNNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQA LSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNHRFLPCENKSKAVEQVK NAFNKLQEKGIYKAMSEFDIFINYIEAYMTMNNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQ LDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDP >SEQ ID NO: 9 ORKMCSF_013 KACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNSNFECEDQDRRQPGTKTTG KVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMED TMRFRDNTPNAIAIVQLQELSLRLKSCFTKDY >SEQ ID NO: 10 ORKMCSF_014KACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECGKPPQYIDRHTQPG QPNATSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDI MEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDY >SEQ ID NO: 11 IL10: SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYL GCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAV EQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN >SEQ ID NO: 50 IL10-HiBit: SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYL GCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAV EQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGVSGWRLFKKIS >SEQ ID NO: 12 CSF1 EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNNSFAECSSQD >SEQ ID NO: 51 CSF1-HiBit EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNNSFAECSSQDGGVSGWRLFKKIS >SEQ ID NO: 13 IL-10-CSF1 SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAV EQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGGSGGSGGSGGSGGSGGSGGS GGSGGSGGGEEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLK KAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLL EKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQD >SEQ ID NO: 52 IL-10-CSF1-HiBit SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLKESLLEDFKGYL GCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAV EQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGGS GGSGGSGGGEEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLK KAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLL EKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQDGGVSGWRLFKKIS >SEQ ID NO: 14 Foldikine10-CSF1 MTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKN AFNKLQEKGIYKAMSEFDIFINYIEAYMTMNNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQL DNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQPDIKAHVNSLGENLKTLRLRLRRNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGGGSG GSGGSGGSGGSGGSGGSGGSGGSGGGEEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFE FVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEH DKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQD >SEQ ID NO: 53 Foldikine10-CSF1-HiBit MTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKN AFNKLQEKGIYKAMSEFDIFINYIEAYMTMNNGGLDYLPNMLRDAFSRVKTFFQMKDQL DNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRL RRNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGGSG GSGGSGGSGGSGGSGGSGGSGGSGGSGGGEEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFE FVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEH DKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQDGGVSGWRL FKKIS >SEQ ID NO: 15 CSF1-IL10 EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEH DKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNNSFAECSSQDGGGGSASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLKESLLEDFKGYL GCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAV EQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN >SEQ ID NO: 54 CSF1-IL10-HiBit EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNSFAECSSQDGGGGGS EQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGVSGWRLFKKIS >SEQ ID NO: 16 CSF1-Foldikine10 (=CSF1-30aa-foldikine10) EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNSFAECSSQDGGGGGSNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLLRLR RNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN >SEQ ID NO: 55 CSF1-Foldikine10-HiBit (=CSF1-30aa-foldikine10 -HiBit) EEVSEYCSHMIGSGHLQSLQSLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNSFAECSSQDGGGGGS FNKLQEKGIYKAMSEFDIFINYIEAYMTMNNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQLD NLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLLR RNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGVSGWRL FKKIS >SEQ ID NO: 17 foldikine10_cut116-CSF1 NKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMNNGGLDYLPNMLRDLRDAFSR VKTFFQMKDQLDNLLKELLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSL GENLKTLRLRLRRRNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTM NNGGLDYPNMLRDLRDAFSRVKTFFQMKDQLDNLLKELLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNHRFLPCEGGGGGSGGSGGSGGSG GSGGSGGSGGSGGGEEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDP VCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYET PLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQD >SEQ ID NO: 56 