Bifunctional csf1-il-10 cytokine

Abstract

The present invention relates to single chain polypeptides having IL-10 activity and CSF1 activity and their use in therapy.

Description

Technical Field

[0001] This invention relates to single-chain polypeptides having IL-10 and CSF1 activity and their use in treatment. Background Technology

[0002] Cytokines are proteins produced by the immune system that act as chemical messengers, transmitting signals between cells of the immune system and between these cells and other cells in the body. Cytokines regulate the entire range of the immune response, including its initiation, type, potency, and duration. They are so important that their unregulated production can trigger a variety of inflammatory and autoimmune diseases, allergic reactions, fibrotic lesions, and even tumors. Therefore, the development of cytokines and their use as drugs to treat diseases has significant medical value.

[0003] 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 in monomeric form by various immune cells, including T cells, B cells, macrophages, and dendritic cells. IL-10 exerts its effects by binding to a receptor complex 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, which is then stabilized by binding to IL-10R2. The binding of IL-10 to its receptor complex leads to the activation of downstream signaling pathways.

[0004] When bound to receptors on myeloid cells, IL-10 exerts potent anti-inflammatory activity, including inhibition of pro-inflammatory mediator production and the expression of MHCII and co-stimulatory molecules. Simultaneously, activation of myeloid cells by IL-10 leads to the expression of anti-inflammatory mediators and promotes the development of tolerant phenotypes, such as M2-like anti-inflammatory macrophages and tolerant dendritic cells. In contrast to its potent anti-inflammatory effects on myeloid cells, IL-10 is a growth factor for B cells and promotes plasma cell differentiation and antibody production. Similarly, IL-10 has a stimulatory effect on CD8+ T cells, inducing CD8+ T cell proliferation, IFNγ production, and cytotoxicity.

[0005] In humans, a significant genetic link exists between the IL-10 signaling pathway and autoimmune diseases such as inflammatory bowel disease. Loss-of-function mutations in IL-10, IL-10RA, or IL-10RB induce early-onset, treatment-resistant, severe enterocolitis. Restoration of IL-10RA or IL-10 expression via hematopoietic stem cell transplantation rapidly alleviates clinical symptoms. Correspondingly, IL-10 or IL-10R deficient mice spontaneously develop colitis, and administration of IL-10 has consistently proven beneficial in various colitis animal models. Finally, spontaneous colitis mediated by the production of the clinically validated target IL-23 also occurs in mice where all cells respond to IL-10 except for specific myeloid cell subsets, including monocytes and macrophages.

[0006] Myeloid cells specifically express the CSF1 receptor, or CSF1R. Stimulation of CSF1R by its ligand cytokine CSF1 is crucial for the differentiation, proliferation, and maintenance of M2-like anti-inflammatory macrophages in the gut. These macrophages play a key role in generating a tolerance-inducing environment by producing IL-10. In autoimmune diseases, M2-like anti-inflammatory macrophages in affected tissues (such as the mucosa of IBD patients) are replaced by pro-inflammatory monocytes that drive disease progression.

[0007] In preclinical studies, IL-10 showed promising results in improving inflammation and tissue damage in a colitis model, but clinical trials investigating its therapeutic potential in colitis patients were unsuccessful. One possible explanation is that the delivery of IL-10 to sites of inflammation may be a limiting factor. However, some strategies designed to enhance IL-10 delivery to sites of inflammation have not shown clinical benefit (AMT-101).

[0008] Another possible explanation is that the anti-inflammatory effect of IL-10 on myeloid cells is offset by pro-inflammatory tissue-damage effects, including B cell activation, differentiation, and antibody induction, as well as the induction of cytotoxic activity of CD8+ T cells.

[0009] To overcome this limitation, there is a need to develop IL-10-based therapies that can selectively activate myeloid cells while avoiding activation of other immune cell subsets. Several strategies have been proposed for developing IL-10-based therapeutics that selectively activate myeloid cells. These strategies include fusing IL-10 with antibodies that bind to receptors on myeloid cells, or encapsulating IL-10 in nanocarriers (liposomes or nanoparticles) that specifically target myeloid cells.

[0010] The present invention aims to overcome or at least alleviate one or more defects in the prior art. Summary of the Invention

[0011] This invention relates to a single-chain polypeptide with IL-10 activity and CSF1 activity.

[0012] The present invention also relates to a pharmaceutical composition comprising a single-chain polypeptide according to the present invention.

[0013] The present invention also relates to the use of the single-chain polypeptide according to the invention as a medicine, particularly for the treatment of inflammatory diseases. Invention Details

[0015] The inventors designed a single-chain peptide combining IL-10 and CSF1 activities. When the N-terminus of IL-10 was linked to the C-terminus of CSF1, the fusion protein combining the IL-10 and CSF1 monomers exhibited reduced IL-10 activity; conversely, when the C-terminus of IL-10 was linked to the N-terminus of CSF1, IL-10 activity was significantly reduced. Surprisingly, the design of the fusion protein combining the single-chain dimer IL-10 and CSF1 monomers made it possible to obtain single-chain peptides exhibiting significant IL-10 and CSF1 activities.

[0016] On one hand, IL-10 monomers pair to form "domain-exchangeable" dimers of the IL-10 protein. In these IL-10-like dimers, the monomeric structure of the standard IL-10 helical bundle cytokine opens and binds to another open IL-10 monomer in an antiparallel manner, together generating two functionally separated cytokine domains, defined as two adjacent 3D domains. On the other hand, CSF1 monomers dimerize, with two CSF1 monomer molecules linked together face-to-face by disulfide bonds.

[0017] Without being limited to any particular theory, the inventors speculate that since IL-10 is an exchange-type dimer and CSF1 dimers, the fusion of IL-10 and CSF1 can lead to the formation of multimers, and this situation will be prevented by replacing the IL-10 monomer with the single-chain dimer IL-10.

[0018] Functional characterization of this bifunctional single-chain polypeptide with IL-10 and CSF1 activities revealed that the bifunctional CSF1-IL-10 cytokine inhibited myeloid cell activation and induced differentiation of monocyte-derived macrophages with a regulatory phenotype (M2 phenotype). Furthermore, this bifunctional CSF1-IL-10 cytokine was specific to monocytes and did not activate CD8 T cells or B cells.

[0019] Functionally, in vivo, in a TNBS-induced mouse model of colitis, the bifunctional CSF1-IL-10 cytokine has been shown to alleviate clinical symptoms of inflammatory disease.

[0020] Single-chain polypeptides with IL-10 and CSF1 activities

[0021] This invention provides a single-chain polypeptide with IL-10 activity and CSF1 activity.

[0022] As used herein, “IL-10 activity” means one or more biological activities mediated by IL-10 through binding to its receptor IL-10R. These activities include or consist of: (i) binding to IL-10R on myeloid cells (especially monocytes), or (ii) anti-inflammatory activity, or preferably both. Anti-inflammatory activities include: (a) inhibiting the production of pro-inflammatory mediators such as pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), IL-1, IL-12, IL-6 and granulocyte-macrophage colony-stimulating factor, inflammatory enzymes such as cyclooxygenase 2 and inducible nitric oxide synthase, chemokines such as RANTES, membrane inflammatory protein-1α (MIP1α), IL-8 and eosinophil chemokine), and / or (b) inhibiting the expression of MHCII and co-stimulatory molecules in myeloid cells, and / or (c) inducing monocytes to differentiate into tolerant macrophages. In some embodiments, the anti-inflammatory activity includes inhibiting the production of TNF-α and / or IL-6 by activated monocytes. In some embodiments, the anti-inflammatory activity includes inducing monocytes to differentiate into tolerant macrophages. Tolerant macrophages have an M2-like phenotype and can be identified by their regulatory phenotype and high levels of expression of CD206 and CD163. In some embodiments, the activity of IL-10 includes or consists of: binding to IL-10R on myeloid cells (especially monocytes), inhibiting the production of TNF-α and / or IL-6 by activated monocytes, and inducing monocytes to differentiate into tolerant macrophages.

[0023] In some implementations, the single-chain polypeptide having IL-10 activity and CSF1 activity has reduced T cell and B cell activation capacity compared to IL-10.

[0024] As used herein, “CSF1 activity” means one or more biological activities mediated by activation of the CSF1 receptor (CSF1-R). These activities include or consist of: (i) activation of CSF1-R on the surface of monocytes or their progenitor cells; and / or (ii) differentiation of monocytes into macrophages, preferably both activities. In some embodiments, activation of CSF1-R is triggered by the binding of CSF1 to CSF1-R.

[0025] In some embodiments, the single-chain polypeptide with both IL-10 and CSF1 activities has a similar or higher affinity for CSF1R compared to wild-type CSF1. In some embodiments, the single-chain polypeptide with both IL-10 and CSF1 activities has a reduced affinity for IL-10 receptor α (IL10RA) and IL-10 receptor β (IL10RB) compared to wild-type IL-10. In some embodiments, the single-chain polypeptide with both IL-10 and CSF1 activities has a similar or higher affinity for CSF1R compared to wild-type CSF1, and a reduced affinity for IL-10 receptor α (IL10RA) and IL-10 receptor β (IL10RB) compared to wild-type IL-10. These properties are expected to facilitate the selective binding of the single-chain polypeptide with both IL-10 and CSF1 activities to myeloid cells that specifically express the CSF1 receptor in the cells of the innate immune system, and subsequently activate them.

[0026] "Similar affinity" in this document means that the affinity is preferably no more than 5 times different (i.e., increased or decreased) from the reference affinity level of wild-type cytokine (CSF1), preferably 4 times, 3 times, 2 times or 1.5 times.

[0027] "Higher" or "lower" in this document means that the affinity is preferably a change (i.e. an increase or decrease) of at least 5 times, preferably 7 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 100 times, or 200 times, compared to the reference affinity level of the wild-type cytokine (CSF1 or IL-10, as the case may be).

[0028] Therefore, in a first aspect, a single-chain polypeptide having IL-10 activity and CSF1 activity is provided, comprising an IL-10 monomer fused to a CSF1 monomer, wherein the N-terminus of the IL-10 is linked to the C-terminus of the CSF1 via a linker.

[0029] In fact, under this orientation, the single-chain polypeptide has reduced IL-10 activity compared to wild-type IL-10, but maintains CSF1 activity similar to wild-type CSF1.

[0030] Accordingly, the single-chain polypeptide with IL-10 and CSF1 activities therefore comprises, in the direction from N-terminus to C-terminus, a CSF1 monomer, a peptide linker, and an IL-10 monomer.

[0031] Preferably, the linker bridging the C-terminus of the CSF1 monomer and the N-terminus of the IL-10 monomer is a (flexible) peptide sequence composed of Gly and Ser residues in varying proportions. Examples of suitable linkers include or consist of the following sequences: GGGSGGSGGSGGSGGSGGSGGSGGG (25 amino acids long, SEQ ID NO: 34), GGGGGSGGSGGSGGSGGSGGSGGSGGSGGSG (30 amino acids long, SEQ ID NO: 35), or GGGGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSG (35 amino acids long, SEQ ID NO: 36).

[0032] CSF1 monomers can be as defined in the following sections.

[0033] The IL-10 monomer can be wild-type mature IL-10 as shown in SEQ ID NO:11, or a mutant thereof, or a polypeptide containing the sequence of mature IL-10 and having 1, 2, 3 or more additional amino acid residues (i.e. A, RA or VRA) consecutively present in the signal peptide of IL-10 at the N-terminus.

[0034] In some embodiments, the single-chain polypeptide having IL-10 and CSF1 activities comprises or consists of sequences of SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, or comprises or consists of sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to these sequences and retain IL-10 and CSF1 activities.

[0035] In a second aspect, a single-chain polypeptide with IL-10 activity and CSF1 activity is provided, comprising a single-chain dimer IL-10 fused to a CSF1 monomer.

[0036] In some embodiments, the single-chain polypeptide having IL-10 and CSF1 activities comprises a single-chain (SC) dimer IL-10-CSF1 monomer fusion protein. In some embodiments, the single-chain polypeptide having IL-10 and CSF1 activities comprises a CSF1 monomer-single-chain dimer IL-10 fusion protein.

[0037] In some embodiments, the single-chain dimer IL-10 is linked to the CSF1 monomer via a linker containing 10 to 45 amino acid residues, preferably 12 to 40 amino acids, more preferably 15 to 40 amino acids, more preferably 20 to 35 amino acids, and even more preferably 25 to 35 amino acids.

