Compounds for immunomodulation

Abstract

The present invention relates to compounds based on the recombinant and oligomerization domains of the CCP6 region of C4BP for use in the prevention and / or treatment of immunological diseases. Furthermore, the present invention relates to methods for obtaining tolerogenic dendritic cells and tolerogenic macrophages using the compounds of the present invention, as well as cells obtained by these methods and their therapeutic uses.

Description

[Technical Field]

[0001] The present invention relates to the field of immunology, more specifically to compounds based on a recombinant form of the CCP6 region of C4BP that are capable of inducing a tolerogenic state in mononuclear phagocytes, and their use for the prevention and / or treatment of diseases characterized by unwanted activation of the immune system. [Background technology]

[0002] The mononuclear phagocyte system (MPS) refers to a network of myeloid cells that share many functional characteristics, including the ability to secrete, inter alia, chemokine and cytokine mediators, and to effect migration and activation of immune and non-immune cells as well as their own migration. The MPS is composed of dendritic cells, macrophages, and monocytes.

[0003] Dendritic cells (DCs) are specialized APCs of the immune system. At their immature stage, DCs ingest extracellular antigens by phagocytosis or pinocytosis, process the antigens into peptides within endocytic compartments such as endosomes and phagosomes, and then bind to MHC class II molecules. They also possess the unique ability to deliver peptides from exogenous proteins to the MHC class I presentation pathway (a process called "cross-presentation"). When provided with appropriate differentiation signals (e.g., microbial products), immature DCs can develop into immunogenic DCs capable of activating both naive and memory T cells. On the other hand, immature DCs can differentiate into tolerogenic phenotypes, which are thought to play an important role in maintaining peripheral tolerance (Steinman, Ann. Rev. Immunol. 2003, 21: 685-711; Morelli, Immunol Rev 2003: 125-146).

[0004] Numerous protocols for generating tolerogenic DCs in vitro have been described (Xiao et al., J. Immunother. 2006 (29) 465-471). The best-characterized methods use pharmacological mediators (e.g., immunosuppressants including vitamin D3 analogs, glucocorticoids, and estrogens), cytokines and growth factors (e.g., IL-10, TGF-β, IL-4, and IFN-γ), or genetic engineering to suppress the expression of T cell costimulatory molecules (e.g., CD86 and CD40) or enhance the expression of T cell inhibitory molecules (e.g., CTLA-4 and indoleamine 2,3-dioxygenase).

[0005] Recently, it has been shown that the physiological isoform of the complement regulatory factor C4b-binding protein, α7β0, induces a semi-mature anti-inflammatory state in dendritic cells (Olivar R. et al. 2013. J. Immunol., 190:2857-2872).

[0006] Meanwhile, several protocols have been described for the generation of tolerogenic or regulatory macrophages in vitro, including the use of glucocorticoids such as dexamethasone, IL-10, or TGF-β, or combinations of M-CSF+IFNγ or M-CSF+LPS, among other compounds (Mosser DM and Edwards JP. 2008. Nat. Rev. Immunol. 8:958-969; Zheng G. et al. 2013. Eur. J. Immunol. 43:219-227; Navarro-Barriuso J. et al. 2018. Front. Immunol. 9:2062; Foey AD. 2014. Immune response activation, Chapter 5: Macrophages: masters of immune activation, suppression, and deviation, Publisher: InTech, Editors: Guy Huynh Thien Duc, pp.121-149).

[0007] Currently, the majority of treatments approved by the US FDA for autoimmune diseases focus on the systemic inhibition of immune inflammatory activity. Although nonspecific immunosuppression is partially effective in inhibiting autoreactive immune cell function, the drugs used to suppress immune responses have numerous side effects, and continuous treatment does not result in long-term survival for treated individuals. Therefore, it is desirable to develop autoantigen-specific treatments that specifically block the harmful effects of autoreactive immune cell function while preserving the immune system's ability to clear infection. Therefore, there is a strong need for methods to generate appropriately equipped DCs that can efficiently induce antigen-specific immune tolerance.

[0008] Furthermore, ex vivo generated DCs with appropriate tolerogenic functions can also be used as therapeutic vaccines for the treatment of allergies and the induction of transplantation tolerance. Similar to immunotherapy for autoimmune diseases, efficient suppression of harmful immune responses involves the induction of tolerance to both CD4+ and CD8+ T cells. Therefore, it is expected that ex vivo generated tolerogenic DCs should have the same properties for the treatment of autoimmune diseases and allergies and the prevention of graft rejection.

[0009] However, new and alternative methods for producing tolerogenic dendritic cells and macrophages with a distinct tolerogenic phenotype and expression of tolerogenic determinants will always be an ongoing goal of research in this field. Summary of the Invention

[0010] In a first aspect, the present invention relates to a recombinant polypeptide comprising the CCP6 domain of the C4BP alpha chain or a functionally equivalent variant thereof, and an oligomerization domain, said recombinant polypeptide not comprising any of the CCP1, CCP2, CCP3, CCP4, CCP5, CCP7 and CCP8 domains of the C4BP alpha chain.

[0011] In a second aspect, the present invention relates to a homo-oligomer of at least six recombinant polypeptides of the first aspect of the invention.

[0012] In a third aspect, the present invention relates to a polynucleotide encoding a recombinant polypeptide of the first aspect of the invention.

[0013] In a fourth aspect, the present invention relates to a vector comprising a polynucleotide according to the third aspect of the invention.

[0014] In a fifth aspect, the present invention relates to a host cell comprising a vector according to the fourth aspect of the invention.

[0015] In a sixth aspect, the present invention relates to a pharmaceutical composition comprising a recombinant polypeptide according to the first aspect of the invention, a homo-oligomer according to the second aspect of the invention, a polynucleotide according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a host cell according to the fifth aspect of the invention, and a pharmaceutically acceptable carrier.

[0016] In a further aspect, the present invention relates to a recombinant polypeptide, homo-oligomer, polynucleotide, vector, host cell or pharmaceutical composition of the invention for use in medicine, in particular for use in the prevention and / or treatment of immunological diseases caused by unwanted activation of the immune system.

[0017] In another aspect, the present invention provides a method for generating a population of tolerogenic dendritic cells in vitro, comprising: (i) incubating a population of dendritic precursor cells under conditions suitable for the formation of a population of immature dendritic cells; and (ii) incubating the population of immature dendritic cells obtained in step (i) under conditions suitable for the formation of mature dendritic cells. Including, The steps (i) and / or (ii) are (a) a polypeptide of the present invention; (b) a homo-oligomer of the present invention; (c) a polynucleotide of the invention, and (d) Vector of the present invention The method is carried out in the presence of a composition comprising a substance selected from the group consisting of:

[0018] In another aspect, the present invention provides a method for generating a population of tolerogenic macrophages in vitro, comprising: (i) incubating a population of macrophage progenitor cells under conditions suitable for the formation of a population of immature macrophages; and (ii) incubating the population of immature macrophages obtained in step (i) under conditions suitable for the formation of mature macrophages. Including, The steps (i) and / or (ii) are (a) a polypeptide of the present invention; (b) a homo-oligomer of the present invention; (c) a polynucleotide of the invention, and (d) Vector of the present invention The method is carried out in the presence of a composition comprising a substance selected from the group consisting of:

[0019] In yet another aspect, the present invention relates to tolerogenic dendritic cells or tolerogenic macrophages obtainable by the methods of the present invention, cell populations comprising at least 80% of such cells, pharmaceutical compositions comprising the cells or populations of the present invention, and the use of the cells of the present invention in medicine, in particular in medicine for the prevention and / or treatment of immunological diseases caused by unwanted activation of the immune system. [Brief explanation of the drawings]

[0020] [Figure 1]Premature sequences of PRP6-HO7 and PRP6-NO. (A) Peptide and DNA premature sequences of PRP6-HO7. The figure shows the sequences of the signal peptide, histidine tag, CCP6 domain, and oligomerization domain. The predicted molecular mass of PRP6-HO7 is 19.6 kDa, and 14.3 kDa without the signal peptide. (B) Peptide and DNA premature sequences of PRP6-NO. The figure shows the sequences of the signal peptide, histidine tag, CCP6 domain, and oligomerization domain. In PRP6-NO, the original oligomerization sequence has been modified by replacing two cysteine ​​residues with alanine (bold) and removing the last 13 amino acids at the C-terminus. The predicted molecular mass of PRP6-NO is 18 kDa, and 12.7 kDa without the signal peptide. [Figure 2] Molecular modeling of PRP6-HO7. Structural prediction of PRP6-HO7 homo-oligomer from monomer sequence / structure using the GalaxyHomomer server (http: / / galaxy.seoklab.org / homomer) (Baek et al. (2017) Nucleic Acids Res. 45: W320-W324). [Figure 3] Visualization of PRP6-HO7 and PRP6-NO proteins by PAGE. (A) 12% SDS-PAGE analysis and Coomassie Blue staining of purified PRP6-HO7 and PRP6-NO proteins under reducing (R) (monomers, 14.3 kDa and 12.7 kDa, respectively) and nonreducing (NR) conditions (oligomers, 100 kDa). (B) Bis-Tris 4-12% SDS-PAGE and Western analysis of purified PRP6-HO7 and PRP6-NO proteins probed with anti-His MoAb under nonreducing (NR; left) conditions and with anti-C4BP α-chain polyclonal antibody under reducing (R; right) conditions. [Figure 4]PRP6-HO7, but not PRP6-NO, down-regulates CD83 and CD86 expression in LPS-activated DCs. Human Mo-DCs were incubated with 12 nM PRP-HO7 and PRP-HE8 (C4BP(β+), corresponding to the inactive isoform of PRP(C4BP)), PRP6-HO7 (32 nM), and PRP6-NO (32 nM and 224 nM) throughout their differentiation process. DC maturation was achieved by LPS treatment (see Materials and Methods for details). Cells were then harvested, washed, and analyzed by flow cytometry for cell surface expression of the activation marker CD83 and the costimulatory molecule CD86. MFI is the median fluorescence intensity of the various surface markers. LPS is lipopolysaccharide. iDCs are untreated immature DCs; mDCs are DCs matured with untreated LPS. Results shown are means ± SD based on six independent experiments (**p<0.01; ****p<0.0001 vs. mDC). [Figure 5] Treatment with PRP6-HO7, but not PRP6-NO, increases the endocytic activity of human iDCs. The endocytic activity of iDCs was assessed by flow cytometry during differentiation, measuring the internalization and processing (receptor-mediated endocytosis) of fluorescent DQ-OVA. Monocytes were either left untreated (iDCs) or differentiated by treatment with PRP-HE8 and PRP-HO7 (both at 12 nM) and PRP6-HO7 and PRP6-NO (both at 32 nM). MFI is the median fluorescence intensity. iDCs are untreated immature DCs. Data shown are the mean MFI ± SD based on five independent experiments (* indicates p < 0.05 for iDCs). [Figure 6]PRP6-HO7, but not PRP6-NO, regulates inflammatory cytokine production in DCs activated with LPS. Mo-DCs treated with various PRP-based proteins: PRP-HE8, PRP-HO7 (all 12 nM; 5 μg / ml), and PRP6-HO7, PRP6-NO (all 32 nM; 3 μg / ml) were matured with LPS, and the concentrations of IL-12p70 and TNF-α in the supernatants were analyzed by ELISA. Results shown are the mean ± SD of five independent experiments performed in duplicate. LPS is lipopolysaccharide; iDCs are untreated immature DCs; mDCs are untreated LPS-matured DCs (*, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. mDCs). [Figure 7]PRP6-HO7 lacks complement inhibitory activity. (A) Schematic of the cofactor activity of PRP-HE8 and PRP-HO7 for factor I-mediated C4b cleavage. Factor I cleavage of C4b requires the presence of a specific cofactor, in this case PRP-HE8 / PRP-HO7. Factor I cleaves the α' chain of C4b at two sites indicated by arrows. Cleavage at either one of the two sites generates the α3-C4d (70 kDa) and α4 (14 kDa) or α3 (25 kDa) and C4d-α4 (59 kDa) fragments, which are visible after SDS-PAGE under reducing conditions. Further cleavage of the α' chain of C4b generates a large, soluble C4c (146 kDa) fragment and a smaller C4d (45 kDa) fragment that maintains target binding. (B) Assay of cofactor activity. The indicated concentrations of PRP-HE8, PRP-HO7, and PRP6-HO7 (lanes 3–7) were incubated with C4b (8.9 μg / ml) followed by the addition of factor I (4.4 μg / ml). Reaction controls included C4b alone and C4b + FI. All reactions were stopped after 30 minutes with SDS-reducing sample buffer. C4b cleavage fragments were separated by 4–12% SDS-PAGE under reducing conditions and then subjected to Western blotting using anti-C4d MoAb. C4b fragments are indicated on the left side of the gel. Cofactor activity was confirmed by the appearance of α3-C4d (70 kDa) or C4d (45 kDa). FI is factor I. Results are representative of three independent experiments. [Figure 8]Expression of surface markers on DCs treated with PRP6-HO7 and activated with LPS. Human Mo-DCs were incubated with 12 nM PRP-HO7 and PRP-HE8, as well as 32 nM PRP6-HO7, throughout their differentiation process. DC maturation was achieved by LPS treatment. Cells were then harvested, washed, and analyzed for cell surface expression of CD83, CD86, HLA-DR, CD40, CD80, and CD14 by flow cytometry. MFI is the median fluorescence intensity of various surface markers. LPS is lipopolysaccharide. iDCs are untreated immature DCs; mDCs are untreated LPS-matured DCs. Results shown are the mean ± SD based on five independent experiments (*, p < 0.05; **, p < 0.01; ***, p < 0.001 for mDCs). [Figure 9] PRP6-HO7 down-regulates CD83 and CD86 expression in LPS-activated DCs grown in 50% human serum. Human Mo-DCs were incubated with 12 nM PRP-HO7 and PRP-HE8, as well as the indicated concentrations of PRP6-HO7, throughout their differentiation process in RPMI1640 cell culture medium supplemented with 50% heat-inactivated human serum. DC maturation was achieved by LPS treatment. Cells were then harvested, washed, and analyzed for cell surface expression of CD83 and CD86 by flow cytometry. MFI is the median fluorescence intensity of various surface markers. LPS is lipopolysaccharide. iDCs are untreated immature DCs; mDCs are untreated LPS-matured DCs. Results shown are the mean ± SD based on five independent experiments (* p < 0.05; ** p < 0.01 for mDCs). [Figure 10]PRP6-HO7 down-regulates the expression of CD83 and CD86 surface markers in Gardiquimod-activated DCs. Human mo-DCs were incubated with 12 nM PRP-HE8 and PRP-HO7, as well as 32 nM PRP6-HO7, throughout their differentiation process. DC maturation was achieved by Gardiquimod (a TLR7 agonist) treatment (see Materials and Methods for details). Cells were then harvested, washed, and analyzed for CD83 and CD86 surface expression by flow cytometry. MFI is the median fluorescence intensity of various surface markers. iDCs are untreated immature DCs; mDCs are untreated Gardiquimod-matured DCs. Results shown are the mean ± SD based on five independent experiments (* p < 0.05; ** p < 0.01 for mDCs). [Figure 11] PRP6-HO7 down-regulates surface marker expression in DCs isolated from patients with lupus nephritis and activated with both LPS and Gardiquimod. Human mo-DCs from patients with lupus nephritis were incubated with 12 nM PRP-HO7 and PRP-HE8, as well as 32 nM PRP6-HO7 throughout their differentiation process. DC maturation was achieved by either (A) LPS treatment or (B) Gardiquimod treatment. Cells were then harvested, washed, and analyzed by flow cytometry for cell surface expression of CD83, CD86, HLA-DR, CD40, and CD80. MFI is the median fluorescence intensity of the various surface markers. LPS is lipopolysaccharide. iDCs are untreated immature DCs; mDCs are DCs matured with untreated LPS or Gardiquimod. Results shown are means ± SD based on four independent experiments (*p<0.05; **p<0.01 vs. mDC). [Figure 12]PRP6-HO7 down-regulates CCR7 expression and alters the chemotaxis of human DCs. (A) Analysis of CCR7 expression in DCs at the translational level. Surface expression of CCR7 on DCs treated with PRP-based proteins and matured with LPS is shown. The MFI of CCR7 cell surface expression is shown. Results shown are means ± SD based on seven independent experiments. (**p<0.01 vs. mDCs). (B) Migration toward the chemokine CCL21 after LPS maturation of untreated DCs, DCs treated with PRP-HE8 (12 nM), DCs treated with PRP-HO7 (12 nM), and DCs treated with PRP6-HO7 (32 nM) was assessed in a transwell assay. Shown is the absolute number of LPS-matured DCs (mDCs) that migrated toward the lower chamber containing CCL21 after 2 hours of incubation (black columns). Spontaneous migration of DCs into the lower chamber without CCL21 was also assessed (gray columns). Results are the mean ± SD based on seven independent experiments performed in duplicate. MFI is the median fluorescence intensity of the CCR7 surface marker. LPS is lipopolysaccharide. iDCs are untreated immature DCs; mDCs are untreated LPS-matured DCs; PRP-HE8, PRP-HO7, and PRP6-HO7 are LPS-matured DCs treated with PRP-based proteins. (**** p < 0.0001 vs. mDCs). [Figure 13]Human DCs exposed to PRP6-HO7 down-regulate PTGER3 and EGLN3 during differentiation. Expression of PTGER3 and EGLN3 transcripts in DCs treated with PRP-HE8, PRP-HO7, or PRP6-HO7 compared with untreated immature DCs (iDCs) on days 4 and 5 of differentiation (n=1). Gene expression data are expressed as Log2FC (FC is fold change). Relative expression data of identified genes were obtained by total RNA extraction and TaqMan RT-qPCR validation. Log2FC values ​​are expressed as mean + SD (2 technical replicates / sample) compared with untreated iDCs. [Figure 14] Olfactory receptors are upregulated during differentiation by human DCs exposed to PRP6-HO7. Expression of "find me" or olfactory receptor CCR2, CX3CR1, CXCR1, FPR2, GPR132, P2RY2, and S1PR1 transcripts in DCs treated with PRP-HE8, PRP-HO7, or PRP6-HO7 compared with untreated immature DCs (iDCs) on day 5 of differentiation. Gene expression data are expressed as Log2FC (FC is fold change). Relative expression data for identified genes were obtained by total RNA extraction and TaqMan RT-qPCR validation. Log2FC values ​​are expressed as the mean + SD based on four independent experiments (* indicates p<0.05 vs. untreated iDCs). [Figure 15]PRP6-HO7 prevents M1 and M2 polarization of human macrophages. (A) Expression of M1 markers. (B) Expression of M2 markers. Human monocytes were incubated with 12 nM PRP-HE8 and PRP-HO7, as well as PRP6-HO7 (32 nM), throughout their differentiation into uncommitted macrophages (M0) and further polarization into inflammatory macrophages (M1) or activated macrophages (M2) (see Materials and Methods for details). Cells were then collected, washed, and analyzed by flow cytometry for cell surface expression of CD64 and CD80 (M1 markers) or CD209 and CD11b (M2 markers). MFI is the median fluorescence intensity of the various surface markers. M0 refers to undifferentiated macrophages; M1 / M2 refers to undifferentiated macrophages polarized to M1 or M2. Results shown are means ± SD based on five independent experiments (*, p<0.05; **, p<0.01; ***, p<0.001 vs. M1 / M2). [Figure 16] Human M0 macrophages exposed to PRP6-HO7 down-regulate ALCAM, SLC16A1, and LMNB1 during differentiation. Expression of ALCAM, LMNB1, and SLC16A1 transcripts in differentiated macrophages treated with PRP-HE8, PRP-HO7, or PRP6-HO7 compared with untreated undifferentiated (M0) macrophages on days 1, 2, 4, and 6 of differentiation. Gene expression data are expressed as Log2FC (FC is fold change). Relative expression data for identified genes were obtained by total RNA extraction and independent validation by TaqMan RT-qPCR. Log2FC values ​​are expressed as the mean + SD based on five independent experiments (*, p<0.05, **, p<0.01, ***, p<0.001, and ****, p<0.0001 relative to untreated M0 macrophages). [Figure 17]Olfactory receptors are upregulated by differentiated human M0 macrophages exposed to PRP6-HO7. Expression of "find me" or olfactory receptor CCR2, CX3CR1, CXCR1, FPR2, GPR132, P2RY2, and S1PR1 transcripts in undifferentiated macrophages treated with PRP-HE8, PRP-HO7, or PRP6-HO7 compared to untreated undifferentiated macrophages (M0) on day 6 of differentiation. Gene expression data are expressed as Log2FC (FC is fold change). Relative expression data for identified genes were obtained by total RNA extraction and TaqMan RT-qPCR validation. Log2FC values ​​are expressed as the mean + SD based on four independent experiments (* p<0.05; *** p<0.001 vs. untreated M0 macrophages). [Figure 18] Comparison of cytokine profiles of M1 macrophages treated with PRP-HO7 and PRP6-HO7. Cell culture supernatants from untreated M1 macrophages (M1), PRP-HO7-treated M1 macrophages, and PRP6-HO7-treated M1 macrophages were incubated with the R&D Systems "Human Cytokine Array Kit" according to the manufacturer's guidelines. The density of each dot was quantified using Quantity One® software and displayed as the normalized mean pixel density for each cytokine and treatment involved. [Figure 19] Production and purification of PRP6-HO7 protein in Pichia pastoris. 12% SDS-PAGE stained with Coomassie blue visualizes PRP6-HO7 after purification by nickel affinity chromatography (His tag). Elution fraction pools were run under reducing (R) and non-reducing (NR) conditions. [Figure 20]Production and purification of PRP6-HO7 protein in E. coli. Purification by nickel affinity chromatography (His tag) followed by 12% SDS-PAGE stained with Coomassie blue to visualize PRP6-HO7 under reducing conditions (R) or Western analysis. Western blots were probed with anti-HisMoAb. [Figure 21] Experimental design and treatment regimen for the mouse dextran sulfate sodium (DSS) colitis model. Routes of administration: minocycline (reference compound, standard of care), oral gavage; PRP-HO7, PRP6-HO7, and DPBS (controls), subcutaneous injection. [Figure 22] ΔBody weight (% body weight loss relative to day 0). Effect of PRP-HO7, PRP6-HO7, and minocycline on body weight changes in a DSS-induced colitis mouse model. Results are expressed as mean ± SEM. **p<0.01, ***p<0.001 vs. 2% DSS-control group (two-way ANOVA). [Figure 23] Effect of PRP-HO7, PRP6-HO7, and minocycline on the progression of clinical colitis scores in mice with DSS-induced colitis. DAI values ​​over the 9-day experimental period based on the criteria proposed in Table 5. Results are expressed as mean ± SEM. *p<0.05, **p<0.01, and ****p<0.0001 relative to the 2% DSS-control group (two-way ANOVA). Detailed Description of the Invention

