Dual il-34-il-10 cytokine

Abstract

The invention relates to dual cytokine single chain polypeptides having IL-10 and IL- 34 activities and to their use in therapy.

Description

[0001] DUAL IL-34-IL-10 CYTOKINE

[0002] Field of the Invention

[0003] The invention relates to single chain dual-cytokine polypeptides having IL-10 and IL-34 activities and to their use in therapy.

[0004] Background of the Invention

[0005] Cytokines are proteins that are produced in the immune system and act as chemical messengers to communicate between cells of the immune system, and also with other cells of the body. Cytokines control the entire range of immune responses, including their initiation, type, potency, and duration. So much so that its unregulated production triggers a wide variety of inflammatory and autoimmune diseases, allergies, fibrosis, and can even lead to cancer. For this reason, there is great medical interest in the development and use of cytokines as drugs to treat diseases.

[0006] 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 that is produced as a monomer by various immune cells, including T cells, B cells, macrophages, and dendritic cells, and it exerts its effects by binding to its receptor complex, which is composed of two subunits, IL-10R1 and IL-10R2. When IL-10 binds to its receptor, IL-10R1 first recognizes and binds to IL-10 with high affinity, forming a complex that is then stabilized by the binding of IL-10R2. The binding of IL- 10 to its receptor complex leads to the activation of downstream signaling pathways.

[0007] When binds to its receptor on myeloid cells, IL-10 exerts potent anti-inflammatory activities, including inhibition of the production of pro-inflammatory mediators, expression of MHCII and costimulatory molecules. Simultaneously, activation of myeloid cells by IL-10 leads to the expression of anti-inflammatory mediators and promotes the development of tolerogenic phenotype, like M2-like anti-inflammatory macrophages and tolerogenic dendritic cells. In contrast to the strong anti-inflammatory activities on myeloid cells, IL- 10 is a growth factor for B cells and promotes plasma cell differentiation and antibody production. Similarly, IL-10 has stimulatory effects on CD8+ T cells, inducing their proliferation, IFNY production, and cytotoxicity.

[0008] In humans, there is a strong genetic association between the IL- 10 pathway and autoimmune diseases, such as inflammatory bowel disease. Loss-of-function mutations in IL- 10, IL-10RA, or IL-10RB result in early-onset, therapy-resistant severe enterocolitis. Restoring IL-10RA or IL-10 expression by hematopoietic stem cell transplantation rapidly alleviates clinical symptoms. In turn, mice deficient in IL-10 or IL-10R develop colitisspontaneously, while the administration of IL-10 in different animal models of colitis has been shown to be consistently beneficial. Finally, mice in which all cells of the organism respond to IL-10, except specific subsets of myeloid cells that includes monocytes and macrophages, also develop spontaneous colitis that is mediated by the production of the clinically validated target IL-23.

[0009] Myeloid cells exclusively express CSF1 receptor, i.e. CSF1R. Stimulation of CSF1R by its ligand, the cytokine CSF1, is essential for the differentiation, proliferation and maintenance of M2-like anti-inflammatory macrophages in the intestine, which play a key role in generating a tolerogenic environment through the production of IL-10. In autoimmune disease, the M2-like anti-inflammatory macrophages of the affected tissues, such as the mucosa of IBD patients, are replaced by inflammatory monocytes that drive disease progression. I L-34 is another ligand of CSF1 R that directs the differentiation of myeloid cells in a tissue-restricted manner, in the skin epidermis and CNS (Wang et al., 2012, Nat. immunol., 24; 13(8): 753-60).

[0010] Although IL-34 and CSF-1 share no sequence homology, they exhibit functional redundancy in supporting the survival, proliferation, and differentiation of human monocytes into macrophages. Notably, IL-34 is more evolutionarily conserved than CSF-1. In addition to CSF1 R, IL-34 also binds two additional co-receptors that seems to play important role in I L-34-signaling through the CSF1-R: the receptor-type protein-tyrosine phosphatase-^ (PTP-Q and CD138 (syndecan-1), suggesting broader roles in immune and tissue-specific processes. IL-34 is primarily expressed in the brain and skin, where it plays a critical role in maintaining Langerhans cells and microglia populations.

[0011] IL-34 has been also found to be involved in the development of intestinal fibrosis, a feature of inflammatory bowel disease patients, through CSF-1 R signaling in fibroblasts (Franzè et al., 2020, Journal of Crohn's and Colitis, Volume 14, Issue 10, Pages 1436-1445). The involvement of IL-34 has been shown in areas as diverse as neuronal protection, autoimmune diseases, infection, cancer, and transplantation (Guillonneau C. Cell Mol Life Sci. 2017). Recent work has also demonstrated a new and possible therapeutic role for IL-34 as a Foxp3+ regulatory T cell-secreted cytokine mediator of transplant tolerance (Bezie el al., JCI, 2015). And clinical studies have indicated a correlation between changes in secreted IL-34 into extracellular biofluids with disease parameters in various pathological conditions such as in rheumatoid arthritis (RA), SLE, heart failure, viral infections, sepsis, periodontal disease, non-alcoholic fatty liver disease (NAFLD), obesity, and type 2 diabetes mellitus (T2DM; Table 1). Secreted levels of IL-34 are reduced to normal levels upon successful treatment of disease, such as in TNF-a-antagonist therapy in RA33 and gastricbypass surgery in obesity. (Baghdadi M, Umeyama Y, Hama N, et al. Interleukin 34, a comprehensive review. J Leukoc Biol. 2018;1-21).

[0012] While in preclinical studies IL-10 has shown promising results in ameliorating inflammation and tissue damage in models of colitis, clinical trials investigating the therapeutic potential of IL-10 in patients with colitis have not been successful. One possible explanation is that the delivery of IL- 10 to the site of inflammation may be a limiting factor. However, some strategies aimed to enhance the delivery of IL-10 at the site of inflammation did not show clinical benefit (AMT-101).

[0013] Another possible explanation is because the anti-inflammatory effects of IL-10 on myeloid cells are counteracted by pro-inflammatory tissue-damaging effects, including activation, differentiation, and induction of antibodies by B cells and induction of cytotoxic activities by CD8+ T cells.

[0014] To address this limitation, there is a need for IL-10-based therapeutics 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 include fusion of IL-10 to antibodies that bind to receptors on myeloid cells, or encapsulation of IL-10 in nanocarriers (liposomes or nanoparticles) that are specifically targeted to myeloid cells.

[0015] The present invention seeks to overcome or at least alleviate one or more of the deficiencies in the prior art.

[0016] Summary of the Invention

[0017] The invention is as defined by the claims.

[0018] It is disclosed a single chain dual-cytokine polypeptide having IL-10 and IL-34 activities.

[0019] IL-34’s characteristics position IL-34 as a strategic targeting domain in a dual-cytokine polypeptide construct, enabling precise delivery of IL-10 signaling to CSF1R+ myeloid cells while leveraging IL-34’s unique tissue-specific and tolerogenic properties. The functional characterization of the single chain dual-cytokine polypeptides having IL-10 and IL-34 activities disclosed herein demonstrate binding to IL-10R and CSF1R, and preferentially targeting of myeloid cells. The dual-cytokine polypeptides reprogram monocytes into regulatory macrophages that suppress inflammatory T cell responses.

[0020] In some embodiments the single chain dual-cytokine polypeptide comprises an IL-10 domain fused to an IL-34 monomer, wherein the IL-10 domain is an IL-10 monomer or a single chain dimeric IL-10.In some embodiments, the single chain dual-cytokine polypeptide comprises an IL-10 monomer fused to an IL-34 monomer through a peptide linker L1.

[0021] In some embodiments, the single chain dual-cytokine polypeptide comprises a single chain dimeric IL-10 fused to an IL-34 monomer through a peptide linker L1.

[0022] In some embodiments of the single chain dual-cytokine polypeptide, one or more of the following conditions are met:

[0023] (i) the IL-34 monomer comprises sequence SEQ ID NO: 12, or a sequence at least 80% identical thereto and that retains IL-34 activity;

[0024] (ii) the IL-10 monomer comprises sequence SEQ ID NO: 11, or a sequence at least 80% identical thereto and that retains IL- 10 activity;

[0025] (iii) the single chain dimeric IL-10 comprises sequence X1-SEQ ID NO: 65-X2- SEQ ID NO: 143-X3-L1.1-Z-SEQ ID NO: 66-X4-SEQ ID NO: 144,

[0026] wherein L1.1 is a peptide linker,

[0027] wherein X1 is absent or present, and where present it consists of M or sequence SPGQG (SEQ ID NO: 81),

[0028] wherein X2 is D or E,

[0029] wherein X3 is absent or present, and where present it consists in one or more amino acids of IL-10 sequence SEQ ID NO: 11 in continuity with the preceding amino acids on its N-terminal side, optionally with a mutation to accommodate the L1.1 linker,

[0030] wherein Z is absent or present, and where present it consists one or more amino acids of the IL-10 sequence SEQ ID NO: 11 in continuity with the preceding amino acids on its C-terminal side, optionally with a mutation to accommodate the L1.1 linker,

[0031] wherein X4 is C or N,

[0032] or a sequence at least 80% identical thereto and that retain at least the same stability, and / or at least the same level of interaction with IL-10 receptor;

[0033] (iv) the single chain dimeric IL-10 comprises sequence SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 1, or a sequence at least 80% identical thereto and that retains IL-10 activity.

[0034] In some embodiments of the single chain dual-cytokine polypeptide, one or more of the following conditions are met:

[0035] (i) the peptide linker L1 comprises from 5 to 50 amino acid residues;

[0036] (ii) the peptide linker L1 is a flexible peptide sequence composed of Gly and Ser residues, in different proportion;

[0037] (iii) the peptide linker L1 comprises sequence SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36,; or

[0038] (iv) the peptide linker L1 comprises sequence (SEQ ID NO:82)n, n being an integer from 1 to 10, preferably from 3 to 6.In some embodiments, the single chain dual-cytokine polypeptide comprises sequence SEQ ID NO: 15 or SEQ ID NO: 16, or a sequence at least 80% identical thereto and that retains IL-10 and IL-34 activities.

[0039] In some embodiments, the single chain dual-cytokine polypeptide comprises sequence SEQ ID NO: 19, SEQ ID NO: 145, SEQ ID NO: 37, or a sequence at least 80% identical thereto and that retains IL- 10 and IL-34 activities.

[0040] In some embodiments, the single chain polypeptide is modified to increase its halflife.

[0041] It is further disclosed a protein comprising the single chain dual-cytokine polypeptide having IL-10 and IL-34 activities fused to an immunoglobulin Fc fragment or to albumin, or to albumin fragment or an anti-albumin antibody, or anti-albumin antibody fragment, or conjugated with a poly(ethylene glycol) (PEG) molecule.

[0042] In some embodiments, the protein is fused to an immunoglobulin Fc fragment, optionally of I gG 1, I gG2, lgG3 or I gG4, silent and non-silent.

[0043] In some embodiments, the protein comprises one single chain polypeptide having IL-10 and IL-34 activities and one immunoglobulin Fc fragment, wherein the single chain polypeptide having IL-10 and IL-34 activities is fused to the immunoglobulin Fc fragment through a peptide linker L2 connecting (i) the C-terminus of the IL-10 domain with the N-terminus of one hinge region of the Fc fragment, or (ii) the C-terminus of the one of the CH3 domains of the Fc fragment with the N-terminus of the IL-34 monomer.

[0044] In some embodiments, the protein comprises two polypeptide chains that each comprise, in the N-terminus to C-terminus direction, (i) an IL-34 monomer, a peptide linker L1, an IL-10 domain which is an IL-10 monomer or a SC dimeric IL-10 monomer, a peptide linker L2, and a Fc domain, or (ii) a Fc domain, a peptide linker L2, an IL-34 monomer, a peptide linker L1, and an IL-10 domain which is an IL-10 monomer or a SC dimeric IL-10 monomer, wherein the two polypeptide chains dimerise through their Fc domains.

[0045] In some embodiments, in the protein, one or more of the following conditions are met: (i) the peptide linker L2 comprises from 3 to 50 amino acid residues;

[0046] (ii) the peptide linker L2 is a flexible peptide sequence composed of Gly and Ser residues; (iii) the peptide linker L2 comprises sequence (SEQ ID NO:82)p, p being an integer from 1 to 8, preferably from 1 to 4; or

[0047] (iv) the peptide linker L2 comprises any one of sequences SEQ ID NO: 30 to SEQ ID NO: 32, SEQ ID NO: 48, and SEQ ID NO: 119 to SEQ ID NO: 131, and SEQ ID NO: 146.

[0048] In some embodiments, the protein comprises sequence SEQ ID NO: 83 or SEQ ID NO: 84, or the combination of sequences SEQ ID NO: 85 and SEQ ID NO:86, or thecombination of sequences SEQ ID NO: 87 and SEQ ID NO: 86, or a sequence at least 80% identical thereto and that retains IL- 10 and IL-34 activities.

[0049] A pharmaceutical composition comprising the single chain polypeptide or the protein, and a pharmaceutically acceptable carrier, is further disclosed.

[0050] The disclosure also relates to the single chain polypeptide or the protein for use as a medicament, in particular for use for treating an auto-immune disease, an inflammatory disease, or a neuroinflammatory disease.

[0051] Detailed description of the invention

[0052] The inventors have designed single chain dual-cytokine polypeptides combining IL-10 and IL-34 activities.

[0053] The functional characterization of the dual single chain polypeptide having IL-10 and IL-34 activities has shown that the dual I L-34-I L-10 cytokine inhibits activation of myeloid cells and induces differentiation of monocyte-derived macrophages that have a regulatory phenotype (M2 phenotype). Furthermore, the dual I L-34-I L-10 cytokine has specificity for monocytes and does not activate CD8 T cells or B cells.

[0054] Main definitions

[0055] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, “a ribonucleotide” is understood to represent one or more ribonucleotides. As such, the terms “a,” “an,” “one or more,” and “at least one” can be used interchangeably herein

[0056] Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is further understood that wherever embodiments are described herein with the language “comprising” or “having,” or grammatical equivalents thereof, otherwise analogous embodiments described in terms of “consisting of” and / or “consisting essentially of” are also provided.