foldikine10_cut116-CSF1-HiBit NKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMNNGGLDYLPNMLRDLRDAFSR VKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSL GENLKTLRLRLRRNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTM NNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFY LEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNHRFLPCEGGGGGSGGSGGSGGSG GSGGSGGSGGSGGGEEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDP VCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYET PLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQDGGGVSGWRLFKKIS >SEQ ID NO: 18 CSF1-foldikine10_cut116 MTAPGAAGRCPPTTWLGSLLLLVCLLASRSITEEVSEYCSHMIGSGHLQSLQRLIDSQMETS CQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTK DYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQDGGGGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGMTQSENSCTHFPGNLPNMLRDLRDAFSRVK TFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQEPDIKAHVNSLGE NLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMNN GGLDYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN >SEQ ID NO: 57 CSF1-foldikine10_cut116-HiBit MTAPGAAGRCPPTTWLGSLLLLVCLLASRSITEEVSEYCSHMIGSGHLQSLQRLIDSQMETS CQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTK DYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQDGGG GGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGMTQSENSCTHFPGNLPNMLRDLRDAFSRVK TFFQMKDQLDNLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQEPDIKAHVNSLGE NLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMNN GGLDYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNRLFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGVSGWRLFKKIS >SEQ ID NO: 19 CSF1-25aa-foldikine10EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNNSFAECSSQDGGGGGS CSF1-25aa-foldikine10-HiBit EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNNSFAECSSQDGGGGSGGGSGGGSGGGSGGGSGGS TQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALS EMIQFYLEEVMPQAENQEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNA FNKLQEKGIYKAMSEFDIFINYIEAYMTMNNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQLD NLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQPDIKAHVNSLGENLKTLRLLRLRRNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGVSGWRL FKKIS >SEQ ID NO: 21 CSF1-35aa-foldikine10 EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNSFAECSSQDGGGGGGSGGGSGGS MKDQLDNLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKT LRLRLRRNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN >SEQ ID NO: 59 CSF1-35aa-foldikine10-HiBit EEVSEYCSHMIGSGHLQSLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNSFAECSSQDGGGGGSGGGSGGSEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMNNGGLDYLPNMLRDLAFSRVKTFFQ MKDQLDNLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKT LRLRLRRNHRLFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGV SGWRLFKKIS >SEQ ID NO: 22 CSF1-15aa-foldikine10 EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNNSFAECSSQDGGSGGSGGSGGSGGMTQSENSCTHFPGNLPN MLRDLRDLAFSRVKTFFQMKDQLDNLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAEN QEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFD IFINYIEAYMTMNNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLG CQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNHRFLPCENKSKAVE QVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN >SEQ ID NO: 60 CSF1-15aa-foldikine10-HiBit EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNNSFAECSSQDGGSGGSGGSGGGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAEN QEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFD IFINYIEAYMTMNNGGLDYLPNMLRDAFSRVKTFFQMKDQLDNLLKELLEDFKGYLG CQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNRLFLPCENKSKAVE QVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGVSGWRLFKKIS >SEQ ID NO: 23 CSF1-15aa-IL10 EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNNSFAECSSQDGGSGGSGGSGGGRASPGQGTQSENSCTHF PGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEV MPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIY KAMSEFDIFINYIEAYMTMKIRN >SEQ ID NO: 61 CSF1-15aa-IL10-HiBit EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNSFAECSSQDGGSGGSGGSGGGRASPGQGTQSENSCTHF PGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIY KAMSEFDIFINYIEAYMTMKIRNGGVSGWRLFKKIS >SEQ ID NO: 24 CSF1-25aa-IL10 EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNNSFAECSSQDGGGSGGSGGSGGSGGSGGSGGSGGGRASPGQ GTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKN AFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN >SEQ ID NO: 62 CSF1-25aa-IL10-HiBit EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNNSFAECSSQDGGGSGGSGGSGGSGGSGGSGGSGGGRASPGQ GTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKN AFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGVSGWRLFKKIS >SEQ ID NO: 25 CSF1-30aa-IL10 EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNNSFAECSSQDGGGGGS ASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYL GCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAV EQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGVSGWRLFKKIS >SEQ ID NO: 26 CSF1-35aa-IL10 EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNSFAECSSQDGGGGGSGGGGSGGSGGSGGSGGSGGSGG SGGGRASPQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLE<h2 style=";text-align:left;direction:ltr">SEQ ID NO: 64 CSF1-35aa-IL10-HiBit EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNNSFAECSSQDGGGGGSGGGGSGGSGGSGGSGGSGGSGGSGG SGGGRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLE DFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCE NKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGVSGWRLFKKIS >SEQ ID NO: 27 Repetition unit GGSGG >SEQ ID NO: 28 MTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKN AFNKLQEKGIYKAMSEFDIFINYIEAYMTMN >SEQ ID NO: 29 LPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQ AENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAM SEFDIFINYIEAYMTMKIRN >SEQ ID NO: 30 encoder Nt Ct NGGLDY >SEQ ID NO: 31 encoder Nt Ct YKTIT >SEQ ID NO: 32 encoder Nt Ct DKDIRDGD >SEQ IDNO: 33 enlazador intermolecular 115 GGSGGSGGS GSGGG >SEQ ID NO: 34 enlazador intermolecular 125 GGSGGSGGS GGSGGSGGSG GSGGG >SEQ ID NO: 35 enlazador intermolecular 130 GGGGGSGGSG GSGGSGGSGG SGGSGGSGGG >SEQ ID NO: 36 enlazador intermolecular 135 GGGGGSGGGG SGGSGGSGGS GGSGGSGGSG GSGGG >SEQ ID NO:371136 CTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFY LEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQE KGIYKAMSEFDIFINYIEA >SEQ ID NO: 38 NMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAE NQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSE FDIFINYIEAYMTM >SEQ ID NO: 39 MTQSENSCTHFOGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQA LSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVK NAFNKLQEKGIYKAMSEFDIFINYIEAMTMN >SEQ ID NO: 40 LPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQ AENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAM SEFDIFINYIEAYMTM >SEQ ID NO: 41 MTQSENSCTHFOGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQA<h2 style=";text-align:left;direction:ltr">LSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVK NAFNKLQEKGIYKAMSEFDIFINYIEAYL >SEQ ID NO: 42 PEFANMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVM PQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYK AMSEFDIFINYIEAYMTM >SEQ ID NO: 43 MTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKN AFNKLQEKGIYKAMSEFDIFINYIEAK >SEQ ID NO: 44 ELANMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMP QAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKA MSEFDIFINYIEAYMTM >SEQ ID NO: 71 NNGGLDYL >SEQ ID NO: 45 NFGGLDYL >SEQ ID NO: 46 LYKTITPEFA >SEQ ID NO: 47 KDKDIRDGDELA >SEQ ID NO: 48 FGGLDY >SEQ ID NO: 65 N-terminal part of SC dimeric IL-10 MTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKN AFNKLQEKGIYKAMSEFDIFINYIEAY >SEQ ID NO: 66 C-terminal part of SC dimeric IL-10<h2 style=";text-align:left;direction:ltr">NMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAE NQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSE FDIFINYIEAYMTMKIRN >SEQ ID NO: 67 MTMN >SEQ ID NO: 68 PEFA >SEQ ID NO: 72 CSF1-Foldikine10-TEV-his-HiBit EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNNSFAECSSQDGGGGGSGGSGGSGGSGGSGGSGGSGGSGGGM TQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALS EMIQFYLEEVMPQAENQEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNA FNKLQEKGIYKAMSEFDIFINYIEAYMTMMNNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQLD NLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLR RNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGENLYFQS GGHHHHHHGGGVSGWRLFKKIS* >SEQ ID NO: 73 CSF1(E82A)-Foldikine10-TEV-his-HiBit EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQALSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNNSFAECSSQDGGGGGSGGSGGSGGSGGSGGSGGSGGSGGGMTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALS EMIQFYLEEVMPQAENQEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNA FNKLQEKGIYKAMSEFDIFINYIEAYMTMNNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQLD NLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLR RNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGENLYFQS GGHHHHHHGGGVSGWRLFKKIS* >SEQ ID NO: 74 CSF1-IL10-TEV-his-HiBit EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIME DTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNET KNLLDKDWNIFSKNCNNSFAECSSQDGGGGGSGGSGGSGGSGGSGGSGGSGGSGGGS PGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLG CQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVE QVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGENLYFQSGGHHHHHHGGGVSGW RLFKKIS*