[0021] The present inventors have surprisingly found that the CCP6 domain of C4BP is sufficient to maintain the tolerogenic and immunomodulatory activity of the C4BP α chain without requiring any of the other CCP domains of the C4BP α chain. Furthermore, the present inventors have surprisingly found that oligomerization of CCP6 is essential for maintaining immunomodulatory activity. As shown in Examples 1-11 of the present invention, homo-oligomers formed by seven recombinant polypeptides containing the CCP6 domain and the oligomerization domain of C4BP, designated PRP6-HO7, have even more tolerogenic activity than the physiological isoform of C4BP lacking the β chain, designated PRP-HO7, as demonstrated by gene expression experiments (Figures 13, 14, 16, 17, and 18) and by conversion into regulatory or tolerogenic macrophages (Figure 15). We successfully expressed and purified PRP6-HO7 in eukaryotic HEK (Expi293) cells, the yeast Pichia pastoris (Example 12 and Figure 19), and bacterial cells (Example 13 and Figure 20). We then performed in vitro studies to demonstrate that this compound can inhibit dendritic cell maturation in the presence of maturation stimuli and promote the generation of dendritic cells exhibiting characteristics of tolerogenic dendritic cells. Specifically, PRP6-HO7 down-regulated activation markers of human Mo-DCs (Examples 2, 4, 5, and 6; Figures 4, 8, 9, 10, 11, and 12), the release of inflammatory cytokines by LPS-matured human Mo-DCs (Example 2 and Figure 6), and the release of key transcripts consistent with the inflammatory molecular signature of Mo-DCs (Example 8 and Figure 13). Furthermore, PRP6-HO7 reduces the chemotaxis of human Mo-DCs (Example 7 and Figure 12) and upregulates the "find me" receptor at the transcriptional level (Example 8 and Figure 14).

[0022] Meanwhile, we have also demonstrated that PRP6-HO7 inhibits macrophage maturation and promotes the generation of macrophages exhibiting characteristics of tolerogenic cells. Specifically, PRP6-HO7 down-regulates the expression of human M1 and M2 activation markers (Example 9 and Figure 15), the release of proinflammatory cytokines by M1 macrophages (Example 11 and Figure 18), and the release of key transcripts corresponding to the molecular signature of monocyte-derived macrophage differentiation (Example 10 and Figure 16). Furthermore, PRP6-HO7 up-regulates the "find me" receptor in M0 macrophages at the transcriptional level (Example 10 and Figure 17).

[0023] The present inventors have shown that PRP6-HO7 is more effective than the physiological isoform of C4BP lacking the β chain, C4BP(β-), called PRP-HO7, in treating immunological diseases caused by undesired activation of the immune system, particularly ulcerative colitis (Example 14).

[0024] The homo-oligomers of the present invention lack complement inhibitory activity (Example 3 and Figure 7); are smaller than antibodies (100 kDa) (Figure 2); have superior stability (extended plasma half-life) compared to antibodies in body fluids; exhibit high avidity interactions with potential surface receptors with Kd in the low nanomolar range; and have high thermodynamic stability due to the C-terminal oligomerization scaffold stabilized by intermolecular disulfide bonds and a layer of electrostatic interactions. Furthermore, unlike its precursor, PRP-HO7, PRP6-HO7 is not glycosylated, allowing for production and further purification in various expression systems (eukaryotic, yeast, or bacterial cells). Finally, due to its simplified structure containing only the CCP6 domain, administration of PRP6-HO7 does not increase infection susceptibility compared to PRP-HO7.

[0025] Recombinant Polypeptides of the Invention We have shown that a recombinant polypeptide containing the CCP6 domain and oligomerization domain of C4BP can form homo-oligomers that are more tolerogenic than the physiological isoform of C4BP, C4BP(β-), which lacks the β chain.

[0026] Thus, in a first aspect, the present invention relates to a recombinant polypeptide comprising the CCP6 domain of the C4BP alpha chain or a functionally equivalent variant thereof, the oligomerization domain, wherein said polypeptide does not comprise any of the CCP1, CCP2, CCP3, CCP4, CCP5, CCP7 and CCP8 domains of the C4BP alpha chain.

[0027] The terms "recombinant polypeptide" and "recombinant protein" are used interchangeably herein to refer to a polypeptide or protein comprising a fusion of the CCP6 domain of C4BP and an oligomerization domain obtained by recombinant DNA technology upon expression of a recombinant polynucleotide. As used herein, "recombinant" means that a particular nucleic acid (DNA or RNA) or vector is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR), and ligation steps that result in a construct with a structural coding sequence that is distinct from the endogenous nucleic acid found in natural systems. A DNA sequence encoding a polypeptide can be assembled from cDNA fragments or a series of synthetic oligonucleotides to provide a synthetic nucleic acid that can be expressed from a recombinant transcription unit contained in a cell or a cell-free transcription and translation system. Genomic DNA containing the relevant sequence can also be used to form a recombinant gene or transcription unit. Thus, the term "recombinant" nucleic acid refers to one that does not exist in nature, e.g., is created by the artificial combination of two otherwise separated sequence segments through human intervention. This artificial combination is often achieved by chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques, to join nucleic acid segments of desired functions together to produce a desired combination of functions. The recombinant polypeptides of the present invention are not deletion mutants obtained by deleting the complete CCP1, CCP2, CCP3, CCP4, CCP5, CCP7, and CCP8 domains of C4BP.

[0028] As used herein, the term "polypeptide" or "protein" refers to a polymer of amino acid residues covalently linked by peptide bonds to form a linear chain. The terminal amino acid at one end of the chain (the amino terminus) has a free amino group, and the terminal amino acid at the other end of the chain (the carboxy terminus) has a free carboxyl group. As used herein, the term "amino terminus" (abbreviated N-terminus) refers to the free amino group on the amino terminal amino acid of a polypeptide, or the amino group when involved in a peptide bond of an amino acid at any other position within a peptide. Similarly, the term "carboxy terminus" refers to the free carboxyl group on the carboxy terminus of a polypeptide, or the carboxyl group of an amino acid at any other position within a peptide. In one embodiment, the polypeptides of the present invention have fewer than 1,000 amino acids, fewer than 900 amino acids, fewer than 800 amino acids, fewer than 700 amino acids, fewer than 600 amino acids, fewer than 500 amino acids, fewer than 475 amino acids, fewer than 450 amino acids, fewer than 425 amino acids, fewer than 400 amino acids, fewer than 375 amino acids, fewer than 350 amino acids, fewer than 325 amino acids, fewer than 300 amino acids, fewer than 275 amino acids, fewer than 250 amino acids, fewer than 225 amino acids, fewer than 200 amino acids, or fewer than 175 amino acids. In a preferred embodiment, the polypeptides of the present invention have 100 to 200 amino acids, preferably 115 to 175 amino acids, more preferably 119 to 173 amino acids, and even more preferably 119 to 125 amino acids. In a preferred embodiment, the polypeptides of the present invention have 119 amino acids.

[0029] As used herein, "amino acid residue" refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimic known in the art. In certain embodiments, the residues of a polypeptide are contiguous, and non-amino acids do not interrupt the sequence of amino acid residues. In other embodiments, the sequence may include one or more non-amino acid moieties. In certain embodiments, the sequence of residues of a polypeptide may be interrupted by one or more non-amino acid moieties (see Li et al. 2013. Molecules, 18(8):9797-981).

[0030] As used herein, the term "C4BP" or "C4b-binding protein" refers to a regulatory component of the classical pathway synthesized primarily by hepatocytes that acts as a cofactor for the factor I-dependent degradation of C3b and C4b, facilitating the decay of the classical pathway C3 / C5-convertase. C4BP circulates in plasma as three isoforms, the proportion of which depends on the relative levels of the C4BP α (70 kDa) and C4BP β (45 kDa) chains. The major isoform of C4BP is composed of seven identical α chains and one β chain (α7β1), and is known as C4BP(α7β1) or C4BP(β + During inflammation, the normally less abundant isoform is upregulated, consisting only of the α chain (α7β0), and is called C4BP(α7β0) or C4BP(β - Furthermore, recombinant expression of the α chain in eukaryotic cells can result in an oligomer containing six α chains (α6β0).

[0031] As used herein, the term "C4BP α chain," also known as PRP or proline-rich protein, refers to the mature, processed form of the human polypeptide defined by NCBI database accession number P04003 (released July 31, 2019), which contains amino acids 49 to 597. The term C4BP α chain also refers to orthologs of the human C4BP α chain, such as the mouse C4BP α chain (amino acids 57 to 469) corresponding to the mature form of the polypeptide defined by NCBI database accession number P08607 (released July 31, 2019), the C4BP α chain (amino acids 14 to 558) corresponding to the mature form of the polypeptide defined by NCBI database accession number Q63514 (released May 8, 2019), and the bovine C4BP α chain (amino acids 49 to 610) corresponding to the mature form of the polypeptide defined by NCBI database accession number Q28065 (released May 8, 2019).

[0032] The C4BP α-chain contains eight complement control protein domains (CCPs) 60 amino acid residues in length, each disulfide-bonded by four cysteine ​​residues in a 1-3 2-4 sequence, and a hydrophobic core built around a nearly invariant tryptophan residue. Thus, the term "CCP domain" as used herein refers to one of the complement control domains found in the C4BP α-chain.

[0033] The expression "CCP6 domain" as used herein corresponds to the region found between amino acids 363 and 424 for the human C4BP α chain as defined in the sequence provided in the NCBI database under accession number P04003 (released July 31, 2019) (SEQ ID NO: 1), which corresponds to the following sequence: LCCPEPKLNNGEITQHRKSRPANHCVYFYGDEISFSCHETSRFSAICQGDGTWSPRTPSCGD (SEQ ID NO: 1).

[0034] Thus, in a preferred embodiment, the CCP6 domain is the CCP6 domain of the human C4BP α chain, more preferably SEQ ID NO: 1. In one embodiment, the CCP6 domain of the C4BP α chain comprises SEQ ID NO: 1. In another embodiment, the CCP6 domain of the C4BP α chain consists of SEQ ID NO: 1.

[0035] The term "CCP6 domain" as used herein also refers to the CCP6 domain of the C4BP α chain of any ortholog of the human C4BP chain, such as the CCP6 domain of the rabbit C4BP α chain (amino acids 410 to 460) (SEQ ID NO: 12) for the sequence provided under NCBI database accession number G1T7A2 (released October 16, 2019) shown in Table 1 below, the CCP6 domain of the rat C4BP α chain (amino acids 327 to 388) (SEQ ID NO: 13) for the sequence provided under NCBI database accession number Q63514 (released May 8, 2019), or the CCP6 domain of the bovine C4BP α chain (amino acids 365 to 427) (SEQ ID NO: 14) for the sequence provided under NCBI database accession number Q28065 (released May 8, 2019).

[0036] [Table 1]

[0037] As used herein, the term "CCP6 domain" is also used to refer to functionally equivalent variants of the naturally occurring CCP6 domain defined above, which result from the substitution, insertion or deletion of one or more amino acids and which substantially preserve the ability of the original polypeptide to induce the tolerogenic phenotype upon oligomerization.

[0038] In preferred embodiments, functionally equivalent variants of the CCP6 domain result from modification of any of the above sequences by substitutions (e.g., conservative amino acid substitutions) and / or insertions (e.g., small, single amino acid insertions, or insertions comprising 2, 3, 4, 5, 10, 15, 20, or more consecutive amino acids) and / or deletions (e.g., small, single amino acid deletions, or deletions of 2, 3, 4, 5, 10, 15, 20, or more consecutive amino acids). Thus, in certain embodiments, variants of the original sequence differ from the naturally occurring sequence by (i) one or more (e.g., 2, 3, 4, 5, 6, or more) conservative amino acid substitutions, (ii) one or more (e.g., 2, 3, 4, 5, 6, or more) amino acid deletions, (iii) one or more (e.g., 2, 3, 4, 5, 6, or more) amino acid insertions, or (iv) combinations thereof. The deleted or inserted amino acids may be contiguous or non-contiguous.

[0039] The hydrophobicity index of amino acids is taken into consideration when making such changes, as it is known that substitution of a particular amino acid with another amino acid having a similar hydrophobicity index or hydrophobicity score will result in a polypeptide with similar biological activity. For example, the relative hydrophobicity properties of amino acid residues affect the secondary and tertiary structure of the resulting polypeptide, thereby determining the polypeptide's interactions with other molecules, such as enzymes, substrates, receptors, antibodies, and antigens. As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Examples of substitutions that take into account the various characteristics discussed above are known to those of skill in the art and are set forth in Table 2 below.

[0040] [Table 2]

[0041] In one embodiment, functionally equivalent variants include an addition of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 amino acids to the N-terminus, C-terminus, or both ends of the CCP6 domain sequence. In one embodiment, functionally equivalent variants include an addition of fewer than 20, fewer than 15, fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, fewer than 2, or 1 amino acid. In another embodiment, functionally equivalent variants include a deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 amino acids to the N-terminus, C-terminus, or both ends of the CCP6 domain sequence. In another embodiment, functionally equivalent variants comprise deletion of fewer than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids. In another embodiment, functionally equivalent variants have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 amino acid substitutions in the sequence of the CCP6 domain. In another embodiment, functionally equivalent variants have fewer than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. In a preferred embodiment, functionally equivalent variants are variants with the addition of a single amino acid, preferably a methionine, to the N-terminus of the CCP6 domain.

[0042] In another embodiment, functionally equivalent variants of the CCP6 domain of the C4BP α-chain contain one or more residues equivalent to cysteine ​​at position 2 of SEQ ID NO: 1, cysteine ​​at position 3 of SEQ ID NO: 1, proline at position 4 of SEQ ID NO: 1, proline at position 6 of SEQ ID NO: 1, isoleucine at position 13 of SEQ ID NO: 1, histidine at position 16 of SEQ ID NO: 1, cysteine ​​at position 25 of SEQ ID NO: 1, tyrosine at position 27 of SEQ ID NO: 1, glycine at position 30 of SEQ ID NO: 1, aspartic acid at position 31 of SEQ ID NO: 1, cysteine ​​at position 37 of SEQ ID NO: 1, cysteine ​​at position 47 of SEQ ID NO: 1, glycine at position 51 of SEQ ID NO: 1, threonine at position 52 of SEQ ID NO: 1, tryptophan at position 53 of SEQ ID NO: 1, proline at position 55 of SEQ ID NO: 1, threonine at position 57 of SEQ ID NO: 1, proline at position 58 of SEQ ID NO: 1, and cysteine ​​at position 60 of SEQ ID NO: 1. Preferably, they contain all of these residues. In a more preferred embodiment, the variant retains the relative spacing between these residues.

[0043] Functionally equivalent variants of the CCP6 domain of the C4BP α-chain include, but are not limited to, naturally occurring polymorphic variants (i.e., allelic variants) and recombinantly engineered or modified variants. CCP6 domain variants suitable for use in the present invention include, but are not limited to, variants that share at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 48%, at least 45%, at least 40%, or at least 35% sequence identity with the naturally occurring CCP6 domain defined above, particularly with naturally occurring CCP6 of human origin.

[0044] The percentage identity of the amino acid sequence of a CCP6 domain variant to the above amino acid sequence can be easily determined by those skilled in the art by sequence comparison. As used herein, two amino acid sequences have 100% amino acid sequence identity if the amino acid residues of the two amino acid sequences are the same when the two amino acid sequences are aligned to maximize correspondence. Sequence comparison of polypeptides and polynucleotides (e.g., polynucleotides encoding the polypeptides described herein) can be performed using any method known to those skilled in the art, such as methods using computer algorithms. Such algorithms include the Align or BLAST algorithm (see, e.g., Altschul, J. Mol. Biol. 219:555-565, 1991; Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919), which are available on the NCBI website (see [online] Internet ncbi.nlm.nih.gov / cgi-bin / BLAST). Default parameters can be used. Additionally, standard software programs such as those included in the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wis.); the CLUSTALW program (Thompson et al., Nucleic Acids Res. 22:4673-80 (1991)); and "GeneDoc" (Nicholas et al., EMBNEW News 4:14 (1991)) can be utilized.Other methods for comparing two amino acid sequences by determining optimal alignment are practiced by those skilled in the art (see, e.g., Peruski and Peruski, The Internet and the New Biology: Tools for Genomic and Molecular Research (ASM Press, Inc. 1997); Wu et al. (eds.), "Information Superhighway and Computer Databases of Nucleic Acids and Proteins," in Methods in Gene Biotechnology, pages 123-151 (CRC Press, Inc. 1997); and Bishop (ed.), Guide to Human Genome Computing, 2nd Ed. (Academic Press, Inc. 1998)).

[0045] As used herein, the phrase "substantially preserves the ability of the original polypeptide to induce the tolerogenic phenotype upon oligomerization" refers to a polypeptide capable of inhibiting dendritic cell and / or macrophage maturation, e.g., as defined in any of Examples 1-11 of the present invention when a CCP6 variant is fused to an oligomerization domain, preferably the oligomerization domain of SEQ ID NO: 2. In particular, functionally equivalent variants exhibit the ability to generate tolerogenic dendritic cells when added to monocytic cells during differentiation into immature dendritic cells and / or when added to immature dendritic cells during maturation into mature dendritic cells. The ability of a variant to promote the generation of tolerogenic dendritic cells can be determined, for example, by measuring the expression levels of maturation markers, such as CD83, CD86, and / or CD80, in dendritic cells matured in the presence of the variant (Example 4 of the present invention). Thus, a polypeptide is considered a functionally equivalent variant of the CCP6 domain of the C4BP α-chain if it exhibits at least 100%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, or at least 50% of the activity of the CCP6 domain of the human C4BP α-chain when fused to an oligomerization domain, particularly the oligomerization domain of SEQ ID NO: 2.

[0046] As used herein, the term "oligomerization domain" refers to a polypeptide domain that has the property that polypeptides containing the domain aggregate to form oligomers, i.e., a region involved in oligomerization between monomers. An oligomerization domain comprises amino acids in a region of one monomer that can interact with amino acids in a region of another monomer to enable oligomerization of the monomers. Suitable oligomerization domains are known in the art. In a preferred embodiment, the oligomerization domain is the oligomerization domain of the C4BP α chain. In another embodiment, the oligomerization domain is a functionally equivalent variant of the oligomerization domain of the C4BP α chain. As used herein, the expression "oligomerization domain of the C4BP α chain" corresponds to the region found between amino acids 541 and 597 (SEQ ID NO: 2) for the human C4BP α chain as defined in the sequence provided in the NCBI database under accession number P04003 (released July 31, 2019), which corresponds to the following sequence: ETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKEL (SEQ ID NO: 2).