[0038] Preferably, the linker bridging the C-terminus of SC dimer IL-10 to the N-terminus of CSF1, or bridging the N-terminus of SC dimer IL-10 to the C-terminus of CSF1, is a (flexible) peptide sequence composed of Gly and Ser residues in different proportions. Examples of suitable linkers include or consist of the following sequences: GGSGGSGGSGGSGGG (15 amino acids long, SEQ ID NO: 33), GGGSGGSGGSGGSGGSGGSGGSGG (25 amino acids long, SEQ ID NO: 34), GGGGGSGGSGGSGGSGGSGGSGGSGGSG (30 amino acids long, SEQ ID NO: 35), or GGGGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSG (35 amino acids long, SEQ ID NO: 36).

[0039] Preferably, when the single-chain polypeptide having IL-10 activity and CSF1 activity contains a CSF1 monomer-single-chain dimer IL-10 fusion protein, the linker bridging the C-terminus of CSF1 and the N-terminus of SC dimer IL-10 contains more than 15 amino acids, preferably at least 20 or 25 amino acids.

[0040] CSF1 monomer

[0041] In the first and second aspects of the present invention, the CSF1 monomer incorporated into the single-chain polypeptide having IL-10 activity and CSF1 activity may be a wild-type CSF1 monomer, or a mutant thereof, or a cyclically arranged CSF1 monomer.

[0042] The polypeptide sequence of wild-type human CSF1 is shown in SEQ ID NO: 12.

[0043] In some embodiments, the CSF1 monomer is a mutant CSF1 or a circularly arranged CSF1 monomer that has similar or higher affinity for CSF1R compared to wild-type CSF1.

[0044] In some embodiments, the CSF1 monomer is a CSF1 monomer mutant comprising a SEQ ID NO:12 sequence modified by at least one of the following substitutions:

[0045] a) Q17R;

[0046] b) V78W;

[0047] c) T124I;

[0048] d) V120I;

[0049] e) Q17R, T124I, and V120I;

[0050] f) V78W, T124I, and V120I; or

[0051] g) Q17R, V78W, T124I and V120I.

[0052] Compared to wild-type CSF1, the Q17R or V78W substitution in CSF1 monomeric mutants was shown to improve EC-50 values ​​(i.e., improved affinity for CSF1R) in HEK293 cells transfected with the CSF1R / Reporter Kit. The T124I or V120I substitutions in the CSF1 monomeric mutants were engineered to increase the stability of the CSF1 monomer in its conformation for binding to the receptor CSF1R, and they increased the maximal response in HEK cells transformed with the CSFR1 Reporter Kit.

[0053] Circular arrangements in protein structures are rearrangements of amino acid sequences that make the original N-terminus and C-terminus of the polypeptide appear to be linked together, and generate new ends at other locations. Therefore, circular arrangements are typically prepared by linking native protein ends or terminal regions via covalent linkers and by introducing new N-terminals and C-terminals by cleaving existing peptide bonds.

[0054] In some embodiments, the CSF1 monomer is a cyclic arrangement of CSF1 generated by linking the amino acids in the N-terminal and C-terminal regions of CSF-1 and by inhibiting the peptide bond consisting of residues 95 to 99 in SEQ ID NO: 12.

[0055] As used herein, each amino acid in the N-terminal and C-terminal regions of CSF-1 is an amino acid located at the N-terminus of CSF-1 (i.e., residue 1 of SEQ ID NO:12) and an amino acid located at the C-terminus of CSF-1 (i.e., residue 150 of SEQ ID NO:12), or an amino acid located at a position at most 2, 3, 4, 5, 6, 7 or 8 residues away from the N-terminus or C-terminus of CSF-1.

[0056] Preferably, the CSF1 monomer is a circular arrangement of CSF1 comprising or composed of the sequence of SEQ ID NO: 9 (referred to as ORKMCSF_013) or the sequence of SEQ ID NO: 10 (referred to as ORKMCSF_014), or a circular arrangement of CSF1 comprising or composed of sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to these sequences and retain CSF1 activity. Both circular arrangements have been shown to activate downstream CSF1 signal cascades with similar EC-50 values ​​and are slightly superior to wild-type CSF1 monomers.

[0057] Single-chain dimer IL-10

[0058] The SC dimer IL-10 incorporated into the single-chain polypeptide having IL-10 and CSF1 activities can be a fusion protein comprising a first IL-10 monomer fragment, a peptide linker, and a second IL-10 monomer fragment or their circular arrangement, wherein the first IL-10 monomer fragment comprises at least IL-10 α-helices A to F, and the second IL-10 monomer fragment comprises at least IL-10 α-helices A to F.

[0059] The folding mechanism of the SC dimer IL-10 enables the formation of "continuous 3D IL-10 domains" ( Figure 2 The left-hand structural domain) and the segmented 3D IL-10 structural domain ( Figure 2 (The right-hand structural domain in the text).

[0060] The polypeptide sequence of wild-type human IL-10 is shown in SEQ ID NO: 11:

[0061]

[0062]

[0063] The position of the helix is ​​indicated by the string "h" in the above sequence. For the α-helix F, depending on the structure assignment of different crystal structures of the same molecule by different software, the helix always extends to M. 154 And it can extend to M 156 Or I 158 (Wave underline character). Therefore, this paper considers that as long as the IL-10 monomer fragment contains at least M... 154 If the amino acid residues are included, then the α-helix F is completely incorporated.

[0064] According to one embodiment, the SC dimer IL-10 comprises or consists of the following sequences:

[0065]

[0066] (Sequence SEQ ID NO: 65-X-(NtCt)-Z-SEQ ID NO: 66)

[0067] Where (NtCt) is the peptide linker.

[0068] X may or may not be present; when present, it consists of one or more amino acids from the original IL-10 sequence, continuous with the amino acid preceding its N-terminus, and optionally has a mutation to accommodate the NtCt linker.

[0069] Wherein Z is absent or present, and when present, it consists of amino acids of one or more of the original IL-10 sequence, continuous with the amino acids preceding its C-terminal side, optionally having a mutation to adapt to the NtCt linker, or a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the sequence and retains at least the same stability and / or at least the same level of interaction with the IL-10 receptor.

[0070] For example, X can represent M, MT, MTM, MTMN (SEQ ID NO: 67), which are present in the original IL-10 sequence and are consecutive to the amino acid preceding its N-terminus. X can also represent L or K, i.e., mutations adapted to the NtCt linker compared to the original IL-10 sequence. Therefore, X is preferably selected from the group consisting of M, MT, MTM, MTMN (SEQ ID NO: 67), L, or K.

[0071] For example, Z can represent LP, which is present in the original IL-10 sequence and continues with the subsequent amino acids on its C-terminal side. Z can also represent PEFA (SEQ ID NO: 68) or ELA. Therefore, Y is preferably selected from the group consisting of LP, PEFA (SEQ ID NO: 68), or ELA.

[0072] The NtCt linker sequence bridging the C-terminus of one IL-10 monomer fragment to the N-terminus of another IL-10 monomer fragment can have any suitable sequence. It preferably contains a sequence that defines a conformationally restricted structure, i.e., a “structured” linker. In this way, engineered linkers help to restrict the 3D domain structure with a suitable / desired structural orientation. Such linkers can have about 3 to about 20 amino acid residues; about 3 to about 16 amino acid residues; about 4 to about 12 amino acid residues; about 4 to about 8 amino acid residues; about 3 to 8 amino acid residues; or about 3 to 6 amino acid residues. Advantageously, such linkers do not contain multiple adjacent Gly and / or Ser residues; for example, advantageously, the linker peptide contains 5 or fewer adjacent Gly and / or Ser residues; suitably 3 or fewer adjacent Gly and / or Ser residues; 2 or fewer Gly and / or Ser residues; only isolated Gly and / or Ser residues; or in some embodiments, no Gly and / or Ser residues at all. In one embodiment, the NtCt linker comprises 3 to 20 amino acid residues and includes no more than 2 adjacent Gly and / or Ser residues.

[0073] According to some implementations, the NtCt connector includes the sequence NGGLDY (SEQ ID NO: 30), FGGLDY (SEQ ID NO: 48), YKTIT (SEQ ID NO: 31), or DKDIRDGD (SEQ ID NO: 32).

[0074] X may be located before the N-terminal side of the NtCt linker, i.e., at amino acid residues 152 through 157 of IL-10, as shown in SEQ ID NO: 11. Alternatively, or additionally, Z may be located after the C-terminal side of the NtCt linker, i.e., at amino acid residues 16 through 20 of IL-10, as shown in SEQ ID NO: 11.

[0075] Therefore, according to some implementations, the NtCt adapter and its N-terminal or C-terminal X or Z sequence (the bolded X and Z sequences have the same structure in IL10 and they correspond to the WT IL10 sequence or may have point mutations to adapt to the adapter) comprise or consist of the following sequences: .

[0076] In some embodiments, the single-chain dimer IL-10 comprises or is composed of SEQ ID NO: 2 (ORK10-002), SEQ ID NO: 3 or SEQ ID NO: 49 (ORK10-003; with or without a HiBit tag sequence for quantification added to the C-terminus of SEQ ID NO: 3), or SEQ ID NO: 4 (ORK10-005), or comprises or is composed of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the said sequence and retains IL-10 activity.

[0077] In some embodiments, the single-chain dimer IL-10 comprises a first IL-10 monomer fragment containing a D84E substitution at the 84th naturally occurring residue of SEQ ID NO:11. The single-chain dimer IL-10 may subsequently comprise or consist of SEQ ID NO:1 or SEQ ID NO:20 (Foldikine10, with or without the HiBit tag sequence for quantification added to the C-terminus of SEQ ID NO:1).

[0078] According to some embodiments, the SC dimer IL-10 consists of a cyclic arrangement of one of the aforementioned SC dimer IL-10. It is considered advantageous to use a cyclic arrangement to minimize masking effects that may result from the connection method between the fused CSF1 monomer and the SC dimer IL-10. Figure 19).

[0079] In some embodiments, the circular assembly comprises: a first IL-10 monomer fragment comprising α-helices E to F containing IL-10, a first SC dimer IL-10 peptide linker, and a second IL-10 monomer fragment comprising α-helices A to F containing at least IL-10, a second peptide linker, and a third IL-10 monomer fragment comprising α-helices A to D containing at least IL-10. In some embodiments, the circular assembly comprises the sequence of SEQ ID NO: 5 (Foldikine10_cut116, also referred to herein as Foldikine10_cp116), or comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the sequence and retains IL-10 activity.

[0080] A single-chain polypeptide containing a single-chain dimer of IL-10 fused to a CSF1 monomer and possessing both IL-10 and CSF1 activities.

[0081] According to some embodiments, the single-chain polypeptide having IL-10 and CSF1 activities therefore comprises or is composed of sequences SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or comprises or is composed of sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the said sequences and retain IL-10 and CSF1 activities.

[0082] Pharmaceutical Composition

[0083] The present invention also relates to a pharmaceutical composition comprising a single-chain polypeptide having IL-10 activity and CSF1 activity and a pharmaceutically acceptable carrier.

[0084] Suitable pharmaceutically acceptable “carriers” (such as diluents, adjuvants, excipients, or mediators) are described in EW Martin’s “Remington’s Pharmaceutical Sciences.” The pharmaceutical compositions of the present invention are formulated to meet regulatory standards and can be administered orally, intravenously, topically, or via other standard routes. As used herein, the term “carrier” includes any and all solvents, dispersion media, mediators, coatings, diluents, antimicrobial and antifungal agents, isotonic and absorption-delaying agents, buffers, carrier solutions, suspensions, colloids, etc. Such media and reagents are well known in the art for use with pharmaceutically active substances. Unless any conventional media or reagent is incompatible with the active ingredient, its use in therapeutic compositions is considered. Additional active ingredients may also be incorporated into the composition. 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 humans or non-human mammals.

[0085] Acceptable pharmaceutical mediators can be liquids, such as water, and oils, including petroleum-derived, animal-derived, plant-derived, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical mediators can also include saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. Additionally, excipients, stabilizers, thickeners, lubricants, and colorants can be used. When administered to a subject, pharmaceutically acceptable mediators are preferably sterile. Water is a suitable mediator, particularly when the compounds of the present invention are administered intravenously. Saline solutions, as well as glucose solutions and glycerol solutions, can also be used as liquid mediators, particularly for injectable solutions. Suitable pharmaceutical mediators also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc. If desired, the compositions of the present invention may also contain small amounts of wetting agents or emulsifiers, or buffers.