[0057] As used herein ‘IL-10 activity” or “IL-10 activities” denotes one or more biological activities mediated by IL-10 through binding to its receptor, IL-10R. These comprise or consist of (i) the binding to IL-1 OR on myeloid cells, in particular monocytes, or (ii) antiinflammatory activities, or preferentially both. Anti-inflammatory activities include (a) the inhibition of production of pro-inflammatory mediators (e.g. pro-inflammatory cytokines suchas tumour necrosis factor-a (TNF-a), IL-1, IL-12, IL-6 and granulocyte–macrophage colony-stimulating factor, inflammatory enzymes such as cyclo-oxygenase 2 and inducible nitric oxide synthase, chemokines such as RANTES, membrane inflammatory protein-1 a (MIP1a), IL-8, and eotaxin), and / or (b) the inhibition of expression of MHCII and costimulatory molecules by myeloid cells, and / or (c) the induction of differentiation of monocytes into tolerogenic macrophages. In some embodiments, anti-inflammatory activities comprise inhibition of production of TNF-a and / or IL-6 by activated monocytes. In some embodiments, anti-inflammatory activities comprise induction of differentiation of monocytes into tolerogenic macrophages. Tolerogenic macrophages have M2-like phenotype and can be identified by their regulatory phenotype and high levels of expression of CD206 and CD163. In some embodiments, IL-10 activities comprise or consist of binding to IL-10R on myeloid cells, in particular monocytes, inhibition of production of TNF-a and / or IL-6 by activated monocytes, and differentiation of monocytes into tolerogenic macrophages.

[0058] As used herein “IL-34 activity” or “IL-34 activities” denotes one or more biological activities mediated by activation of CFS1-R by IL-34. These comprise or consist of (i) the activation of CFS1-R at the surface of monocytes or progenitors thereof, and / or (ii) the differentiation of monocytes into macrophages, preferentially both. According to the invention, activation of CSF1-R is triggered by binding of IL-34 to CSF1-R.

[0059] An “immunoglobulin Fc fragment” or “Fc fragment” consists of two polypeptides linked by disulfide bonds, each polypeptide, from the N-terminal to C-terminal, being composed of a hinge region, a CH2 domain and a CH3 domain. The structure of the Fc fragment is nearly identical across all subtypes of human immunoglobulin. As used herein “an immunoglobulin Fc domain” or “Fc domain” denotes the monomeric polypeptide comprising immunoglobulin hinge and CH2, CH3 immunoglobulin heavy chain constant domains. Fc domain as used herein encompasses native Fc and Fc variants. The term "native Fc" as used herein refers to a molecule comprising the sequence of a non-antigen-binding fragment resulting from digestion of an antibody, or produced by other means, and that contain the hinge region. The original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins, of isotype as IgM, I g D, IgG, IgA, or IgE, IgG has several subclasses, including, but not limited to, lgG1, lgG2, lgG3, and lgG4. The term " Fc variant" as used herein refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants, and their interaction with the salvage receptor, are known in the art. Thus, the term " Fc variant" can comprise a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fccomprises regions that can be removed because they provide structural features or biological activity that are not required for the antibody-like binding proteins of the invention. Thus, the term " Fc variant" comprises a molecule or sequence that lacks one or more native Fc sites or residues, or in which one or more Fc sites or residues has be modified, that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0060] Single chain polypeptide having IL- 10 and IL-34 activities

[0061] A single chain dual-cytokine polypeptide having IL-10 and IL-34 activities is thus provided.

[0062] As used herein ‘IL-10 activity” or “IL-10 activities” denotes one or more biological activities mediated by IL-10 through binding to its receptor, IL-10R. These comprise or consist of (i) the binding to IL-1 OR on myeloid cells, in particular monocytes, or (ii) antiinflammatory activities, or preferentially both. Anti-inflammatory activities include (a) the inhibition of production of pro-inflammatory mediators (e.g. pro-inflammatory cytokines such as tumour necrosis factor-a (TNF-a), IL-1, IL-12, IL-6 and granulocyte–macrophage colony-stimulating factor, inflammatory enzymes such as cyclo-oxygenase 2 and inducible nitric oxide synthase, chemokines such as RANTES, membrane inflammatory protein-1 a (MIP1a), IL-8, and eotaxin), and / or (b) the inhibition of expression of MHCII and costimulatory molecules by myeloid cells, and / or (c) the induction of differentiation of monocytes into tolerogenic macrophages. In some embodiments, anti-inflammatory activities comprise inhibition of production of TNF-a and / or IL-6 by activated monocytes. In some embodiments, anti-inflammatory activities comprise induction of differentiation of monocytes into tolerogenic macrophages. Tolerogenic macrophages have M2-like phenotype and can be identified by their regulatory phenotype and high levels of expression of CD206 and CD163. In some embodiments, IL-10 activities comprise or consist of binding to IL-10R on myeloid cells, in particular monocytes, inhibition of production of TNF-a and / or IL-6 by activated monocytes, and differentiation of monocytes into tolerogenic macrophages.

[0063] In some embodiments, the single chain polypeptide having IL-10 and IL-34 activities has reduced T cells and B cell activation capacities compared to IL-10.

[0064] As used herein “IL-34 activity” or “IL-34 activities” denotes one or more biological activities mediated by activation of CFS1-R by IL-34. These comprise or consist of (i) the activation of CFS1-R at the surface of monocytes or progenitors thereof, and / or (ii) thedifferentiation of monocytes into macrophages, preferentially both. According to the invention, activation of CSF1-R is triggered by binding of IL-34 to CSF1-R.

[0065] In some embodiments, the single chain polypeptide having IL-10 and IL-34 activities has similar or higher affinity to CSF1R compared to wild-type IL-34. In some embodiments, the single chain polypeptide having IL- 10 and IL-34 activities has reduced affinity to IL- 10 receptor a (IL10RA) and IL-10 receptor p (IL10RB) compared to wild-type IL-10. In some embodiments, the single chain polypeptide having IL-10 and IL-34 activities has similar or higher affinity to CSF1R compared to wild-type IL-34 and has reduced affinity to IL- 10 receptor a (IL10RA) and IL-10 receptor (IL10RB) compared to wild-type IL-10. These properties are expected to favor selective binding of the single chain polypeptide having IL-10 and IL-34 activities to myeloid cells that exclusively express CSF1 receptor, among cells of the innate immune system, and subsequent activation thereof.

[0066] By ‘similar affinity’ it is meant herein the affinity preferably does not vary (i.e. increase or decrease) by more than a 5-fold, preferably 4-fold, 3-fold, 2-fold, or 1.5-fold factor compared to the reference level of affinity of the wild-type cytokine (IL-34).

[0067] By ‘higher’ or ‘reduced’ it is meant herein the affinity preferably varies (i.e. increase or decrease) by at least a 5-fold, preferably, 7-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold factor compared to the reference level of affinity of the wild-type cytokine (IL-34 or IL-10 as appropriate).

[0068] The single chain polypeptide having IL-10 and IL-34 activities comprises an IL-10 domain fused to an IL-34 monomer, wherein the IL-10 domain is an IL-10 monomer or a single chain dimeric IL-10.

[0069] In some embodiments, the single chain polypeptide having IL-10 and IL-34 activities comprises in the N-terminus to C-terminus direction, an IL-34 monomer, a peptide linker L1, and an IL-10 domain, wherein the IL-10 domain is an IL-10 monomer or a single chain dimeric IL-10.

[0070] In some embodiments, the single chain polypeptide having IL-10 and IL-34 activities comprises in the N-terminus to C-terminus direction, an IL-10 domain, a peptide linker L1, and an IL-34 monomer, wherein the IL-10 domain is an IL-10 monomer or a single chain dimeric IL-10.

[0071] In a first aspect, the single chain polypeptide having IL-10 and IL-34 activities is thus provided that comprises an IL- 10 monomer fused to an IL-34 monomer through a peptide linker L1.According to an embodiment, the single chain polypeptide having IL-10 and IL-34 activities comprises, in the N-terminus to C-terminus direction, an IL-34 monomer, a peptide linker L1, and an IL-10 monomer. According to another embodiment, the single chain polypeptide having IL-10 and IL-34 activities comprises, in the N-terminus to C-terminus direction, an IL-10 monomer, a peptide linker L1, and an IL-34 monomer.

[0072] In some embodiments the IL-10 and IL-34 monomers are linked through a peptide linker L1 that comprises from (or consists of) 5 to 50, preferably from 10 to 45 amino acid residues, preferably 12 to 40 amino acids, still preferably 15 to 40 amino acids, still preferably 20 to 35 amino acids, even more preferably 25 to 35 amino acids.

[0073] In some embodiments, the peptide linker L1 connecting the IL-34 monomer and the IL-10 monomer is a (flexible) peptide sequence composed of Gly and Ser residues, in different proportions. Examples of suitable linkers comprise or consist of GGGSGGSGGSGGSGGSGGSGGSGGG (25 amino acid long, SEQ ID NO: 34), GGGGGSGGSGGSGGSGGSGGSGGSGGSGGG (30 amino acid long, SEQ ID NO: 35), or GGGGGSGGGGSGGSGGSGGSGGSGGSGGSGGSGGG (35 amino acid long, SEQ ID NO: 36), or SEQ ID NO: 33. In some embodiments the peptide linker Llcomprises or consists of a sequence (GGGGS, SEQ ID NO:82)n, n being an integer from 1 to 10, such as from 2 to 10, preferably from 3 to 6.

[0074] The IL-34 monomer may be as defined in the below section.

[0075] The IL-10 monomer may be wild-type mature IL-10 and comprises or consists in SEQ ID NO:11, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and that retains IL-10 activity, e.g. a mutant thereof, or a polypeptide comprising the sequence of mature IL-10 and on the N-terminal end 1, 2, 3 or more additional amino acids residues consecutively present in the signal peptide of IL-10 (i.e. A, RA or VRA). In some embodiments the IL-10 monomer comprises sequence SEQ ID NO:11 modified by substitution D84E, or C108N in the second 11-10 monomer, or by substitutions D84E and C259N, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and that retains IL-10 activity.

[0076] In some embodiments the IL-10 single-chain comprises sequence SEQ ID NO: 1 which has been modified compared with WT sequenceby substitutions D84E and C108N (2nd monomer), or SEQ ID NO: 88 which is a single-chain IL-10, or SEQ ID NO: 89 which is preferred single chain without methionine, C108N (2nd monomer)mutation and completed IL-10 N terminal, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and that retains IL-10 activityIn some embodiments, the single chain polypeptide having IL-10 and IL-34 activities comprises or consists of sequence SEQ ID NO: 15 or SEQ ID NO: 16, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and that retains IL- 10 and IL-34 activities.

[0077] In a second aspect, a single chain polypeptide having IL- 10 and IL-34 activities is provided that comprises a single chain dimeric IL-10 fused to an IL-34 monomer.

[0078] In some embodiments, the single chain polypeptide having IL-10 and IL-34 activities comprises a single chain (SC) dimeric IL-10-IL-34 monomer fusion protein. In some embodiments, the single chain polypeptide having IL-10 and IL-34 activities comprises an IL-34 monomer-single chain dimeric IL-10 fusion protein.

[0079] In some embodiments the single chain dimeric IL-10 and IL-34 monomer are linked through a peptide linker L1 that comprises from (or consists of) 5 to 50 amino acid residues, preferably 10 to 45 amino acid residues, preferably 12 to 40 amino acids, still preferably 15 to 40 amino acids, still preferably 20 to 35 amino acids, even more preferably 25 to 35 amino acids.

[0080] In some embodiments, the peptide linker L1 connecting the IL-34 monomer and the SC dimeric IL-10 (i.e. bridging the C-terminus of SC dimeric IL-10 to the N-terminus of IL-34, or bridging the N-terminus of SC dimeric IL-10 to the C-terminus of IL-34), is a (flexible) peptide sequence composed of Gly and Ser residues, in different proportions. The flexible linker L1 may comprise from (or consists of) 10 to 45 amino acid residues, preferably 12 to 40 amino acids, still preferably 15 to 40 amino acids, still preferably 20 to 35 amino acids, even more preferably 25 to 35 amino acids. Examples of suitable linkers comprise or consist of sequence GGSGGSGGSGGSGGG (15 amino acid long, SEQ ID NO: 33), GGGSGGSGGSGGSGGSGGSGGSGGG (25 amino acid long, SEQ ID NO: 34), GGGGGSGGSGGSGGSGGSGGSGGSGGSGGG (30 amino acid long, SEQ ID NO: 35), or GGGGGSGGGGSGGSGGSGGSGGSGGSGGSGGSGGG (35 amino acid long, SEQ ID NO: 36). In some embodiments the peptide linker L1 comprises or consists of a sequence (GGGGS, SEQ ID NO:82)n, n being an integer from 2 to 10, preferably from 3 to 6.

[0081] In some embodiments, the single chain polypeptide having IL-10 and IL-34 activities comprises an IL-34 monomer-single chain dimeric IL-10 fusion protein, and the peptide linker L1 bridging the C-terminus of IL-34 to the N-terminus of SC dimeric IL-10 comprises more than 15 amino acids, preferably at least 20 or 25 amino acids, and optionally no more than 40 or 35 amino acids. In some embodiments, the single chain polypeptide having IL-10 and IL-34 activities comprises a single chain dimeric IL-10-IL-34 monomer fusion protein, and the peptide linker L1 bridging the C-terminus of the SC dimeric IL-10 to the N-terminusof the IL-34 monomer comprises more than 15 amino acids, preferably at least 20 or 25 amino acids, and optionally no more than 40 or 35 amino acids.

[0082] IL-34 monomer

[0083] In said first and second aspects of the invention, the IL-34 monomer incorporated into the single chain polypeptide having IL-10 and IL-34 activities may be a wild-type IL-34 monomer, a fragment, or a mutant thereof.

[0084] A canonical sequence of human IL-34 is available from the database UniProtKB under accession number Q6ZMJ4-1 (entry version 144 of 27 November 2024). The sequence Q6ZMJ4-1 of human IL-34 includes a signal peptide of 20 amino acids (SEQ ID NO: 133) and a 222 amino acid long mature polypeptide.

[0085] In some embodiments, the IL-34 monomer is a polypeptide that comprises or consists of sequence SEQ ID NO: 12 (amino acids N1-P222 of mature human IL-34 protein), or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and that retains IL-34 activity.

[0086] In some embodiments, the IL-34 monomer is a mutant IL-34 or a circular permutant IL-34 monomer that has similar or higher affinity to CSF1R compared to wild-type IL-34.

[0087] Single chain dimeric IL- 10

[0088] The single chain (SC) dimeric IL-10 incorporated into the single chain polypeptide having IL-10 and IL-34 activities may be a fusion protein comprising a first IL-10 monomer fragment comprising at least a-helices A F of IL-10, a peptide linker, and a second IL-10 monomer fragment comprising at least a-helices A to F of IL-10. The folding of the SC dimeric IL-10 is such that a ‘continuous 3D IL-10 domain’ is formed, and a split 3D IL-10 domain is formed, as disclosed in the international patent application published under number WO2023144393 A1 (see e.g. figure 28 of WO2023144393 A1).