[0047] Thus, in a preferred embodiment, the oligomerization domain is the oligomerization domain of the human C4BP α-chain or a functionally equivalent variant thereof; more preferably SEQ ID NO: 2 or a functionally equivalent variant thereof; even more preferably SEQ ID NO: 2. In one embodiment, the oligomerization domain comprises SEQ ID NO: 2 or a functionally equivalent variant thereof, preferably SEQ ID NO: 2. In another embodiment, the oligomerization domain consists of SEQ ID NO: 2 or a functionally equivalent variant thereof, preferably SEQ ID NO: 2.

[0048] As used herein, the term "oligomerization domain of the C4BP α chain" also refers to the oligomerization domain of any ortholog of the human C4BP α chain, such as the oligomerization domain of the mouse C4BP α chain (amino acids 416 to 469) (SEQ ID NO: 15) for the sequence provided by NCBI database accession number P08607 (released July 31, 2019) shown in Table 3 below, the oligomerization domain of the rat C4BP α chain (amino acids 504 to 558) (SEQ ID NO: 16) for the sequence provided by NCBI database accession number Q63514 (released May 8, 2019), or the oligomerization domain of the bovine C4BP α chain (amino acids 544 to 610) (SEQ ID NO: 17) for the sequence provided by NCBI database accession number Q28065 (released May 8, 2019). Other mammalian orthologue C4BP α-chain oligomerization domains useful in the present invention are SEQ ID NOS: 18-33 shown in Table 3. Functionally equivalent variants of the human C4BP α-chain oligomerization domain useful in the present invention are SEQ ID NOS: 34-41. Examples of avian orthologue oligomerization domains described in PCT patent application WO2007 / 062819 include the chicken C4BP α-chain oligomerization domain (SEQ ID NOS: 42) or the zebra finch avian homolog (SEQ ID NOS: 43). Functionally equivalent variants of the C4BP oligomerization domain disclosed in Table 1 of WO2007 / 062819 are also included (SEQ ID NOS: 44-55).

[0049] [Table 3] TIFF0007778070000004.tif239163TIFF0007778070000005.tif109163

[0050] In a preferred embodiment, the oligomerization domain is the oligomerization domain of a mammalian C4BP α chain, more preferably a human, mouse or rat C4BP α chain, most preferably a human C4BP α chain.

[0051] In a preferred embodiment, the oligomerization domain is an oligomerization domain selected from the group consisting of SEQ ID NO: 2 and any one of SEQ ID NOs: 15 to 55; preferably an oligomerization domain selected from the group consisting of SEQ ID NO: 2 and any one of SEQ ID NOs: 15 to 41; more preferably an oligomerization domain selected from the group consisting of SEQ ID NO: 2 and any one of SEQ ID NOs: 15 to 33; even more preferably an oligomerization domain selected from the group consisting of SEQ ID NO: 2 and any one of SEQ ID NOs: 15 to 32; even more preferably an oligomerization domain selected from the group consisting of SEQ ID NO: 2 and any one of SEQ ID NOs: 15 to 17; and even more preferably an oligomerization domain of SEQ ID NO: 2.

[0052] The oligomerization domain of the C4BP α-chain also includes other mammalian and non-mammalian homologs of these sequences. Means for obtaining such homologs are routine techniques available to those skilled in the art. Essentially, such techniques involve using nucleic acids encoding any of the oligomerization domain sequences of the present invention, or fragments thereof, as probes to retrieve and determine the sequences of C4BP homologs in other species. A variety of techniques are available for this, including PCR amplification and cloning of homologs using an appropriate mRNA source (e.g., from an embryo or actively dividing differentiated or tumor cells), or methods involving obtaining a cDNA library from an animal, e.g., from one of the sources listed above, probing the library under stringent conditions with nucleic acids encoding any of the oligomerization domains of the present invention, and retrieving cDNAs encoding all or part of the animal homolog. Here, partial cDNAs can be obtained and the full-length coding sequence can be determined by primer extension techniques. Alternatively, if all or part of the animal's genomic sequence is available, suitable homologs can be determined using homology searches using the sequences of the present invention.

[0053] The term "oligomerization domain of the C4BP α chain" as used herein also refers to any functionally equivalent variant of the naturally occurring oligomerization domain of the C4BP α chain defined above, by substitution, insertion or deletion of one or more amino acids, which, when forming part of a recombinant polypeptide of the present invention, substantially retains the ability of the original polypeptide to form oligomers.

[0054] Thus, functionally equivalent variants may be fragments of the oligomerization domain in which amino acids not involved in oligomerization ability have been deleted but amino acids involved in oligomerization have been preserved, or variants in which amino acids not involved in oligomerization have been mutated.

[0055] In preferred embodiments, functionally equivalent variants of the oligomerization domain of the C4BP α-chain are produced by modifying any of the above-mentioned sequences by substitutions (e.g., conservative amino acid substitutions) and / or insertions (e.g., small, single amino acid insertions or insertions involving 2, 3, 4, 5, 10, 15, 20, or more consecutive amino acids) and / or deletions (e.g., small, single amino acid deletions or deletions involving 2, 3, 4, 5, 10, 15, 20, or more consecutive amino acids). Thus, in certain embodiments, variants of a native sequence differ from the naturally occurring sequence by (i) one or more (e.g., 2, 3, 4, 5, 6, or more) conservative amino acid substitutions, (ii) one or more (e.g., 2, 3, 4, 5, 6, or more) amino acid deletions, (iii) one or more (e.g., 2, 3, 4, 5, 6, or more) amino acid insertions, or (iv) combinations thereof. The deleted or inserted amino acids may be contiguous or non-contiguous.

[0056] As described for functionally equivalent variants of the CCP6 domain, such changes take into account the hydrophobicity index of amino acids. Table 2, which lists example substitutions, is also applicable to functionally equivalent variants of the oligomerization domain of the C4BP α chain.

[0057] In one embodiment, functionally equivalent variants include an addition of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 amino acids at the N-terminus, C-terminus, or both ends of the oligomerization domain sequence. In one embodiment, functionally equivalent variants include an addition of fewer than 20, fewer than 15, fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, fewer than 2, or 1 amino acid. In another embodiment, functionally equivalent variants include a deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 amino acids at the N-terminus, C-terminus, or both ends of the oligomerization domain sequence. In another embodiment, functionally equivalent variants comprise deletion of fewer than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids. In another embodiment, functionally equivalent variants have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 amino acid substitutions in the sequence of the oligomerization domain. In another embodiment, functionally equivalent variants have fewer than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. In a preferred embodiment, functionally equivalent variants have an addition of a single amino acid, preferably a methionine, to the N-terminus of the oligomerization domain.

[0058] In one embodiment, a fragment of the oligomerization domain of the C4BP α-chain capable of forming oligomers may comprise at least 47 amino acids, preferably at least 50 amino acids.

[0059] In another embodiment, a variant of the oligomerization domain of C4BP α-chain contains one or more residues equivalent to glutamic acid at position 1 of SEQ ID NO:2, glycine at position 12 of SEQ ID NO:2, glutamine at position 17 of SEQ ID NO:2, valine at position 25 of SEQ ID NO:2, lysine at position 26 of SEQ ID NO:2, alanine at position 28 of SEQ ID NO:2, leucine at position 29 of SEQ ID NO:2, glutamic acid at position 30 of SEQ ID NO:2, tyrosine at position 32 of SEQ ID NO:2, lysine at position 33 of SEQ ID NO:2, leucine at position 34 of SEQ ID NO:2, leucine at position 36 of SEQ ID NO:2, glutamic acid at position 37 of SEQ ID NO:2, and leucine at position 41 of SEQ ID NO:2. Preferably, the variant contains all of these residues. More preferably, the variant retains the relative spacing between these residues.

[0060] In a preferred embodiment, the variant contains residues equivalent to glycine at position 12 of SEQ ID NO: 2, alanine at position 28 of SEQ ID NO: 2, leucines at positions 29, 34, 36 and 41 of SEQ ID NO: 2, tyrosine at position 32 of SEQ ID NO: 2, and lysine at position 33 of SEQ ID NO: 2. Preferably, the variant retains the relative spacing between these residues.

[0061] In another preferred embodiment, a variant of the oligomerization domain of C4BP α-chain contains residues equivalent to the glycine at position 12 of SEQ ID NO: 2, the alanine at position 28 of SEQ ID NO: 2, the leucines at positions 29, 34, 36, and 41 of SEQ ID NO: 2, the tyrosine at position 32 of SEQ ID NO: 2, the lysine at position 33 of SEQ ID NO: 2, and the two cysteine ​​residues at positions 6 and 18 of SEQ ID NO: 2. Preferably, the relative spacing between these residues is maintained in the variant.

[0062] Functionally equivalent variants of the oligomerization domain of the C4BP α-chain used in the present invention include, but are not limited to, naturally occurring polymorphic variants (i.e., allelic variants) and recombinantly engineered or modified variants. Variants of the oligomerization domain suitable for use in the present invention include, but are not limited to, variants having at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, or at least 35% sequence identity with the naturally occurring oligomerization domain defined above, particularly the naturally occurring oligomerization domain of the C4BP α-chain of human origin.

[0063] Methods for determining the percent identity of the amino acid sequences of variants of the oligomerization domain are as described above for the CCP6 domain.

[0064] As used herein, the phrase "substantially preserves the ability of the original polypeptide to form oligomers when forming part of a recombinant polypeptide of the invention" refers to a polypeptide that can form oligomers, for example, as shown in Example 1 of the present invention and in Figure 3 when the oligomerization domain mutant is fused to a CCP6 domain, preferably the CCP6 domain of SEQ ID NO: 1. Methods for determining whether a polypeptide is capable of forming oligomers are available to those skilled in the art, such as the method described by Blom et al. (J. Biol. Chem. 2001, 276: 27136-27144) based on the analysis by polyacrylamide gel electrophoresis under native conditions of purified C4BP obtained by recombinant expression of a mutant α-chain in eukaryotic cells (e.g., 293 cells), followed by affinity purification using an antibody specific for the CCP6 region. Alternatively, the ability of oligomerization domain mutants to form oligomers can be tested in the present invention by expressing the mutants in prokaryotic or eukaryotic host cells and analyzing the recovered mutants by polyacrylamide gel electrophoresis under reducing and non-reducing conditions and Coomassie blue staining (i.e., comparing the behavior of the mutants on an SDS-PAGE gel in the presence and absence of the reducing agent β-mercaptoethanol), or by performing Western blots probed with anti-tag antibodies or anti-C4BP α-chain under reducing or non-reducing conditions, as described in Example 1 or Figure 3. Alternatively, the ability of oligomerization domain mutants to form oligomers can be tested in the present invention by expressing the mutants in prokaryotic host cells and recovering the mutants under conditions that result in oligomerization of the complete 57-amino acid oligomerization domain of human C4BP α-chain, and determining whether the mutants form oligomers, for example, by gel filtration.

[0065] Thus, a polypeptide is considered a functionally equivalent variant of the oligomerization domain of the C4BP α-chain if it exhibits at least 100%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, or at least 50% of the ability of the oligomerization domain of the human C4BP α-chain to form oligomers when fused to a CCP6 domain, particularly the CCP6 domain of SEQ ID NO: 1.

[0066] Functionally equivalent variants of the oligomerization domain of the human C4BP α-chain preferably have: (a) a cysteine ​​residue equivalent to the cysteine ​​residue at position 498 of the mature form of the human C4BP α-chain (i.e., a form of the human C4BP α-chain lacking the signal peptide), which corresponds to amino acid 546 of the immature form as defined in NCBI database accession number P04003 (released July 31, 2019); (b) a cysteine ​​residue equivalent to the cysteine ​​residue at position 498 of the mature form of the human C4BP α-chain (i.e., a form of the human C4BP α-chain lacking the signal peptide), which corresponds to amino acid 546 of the immature form as defined in NCBI database accession number P04003 (released July 31, 2019); and / or (c) the C-terminal amphipathic α-helical region, more preferably the last 13 residues equivalent to residues 537-549 of the mature human C4BP α-chain, corresponding to amino acids 585-597 of the immature form as defined in NCBI database accession number P04003. For intracellular oligomerization of the molecule, functionally equivalent variants should preferably maintain (c); functionally equivalent variants should more preferably maintain (a), (b), and (c).

[0067] Recombinant polypeptides of the present invention containing the CCP6 domain of the C4BP α chain do not contain any of the other CCP domains found in the C4BP α chain. Thus, the polypeptides of the present invention do not contain the CCP1, CCP2, CCP3, CCP4, CCP5, CCP7, and CCP8 domains of the C4BP α chain. When referring to the human C4BP α chain, the CCP1 domain corresponds to amino acids 49 to 110 of the polypeptide shown in NCBI database accession number P04003 (released July 31, 2019), the CCP2 domain corresponds to amino acids 111 to 172 of the polypeptide shown in NCBI database accession number P04003 (released July 31, 2019), the CCP3 domain corresponds to amino acids 173 to 236 of the polypeptide shown in NCBI database accession number P04003 (released July 31, 2019), and the CCP4 domain corresponds to amino acids 174 to 236 of the polypeptide shown in NCBI database accession number P04003 (released July 31, 2019). The CCP5 domain corresponds to amino acids 237-296 of the polypeptide shown in NCBI database accession number P04003 (released July 31, 2019), the CCP5 domain corresponds to amino acids 297-362 of the polypeptide shown in NCBI database accession number P04003 (released July 31, 2019), the CCP7 domain corresponds to amino acids 425-482 of the polypeptide shown in NCBI database accession number P04003 (released July 31, 2019), and the CCP8 domain corresponds to amino acids 483-540 of the polypeptide shown in NCBI database accession number P04003 (released July 31, 2019). Those skilled in the art will know how to determine equivalent CCPs to the human C4BP α chain in other C4BP α chains.

[0068] In one embodiment, the recombinant polypeptide consists of the CCP6 domain of the C4BP α chain or a functionally equivalent variant thereof and the oligomerization domain; preferably, it consists of the CCP6 domain of the C4BP α chain and the oligomerization domain of the C4BP α chain; more preferably, it consists of the CCP6 domain of the human C4BP α chain and the oligomerization domain of the human C4BP α chain. Both domains can be in any order.

[0069] In a preferred embodiment of the polypeptide of the invention, the CCP6 domain of the C4BP α-chain and the oligomerization domain, preferably the oligomerization domain of the C4BP α-chain, are associated with the same species.

[0070] In another embodiment, the recombinant polypeptide consists of (a) a methionine, (b) the CCP6 domain of the C4BP α chain or a functionally equivalent variant thereof, and (c) the oligomerization domain; preferably, it consists of (a) a methionine, (b) the CCP6 domain of the C4BP α chain, and (c) the oligomerization domain of the C4BP α chain; more preferably, it consists of (a) a methionine, (b) the CCP6 domain of the human C4BP α chain, and (c) the oligomerization domain of the human C4BP α chain. The methionine is at the N-terminus of the recombinant polypeptide, and the domains can be arranged in any order.

[0071] In a preferred embodiment, the recombinant polypeptide of the present invention comprises SEQ ID NO: 3 or a functionally equivalent variant thereof, preferably SEQ ID NO: 3. In a more preferred embodiment, the recombinant polypeptide of the present invention consists of SEQ ID NO: 3 or a functionally equivalent variant thereof, preferably SEQ ID NO: 3. LCCPEPKLNNGEITQHRKSRPANHCVYFYGDEISFSCHETSRFSAICQGDGTWSPRTPSCGDETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKEL (SEQ ID NO: 3)

[0072] In another embodiment, a recombinant polypeptide of the present invention consists of the sequence of SEQ ID NO: 3 and an additional methionine at the N-terminus of the polypeptide.

[0073] In one embodiment, a polypeptide of the invention does not include a region of a protein other than C4BP, e.g., a polypeptide of the invention cannot be a fusion protein that includes a region that forms part of a protein other than C4BP.

[0074] In another embodiment, the polypeptides of the present invention include polypeptides that do not form part of C4BP. Polypeptides that do not form part of C4BP are considered heterologous polypeptides, i.e., polypeptides derived from a gene different from that encoding C4BP.

[0075] The recombinant polypeptides of the present invention may contain the CCP6 domain and the oligomerization domain in any order. From the amino terminus to the carboxy terminus, the recombinant polypeptides of the present invention may contain (a) a region containing the CCP6 domain of the C4BPα chain and (b) a region containing the oligomerization domain. Alternatively, from the amino terminus to the carboxy terminus, the recombinant polypeptides of the present invention may contain (a) a region containing the oligomerization domain and (b) a region containing the CCP6 domain of the C4BPα chain. Those skilled in the art will understand that the recombinant polypeptides of the present invention may contain additional amino acids between the CCP6 domain and the oligomerization domain. In a preferred embodiment, the C-terminus of the CCP6 domain of the C4BPα chain is directly fused to the N-terminus of the oligomerization domain, i.e., no additional amino acids or linker are present between the two domains. In another embodiment, the C-terminus of the oligomerization domain is directly fused to the N-terminus of the CCP6 domain of the C4BPα chain. In another embodiment, the C-terminus of the CCP6 domain of the C4BPα chain is separated from the N-terminus of the oligomerization domain by additional amino acids, i.e., amino acids not present in the naturally occurring domain from which it is derived. In another embodiment, the C-terminus of the oligomerization domain is separated from the N-terminus of the CCP6 domain of the C4BPα chain by additional amino acids. In a preferred embodiment, a polypeptide of the invention comprises, from amino- to carboxy-terminal, (a) a methionine, (b) a region comprising the CCP6 domain of the C4BPα chain, and (c) a region comprising the oligomerization domain. In another embodiment, a polypeptide of the invention comprises, from amino- to carboxy-terminal, (a) a methionine, (b) a region comprising the oligomerization domain, and (c) a region comprising the CCP6 domain of the C4BPα chain. In another embodiment, a recombinant polypeptide of the invention consists, from amino- to carboxy-terminal, of (a) the CCP6 domain of the C4BPα chain and (b) the oligomerization domain of the C4BPα chain.In another embodiment, a recombinant polypeptide of the invention consists, from amino to carboxy terminus, of (a) the oligomerization domain of the C4BP α chain and (b) the CCP6 domain of the C4BP α chain. All of these embodiments apply equally when the polypeptide of the invention is formed by a functionally equivalent variant of the CCP6 domain and / or a functionally equivalent variant of the oligomerization domain of the C4BP α chain.

[0076] The recombinant polypeptides of the present invention may be in the form of a precursor. The term "precursor" refers to a polypeptide that can be processed to give rise to the mature form of the polypeptide. A precursor is a polypeptide that includes a signal peptide for transport of the polypeptide to the extracellular medium when the host cell is a eukaryotic cell.

[0077] In another embodiment, the recombinant polypeptide of the present invention further comprises a signal peptide. As used herein, the term "signal peptide" refers to a signal sequence located at the N-terminus of a polypeptide. As used herein, the terms "signal peptide" or "signal sequence" or "secretory signal peptide" or "secretory signal sequence" refer to a relatively short peptide, generally 5 to 30 amino acid residues, that directs a protein synthesized within a cell toward the secretory pathway. Signal peptides typically contain a stretch of hydrophobic amino acids with a secondary alpha-helical structure. In addition, many peptides contain a stretch of positively charged amino acids that can help the protein adopt the correct topology for translocation. Signal peptides tend to have a motif at their carboxyl terminus for recognition by peptidases, which can hydrolyze the signal peptide to produce the free signal peptide and mature protein. The signal peptide can be cleaved once the protein of interest has reached the appropriate location. Any signal peptide can be used in the present invention. The signal sequence can be from the same species of organism being transformed or from a different species. As illustrative, non-limiting examples, the signal peptide from carbonic anhydrase (CAH1) of Chlamydomonas reinhardtii, the signal peptide from periplasmic arylsulfatase (ARS1) of Chlamydomonas reinhardtii, or the signal peptide from gametolysin M11 of Chlamydomonas reinhardtii can be used. A signal peptide useful for expression in bacteria can be the Sec signal peptide or the Tat signal peptide. Signal peptides particularly for expression in yeast can be the signal peptide from the S. cerevisiae α-mating factor prepro peptide, the signal peptide from the P. pastoris acid phosphatase gene (PHO1), and the extracellular protein X (EPX1). In a preferred embodiment, the signal peptide is the signal peptide from the α-factor mating peptide. A signal peptide, preferably the α-factor conjugation peptide from Saccharomyces cerevisiae, is useful. Signal peptides useful for expression in mammalian eukaryotic cells include, but are not limited to, signal peptides from human OSM, VSV-G, mouse Igκ, human IgG2 H, BM40, secretome, human IgKVIII, CD33, tPA, human chymotrypsinogen, human trypsinogen-2, human IL-2, Gaussia luc, albumin (HSA), influenza hemagglutinin, human insulin, or silkworm fibroin LC.