[0086] The pharmaceuticals and pharmaceutical compositions of the present invention may be in the form of liquids, solutions, suspensions, lotions, gels, tablets, pills, granules, powders, modified release formulations (such as slow-release or sustained-release), suppositories, emulsions, aerosols, sprays, capsules (e.g., capsules containing liquid or powder), liposomes, microparticles, or any other suitable dosage form known in the art. Examples of other suitable pharmaceutical mediators are described in Remington's Pharmaceutical Sciences, edited by Alfonso R. Gennaro, Mack Publishing Co. Easton, Pa., 19th edition, 1995, see, for example, pages 1447-1676.

[0087] In some embodiments, the therapeutic compositions or pharmaceuticals of the present invention are formulated according to conventional procedures into pharmaceutical compositions suitable for oral administration (more suitable for humans). Compositions for oral delivery may be in the form of, for example, tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. Therefore, in various embodiments, a pharmaceutically acceptable medium may be capsules, tablets, or pills.

[0088] Orally administered compositions may contain one or more agents, such as sweeteners like fructose, aspartame, or saccharin; flavoring agents like peppermint, wintergreen oil, or cherry; coloring agents; and preservatives to provide a pharmaceutically palatable formulation. When the composition is in tablet or pill form, it may be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing sustained release of the active agent over an extended period. Selective permeation membranes surrounding the osmotically driven compound are also suitable for orally administered compositions. In these dosage forms, liquid from the environment surrounding the capsule is absorbed by the driven compound, causing the compound to swell and displace the agent or reagent composition through the pores. These dosage forms provide a substantially zero-order release profile, in contrast to the spiked profile of immediate-release formulations. Time-delaying materials such as glyceryl monostearate or glyceryl stearate may also be used. Orally administered compositions may contain standard mediators such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such mediators are preferably pharmaceutical grade. For oral formulations, the release site can be the stomach, small intestine (duodenum, jejunum, or ileum), or large intestine. Those skilled in the art can prepare formulations that are insoluble in the stomach but will release material into the duodenum or other parts of the intestine. Suitably, the release will avoid the harmful effects of the gastric environment, either by protecting the peptide (or its derivatives) or by releasing the peptide (or its derivatives) outside the gastric environment, such as in the intestine. To ensure complete resistance to gastric acid, a coating that is at least impermeable at pH 5.0 is essential. Examples of more common inert ingredients used as enteric coatings include 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.

[0089] To aid in the dissolution of therapeutic agents in an aqueous environment, surfactants may be added as wetting agents. Surfactants may include anionic detergents such as sodium dodecyl sulfate, sodium dioctyl sulfosuccinate, and sodium octyl sulfosuccinate. Cationic detergents may be used, including benzalkonium chloride or benzoyl chloride. Potential nonionic detergents that may be included as surfactants in formulations include: lauryl ether 400, polyoxyethylene 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glyceryl monostearate, polysorbate 20, 40, 60, 65, and 80, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. When using these surfactants, they may be present alone or in mixtures of varying proportions in formulations of peptides or nucleic acids or their derivatives.

[0090] Typically, compositions for intravenous administration contain a sterile isotonic buffer solution. If necessary, the composition may also contain a solubilizer.

[0091] The polypeptide mixtures described herein can be prepared in water with one or more excipients, carriers, or diluents appropriately mixed. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, mixtures thereof, and oils. Under normal storage and use conditions, these formulations may contain preservatives to prevent microbial growth. Suitable pharmaceutical forms for injection include sterile aqueous solutions or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In all cases, the form can be sterile and sufficiently fluid for injection using a suitable syringe. Suitably, the composition is stable under the conditions of manufacture and storage and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Appropriate flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Microbial action can be prevented by various antimicrobial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, isotonic agents, such as sugar or sodium chloride, are preferred. The absorption of the injectable composition can be prolonged by using delaying absorption agents such as aluminum monostearate and gelatin in the composition.

[0092] For parenteral administration of aqueous solutions, the solution may be appropriately buffered if necessary, and the liquid diluent should first be isotonic with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, intratumoral, and intraperitoneal administration. Those skilled in the art will recognize the sterile aqueous media that can be used in this regard.

[0093] Another suitable route of administration for the pharmaceutical composition of the present invention is by pulmonary delivery or nasal delivery.

[0094] It may contain additives to enhance cellular uptake of the therapeutic agents of the present invention, such as fatty acids, oleic acid, linoleic acid and linolenic acid.

[0095] Therapeutic applications

[0096] The present invention relates to a single-chain polypeptide having IL-10 activity and CSF1 activity, or a pharmaceutical composition comprising it, which is used as a drug.

[0097] Therefore, the present invention also relates to a treatment method comprising administering the single-chain polypeptide of the present invention having IL-10 activity and CSF1 activity to a subject in need.

[0098] The subjects can be humans or non-human mammals, such as rodents (mice or rats), primates (e.g., monkeys), pigs, etc.

[0099] Subjects in need, particularly those with inflammatory diseases. Single-chain peptides with IL-10 and CSF1 activity are engineered to preferentially activate myeloid cells (monocytes, macrophages, dendritic cells) that co-express CSF1R and IL-10R—and induce an anti-inflammatory response—rather than IL-10R-expressing CD8 T cells and B cells that, upon activation, can induce cytotoxic responses and autoantibodies.

[0100] Therefore, the present invention also relates to the use of the single-chain polypeptide according to the invention for the treatment of inflammatory diseases. A method for treating inflammatory diseases is also provided, comprising administering to a subject in need the single-chain polypeptide of the invention having IL-10 and CSF1 activity.

[0101] In some implementations, the inflammatory disease is an inflammatory bowel disease, such as inflammatory bowel disease, including Crohn's disease and colitis.

[0102] Treatment options can vary and are typically determined by the type and / or location of the disease, its stage / progression, and / or the patient's health condition and age. A skilled clinician will be able to determine the appropriate treatment option in each case.

[0103] The present invention will be further illustrated by the following figures and embodiments. Attached Figure Description

[0104] Figure 1 Decoupling of IL-10's pro-inflammatory and anti-inflammatory functions. This figure shows an engineered single molecule that combines IL-10 and CSF1 activities, in which the binding affinity to the IL-10 receptor is weakened, resulting in IL-10 activation only in cells expressing the CSF1 receptor.

[0105] Figure 2Schematic diagram of foldikine formation based on exchange-domain dimers of class II cytokines (e.g., IL-10): (i) Wild-type domain exchange-domain dimers, showing α-helices D, E, and F of each IL-10 monomer unit aligned with the opposing IL-10 monomer α-helices A, B, and C to form adjacent 3D IL-10 domains containing α-helices A, B, and C of the first IL-10 monomer and α-helices D, E, and F of the second IL-10 monomer; (ii) Single-chain foldikine-10 based on fused wild-type IL-10 monomers, wherein the monomer sequences are linked to form a single-chain polypeptide by connecting the C-terminus of one IL-10 monomer sequence to the N-terminus of the second monomer sequence using a peptide linker (dashed line). Wild-type linkers are shown in solid lines. Boxes indicate the α-helices forming each of the left and right 3D IL-10 domains (continuous IL-10 domains and segmented IL-10 domains).

[0106] Figure 3 Analysis of different versions of Foldikine-10. All versions were unlabeled and quantified by ELISA (IL-10). The activity of the designed molecule was assessed in commercially available HEK-Blue 10 cells in a dose-response assay, and the data were fitted to an S-shaped dose-response curve to determine the EC-50 (see Methods). We fixed the baseline 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) were analyzed.

[0107] Figure 4 . Circular arrangement of Foldikine-10. A schematic diagram depicting the circular arrangement of foldikine-10 is shown. Wild-type connectors are shown in solid lines. Boxes indicate α-helices forming each of the left and right 3D IL-10 domains (continuous and segmented IL-10 domains). Discontinuities represent engineered NtCt connectors. The new Nt and new Ct resulting from the open loop 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.

[0108] Figure 5 CSF1 dimer structure. The CSF1 dimer complex, with the two subunits shown in different shades of gray. The positions of the disulfide bonds are shown in black.

[0109] Figure 6 . Circular arrangements of CSF1. The natural Nt-Ct of CSF1 and two residues (Y95, K100) of which we open the ring. Two circular arrangements (ORKMCSF_013 and ORKMCSF_014) are generated by connecting the natural Nt-Ct with two different linkers.

[0110] Figure 7 The circular arrangement of CSF1 was evaluated in HEK293 cells transfected with a reporter cell kit to measure the downstream MAPK / ERK signaling cascade following CSF1 / CSF1R activation. CSF1 (1-190) HiBit corresponds to SEQ ID NO: 51. ORKMCSF_013 is SEQ ID NO: 9, and ORKMCSF_014 is SEQ ID NO: 10.

[0111] Figure 8 Dose-response analysis of IL-10 variants with engineered affinity for IL10RA in HEK-Blue10 cells. All mutations were introduced only into half (continuous domains) of the ORK10-003b foldikine scaffold (SEQ ID NO: 49). Data were fitted to sigmoid dose-response curves to determine EC-50 (see Methods). We fixed the baseline and saturation values. Foldikine10 (ORK10-003REF is SEQ ID NO: 49 ORK10-003-HiBit). Mutants were numbered based on SEQ ID NO: 50. In ORK10-003b, R24 was R163, K34 was 173, Q38 was 177, D44 was 183, and E50 was 189. We fixed the baseline at 0.27 and the Hill slope at -1.6.

[0112] Figure 9 Dose-response analysis of IL-10 variants with engineered affinity for IL10RB in HEK-Blue10 cells. All mutations were introduced only into half (continuous domain) of the ORK10-003b foldikine scaffold (SEQ ID NO: 49). Data were fitted to sigmoid dose-response curves to determine EC-50 (see Methods). We fixed baseline and saturation values. Mutants were numbered based on SEQ ID NO: 50. In ORK10-003b, N92 was 231. We fixed the baseline at 0.12 and the last two rows at -1.9.

[0113] Figure 10 Dose-response analysis of the CSF1 (SEQ ID NO: 12) mutant with predicted improved affinity in HEK reporter cells. We present the titration curves, with a table of EC-50 values ​​at the bottom. The mutants are numbered based on SEQ ID NO: 12. Data were fitted to an sigmoid dose-response curve to determine EC-50 (see Methods). We fixed the baseline values ​​and the Hill slope.

[0114] Figure 11 Dose-response analysis of a CSF1 variant (SEQ ID NO: 12) designed to enhance CSF1 stability. Analysis was performed in HEK293 reporter cells, and curve fitting was derived. The mutant was numbered based on SEQ ID NO: 12. Data were fitted to an sigmoid dose-response curve to determine EC-50 (see Methods). We fixed baseline values ​​and the Hill slope. The Hill slope was fixed at -1.7, and the baseline was fixed at 0.3.

[0115] Figure 12. Schematic diagram showing possible fusion proteins of IL-10 and CSF1 (A), and possible fusion proteins of Foldikine10 and CSF1 (B). (C): Showing how IL-10 WT monomers can aggregate or form multimers after fusion with CSF1.

[0116] Figure 13 EC-50 of orientation A fusion proteins of IL-10 or Foldikine10 with CSF1 was determined. Titration curves of HEKblue cells IL10R (upper half) and CSF1R reporter cells (lower half) were obtained by increasing candidate variant concentrations while maintaining orientation A (IL-10 variant-adaptor-CSF variant). Adaptor length and Figure 18 Same (30aa). CSF1 (REF) is SEQ ID NO:12. Data were fitted to an S-shaped dose-response curve to determine EC-50 (see Methods). For the upper plot, we fixed the baseline at 0.08 and the Hill slope at -1.38. For the lower plot, we fixed the baseline 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), and Foldikine10-CSF1 is Foldikine10-CSF1-HiBit (SEQ ID NO: 53).

[0117] Figure 14EC-50 of orientation B IL10 or Foldikine10 fusion protein with CSF1 was determined. Titration curves of HEKblue cells IL10R reporter cells (left half) and CSF1R reporter cells (right half) were obtained by increasing candidate variant concentrations while maintaining orientation B (CSF variant-adaptor-IL10 variant). Adaptor length and Figure 17 Same (30aa). CSF1 (REF) is SEQ ID NO: 12. The data were fitted to an S-shaped dose-response curve to determine EC-50 (see Methods). For the upper plot, we fixed the baseline at 0.08 and the Hill slope at -1.38. For the lower plot, we fixed the baseline 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), and CSF1-Foldikine10 is CSF1-Foldikine10-HiBit (SEQ ID NO: 55).

[0118] Figure 15 EC-50 of the fusion protein of Foldikine10_cut116 and CSF1 was determined. HEKBlue IL10R reporter cell titration curves of the Foldikine10_cut116-CSF1 fusion were obtained. Data were fitted to an sigmoid dose-response curve to determine EC-50 (see Methods). Baseline and Hill slope values ​​were fixed. 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), and foldikine10_cut116 is SEQ ID NO: 5.