[0089] A polypeptide sequence of mature (without Met1) wild-type human IL-10 is shown in SEQ ID NO: 11:

[0090] SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQ

[0091] hhh hhhhhhhhhhh hhhhhh hhhhhhhhhhhhhhhhhhh HA HB AENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0092] hhh hhhhhhhhhhhhhhhhhhh hhhhhhhhhhhh hhhhhhh hhhhhhhhhhhhhhh HC HD HE HF

[0093] Positions of the helices are represented by strings of ‘h’ in the above sequence. For a-helix F, depending of the structural assignments done by different software on different crystal structures of the same molecule, the helix consistently extends up to M₁₅₄, and mayextends up to M₁₅₆ or I₁₅₈ (characters underlined weavy). Accordingly, it is herein considered that a-helix F is entirely incorporated as long as the IL-10 monomer fragments comprise amino acid residues extending at least up to M₁₅₄.

[0094] According to an embodiment, the SC dimeric IL-10 comprises or consists of sequence X1TQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQ hhh hhhhhhhhhhh hhhhhh hhhhhhhhhhhhhhhhhhh HA HB AENQX2PD IKAH VNSLGENLKTLRLRLRRCHRFLPCENKSKAVE QVKNAFNKLQE KGI YKAMS E FD I F I NY hhh hhhhhhhhhhhhhhhhhhh hhhhhhhhhhhh hhhhhhh hhhhhh HC HD HE HF IEAY 3(L1.1) ZNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEV hhhh hhhhhhhhhhh hhhhhh hhhhhhhhhhhhhhhh HF HA HB

[0095] V

[0096]

[0097] MPQAENQDPDIKAHVNSLGENLKTLRLRLRRX4HRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIF hhhhhh hhhhhhhhhhhhhhhhhhh hhhhhhhhhhhh hhhhhhh hhh HC HD HE IN Y IE A YMTMKIRN

[0098] hhhhhhhhhhhh

[0099] HF

[0100] (sequence X1-SEQ ID NO: 65-X2-SEQ ID NO: 143-X3-L1.1-Z-SEQ ID NO: 66-X4-SEQ ID NO: 144),

[0101] wherein L1.1 is a peptide linker,

[0102] wherein X1 is absent or present, and where present it consists of M or sequence SPGQG (SEQ ID NO: 81),

[0103] wherein X2 is D (the residue naturally present at position 84 of SEQ ID NO:11) or E, wherein X3 is absent or present, and where present it consists in one or more amino acids of IL-10 sequence SEQ ID NO: 11 in continuity with the preceding amino acids on its N-terminal side, optionally with a mutation to accommodate the L1.1 linker,

[0104] wherein Z is absent or present, and where present it consists one or more amino acids of the IL-10 sequence SEQ ID NO: 11 in continuity with the preceding amino acids on its C-terminal side, optionally with a mutation to accommodate the L1.1 linker,

[0105] wherein X4 is C (the residue naturally present in SEQ ID NO: 11) or N,

[0106] or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and that retain at least the same stability, and / or at least the same level of interaction with IL-10 receptor.

[0107] In some embodiments, X3 represents M, MT, MTM, MTMN (SEQ ID NO: 67), or MTMKIRN (SEQ ID NO: 76) that are present in the original IL- 10 sequence in continuity with the preceding amino acids on its N-terminal side. X may also represent L or K, i.e. mutation compared to the original IL-10 sequence to accommodate the L1.1 linker. Accordingly, X3 is preferably selected from the group consisting of M, MT, MTM, MTMN (SEQ ID NO: 67), MTMKIRN (SEQ ID NO: 76), L and K, still preferably it consists of MTMKIRN (SEQ ID NO: 76).In some embodiments, Z represents LP that are present in the original IL- 10 sequence in continuity with the following amino acids on its C-terminal side. Z may also represent PEFA (SEQ ID NO: 68) or ELA. Z is preferably selected from the group consisting of LP, PEFA (SEQ ID NO: 68) or ELA.

[0108] In some embodiments X2 is D, and X4 is C. In some embodiments X2 is E, and X4 is N. In some embodiments X2 is D, and X4 is N.

[0109] Preferably X1 is present and consists of sequence SPGQG (SEQ ID NO: 81).

[0110] The L1.1 linker sequence bridging the C-terminus of one IL-10 monomer fragment and the N-terminus of the other IL- 10 monomer fragment may have any suitable sequence. It preferentially comprises a sequence that defines a conformationally restrained structure, i.e. a ‘structured’ linker. In this way, the engineered linker helps to conformationally restrain the 3D domain structures in an appropriate / desired structural orientation. Such linkers may have from about 3 to about 20 amino acid residues; from about 3 to about 16 amino acid residues; from about 4 to about 12 amino acid residues, from about 4 to about 8 amino acid residues, from about 3 to 8 amino acid residues or from about 3 to 6 amino acid residues. Beneficially, such linkers do not contain a plurality of Gly and / or Ser residues adjacent each other; for example, beneficially the linker peptide contains 5 or less adjacent Gly and / or Ser residues; suitably 3 or less adjacent Gly and / or Ser residues; 2 or less Gly and / or Ser residues; contains only isolated Gly and / or Ser residues; or in some embodiments contains no Gly and / or Ser residues. In an embodiment, the L1.1 linker comprises from 3 to 20 amino acid residues and comprises no more than 2 adjacent Gly and / or Ser residues.

[0111] According to some embodiments, the L1.1 linker comprises sequence NGGLDY (SEQ ID NO: 30), FGGLDY (SEQ ID NO: 48), YKTIT (SEQ ID NO: 31), or DKDIRDGD (SEQ ID NO: 32).

[0112] The L1.1 linker may be preceded, at its N-terminal end, by X3, i.e. amino acid residue(s) that are naturally and consecutively present at position(s) 152 up to 157 of IL- 10 as shown in SEQ ID NO: 11. Alternatively, or additionally the L1.1 linker may be followed, at its C-terminal end, by Z, i.e. amino acid residue(s) that are naturally and consecutively present at position(s) 16 up to 20 of IL- 10 as shown in SEQ ID NO: 11.

[0113] Hence, according to some embodiments, the L1.1 linker and the X3 or Z sequence at its N-terminal side or C-terminal side (X3 and Z sequences in bold have the same structure as in IL10 and they correspond to the WT IL10 sequence or could have point mutations to accommodate the linker) comprises or consists of sequence NNGGLDYL (SEQ ID NO: 71), NFGGLDYL (SEQ ID NO: 45), LYKTITPEFA (SEQ ID NO: 46), KDKDIRDGDELA (SEQ ID NO: 47), MTMKIRNNNGGLDYLP (SEQ ID NO: 77), or MTMNNGGLDYLP (SEQ ID NO: 70). Thus, in some embodiments the SC dimeric IL-10 comprises or consists of sequenceX1-SEQ ID NO: 65-X2-SEQ ID NO: 143-X3-L1.1-Z-SEQ ID NO: 66-X4-SEQ ID NO: 144, wherein X3- L1.1-Z consists of SEQ ID NO: 71, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 77, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to X1-SEQ ID NO: 65-X2-SEQ ID NO: 143-X3 L1.1-Z-SEQ ID NO: 66-X4-SEQ ID NO: 144 and that retains at least the same stability, and / or at least the same level of interaction with IL-10 receptor.

[0114] According to an embodiment, the L1.1 linker comprises or consists of GGGSGGGGS (SEQ ID NO: 147). Preferably, in this embodiment, the X3-L1.1-Z motif consists of sequence MTMKIRNGGGSGGGGSSPGQGTQSENSCTHFPGNLPN (SEQ ID NO: 148).

[0115] In some embodiments, the single chain dimeric IL-10 comprises or consists of SEQ ID NO: 2 (ORK10-002), SEQ ID NO: 3 or SEQ ID NO: 49 (ORK10-003; with or without the addition of a HiBit Tag sequence for quantification at the C-terminus of SEQ ID NO:3), or SEQ ID NO: 4 (ORK10-005), or SEQ ID NO:88 (single-chain IL-10), or SEQ ID NO: 89, or SEQ ID NO: 90; or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and that retains IL-10 activity.

[0116] In some embodiments, the single chain dimeric IL-10 comprises a first IL-10 monomer fragment that includes a substitution D84E at the residue naturally present at position 84 of SEQ ID NO:11. The single chain dimeric IL-10 may then comprise or consist of SEQ ID NO: 1 (Foldikinel 0), or SEQ ID NO: 89, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and that retains IL-10 activity

[0117] According to some embodiments, the SC dimeric IL-10 consists of a circular permutant of one of the above SC dimeric IL-10. It is thought that the use of a circular permutant could be advantageous in order to minimize a possible masking effect due to the way the fused IL34 monomer is connected to the SC dimeric IL-10 (Figure 19).

[0118] In some embodiments, the circular permutant comprises a first IL-10 monomer fragment comprising a-helices E to F of IL-10, a first SC dimeric IL-10 peptide linker, and a second IL-10 monomer fragment comprising at least a-helices A to F of IL-10, a second peptide linker, and a third IL- 10 monomer fragment comprising at least a-helices A to D of IL-10. In some embodiments, the circular permutant comprises sequence SEQ ID NO: 5 (Foldikinel 0_cut116, also called herein Foldikinel 0_cp116), or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and that retains IL- 10 activity.Resulting single chain polypeptide comprising a single chain dimeric IL- 10 fused to a IL-34 monomer and having IL-10 and IL-34 activities

[0119] According to some embodiments, the IL-10 domain is an IL-10 monomer and the single chain polypeptide having IL-10 and IL-34 activities thus comprises or consists of sequence SEQ ID NO: 15, or SEQ ID NO:16, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and that retains IL-10 and IL-34 activities.

[0120] According to some embodiments, the single chain polypeptide having IL-10 and IL-34 activities comprises, in the N-terminus to C-terminus direction, an IL-34 monomer comprising of consisting of sequence SEQ ID NO: 12, a peptide linker L1 as defined above, and a single chain dimeric IL-10 that comprises or consists of sequence X1-SEQ ID NO: 65-X2-SEQ ID NO: 143-X3-L1,1-Z-SEQ ID NO: 66-X4-SEQ ID NO: 144, wherein X3L1.1-Z consists of SEQ ID NO: 71, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 77, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and that retains IL-10 and IL-34 activities.

[0121] According to some embodiments, the single chain polypeptide having IL-10 and IL-34 activities comprises, in the N-terminus to C-terminus direction, a single chain dimeric IL-10 that comprises or consists of sequence X1-SEQ ID NO: 65-X2-SEQ ID NO: 143-X3-L1.1-Z-SEQ ID NO: 66-X4-SEQ ID NO: 144, wherein X3-L1.1-Z consists of SEQ ID NO: 71, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 77, or SEQ ID NO: 148, a peptide linker L1 as defined above, and an IL-34 monomer comprising of consisting of sequence SEQ ID NO:12, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and that retains IL-10 and IL-34 activities.

[0122] According to some embodiments, the single chain polypeptide having IL-10 and IL-34 activities comprises sequence SEQ ID NO: 19, SEQ ID NO: 145, SEQ ID NO: 37, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and that retains IL-10 and IL-34 activities. In some embodiments, the single chain polypeptide having IL-10 and IL-34 activities comprises sequence SEQ ID NO: 19, SEQ ID NO: 145, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and that retains IL-10 and IL-34 activities.

[0123] Modified dual-cytokine single chain polypeptide having IL-10 and IL-34 activities and increased half-life

[0124] According to some embodiments the single chain dual-cytokine polypeptide having IL- 10 and IL-34 activities is modified to increase its half-life. In some embodiments, saidmodification comprises one or more alterations selected from the group consisting of a fusion to human Fc antibody fragment, a fusion to albumin, and a PEGylation.

[0125] Accordingly, the invention further relates to a protein comprising the single chain dualcytokine polypeptide having IL- 10 and IL-34 activities as herein defined fused to a human immunoglobulin Fc fragment or to albumin, or to albumin fragment or an anti-albumin antibody, or anti-albumin antibody fragment, or conjugated to a poly(ethylene glycol) (PEG) molecule.

[0126] In some embodiments, in the protein, the single chain polypeptide having IL-10 and IL-34 activities comprises in the N-terminus to C-terminus direction, an IL-34 monomer, a peptide linker L1, and an IL-10 domain, wherein the IL-10 domain is an IL-10 monomer or a single chain dimeric IL-10. In some aspects the IL-10 domain is a single chain dimeric IL-10.

[0127] The single chain polypeptide having IL-10 and IL-34 activities, IL-10 monomer, single chain dimeric IL-10, IL-34 monomer, and peptide linker L1 are as disclosed above.

[0128] In some embodiments, in the protein, the IL-34 monomer comprises or consists of sequence SEQ ID NO: 12, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and that retains IL-34 activity.

[0129] In some embodiments, the IL-10 domain is an IL-10 monomer as defined above. In some embodiments, in the protein, the single chain polypeptide having IL-10 and IL-34 activities comprises or consists of sequence SEQ ID NO: 15 or SEQ ID NO: 16, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical thereto and that retains IL-10 and IL34 activities.

[0130] In some embodiments, the IL-10 domain is a single chain dimeric IL-10 as defined above. In some embodiments, in the protein, the single chain dimeric IL-10 comprises or consists of sequence X1 -SEQ ID NO: 65-X2-SEQ ID NO: 143— X3-L1.1-Z-SEQ ID NO: 66-X4-SEQ ID NO: 144, as defined above, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and that retain at least the same stability, and / or at least the same level of interaction with IL-10 receptor, as defined above.

[0131] According to some embodiments, in the protein, the single chain polypeptide having IL-10 and IL-34 activities comprises sequence SEQ ID NO: 19, SEQ ID NO: 145, SEQ ID NO: 37, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and that retains IL-10 and IL-34 activities.

[0132] In some embodiments, the protein comprises a single chain polypeptide having IL-10 and IL-34 activities and an immunoglobulin Fc fragment, preferably a humanimmunoglobulin Fc fragment. In some embodiments, the immunoglobulin Fc fragment is a human IgG 1 or lgG4 Fc fragment.

[0133] In some embodiments, the protein comprises a single chain polypeptide having IL-10 and IL-34 activities and albumin or an albumin fragment, preferably human albumin or a fragment thereof. In some embodiments, the protein comprises a single chain polypeptide having IL-10 and IL-34 activities and an anti-albumin antibody or a fragment thereof, preferably an anti-human albumin antibody or a fragment thereof.

[0134] In some embodiments, the protein comprises a single chain polypeptide having IL-10 and IL-34 activities conjugated to PEG. Process for protein pegylation has been reviewed for instance by Pfister and Morbidelli (Journal of Controlled Release, Volume 180, 2014, Pages 134-149).