Claims

1. Single chain polypeptide having IL-10 and CSF1 activity.

2. Single chain polypeptide according to claim 1, comprising in the N-terminal to C-terminal direction, a CSF1 monomer, a peptide linker and an IL-10 monomer.

3. Single chain polypeptide according to claim 1 or 2, comprising the sequence SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or a sequence at least 80% identical thereto and retaining IL-10 and CSF1 activities.

4. Single chain polypeptide according to claim 1, comprising a single chain dimeric IL-10 fused to a CSF1 monomer, in particular a single chain dimeric IL-10-CSF1 monomer fusion protein, or a CSF1 monomer-single chain dimeric IL-10 fusion protein. 5.Single chain polypeptide according to any one of claims 1 to 4, wherein the CSF1 monomer is a wild-type CSF1 monomer, or a mutant thereof, or a circular permutant CSF1 monomer.

6. Single chain polypeptide according to any one of claims 1 to 5, which has a similar or greater affinity to CSF1R compared to wild-type CSF1.

7. Single chain polypeptide according to any one of claims 1 to 6, wherein the CSF1 monomer is a mutant of the CSF1 monomer comprising the sequence SEQ ID NO:12 modified by at least the substitutions: a) Q17R; b) V78W; c) T124I; d) V120I; e) Q17R, T124I and V120I; f) V78W, T124I and V120I; og) Q17R, V78W, T124I and V120I.

8. Single chain polypeptide according to any one of claims 1 to 6, wherein the CSF1 monomer is a circular permutant of CSF1:. a) generated by joining an amino acid into each of the N-terminal and C-terminal regions of CSF-1 and by deleting the peptide bond consisting of residues at positions 95 to 99 or SEQ ID NO: 12; or b) comprising the sequence SEQ ID NO: 9, or SEQ ID NO: 10, or a sequence at least 80% identical thereto and retaining CSF-1 activity.

9. The single-chain polypeptide of any one of claims 1 and 4 to 8, wherein the single-chain dimeric IL-10 is a fusion protein comprising a first IL-10 monomeric fragment comprising at least IL-10 α-helices A to F, a peptide linker, and a second IL-10 monomeric fragment comprising at least IL-10 α-helices A to F, or a circular permutant thereof. 10.Single chain polypeptide according to any one of claims 1 and 4 to 9, wherein the single chain dimeric IL-10 sequence is SEQ ID NO: 65-X-(NtCt)-Z-SEQ ID NO: 66, wherein (NtCt) is a peptide linker, wherein X is absent or present, and when present consists of one or more amino acids of the original IL-10 sequence in continuity with preceding amino acids on its N-terminal side, optionally with a mutation to accommodate the NtCt linker, wherein Z is absent or present, and when present consists of one or more amino acids of the original IL-10 sequence in continuity with preceding amino acids on its C-terminal side, optionally with a mutation to accommodate the NtCt linker, or an at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical sequence. to the same and that retains at least the same stability, and / or at least the same level of interaction with the IL-10 receptor. 11.Single chain polypeptide according to claim 9 or 10, wherein the peptide linker comprises 3 to 20 amino acid residues and does not comprise more than 2 adjacent Gly and / or Ser residues.

12. Single chain polypeptide according to any one of claims 1 and 4 to 11, wherein the single chain dimeric IL-10 comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or a sequence at least 80% identical thereto and which retains IL-10 activity.

13. Single chain polypeptide according to any one of claims 1 and 4 to 8, wherein the single chain dimeric IL-10 is a circular permutant: a) comprising a first IL-10 monomeric fragment comprising IL-10 α-helices E to F, a first peptide linker, and a second IL-10 monomeric fragment comprising at least IL-10 α-helices A to F, a second peptide linker, and a third IL-10 monomeric fragment comprising at least IL-10 α-helices A to D, and / or b) comprising the sequence SEQ ID NO: 5, or a sequence at least 80% identical thereto and retaining IL-10 activity.

14. A single-chain polypeptide according to any one of claims 1 and 4 to 13, wherein the single-chain dimeric IL-10 and the CSF1 monomer are linked via a linker comprising 12 to 40 amino acid residues.Single chain polypeptide according to any one of claims 1 and 4 to 14, comprising the sequence SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 1, SEQ ID NO: 21, SEQ ID NO: 22, or a sequence at least 80% identical thereto and retaining IL-10 and CSF1 activities.

16. Single chain polypeptide according to any one of claims 1 to 15, having reduced affinity to the IL-10 receptor α (IL10RA) and the IL-10 receptor β (IL10RB) as compared to wild-type IL-10.

17. Pharmaceutical composition comprising the single chain polypeptide according to any one of claims 1 to 16, and a pharmaceutically acceptable carrier.

18. Single-chain polypeptide according to any one of claims 1 to 16, for use as a medicament, in particular for use in the treatment of an inflammatory disease.