[0119] Figure 16The EC-50 of the fusion protein of Foldikine10_cut116 and CSF1 was determined. A dose-response curve was generated using the CSF1R reporter kit to transform HEK. Data were fitted to an sigmoid dose-response curve to determine the EC-50 (see Methods). We fixed the baseline 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), and CSF1-Foldikine10 is CSF1-Foldikine10-HiBit (SEQ ID NO: 55).

[0120] Figure 17 Connector length and protein function for orientation B. Data from HEKBlue IL-10 R reporter cells. Data were fitted to an sigmoid dose-response curve to determine EC-50 (see Methods). We fixed the baseline 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, and CSF1-35aa-IL10 is SEQ ID NO: 64. The hill slope is fixed at -1.2, and the baseline is fixed at 0.40.

[0121] Figure 18Linker length and protein function for orientation B. Data from HEK CSF1R reporter cells. Protein concentrations of IL-10 variants with linkers of 15 and 25 amino acids (aa) were below the dynamic range of HEK CSF1R 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, and CSF1-35aa-IL10 is SEQ ID NO: 64. The protein expression of the two mutants (CSF1-15AA-IL10 and CSF1-25AA-IL10) was below the dynamic range of CSFF1R HEK reporter cells. The Hill slope was fixed at -1.06 and the baseline was fixed at 1.2.

[0122] Figure 19 The diagrams illustrate the masking effect that can result from the way fused CSF1 protein binds to Foldikine 10 or its circular arrangement. A) The diagram shows the binding of Foldikine 10 to its two receptors, where the native Nt and Ct point towards the cell membrane. B) The diagram shows the binding of the native Nt of Foldikine 10 to the Ct of CSF1 (the same would occur if fused to the Ct of Foldikine 10). In this case, the presence of CSF1 fused to Nt affects the binding of the left receptor, leading to a masking effect and reducing the binding affinity of Foldikine 10. C) The diagram shows the binding of a new Nter generated by opening the loop pointing away from the membrane to the Ct of CSF1 (the same would occur if fused to the Ct of the circular arrangement). In this case, the presence of CSF1 fused to Nt does not affect the binding of the left receptor.

[0123] Figure 20. FLDK-1 and CSF1-IL10, similar to IL-10, inhibit monocyte activation. (A) Total PBMCs from healthy donors were stimulated with LPS in the presence or absence of different concentrations of rIL-10, FLDK-1, or CSF1-IL10. (B) TNFα levels in the supernatant of PBMCs treated with rIL10 or FLDK1 were measured by ELISA 24 hours after stimulation. Each data point represents an independent experiment (n=3). (C) TNFα levels in the supernatant of PBMCs treated with CSF1-IL10 were measured by ELISA 24 hours after stimulation. Each data point represents an independent experiment (n=3).

[0124] Figure 21. FLDK1, similar to CSF1, induces differentiation of macrophages derived from monocytes. (A) PBMCs were isolated from human erythrocyte sedimentation rate (ESR) amber layer and CD14+ cells were purified by positive microbead selection. Monocytes were cultured for 7 days in the presence or absence of rIL-10, rCSF1, FLDK1, or untreated. (BC) Viable cells were counted on day 7, and the expression levels of macrophage markers (CD14, MHCII, CD206, CD163, and CD86) were determined by FACS (flow cytometry).

[0125] Figure 22 FLDK1-induced macrophages exhibit a regulatory phenotype. (A) PBMCs were isolated from human erythrocyte sedimentation rate (ESR) amber layer 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 supernatant were measured by ELISA 17 hours after stimulation.

[0126] Figure 23 FLDK1-induced macrophages exhibit a regulatory phenotype. (A) Monocytes were isolated from human erythrocyte sedimentation rate (ESR) amber layer samples by selecting positive CD14 cells. Monocytes were cultured for 7 days in the presence or absence of rCSF1 or FLDK1. On day 7, cells were detached from plates and counted, and the same number of macrophages were replated and stimulated with LPS for 4 hours. CD3+ cells from different donors were purified, counted, and added to macrophages differentiated by CSF1 or FLDK1. Cytostim (Miltenyi) was also added to the co-culture to enhance the interaction between the two cell types. The supernatant was harvested and cells were counted at 48 hours after 5 days of co-culture. (B) Data are presented as a summary of 4–6 different experiments, expressed as mean + SEM.

[0127] Figure 24. FLDK-1 showed selectivity for myeloid cells compared to lymphocytes. (A) PBMCs from healthy donors were seeded in 96-well plates and stimulated with rIL10 or FLDK1 at 37°C for 20 min the next day. Cells were stained with CD14 Alexa647 and CD3APC / Cy7, fixed with 4% PFA, and permeabilized with 80% MetOH for intracellular staining. Cells were stained with AlexaFluor488pSTAT3 (pY705). (BC) Samples were analyzed using LSRII flow cytometry, and MFI values ​​obtained from STAT3 activation were plotted using GraphPad Prism software. A representative result is shown with two technical replicates, expressed as mean, with error bars depicting standard errors. Each biological replicate was normalized by specifying the highest MFI value of the highest concentration of IL10 as 100% and the lowest MFI value of the untreated control as 0%. The MFI of samples treated with FLDK1 was normalized accordingly.

[0128] Figure 25. FLDK-1 and CSF1-IL10 were less effective than IL-10 in activating CD8 T cells. (A) CD8 T cells were isolated from human PBMCs using positive microbead selection (Miltenyi). Lymphocytes were stimulated with anti-CD3 and anti-CD28 for 72 hours. The cells were then divided into two aliquots and treated with (B) IL10 or FLDK1 or (C) IL10 or CSF1-IL10 plus IL-2 for 48 hours. Cell supernatants were obtained and Gzmb and IFNγ were detected by ELISA. The figure shows the pooled normalized data from four independent experiments. t-test: IL-10 vs FLDK1 p < 0.05.

[0129] Figure 26. FLDK-1 and CSF1-IL10 were less effective than IL-10 in activating B cells. (A) B cells were isolated from human PBMCs using positive microbeads. Isolated lymphocytes were stimulated on days 0 and 2 with CpG (InvivoGen) and (B) IL10 or FLDK1, or (C) CSF1-IL10 or IL10. IgG levels in the supernatant were measured by ELISA after 7 days. The figure shows the pooled data from four independent experiments. t-test: IL-10 vs FLDK1 p < 0.05.

[0130] Figure 27. FLDK1 protects mice from chronic TNBS-induced colitis. The therapeutic efficacy of FLDK1 compared to IL-10 was evaluated in a TNBS-induced chronic IBD experimental model (three doses: 1 µg, 3 µg, or 10 µg per mouse). Mice treated with FLDK1 experienced less weight loss after 30 days of treatment compared to mice treated with IL10 or the mediator.

[0131] Figure 28 Structure-activity relationship. (A) PBMCs from healthy donors were plated in 96-well plates and stimulated the next day with rIL10, FLDK1, or FLDK1-E82A. (B) Samples were analyzed using LSRII flow cytometry, and MFI values ​​obtained from STAT3 activation were plotted using GraphPad Prism software.

[0132] Figure 29 Structure-activity relationship. (A) PBMCs from healthy donors were seeded in 96-well plates and stimulated the next day with rIL10, FLDK1, or saturated concentrations of CSF1 + FLDK1. (B) Samples were analyzed using LSRII flow cytometry, and MFI values ​​obtained from STAT3 activation were plotted using GraphPadPrism software.

[0133] Example

[0134] Example 1: Generating CSF1-IL10 co-biological activity in single-chain proteins

[0135] Our goal was to create a bifunctional molecule that could activate IL-10 signaling only in cells expressing CSF1R (primarily myeloid cells, rather than other cell types such as CD8+ T cells). To achieve this, we engineered IL-10 variants with reduced affinity for IL10RA and IL10RB and / or Foldikine-10 variants and linked them to a CSF1 monomer. The CSF1 component served as a tool for targeting circulating monocytes, promoting preferential binding of IL-10 to these cells (see [link to documentation]). Figure 1 ).

[0136] Engineered Foldikine10 chassis

[0137] The proposed product Folkidine TM -1 relates to variants of IL-10 and CSF1 connected at specific distances and orientations. To engineer the IL-10 variant, we designed an Nter-Cter closed joint (internal joint) within the exchange structure domain of IL-10, ultimately forming a single chain (Foldikine). TM support Figure 2 (Foldikine10, based on the so-called MutSC1 construct in 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 molecules ORK10-002, ORK10-003, ORK10-005).

[0138] Foldikine10 (SEQ ID NO: 1) contains amino acids 5 through 157 of IL-10 (SEQ ID NO: 11), which has G5M, D84E, and K157N substitutions (SEQ ID NO: 28), an Nt-Ct linker peptide composed of NGGLDY (SEQ ID NO: 30), and amino acids 19 through 160 of IL-10 (i.e., SEQ ID NO: 29). SEQ ID NO: 20 (foldikine10-HiBit) is identical to SEQ ID NO: 1 except that a HiBit tag for protein quantification is added to the C-terminus.

[0139] The difference between ORK10-002 (SEQ ID NO: 2) and Foldikine10 (SEQ ID NO: 1) lies in the N154F substitution and the E80D reverse substitution (i.e., ORK10-002 contains the Asp(D) residue present at position 84 of IL-10 as shown in sequence SEQ ID NO: 11). ORK10-002 contains amino acids from positions 5 to 152 of IL-10, has a G5M substitution (SEQ ID NO: 39), an Nt-Ct linker peptide composed of FGGLDY (SEQ ID NO: 46), and amino acids from positions 19 to 156 of IL-10 (the sequence of SEQ ID NO: 40).

[0140] ORK10-003 and ORK10-003-HiBit (SEQ ID NO: 3 and SEQ ID NO: 49) and ORK10-005 (SEQ ID NO: 4) also contain E80D reverse substitution compared to Foldikine10 (SEQ ID NO: 1), and are further distinguished by the Nt-Ct linkage of the IL-10 monomer and the engineering of surrounding residues.

[0141] ORK10-003 (SEQ ID NO: 3) contains amino acids 5 through 154 of IL-10 (SEQ ID NO: 11), with a G5M substitution (SEQ ID NO: 39) and an M154 mutation to L (SEQ ID NO: 41), an Nt-Ct linker peptide composed of YKTIT (SEQ ID NO: 31), and amino acids 17 through 156 of IL-10 (SEQ ID NO: 42), wherein the residue GNLP is mutated to PEFA. SEQ ID NO: 49 (ORK10-003-HiBit) is identical to SEQ ID NO: 3 except that a HiBit tag for protein quantification is added to the C-terminus.

[0142] ORK10-005 (SEQ ID NO: 4) contains amino acids 5 to 153 of IL-10 (SEQ ID NO: 43 sequence), an Nt-Ct linker peptide composed of DKDIRDGD (SEQ ID NO: 32), and amino acids 18 to 156 of IL-10 (SEQ ID NO: 44 sequence).

[0143] EC-50 comparisons of Foldikine10 (SEQ ID NO: 1), ORK10-002 (SEQ ID NO: 2), ORK10-003 (SEQ ID NO: 3), and ORK10-005 (SEQ ID NO: 4) showed that, compared to Foldikine-10, molecule ORK10-003 had an improved EC-50. Figure 3 (Table 1).

[0144] Table 1. EC-50 values ​​and associated errors of different IL-10 foldikine variants

[0145]

[0146] For all variants, the baseline was fixed at 0.27 and the Hill slope was fixed at -1.6, except for ORK10-005, which had a significantly different slope.

[0147] Circular arrangement of Foldikine 10

[0148] When cytokine molecules bind to IL10RA and IL10RB receptors, the natural Nt and Ct of Foldikine10 and different ORK-10s point towards the cell membrane. Figure 4When fused with other cytokines such as CSF1, this can lead to a masking effect due to the steric hindrance of the fusion molecule to the cell membrane or receptor. To mitigate this effect, we circularized Foldikine10 by exploring structural openings connecting all the flexible regions of the α-helix (thus generating non-natural Nt and Ct), while simultaneously linking the free Nt and Ct to the same linkers used for engineering 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 different versions and quantified them by ELISA. Only Foldikine10_cut116 provided sufficient protein for testing in HEK reporter cells. The molecule (Foldikine10_cut116) is active (cp116), but its EC-50 is reduced (in HEK reporter cell IL-10, the EC50 of Foldikine10 is 6.04E-10, while that of Foldikine10_cut116 is 1.67E-09). This molecule involves opening at position 116 in the junction region of two 3D IL-10 domains (see [link to IL-10]). Figure 4 Cleavage at this site should allow for attachment to other molecules without causing a masking effect (see below), because the new Nt and Ct point in the opposite direction to the membrane and away from the receptor (see below). Figure 3 ).