[0135] Protein comprising one single chain polypeptide having IL- 10 and IL-34 activities and one immunoglobulin Fc fragment

[0136] In some embodiments, the protein comprises one SC polypeptide having IL-10 and IL-34 activities and one immunoglobulin Fc fragment.

[0137] In some embodiments, the protein comprises one SC polypeptide having IL-10 and IL-34 activities and one immunoglobulin Fc fragment, wherein the SC polypeptide having IL- 10 and IL-34 activities is fused to the immunoglobulin Fc fragment through a peptide linker L2 connecting the C-terminus of the IL-10 domain with the N-terminus of one hinge region of the Fc fragment (format ORK6-Fc-03 as shown in Figure 6). In some aspects the IL-10 domain is a SC dimeric IL-10. According to these embodiments the protein then comprises two polypeptide chains comprising or consisting of formula (I):

[0138] IL-34 monomer-L1-IL-10 domain-L2-Fc1, wherein Fc1 is a Fc domain comprising hinge-CH2-CH3, and

[0139] Fc2, wherein Fc2 is a Fc domain comprising hinge-CH2-CH3,

[0140] wherein the two polypeptide chains dimerise through the Fc1 and Fc2 domains.

[0141] In some embodiments, the protein comprises one SC polypeptide having IL-10 and IL-34 activities and one immunoglobulin Fc fragment, wherein the SC polypeptide having IL- 10 and IL-34 activities is fused to the immunoglobulin Fc fragment through a peptide linker L2 connecting the C-terminus of the one of the CH3 domains of the Fc fragment with the N-terminus of the IL-34 monomer (format ORK6-Fc-04 as shown in Figure 6). In some aspects the IL-10 domain is a SC dimeric IL-10. According to these embodiments the protein then comprises two polypeptide chains comprising or consisting of formula (II):Fc1-L2-IL-34 monomer-L1-IL-10 domain, wherein Fc1 is a Fc domain comprising hinge-CH2-CH3, and

[0142] Fc2, wherein Fc2 is a Fc domain comprising hinge-CH2-CH3,

[0143] wherein the two polypeptide chains dimerise through the Fc1 and Fc2 domains.

[0144] In some embodiments, the linker L2 connecting the single chain polypeptide having IL-10 and IL-34 activities and the immunoglobulin Fc fragment is preferably a peptide sequence that comprises from, or consists of, 3 to 50 amino acid residues, preferably 3 to 45 amino acid residues, preferably 5 to 40 amino acid residues, preferably 5 to 25 amino acids, still preferably 10 to 25 amino acids.

[0145] The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, but linkers comprising amino acids randomly selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine, and proline may also be suitable. A well-suited linker L2 according to the present disclosure contains or consists of glycine and serine residues and is for example of the format (GGGGS, SEQ ID NO: 82)p, wherein p is an integer from 1 to 8, notably from 1 to 4, advantageously 2, 3 or 4. In some embodiments, the linker L2 is (GGGGS)3(SEQ ID NO: 119). In some embodiments, the peptide linker L2 is a rigid peptide sequence composed of Glu, Ala, and Lys residues. In some embodiments, the linker is selected from the group consisting of the amino acid sequences SGGGGSGGGGS (SEQ ID NO: 120), SGGGGSGGGGSAP (SEQ ID NO: 121), NFSQP (SEQ ID NO: 122), KRTVA (SEQ ID NO: 123), GGGSGGGG (SEQ ID NO: 124), GGGGSGGGGS (SEQ ID NO: 125), GGGGSGGGGSGGGGS (SEQ ID NO: 119), THTCPPCPEPKSSDK (SEQ ID NO: 126), GGGS (SEQ ID NO: 127), EAAKEAAKGGGGS (SEQ ID NO: 128), EAAKEAAK (SEQ ID NO: 129), GGGGS (SEQ ID NO: 82), (SG)m where m is an integer comprised between 1 and 7, GGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGG (SEQ ID NO: 130), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 131), GGGGGSGGGGSGGGGS (SEQ ID NO: 146).

[0146] In some embodiments the linker L1 comprises or consist of sequence SEQ ID NO: 35, and the linker L2 comprises or consist of sequence SEQ ID NO: 146.

[0147] In some embodiments, the protein comprises SEQ ID NO: 85 (ORK6_P_13) or SEQ ID NO: 87 (ORK6_P_14), or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and that retains IL-10 and IL-34 activities.In some embodiments, the protein comprises a Fc2 domain comprising or consisting of SEQ ID NO: 86, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto.

[0148] In some embodiments, the protein comprises:

[0149] a) a first polypeptide chain comprising any one of SEQ ID NO: 85 or SEQ ID NO:

[0150] 87, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto, and

[0151] b) a second polypeptide chain comprising SEQ ID NO: 86, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto,

[0152] wherein the first and second polypeptides dimerise, and

[0153] wherein said protein retains IL-10 and IL-34 activities.

[0154] In some embodiments, the protein comprises:

[0155] a) a first polypeptide chain comprising SEQ ID NO: 85 or SEQ ID NO: 87, and b) a second polypeptide chain comprising SEQ ID NO: 86,

[0156] wherein the first and second polypeptides dimerise.

[0157] Protein comprising two single chain polypeptides having IL-10 and IL-34 activities and one immunoglobulin Fc fragment

[0158] In some embodiments, the protein comprises two single chain polypeptides having IL-10 and IL-34 activities and one immunoglobulin Fc fragment.

[0159] Preferably, the two SC polypeptides having IL-10 and IL-34 activities are identical. In some embodiments, the protein comprises two polypeptide chains that each comprise, in the N-terminus to C-terminus direction, an IL-34 monomer, a peptide linker L1, an IL-10 domain which is an IL-10 monomer or a SC dimeric IL-10 monomer, a peptide linker L2, and a Fc domain, wherein the two polypeptide chains dimerise through their Fc domains, thereby forming a Fc fragment (format QRK6-Fc-02 as shown in Figure 6). In some embodiments the peptide linker L2 connects the C-terminus of the IL-10 domain with the hinge of the Fc domain; the protein then comprises two polypeptide chains comprising or consisting of formula (III):

[0160] (i) IL-34 monomer-L1-IL-10 domain-L2-Fc1 wherein Fc1 is a Fc domain comprising hinge-CH2-CH3, and

[0161] (ii) IL-34 monomer-L1-IL-10 domain-L2-Fc2 wherein Fc2 is a Fc domain comprising hinge-CH2-CH3,

[0162] wherein the two polypeptide chains dimerise through the Fc1 and Fc2 domains.In some embodiments, the protein comprises two polypeptide chains that each comprise, in the N-terminus to C-terminus direction, a Fc domain, a peptide linker L2, an IL-34 monomer, a peptide linker L1, and an IL-10 domain which is an IL-10 monomer or a SC dimeric IL-10 monomer, wherein the two polypeptide chains dimerise through their Fc domains, thereby forming a Fc fragment (format ORK6-Fc-01 as shown in Figure 6). In some embodiments the peptide linker L2 connects the C-terminus of the Fc fragment with the N-terminus of the IL-34 monomer; the protein then comprises two polypeptide chains comprising or consisting of formula (IV):

[0163] (i) Fc1-L2-IL-34 monomer-L1-IL-10 domain, wherein Fc1 is a Fc domain comprising hinge-CH2-CH3, and

[0164] (ii) Fc2-L2-IL-34 monomer-L1-IL-10 domain, wherein Fc2 is a Fc domain comprising hinge-CH2-CH3

[0165] wherein the two polypeptide chains dimerise through the Fc1 and Fc2 domains.

[0166] In some embodiments, the linker L2 connecting the single chain polypeptide having IL- 10 and IL-34 activities and the immunoglobulin Fc fragment is preferably a peptide sequence that comprises from, or consists of, 3 to 45 amino acid residues, preferably 5 to 40 amino acid residues, preferably 5 to 25 amino acids, still preferably 10 to 25 amino acids.

[0167] The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, but linkers comprising amino acids randomly selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine, and proline may also be suitable. A well-suited linker L2 according to the present disclosure contains or consists of glycine and serine residues and is for example of the format (GGGGS, SEQ ID NO: 82)p, wherein p is an integer from 1 to 8, notably from 1 to 4, advantageously 2, 3 or 4. In some embodiments, the linker L2 is (GGGGS)3(SEQ ID NO: 119). In some embodiments, the linker is selected from the group consisting of the amino acid sequences SGGGGSGGGGS (SEQ ID NO: 120), SGGGGSGGGGSAP (SEQ ID NO: 121), NFSQP (SEQ ID NO: 122), KRTVA (SEQ ID NO: 123), GGGSGGGG (SEQ ID NO: 124), GGGGSGGGGS (SEQ ID NO: 125), GGGGSGGGGSGGGGS (SEQ ID NO: 119), THTCPPCPEPKSSDK (SEQ ID NO: 126), GGGS (SEQ ID NO: 127), EAAKEAAKGGGGS (SEQ ID NO: 128), EAAKEAAK (SEQ ID NO: 129), GGGGS (SEQ ID NO: 82), (SG)m where m is an integer comprised between 1 and 7, GGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGG (SEQ ID NO: 130), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 131), or GGGGGSGGGGSGGGGS (SEQ ID NO: 146)In some embodiments the linker L1 comprises or consist of sequence SEQ ID NO: 35, and the linker L2 comprises or consist of sequence SEQ ID NO: 146.

[0168] In some embodiments, the protein comprises sequence SEQ ID NO: 83 (ORK6_P_11), SEQ ID NO: 84 (ORK6_P_12), or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and that retains IL-10 and IL-34 activities.

[0169] Fc fragment or Fc domain

[0170] In some embodiments, the immunoglobulin Fc fragment or Fc domain is of the isotype IgG, such as lgG1, lgG2, lgG3, and lgG4, preferably lgG1 or lgG4.

[0171] Accordingly, in some embodiments the protein comprises a Fc fragment that comprises two polypeptides that comprise a hinge region, an IgG CH2 domain and an IgG CH3 domain. Accordingly, in some embodiments the protein comprises two polypeptide chains that each comprise a Fc domain that comprise a hinge region, an IgG CH2 domain and an IgG CH3 domain.

[0172] Preferably the Fc fragment is silenced, i.e. the Fc fragment is mutated to reduce or eliminate binding to Fc gamma receptors and / or complement protein C1q and thereby reduce or abolish immune effector functions.

[0173] In certain embodiments, a human IgG heavy chain Fc fragment or domain extends from Cys226 to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc fragment or domain may or may not be present, without affecting the structure or stability of the Fc fragment. Unless otherwise specified herein, numbering of amino acid residues in the IgG or Fc region is according to the EU numbering system for antibodies, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0174] In certain embodiments, Fc fragment or domain refers to an immunoglobulin IgG heavy chain constant region comprising a hinge region (starting at Cys226), an IgG CH2 domain and CH3 domain. The term “hinge region” or “hinge sequence” as used herein refers to the amino acid sequence that is, in some instance, located between the linker L2 and the CH2 domain. In certain embodiments, the hinge region comprises the amino acid sequence CPPCP (SEQ ID NO: 21), a sequence found in the native lgG1 or lgG2 hinge region at positions 239-243, sequence CPRCP (SEQ ID NO: 22), a sequence found in the native lgG3 hinge region at positions 239-241 B, sequence CPSCP (SEQ ID NO: 23), a sequence found in the native lgG4 hinge region at positions 239-243, to facilitate dimerization. In certain other embodiments, the Fc fragment or domain starts at the hingeregion and extends to the C-terminus of the IgG heavy chain. In certain particular embodiments, the Fc fragment or domain comprises the Fc fragment or domain of human lgG1, lgG2, lgG3 or lgG4. In certain particular embodiments, the Fc fragment or domain comprises the CH2 and CH3 domain of lgG4. In certain other particular embodiments, the Fc fragment or domain comprises the CH2 and CH3 domain of lgG1. In certain embodiments, the IgG CH2 domain starts at Ala 231. In certain other embodiments, the CH3 domain starts at Gly 341. It is understood that the C-terminus Lys residue of human IgG can be optionally absent. In some embodiments, the Fc fragment or domain is from human lgG1 heavy chain as shown in SEQ ID NO: 151. The lgG1 Fc fragment typically comprises or consists of sequence SEQ ID NO: 152, or amino acids 1-226 or 1-225 of SEQ ID NO: 152 (Fc with absent C-terminal Lys, or Gly-Lys), or a silenced variant thereof.

[0175] In certain embodiments, the Fc fragment is a human lgG4 Fc comprising substitution S228P in the hinge region and R409K in the CH3 region. In certain embodiments, the Fc fragment is engineered to modulate Fc mediated antibody functions. In some embodiments, the Fc fragment of the present disclosure does not induce antibody dependent cellular cytotoxicity (ADCC) and / or is an “Fc silent” antibody. Examples of silent lgG4 Fc fragments comprise mutations at positions 234, 235, 236, 237, 238, 265 and 329 in the lgG4 Fc amino acid sequence (Ell numbering). For example, lgG4 Fc silent fragment may include any one of the following mutations or combinations of mutations: F234A / L235A (FALA); L235E; F234A / L235A / G237A / P238S; and F234S / L235T / G236R (STR).

[0176] Examples of silent IgG1 Fc fragments comprise mutations at positions 234, 235, 236, 239, 265, 297, 329 and / or 331 in the lgG1 Fc amino acid sequence (Ell numbering) corresponding to positions 14, 15, 16, 19, 45, 77, 109 and / or 112 of SEQ ID NO: 152. Another silent lgG1 Fc fragment comprises the N297A mutation, which results in aglycosylated or non-glycosylated Fc fragments. For example, lgG1 Fc silent fragment may include any one of the following mutations or combinations of mutations: D265A / P329A; L234F / L235Q / K322Q (FQQ); L234A / L235A (LALA); L234A / L235A / K322A (LALAKA); N297Q (aglycosyl); L234F / L235E / P331S (FES); L234A / L235A / G237A (LALAGA); L234A / L235E; L234A / L235A / G237A / P238S / H268A / A330S / P331 S; L234A / L235A / P329G (LALAPG), e.g. an lgG1 Fc comprising or consisting of SEQ ID NO: 156 or SEQ ID NO: 157; F234S / L235T / G236R (STR), e.g. an lgG1 Fc comprising or consisting of SEQ ID NO: 158, SEQ ID NO: 159, or SEQ ID NO: 160; L234A / L235A / P329S (LALAPS) e.g. an lgG1 Fc comprising or consisting of SEQ ID NO: 161 or SEQ ID NO: 162, L234A / L235A / D265 (LALADS), e.g. an lgG1 Fc comprising or consisting of SEQ ID NO: 163 or SEQ ID NO: 164, and G236R / L328R.For certain proteins whose Fc Region-containing first and second polypeptide chains are not identical, it is desirable to reduce or prevent homodimerization from occurring between the CH2-CH3 domains of two first polypeptide chains or between the CH2-CH3 domains of two third polypeptide chains. The CH2 and / or CH3 Domains of such polypeptide chains need not to be identical in sequence, and advantageously are modified to foster complexing between the two polypeptide chains. For example, an amino acid substitution (preferably a substitution with an amino acid comprising a bulky side group forming a "knob", e.g., tryptophan) can be introduced into the CH2 or CH3 Domain such that steric interference will prevent interaction with a similarly mutated domain and will obligate the mutated domain to pair with a domain into which a complementary, or accommodating mutation has been engineered, i.e., "the hole" {e.g., a substitution with serine, alanine or valine). Such sets of mutations can be engineered into any pair of polypeptides comprising CH2-CH3 domains that forms a Fc Region to foster heterodimerization (also called knobs-in-holes (KIH) Fc heterodimerization). Methods of protein engineering to favor heterodimerization over homodimerization are well known in the art, in particular with respect to the engineering of immunoglobulin-like molecules, and are encompassed herein (see e.g., Ridgway et al. (1996), Protein Engr. 9:617-621, Atwell et al. (1997), J. Mol. Biol.