[0078] In a preferred embodiment, the signal peptide is a human signal peptide. In a preferred embodiment, the signal peptide is the signal peptide of the C4BP α chain, preferably the human C4BP α chain, more preferably SEQ ID NO: 4 or a functionally equivalent variant thereof; more preferably SEQ ID NO: 4. MHPPKTPSGALHRKRKMAAWPFSRLWKVSDPILFQMTLIAALLPAVLG (SEQ ID NO: 4)

[0079] Thus, in one embodiment, the signal peptide comprises SEQ ID NO: 4. In another embodiment, the signal peptide consists of SEQ ID NO: 4.

[0080] Fusing a signal peptide to the polypeptide is a preferred strategy because it allows the fusion protein to be secreted into the medium, allowing for easy and efficient purification from the extracellular medium.

[0081] In one embodiment, a polypeptide of the present invention is a fusion protein comprising, from amino to carboxy terminus, (a) a signal peptide, (b) a region comprising the CCP6 domain of the C4BP α chain, and (c) a region comprising the oligomerization domain. Alternatively, a polypeptide of the present invention is a fusion protein comprising, from amino to carboxy terminus, (a) a signal peptide, (b) a region comprising the oligomerization domain, and (c) a region comprising the CCP6 domain of the C4BP α chain. All of these embodiments are equally applicable when a polypeptide of the present invention comprises a signal peptide, a region comprising the CCP6 domain of the C4BP α chain, and a region comprising the oligomerization domain. All of these embodiments are equally applicable when a polypeptide of the present invention is formed by a functionally equivalent variant of the CCP6 domain of the C4BP α chain and / or a functionally equivalent variant of the oligomerization domain.

[0082] In one embodiment, the recombinant polypeptide of the invention comprises SEQ ID NO: 5 or a functionally equivalent variant thereof, preferably SEQ ID NO: 5. In another embodiment, the recombinant polypeptide of the invention consists of SEQ ID NO: 5 or a functionally equivalent variant thereof, preferably SEQ ID NO: 5. MHPPKTPSGALHRKRKMAAWPFSRLWKVSDPILFQMTLIAALLPAVLGLCCPEPKLNNGEITQHRKSRPANHCVYFYGDEISFSCHETSRFSAICQGDGTWSPRTPSCGDETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKEL (SEQ ID NO: 5)

[0083] In another embodiment, the polypeptides of the invention do not include a signal peptide.

[0084] In another embodiment, the recombinant polypeptide further comprises a peptide that is not part of the C4BP α chain.

[0085] In one embodiment, a polypeptide of the invention is a fusion protein comprising a region comprising the CCP6 domain of the C4BP α chain, a region comprising the oligomerization domain, and one or more regions comprising sequences not forming part of C4BP, any of which may be in any order relative to one another.

[0086] An example of a recombinant polypeptide comprising a peptide that is not part of the C4BP α-chain can be, for example, a fusion protein that improves pharmacokinetic properties.

[0087] The recombinant polypeptides of the present invention can be modified to adjust receptor affinity, circulating half-life, therapeutic half-life, polypeptide stability, protease cleavage, dosage, release or bioavailability, facilitate purification, or improve or alter specific administration routes. Similarly, the polypeptides can contain protease cleavage sequences, reactive groups, or other molecules that improve detection, purification, or other properties of the polypeptide. Preferably, the polypeptides contain a protease-cleavable MBP sequence or an AFV, slyD, tsf, SUMO, Bla, or GST sequence.

[0088] In a preferred embodiment, the peptide that is not part of the C4BP α chain is a tag peptide.

[0089] As used herein, the expression "tag peptide" refers to a peptide suitable for the detection, isolation and / or purification of a recombinant polypeptide. Non-limiting examples of tags include affinity purification tags such as polyhistidine [poly(His)] sequences; peptide sequences that can be recognized by antibodies that can be used to purify the resulting fusion protein by immunoaffinity chromatography, such as an epitope derived from the hemagglutinin of a fever virus, c-myc-tag (recognized by anti-c-myc antibodies); streptavidin-binding peptide tags or SBP-tags; S-tags; calmodulin-binding peptides (CBPs); cellulose-binding domains; chitin-binding domains (CBDs); glutathione S-transferase-tags; maltose-binding protein (MBP); 3xHA-tags or hemagglutinin-tags; NusA; TrxA; DsbA; Avi-tags; Strep-tags; arginine-tags (Arg-tags); FLAG-tags, etc. (Zhao et al. 2013. J. Anal. Methods Chemistry, 2013:581093; Terpe K. 2003. Appl. Microbiol. Biotechnol. 60(5):523-533). The affinity purification tag can be fused directly to the monomer polypeptide in tandem, or can be fused to the monomer polypeptide via a cleavable linker, i.e., a peptide segment comprising an amino acid sequence that is specifically cleaved by enzymatic or chemical means (i.e., a recognition / cleavage site). In certain embodiments, the cleavable linker comprises an amino acid sequence that is cleavable by a protease (or protease recognition site) once the protein is translated.For example, the cleavable linker may be an amino acid sequence cleavable by a protease such as enterokinase, Arg C endoprotease, Glu C endoprotease, Lys C endoprotease, Factor Xa, thrombin, TEV (tobacco etch virus) protease, human rhinovirus 3C protease, sortase A, PreScission protease (Zhao et al. 2013. J. Anal. Methods Chem. 2013:581093), SUMO protease (Butt TR. et al. 2005. Protein Expr. Purif. 43(1):1-9), etc.; alternatively, in another specific embodiment, the cleavable linker comprises an amino acid sequence cleavable by a chemical reagent, such as cyanogen bromide, which cleaves methionine residues, or any other suitable chemical reagent. In another embodiment, the cleavable linker is an intein. In a preferred embodiment, the cleavable linker is an amino acid sequence cleavable by enterokinase, preferably the sequence DDDDK (SEQ ID NO: 59). Cleavable linkers are useful when subsequent removal of the affinity purification tag is desired. The tag can be placed at any position of the monomer, particularly at the C-terminus or N-terminus of the CCP6 domain and oligomerization domain of the C4BP α chain. In a more preferred embodiment, the tag peptide is linked to the N-terminus of the CCP6 domain of the C4BP α chain. In a preferred embodiment, the tag is linked to the polypeptide via a linker cleavable by TEV protease. In another embodiment, the tag is linked to the polypeptide via a linker cleavable by enterokinase. In another embodiment, the tag is fused directly in tandem with the polypeptide. In a more preferred embodiment, the tag is a poly(His) tag, preferably a poly(His) tag having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 histidines. In a preferred embodiment, the tag is a hexahistidine tag or a His6-tag.In another embodiment, the poly(His) tag has at least two histidines, preferably at least five histidines, more preferably at least six histidines.

[0090] Preferably, the tag is a poly(His) tag, more preferably a His6-tag, and the tag is linked to the polypeptide via a linker that is cleavable by TEV protease.

[0091] In another embodiment, the tag is a Strep-tag II peptide of SEQ ID NO:60. WSHPQFEK (SEQ ID NO: 60)

[0092] Those skilled in the art will understand that the polypeptide of the present invention can contain two or more tags. Thus, the recombinant polypeptide of the present invention can have one, two, three, four, five, six or more tags, preferably two tags, more preferably one tag. In a preferred embodiment, the recombinant polypeptide of the present invention has a poly(His) tag and a Strep tag II.

[0093] In one embodiment, a polypeptide of the present invention is a fusion protein comprising a region comprising the CCP6 domain of the C4BPα chain, a region comprising the oligomerization domain, and one or more tag peptides. These regions can be arranged in any order relative to each other. Thus, in one embodiment, a polypeptide of the present invention is a fusion protein comprising, from amino to carboxy terminus, (a) at least one tag peptide, (b) a region comprising the CCP6 domain of the C4BPα chain, and (c) a region comprising the oligomerization domain. In another embodiment, a polypeptide of the present invention is a fusion protein comprising, from amino to carboxy terminus, (a) a region comprising the CCP6 domain of the C4BPα chain, (b) a region comprising the oligomerization domain, and (c) at least one tag peptide. In another embodiment, a polypeptide of the present invention is a fusion protein comprising, from amino to carboxy terminus, (a) a region comprising the CCP6 domain of the C4BPα chain, (b) at least one tag peptide, and (c) a region comprising the oligomerization domain. In another embodiment, a polypeptide of the present invention is a fusion protein comprising, from amino to carboxy terminus, (a) at least one tag peptide, (b) a region comprising an oligomerization domain, and (c) a region comprising the CCP6 domain of the C4BP α chain. In another embodiment, a polypeptide of the present invention is a fusion protein comprising, from amino to carboxy terminus, (a) a region comprising an oligomerization domain, (b) a region comprising the CCP6 domain of the C4BP α chain, and (c) at least one tag peptide. In another embodiment, a polypeptide of the present invention is a fusion protein comprising, from amino to carboxy terminus, (a) a region comprising an oligomerization domain, (b) at least one tag peptide, and (c) a region comprising the CCP6 domain of the C4BP α chain. Those skilled in the art will understand that reference to a "tag peptide" as used herein refers both to a tag comprising a cleavable linker or to a tag directly fused to the recombinant polypeptide.Those skilled in the art will understand that the phrase "at least one tag peptide" means that the recombinant polypeptide may have one, two, three, four, five, six, or more tag peptides, preferably two tag peptides, and more preferably one tag peptide. Those skilled in the art will understand that all of these embodiments may include an additional methionine at the N-terminus of the polypeptide. All of these embodiments are equally applicable when the polypeptide of the invention comprises at least one tag peptide, a region consisting of the CCP6 domain of the C4BP α chain, and a region consisting of the oligomerization domain. All of these embodiments are equally applicable when the polypeptide of the invention is formed by a functionally equivalent variant of the CCP6 domain of the C4BP α chain and / or a functionally equivalent variant of the oligomerization domain.

[0094] In one embodiment, a polypeptide of the present invention is a fusion protein comprising a region comprising the CCP6 domain of the C4BP α chain, a region comprising the oligomerization domain, at least one tag peptide, and a signal peptide. These regions can be arranged in any order relative to each other. In one embodiment, a polypeptide of the present invention is a fusion protein comprising, from amino to carboxy terminus, (a) a signal peptide, (b) at least one tag peptide, (c) a region comprising the CCP6 domain of the C4BP α chain, and (d) a region comprising the oligomerization domain. In one embodiment, a polypeptide of the present invention is a fusion protein comprising, from amino to carboxy terminus, (a) a signal peptide, (b) at least one tag peptide, (c) a region comprising the oligomerization domain, and (d) a region comprising the CCP6 domain of the C4BP α chain. In one embodiment, a polypeptide of the present invention is a fusion protein comprising, from amino to carboxy terminus, (a) a signal peptide, (b) a region comprising the oligomerization domain, (c) at least one tag peptide, and (d) a region comprising the CCP6 domain of the C4BP α chain. In one embodiment, a polypeptide of the present invention is a fusion protein comprising, from amino to carboxy terminus, (a) a signal peptide, (b) a region comprising the CCP6 domain of the C4BP α chain, (c) at least one tag peptide, and (d) a region comprising the oligomerization domain. In one embodiment, a polypeptide of the present invention is a fusion protein comprising, from amino to carboxy terminus, (a) a signal peptide, (b) a region comprising the CCP6 domain of the C4BP α chain, (c) a region comprising the oligomerization domain, and (d) at least one tag peptide. In one embodiment, a polypeptide of the present invention is a fusion protein comprising, from amino to carboxy terminus, (a) a signal peptide, (b) a region comprising the oligomerization domain, (c) a region comprising the CCP6 domain of the C4BP α chain, and (d) at least one tag peptide.Those skilled in the art will understand that the reference to a "tag peptide" as used herein refers to both tag peptides with cleavable linkers or directly fused to a recombinant polypeptide. Those skilled in the art will understand that the phrase "at least one tag peptide" means that the tag peptide of a recombinant polypeptide can be one, two, three, four, five, six, or more tag peptides, preferably two tag peptides, and more preferably one tag peptide. All of these embodiments are equally applicable when the polypeptide of the invention comprises a signal peptide, at least one tag peptide, a region consisting of the CCP6 domain of the C4BP α chain, and a region consisting of the oligomerization domain. All of these embodiments are equally applicable when the polypeptide of the invention is formed by a functionally equivalent variant of the CCP6 domain of the C4BP α chain and / or a functionally equivalent variant of the oligomerization domain.

[0095] In a preferred embodiment, the recombinant polypeptide of the present invention comprises SEQ ID NO: 6 or a functionally equivalent variant thereof, preferably SEQ ID NO: 6. In a preferred embodiment, the recombinant polypeptide of the present invention consists of SEQ ID NO: 6 or a functionally equivalent variant thereof; more preferably consists of SEQ ID NO: 6. HHHHHHLCCPEPKLNNGEITQHRKSRPANHCVYFYGDEISFSCHETSRFSAICQGDGTWSPRTPSCGDETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKEL (SEQ ID NO: 6)

[0096] In another embodiment, a recombinant polypeptide of the present invention consists of the sequence of SEQ ID NO: 6 and an additional methionine at the N-terminus of the polypeptide.

[0097] In another embodiment, the polypeptide of the invention comprises SEQ ID NO: 7 or a functionally equivalent variant thereof, preferably SEQ ID NO: 7. In a preferred embodiment, the polypeptide of the invention consists of SEQ ID NO: 7 or a functionally equivalent variant thereof; more preferably consists of SEQ ID NO: 7. MHPPKTPSGALHRKRKMAAWPFSRLWKVSDPILFQMTLIAALLPAVLGHHHHHHLCCPEPKLNNGEITQHRKSRPANHCVYFYGDEISFSCHETSRFSAICQGDGTWSPRTPSCGDETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKEL (SEQ ID NO: 7)

[0098] In another embodiment, the recombinant polypeptide of the present invention does not contain a tag peptide; preferably does not contain a tag peptide and a signal peptide.

[0099] In a preferred embodiment, a polypeptide of the present invention comprises or consists, preferably consists of, from amino to carboxy terminus: (a) a signal peptide, (b) at least one tag peptide, (c) a cleavable linker, (d) the CCP6 domain of the C4BP α chain, and (e) an oligomerization domain. In a preferred embodiment, a polypeptide of the present invention comprises or consists, preferably consists of, from amino to carboxy terminus: (a) a signal peptide, (b) one tag peptide, (c) a cleavable linker, (d) the CCP6 domain of the C4BP α chain, and (e) an oligomerization domain. In a preferred embodiment, a polypeptide of the present invention comprises or consists, preferably consists of, from amino to carboxy terminus: (a) a signal peptide of the CB4P α chain, (b) a poly(His) tag, (c) a TEV-cleavable linker, (d) the CCP6 domain of the C4BP α chain, and (e) an oligomerization domain. In a preferred embodiment, the polypeptide of the invention comprises or consists, preferably consists of SEQ ID NO:56. MHPPKTPSGALHRKRKMAAWPFSRLWKVSDPILFQMTLIAALLPAVLGHHHHHHENLYFQGLCCPEPKLNNGEITQHRKSRPANHCVYFYGDEISFSCHETSRFSAICQGDGTWSPRTPSCGDETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKEL (SEQ ID NO: 56)

[0100] In another preferred embodiment, a recombinant polypeptide of the present invention comprises or consists, preferably consists of, from amino to carboxy terminus: (a) at least one tag peptide, (b) a cleavable linker, (c) the CCP6 domain of the C4BP α chain, and (d) an oligomerization domain. In another preferred embodiment, a recombinant polypeptide of the present invention comprises or consists, preferably consists of, from amino to carboxy terminus: (a) one tag peptide, (b) a cleavable linker, (c) the CCP6 domain of the C4BP α chain, and (d) an oligomerization domain. In a preferred embodiment, a recombinant polypeptide of the present invention comprises or consists, preferably consists of, from amino to carboxy terminus: (a) a poly(His) tag, (b) a TEV-cleavable linker, (c) the CCP6 domain of the C4BP α chain, and (d) an oligomerization domain of the C4BP α chain. Those skilled in the art will understand that all of these embodiments can be applied to recombinant polypeptides having an N-terminal methionine. In a preferred embodiment, the polypeptide of the invention comprises or consists, preferably consists of SEQ ID NO:57. HHHHHHENLYFQGLCCPEPKLNNGEITQHRKSRPANHCVYFYGDEISFSCHETSRFSAICQGDGTWSPRTPSCGDETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKEL (SEQ ID NO: 57)

[0101] In another embodiment, a recombinant polypeptide of the present invention consists of the sequence of SEQ ID NO: 57 and an additional methionine at the N-terminus of the polypeptide.

[0102] In another preferred embodiment, a recombinant polypeptide of the invention comprises or consists, preferably consists of, from amino to carboxy terminus: (a) a glycine residue, (b) the CCP6 domain of the C4BP α chain, and (c) an oligomerization domain, preferably the oligomerization domain of the C4BP α chain. In a preferred embodiment, a polypeptide of the invention comprises or consists, preferably consists of SEQ ID NO:58. GLCCPEPKLNNGEITQHRKSRPANHCVYFYGDEISFSCHETSRFSAICQGDGTWSPRTPSCGDETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKEL (SEQ ID NO: 58)

[0103] In a preferred embodiment, the recombinant polypeptide of the present invention comprises or consists, preferably consists of, from amino to carboxy terminus: (a) a methionine or signal peptide, (b) two tag peptides, (c) a cleavable linker, (d) the CCP6 domain of the C4BP α chain, and (e) the oligomerization domain. In a more preferred embodiment, the recombinant polypeptide of the present invention comprises or consists, preferably consists of, from amino to carboxy terminus: (a) a methionine or signal peptide, (b) a His6-tag, (c) a Strep-tag II, (d) an enterokinase-cleavable linker, (e) the CCP6 domain of the human C4BP α chain, and (f) the oligomerization domain of the human C4BP α chain. In a preferred embodiment, a recombinant polypeptide of the present invention comprises or consists, preferably consists of, from amino to carboxy terminus: (a) methionine, (b) a His6-tag, (c) a Strep-tag II of SEQ ID NO: 60, (d) an enterokinase-cleavable linker of SEQ ID NO: 59, (e) the CCP6 domain of the human C4BP α chain, and (f) the oligomerization domain of the human C4BP α chain. In another preferred embodiment, a recombinant polypeptide of the present invention comprises or consists, preferably consists of, from amino to carboxy terminus: (a) a signal peptide, (b) a His6-tag, (c) a Strep-tag II of SEQ ID NO: 60, (d) an enterokinase-cleavable linker of SEQ ID NO: 59, (e) the CCP6 domain of the human C4BP α chain, and (f) the oligomerization domain of the human C4BP α chain. In a preferred embodiment, a recombinant polypeptide of the present invention comprises SEQ ID NO: 61. In a more preferred embodiment, a recombinant polypeptide of the present invention consists of SEQ ID NO: 61. MHHHHHHWSHPQFEKDDDDKLCCPEPKLNNGEITQHRKSRPANHCVYFYGDEISFSCHETSRFSAICQGDGTWSPRTPSCGDETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKEL (SEQ ID NO: 61)

[0104] Preferably, the recombinant polypeptide of the present invention has a size of 75 to 1,000 amino acids, more preferably 100 to 800 amino acids, more preferably 100 to 600 amino acids, more preferably 110 to 550 amino acids, even more preferably 115 to 550 amino acids, and even more preferably 119 to 550 amino acids. In one embodiment, the recombinant polypeptide of the present invention preferably has at least 90 amino acids, at least 100 amino acids, at least 110 amino acids, at least 115 amino acids, at least 119 amino acids, at least 120 amino acids, at least 130 amino acids, or at least 150 amino acids. In a preferred embodiment, the recombinant polypeptide of the present invention has a size of 115 to 650 amino acids, preferably 119 to 550 amino acids.

[0105] In a preferred embodiment, when an insertion is made, the size of the polypeptide preferably does not exceed 20 amino acids, preferably does not exceed 15 amino acids, and more preferably does not exceed 10 amino acids relative to the length of the wild-type sequence of the C4BP α-chain. Thus, for example, when modified by an insertion, a protein comprising the CCP6 domain of the human C4BP α-chain desirably is 617 amino acids or less.