[0149] CSF1 structure and CSF1 ring arrangement

[0150] CSF1 naturally dimers in two monomer molecules facing each other and linked together by disulfide bonds (see [link]). Figure 4 ).

[0151] A circular arrangement of CSF1 was generated, in which the native Nt-Ct are linked by an engineered linker, and the new Nt and Ct are linked by a region of protein interaction with the receptor that does not disrupt the protein's interaction (between residues 95 and 100). Figure 6 The peptide bonds are broken to form two new molecules (ORKMCSF_013 (SEQ ID NO: 9) and ORKMCSF_014 (SEQ ID NO: 10)). Figure 7 All of these can activate the downstream CSF1 signal cascade ( Figure 8 They have similar EC-50 values ​​and are slightly better than the monomer CSF1 molecule (Table 2).

[0152] Table 2. EC-50 values ​​and associated errors for different CSF1 variants

[0153]

[0154] Engineered IL-10 affinity

[0155] We engineered a group of mutants to reduce IL10RA affinity based on the ORK10-003-HiBit foldikine10 active scaffold (SEQ ID NO: 49). We explored 11 mutants using FoldX and sequence conservation analysis, which identified a series of mutations that did not impair protein expression but reduced EC-50 (R24A, E50A, D44N, K34A, Q38A, D44A, numbered based on SEQ ID NO: 11b IL10b, see Methods). In ORK10-003-HiBit, R24 is R163, K34 is 173, Q38 is 177, D44 is 183, and E50 is 189. These mutations are point mutations integrated into a continuous domain of Foldikine10. These mutants exhibited varying degrees of HEKblue cell reporter activation and EC-50 (see Methods). Figure 8 (Table 3). Mutations weakening IL10RB interactions were also identified (N92E; numbered based on SEQ ID NO: 11 IL10, N92 is 231 in ORK10-003-HiBit) (see Table 3). Figure 9 (Table 3).

[0156] Table 3. EC-50 values ​​and their related errors

[0157]

[0158] In the fitting, we fixed the baseline at -0.27, the Hill slope for the first 7 rows at -1.6, and the baseline for the last two rows at 0.12, with the Hill slope at -1.9.

[0159] mutants that increase CSF1 and CSF1 receptor EC-50

[0160] For CSF1, we engineered a set of single- or double-point mutations to increase affinity for CSF1R (Table 4). Based on FoldX-predicted changes in the complex interaction energy with the receptor and the stability of the mutated CSF1, and through visual examination of the complex interface, we selected a set of conserved and non-conserved residues (bold indicates conserved residues (allowing one mismatch) between CSF1 sequences in mice, hamsters, humans, pigs, cattle, sheep, alpacas, camels, foxes, dogs, mink, walruses, otters, polar bears, cats, cougars, dolphins, bats, raccoons, blue whales, and horses; asterisks indicate...). Residues mutated to improve binding are indicated; residues marked with an "x" are mutated to increase CSF1 stability.

[0161]

[0162] 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 prediction).

[0163]

[0164] To increase affinity, we selected the mutations H9F, Q17R, S18Q, V78W, Q79L, Q81F, Q81Y, and E82K. In the second round of screening based on sequence conservation, we selected V78F, S18F-R21Q, Q17V / L, S18F, V78F / L / Y, Q79T / K, K93E, T93E, T34L, and M10L.

[0165] exist Figure 10 In the figure, we present titration curves and EC-50 values ​​and their associated errors for different CSF1 mutants. These variants were evaluated in HEK293 cells transfected with the CSF1R / Reporter Kit (see Methods). Among all mutants, two (Q17R and V78W) had similar EC-50 values ​​to CSF1 WT, and performed better in one of the two biological replicates.

[0166] Table 5. EC-50 values ​​and their related errors

[0167]

[0168] The hill slope is fixed at -1.7, and the baseline is fixed at 0.3.

[0169]

[0170] The hill slope is fixed at -1.7, and the baseline is fixed at 0.45.

[0171] Meanwhile, as described above, we designed different variants to increase the conformational stability of CSF1 in the CSF1R-bound state. These variants were evaluated in HEK293 cells transfected with the CSF1R / reporter kit (see Methods) (see Table 6). Mutants were selected to involve residues that do not directly interact with the CSFR1 receptor, as well as residues partially or completely embedded in the structure. In the mutants, we observed a slight increase in binding affinity of mutant V117L in one biological replicate, while the T124I value was similar to WT in both biological replicates. More interestingly, we found an increase in maximum activation values ​​in HEK293 cells transfected with the CSF1R / reporter kit in both replicates, but this was statistically significant only in the second replicate.

[0172] Table 6. This table shows the EC50 and maximum response values ​​measured in HEK cells transformed with the CSFR1 reporter kit.

[0173]

[0174] The hill slope is fixed at -1.2, and the baseline is fixed at 0.33.

[0175]

[0176] The hill slope is fixed at -1.33, and the baseline is fixed at 0.12.

[0177] Based on this analysis, we propose mutant combinations that, within the margin of error, can improve the binding affinity (Q78R, V78W, V120I) and / or signal range (V117L, V120I, T124I) of CSF1 to its receptor:

[0178] i) Q17R, T124I and V120I

[0179] ii) V78W, T124I and V120I

[0180] iii) Q17R, V78W, T124I and V120I

[0181] iv) V117L, T124I.

[0182] Example 2: Generation of bifunctional CSF1-IL10 molecules

[0183] Determination of the optimal orientation of CSF1-IL10 products

[0184] Engineering of Foldikine-10 with CSF1 or Foldikine-CSF1

[0185] To assess whether fusing Foldikine-10 with CSF1 would maintain the activity of both cytokines, we first tested IL10 activity using HEKBlue 10 reporter cells and CSF1 activity using HEK293 cells transfected with the CSF1R reporter kit. There are two possibilities for preparing the fusion protein: either linking the Ct of IL-10 or Foldikine10 to the Nt of CSF1, or fusing the Nt of IL-10 or Foldikine10 to the Ct of CSF1 (this also applies to the circular arrangement of Foldikine10 and CSF1) (see Figure 12). Because IL-10 is an interchangeable dimer and CSF1 dimers (see Figure 12), fusing IL-10 with CSF1 can lead to multimer formation. Other fusions do not produce this result. Due to CMC considerations, we decided to continue using foldikine-10 instead of ORK10-003 for protein fusion.

[0186] Orientation A – IL10-CSF1

[0187] In this orientation, we fused the Ct of IL-10 or Foldikine10 with the Nt of CSF1 to form IL-10-CSF1 (SEQ ID NO:52) or Foldikine10-CSF1 (SEQ ID NO:53). At the functional level, the EC50 of IL-10 WT was affected upon fusion with CSF1. The EC50 of Foldikine-10 was also affected, but to a lesser extent than that of IL-10 WT. Regarding the function of CSF1, its activity was lost upon fusion with the C-terminus of IL-10, while the loss was less significant upon fusion with Foldikine-10 (see [link to relevant documentation]). Figure 13 (and Tables 7 and 8).

[0188] Orientation B – CSF1-IL10

[0189] In this orientation, we fused the Ct of CSF1 with the Nt of either IL-10 or Foldikine10 to form CSF1-IL-10 (SEQ ID NO: 54) or CSF1-Foldikine10 (SEQ ID NO: 55). Upon fusion with CSF1, the WT of IL-10 and the EC50 of Foldikine10 were both affected. Regarding the function of CSF1, its activity was lost upon fusion with IL-10, but not upon fusion with Foldikine10 as in orientation A (see [link to CSF1-Foldikine10]). Figure 14 (and Tables 7 and 8).

[0190] Table 7. IL-10 EC-50 values ​​and the errors of these EC-50 values

[0191]

[0192] The baseline was fixed at 0.08, and the hill slope was fixed at -1.38.

[0193] The concentration of IL10 in the active dimer form, estimated by Hibit, is twice that of Foldikine10, therefore we divide EC50 by 2.

[0194] Table 8. CSF1 EC-50 values ​​and the errors of these EC-50 values

[0195]

[0196] The baseline was fixed at 0.3, and the hill slope was fixed at -1.7.

[0197] Testing the activity of CSF1 fused with the cyclic arrangement of Foldikine10

[0198] As described above, fusing Foldikine10 to CSF1 at its N-terminus or C-terminus increases its EC-50. This could be due to the steric hindrance effect that may exist because the native Nt and Ct of Foldikine10 point towards the cell membrane in the complex with receptors IL10RA and IL10RB. To address this issue, we designed different circular arrangements of Foldikine10 and found one that expressed at WT levels as described above and had an EC-50 approximately three-fold lower (Foldikine10_cut116 (SEQ ID NO: 5)) (see [link to documentation]). Figure 4 In this ring-shaped arrangement, Nt and Ct are not pointed towards the membrane and are far from the receptor, so we expect no steric hindrance when fusing with CSF1. To verify this, we fused Foldikine10_cut116 with CSF1 in two orientations (N-terminus and C-terminus: Foldikine10_cut116-CSF1-HiBit (SEQ ID NO: 56); CSF1-Foldikine10_cut116-HiBit (SEQ ID NO: 57)). Compared with the CSF1-Foldikine10 scaffold, the fusion of the ring-shaped arrangement produced similar CSF1 EC-50 values ​​in both orientations (see [link to documentation]). Figure 15-16We observed that IL10 with standalone cyclic substitution of Foldikine (Foldikine_cut116) or IL10 combined with CSF1 had similar EC-50 values, which contrasts with the behavior of Foldikine10 that loses its activity when fused to CSF1, supporting the hypothesis of steric hindrance effect mentioned above (see Table 9).

[0199] Table 9. EC-50 values ​​and related errors of CSF1 and IL10 activities

[0200]

[0201] Effect of linker length on protein function

[0202] We tested orientation B (CSF-linker-IL10) to determine whether shorter or longer linkers could interfere with protein function. While CSF1-Foldikine10 showed IL-10 signaling in the linker length range of 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 inactive. CSF1-IL-10 with linker lengths of 25 to 35 amino acids exhibited 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)). Regarding CSF1 activity, the two mutants (CSF1-15AA-IL10-HiBit (SEQ ID NO: 61) and CSF1-25AA-IL10-HiBit (SEQ ID NO: 62)) showed lower protein expression than the dynamic range of CSF1R HEK reporter cells. The fusion protein CSF1-Foldikine10 supports different linker lengths, and IL-10 either loses activity or expression in the presence of short linkers. See also Figure 17-18 And Tables 10 and 11.

[0203] Table 10. EC-50 values ​​and related errors of IL10 activity fused with connectors of different lengths.

[0204]

[0205] The hill slope is fixed at -1.2, and the baseline is fixed at 0.40. The concentration of IL10 in the active dimer form estimated by Hibit is twice that of Foldikine10, so we divide EC50 by 2.

[0206] Table 11. EC-50 values ​​and related errors of CSF1 activity fused with joints of different lengths.

[0207]

[0208] The hill slope is fixed at -1.06, and the baseline is fixed at 1.2.

[0209] Example 2: Method

[0210] Generating cyclically arranged molecules using FoldX-ModelX software

[0211] The cyclic arrangement is generated in two steps: first, an endless cyclic model is created by linking native Nt-Ct bonds; second, peptide bonds are broken in the flexible regions of the model. The linkers between Nt-Ct bonds are engineered using ModelX's Bridging feature. FoldX's AlaScan helps select cleaved residues and predict those less important for protein stability.

[0212] Single-chain (SC) molecules were generated using ModelX software.