[0177] 270: 26-35, and Xie etal. (2005), J. Immunol. Methods 296:95-101; each of which is hereby incorporated herein by reference in its entirety).

[0178] A preferred knob is created by modifying an IgG Fc Region to contain the modification T366W. A preferred hole may also be created by modifying an IgG Fc Region to contain the modification T366S, L368A and Y407V.

[0179] According to some embodiments of the invention, the first Fc fragment comprises an S354C or Y349C mutation and the second heavy chain comprises a Y349C or S354C mutation.

[0180] In some embodiments, the Fc fragment or Fc pair comprises or consists of sequence SEQ ID NO: 24 (lgG4 Fc (knob)) and / or SEQ ID NO: 165 (lgG4 Fc FALA (hole)), or SEQ ID NO: 153 (lgG4 silent FALA (R409K mutation, knob) and / or SEQ ID NO: 155 (lgG4 silent FALA (R409K mutation, hole).

[0181] In some embodiments, the Fc fragment or Fc pair comprises or consist of sequence SEQ ID NO: 171 (lgG1 murine silent (knob)) and / or SEQ ID NO: 172 (lgG1 murine silent (hole)).

[0182] In some embodiments, the lgG1 Fc fragment or Fc pair comprises or consist of sequence SEQ ID NO: 158 (lgG1 silent STR (knob)) and / or SEQ ID NO: 159 (lgG1 silent STR (hole)); SEQ ID NO: 156 (lgG1 silent LALAPG (knob)) and / or SEQ ID NO: 157 (lgG1 silent LALAPG (hole)); SEQ ID NO: 161 (lgG1 silent LALAPS (knob)) and / or SEQ ID NO:162 (lgG1 silent LALAPS (hole)); or SEQ ID NO: 163 (lgG1 silent LALADS (knob)) and / or SEQ ID NO: 164 (lgG1 silent LALADS (hole)).

[0183] Pharmaceutical composition

[0184] The invention further relates to a pharmaceutical composition comprising the single chain polypeptide having IL-10 and IL-34 activities and a pharmaceutically acceptable carrier.

[0185] A suitable pharmaceutically acceptable ‘carrier’ (such as diluents, adjuvants, excipients or vehicles) is described in " Remington's Pharmaceutical Sciences" by E. W. Martin. Pharmaceutical compositions of the invention are formulated to conform to 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, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The phrase ‘pharmaceutically acceptable’ or ‘pharmacologically-acceptable’ refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human or a non-human mammal.

[0186] Acceptable pharmaceutical vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical vehicles can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilising, thickening, lubricating and colouring agents may be used. When administered to a subject, the pharmaceutically acceptable vehicles are preferably sterile. Water is a suitable vehicle particularly when the compound of the invention is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles, particularly for injectable solutions. Suitable pharmaceutical vehicles also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or buffering agents.The medicaments and pharmaceutical compositions of the invention can take the form of liquids, solutions, suspensions, lotions, gels, tablets, pills, pellets, powders, modified-release formulations (such as slow or sustained-release), suppositories, emulsions, aerosols, sprays, capsules (for example, capsules containing liquids or powders), liposomes, microparticles or any other suitable formulations known in the art. Other examples of suitable pharmaceutical vehicles are described in Remington's Pharmaceutical Sciences, Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th ed., 1995, see for example pages 1447-1676.

[0187] In some embodiments the therapeutic compositions or medicaments of the invention are formulated in accordance with routine procedures as a pharmaceutical composition adapted for oral administration (more suitably for human beings). Compositions for oral delivery may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Thus, in various embodiments, the pharmaceutically acceptable vehicle may be a capsule, tablet or pill.

[0188] Orally administered compositions may contain one or more agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavouring agents such as peppermint, oil of Wintergreen, or cherry; colouring agents; and preserving agents, to provide a pharmaceutically palatable preparation. When the composition is in the form of a tablet or pill, the compositions may be coated to delay disintegration and absorption in the gastrointestinal tract, so as to provide a sustained release of active agent over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compositions. In these dosage forms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These dosage forms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A time delay material such as glycerol monostearate or glycerol stearate may also be used. Oral compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such vehicles are preferably of pharmaceutical grade. For oral formulations, the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine. The person skilled in the art is able to prepare formulations that will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Suitably, the release will avoid the deleterious effects of the stomach environment, either by protection of the peptide (or derivative) or by release of the peptide (or derivative) beyond the stomach environment, such as in the intestine. To ensure full gastric resistance a coating impermeable to at leastpH 5.0 would be essential. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac, which may be used as mixed films.

[0189] To aid dissolution of the therapeutic agent(s) into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride. Potential nonionic detergents that could be included in the formulation as surfactants include: lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 20, 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants, when used, could be present in the formulation of the peptide or nucleic acid or derivative either alone or as a mixture in different ratios.

[0190] Typically, compositions for intravenous administration comprise sterile isotonic aqueous buffer. Where necessary, the compositions may also include a solubilising agent.

[0191] Mixtures of the polypeptides as described herein may be prepared in water suitably mixed with one or more excipients, carriers, or diluents. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form may be sterile and may be sufficiently fluid to enable injection by an appropriate syringe. Suitably, the composition is stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0192] For parenteral administration in an aqueous solution, for example, the solution may be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, subcutaneous, and intracerebroventricular, intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art.

[0193] Another suitable route of administration for the pharmaceutical compositions of the invention is via pulmonary or nasal delivery.

[0194] Additives may be included to enhance cellular uptake of a therapeutic agent of the invention, such as the fatty acids, oleic acid, linoleic acid and linolenic acid.

[0195] Therapeutic applications

[0196] The single chain polypeptide having IL-10 and IL-34 activities of the invention, or pharmaceutical composition comprising it, is for use as a medicament.

[0197] Accordingly, the invention further relates to a method of treatment which comprises administering the single chain polypeptide having IL-10 and IL-34 activities of the invention to a subject in need thereof.

[0198] The subject may be a human or non-human mammal such as a rodent (mouse or rat), a primate (e.g. a monkey), a pig, etc.

[0199] The subject in need thereof is in particular a subject suffering from an inflammatory and / or autoimmune and / or neuroinflammatory disease. The single chain polypeptide having IL- 10 and IL-34 activities is designed to preferentially activate myeloid cells (monocytes, macrophages, dendritic cells) that co-express CSF1R and IL-1 OR - and that induce antiinflammatory responses - over CD8 T cells and B cells expressing IL-1 OR that could induce cytotoxic responses and auto-antibodies when activated.

[0200] Accordingly, the invention further relates to a single chain polypeptide according the invention for use for treating an inflammatory and / or autoimmune and / or neuroinflammatory disease. Also provided is a method of treating an inflammatory and / or autoimmune and / or neuroinflammatory disease which comprises administering the single chain polypeptide having IL- 10 and IL-34 activities of the invention to a subject in need thereof.

[0201] In some embodiments, the inflammatory and / or autoimmune disease is a disease of the central nervous system (CNS) such as an infectious inflammatory disease (e.g. meningitis, ventriculitis, cerebritis / brain abscesses, subdural empyema, epidural empyema, and encephalitis) or a non infectious inflammatory disease (e.g. autoimmune encephalitis,neuromyelitis optica spectrum disorders (NMOSD), anti-GQ1b IgG antibody syndrome, sarcoidosis or vasculitis, may also affect the brain and spinal cord.

[0202] Treatment regimens may vary, and often depend on the type and / or location of the disease, the stage I progression of the disease, and / or the health and age of the patient. The skilled clinician will be able to determine a suitable therapeutic treatment regime under each circumstance.

[0203] The invention will be further illustrated in view of the following figures and examples. For clarification, ORK6_P_Number from Table 1 is referred in Figures and example experiments by ORK6-Number

[0204] FIGURES

[0205] Figure 1. ORK6 - shows selectivity towards myeloid cells compared to lymphoid cells. (A) PBMC from healthy donors were plated o / n in 96-well plates and stimulated next day with IL10, ORK1 (ORK1-068) or ORK6 (GRK6-004) for 20 min at 37°C. Cells were stained with CD14 Alexa647 (Biolegend) and CD3 APC / Cy7 (Biolegend), fixed with 4% PFA and permeabilized for intracellular staining with 80% MetOH. Cells were stained with AlexaFluor488 pSTAT3 (pY705) (BD) for 1h. (B) Samples were analyzed with LSRII cytometer and MFI values obtained from STAT3 activation were plotted using GraphPad Prism software. One representative result with two technical replicates is shown as mean with error bar depicting the SEM, similar results were obtained from 4-5 different donors. Each biological replicate was normalized by assigning the highest I L10 M Fl value of the top concentration as 100% and the lowest MFI value of an untreated control as 0%. The MFI from the samples treated with ORK1 and ORK6 was normalized accordingly.

[0206] Figure 2. ORK6 is less potent than IL-10 at activating B cells. (A) B cells were isolated from human PBMCs using positive microbeads selection (Miltenyi). The isolated lymphocytes were stimulated with CpG (T-independent conditions) and IL10, ORK1-068 or ORK6-004 at dO and d2. (B), (C) After 7 days cell supernatants were obtained for the determination of IgG levels by ELISA and analysis of plasma cell differentiation (PCs) by flow cytometry. Similar results were obtained when stimulating with CD40L (T-dependent conditions). Independent data of 3 independent experiment are shown in the graph.

[0207] Figure 3. ORK6 is less potent than IL-10 at activating CD8 T cells. (A) CD8 T cells were isolated from human PBMCs using positive microbeads selection (Miltenyi). The lymphocytes were stimulated with anti-CD3 and anti-CD28 for 72h. Then, cells were counted, replated and treated with IL10, ORK1-068 and ORK6-004 with IL-2 for 48h. (B)For cytokine measurement cell supernatants were obtained and GzmB was detected by ELISA. Independent data of 3 experiments is shown in the graph.

[0208] Figure 4. ORK6 induces the differentiation of monocyte-derived macrophages. (A) PBMCs were isolated from human buffy coats and CD14+ cells were purified by positive microbead selection (Miltenyi). Monocytes were cultured for 7 days in the presence or absence of: CSF1, IL-34, ORK1-068 or ORK6-004. Cytokines were added every 2 days.

[0209] (B) (C) At day 7 cells were counted and characterized by FACS (CD163, CD86).

[0210] Figure 5. ORK6 induces regulatory macrophages. (A) PBMCs were isolated from human buffy coats and CD14+ cells were purified by positive microbead selection (Miltenyi). Monocytes were cultured for 7 days in the presence or absence of: CSF1, IL-34, ORK1-068 or ORK6-004. (B) Cytokines were added every 2 days. At day 7 cells were stimulated with LPS±IFNg and TNFa levels in the supernatants were determined by ELISA 17h after stimulation.

[0211] Figure 6. Structural organisation of different proteins comprising a single chain polypeptide having IL-10 and IL-34 activities and an immunoglobulin Fc fragment.

[0212] Symmetric molecules comprising two single chain polypeptides having IL-10 and IL-34 activities and one immunoglobulin Fc fragment (A) Format ORK6-Fc-O1 from ORK6_P_011, Format ORK6-FC-02 from ORK6_P_012 and asymmetric molecules comprising one single chain polypeptide having IL-10 and IL-34 activities and one immunoglobulin Fc fragment (B) Format ORK6-Fc-03 from ORK6_P_013, Format ORK6-Fc-04 from ORK6_P_014. Fc fragment is from lgG4. In asymmetric molecules, Fc fragment comprises stabilization mutation S228P and silent FALA.

[0213] Figure 7. ORK6 shows selectivity towards myeloid cells compared to lymphoid cells. (A) PBMC from healthy donors were plated o / n in 96-well plates and stimulated next day with IL10, ORK6-004, ORK6-013 or ORK6-014 for 20 min at 37°C. Cells were stained with CD14 Alexa647 (Biolegend) and CD3 APC / Cy7 (Biolegend), fixed with 4% PFA and permeabilized for intracellular staining with 80% MetOH. Cells were stained with AlexaFluor488 pSTAT3 (pY705) (BD) for 1h. (B) Samples were analyzed with LSRII cytometer and MFI values obtained from STAT3 activation were plotted using GraphPad Prism software. One representative result with two technical replicates is shown as mean with error bar depicting the SEM. Each biological replicate was normalized by assigning the highest IL10 MFI value of the top concentration as 100% and the lowest MFI value of an untreated control as 0%. The MFI from the samples treated with ORK1 and ORK6 was normalized accordingly.

[0214] Figure 8. ORK6-13 Binding to Monocytes is CSF1 -Dependent. (A) PBMC from healthy donors were plated o / n in 96-well plates and treated when specified with saturatingconcentrations of rCSF1, rlL-10 or both (5 min, 37°C), after that cells were washed and resuspend in buffer containing sodium azide 0,1%. Then, ORK6-013 is added and incubated 20’ on ice. Cells were washed and resuspended in stain buffer containing the mix of antibodies: CD14 Alexa 647, CD3 APC / Cy7 and anti-human lgG4-FC PE. Finally, cells were resuspended in stain buffer. (B) Samples were analysed with LSRII cytometer and MFI values obtained from anti-human lgG4-FC PE were plotted using GraphPad Prism software. Flow cytometry analysis shows dose-dependent binding of ORK6-013 to CD14+ monocytes. Binding is reduced upon CSF1R blocking, confirming specificity.