[0106] Those skilled in the art will understand that different domains of the recombinant polypeptide of the present invention can be linked by a spacer. As disclosed herein, a spacer is an insert that connects or links peptides of appropriate length and properties. Generally, the spacer functions as a hinge region between the domains, allowing them to move independently of each other while maintaining their three-dimensional shape. In this sense, a preferred spacer may be a hinge region characterized by structural ductility or flexibility that allows this movement. The length of the spacer can vary. Typically, the number of amino acids in the spacer is 100 or less amino acids, preferably 50 or less amino acids, more preferably 40 or less amino acids, even more preferably 30 or less amino acids, and even more preferably 20 or less amino acids.

[0107] Alternatively, a suitable spacer may be based on the 10 amino acid residue sequence of the upper hinge region of mouse IgG3, which is used in the production of coiled-coil dimerized antibodies and may be useful as a spacer peptide in the present invention (Pack P. and Pluckthum, A., 1992, Biochemistry 31:1579-1584). It may also be the corresponding sequence of the upper hinge region of human IgG3 or other human Ig subclasses (IgG1, IgG2, IgG4, IgM, and IgA). Human Ig sequences are not predicted to be immunogenic in humans. Additional spacers that may be used in the present invention include peptides with the amino acid sequence GAP, AAA.

[0108] In certain embodiments, the spacer is a peptide with structural flexibility (i.e., a flexible connecting peptide or "flexible linker"). Generally, such linkers are several amino acids long, such as 1 to 20 amino acids in length, e.g., 2 to 10 amino acids in length. These amino acids are selected from the group consisting of glycine, serine, alanine, and threonine. In another particular embodiment, the flexible linker is a peptide comprising repeats of amino acid residues, particularly Gly and Ser, or any other suitable repeat of amino acid residues. Virtually any flexible linker can be used as a spacer according to the present invention. One such linker is (Gly m -Ser) n is a linker, and m and n are each independently 1 to 4. These are used in the art to attach protein domains to each other. Thus, a first component can be linked to a second component by such a linker.

[0109] In a preferred embodiment, the CCP6 domain and the oligomerization domain of the C4BP α chain are directly linked. In another embodiment, they are linked via a flexible linker. In a more preferred embodiment, the flexible linker is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 residues.

[0110] It will be appreciated that it may be necessary to incorporate a sequence of amino acids suitable for protein expression, including at least an N-terminal methionine. The N-terminal sequence may include a cleavage site for chemical or enzymatic removal of all or part of the sequence.

[0111] Recombinant polypeptides of the present invention can be modified to contain any of a variety of known chemical groups or molecules, including, but not limited to, glycosylation, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment to polyethylene glycol (e.g., PEGylation), covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, formation of a GPI anchor, hydroxylation, iodination, methylation, esterification, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, ubiquitination, modification with fatty acids, transfer-RNA-mediated addition of amino acids to proteins such as arginylation, and the like. Also included are peptides containing analogs of amino acids (including unnatural amino acids) and peptides with substituted bonds.

[0112] The recombinant polypeptides of the present invention can be obtained by recombinant DNA techniques known to those skilled in the art. Briefly, a recombinant nucleic acid encoding the CCP6 domain and oligomerization domain of the C4BP α chain is expressed in eukaryotic or prokaryotic cells and the product is recovered.

[0113] It will be appreciated that expression of a polypeptide is often accompanied by post-translational modifications, such as cleavage of a signal sequence and / or cleavage of a tag peptide.

[0114] Those skilled in the art will understand that the recombinant polypeptide of the present invention as a whole must substantially preserve the ability of the original CCP6 domain of the C4BP α chain to induce the tolerogenic phenotype and the ability of the original oligomerization domain to form oligomers, specifically the ability of the CCP6 domain of the human C4BP α chain to induce the tolerogenic phenotype and the ability of the oligomerization domain of the C4BP α chain to form oligomers, more specifically the ability to induce the tolerogenic phenotype and the ability of the recombinant polypeptide of SEQ ID NO: 6 to form oligomers.

[0115] All embodiments referring to the CCP6 domain or the oligomerization domain of the C4BP α-chain apply equally to functionally equivalent variants of the CCP6 domain and to functionally equivalent variants of the oligomerization domain of the C4BP α-chain.

[0116] Homo-oligomers of the present invention The present inventors have found that the recombinant polypeptides of the present invention oligomerize to form homo-oligomers of seven identical monomers, i.e., seven recombinant polypeptides of the present invention. As shown in gene expression experiments (Figures 13, 14, 16, 17, and 18) and the conversion into regulatory or tolerogenic macrophages (Figure 15), the homo-oligomers of the present invention can regulate the maturation of mononuclear phagocytes, mainly dendritic cells (DCs) and macrophages, with high specificity and superior efficiency compared to the physiological isoform of C4BP (C4BP(β)). The present inventors have found that the homo-oligomers of the present invention are potentially very attractive as pharmaceutical active principles.

[0117] Thus, in one embodiment, the present invention relates to a homo-oligomer of at least six recombinant polypeptides comprising the CCP6 domain of the C4BP alpha chain or a functionally equivalent variant thereof and an oligomerization domain, wherein the polypeptides do not comprise the CCP1, CCP2, CCP3, CCP4, CCP5, CCP7 and CCP8 domains of the C4BP alpha chain.

[0118] As used herein, the term "homo-oligomer" refers to an oligomer formed by identical monomers. As used herein, an oligomer is a macromolecular complex formed by the non-covalent bonding of proteins or polypeptides consisting of several repeating units. Each repeating unit is a monomer. In particular, the homo-oligomers of the present invention are hexamers or heptamers, each consisting of six or seven monomers. Each monomer of the homo-oligomer of the present invention is a recombinant polypeptide of the present invention.

[0119] Thus, homo-oligomers useful in the present invention include any homo-oligomers resulting from the association of multiple recombinant polypeptides of the present invention and lacking β-strands. For example, a homo-oligomer of the present invention can contain at least six, at least seven, or at least eight monomers, i.e., recombinant polypeptides of the present invention. In one embodiment, a homo-oligomer is formed by seven recombinant polypeptides of the present invention.

[0120] In one embodiment, a homo-oligomer of the invention comprises at least six recombinant polypeptides of the invention; particularly six, seven or eight polypeptides of the invention; particularly six or seven polypeptides of the invention, more particularly seven polypeptides of the invention.

[0121] In a preferred embodiment, the homo-oligomer of the present invention consists of 6, 7 or 8 polypeptides of the present invention. In a more preferred embodiment, the homo-oligomer of the present invention consists of 6 or 7 polypeptides of the present invention.

[0122] In one embodiment, the homo-oligomer of the invention consists of six monomer chains, ie, six recombinant polypeptides of the invention.

[0123] In a preferred embodiment, the homo-oligomer of the invention consists of seven monomer chains, ie, seven polypeptides of the invention.

[0124] In one embodiment, the homo-oligomer consists of seven recombinant polypeptides of the invention, each comprising, from amino to carboxy terminus, the CCP6 domain of the C4BP α chain or a functionally equivalent variant thereof and the oligomerization domain. In a preferred embodiment, the homo-oligomer consists of seven recombinant polypeptides of the invention, each comprising, from amino to carboxy terminus, the CCP6 domain of the C4BP α chain and the oligomerization domain of the C4BP α chain. In a more preferred embodiment, the homo-oligomer consists of seven recombinant polypeptides of the invention, each comprising, from amino to carboxy terminus, the CCP6 domain of the human C4BP α chain and the oligomerization domain of the human C4BP α chain. In this last embodiment, the CCP6 domain and the oligomerization domain are directly linked.

[0125] In a preferred embodiment, the polypeptide forming the homo-oligomer is a polypeptide consisting of SEQ ID NO: 6 or a functionally equivalent variant thereof, specifically a polypeptide consisting of SEQ ID NO: 6.

[0126] All terms and embodiments previously described for the recombinant polypeptides of the invention are equally applicable to the homo-oligomers of the invention.

[0127] To obtain a homo-oligomer, for example, a recombinant sequence encoding the CCP6 domain of the C4BP α chain and an oligomerization domain in which the 3' end of the DNA sequence encoding the CCP6 domain is ligated to the 5' end of a DNA sequence encoding the oligomerization domain is prepared. Upon expression in a suitable host, this hybrid DNA sequence produces the recombinant polypeptides of the invention assembled into oligomers, specifically homo-oligomers, more specifically heptamers formed by seven identical recombinant polypeptides.

[0128] In a preferred embodiment, the recombinant polypeptides forming the homo-oligomers of the present invention do not contain regions of proteins other than C4BP, e.g., the polypeptides of the present invention should not be fusion proteins containing regions forming part of proteins other than C4BP.

[0129] Those skilled in the art will understand that the homo-oligomers of the present invention are typically formed by the mature form of the recombinant polypeptide of the present invention, i.e., the signal peptide of the recombinant polypeptide of the present invention is typically cleaved before secretion. Thus, in a preferred embodiment, the homo-oligomers of the present invention are formed by the recombinant polypeptide without the signal polypeptide.

[0130] Alternatively, the homo-oligomers of the present invention can be formed by recombinant polypeptides that maintain the tag peptide. This is the case for small tags such as His(6), FLAG, Strep II, and CBP, which do not usually need to be removed. Thus, in one embodiment, the homo-oligomers of the present invention are formed by recombinant polypeptides that contain at least one tag peptide.

[0131] However, in some cases, it may be preferable to remove the tag peptide. Preferably, the tag peptide is cleaved in the mature form of the recombinant polypeptide that forms the homo-oligomer. Thus, in another embodiment, the homo-oligomer of the present invention is formed by a recombinant polypeptide that does not contain a tag peptide. For example, if a poly(His) tag and the CCP6 domain of the C4BP α chain are fused via a linker cleavable by TEV protease and the sequence recognized by the protease is ENLYFQ / G (SEQ ID NO: 59), the mature form of the recombinant polypeptide of the present invention that forms the homo-oligomer of the present invention may have a glycine residue at its N-terminus, since TEV protease cleaves between the Q and G residues. This glycine residue does not affect the activity of the recombinant polypeptide of the present invention. Thus, the recombinant polypeptide of the present invention that forms the homo-oligomer of the present invention may have an additional amino acid (e.g., an additional methionine) at its N-terminus that does not affect its activity or that results from cleavage of the cleavable linker added to enable its expression in bacteria.

[0132] Thus, in one embodiment, a homo-oligomeric recombinant polypeptide of the invention has an additional amino acid at its N-terminus.

[0133] In one embodiment, the homo-oligomeric recombinant polypeptide of the present invention does not include a signal peptide or a tag peptide.

[0134] The homo-oligomers of the invention spontaneously assemble in the recombinant expression system used. Methods for assessing whether a complex is a homo-oligomer of the invention have been previously disclosed in the context of mutants of the oligomerization domain.

[0135] Polynucleotides, Vectors and Host Cells of the Invention The present invention also provides polynucleotides encoding the polypeptides of the present invention. Accordingly, in another aspect, the present invention relates to a polynucleotide encoding a recombinant polypeptide comprising the CCP6 domain of the C4BP α chain or a functionally equivalent variant thereof and the oligomerization domain, said polypeptide not comprising the CCP1, CCP2, CCP3, CCP4, CCP5, CCP7 and CCP8 domains of the C4BP α chain.

[0136] The terms "polynucleotide," "nucleic acid," and "nucleic acid molecule" are used interchangeably and refer to a polymeric form of nucleotides of any length. Polynucleotides can contain deoxyribonucleotides, ribonucleotides, and / or their analogs. Nucleotides can have any three-dimensional structure and can perform any function, known or unknown. The term "polynucleotide" includes, for example, single-stranded, double-stranded, and triple-helical molecules, genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. In addition to naturally occurring nucleic acid molecules, the nucleic acid molecules of the present invention can also include modified nucleic acid molecules. As used herein, mRNA refers to RNA that can be translated within a cell.

[0137] A polynucleotide of the present invention may further comprise a single promoter region that controls transcription of the region encoding the polypeptide of the present invention, provided that the promoter is compatible with the cell in which the polypeptide is to be expressed. A polynucleotide encoding a polypeptide of the present invention may be isolated by itself or may be found to form part of a vector, allowing propagation of the polynucleotide in a suitable host cell.

[0138] Therefore, in another aspect, the present invention relates to a vector comprising a polynucleotide of the present invention.

[0139] The choice of expression vector depends on the choice of host. Suitable vectors for inserting polynucleotides are pUC18, pUC19, Bluescript and its derivatives, pET and its derivatives, mp18, mp19, pBR322, pMB9, Co1E1, pCR1, RP4, phage and "shuttle" vectors such as pSA3 and pAT28, yeast expression vectors such as 2-micron plasmid type vectors, insect cell expression vectors such as integrative plasmids, YEP vectors, pBGZα vectors, centromeric plasmids, pAC series vectors and pVL vectors, plant expression vectors such as pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series, and eukaryotic expression vectors including baculoviruses suitable for transfection of insect cells using commercially available baculovirus systems. Vectors for eukaryotic cells preferably include viral vectors (adenovirus, adenovirus-associated virus (AAV), retrovirus, especially lentivirus), and non-viral vectors such as pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1 / hyg, pHMCV / Zeo, pCR3.1, pEFI / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAX1, pZeoSV2, pCI, pSVL, and PKSV-10, pBPV-1, pML2d, and pTDT1. In a preferred embodiment, the vector is pcDNA3.1.

[0140] The vector may also contain a reporter or marker gene that allows identification of cells that have incorporated the vector after contact with the vector. Reporter genes useful in the present invention include lacZ, luciferase, thymidine kinase, GFP, and the like. Marker genes useful in the present invention include, for example, the neomycin resistance gene that confers resistance to the aminoglycoside G418; the hygromycin phosphotransferase gene that confers resistance to hygromycin; the ODC gene that confers resistance to inhibitors of ornithine decarboxylase (2-(difluoromethyl)-DL-ornithine (DFMO)); the dihydrofolate reductase gene that confers resistance to methotrexate; the puromycin-N-acetyltransferase gene that confers resistance to puromycin; the ble gene that confers resistance to zeocin; and the 9-β These include the adenosine deaminase gene, which confers resistance to -D-xylofuranose adenine; the cytosine deaminase gene, which allows cells to grow in the presence of N-(phosphonacetyl)-L-aspartate; the thymidine kinase gene, which allows cells to grow in the presence of aminopterin; the xanthine-guanine phosphoribosyltransferase gene, which allows cells to grow in the presence of xanthine but not guanine; the E. coli trpB gene, which allows cells to grow in the presence of indole but not tryptophan; and the E. coli hisD gene, which allows cells to utilize histidinol but not histidine. The selection gene is incorporated into a plasmid that may further contain a promoter suitable for gene expression in eukaryotic cells (e.g., the CMV or SV40 promoter), an optimized translation initiation site (e.g., a site following the so-called Kozak rule or an IRES), a polyadenylation site such as the SV40 polyadenylation or phosphoglycerate kinase site, and an intron such as the β-globin gene intron. Alternatively, it is possible to use both a reporter gene and a marker gene simultaneously in the same vector.

[0141] The present invention also provides vectors containing both a promoter and a cloning site to which a polynucleotide of the present invention can be operably linked. Such vectors are capable of transcribing RNA in vitro or in vivo. To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' and / or 3' untranslated portions of the clone to remove redundant and potentially inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression at the transcriptional or translational level. Alternatively, a consensus ribosome binding site may be inserted immediately 5' of the initiation codon to enhance expression. Gene delivery vehicles also include some non-viral vectors, including DNA / liposome complexes and targeted viral protein-DNA complexes. Liposomes that also contain targeting antibodies or fragments thereof can be used in the methods of the present invention. To enhance delivery to cells, the nucleic acids or proteins of the present invention can be conjugated to antibodies or binding fragments thereof that bind to cell surface antigens.

[0142] As is known in the art, for expression of a DNA sequence of the present invention, the DNA sequence must be operably linked to an expression control sequence in an appropriate expression vector and used to transform a suitable unicellular host. Of course, such operably linking a DNA sequence of the present invention to an expression control sequence includes providing a translation initiation signal in the correct reading frame upstream of the DNA sequence. If the particular DNA sequence to be expressed does not begin with methionine, the initiation signal will place an additional amino acid (methionine) at the N-terminus of the product. While such methionyl-containing products can be used directly in the methods of the present invention, it is usually more desirable to remove the methionine before use. Methods for removing such N-terminal methionine from polypeptides expressed therewith are known to those skilled in the art. For example, certain hosts and fermentation conditions allow for the removal of substantially all N-terminal methionine in vivo. Other hosts require in vitro removal of N-terminal methionine. However, such in vitro and in vivo methods are known in the art.

[0143] In another aspect, the present invention relates to a host cell comprising the vector as described above. The term "host cell" is used to refer not only to the specific subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term as used herein.

[0144] The recombinant polypeptides of the present invention can be expressed in any of the known recombinant polypeptide production systems.

[0145] A wide variety of unicellular host cells are also useful for expressing the DNA sequences of the present invention. In preferred embodiments, the host cell is selected from bacteria, yeast, and mammalian eukaryotic cells. These hosts include known eukaryotic and prokaryotic hosts such as E. coli, strains of Pseudomonas, Bacillus, Lactobacillus, Thermophilus, Salmonella, Enterobacteriaceae, or Streptomyces, yeast, and animal cells such as CHO and mouse cells, African green monkey cells such as cos-1, COS-7, BSC1, BSC40, and BMT10, insect cells, and human and plant cells in tissue culture. For expression in animal cells, CHO, COS-7 cells, and HEK293 cells are preferred, with HEK293 cells being more preferred.

[0146] For example, when E. coli of the genus Escherichia is used in the methods of the present invention, preferred strains of this bacterium to be used include BL21(DE3) and its derivatives, including C41(DE3), C43(DE3) or C0214(DE3), C3030H, or other strains that are resistant to the toxicity of recombinant protein expression. Even more preferably, when the promoter is not a T7 promoter, derivatives of these strains lacking prophage DE3 can be used.

[0147] In a preferred embodiment, the expression cassette contains, from 5' to 3' of the same open reading frame, a nucleotide sequence encoding the CCP6 domain and a nucleotide sequence encoding the oligomerization domain of the C4BP α chain. As used herein, the term "open reading frame" or "ORF" refers to a length of nucleic acid, either DNA, cDNA, or RNA, that includes a translation initiation signal or start codon, such as ATG or AUG, and a stop codon, and that can be translated into a polypeptide sequence. The above DNA sequences do not contain internal stop codons and are generally translatable into peptides.

[0148] In a preferred embodiment, the expression cassette comprises, from 5' to 3', a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding a first tag, a nucleotide sequence encoding a selectable marker, a nucleotide sequence encoding a second tag, a nucleotide sequence encoding the CCP6 domain of the C4BP α chain, and a nucleotide sequence encoding the oligomerization domain. In another embodiment, the expression cassette comprises, from 5' to 3', a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding a first tag, a nucleotide sequence encoding a second tag, a nucleotide sequence encoding a cleavable linker, a nucleotide sequence encoding the CCP6 domain of the C4BP α chain, and a nucleotide sequence encoding the oligomerization domain. In another embodiment, the expression cassette comprises, from 5' to 3', a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding a tag, a nucleotide sequence encoding a cleavable linker, a nucleotide sequence encoding the CCP6 domain of the C4BP α chain, and a nucleotide sequence encoding the oligomerization domain. In another embodiment, the expression cassette comprises, from 5' to 3', a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding a tag, a nucleotide sequence encoding the CCP6 domain of the C4BP α chain, and a nucleotide sequence encoding the oligomerization domain. In a more preferred embodiment, all elements are in the same open reading frame. In an even more preferred embodiment, all elements are under the operational control of a regulatory nucleotide sequence. In a preferred embodiment, the first tag is different from the second tag.

[0149] In a preferred embodiment, the polynucleotide encoding the polypeptide of SEQ ID NO:7 is the polynucleotide of SEQ ID NO:9.

[0150] Methods for cloning nucleotide sequences into vectors and for expressing and purifying recombinant polypeptides of the invention are known in the art. Exemplary conditions for expression and purification are provided in the Materials and Methods section of the Examples of the present invention.

[0151] All terms and embodiments previously described for the recombinant polypeptides of the invention and the homo-oligomers of the invention are equally applicable to the nucleotides, vectors and host cells of the invention.