[0213] The SC IL-10 variant was generated by reconnecting X-ray IL-10 dimer structures (PDB: 1y6k, 2ilk). The novel connectivity was designed using the ModelX toolkit. The ModelX Bridging command (crosslinking mode) was used, which linked a pair of residues selected as anchor points with all geometrically compatible peptide fragments from a custom protein fragment library (PepXDB). 120k structures from PepXDB were extracted from the PDB and digested into peptides of varying lengths (3–45 aa), including the geometric descriptors required by the Bridging algorithm. The Bridging command allows the user to select different peptide lengths; the output is a set of bridging models with different conformations, sequences, and lengths, where connectors / connections with forbidden phi and psi dihedral angles in the Ramachandran diagram are discarded. Once the bridging mode was created, an extensive connector screening was performed using the Bridging algorithm, exhaustively combining anchor points in overlapping sliding windows near the flanks of the region to be connected. Each window queried a different peptide length (6–20 aa). The linkers on the bridging model contain the same side-chain rotomers as those in the PDB structures they were digested; therefore, the RepairPDB command of FoldX (Montero-Blay et al., 2023, Molecular Systems Biology, 19:e11037) was used to adapt to the new SC-IL10 environment. RepairPDB identifies residues with poor torsion angles, van der Waals collisions, or total energies and mutates them and their neighbors to themselves, exploring different combinations of rotomers to find new energy minimums. The resulting model is sorted by global energy (FoldX Stability command). When the peptide length exceeds the numerical position between anchor points, Bridging renumbers the redundant residues with res codes that FoldX cannot recognize in subsequent modeling steps. Therefore, SC-IL10 design requires a numerical rearrangement of monomeric protein residues in the dimer to allow for 18 aa-long numerical gaps between regions to be linked. This is crucial for the extreme case of anchoring residues on the flanking sides of numerical gaps. To cover this situation, during renumbering, gaps of 18 numerical positions are created around the region to be connected. This is sufficient to accept fragments of 6–20 residues in length without using residue codes. The difference between 18 and 20 is that the algorithm requires anchor points to locate the bridge, but these are replaced by the terminal residues of the found peptide. Bridging also replaces all edge residues between anchor points with residues from the peptide. For clarity, the template generated before using Bridging is called the "stitching pattern." Because the numerical gaps are not spatial gaps, Bridging can also accept smaller peptide fragments, resulting in discontinuous numbering.

[0214] Connector generation for Foldikine10 variant and CSF1

[0215] To generate the linker between the Foldikine10 variant and CSF1, we first observed the 3D structures of the complexes of IL-10 with IL-10 RA and RB (PDB 6x93; 3.5 Å) and the 3D structure of the complex of CSF1 with its receptor (PDB 4wrm; 6.5 Å), placing the receptors in the orientation we expected them to have on the membrane, and determining the distance between the native Nt or CT of IL10 and the corresponding Ct or NT of CSF1. Using this distance as a reference, we estimated the need for a random linker of approximately 30 amino acids, consisting of repeating GGSGG (SEQ ID NO: 27) units, between CSF1 Cys146 and IL10 Cys12 aa.

[0216] Affinity mutants of IL-10 and CSF1 were generated using FoldX software.

[0217] A) IL-10. To generate an IL-10 version with reduced affinity for R1, we used hIL-10 molecules crystallized with the IL-10RA complex, which had the best crystal resolution (PDB 1y6k; 2.5Å). We then used the BuildModel FoldX command to perform a computer-simulated mutation scan of the complex interface residues, which sequentially mutates all residues of IL-10 to 19 other natural amino acids (Schymkowitz et al., 2005), and used the AnalyseComplex FoldX command to estimate the change in interaction energy for each mutation. The selection of mutants was based on the FoldX-predicted interaction energy (ΔΔG) and stability of the complex, as well as visual inspection of the complex interface. From this, we selected R24A, Q34A, M39A, D44N, D44A, L46A, E50A, E50Y, E151R, and D144Q.

[0218] B) CSF1. To generate a CSF1 mutant with increased affinity for its receptor, we used CSF1 crystallized with CSF1 (PDB 4wrl; 2.8 Å). A computer-simulated mutation scan of the complex interface residues was then performed using the BuildModel FoldX command, which mutates all CSF1 residues one by one to 19 other native amino acids (Schymkowitz et al., 2005, PNAS, 102(29), 10147-10152), and the change in interaction energy for each mutation was estimated using the AnalyseComplex FoldX command. Based on the complex interaction energies (ΔΔG) and stability predicted by FoldX, and visual inspection of the complex interface, we selected a set of conserved and non-conserved residues. From these, we selected H9F, Q17R, S18Q, V78W, Q79L, Q81F, Q81Y, and E82K in the first round of mutations, and V78F, S18F-R21Q, Q17V / L, S18F, V78F / L / Y, Q79T / K, K93E, T93E, T34L, and M10L in the second round of mutations.

[0219] Stable mutants of CSF1 were generated using FoldX software.

[0220] We used CSF1 crystallized with CSF1 (PDB 4wrl; 2.8 Å) to generate a mutant of CSF1 with increased stability against CSF1R in the bound state. We then used the BuildModel FoldX command to perform a computer-simulated mutation scan of the complex interface residues, which sequentially mutated all CSF1 residues to 19 other native amino acids (Schymkowitz et al., 2005, PNAS, 102(29), 10147-10152). Based on FoldX-predicted complex stability and visual inspection, we selected A76I, V117I, V117L, V120I, and T124I.

[0221] Protein quantification

[0222] Because we generate various point mutations to improve or reduce affinity for the receptor, and break loops or create new linkers in some constructs, there is a risk that the resulting molecules may not be recognized by antibodies used in commercial ELISA kits. To avoid this problem, we use the HIBIT method to quantify all of our constructs (https: / / www.promega.es / en / resources / technologies / hibit-protein-tagging-system / ). Essentially, this involves fusing peptide tags separated by random linkers to Nt or CT. The Nano-Glo® HiBiT extracellular detection system is a method for detecting HiBiT-tagged proteins secreted into the extracellular medium. Detection is accomplished by adding a non-cleavage assay reagent containing substrate and large BiT (LgBiT). HiBiT binds tightly to LgBiT, promoting complex formation and producing a bright luminescent enzyme. The amount of luminescence produced is proportional to the amount of HiBiT-tagged protein present, and the system can detect protein amounts spanning seven orders of magnitude. The detection process is simple, requiring only an add-mix-read assay protocol. The expression of any protein can be quantified extremely easily using a peptide reference with a known concentration.

[0223] plasmid construction

[0224] All plasmids generated in this work were assembled according to the Gibson method (Gibson DG et al., Nat Methods. May 2009; 6(5):343-5). Gene synthesis (gBlock double-stranded fragments) and oligonucleotide synthesis were performed by Integrated DNA Technologies (IDT) when necessary. Gene amplification was performed using Phusion DNA polymerase (Thermo Fisher Scientific) and transformed into *E. coli* (…). Escherichia coli In DH5-alpha competent cells (NEB).

[0225] The vector used for mammalian protein expression in ExpiCHO cells was pcDNA3.1 (V790-20, Invitrogen). All plasmids were validated by Sanger sequencing (Eurofins Genomics).

[0226] Expression in CHO cells

[0227] Cells for the ExpiCHO protein expression kit were purchased from Thermofisher (A29133). For small-scale production (2.5 ml), 24-well deep-well plates (AXYPDW10ML24CS, Merck) were used and covered with a breathable membrane (ThermoFisher). On day -1 of production, ExpiCHO cells were aliquoted to a final density of 3 x 10⁻⁶ cells / well. 6 Viable cells / ml. Cultured at 37°C, 8% CO2, and 110 rpm with shaking. On day 0 of production, ExpiCHO cells were aliquoted to a final density of 3 x 10-1 per sample. 6 1 live cells / ml, aliquot 2.5 mL of cell suspension into one well of the plate. For each sample, add 2 μg DNA to 200 μL of cold OptiPRO SFM and 9.0 μL of ExpiFectamine CHO reagent. Then, add 200 μL of the complex mixture to each relevant well. Cover the entire plate with a breathable sealing membrane and incubate at 37°C, 8% CO2, and 225 rpm. On Day 1, 18–22 hours post-transfection, add ExpiFectamine CHO enhancer and ExpiCHO feed to each well. For the entire 24-well plate, mix 400 μL of ExpiFectamine CHO enhancer and 16 mL of ExpiCHO feed in a conical tube. From this mixture, add 600 μL to each well of the plate. Incubate at 37°C, 8% CO2, and 225 rpm. Harvest samples after 4 days. Centrifuge the plate at 4600 rpm for 5 minutes at room temperature. The supernatant was dispensed, flash-frozen in liquid nitrogen, and stored at -80°C.

[0228] Cell culture

[0229] HEK-Blue carrying the SEAP report subconstruct TM Cell lines were purchased from InvivoGen (InvivoGen, San Diego, CA, USA). In this study, we used human IL-10 reporter cells (hkb-il10). HEK293 cells were purchased directly from InvivoGen. HEK-Blue TM Both cell lines and HEK293 cells were cultured in DMEM (Lonza, BE12-604F) supplemented with 10% FBS, 2 mM L-glutamine, and the manufacturer-specified antibiotic for each reporter cell line. Cells were passaged at 70% confluence, following the manufacturer's recommendations.

[0230] We cultured THP-1 (ATCC, batch: 70047949) cells in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2-mercaptoethanol to a final concentration of 0.05 mM, and penicillin-streptomycin (Gibco / 15140122) and incubated at 37°C in a CO2 incubator. We maintained cell concentrations between 0.2 and 1e6 cells / ml.

[0231] HEK-Blue TM Colorimetric analysis of Foldikine-10 activity in cells

[0232] After quantification of the supernatant, samples were adjusted to the dynamic range of HEK reporter cells using different dilutions in DMEM medium. A maximum concentration was set for each reporter cell type to reach saturation, and each sample was serially diluted 8 times (0.5-fold each time). HEK reporter cells were then prepared according to the manufacturer's instructions. Briefly, 180 µL of cells at a concentration of 280,000 cells / ml was seeded per well in a 96-well plate (Nunc Microwell, ThermoFisher Scientific, #167008). From there, 20 µL of each adjusted supernatant was added, and the cells were incubated at 37°C and 5% CO2 for 20–24 hours (induced HEK-Blue). TM Subsequently, 180 µL of QUANTI-Blue solution (alkaline phosphatase assay medium, #repqbs, InvivoGen) was mixed with 20 µL of induced HEK-Blue in a new 96-well plate. TM Cells were mixed. Subsequently, the cells were incubated at 37°C for 60 minutes, and the absorbance (630 nm) was measured using a Tecan i-control 2.0.10.0 spectrophotometer.

[0233] HEK293 CSF1 reporter cells

[0234] HEK-Blue TM Colorimetric analysis of CSF1 activity in cells. The CSF1R / SER reporter kit (MAPK / ERK signaling pathway, #79379) was purchased from BPS Bioscience (BPS Bioscience Inc., San Diego, CA, USA). HEK293T cells (3 × 10⁶ cells per well) were transfected one day prior to DNA transfection. 4Cells were seeded in 96-well multi-well plates (Nunc Microwell, ThermoFisherScientific, #167008), with 100 μL of DMEM (Lonza, BE12-604F) supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin / streptomycin per well, and incubated at 37°C and 5% CO2 for 20–24 hours. The next day, 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) were prepared. Simultaneously, 30 μL of Lipofectamine 2000 (Invitrogen, #11668019) was diluted in 1500 μL of Opti-MEM (GIBCO, #31985-070). After 5 minutes of incubation, the diluted DNA was combined with diluted Lipofectamine 2000, gently mixed, and incubated at room temperature for 25 minutes to form a complex. Then, 30 μL of the complex was added to each well containing cells. Cells were incubated at 37°C in a 5% CO2 incubator. Approximately 5 to 6 hours after transfection, the medium was replaced with growth medium containing 0.5% serum. Cells were incubated overnight at 37°C in a 5% CO2 incubator. The following day, the SRE reporter was induced by activating the CSFR1R signaling cascade with our CSF1-IL10 fusion variant. After 6 hours of treatment, the medium was removed, and 50 μL of diluted passive lysis buffer 5X (Promega, #E1941) was added. 100× firefly luciferase reagent substrate (component B) was diluted in firefly luciferase reagent buffer (component A). 20 μL of this mixture was added to 20 μL of the lysate placed in a white 96-well multi-well plate (PerkinElmer, #6005290), and the plate was incubated at room temperature for 15 minutes. Firefly bioluminescence was then measured using a luminometer. Subsequently, 20 μL of diluted 100× Renida luciferase reagent substrate (component D) was added to the Renida luciferase reagent buffer (component C) and then to the wells, and bioluminescence was immediately measured using a luminometer. To obtain the normalized luciferase activity of the SRE reporter, we calculated the ratio of firefly bioluminescence to Renida bioluminescence and deduced the dose-response linear fit (Hill slope), as shown below.

[0235] EC-50 data inference

[0236] To infer EC-50, we performed dose-response analysis by tracking absorbance (HEKBlue10) or fluorescence (HEK293-CSF1 reporter cells).

[0237] The following equation was used to fit the variation in absorbance at 630 nm caused by different IL-10 test concentrations to the saturated binding model:

[0238] Y = baseline + (maximum value of B - baseline) / (1 + (X / EC50)^-Hill slope)

[0239] 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 the maximum value of B is the saturation signal. This equation assumes only specific binding; all non-specific signals are subtracted. For the EC-50 calculated in Tables 3 and 6, the equation used is log(agonist) vs. response-variable slope (Y = bottom + (top - bottom) / (1 + 10^(LogEC50 - X))) in Prism 9 software. Hill slope).