[0215] Figure 9. ORK6 induces the differentiation of monocyte-derived macrophages. (A) PBMCs were isolated from human buffy coats and CD14+ cells were purified by positive microbead selection (Miltenyi). Monocytes were cultured for 7 days in the presence or absence of: CSF1, IL-34, ORK6-004 or ORK6-013. Cytokines were added every 2 days. At day 7 cells were counted (B) and characterized by LSRII cytometer (CD163 and CD86) (C).

[0216] Figure 10. ORK6 induces regulatory macrophages.

[0217] (A) PBMCs were isolated from human buffy coats and CD14+ cells were purified by positive selection (Miltenyi). Monocytes were cultured for 7 days in the presence or absence of: CSF1, IL-34, ORK6-004 or ORK6-013. Cytokines were added every 2 days. (B) At day 7 cells were stimulated overnight with LPS±IFNg and TNFa levels in the supernatants were determined by ELISA.

[0218] Figure 11. ORK6 is less potent than IL-10 at activating B cells. (A) B cells were isolated from human PBMCs using positive microbeads selection (Miltenyi). The isolated lymphocytes were stimulated with CpG (T-independent conditions) and IL-10, ORK6-004 or ORK6-013 at dO and d2. (B) After 7 days cells were harvested for the analysis of plasma cell differentiation (PCs) by flow cytometry. Similar results were obtained when stimulating with CD40L (T-dependent conditions).

[0219] Figure 12. ORK6 is less potent than IL-10 at activating CD8 T cells.

[0220] (A) CD8 T cells were isolated from human PBMCs using positive microbeads selection (Miltenyi). The lymphocytes were stimulated with soluble anti-CD3 and anti-CD28 for 72h. Then, cells were counted, replated and treated with IL- 10, ORK6-004 or ORK6-013 with IL-2 for 48h. (B) For cytokine measurement cell supernatants were obtained and GzmB was detected by ELISA.

[0221] Figure 13. ORK6-lnduced Macrophages suppress T cell activation.

[0222] (A) In a macrophage-T cell co-culture assay, ORK6-differentiated macrophages suppress TNFa, I FNy, and GzmB in activated T cells. CD14+ cells were purified from total PBMC of healthy donors. Monocytes were cultured for 5 days in the presence or absence of: CSF1, IL-34, ORK6-004 or ORK6-013 at 20ng / ml. Cytokines were added every 2 days. At day 5cells were counted and replated to a 96 round well plate (40.000 cells / well) and last dose of cytokines was added. After 2 days CD3 T cells from another donor were purified and added to macrophages after refreshing media. 120.000 CD3 T cells were added per well for a final ratio of 1:3, macrophage: T cell. (B) Macrophages were activated with LPS and after 2 days supernatants were collected and I FNg, GzmB, and TNFa production in cocultures were determined by ELISA.

[0223] EXAMPLES

[0224] Our goal is to create a dual molecule that can activate IL- 10 signaling exclusively in cells expressing CSF1R, which are predominantly myeloid cells and no other cells such as CD8+ T cells. To achieve this, we have engineered variants of IL-10 and / or Foldikine-10 with reduced affinity to I L1 ORA and I L1 ORB linked to a CSF 1 or I L-34 monomer. The CSF1 R targeting component acts as a targeting tool to circulating monocytes, facilitating preferential binding of IL-10 to these cells.

[0225] Example 1: Methods

[0226] Plasmid generation

[0227] All plasmids generated in this work were assembled following the Gibson method (Gibson DG et al. Nat Methods. 2009 May;6(5):343-5). When required, Integrated DNA Technologies (IDT) Corporation performed gene synthesis (gBIock double-stranded fragments) and oligonucleotides synthesis. Gene amplification was carried out with Phusion DNA polymerase (Thermo Fisher Scientific) and transformed in Escherichia coli DH5-alpha competent cells (NEB).

[0228] For mammalian protein expression in ExpiCHO cells, the vector used was pcDNA3.1 (V790-20, Invitrogen). All the plasmids were verified by Sanger sequencing (Eurofins Genomics).

[0229] Expression in CHO cells

[0230] ExpiCHO protein expression kit cells were purchased from Thermofisher (A29133). For a low-scale production (2.5 mL), 24 deep well plates were used (AXYPDW10ML24CS, Merck) and covered by gas-permeable film (ThermoFisher). The day -1 of production, ExpiCHO-cells were split to a final density of 3 x 106viable cells / mL. Grown at 37C, 8% CO2 and shaking at 110rpm. The Day 0 of production, ExpiCHO cells were split to a final density of 3 x 106viable cells / mL per sample aliquot 2.5mL of the cell suspension into one well of the plate. Per sample, 2ug of DNA to 200ul of cold OptiPRO SFM & 9.0 pL ExpiFectamine CHO Reagent was added. Then, 200 pL of the complexation mixture wasadded to each relevant well. All the plate was covered with a gas permeable seal and placed at 37°C, 8% CO2 and 225rpm. The day 1 after 18–22 hours post-transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed was added to each well. For an entire plate of 24 wells, 400 pL ExpiFectamine CHO Enhancer and 16 mL ExpiCHO Feed in a conical tube were mix. From this mixture, 600 pLwas added to each well of the plate. Place at 37C, 8% CO2 and 225rpm. After 4 days the samples were harvested. Plates were centrifuged 5 minutes at 4600 rpm at room temperature. Supernatants were aliquoted, snap frozen liquid nitrogen and stored at -80°C.

[0231] Protein purification

[0232] Affinity purification of Fc proteins was performed with HiTrap MabSelect PrismA. Binding buffer was PBS, 850 mM NaCI. Elution was performed with the following buffer: 0.1 M Na-citrate pH 3.3. Samples were neutralized right after elution with 1 M Tris-HCI, pH 9 (10% v / v). Purity, as defined by the fraction of Fc proteins, was determined by HPLC-SEC. SDS-PAGE of purified products was performed to verify the fragmentation and / or aggregation status of the final material.

[0233] Example 2: in v / 'tro functional evaluation of the dual IL34-IL10 cytokine PBMCs were isolated from human buffy coats and CD14+ cells were purified by positive microbead selection (Miltenyi). Monocytes (0.5 million / ml) were cultured for 7 days in the presence or absence of: rCSF1, IL-34, ORK1-068 or ORK6-004 (1,08nM per treatment) or untreated condition. Cytokines were added every 2 days. At day 7 alive cells were counted and characterized by FACS (CD163, CD86). The results show that ORK6 in comparison with IL-34, similarly to ORK1 in comparison with CSF1, induces the differentiation of regulatory monocyte-derived macrophages, as identified by the CD163 expression (Figure 4). Further characterization was performed to determine the phenotype of the induced monocyte-derived macrophages.

[0234] PBMCs were isolated from human buffy coats and CD14+ cells were purified by positive selection (Miltenyi). Monocytes (0.5 million / ml) were cultured for 7 days in the presence or absence of: rCSF1, IL-34, ORK1-068 or ORK6-004 (1,08nM). Cytokines were added every 2 days. At day 7, cells were harvested and stimulated with LPS, with or without IFNy. TNF-a levels in the supernatants were determined by ELISA, 17h after stimulation. CSF-1 -differentiated macrophages produce TNF-a, which is further enhanced by the addition of IFNy. In contrast, ORK1 and ORK6-differentiated macrophages produce less TNF-a, even when stimulated with LPS+IFNy (Figure 5). These results show that ORK6-induced macrophages have a regulatory phenotype.

[0235] PBMC (150,000 cells / well) from healthy donors were plated on in 96-well plates and stimulated next day with rl L10, ORK1-068, or ORK6-004 (15 nM per treatment, 8 dilutions 1 / 3) for 20 min at 37°C. Cells were stained with CD14 Alexa647 (Biolegend) and CD3 APC / Cy7 (Biolegend), fixed with 4% PFA and permeabilized for intracellular staining with 80% MetOH. Cells were stained with AlexaFluor488 pSTAT3 (pY705) (BD) for 1h. Samples were analyzed with LSRII cytometer and MFI values obtained from STAT3 activation were plotted using GraphPad Prism software. One representative result with two technical replicates is shown as mean with error bar depicting the SEM. Each biological replicate was normalized by assigning the highest IL10 MFI value of the top concentration as 100% and the lowest MFI value of an untreated control as 0%. The MFI from the samples treated with ORK1 and ORK6 variants was normalized accordingly. These results show that ORK6 variants shows selectivity towards myeloid cells compared to lymphoid cells, in contrast with IL-10 (Figure 1).

[0236] ORK6, ORK1 and IL- 10, respective potencies at activating CD8 T and B cells were further characterized.

[0237] T cells were isolated from human PBMCs using positive microbeads selection (Miltenyi). The lymphocytes (1.5 million / ml) were stimulated with anti-CD3 (ThermoFisher) (1 pg / ml) and anti-CD28 (ThermoFisher) (3 pg / ml) for 72h. Then, cells were splited, replated and treated with IL10, ORK1-068, ORK6-004 10 nM per treatment, 6 dilutions 1 / 4) with IL-2 (5 ng / ml) for 48h. Cell supernatants were obtained, and Granzyme B (GzmB) was detected by ELISA. The results show that ORK6-004 is less potent than IL-10 and ORK1-068 at activating CD8 T cells (Figure 3).

[0238] B cells were isolated from human PBMCs using positive microbeads selection (Miltenyi). The isolated lymphocytes (50,000 cells / well) were stimulated with CpG (InvivoGene) (1 pg / ml) and IL10, ORK1-068 orORK6-004 (10 nM per treatment, 8 dilutions 1 / 3) at dO and d2. After 7 days cell supernatants were obtained for the determination of IgG levels by ELISA and PCs by flow cytometry. Similar results were obtained when stimulating with CD40L. The results show that ORK6-004 is less potent than ORK1-068 and IL-10 at activating B cells (Figure 2).Example 3: Further characterization of receptor binding and selectivity of the dual cytokine single chain IL34-IL10 polypeptide, with or without an Fc

[0239] Flow cytometry analyses were conducted with ORK6 showing dose-dependent binding of ORK6-013 to CD14+ monocytes, and reduction of binding upon CSF1 R blocking, thereby confirming that ORK6 binding to monocytes is CSF1 -dependent, indicating functional CSF1R engagement (Figure 8).

[0240] Functional validation of the dual activity of the dual cytokine single chain IL34-IL10 polypeptide was further performed.

[0241] Induction of regulatory macrophages was analyzed by cultivating CD14+ monocytes treated with CSF1, IL-34, ORK6-004 orORK6-013 over 7 days. Cytokine production (TNFa) was analyzed by ELISA, and phenotypic markers were measured by flow cytometry. The results demonstrate that ORK6 (ORK6-004 or ORK6-013) induce macrophage differentiation (N=3-5 donors), with increased CD163 (and CD206) and reduced CD86 (and H LA-DR). The phenotype of the induced macrophages is consistent with the phenotype of regulatory macrophages (Figure 9). Differentiated macrophages were further stimulated by LPS and IFNy: TNFa production was found decreased in ORK6-004 and ORK6-013 induced macrophages, compared to IL-34 or CSF-1 induced macrophages (Figure 10).

[0242] ORK6 induced macrophages were further shown to suppress T cell activation (Figure 13). ORK6 (ORK6-004 or ORK6-013), CSF-1 or IL-34-differentiated macrophages, stimulated by LPS, were co-cultured with CD3+ T cells at 1:3 (macrophage: T cell) ratio. T cell cytokine production (TNFa, IFNy, GzmB) and were analyzed after 48 hours. Data from 3 independent donors show consistently that ORK6-differentiated macrophages suppress TNFa, IFNy, and GzmB in activated T cells, hence that ORK6-differentiated macrophages reduce inflammatory T cell responses. Microscopy images illustrate the reduction in T-cell proliferation (data not shown).

[0243] The safety profile of ORK6 is favorable as ORK6 (ORK6-004 or ORK6-013), is less potent than IL-10 in promoting plasma cell differentiation (P = 0.0473) and therefore induce reduced B cell activation compared to IL-10 (Figure 11). Furthermore, GzmB production measured by ELISA in aTCR-stimulated CD8+ T cells treated with ORK6 (ORK6-004 or ORK6-013) demonstrates attenuated CD8 activation (P = 0.0075-0.0076; Figure 12).

[0244] Example 4: Generation of a dual cytokine single chain IL34-IL10 polypeptide The principles of protein expression are as follows.

[0245] In bold is highlighted the signal peptide (SP). Signal peptide is NOT included in final ORF as it is processed. In italic is included the tag used for purification (TEV-his-hibit). Tagis included in the ORF. Linkers are underlined. Some proteins are the result of the cotransfection of two vectors. The two vectors are displayed. Knob chain or hole chain is specified. For context. Specified origin of the signal peptide at the end of the document. For Fc-related constructs the signal peptide used in all cases MEWSWVFLFFLSVTTGVHS

[0246] > SEQ ID NO: 134 ORK6_001(IL10-IL34)-tev-his-hibit MEWSWVFLFFLSVTTGVHSSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQ MKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGEN LKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKI RNGGGGGSGGSGGSGGSGGSGGSGGSGGSGGGNEPLEMWPLTQNEECTVTGFLRD KLQYRSRLQYMKHYFPINYKISVPYEGVFRIANVTRLQRAQVSERELRYLWVLVSLSATE SVQDVLLEGHPSWKYLQEVETLLLNVQQGLTDVEVSPKVESVLSLLNAPGPNLKLVRPK ALLDNCFRVMELLYCSCCKQSSVLNWQDCEVPSPQSCSPEPSLQYAATQLYPPPPWSP SSPPHSTGSVRPVRFQGEGLLPGENLYFQSGGHHHHHHGGGVSGWRLFKKIS* > SEQ ID NO: 135 ORK6_005- tev-his-hibit MPRGFTWLRYLGIFLGVALGNEPLEMWPLTQNEECTVTGFLRDKLQYRSRLQYMKHYF PINYKISVPYEGVFRIANVTRLQRAQVSERELRYLWVLVSLSATESVQDVLLEGHPSWKY LQEVETLLLNVQQGLTDVEVSPKVESVLSLLNAPGPNLKLVRPKALLDNCFRVMELLYCS CCKQSSVLNWQDCEVPSPQSCSPEPSLQYAATQLYPPPPWSPSSPPHSTGSVRPVRA QGEGLLPGGGGGSGGSGGSGGSGGSGGSGGSGGSGGGMTQSENSCTHFPGNLPNM LRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAE NQEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMS EFDIFINYIEAYMTMNNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFK GYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNHRFLPCEN KSKAVEQVKNAFN KLQEKGI YKAMSEFDI Fl NYI EAYMTM K\RNGENLYFQSGGHHHHHH GGGVSGWRLFKKIS*

[0247] > ORK6_014_P = co- transfection of two vectors (ORK6_10_V I ORK1_287_V)

[0248] > SEQ I D NO: 136 ORK6_10_V (knob chain) MEWSWVFLFFLSVTTGVHSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEG N VFSCSVM H EALH N HYTQKSLSLSLGGGGGGSGGGGSGGGGSN EPLEM W PLTQNEECTVTGFLRDKLQYRSRLQYMKHYFPINYKISVPYEGVFRIANVTRLQRA QVSERELRYLWVLVSLSATESVQDVLLEGHPSWKYLQEVETLLLNVQQGLTDVEV SPKVESVLSLLNAPGPNLKLVRPKALLDNCFRVMELLYCSCCKQSSVLNWQDCEV PSPQSCSPEPSLQYAATQLYPPPPWSPSSPPHSTGSVRPVRAQGEGLLPGGGGG SGGSGGSGGSGGSGGSGGSGGSGGGTQSENSCTHFPGNLPNMLRDLRDAFSR VKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQEPDIK AHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEF DIFINYIEAYMTMNNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLE DFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNH RFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN*

[0249] > SEQ ID NO: 137 ORK1_287_V (hole chain) MEWSWVFLFFLSVTTGVHSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLG*

[0250] Signal peptide context:

[0251] MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 132) (Taken from antibodies. Used as universal in different constructs).