[0152] Pharmaceutical compositions of the present invention A further aspect of the present invention is a pharmaceutical composition comprising a recombinant polypeptide of the invention, a homo-oligomer of the invention, a polynucleotide of the invention, a vector of the invention, a host cell of the invention, a tolerogenic dendritic cell of the invention, a tolerogenic macrophage of the invention or a cell population of the invention and a pharmaceutically acceptable carrier.

[0153] The therapeutic methods of the present invention involve treating a patient with these compositions in a pharmaceutically acceptable manner. These compositions can be used to treat any mammal, including humans.

[0154] The pharmaceutical compositions of the present invention may take a variety of forms. These forms include solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, liposomes, suppositories, injectable and infusible solutions, and sustained-release forms. In general, the pharmaceutical compositions of the present invention can be formulated and administered using methods and compositions similar to those used for pharmaceutically important polypeptides, such as alpha interferon. Thus, the recombinant proteins of the present invention can be stored in lyophilized form, reconstituted with sterile water immediately before administration, and administered by conventional routes of administration, such as parenteral, subcutaneous, intravenous, intramuscular, or intralesional routes. In a preferred embodiment, the compounds of the present invention are administered subcutaneously.

[0155] The composition may be a pharmaceutical composition that is a sterile aqueous or non-aqueous solution, suspension, or emulsion, and further comprises a physiologically acceptable or suitable carrier. Pharmaceutically acceptable or suitable carriers may include (or refer to) excipients (i.e., non-toxic substances that do not interfere with the activity of the active ingredient) and / or diluents. Such compositions may be in solid, liquid, or gaseous (aerosol) form. Alternatively, the compositions described herein may be formulated as lyophilizates, or the compounds may be encapsulated in liposomes using techniques known in the art. Pharmaceutical compositions may also contain other biologically active or inactive components. Such components include, but are not limited to, buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose, or dextrans), mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, stabilizers, dyes, flavoring agents, and suspending and / or preservatives.

[0156] Any suitable excipient or carrier known to those skilled in the art for use in pharmaceutical compositions can be used in the compositions described herein. Excipients for therapeutic use are known and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro ed. 1985). Generally, the type of excipient is selected based on the method of administration. Pharmaceutical compositions can be formulated for any suitable method of administration, including, for example, topical, oral, intranasal, intrathecal, rectal, vaginal, intraocular, subconjunctival, sublingual, or parenteral administration, subcutaneous, intravenous, intramuscular, intrasternal, intracavernous, intraductal, or intraurethral injection or infusion. For parenteral administration, the carrier preferably comprises water, saline, alcohol, fat, wax, or buffer. For oral administration, any of the above excipients or solid excipients or carriers may be used, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, kaolin, glycerin, starch dextrin, sodium alginate, carboxymethylcellulose, ethylcellulose, glucose, sucrose, and / or magnesium carbonate.

[0157] Pharmaceutical compositions (e.g., for oral administration or delivery by injection) may be in liquid form. Liquid pharmaceutical compositions may contain, for example, one or more of the following: water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, sterile diluents such as fixed oils, polyethylene glycol, glycerin, propylene glycol, or other solvents that can function as solvents or suspending agents; antibacterial agents; antioxidants; chelating agents; buffers and reagents for adjusting tonicity, such as sodium chloride and dextrose. Parenteral preparations can be packaged in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic. Physiological saline is preferably used, and injectable pharmaceutical compositions are preferably sterile.

[0158] The agents described herein, including polypeptides, homo-oligomers, polynucleotides, vectors, host cells, tolerogenic dendritic cells, or tolerogenic macrophages, may be formulated for sustained or sustained release. Such compositions are generally prepared using known techniques and can be administered, for example, by oral, rectal, or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may contain the agent dispersed in a carrier matrix and / or contained within a reservoir surrounded by a rate-controlling membrane. Excipients for use within such formulations are biocompatible and may also be biodegradable; preferably, the formulation provides a relatively constant level of active ingredient release. The amount of active compound contained in a sustained-release formulation depends on the site of implantation, the rate and expected duration of release, and the nature of the condition being treated or prevented.

[0159] The pharmaceutical composition can be administered in a manner appropriate to the disease to be treated (or prevented), as determined by those skilled in the medical art. The appropriate dose and the appropriate duration and frequency of administration are determined by factors such as the patient's condition, the type and severity of the patient's disease, the specific form of the active ingredient, and the method of administration. In general, an appropriate dose and treatment regimen provides a sufficient amount of the composition to provide therapeutic and / or prophylactic benefits (e.g., more frequent complete or partial remission, or longer disease-free and / or overall survival, or reduced severity of symptoms). For prophylactic use, the dose should be sufficient to prevent, delay the onset of, or reduce the severity of diseases associated with immunological diseases or disorders.

[0160] Optimal dosages can generally be determined using experimental models and / or clinical trials. The optimal dosage will vary depending on the patient's mass, body weight, or blood volume. Generally, the amount of polypeptide present in a given dosage, or produced in situ by DNA present in a given dosage, ranges from about 0.01 to about 1000 μg per kg of host. Use of the minimum dosage sufficient to provide effective treatment is generally preferred. Patients are generally monitored for therapeutic or prophylactic effectiveness using assays appropriate for the condition being treated or prevented, which will be familiar to those skilled in the art. When administered in liquid form, appropriate dose sizes vary depending on the patient's size, but typically range from about 1 to 500 ml (containing about 0.01 to about 1000 μg per kg) for a 10-60 kg subject.

[0161] In a preferred embodiment, the pharmaceutical composition contains 0.45 to 18.90 mg, more preferably 0.45 to 9.45 mg, more preferably 0.56 to 8.51 mg, more preferably 0.75 to 8.13 mg, and even more preferably 0.79 to 8.05 mg of the homo-oligomer of the present invention. In a preferred embodiment, the dose is 0.79 mg. In another preferred embodiment, the dose is 0.8 mg. In another preferred embodiment, the dose is 8 mg. In another preferred embodiment, the dose is 8.05 mg.

[0162] In one embodiment, the pharmaceutical composition contains 7.56 to 18.90 mg, preferably 9.45 to 15.13 mg, preferably 11.35 to 15.13 mg, preferably 13.24 to 15.13 mg of the compound of the present invention.

[0163] In one embodiment, the pharmaceutical composition contains 1.89 to 17.02 mg, preferably 3.78 to 15.13 mg, preferably 5.67 to 13.24 mg, more preferably 7.56 to 11.35 mg of the compound of the present invention.

[0164] In one embodiment, the pharmaceutical composition contains 0.5 to 9 mg, more preferably 0.75 to 8.5 mg, of the compound of the present invention. In another embodiment, the composition contains 7 to 18.90 mg, more preferably 7.5 to 18 mg, more preferably 9 to 15 mg, more preferably 11 to 15 mg, and even more preferably 13 to 15 mg, of the compound of the present invention. In another embodiment, the pharmaceutical composition contains 1 to 17 mg, preferably 3 to 15 mg, preferably 5 to 13 mg, and more preferably 7 to 11 mg of the compound of the present invention.

[0165] In the case of pharmaceutical compositions containing drugs that are nucleic acid molecules, including aptamers, the nucleic acid molecules can be present in a variety of delivery systems known to those skilled in the art, including bacterial, viral, and mammalian expression systems, such as nucleic acids and recombinant expression constructs as provided herein. Techniques for incorporating DNA into such expression systems are known to those skilled in the art. The DNA can also be "naked," as reviewed, for example, by Ulmer et al., Science 259:1745-49, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA can be increased by coating it onto biodegradable beads, which are efficiently transported into cells.

[0166] Nucleic acid molecules can be delivered to cells by any one of several methods described in the art (e.g., Akhtar et al., Trends Cell Bio. 2:139 (1992); Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al., Mol. Membr. Biol. 16:129-40 (1999); Hofland and Huang, Handb. Exp. Pharmacol. 137:165-92 (1999); Lee et al., ACS Symp. Ser. 752:184-92 (2000); U.S. Pat. No. 6,395,713; International Publication No. WO 94 / 02595); Selbo et al., Int. J. Cancer 87:853-59 (2000); Selbo et al., Tumour Biol. 23:103-12 (2002); see U.S. Patent Application Publication Nos. 2001 / 0007666 and 2003 / 077829). Such delivery methods known to those skilled in the art include, but are not limited to, encapsulation in liposomes, iontophoresis, or incorporation into other vehicles such as biodegradable polymers; hydrogels; cyclodextrins (see, e.g., Gonzalez et al., Bioconjug. Chem. 10: 1068-74 (1999); Wang et al., International Application Publication Nos. WO 03 / 47518 and WO 03 / 46185); poly(lactic-co-glycolic) acid (PLGA) and PLCA microspheres (also useful for delivery of peptides and polypeptides and other substances) (see, e.g., U.S. Pat. No. 6,447,796; U.S. Patent Application Publication No. 2002 / 130430); biodegradable nanocapsules; and bioadhesive microspheres, or proteinaceous vectors (International Publication No. WO 00 / 53722).In another embodiment, nucleic acid molecules used to alter (suppress or enhance) immune responses in immune cells and to treat immunological diseases or disorders may also be formulated or complexed with polyethyleneimine and its derivatives, such as polyethyleneimine-polyethylene glycol-N-acetylgalactosamine (PEI-PEG-GAL) derivatives or polyethyleneimine-polyethylene glycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives (see also, e.g., U.S. Patent Application Publication No. 2003 / 0077829).

[0167] The pharmaceutical compositions / medicaments of the present invention may further comprise, for example, active ingredients as described above, such as other immunomodulatory antibodies, e.g., anti-ICOS antibodies, anti-CD154 antibodies, anti-CD134L antibodies, or recombinant proteins, e.g., rCTLA-4 (CD152), rOX40 (CD134), or anti-inflammatory agents, or immunomodulatory compounds, e.g., but not limited to, cyclosporin A, FTY720, RAD, rapamycin, FK506, 15-deoxyspergualin, steroids, etc.

[0168] Furthermore, DNA sequences encoding the recombinant polypeptides of the present invention can be used in somatic cell gene therapy. This involves, for example, inserting the DNA sequence into a retroviral-based vector suitable for infecting human somatic cells (A. Kasid et al. 1990. "Human Gene Transfer: Characterization of human tumor-infiltrating lymphocytes as vehicles for retroviral-mediated gene transfer", Proc. Natl. Acad. Sci., USA, 87:473-477). For example, a patient with an immunoinflammatory disease can be treated as follows: First, a retrovirus featuring a DNA sequence encoding the recombinant polypeptide of the present invention is prepared. Cells are then isolated from the patient and infected in vitro with the retrovirus. These cells are then reintroduced into the patient, whereupon the vector is expressed and the cells secrete the recombinant polypeptide of the present invention, which then joins to form the homo-oligomers of the present invention.

[0169] All terms and embodiments described for the remaining aspects of the invention are equally applicable to the pharmaceutical compositions of the invention.

[0170] Therapeutic Uses of the Polypeptides, Homo-Oligomers, Polynucleotides, Vectors, Host Cells and Pharmaceutical Compositions of the Invention Therefore, in another aspect, the present invention relates to a recombinant polypeptide of the invention, a homo-oligomer of the invention, a polynucleotide of the invention, a vector of the invention, a host cell of the invention or a pharmaceutical composition of the invention for use in medicine.

[0171] In another aspect, the present invention relates to the use of a recombinant polypeptide of the present invention, a homo-oligomer of the present invention, a polynucleotide of the present invention, a vector of the present invention, a host cell of the present invention, or a pharmaceutical composition of the present invention for the manufacture of a medicament.

[0172] In another aspect, the present invention relates to a recombinant polypeptide of the present invention, a homo-oligomer of the present invention, a polynucleotide of the present invention, a vector of the present invention, a host cell of the present invention, or a pharmaceutical composition of the present invention for use in the prevention and / or treatment of an immunological disease caused by undesired activation of the immune system.

[0173] In another aspect, the present invention relates to the use of a recombinant polypeptide of the present invention, a homo-oligomer of the present invention, a polynucleotide of the present invention, a vector of the present invention, a host cell of the present invention, or a pharmaceutical composition of the present invention for the manufacture of a medicament for the prevention and / or treatment of an immunological disease caused by undesired activation of the immune system.

[0174] In another aspect, the present invention relates to a method for preventing and / or treating an immunological disease caused by unwanted activation of the immune system in a subject in need thereof, the method comprising administering to said subject a recombinant polypeptide of the invention, a homo-oligomer of the invention, a polynucleotide of the invention, a vector of the invention, a host cell of the invention or a pharmaceutical composition of the invention.

[0175] In another aspect, the present invention relates to a method for increasing the population of tolerogenic dendritic cells and / or tolerogenic macrophages in a subject in need thereof, the method comprising administering to said subject a polypeptide of the invention, a homo-oligomer of the invention, a polynucleotide of the invention, a vector of the invention, a host cell of the invention or a pharmaceutical composition of the invention.

[0176] The term "prevention" as used herein refers to the administration of a compound of the present invention at an incipient or early stage of a disease or to prevent its onset.

[0177] The term "treatment" is used to refer to the administration of the compounds of the present invention to control the progression of disease before or after the appearance of clinical signs. Controlling the progression of disease is understood to mean beneficial or desirable clinical results, including, but not limited to, alleviating symptoms, shortening the duration of disease, stabilizing the pathological condition (especially avoiding additional disorders), delaying the progression of disease, improving the pathological condition, and remission (both partial and complete). Controlling the progression of disease also involves prolonging survival compared to the expected survival if no treatment is applied.

[0178] As used herein, the expression "immunological disease caused by unwanted activation of the immune system" refers to any disease caused by unwanted activation of the immune system, including the innate or adaptive immune system and the humoral or cellular immune system. Preferably, the immunological disease in the present invention is a disease in which the immune system is activated in response to alloantigens or autoantigens. Therefore, immunological diseases in which the immune system is suppressed are not included in the present invention.

[0179] In a preferred embodiment, the immunological disease is selected from the group consisting of immunoinflammatory diseases, sepsis, autoimmune diseases, transplant rejection, graft-versus-host disease, and hypersensitivity diseases.

[0180] As used herein, the term "immunoinflammatory disease" refers to inflammatory diseases and disorders in which immune cells and / or cytokines are involved in the pathophysiology of the disease or disorder. Examples of immunoinflammatory diseases include conditions such as rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, acute respiratory distress syndrome, and asthma. The term immunoinflammatory disease includes both acute and chronic inflammatory diseases. The term "acute inflammatory disorder" is intended to include disorders and episodes of disorders characterized by the rapid onset and relatively short duration of symptoms associated with an inflammatory response, while "chronic inflammatory disorder" is intended to include disorders characterized by the continued presence of symptoms associated with an inflammatory response and the duration of ongoing symptoms. Immunoinflammatory diseases that can be treated by the methods of the present invention include, but are not limited to, cardiovascular diseases such as infarction or stroke, atherosclerosis, pulmonary fibrosis, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, acute respiratory distress syndrome, asthma, and cancer. Immunoinflammatory disorders that can be treated by the present invention also include disorders that occur during pregnancy, such as preeclampsia and eclampsia. Preeclampsia is a pregnancy-related disorder characterized by high blood pressure, proteinuria, and edema. Preeclampsia is understood and defined herein as being encompassed within and belonging to the spectrum of preeclamptic disorders, which include placental insufficiency, intrauterine growth restriction, early miscarriage, preterm birth, intrauterine death, and eclampsia.

[0181] As used herein, the term "sepsis" refers to a systemic host response to microorganisms in previously sterile tissues characterized by end-organ dysfunction distant from the primary site of infection. To be considered sepsis, infection must be suspected or proven (by culture, staining, or polymerase chain reaction (PCR)) or a clinical syndrome consistent with infectious etiology must be present. Specific evidence of infection includes white blood cells (WBCs) in normally sterile fluids (e.g., urine or cerebrospinal fluid (CSF)), evidence of perforated viscus (free air on abdominal x-ray or CT scan, signs of acute peritonitis), an abnormal chest x-ray consistent with pneumonia (with focal opacities), or petechiae, purpura, or purpura fulminans. More important subsets of sepsis are severe sepsis (sepsis with acute organ failure) and septic shock (sepsis with refractory arterial hypotension). Alternatively, patients may simply be diagnosed with "SIRS" if two or more criteria for systemic inflammatory response syndrome are met without evidence of infection. Patients with SIRS and acute organ failure may be referred to as "severe SIRS." Patients are defined as having "severe sepsis" if they exhibit sepsis plus signs of systemic circulatory failure: end-organ failure or serum lactate greater than 4 mmol / dL. Other signs include petechiae and altered mental status. Patients are defined as having septic shock if sepsis and hypotension (usually greater than 6 liters or 40 ml / kg of crystalloids) occur after aggressive fluid resuscitation. Examples of end-organ failure include acute lung injury or acute respiratory distress syndrome, encephalopathy, or dysfunction affecting the liver (impaired protein synthesis and metabolic function), kidneys (oliguria and anuria, electrolyte abnormalities, volume overload), and heart (systolic and diastolic heart failure).

[0182] Suitable sepsis conditions that can be treated with the compounds of the present invention include, but are not limited to, severe sepsis and septic shock. In one embodiment, the condition associated with sepsis syndrome is selected from the group consisting of organ failure, preferably renal or hepatic failure, multiple organ dysfunction syndrome (MODS), acute respiratory distress syndrome (ARDS), and disseminated intravascular coagulation (DIC).

[0183] Sepsis can be induced by one or more bacteria selected from the group consisting of gram-negative and gram-positive bacteria. Preferably, the gram-negative bacteria are selected from the group consisting of Escherichia coli, Klebsiella species, Serratia species, Enterobacter species, Proteus species, Pseudomonas aeruginosa, Haemophilus influenzae, Neisseria species, and Listeria species. Also, the Gram-positive bacteria are selected from the group consisting of Staphylococcus aureus, Streptococcus pneumoniae, coagulase-negative Staphylococci, Enterococcus species, Streptococcus pyogenes, and Streptococcus viridans. In one embodiment, the sepsis syndrome is induced by LPS. In yet another embodiment, the sepsis is induced by one or more microorganisms selected from the group consisting of anaerobic bacteria, fungi, rickettsia, chlamydia, mycoplasma, spirochetes, and viruses.

[0184] In a preferred embodiment, the immunological disease is an autoimmune disease.

[0185] Throughout this specification, the terms "autoimmune disease," "disease associated with immune dysfunction / dysregulation," or "immunoinflammatory disease" are used to refer to pathological conditions in which a patient's immune system produces disease due to self-antigens (autoimmunity) or foreign antigens (immune dysfunction / dysregulation or immune inflammatory disease). Autoimmunity exists to some degree in all people. It is usually harmless and is likely a universal phenomenon in vertebrate life. However, autoimmunity can be the cause of a wide range of human illnesses known as autoimmune diseases. This concept of autoimmunity as a cause of human disease is relatively new and was not accepted as mainstream medical thinking until the 1950s and 1960s. Thus, autoimmune disease is defined as the progression from benign to pathogenic autoimmunity. This progression is determined by both genetic influences and environmental triggers. The concept of autoimmunity as the actual cause of human disease (rather than a consequence or harmless concomitant) can be used to establish criteria for defining a disease as an autoimmune disease. Autoimmune diseases or diseases characterized by immune dysfunction or dysregulation (immunoinflammatory diseases) that can be treated by the present invention include the diseases systemic lupus erythematosus (SLE), lupus nephritis, central nervous system (CNS) lupus, diabetes mellitus (Type I), asthma, ulcerative colitis, Crohn's disease, Grave's disease, arthritis including rheumatoid arthritis and osteoarthritis, pernicious anemia, inflammatory bowel disease, and multiple sclerosis, among a variety of other diseases.The methods of the present invention can be used to treat, among other diseases, autoimmune blood disorders including pernicious anemia, autoimmune hemolytic anemia, aplastic anemia, idiopathic thrombocytopenic purpura, ankylosing spondylitis; autoimmune diseases of muscle tissue including polymyositis and dermatomyositis; autoimmune diseases of the ear including autoimmune hearing loss and Meniere's syndrome; autoimmune eye diseases including Mooren's disease, Reiter's syndrome, and Vogt-Koyanagi-Harada disease; autoimmune diseases of the kidney including glomerulonephritis, IgA nephropathy, and lupus nephritis; diabetes mellitus (Type 1); autoimmune skin diseases including pemphigus vulgaris, pemphigus foliaceus, pemphigus erythematosus, bullous pemphigoid, vitiligo, epidermolysis bullosa acquisita, psoriasis, and alopecia; cardiovascular autoimmune diseases including autoimmune myocarditis; Vasculitis, including Strauss syndrome, giant cell arteritis, Kawasaki disease, polyarteritis nodosa, Takayasu's arteritis, and Wegener's granulomatosis; endocrine autoimmune diseases, including Addison's disease, autoimmune hypoparathyroidism, autoimmune hypophysitis, autoimmune oophoritis, autoimmune orchitis, Grave's disease, Hashimoto's thyroiditis, polyglandular autoimmune syndrome type 1 (PAS-1), polyglandular autoimmune syndrome type 2 (PAS-2), and polyglandular autoimmune syndrome type 3 (PAS-3); autoimmune gastrointestinal diseases, including autoimmune hepatitis, primary biliary cirrhosis, inflammatory bowel disease, celiac disease, and Crohn's disease; autoimmune neurological diseases, including multiple sclerosis, myasthenia gravis, Guillain-Barré syndrome, and chronic inflammatory demyelinating neuropathy; and systemic lupus erythematosus (SLE). Many autoimmune diseases can be treated, including systemic autoimmune diseases including lupus erythematosus, antiphospholipid syndrome, autoimmune lymphoproliferative disorders, autoimmune polyendocrinopathy, Behcet's disease, Goodpasture's disease, arthritis including rheumatoid arthritis, osteoarthritis and septic arthritis, sarcoidosis, scleroderma and Sjogren's syndrome, and psoriasis.