[0240] For CSF1 analysis, we measured the changes in the firefly / kidney luminescence ratio.

[0241] Different experiments were analyzed independently and fitted using GraphPad Prism 9 software. For the IL-10 and Foldikine10 variants, in experiments where the baseline or terminal line was not defined for some mutants, we used the mean of mutants with lower EC-50 (terminal value at saturation) or higher EC-50 (initial value without ligand). In the case of CSF1, since we could not determine whether the transformation efficiency of the CSFR1 kit was the same in all cases, we fixed the initial values ​​as indicated above and fixed the Hill slope to the mean of mutants with lower EC-50. The fitting method is indicated in each graph.

[0242] Testing the activity of CSF1-Foldikine in THP-1 cells

[0243] Thrp1 cells were prepared to a viable cell concentration of 1e6 / ml. 100 μl cells were seeded per well in 96-well U-plates and incubated for 15 min. Simultaneously, a supernatant at the target dilution (60 nM) was prepared in preheated 96-well plates using RPMI medium. Next, 20 μl of the dilution was added to 100 μl of cells and incubated for 20 min. Cells were then immediately fixed to maintain phosphorylation by adding an equal volume of preheated BD Cytofix buffer to the cell suspension, resulting in a total volume of 240 μL. Cells were incubated at 37°C for 10 min and then centrifuged. Subsequently, cells were separated by adding 100 μl of BD™ Phosflow Perm Buffer III (for 1×10⁻⁶ cells / ml). 6Cells were permeabilized by incubating on ice (protected from light) for 30 minutes. Note: Prolonged incubation in this permeabilization buffer can reduce the signal intensity of 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 fluorescent dye-conjugated antibody Alexa Fluor 488 pSTAT3 Y407 was added to each well of a U-bottom 96-well plate. The plate was incubated in the dark at room temperature for 60 minutes. Finally, cells were washed and resuspended in PBS for flow cytometry analysis.

[0244] Example 3: In vitro functional evaluation of bifunctional CSF1-IL10 cytokines

[0245] For the experiments in Examples 3 and 4, FLDK1 (SEQ ID NO:72, i.e. SEQ ID NO:16 or 55 with the HisHiBit tag) and / or CSF1-IL-10 (SEQ ID NO:74) were used as exemplary bifunctional CSF1-IL10 cytokines.

[0246] PBMCs (150,000 cells per 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 CSF1-IL-10 (Fig. 20 A). TNFα levels measured in the presence of rIL-10 or FLDK-1 showed that FLDK-1, similar to IL-10, inhibited monocyte activation (dashed lines indicate LPS levels alone) (Fig. 20 B). CSF1-IL-10 was also found to inhibit monocyte activation (Fig. 20 C).

[0247] Similar results were obtained when using IL-6 and when using isolated monocytes (CD14+ cells) (data not shown).

[0248] In addition, PBMCs were isolated from the brown-yellow layer of human erythrocyte sedimentation rate (ESR), and CD14+ cells were purified by positive microbead selection (Miltenyi). Monocytes (500,000 / 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. Viable cells were counted on day 7 and characterized by FACS (CD163, CD206, MHCII, CD14, CD86) (Figure 21A). Results showed that, similar to CSF1, FLDK1 induced macrophage differentiation from monocytes, as identified by CD163 expression (Figure 21B).

[0249] Further characterization was performed to determine the phenotype of macrophages derived from induced monocytes.

[0250] PBMCs were isolated from the brown-yellow layer of human erythrocyte sedimentation rate (ESR) and CD14+ cells were purified by positive selection (Miltenyi). Monocytes (500,000 / ml) were cultured for 7 days 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 in the presence or absence of IFNγ. Figure 22 A). Seventeen hours after stimulation, TNF-α levels in the supernatant were measured by ELISA. CSF-1 differentiated macrophages produced TNF-α, which was further enhanced upon the addition of IFNγ. Conversely, FLDK1 differentiated macrophages did not produce TNF-α even upon stimulation with LPS+IFNγ. These results suggest that FLDK1-induced macrophages exhibit a regulatory phenotype ( Figure 22 B).

[0251] In addition, monocytes were isolated from human erythrocyte sedimentation rate (ESR) amber layer samples by selecting positive CD14 cells (Miltenyi). Monocytes (500,000 / ml) were cultured for 7 days with or without rCSF1 or FLDK1 (10 ng / ml per treatment). On day 7, cells were detached from the plate and counted, and the same number of macrophages (40,000 cells) were replated and stimulated with LPS (Sigma) (10 ng / ml) for 4 hours. CD3+ cells were purified from different donors, counted (120,000 cells), and added to macrophages differentiated from CSF1 or FLDK1. Cytostim (Miltenyi) (2 µl per million cells) was also added to the co-culture to enhance the interaction between the two cell types. Cytokines in the supernatant were analyzed by ELISA at 48 hours of co-culture, and T cell proliferation was analyzed after 5 days of co-culture. Figure 23 A). FLDK1-differentiated macrophages inhibited T cell proliferation and TNF-α and IFNγ production in these co-cultures, demonstrating their regulatory phenotype ( Figure 23 B).

[0252] PBMCs (150,000 cells per well) from healthy donors were seeded in 96-well plates and stimulated for 20 min at 37°C the next day with rIL10 or FLDK1 (10 nM per treatment, 8 times 1 / 3 dilution). Cells were stained with CD14 Alexa647 (Biolegend) and CD3 APC / Cy7 (Biolegend), fixed with 4% PFA, and permeabilized with 80% MetOH for intracellular staining. Cells were stained with AlexaFluor488 pSTAT3 (pY705) (BD) for 1 h (Fig. 24A). Samples were analyzed by LSRII flow cytometry, and MFI values ​​obtained from STAT3 activation were plotted using GraphPad Prism software. A representative result is shown with two technical replicates, expressed as mean, with error bars depicting standard errors (Fig. 24 BC). Similar results were obtained from 10 different donors. Each biological replicate was normalized by designating the highest MFI value of IL-10 at the highest concentration as 100% and the lowest MFI value of the untreated control as 0%. The MFI of samples treated with FLDK1 was normalized accordingly. These results indicate that monocytes and T cells respond to IL-10 in a similar manner. Conversely, the bifunctional CSF1-IL10 cytokine FLDK-1 preferentially activates monocytes compared to T cells (Fig. 24 BC). These results show that, unlike IL-10, FLDK1 exhibits selectivity for myeloid cells relative to lymphocytes.

[0253] Further characterization of the efficacy of FLDK-1 and IL-10, as well as CSF1-IL-10 and IL-10 in activating CD8 T cells and B cells.

[0254] T cells were isolated from human PBMCs using positive microbead selection (Miltenyi). Lymphocytes were stimulated for 72 hours with anti-CD3 (ThermoFisher) (1 µg / ml) and anti-CD28 (ThermoFisher) (3 µg / ml) at a concentration of 1.5 million / ml. Cells were then divided into two aliquots and treated with IL-10 or FLDK1 or CSF1-IL-10 (10 nM each) plus IL-2 (5 ng / ml) for 48 hours. Cell supernatants were obtained, and granzyme B (Gzmb) and IFNγ were detected by ELISA (Figure 25 A). A Gzmb plot showing the pooled normalized data from four independent experiments is presented. A t-test was performed for IL-10 vs FLDK1 with a p-value < 0.05. The results showed that FLDK-1 (Figure 25 B) or CSF1-IL-10 (Figure 25 C) was less effective than IL-10 in activating CD8 T cells.

[0255] B cells were isolated from human PBMCs using positive microbead selection (Miltenyi). Isolated lymphocytes (50,000 cells per well) were stimulated on days 0 and 2 with CpG (InvivoGene) (1 µg / ml) and either IL-10 or FLDK1 or CSF1-IL-10 (10 nM per treatment). Cell supernatant was obtained after 7 days for IgG level determination by ELISA (Fig. 26 A). Similar results were obtained with CD40L stimulation. The figure shows the pooled data from four independent experiments (Fig. 26 B). The t-test for IL-10 vs FLDK1 showed a p-value < 0.05. The results indicated that FLDK-1 (Fig. 26 B) or CSF1-IL-10 (Fig. 26 C) was less effective than IL-10 in activating B cells.

[0256] Example 4: In vivo, bifunctional CSF1-IL10 cytokines alleviate clinical symptoms of a mouse model of colitis and other inflammatory diseases.

[0257] The therapeutic efficacy of FLDK1 (three doses) was evaluated and compared with a reference molecule (recombinant IL-10) in a TNBS-induced chronic IBD experimental model (a trinitrobenzenesulfonic acid (TNBS)-induced colitis mouse model).

[0258] TNBS was administered rectally to 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 was measured daily.

[0259] Each experimental group consisted of 10 mice, and the groups were as follows:

[0260] 1. 50% ethanol in the rectum

[0261] 2. Rectal TNBS / 50% ethanol + mediator treatment

[0262] 3. Rectal treatment with TNBS / 50% ethanol + FLDK1 (dose 1 µg / mouse)

[0263] 4. Rectal treatment with TNBS / 50% ethanol + FLDK1 (3 µg / mouse)

[0264] 5. Rectal treatment with TNBS / 50% ethanol + FLDK1 (dose 10 µg / mouse)

[0265] 6. Rectal treatment with TNBS / 50% ethanol + rIL10 (dose 1 µg / mouse)

[0266] 7. Rectal treatment with TNBS / 50% ethanol + rIL10 (dose 3 µg / mouse)

[0267] 8. Rectal treatment with TNBS / 50% ethanol + rIL10 (dose 10 µg / mouse)

[0268] Both FLDK1 and rIL10 are administered intraperitoneally at each dose three times a week (once every other day, starting from the day of each TNBS infusion).

[0269] The results showed that after 30 days of treatment, mice treated with FLDK1 experienced less weight loss compared to mice treated with IL10 or the medium, showing a dose-response relationship (Figure 27 AC).

[0270] Example 5: The selectivity of bifunctional CSF1-IL10 cytokines for monocytes relative to T cells is mediated by the CSF1 portion of the cytokine.

[0271] The structure-property relationship of FLDK1 was evaluated using the following method.

[0272] PBMCs (150,000 cells per well) from healthy donors were seeded in 96-well plates and stimulated the next day at 37°C for 20 minutes with FLDK1, IL-10, or FLDK1-E82A (SEQ ID NO: 73) (10 nM per treatment) (a mutant version of FLDK1 in which CSF1 cannot bind to CSF1-R due to the E82A mutation). Figure 28 A). Cells were stained with CD14 Alexa647 (Biolegend) and CD3 APC / Cy7 (Biolegend), fixed with 4% PFA, and permeabilized with 80% MetOH for intracellular staining. Cells were stained with AlexaFluor488 pSTAT3 (pY705) (BD) for 1 hour. Samples were analyzed by LSRII flow cytometry, and MFI values ​​obtained from STAT3 activation were plotted using GraphPad Prism software. A representative result is shown with two technical replicates, expressed as mean, with error bars depicting standard errors. Each condition was normalized by specifying the highest IL10 MFI value at the highest concentration as 100% and the lowest MFI value of the untreated control as 0%. The MFI of the remaining treated samples were normalized accordingly.

[0273] The results showed that the selective activation of monocytes by FLDK1 was mediated by the CSF-1 portion of the cytokine, as the mutant FLDK1-E82A showed significantly reduced selective activation of monocytes relative to T cells. Figure 28 B).

[0274] PBMCs (150,000 cells per well) from healthy donors were also seeded in 96-well plates and treated the next day with rIL10, FLDK1 (10 nM per treatment) or saturated concentration of CSF1 (100 nM) (2 min), followed by FLDK1 (10 nM) stimulation at 37°C for 20 min. Figure 29 A). Cells were stained with CD14 Alexa647 (Biolegend) and CD3 APC / Cy7 (Biolegend), fixed with 4% PFA, and permeabilized with 80% MetOH for intracellular staining. Cells were stained with AlexaFluor488 pSTAT3 (pY705) (BD) for 1 hour. Samples were analyzed by LSRII flow cytometry, and MFI values ​​obtained from STAT3 activation were plotted using GraphPad Prism software. A representative result is shown with two technical replicates, expressed as mean, with error bars depicting standard errors. Each condition was normalized by specifying the highest IL10 MFI value at the highest concentration as 100% and the lowest MFI value of the untreated control as 0%. The MFI of the remaining treated samples were normalized accordingly.