[0252] MPRGFTWLRYLGIFLGVALG SEQ ID NO: 133) (From IL34 human).

[0253] The dual cytokine single chain I L34-IL10 polypeptides as disclosed in Table 1 were constructed.Table 1: details of the dual cytokine single chain IL34-IL10 polypeptides (0RK6) ORK ID General IL-10 IL34 Type of Linker Linker Protein Protein protein description descript Fc 1 (L1) 2 (L2) sequen sequence description ion ce 1 1D 2 ID and format

[0254] ORK6_P_ human SEQ ID SEQ ID

[0255] IL10-IL34 human WT No Fc N / A N / A 001 WT NO: 35 NO: 15 ORK6_P_ human SEQ ID SEQ ID

[0256] IL34-IL10 human WT No Fc N / A N / A 002 WT NO: 35 NO: 16

[0257] C108N (2nd

[0258] ORK6_P_ IL10 domain), human SEQ ID SEQ ID FldklO -IL34 No Fc N / A N / A 003 D84E, Short WT NO: 35 NO: 145

[0259] Nter

[0260] C108N (2nd

[0261] ORK6_P_ IL10 domain), human SEQ ID SEQ ID

[0262] IL34-Fldk10 No Fc N / A N / A 004 D84E, Short WT NO: 35 NO: 19

[0263] Nter

[0264] human

[0265] C108N (2nd lgG4

[0266] SEQ ID ORK6_P_ ORK6-FC- IL10 domain) human silent SEQ ID SEQ ID

[0267] NO: N / A 011 001 D84E, Short WT FALA NO: 35 NO: 83

[0268] 119

[0269] Nter (symmetri

[0270] c)

[0271] human

[0272] C108N (2nd lgG4

[0273] SEQ ID ORK6_P_ ORK6-FC- IL10 domain) human silent SEQ ID SEQ ID

[0274] NO: N / A 012 002 D84E, Short WT FALA NO: 35 NO: 84

[0275] 119

[0276] Nter (symmetri

[0277] c)

[0278] C108N (2nd Human

[0279] SEQ ID ORK6_P_ ORK6-FC- IL10 domain) human lgG4 SEQ ID SEQ ID SEQ ID NO:

[0280] 013 003 D84E, Short WT FALA NO: 35 NO: 85 NO: 86

[0281] 119

[0282] Nter (KIH)

[0283] C108N (2nd Human

[0284] SEQ ID ORK6_P_ ORK6-FC- IL10 domain) human lgG4 SEQ ID SEQ ID SEQ ID NO:

[0285] 014 004 D84E, Short WT FALA NO: 35 NO: 87 NO: 86

[0286] 119

[0287] Nter (KIH)

[0288] Human

[0289] SEQ ID ORK6_P_ ORK6-FC- C259N, long murine lgG4 SEQ ID SEQ ID SEQ ID NO:

[0290] 015 004 Nter WT FALA NO: 35 NO: 166 NO: 86

[0291] 119

[0292] (KIH)

[0293] Human

[0294] SEQ ID ORK6_P_ ORK6-FC- murine lgG4 SEQ ID SEQ ID SEQ ID sclL10 murine NO:

[0295] 016 004 WT FALA NO: 35 NO: 168 NO: 86

[0296] 119

[0297] (KIH)

[0298] SEQ ID ORK6_P_ ORK6-FC- murine Murine SEQ ID SEQ ID SEQ ID sclL10 murine NO:

[0299] 017 004 WT lgG1 NO: 35 NO: 169 NO: 170

[0300]

[0301] 119

[0302] 0RK6_P_xxx proteins are also designated 0RK6_xxx proteins throughout the description.The ORK1 construct used in the experiments (ORK1-068) consists of a dual CSF1-IL-10 cytokine comprising, from the N-terminus to the C-terminus, amino acids E1-D150 of mature human CSF1 protein (reference sequence UniProtKB under accession number P09603-1, entry version 234 of 27 November 2024), a 25 amino acid long peptide linker composed of Gly and Ser residues (SEQ ID No:139), and a single chain dimeric IL-10. The overall sequence of ORK1-068 is shown in SEQ ID NO: 140.

[0303] SEQUENCES

[0304] Signal peptides used to secrete the proteins which are cleaved upon secretion are not shown.

[0305] We used for proteins starting by IL10 and FoldikinelO the signal sequence: MHSSALLCCLVLLTGVRA (SEQ ID NO: 69).

[0306] > SEQ ID NO: 1 foldikinelO:

[0307] TQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVK NAFNKLQEKGI YKAMSEFDI FINYIEAYMTMNNGGLDYLPNMLRDLRDAFSRVKTFFQMK DQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKT LRLRLRRNHRFLPCENKSKAVEQVKNAFNKLQEKGI YKAMSEFDI FINYIEAYMTMKIRN

[0308] > SEQ ID NO: 2 ORK10-002 TQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVK NAFNKLQEKGI YKAMSEFDI FINYIEAYMTMNFGGLDYLPNMLRDLRDAFSRVKTFFQMK DQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKT LRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGI YKAMSEFDI FINYIEAYMTM

[0309] > SEQ ID NO: 30RK10-003 TQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVK NAFNKLQEKG I YKAMS E ED I F I NY I EAYLYKTITPE FANMLRDLRDAFS RVKT FFQMKDQ LDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLR LRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGI YKAMSEFDI FINYIEAYMTM

[0310] > SEQ ID NO: 40RK10-005 TQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVK NAFNKLQEKGI YKAMSEFDI FINYIEAKDKDIRDGDELANMLRDLRDAFSRVKTFFQMKD QLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTL RLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGI YKAMSEFDI FINYIEAYMTM

[0311] > SEQ ID NO: 11 IL10:

[0312] SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYL GCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKA VE QVKNAFNKL QE KG I YKAMS E ED I F I NY I E AYMTMK I RN> SEQ ID NO: 12 IL-34 (UniProtKB Q6ZMJ4-1 without signal peptide): NEPLEMWPLTQNEECTVTGFLRDKLQYRSRLQYMKHYFPINYKISVPYEGVFRIANVTRL QRAQVSERELRYLWVLVSLSATESVQDVLLEGHPSWKYLQEVETLLLNVQQGLTDVEVSP KVESVLSLLNAPGPNLKLVRPKALLDNCFRVMELLYCSCCKQSSVLNWQDCEVPSPQSCS PEPSLQYAATQLYPPPPWSPSSPPHSTGSVRPVRAQGEGLLP

[0313] > SEQ ID NO: 13 IL10-IL34 ORK6_P_001 SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYL GCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKA VE QVKNAFNKL QE KG I YKAMS E ED I F I NY I E AYMTMK I RNGGGGGSGGSGGSGGSGGSGG SGGSGGSGGGNEPLEMWPLTQNEECTVTGFLRDKLQYRSRLQYMKHYFPINYKISVPYEG VFRIANVTRLQRAQVSERELRYLWVLVSLSATESVQDVLLEGHPSWKYLQEVETLLLNVQ QGLTDVEVSPKVESVLSLLNAPGPNLKLVRPKALLDNCFRVMELLYCSCCKQSSVLNWQD CEVPSPQSCSPEPSLQYAATQLYPPPPWSPSSPPHSTGSVRPVRAQGEGLLP

[0314] > SEQ ID NO: 14 IL34-IL10 ORK6_P_002 NEPLEMWPLTQNEECTVTGFLRDKLQYRSRLQYMKHYFPINYKISVPYEGVFRIANVTRL QRAQVSERELRYLWVLVSLSATESVQDVLLEGHPSWKYLQEVETLLLNVQQGLTDVEVSP KVESVLSLLNAPGPNLKLVRPKALLDNCFRVMELLYCSCCKQSSVLNWQDCEVPSPQSCS PEPSLQYAATQLYPPPPWSPSSPPHSTGSVRPVRAQGEGLLPGGGGGSGGSGGSGGSGGS GGSGGSGGSGGGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLL KESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRC HRFL PCENKS KAVE QVKNAFNKLQEKG I YKAMS E ED I F I NY I EAYMTMK I RN

[0315] > SEQ ID NO: 15 IL10-IL34 ORK6_P_001 SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYL GCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKA VE QVKNAFNKLQEKG I YKAMS E ED I F I NY I EAYMTMK I RNGGGGGSGGSGGSGGSGGSGG SGGSGGSGGGNEPLEMWPLTQNEECTVTGFLRDKLQYRSRLQYMKHYFPINYKISVPYEG VFRIANVTRLQRAQVSERELRYLWVLVSLSATESVQDVLLEGHPSWKYLQEVETLLLNVQ QGLTDVEVSPKVESVLSLLNAPGPNLKLVRPKALLDNCFRVMELLYCSCCKQSSVLNWQD CEVPSPQSCSPEPSLQYAATQLYPPPPWSPSSPPHSTGSVRPVRAQGEGLLP

[0316] > SEQ ID NO: 16 IL34-IL10 ORK6_P_002 NEPLEMWPLTQNEECTVTGFLRDKLQYRSRLQYMKHYFPINYKISVPYEGVFRIANVTRL QRAQVSERELRYLWVLVSLSATESVQDVLLEGHPSWKYLQEVETLLLNVQQGLTDVEVSP KVESVLSLLNAPGPNLKLVRPKALLDNCFRVMELLYCSCCKQSSVLNWQDCEVPSPQSCS PEPSLQYAATQLYPPPPWSPSSPPHSTGSVRPVRAQGEGLLPGGGGGSGGSGGSGGSGGS GGSGGSGGSGGGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLL KESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRC HRFL PCENKS KAVE QVKNAFNKLQEKG I YKAMS E ED I F I NY I EAYMTMK I RN

[0317] > SEQ ID NO: 19 I L34-Foldikine10 ORK6_P_004 NEPLEMWPLTQNEECTVTGFLRDKLQYRSRLQYMKHYFPINYKISVPYEGVFRIANVTRL QRAQVSERELRYLWVLVSLSATESVQDVLLEGHPSWKYLQEVETLLLNVQQGLTDVEVSP KVESVLSLLNAPGPNLKLVRPKALLDNCFRVMELLYCSCCKQSSVLNWQDCEVPSPQSCS PEPSLQYAATQLYPPPPWSPSSPPHSTGSVRPVRAQGEGLLPGGGGGSGGSGGSGGSGGS GGSGGSGGSGGGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLL EDFKGYLGCQALSEMIQFYLEEVMPQAENQEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGI YKAMSEFDI FINYIEAYMTMNNGGLDYLPNMLRDLRD AFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIK AHVNSLGENLKTLRLRLRRNHRFLPCENKSKAVEQVKNAFNKLQEKGI YKAMSEFDI FIN YIEAYMTMKIRN

[0318] > SEQ ID NO: 28 TQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQF YLEEVMPQAENQEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIY KAMSEFDIFINYIEAYMTMN

[0319] > SEQ ID NO: 29 Foldikine10 L34 ORK6_P_003 TQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQF YLEEVMPQAENQEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIY KAMSEFDIFINYIEAYMTMNNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNHRFLPCENKSKAVEQV KNAFNKLQ E KG I Y KAMS E FDIFINYI E AYMTMKI RNGGGGGSGGSGGSGGSGGSGGSGGSGGSGGG NEPLEMWPLTQNEECTVTGFLRDKLQYRSRLQYMKHYFPINYKISVPYEGVFRIANVTRLQRAQVS ERELRYLWVLVSLSATESVQDVLLEGHPSWKYLQEVETLLLNVQQGLTDVEVSPKVESVLSLLNAP GPNLKLVRPKALLDNCFRVMELLYCSCCKQSSVLNWQDCEVPSPQSCSPEPSLQYAATQLYPPPPW SPSSPPHSTGSVRPVRAQGEGLLP

[0320] > SEQ ID NO: 30

[0321] NGGLDY

[0322] > SEQ ID NO: 31

[0323] YKTIT

[0324] > SEQ ID NO: 32

[0325] DKDIRDGD

[0326] > SEQ ID NO: 138 linker L1 15

[0327] GGSGGSGGSGGSGGG

[0328] > SEQ ID NO: 34 linker L1 25

[0329] GGGSGGSGGSGGSGGSGGSGGSGGG

[0330] > SEQ ID NO: 35 linker L1 30

[0331] GGGGGSGGSGGSGGSGGSGGSGGSGGSGGG

[0332] > SEQ ID NO: 36 linker L1 35

[0333] GGGGGSGGGGSGGSGGSGGSGGSGGSGGSGGSGGG

[0334] > SEQ ID NO: 37 IL34- sc lL10 NEPLEMWPLTQNEECTVTGFLRDKLQYRSRLQYMKHYFPINYKISVPYEGVFRIANVTRLQRAQVS ERELRYLWVLVSLSATESVQDVLLEGHPSWKYLQEVETLLLNVQQGLTDVEVSPKVESVLSLLNAP GPNLKLVRPKALLDNCFRVMELLYCSCCKQSSVLNWQDCEVPSPQSCSPEPSLQYAATQLYPPPPW SPSSPPHSTGSVRPVRAQGEGLLPGGGGGSGGSGGSGGSGGSGGSGGSGGSGGGSPGQGTQSENSC THFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMP QAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFD IFINYIEAYMTMKIRNGGGSGGGGSSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKD QLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRR CHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN> SEQ ID NO: 42 PEFANMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAEN QDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFIN YIEAYMTM