[0186] In one embodiment, the autoimmune disease is lupus erythematosus. As used herein, the term "lupus erythematosus" refers to a group of autoimmune diseases with common symptoms affecting the joints, skin, kidneys, blood cells, heart, and lungs. Lupus erythematosus can manifest as a systemic disease or as a purely cutaneous form, also known as incomplete lupus erythematosus. There are four major types of lupus: systemic, discoid, drug-induced, and neonatal. In the context of the present invention, the term "lupus erythematosus" includes, but is not limited to, acute cutaneous lupus erythematosus, subacute cutaneous lupus erythematosus, discoid lupus erythematosus (chronic cutaneous), childhood discoid lupus erythematosus, generalized discoid lupus erythematosus, localized discoid lupus erythematosus, chilblain lupus erythematosus (Hutchinson), lupus erythematosus-lichen planar overlap syndrome, lupus erythematosus panniculitis (lupus erythematosus profundus), tumid lupus erythematosus, verrucous lupus erythematosus (hypertrophic lupus erythematosus), cutaneous lupus mucinosis, complement deficiency syndrome, drug-induced lupus erythematosus, neonatal lupus erythematosus, and systemic lupus erythematosus. The most common severe form is systemic lupus erythematosus.

[0187] In a preferred embodiment, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, inflammatory bowel disease, and ulcerative colitis; more preferably, selected from the group consisting of systemic lupus erythematosus, lupus nephritis, inflammatory bowel disease, and rheumatoid arthritis; more preferably, selected from the group consisting of systemic lupus erythematosus, lupus nephritis, and rheumatoid arthritis. In a preferred embodiment, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus and lupus nephritis.

[0188] In a preferred embodiment, the autoimmune disease is systemic lupus erythematosus.

[0189] As used herein, the terms "systemic lupus erythematosus" or "SLE" refer to a systemic autoimmune disease in which the body's immune system mistakenly attacks healthy tissues throughout the body. Symptoms vary from person to person and can range from mild to severe. Common symptoms include joint pain and swelling, fever, chest pain, hair loss, mouth sores, swollen lymph nodes, fatigue, and a red rash, most commonly seen on the face. Patients often experience periods of illness called flares and periods of remission with few symptoms. Almost all people with SLE experience joint pain and swelling. Some may also develop arthritis. SLE commonly affects the joints of the fingers, hands, wrists, and knees. Kidney disease in SLE causes significant morbidity and mortality. Acute or chronic kidney dysfunction can occur with lupus nephritis, potentially leading to acute or end-stage renal failure.

[0190] In a preferred embodiment, the autoimmune disease is lupus nephritis.

[0191] "Lupus nephritis," also known as SLE nephritis, or "LN," is inflammation of the kidneys caused by systemic lupus erythematosus (SLE). It is a type of glomerulonephritis in which the glomeruli become inflamed. As a result of SLE, glomerulonephritis is said to be secondary in cause and shows a different pattern and outcome than conditions with a primary kidney-related cause. Common symptoms of lupus nephritis include fever, edema, high blood pressure, joint pain, muscle pain, malar erythema, and foamy urine.

[0192] In another preferred embodiment, the autoimmune disease is inflammatory bowel disease, more preferably ulcerative colitis.

[0193] As used herein, the term "inflammatory bowel disease" refers to a group of inflammatory conditions of the colon and small intestine. Crohn's disease and ulcerative colitis are the main types of inflammatory bowel disease. Crohn's disease affects the small intestine and large intestine, as well as the mouth, esophagus, stomach and anus; while ulcerative colitis mainly affects the colon and rectum. Symptoms include abdominal pain, diarrhea, rectal bleeding, severe internal cramps / muscle spasms in the pelvic region, and weight loss.

[0194] In another preferred embodiment, the autoimmune disease is rheumatoid arthritis.

[0195] As used herein, the terms "rheumatoid arthritis" or "RA" refer to a long-term, systemic autoimmune disorder characterized by chronic inflammation of the joints and subsequent destruction of cartilage and bone. Joints are typically hot, swollen, and painful. Pain and stiffness often worsen after rest. It most commonly occurs in the wrists and hands, usually in the same joints on both sides of the body. The disease can affect other parts of the body as well. This can lead to a decrease in red blood cell count, inflammation around the lungs, and inflammation around the heart. Fever and loss of energy may also occur. Symptoms often develop gradually over weeks to months. RA primarily begins as a state of persistent cellular activation, resulting in autoimmunity and immune complexes in both the joints and other organs where it manifests. The initial site of disease is the synovium, where swelling and congestion lead to infiltration by immune cells. The various stages of RA progression are: Initiation stage caused by non-specific inflammation T cell activation and amplification stage A chronic inflammatory phase accompanied by tissue damage due to cytokines IL-1, TNF-α, and IL-6 is.

[0196] As used herein, the term "graft rejection" refers to an immune condition in which transplanted cells, tissues, or organs are not accepted by the body of the transplant recipient. Expression transplant rejection includes both acute and chronic transplant rejection.

[0197] "Acute rejection" or "AR" is a rejection reaction by the immune system of a tissue transplant recipient when the transplanted tissue is immunologically foreign. Acute rejection is characterized by infiltration of the transplanted tissue by the recipient's immune cells, which carry out effector functions and destroy the transplanted tissue. The onset of acute rejection is rapid, generally occurring in humans within a few weeks after transplant surgery.

[0198] "Chronic transplant rejection" or "CR" generally occurs in humans within months to years after transplantation, even when immunosuppression of acute rejection is successful. Fibrosis is a common factor in chronic rejection of all types of organ transplants. Chronic rejection can usually be described by a series of specific insults characteristic of a particular organ. For example, in lung transplants, such insults include fibroproliferative destruction of the airways (bronchiolitis obliterans); in heart transplants or cardiac tissue transplants, such as valve replacements, such insults include fibroatherosclerosis; in kidney transplants, such insults include obstructive nephropathy, nephrosclerosis, and tubulointerstitial nephropathy; and in liver transplants, such insults include vanishing bile duct syndrome. Chronic rejection can also be characterized by ischemic attacks, denervation of the transplanted tissue, hyperlipidemia, and hypertension associated with immunosuppressive drugs.

[0199] As known in the field of transplantation, the organ, tissue or cell to be transplanted can be allogeneic or xenogeneic, so that the transplant can be an allograft or a xenograft.The characteristic of the graft resistance phenotype detected or identified by the subject method is that it occurs without immunosuppressive therapy, that is, the phenotype exists in a host that is not administered immunosuppressive drugs and is not receiving immunosuppressive therapy.The transplant graft can be any solid organ and skin transplant.Examples of organ transplants that can be treated by the method described herein include, but are not limited to, kidney transplant, pancreas transplant, liver transplant, heart transplant, lung transplant, intestine transplant, pancreas after kidney transplant, and simultaneous pancreas and kidney transplant.

[0200] The method of the present invention is also suitable for preventing and / or treating delayed graft function (DGF) caused by ischemic reperfusion injury. As used herein, the term "delayed graft function" refers to a form of acute renal failure that results in post-transplant oliguria, increased immunogenicity of the allograft, and the risk of acute rejection episodes, as well as reduced long-term survival. DGF can be caused by various donor-related factors and recipient-related pre-renal, renal, or post-renal transplant factors. However, the primary cause of delayed graft function is the restoration of ischemia and blood flow in the ischemic-damaged kidney after hypothermic preservation.

[0201] As used herein, the term "graft-versus-host disease" or GVHD refers to a condition that occurs when T cells present in donor tissue attack the host or recipient of the transplanted cells or tissue. Any type of GVHD, including acute and chronic GVHD, can be treated with the therapeutic agents of the present invention.

[0202] The term "hypersensitivity disorder" refers to a condition in which a subject has abnormal sensitivity to harmless agents known as allergens. Hypersensitivity disorders are classified into four types: Type I, Type II, Type III, and Type IV. Type I is described as atopic or anaphylactic, resulting from the release of mediators from IgE-sensitized basophils and mast cells. Type II is described as cytotoxic, involving complement-fixing antibodies with cytolysis or antibody-dependent cellular cytotoxicity. Type III is described as immune complex-mediated, involving soluble antigen-antibody complexes. Type IV is described as cell-mediated or delayed hypersensitivity, resulting from the release of lymphokines by sensitized T lymphocytes after contact with antigen.

[0203] In a preferred embodiment, the immunological disorder is inflammatory bowel disease, more preferably ulcerative colitis, hi another embodiment, the immunological disorder is Crohn's disease.

[0204] Prevention and / or treatment of immunological diseases is achieved through expansion of tolerogenic dendritic cell and / or tolerogenic macrophage populations.

[0205] The phrase "increasing the tolerogenic dendritic cell population and / or the tolerogenic macrophage population" is understood to mean that administration of a polypeptide of the present invention, a homo-oligomer of the present invention, a polynucleotide encoding the polypeptide or homo-oligomer of the present invention, a vector containing the polynucleotide, a host cell containing the vector, or a pharmaceutical composition containing them results in an increase in the number of tolerogenic dendritic cells and / or tolerogenic macrophages in an untreated subject. The tolerogenic dendritic cell population and / or the tolerogenic macrophage population is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% compared to an untreated subject. The ability of a compound of the present invention to increase the tolerogenic dendritic cell population and / or the tolerogenic macrophage population can be determined, for example, as described in Examples 1 to 11.

[0206] In a preferred embodiment, the compound of the present invention is administered subcutaneously. In another preferred embodiment, the compound of the present invention is administered in a regimen comprising multiple doses, wherein the compound is administered no more than once a week. In a more preferred embodiment, the compound of the present invention is administered subcutaneously in a regimen comprising multiple doses, wherein the compound is administered no more than once a week.

[0207] Multiple administration means at least two administrations.Thus, in a preferred embodiment, the regimen comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least thirty, at least fifty, at least one hundred or more administrations.Preferably, the compound is administered for a long period of time.Preferably, the compound is administered for at least one year, at least two years, at least five years or more.

[0208] The phrase "the compound is administered no more than once a week" means that the maximum number of administrations per week is one. This means that if the compound is administered on day 1, a subsequent administration cannot be administered on days 2, 3, 4, 5, 6, or 7. However, for example, the compound may be administered once every two weeks, so the phrase "the compound is administered no more than once a week" includes the possibility that no administration occurs per week. Therefore, according to the present invention, one administration is separated from another administration by at least 7 days. Thus, in a preferred embodiment, each administration is administered at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 30 days, at least 31 days, at least 32 days, at least and are separated by 33 days, at least 34 days, at least 35 days, at least 36 days, at least 37 days, at least 38 days, at least 39 days, at least 40 days, at least 41 days, at least 42 days, at least 43 days, at least 44 days, at least 45 days, at least 46 days, at least 47 days, at least 48 days, at least 49 days, at least 50 days, at least 51 days, at least 52 days, at least 53 days, at least 54 days, at least 55 days, at least 56 days, at least 57 days, at least 58 days, at least 59 days, or at least 60 days or more.

[0209] In a preferred embodiment, the compound is administered once weekly. In another preferred embodiment, the compound is administered once every two weeks. In another embodiment, the compound is administered once every three weeks. In another embodiment, the compound is administered once every four weeks. In another embodiment, the compound is administered once every five weeks. In another embodiment, the compound is administered once every six weeks. In another embodiment, the compound is administered once every seven weeks. In another embodiment, the compound is administered once every eight weeks. In another embodiment, the compound is administered once every nine weeks. In another embodiment, the compound is administered once every ten weeks. In another embodiment, the compound is administered once every eleven weeks. In another embodiment, the compound is administered once every twelve weeks. In one embodiment, the compound is administered monthly. In another embodiment, the compound is administered once every two months.

[0210] The compounds of the present invention are effective at low doses. Therefore, in a preferred embodiment, the dose of each administration is 0.24 to 9.99 mg / m 2 In a more preferred embodiment, the dose for each administration is 0.24 to 5 mg / m 2 in the range of 0.3 to 4.5 mg / m 2 in the range of 0.4 to 4.3 mg / m 2 more preferably in the range of 0.42 to 4.26 mg / m 2 In a preferred embodiment, the dose is 0.42 mg / m 2 In another preferred embodiment, the dose is 4.26 mg / m 2 is.

[0211] In one embodiment, the dose in each administration is 4 to 9.99 mg / m 2 in the range of 5 to 8 mg / m 2 in the range of 6 to 8 mg / m 2 in the range of 7 to 8 mg / m 2 The range is.

[0212] In one embodiment, the dose of each administration is 1 to 9 mg / m 2in the range of 2 to 8 mg / m 2 in the range of 3 to 7 mg / m 2 in the range of 4 to 6 mg / m 2 The range is.

[0213] In another embodiment, the compound is administered at a dose of 0.24 μg / m 2 ~0.24mg / m 2 , preferably 1 μg / m 2 ~0.24mg / m 2 , preferably 10 μg / m 2 ~0.24mg / m 2 , preferably 50 μg / m 2 ~0.24mg / m 2 , more preferably 100 μg / m 2 ~0.24mg / m 2 , more preferably 150 μg / m 2 ~0.24mg / m 2 , more preferably 200 μg / m 2 ~0.24mg / m 2 is administered at a dose of

[0214] However, the present invention also encompasses administration of the compounds at higher doses.

[0215] The subject in need of such treatment may be a human, or may be a non-human primate or other animal (i.e., for veterinary use) that is experiencing symptoms of or at risk of developing an immunological disease. Examples of non-human primates and other animals include, but are not limited to, livestock, pets, and zoo animals (e.g., horses, cows, buffalo, llamas, goats, rabbits, cats, dogs, chimpanzees, orangutans, gorillas, monkeys, elephants, bears, big cats, etc.). In a preferred embodiment, the compound is administered to a mammal, preferably a human.

[0216] The treatment of the present invention may also include a pre-administration step, which does not need to be separated by 7 days from the subsequent administration.In certain cases, this step can be considered as an induction step.Therefore, in one embodiment, the administration further includes a pre-administration step of subcutaneously administering the compound, which is separated by less than 7 days from the subsequent administration.Preferably, the pre-administration step is separated by less than 6 days, more preferably less than 5 days, more preferably less than 4 days, more preferably less than 3 days, more preferably less than 2 days, and preferably less than 1 day from the subsequent administration.

[0217] In a more preferred embodiment, the prior step of subcutaneous administration is separated from the subsequent step by 2 days. Preferably, in the case of the treatment of rheumatoid arthritis, the prior step is separated from the subsequent step by less than 3 days, preferably 2 days, preferably less than 2 days, preferably 1 day, preferably less than 1 day.

[0218] In a preferred embodiment, the dose administered in the pre-step and each subsequent administration is the same.

[0219] In another preferred embodiment, the dose administered in the pre-step is greater than the dose administered in each subsequent administration. In a preferred embodiment, the dose is 40-45 mg / m 2 and preferably 42.84 mg / m 2 Preferably, said dose is administered in the treatment of rheumatoid arthritis. In another preferred embodiment, said dose is administered in the treatment of systemic lupus erythematosus or lupus nephritis.

[0220] In another embodiment, the compound is administered in combination with one or more therapeutic agents useful in the treatment of immunological diseases caused by unwanted activation of the immune system, preferably said therapeutic agents being cyclosporine A, tacrolimus, methotrexate, thiopurines, anti-TNF agents, infliximab, adalimumab, certolizumab, golimumab, etanercept, rituximab, epratuzumab, belimumab, rapamycin, anti-interferon antibodies, tocilizumab, laquinimod, tabacum, fluoxetine, fluoxetine, fluoxetine-10 ... lumab, ofatumumab, ixekizumab, brodalumab, briakinumab, sarilumab, rilonacept, anifrolumab, cyclophosphamide, mycophenolate mofetil, azathioprine, anticalcineurinics, prednisolone, methylprednisolone, vitamin D, vasoactive intestinal peptide, hydroxychloroquine, chloroquine, ocrelizumab, atacicept, abatacept, alemtuzumab, sirukumab, eculizumab and T-cell vaccines.

[0221] All embodiments disclosed so far for this aspect of the invention may also be applied to this aspect.

[0222] Methods for generating a population of tolerogenic dendritic cells obtained using the polypeptides, homo-oligomers, polynucleotides or vectors of the invention The present inventors have observed that dendritic cells contacted with either a polypeptide of the invention, a homo-oligomer of the invention, a polynucleotide of the invention or a vector of the invention exhibit a tolerogenic phenotype.

[0223] Thus, in another aspect, the present invention provides an in vitro method for generating a population of tolerogenic dendritic cells, comprising: (i) incubating a population of dendritic precursor cells under conditions suitable for the formation of a population of immature dendritic cells; and (ii) incubating the population of immature dendritic cells obtained in step (i) above under conditions suitable for the formation of mature dendritic cells. Including, The above steps (i) and / or (ii) (a) a polypeptide of the present invention; (b) a homo-oligomer of the present invention; (c) a polynucleotide of the invention, and (d) Vector of the present invention The present invention relates to a method of treating a cancer cell in the presence of a composition of matter selected from the group consisting of:

[0224] Thus, the polypeptide of the present invention, the homo-oligomer of the present invention, the polynucleotide of the present invention or the vector of the present invention can be contacted with cells during the differentiation stage (i.e., until dendritic precursor cells differentiate into immature dendritic cells), during the maturation stage (i.e., until immature dendritic cells mature into dendritic cells), or during both stages.

[0225] As used herein, the term "dendritic cell" refers to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. Dendritic cells are a type of "professional" antigen-presenting cell with a high capacity to sensitize MHC-restricted T cells. Dendritic cells can be recognized by function or phenotype, particularly by cell surface phenotype. These cells are characterized by their unique morphology, medium- to high-level expression of surface MHC class II, and ability to present antigens to T cells, particularly naive T cells (Steinman et al. (1991) Ann. Rev. Immunol. 9:271; the description of such cells is incorporated herein by reference). Dendritic cells affected by the methods of the present invention can be selected to be immature or mature dendritic cells.

[0226] Dendritic cells include a group of bone marrow-derived cells with dendritic morphology distributed throughout various tissues and organs in the body, as well as a group of cells with dendritic morphology distributed throughout various organs and tissues in the body obtained by in vitro differentiation using cytokines or derived from bone marrow- or blood-derived stem cells and equivalent cells. Specifically, dendritic cells include lymphoid dendritic cells (including Th2 or tolerance-inducing cells), myeloid dendritic cells (commonly used dendritic cells, including immature and mature dendritic cells), Langerhans cells (dendritic cells important as antigen-presenting cells in the skin), interdigitating cells (distributed in the T cell areas of lymph nodes and the spleen and thought to function in antigen presentation to T cells), and follicular dendritic cells (important as antigen-presenting cells for B cells). Dendritic cells have a non-flat cell surface with characteristic veil-like projections and express the cell surface marker CD1a. + , CD4 + , CD86 + or HLA-DR + Mature dendritic cells are characterized by the expression of CD11c + However, the precursor cells of dendritic cells express CD11c - , IL-3Rα low Those with the phenotype of CD11c - IL-3Rα high In vivo treatment with GM-CSF induces the expression of CD11b high , CD11c high DCs preferentially expand, and Flt-3 ligand promotes CD11c + IL-3Rα low DCs and CD11c - IL-3Rα high It has been shown to expand DC precursors.