[0275] The results showed that the selective activation of FLDK1 on monocytes was mediated by the CSF-1 component of the cytokine, because saturated concentrations of CSF-1 prevented the selective activation of FLDK1 on monocytes relative to T cells. Figure 29 B).

[0276] sequence

[0277] NtCt linkers are underlined. Intermolecular linkers are underlined and italicized. Mutations are indicated in bold. Tags used for protein quantification, such as HiBit or TevHisHibit, are indicated in bold italics and have a Gly linker between the tag and the protein. Signal peptides for secreted proteins are cleaved after secretion and are not shown. For proteins starting with IL10 and Foldikine10, the signal sequence we used is: MHSSALLCCLVLLTGVRA (SEQ ID NO: 69). For proteins starting with CSF1, the signal sequence we used is: MTAPGAAGRCPPTTWLGSLLLLVCLLASRSIT (SEQ ID NO: 70).

[0278] >SEQ ID NO: 1 foldikine10:

[0279]

[0280] >SEQ ID NO: 20 foldikine10-HiBit:

[0281]

[0282] >SEQ ID NO: 2 ORK10-002

[0283]

[0284] >SEQ ID NO: 3 ORK10-003

[0285]

[0286] >SEQ ID NO: 49 ORK10-003-HiBit

[0287]

[0288] >SEQ ID NO: 4 ORK10-005

[0289]

[0290] >SEQ ID NO: 5 Foldikine_cut116

[0291]

[0292] >SEQ ID NO: 6 Foldikine_cut133

[0293]

[0294] >SEQ ID NO: 7 Foldikine_cut209

[0295]

[0296] >SEQ ID NO: 8 Foldikine_cut237

[0297]

[0298] >SEQ ID NO: 9 ORKMCSF_013

[0299]

[0300] >SEQ ID NO: 10 ORKMCSF_014

[0301]

[0302] >SEQ ID NO: 11 IL10:

[0303] SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0304] >SEQ ID NO: 50 IL10-HiBit:

[0305]

[0306] >SEQ ID NO: 12 CSF1

[0307] EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQD

[0308] >SEQ ID NO: 51 CSF1-HiBit

[0309]

[0310] >SEQ ID NO: 13 IL-10-CSF1

[0311] SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN GGGGGSGGSGGSGGSGGSGGSGGSGGSGGG EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQD

[0312] >SEQ ID NO: 52 IL-10-CSF1-HiBit

[0313]

[0314] >SEQ ID NO: 14 Foldikine10-CSF1

[0315]

[0316] >SEQ ID NO: 53 Foldikine10-CSF1-HiBit

[0317]

[0318] >SEQ ID NO: 15 CSF1-IL10

[0319] EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQD GGGGGSGGSGGSGGSGGSGGSGGSGGSGGG SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0320] >SEQ ID NO: 54 CSF1-IL10-HiBit

[0321]

[0322] >SEQ ID NO: 16 CSF1-Foldikine10 (=CSF1-30aa-foldikine10)

[0323] EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQD GGGGGSGGSGGSGGSGGSGGSGGSGGSGGG MTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMN NGGLDY LPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNRLFCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0324] >SEQ ID NO: 55 CSF1-Foldikine10-HiBit (=CSF1-30aa-foldikine10 -HiBit)

[0325]

[0326] >SEQ ID NO: 17 foldikine10_cut116-CSF1

[0327] NKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMN NGGLDY LPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMN NGGLDY LPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNHRFLPCE GGGGGSGGSG GSGGSGGSGGSGGSGGSGGG EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQD

[0328] >SEQ ID NO: 56 foldikine10_cut116-CSF1-HiBit

[0329]

[0330] >SEQ ID NO: 18 CSF1-foldikine10_cut116

[0331] MTAPGAAGRCPPTTWLGSLLLLVCLLASRSITEEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQD GGGGGSGGSGGSGGSGGSGGSGGSGGSGGG MTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMN NGGLDY LPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0332] >SEQ ID NO: 57 CSF1-foldikine10_cut116-HiBit

[0333]

[0334] >SEQ ID NO: 19 CSF1-25aa-foldikine10

[0335] EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQD GGGGGSGGSGGSGGSGGSGGSGGSGGSGGG MTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMN NGGLDY LPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0336] >SEQ ID NO: 58 CSF1-25aa-foldikine10-HiBit

[0337]

[0338] >SEQ ID NO: 21 CSF1-35aa-foldikine10

[0339] EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQD GGGGGSGGGGSGGSGGSGGSGGSGGSGGSGGSGGG MTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMN NGGLDY LPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNRLFCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0340] >SEQ ID NO: 59 CSF1-35aa-foldikine10-HiBit

[0341]

[0342] >SEQ ID NO: 22 CSF1-15aa foldikin10

[0343] EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQD GGSGGSGGSGGSGGG MTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMN NGGLDY LPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNRLFCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0344] >SEQ ID NO: 60 CSF1-15aa-foldikine10-HiBit

[0345]

[0346] >SEQ ID NO: 23 CSF1-15aa-IL10

[0347] EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQD GGSGGSGGSGGSGGG RASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0348] >SEQ ID NO: 61 CSF1-15aa-IL10-HiBit

[0349]

[0350] >SEQ ID NO: 24 CSF1-25aa-IL10

[0351] EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQD GGGSGGSGGSGGSGGSGGSGGSGGG RASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0352] >SEQ ID NO: 62 CSF1-25aa-IL10-HiBit

[0353]

[0354] >SEQ ID NO: 25 CSF1-30aa-IL10

[0355] EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQD GGGGGSGGSGGSGGSGGSGGSGGSGGSG GRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0356] >SEQ ID NO: 63 CSF1-30aa-IL10-HiBit

[0357]

[0358] >SEQ ID NO: 26 CSF1-35aa-IL10

[0359] EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQD GGGGGSGGGGSGGSGGSGGSGGSGGSGGSGGSGGG RASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0360] >SEQ ID NO: 64 CSF1-35aa-IL10-HiBit

[0361]

[0362] >SEQ ID NO: 27 Repeat unit

[0363] GGSGG

[0364] >SEQ ID NO: 28

[0365]

[0366] >SEQ ID NO: 29

[0367] LPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0368] >SEQ ID NO: 30 Nt Ct linker

[0369] NGGLDY

[0370] >SEQ ID NO: 31 Nt Ct linker

[0371] YKTIT

[0372] >SEQ ID NO: 32 Nt Ct linker

[0373] DKDIRDGD

[0374] >SEQ ID NO: 33 Molecular indirect linker 1 15

[0375] GGSGGSGGS GSGGG

[0376] > SEQ ID NO: 34 Molecular linker 1 25

[0377] GGSGGSGGS GGSGGSGGSG GSGGG

[0378] > SEQ ID NO: 35 Molecular linker 1 30

[0379] GGGGGSGGSG GSGGSGGSGG SGGSGGSGGG

[0380] > SEQ ID NO: 36 Molecular linker 1 35

[0381] GGGGGSGGGG SGGSGGSGGS GGSGGSGGSG GSGGG

[0382] > SEQ ID NO:37 1 136

[0383] CTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQ D PDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEA

[0384] > SEQ ID NO: 38

[0385] NMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTM

[0386] > SEQ ID NO: 39

[0387]

[0388] > SEQ ID NO: 40

[0389] LPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTM

[0390] >SEQ ID NO: 41

[0391]

[0392] >SEQ ID NO: 42

[0393]

[0394] >SEQ ID NO: 43

[0395]

[0396] >SEQ ID NO: 44

[0397]

[0398] >SEQ ID NO: 71

[0399]

[0400] >SEQ ID NO: 45

[0401]

[0402] >SEQ ID NO: 46

[0403]

[0404] >SEQ ID NO: 47

[0405]

[0406] >SEQ ID NO: 48

[0407] FGGLDY

[0408] >SEQ ID NO: 65 N-terminal portion of SC dimer IL-10

[0409] MTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAY

[0410] >SEQ ID NO: 66 C-terminal portion of SC dimer IL-10

[0411] NMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0412] >SEQ ID NO: 67

[0413] MTMN

[0414] >SEQ ID NO: 68

[0415] PEFA

[0416] >SEQ ID NO: 72 CSF1-Foldikine10-TEV-his-hibit

[0417]

[0418] >SEQ ID NO: 73 CSF1(E82A)-Foldikine10-TEV-his-hibit

[0419]

[0420] >SEQ ID NO: 74 CSF1-IL10-TEV-his-hibit

[0421]

Claims

1. A single-chain polypeptide with IL-10 activity and CSF1 activity.

2. The single-chain polypeptide according to claim 1, comprising CSF1 monomer, peptide linker and IL-10 monomer in the direction from N-terminus to C-terminus.

3. The single-chain polypeptide according to claim 1 or 2, comprising the sequences of SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, or comprising a sequence that is at least 80% identical to the sequences and retains IL-10 and CSF1 activities.

4. The single-chain polypeptide according to claim 1, comprising a single-chain dimer IL-10 fused to a CSF1 monomer, particularly a single-chain dimer IL-10-CSF1 monomer fusion protein, or a CSF1 monomer-single-chain dimer IL-10 fusion protein.

5. The single-chain polypeptide according to any one of claims 1 to 4, wherein the CSF1 monomer is a wild-type CSF1 monomer, a mutant thereof, or a cyclically arranged CSF1 monomer.

6. The single-chain polypeptide according to any one of claims 1 to 5, having a similar or higher affinity for CSF1R compared to wild-type CSF1.

7. The single-chain polypeptide according to any one of claims 1 to 6, wherein the CSF1 monomer is a CSF1 monomer mutant comprising the sequence of SEQ ID NO:12 and modified by at least one of the following 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.

8. The single-chain polypeptide according to any one of claims 1 to 6, wherein the CSF1 monomer is a cyclic arrangement of CSF1: a) Generated by linking the amino acids in the N-terminal and C-terminal regions of CSF-1 and by inhibiting the peptide bond consisting of residues 95 to 99 in SEQ ID NO: 12; or b) It contains a sequence of SEQ ID NO: 9 or SEQ ID NO: 10, or contains a sequence that is at least 80% identical to the said sequence and retains CSF1 activity.

9. The single-chain polypeptide according to any one of claims 1 to 8, wherein the single-chain dimer IL-10 is a fusion protein comprising a first IL-10 monomer fragment, a peptide linker, and a second IL-10 monomer fragment or a circular arrangement thereof, wherein the first IL-10 monomer fragment comprises at least IL-10 α-helices A to F, and the second IL-10 monomer fragment comprises at least IL-10 α-helices A to F.

10. The single-chain polypeptide according to any one of claims 1 to 9, wherein the sequence of the single-chain dimer IL-10 is SEQ ID NO: 65-X-(NtCt)-Z-SEQ ID NO:

66. Where (NtCt) is the peptide linker. Where X is absent or present, and when present, it consists of one or more amino acids from the original IL-10 sequence, continuous with the amino acid preceding its N-terminus, optionally having a mutation to accommodate the NtCt linker. Wherein Z is absent or present, and when present, it consists of amino acids of one or more original IL-10 sequences, continuous with the amino acids preceding its C-terminal side, optionally having mutations to adapt to the NtCt linker, or a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the sequence and retains at least the same stability and / or at least the same level of interaction with the IL-10 receptor.

11. The single-chain polypeptide according to claim 9 or 10, wherein the peptide linker comprises 3 to 20 amino acid residues and comprises no more than 2 adjacent Gly residues and / or Ser residues.

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

13. The single-chain polypeptide according to any one of claims 1 to 8, wherein the single-chain dimer IL-10 is a cyclic arrangement: a) It comprises a first IL-10 monomer fragment, a first peptide linker, a second IL-10 monomer fragment, a second peptide linker, and a third IL-10 monomer fragment, wherein the first IL-10 monomer fragment comprises α-helices E to F of IL-10, the second IL-10 monomer fragment comprises at least α-helices A to F of IL-10, and the third IL-10 monomer fragment comprises at least α-helices A to D of IL-10, and / or b) It contains the sequence of SEQ ID NO: 5, or a sequence that is at least 80% identical to the sequence and retains IL-10 activity.

14. The single-chain polypeptide according to any one of claims 1 to 13, wherein the single-chain dimer IL-10 and CSF1 monomers are linked by a linker comprising 12 to 40 amino acid residues.

15. The single-chain polypeptide according to any one of claims 1 to 14, comprising the sequences SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or a sequence that is at least 80% identical to said sequences and retains IL-10 activity and CSF1 activity.

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

17. A pharmaceutical composition comprising a single-chain polypeptide according to any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

18. The single-chain polypeptide according to any one of claims 1 to 16, used as a medicine, particularly for treating inflammatory diseases.

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