[0335] > SEQ ID NO: 43 TQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQF YLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIY KAMSEFDIFINYIEAK

[0336] > SEQ ID NO: 44 ELANMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQ DPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDI FINY IEAYMTM

[0337] > SEQ ID NO: 65 N-terminal part of SC dimeric IL-10 X1TQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMI QFYLEEVMPQAENQX2PDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEK GIYKAMSEFDIFINYIEAYX3

[0338] > SEQ ID NO: 66 C-terminal part of SC dimeric IL-10 NMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPD IKAHVNSLGENLKTLRLRLRRX4HRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIE AYMTMKIRN

[0339] > SEQ ID NO: 83 ORK6_P_11 ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSNEPLEMWPLTQNEECTVTGFL RDKLQYRSRLQYMKHYFPINYKISVPYEGVFRIANVTRLQRAQVSERELRYLWVLVSLSATESVQD VLLEGHPSWKYLQEVETLLLNVQQGLTDVEVSPKVESVLSLLNAPGPNLKLVRPKALLDNCFRVME LLYCSCCKQSSVLNWQDCEVPSPQSCSPEPSLQYAATQLYPPPPWSPSSPPHSTGSVRPVRAQGEG LLPGGGGGSGGSGGSGGSGGSGGSGGSGGSGGGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQ MKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQEPDIKAHVNSLGENLKTLRLR LRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMNNGGLDYLPNMLRD LRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHV NSLGENLKTLRLRLRRNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMK IRN*

[0340] > SEQ ID NO: 84 ORK6_P_12 NEPLEMWPLTQNEECTVTGFLRDKLQYRSRLQYMKHYFPINYKISVPYEGVFRIANVTRLQRAQVS ERELRYLWVLVSLSATESVQDVLLEGHPSWKYLQEVETLLLNVQQGLTDVEVSPKVESVLSLLNAP GPNLKLVRPKALLDNCFRVMELLYCSCCKQSSVLNWQDCEVPSPQSCSPEPSLQYAATQLYPPPPW SPSSPPHSTGSVRPVRAQGEGLLPGGGGGSGGSGGSGGSGGSGGSGGSGGSGGGTQSENSCTHFPG NLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQ EPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDI FINY IEAYMTMNNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQ FYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNHRFLPCENKSKAVEQVKNAFNKLQEKGI YKAMSEFDIFINYIEAYMTMKIRNGGGGSGGGGSGGGGSESKYGPPCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLG*

[0341] > SEQ ID NO: 85 ORK6_P_13 sequence encoded by plasmid 1 NEPLEMWPLTQNEECTVTGFLRDKLQYRSRLQYMKHYFPINYKISVPYEGVFRIANVTRLQRAQVS ERELRYLWVLVSLSATESVQDVLLEGHPSWKYLQEVETLLLNVQQGLTDVEVSPKVESVLSLLNAP GPNLKLVRPKALLDNCFRVMELLYCSCCKQSSVLNWQDCEVPSPQSCSPEPSLQYAATQLYPPPPW SPSSPPHSTGSVRPVRAQGEGLLPGGGGGSGGSGGSGGSGGSGGSGGSGGSGGGTQSENSCTHFPG NLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQ EPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDI FINY IEAYMTMNNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQ FYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNHRFLPCENKSKAVEQVKNAFNKLQEKGI YKAMSEFDIFINYIEAYMTMKIRNGGGGGSGGGGSGGGGSESKYGPPCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS LSLG*

[0342] > SEQ ID NO: 86 ORK6_P_13 / ORK6_P_14 sequence encoded by plasmid 2 ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCT LPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0343] > SEQ ID NO: 87 ORK6_P_14 sequence encoded by plasmid 1 ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGGSGGGGSGGGGSNEPLEMWPLTQNEECTVTGF LRDKLQYRSRLQYMKHYFPINYKISVPYEGVFRIANVTRLQRAQVSERELRYLWVLVSLSATESVQ DVLLEGHPSWKYLQEVETLLLNVQQGLTDVEVSPKVESVLSLLNAPGPNLKLVRPKALLDNCFRVM ELLYCSCCKQSSVLNWQDCEVPSPQSCSPEPSLQYAATQLYPPPPWSPSSPPHSTGSVRPVRAQGE GLLPGGGGGSGGSGGSGGSGGSGGSGGSGGSGGGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFF QMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQEPDIKAHVNSLGENLKTLRL RLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDI FINYIEAYMTMNNGGLDYLPNMLR DLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAH VNSLGENLKTLRLRLRRNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTM KIRN*

[0344] > SEQ ID NO: 88 Single chain IL-10 SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALS EMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQ EKGIYKAMSEFDIFINYIEAYMTMKIRNGGGSGGGGSSPGQGTQSENSCTHFPGNLPNMLRDLRDA FSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLG ENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0345] > SEQ ID NO: 89 Foldikine 10 (without Methionine, only C259N mutation, and N-terminal completed) SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALS EMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQ EKGIYKAMSEFDIFINYIEAYMTMNNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLL EDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNHRFLPCENKSK AVEQVKNAFNKLQEKGIYKAMSEFDI FINY IEAYMTMKIRN> SEQ ID NO: 119 (L2 linker)

[0346] GGGGSGGGGSGGGGS

[0347] > SEQ ID NO: 131 (L2 linker)

[0348] GGGGSGGGGSGGGGSGGGGS

[0349] > SEQ ID NO: 140 ORK1_P_068 EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMR FRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDW NIFSKNCNNSFAECSSQD GGGGGSGGSGGSGGSGGSGGSGGSGGSGGGMT QSENSCTHFPGNLPNM LRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQEPDIK AHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYM TMNNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEE VMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMS EFDIFINYIEAYMTMKIRN

[0350] > SEQ ID NO: 145 Foldikine10 L34 ORK6-003_P TQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQF YLEEVMPQAENQEPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIY KAMSEFDIFINYIEAYMTMNNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRNHRFLPCENKSKAVEQV KNAFNKLQ E KG I Y KAMS E FDIFINYI E AYMTMKI RNGGGGGSGGSGGSGGSGGSGGSGGSGGSGGG NEPLEMWPLTQNEECTVTGFLRDKLQYRSRLQYMKHYFPINYKISVPYEGVFRIANVTRLQRAQVS ERELRYLWVLVSLSATESVQDVLLEGHPSWKYLQEVETLLLNVQQGLTDVEVSPKVESVLSLLNAP GPNLKLVRPKALLDNCFRVMELLYCSCCKQSSVLNWQDCEVPSPQSCSPEPSLQYAATQLYPPPPW SPSSPPHSTGSVRPVRAQGEGLLP

[0351] > SEQ ID NO: 166 Protein 1 of ORK6-P-015 ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGGSGGGGSGGGGSNENLEI TLTQDKECDLTGY LRGKLQYKNRLQYMKHYFPINYRIAVPYEGVLRVANITRLQKAHVSERELRYLWVLVSLNATESVM DVLLEGHPSWKYLQEVQTLLENVQRSLMDVEIGPHVEAVLSLLSTPGLSLKLVRPKALLDNCFRVM ELLYCSCCKQSPILKWQDCELPRLHPHSPGSLMQCTATNVYPLSRQTPTSLPGSPSSSHGSLPGGG GGSGGSGGSGGSGGSGGSGGSGGSGGGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQM KDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRL RRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDI FINYIEAYMTMNNGGLDYLPNMLRDL RDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVN SLGENLKTLRLRLRRNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKI RN

[0352] > SEQ ID NO: 168 Protein 1 of ORK6-P-016 ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGGSGGGGSGGGGSNENLEI TLTQDKECDLTGY LRGKLQYKNRLQYMKHYFPINYRIAVPYEGVLRVANITRLQKAHVSERELRYLWVLVSLNATESVM DVLLEGHPSWKYLQEVQTLLENVQRSLMDVEIGPHVEAVLSLLSTPGLSLKLVRPKALLDNCFRVM ELLYCSCCKQSPILKWQDCELPRLHPHSPGSLMQCTATNVYPLSRQTPTSLPGSPSSSHGSLPGGG GGSGGSGGSGGSGGSGGSGGSGGSGGGSRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRL RRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDI FINCIEAYMMIKMKSGGGSGGGGSSR GQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEM IQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQ GVYKAMNEFDIFINCIEAYMMIKMKS

[0353] > SEQ ID NO: 169 Protein 1 of ORK6-P-017 VPRDCGCKPCICTVKEVSKVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHT AQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVCTIP PPKEQMAKDKVSLWCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWE AGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGGGSGGGGSGGGGSNENLEI TLTQDKECDLTGY LRGKLQYKNRLQYMKHYFPINYRIAVPYEGVLRVANITRLQKAHVSERELRYLWVLVSLNATESVM DVLLEGHPSWKYLQEVQTLLENVQRSLMDVEIGPHVEAVLSLLSTPGLSLKLVRPKALLDNCFRVM ELLYCSCCKQSPILKWQDCELPRLHPHSPGSLMQCTATNVYPLSRQTPTSLPGSPSSSHGSLPGGG GGSGGSGGSGGSGGSGGSGGSGGSGGGSRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQT KDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRL RRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDI FINCIEAYMMIKMKSGGGSGGGGSSR GQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEM IQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQ GVY KAMNE FD I F INC I E AYMMI KMKS

[0354] > SEQ ID NO: 170 Protein 2 of ORK6-P-017 VPRDCGCKPCICTVKEVSKVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHT AQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIP PPKCQMAKDKVSLSCAITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVVSKLNVQKSNWE AGNTFTCSVLHEGLHNHHTEKSLSHSPGK

Claims

CLAIMS1. A single chain dual-cytokine polypeptide having IL-10 and IL-34 activities.

2. The single chain dual-cytokine polypeptide according to claim 1, which comprises an IL-10 domain fused to an IL-34 monomer, wherein the IL-10 domain is an IL-10 monomer or a single chain dimeric IL-10.

3. The single chain dual-cytokine polypeptide according to claim 1 or 2, which comprises an IL-10 monomer fused to an IL-34 monomer through a peptide linker L1.

4. The single chain dual-cytokine according to claim 1, which comprises a single chain dimeric IL-10 fused to an IL-34 monomer through a peptide linker L1.

5. The single chain polypeptide according to any one of claims 1 to 4, wherein one or more of the following conditions are met:(i) the IL-34 monomer comprises sequence SEQ ID NO: 12, or a sequence at least 80% identical thereto and that retains IL-34 activity;(ii) the IL-10 monomer comprises sequence SEQ ID NO: 11 or a sequence at least 80% identical thereto and that retains IL- 10 activity;(iii) the single chain dimeric IL-10 comprises sequence SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 1, or a sequence at least 80% identical thereto and that retains IL-10 activity.

6. The single chain polypeptide according to any one of claims 1 to 4, wherein one or more of the following conditions are met:(i) the peptide linker L1 comprises from 5 to 50 amino acid residues;(ii) the peptide linker L1 is a flexible peptide sequence composed of Gly and Ser residues, in different proportion;(iii) the peptide linker L1 comprises sequence SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36; or(iv) the peptide linker L1 comprises sequence (SEQ ID NO:82)n, n being an integer from 1 to 10.

7. The single chain dual-cytokine polypeptide according to any one of claims 1 to 6, which comprises sequence SEQ ID NO: 15 or SEQ ID NO: 16, or a sequence at least 80% identical thereto and that retains IL-10 and IL-34 activities.

8. The single chain dual-cytokine polypeptide according to any one of claims 1 to 6, which comprises sequence SEQ ID NO: 19, SEQ ID NO: 145, SEQ ID NO: 37, or a sequence at least 80% identical thereto and that retains IL-10 and IL-34 activities.

9. The single chain polypeptide according to any one of claims 1 to 8, which is modified to increase its half-life.

10. A protein comprising the single chain dual-cytokine polypeptide having IL-10 and IL-34 activities as defined in any one of claims 1 to 9 fused to an immunoglobulin Fc fragment or to albumin, or conjugated with a poly(ethylene glycol) (PEG) molecule.

11. The protein according to claim 10, which is fused to an immunoglobulin Fc fragment, optionally of lgG1, lgG2, lgG3 or lgG4, silent and non-silent.

12. The protein according to claim 10 or 11, which comprises one single chain polypeptide having IL-10 and IL-34 activities and one immunoglobulin Fc fragment, wherein the single chain polypeptide having IL-10 and IL-34 activities is fused to the immunoglobulin Fc fragment through a peptide linker L2 connecting (i) the C-terminus of the IL-10 domain with the N-terminus of one hinge region of the Fc fragment, or (ii) the C-terminus of the one of the CH3 domains of the Fc fragment with the N-terminus of the IL-34 monomer.

13. The protein according to claim 10 or 11, which comprises two polypeptide chains that each comprise, in the N-terminus to C-terminus direction, (i) an IL-34 monomer, a peptide linker L1, an IL-10 domain which is an IL-10 monomer or a SC dimeric IL-10 monomer, a peptide linker L2, and a Fc domain, or (ii) a Fc domain, a peptide linker L2, an IL-34 monomer, a peptide linker L1, and an IL-10 domain which is an IL-10 monomer or a SC dimeric IL-10 monomer, wherein the two polypeptide chains dimerise through their Fc domains.

14. The protein according to claim 12 or 13, wherein one or more of the following conditions are met:(i) the peptide linker L2 comprises from 3 to 50 amino acid residues;(ii) the peptide linker L2 is a flexible peptide sequence composed of Gly and Ser residues; (iii) the peptide linker L2 comprises sequence (SEQ ID NO:82)p, p being an integer from 1 to 8;(iv) the peptide linker L2 comprises any one of sequences SEQ ID NO: 119 to SEQ ID NO: 131, and SEQ ID NO: 146.

15. The protein according to any one of claims 10 to 14, which comprises sequence SEQ ID NO: 83 or SEQ ID NO: 84, or the combination of sequences SEQ ID NO: 85 and SEQ ID NO:86, or the combination of sequences SEQ ID NO: 87 and SEQ ID NO: 86, or a sequence at least 80% identical thereto and that retains IL-10 and IL-34 activities.

16. A pharmaceutical composition comprising the single chain polypeptide according to any one of claims 1 to 9, or the protein according to any one of claims 10 to 15, and a pharmaceutically acceptable carrier.

17. A single chain polypeptide according to any one of claims 1 to 9, or a protein according to any one of claims 10 to 15, or the pharmaceutical composition according toclaim 16, for use as a medicament, in particular for use for treating an auto-immune disease, an inflammatory disease or a neuroinflammatory disease.

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