[0227] "Tolerogenic dendritic cells" refer to dendritic cells derived from immature dendritic cells that have been exposed to differentiation stimuli, which can be a combination of cytokines, hormones, vitamins, and other biological factors, thereby acquiring the ability to induce tolerance. Tolerogenic dendritic cells have a low ability to activate effector T cells but a high ability to induce and activate regulatory T cells. Tolerogenic dendritic cells can be considered mature resistant cells that function as "immature DCs" with a stable phenotype that is maintained even in the presence of proinflammatory signals. Tolerogenic dendritic cells secrete anti-inflammatory cytokines.

[0228] In a first step, the method for obtaining a population of tolerogenic dendritic cells comprises incubating a population of dendritic precursor cells under conditions suitable for the formation of a population of immature dendritic cells.

[0229] As used herein, the term "dendritic cell precursor" refers to any cell that can differentiate into an immature dendritic cell in the presence of appropriate cytokines (specifically, G-CSF, GM-CSF, TNF-α, IL-4, IL-13, SCF (c-kit ligand), Flt-3 ligand, or a combination thereof), preferably within 4 weeks, more preferably within 20 days, even more preferably within 18 days, and even more preferably within 16 days. Examples of dendritic precursor cells include, but are not limited to, myeloid dendritic precursor cells, lymphoid dendritic precursor cells, and plasmacytoid dendritic precursor cells. Phenotypic surface markers expressed by various subsets of dendritic precursor cells are known in the art and can be used for identification purposes, for example, by flow cytometry or by using immunohistochemical techniques.

[0230] In a preferred embodiment, the population of dendritic precursor cells is a population of monocytic dendritic precursor cells. As used herein, "monocytic dendritic cell precursors" includes monocytes that have GM-CSF receptors on their surface and other bone marrow precursor cells that respond to GM-CSF. Cells can be obtained from any tissue in which they reside, particularly lymphoid tissues such as the spleen, bone marrow, lymph nodes, and thymus. Monocytic dendritic cell precursors can also be isolated from the circulatory system. Peripheral blood is an easily accessible source of monocytic dendritic cell precursors. Umbilical cord blood is another source of monocytic dendritic cell precursors.

[0231] Monocytic dendritic cell precursors can be obtained from peripheral blood mononuclear cells (PBMCs), either from whole blood diluted 1:1 with buffered saline, or from leukocyte concentrates ("buffy coat" fraction, MSKCC Blood Bank) by standard centrifugation over Ficoll-Paque PLUS (endotoxin-free, #17-1440-03, Amersham Pharmacia Biotech AB, Uppsala, Sweden). MoDC precursors are PBMCs adherent to tissue culture plastic (#35-3003; Falcon, Becton-Dickinson Labware, Franklin Lakes, NJ) and can be cultured in complete RPMI 1640 containing 1% normal human serum (NHS) in the presence of GM-CSF (1000 IU / ml) and IL-4 (500 IU / ml), replenished every 2 days, as described (Thurner B et al., 1999, J. Immunol. Meth. 223:1-15 and Ratzinger G. et al., 2004, J. Immunol. 173:2780-2791).

[0232] Generally, monocytic dendritic cell precursors can be identified by the expression of markers such as CD13 and CD33. Myeloid dendritic precursors can differentiate into dendritic cells via the CD14 or CD1a pathway. Thus, the dendritic precursor cells of the present invention are CD14 + CD1a -Dendritic precursor cells or CD14 - CD1a + In certain embodiments of the present invention, myeloid dendritic precursor cells may be CD34 + CD33 + CD7 - CD10 - In a preferred embodiment, myeloid dendritic precursor cells may be characterized by a CD14 phenotype. + CD14 is a monocyte. + Monocytes may also express GM-CSF receptors.

[0233] The dendritic precursor cells used as the starting material for the methods of the present invention can be autologous to the subject being treated. In another embodiment, the dendritic cells used as the starting material for the methods of the present invention are xenogeneic dendritic cells. For example, when treating graft-versus-host disease, the dendritic cells used as the starting material are dendritic cells obtained from a donor. The subject can be, for example, a mouse, rat, dog, chicken, horse, goat, donkey, or primate. Most preferably, the subject is a human.

[0234] As used herein, the phrase "conditions suitable for forming a population of immature dendritic cells" refers to conditions that result in the differentiation of dendritic precursor cells into immature precursor cells. Suitable conditions include, for example, culturing for about three days in the presence of SCF (50 ng / ml), GM-CSF (500 U / ml), and TNF-α (50 ng / ml), followed by culturing in the presence of SCF (50 ng / ml), GM-CSF (500 U / ml), IL-4 (250 U / ml), and TNF-α (50 ng / ml), more preferably in the presence of GM-CSF (20 ng / ml) and IL-4 (20 ng / ml), or in the presence of GM-CSF (20 ng / ml) and SCF (10 ng / ml). In a preferred embodiment, the cells are cultured in the presence of GM-CSF (800 UI / ml) and IL-4 (500 UI / ml).

[0235] This treatment generates immature dendritic cells. "Immature dendritic cells" refer to dendritic cells with significantly lower T cell activation capacity than mature dendritic cells. Specifically, immature dendritic cells may have an antigen-presenting capacity that is less than half, preferably less than one-quarter, of that of dendritic cells induced to mature by adding LPS (1 μg / ml) and culturing for two days. Antigen-presenting capacity can be quantified, for example, using allogeneic T cell activation capacity (mixed lymphocyte test: allogeneic T cells and dendritic cells are co-cultured at a dendritic cell:T cell ratio of 1:10, 1:40, 1:80, or 1:160; preferably, a dendritic cell:T cell ratio of 1:40, 1:80, or 1:160; 3H-thymidine is added 8 hours before the end of the culture, and T cell proliferation capacity is evaluated based on the amount of 3H-thymidine incorporated into the DNA of T cells (see GeneTherapy 7; 249-254 (2000))). Alternatively, peptides can be used to test their ability to induce specific cytotoxic T cells (CTLs), in which a known class I-restricted peptide of a specific antigen is added to dendritic cells; the dendritic cells are co-cultured with T cells obtained from the peripheral blood of the same healthy donor from which the dendritic cells were collected (with 25 U / ml or preferably 100 U / ml IL-2 from day 3 onwards). T cells are preferably stimulated with dendritic cells three times over a 21-day period, more preferably twice over a 14-day period. The resulting effector cells are co-cultured with 51Cr-labeled target cells (peptide-restricted class I-positive tumor cells) at a ratio of 100:1 to 2.5:1 (100:1, 50:1, 25:1, 20:1, 12.5:1, 10:1, 5:1, or 2.5:1), preferably at a ratio of 10:1, for 4 hours; the 51Cr released from the target cells is quantified (see Arch Dermatol Res 292:325-332 (2000)). Furthermore, immature dendritic cells preferably have the ability to phagocytose antigens and more preferably exhibit low (e.g., significantly lower than mature DCs induced by LPS) or negative expression of receptors that induce costimulation of T cell activation.

[0236] In a preferred embodiment, the first step of the method comprises: (i) a polypeptide of the present invention; (ii) a homo-oligomer of the present invention; (iii) a polynucleotide of the invention, and (iv) Vector of the Present Invention The method is carried out in the presence of a composition of matter selected from the group consisting of:

[0237] In a more preferred embodiment, the composition of matter is present during the first step of the method of the invention but not during the second step. Preferably, the composition of matter is a homo-oligomer of the invention.

[0238] The terms "polypeptide of the invention", "homo-oligomer of the invention", "polynucleotide of the invention" and "vector of the invention" have been explained in detail above.

[0239] The step of carrying out the ...

[0240] In a second step, the immature dendritic cells isolated in the first step are then incubated under conditions suitable for the immature dendritic cells to mature into tolerogenic mature dendritic cells, and a composition highly enriched in tolerogenic dendritic cells is achieved by this method.

[0241] Mature human dendritic cells are cells that are positive for the expression of CD40, CD80, CD83, CD86, and HLA class II. Immature dendritic cells can be distinguished from mature dendritic cells based on markers selected from the group consisting of CD83 and CD86. Immature dendritic cells are weakly positive or preferably negative for the markers CD40, CD80, CD83, and CD86, while mature dendritic cells are positive.

[0242] Mature DCs lose the ability to take up antigens, and the cells exhibit upregulated expression of costimulatory cell surface molecules and secrete a variety of cytokines. Specifically, mature DCs typically express high levels of MHC class I and II antigens and generally express CD80. + , CD83 + and CD86 + Higher MHC expression leads to increased antigen density on the DC surface, and upregulation of the costimulatory molecules CD80 and CD86 enhances T cell activation signals via their counterparts on T cells, such as CD28.

[0243] As used herein, the phrase "conditions suitable for the maturation of immature dendritic cells into tolerogenic mature dendritic cells" refers to a method that allows immature dendritic cells to mature into tolerogenic mature dendritic cells. Mature dendritic cells can be prepared (i.e., matured) by contacting an effective amount or concentration of immature dendritic cells with a dendritic cell maturation agent. Examples of dendritic cell maturation agents include BCG; IFNγ; a TLR4 ligand such as LPS; TNFα; or a TLR7 ligand such as Gardiquimod. In a preferred embodiment, the dendritic cell maturation agent is a TLR4 ligand, preferably LPS. In another embodiment, the dendritic cell maturation agent is a TLR7 ligand, preferably Gardiquimod. An effective amount of BCG is typically about 10 per ml of tissue culture medium. 5 ~10 7cfu. The effective amount of IFNγ is typically in the range of approximately 100-1000 U per ml of tissue culture medium. Bacillus Calmette-Guerin (BCG) is a non-pathogenic strain of M. bovis. As used herein, BCG refers to whole BCG as well as cell wall components, BCG-derived lipoarabidomannan, and other BCG components associated with the induction of type 2 immune responses. BCG is optionally inactivated, such as heat-inactivated BCG or formalin-treated BCG. In a preferred embodiment, LPS is added to tissue culture medium at 5 μg / ml. In another embodiment, Gardiquimod is added to tissue culture medium at 10 μg / ml.

[0244] Immature DCs are typically contacted with an effective amount of BCG and IFNγ, or LPS, or Gardiquimod for about 1 hour to about 48 hours. Immature dendritic cells can be cultured and matured under appropriate maturation culture conditions. Suitable tissue culture media include AIM-V, RPMI1640, DMEM, X-VIVO15™, and the like. To promote cell maturation, tissue culture media can be supplemented with amino acids, vitamins, cytokines such as GM-CSF, divalent cations, and the like. Typically, about 500 units / ml of GM-CSF is used.

[0245] In a preferred embodiment, the second step of the method comprises: (i) a polypeptide of the present invention; (ii) a homo-oligomer of the present invention; (iii) a polynucleotide of the invention, and (iv) Vector of the Present Invention The method is carried out in the presence of a composition of matter selected from the group consisting of:

[0246] The maturation of dendritic cells can be monitored by methods known in the art for dendritic cells. Cell surface markers can be detected by assays known in the art, such as flow cytometry, immunohistochemistry, etc. Cells can also be monitored for cytokine production (for example, by ELISA, other immunoassays, or by using oligonucleotide arrays). Mature DCs of the present invention also lose the ability to uptake antigens, which can be analyzed by uptake assays known to those skilled in the art.

[0247] The term "mature dendritic cells" refers to dendritic cells that have significantly stronger antigen-presenting ability to T cells and the like compared with immature dendritic cells. Specifically, mature dendritic cells can have at least half, and preferably equal to or greater, the antigen-presenting ability of dendritic cells induced to mature by adding LPS (1 μg / ml) and culturing for two days. Furthermore, mature dendritic cells preferably have weak or no phagocytic ability against antigens and are more preferably positive for the expression of receptors that induce costimulation of T cell activation. Dendritic cell activation refers to the transition from immature dendritic cells to mature dendritic cells; activated dendritic cells include mature dendritic cells and dendritic cells in the transitional process, and the expression of CD80 and CD86, which induce costimulatory signals, is increased by activation stimuli.

[0248] The tolerogenic cell population may comprise approximately 50% or more of the cell composition, preferably approximately 75% or more, and even 90% or more. The cells of interest are identified by their surface phenotype, ability to induce tolerance, etc. The enriched cell population may be used immediately or may be frozen at liquid nitrogen temperature for long-term storage and thawed for reuse. Cells are typically stored in 10% DMSO, 50% FCS, and 40% RPMI 1640 medium. The tolerogenic dendritic cell enriched cell population can be used in various screening assays and cultures, as described below.

[0249] DC can optionally be further purified by using antibody against DC marker to sort fluorescently labeled cells.DC can also be isolated by using antibody against DC, and antibody is linked to magnetic beads.In certain embodiments, DC that co-expresses CD32a and CD32b is isolated by FACS.

[0250] The separated cells can be collected in any suitable medium that maintains cell viability, usually with a cushion of serum at the bottom of the collection tube. Various media are commercially available and can be used depending on the nature of the cells. The medium can be liquid or semi-solid, including, for example, agar, methylcellulose, etc. The cell population can be appropriately suspended in an appropriate nutrient medium, such as Iscove's modified dMEM, HBSS, dPBS, RPMI, Iscove's medium, DMEM, or RPMI-1640, typically supplemented with fetal bovine serum (approximately 5-10%), L-glutamine, thiols (especially 2-mercaptoethanol), and antibiotics, such as penicillin and streptomycin.

[0251] Optionally, the presence of dendritic cells can be confirmed using standard techniques, such as morphological observation and immunochemical staining. For example, dendritic cell purity can be assessed by flow cytometry using fluorochrome-labeled antibodies against one or more characteristic cell surface markers.

[0252] In some embodiments, tolerogenic dendritic cells are used to produce regulatory T cells (see, e.g., U.S. Patent Application No. 10 / 661,804). Briefly, regulatory T cells can be produced from a population containing CD4+ T cells and / or CD8+ T cells. These T cell populations can be isolated from a subject or cultured. Subpopulations of T cells, such as populations selected by cell surface markers to contain enriched populations of specific cells (e.g., CD4+CD25+ T cells or CD4+CD25- T cells), can also be used. T cells are then cultured or incubated with the tolerogenic dendritic cells of the present invention. When regulatory T cells are produced in vitro, the tolerogenic dendritic cells can be allogeneic or syngeneic with the T cells. In some embodiments, tolerogenic DCs are loaded with antigen (e.g., stimulated with antigen or transfected with RNA encoding the antigen). In such embodiments, the tolerogenic DCs can present the antigen to T cells. During the production of regulatory T cells, T cells can be exposed to and / or cultured with tolerogenic DCs one or more times. For example, T cells can be cultured with DCs for several days or weeks; DCs in the mixed culture can be replenished as needed. In some embodiments, the culture is continued until a therapeutic amount of regulatory T cells is obtained. Other culture techniques and / or additives can be used to improve the results obtained; for example, the culture medium can contain cytokines such as IL-2.

[0253] Regulatory T cells generally secrete IL-10 and / or TGF-β (see, e.g., Walsh et al., 2004, J. Clin. Invest. 114: 1398-1403); assays for confirming secretion of these cytokines are known in the art. In some embodiments, regulatory T cells inhibit a mixed lymphocyte reaction by at least 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or 100%. Mixed lymphocyte reaction assays are known in the art. Regulatory T cells of the present invention can be antigen-specific. CD4+CD25+ regulatory T cells generally express characteristic cell surface markers, including CD4 and CD25, and certain intracellular markers, such as Foxp3; assays for cell surface and intracellular markers are known in the art.

[0254] All embodiments disclosed so far for this aspect of the invention may also be applied to this aspect.

[0255] Methods for generating populations of tolerogenic macrophages obtained using the polypeptides, homo-oligomers, polynucleotides or vectors of the invention The inventors have observed that macrophages contacted with either a polypeptide of the invention, a homo-oligomer of the invention, a polynucleotide of the invention or a vector of the invention exhibit a tolerogenic phenotype.

[0256] Thus, in another aspect, the present invention provides an in vitro method for generating a population of tolerogenic macrophages, comprising: (i) incubating a population of macrophage progenitor cells under conditions suitable for the formation of a population of immature macrophages; and (ii) incubating the population of immature macrophages obtained in step (i) above under conditions suitable for the formation of mature macrophages. Including, The above steps (i) and / or (ii) (a) a polypeptide of the present invention; (b) a homo-oligomer of the present invention; (c) a polynucleotide of the invention, and (d) Vector of the present invention The present invention relates to a method of treating a cancer cell in the presence of a composition of matter selected from the group consisting of:

[0257] Thus, the polypeptide of the present invention, the homo-oligomer of the present invention, the polynucleotide of the present invention, or the vector of the present invention can be contacted with cells during the differentiation stage (i.e., until macrophage precursor cells differentiate into immature macrophages), during the maturation stage (i.e., until immature macrophages mature into mature macrophages), or during both stages.

[0258] As used herein, the term "macrophage" refers to a mononuclear phagocyte, a type of white blood cell of the immune system that engulfs and digests cellular debris, foreign particles, microorganisms, cancer cells, etc., in a process called phagocytosis. These large phagocytes are found in virtually all tissues. They have various names and morphologies throughout the body (e.g., histiocytes, Kupffer cells, alveolar macrophages, microglia, etc.), but all are part of the mononuclear phagocyte system. As used herein, the term "macrophage" encompasses any known type of macrophage. In addition to phagocytosis, they play an important role in nonspecific defense (innate immunity) and help initiate specific defense mechanisms (adaptive immunity) by recruiting other immune cells, such as lymphocytes. Macrophages also play an important anti-inflammatory role and may dampen immune responses through the release of cytokines. Macrophages are a type of "professional" antigen-presenting cell. Macrophages can be recognized by function or by phenotype, particularly their cell surface phenotype. The macrophages affected by the methods of the present invention may be selected to be immature or mature macrophages.

[0259] Macrophages include a group of cells distributed throughout various tissues and organs in the body, which arise as a result of in vitro differentiation using cytokines, etc., or are derived from stem cells and equivalent cells derived from bone marrow or blood. Each type of macrophage has a specific name determined by its location. Specifically, macrophages include, for example, adipose tissue macrophages, Kupffer cells (located in the liver), sinus histiocytes (located in lymph nodes), alveolar macrophages (located in the alveoli of the lungs), tissue macrophages (tissue cells) that give rise to giant cells (located in connective tissue), microglia (located in the central nervous system), Hofbauer cells (located in the placenta), intraglomerular mesangial cells (located in the kidney), osteoclasts (located in bone), epithelioid cells (located in granulomas), red pulp macrophages (located in the red pulp of the spleen), white pulp macrophages (located in the white pulp of the spleen), marginal zone macrophages and metallophilic macrophages (located in the splenic region), peritoneal macrophages (located in the peritoneal cavity), and LysoMacs (located in Peyer's patches). There are many commonly used macrophage markers, including CD14, CD16, CD64, CD68, CD71, and CCR5; the exact markers used vary depending on the macrophage subset and the state of their local environment. Mature M1 macrophages are typically identified by the markers CD86, CD80, CD68, MHCII, IL-1R, TLR2, TLR4, iNOS, and / or SOCS3. M2 macrophages, particularly those activated by IL-4, are typically identified by the markers CD163, MHCII, SR, MMR / CD206, CD200R, TGM2, DecoyR, and / or IL-1RII. Macrophage precursors have the phenotype IL3Ra. low CD11b - CD34 + c-KIT + FLT3 + and IL3Ra high CD11b - CD34 + c-KIT + FLT3 +(Xiao et al. (2015) Stem Cell Reports 4: 984-994).

[0260] As used herein, the term "tolerogenic macrophages" is used interchangeably with "regulatory macrophages" or "Mregs" and "rested macrophages." Tolerogenic, regulatory, or rested macrophages are derived from immature macrophages exposed to differentiation stimuli, which are combinations of cytokines, hormones, vitamins, and other biological factors, that result in the macrophages acquiring the ability to induce tolerance. Tolerogenic macrophages have a low ability to activate effector T cells but a high ability to induce and activate regulatory T cells. Tolerogenic macrophages can be considered mature resistant cells that function as "immature macrophages" with a stable phenotype that is maintained even in the presence of proinflammatory signals. Tolerogenic macrophages are able to induce regulatory T cells through the production of IL-10 and limited or absent secretion of IL-12. H Furthermore, tolerogenic macrophages have increased expression of HLA class II and B7 costimulatory molecules, and improved antigen-presenting function.

[0261] In a first step, the method for obtaining a population of tolerogenic macrophages comprises incubating a population of macrophage precursor cells under conditions suitable for the formation of a population of immature macrophages.

[0262] As used herein, the term "macrophage progenitor cells" refers to any cells that can differentiate into immature macrophages in the presence of appropriate cytokines (specifically, G-CSF, GM-CSF, M-CSF, TNF-α, IL-4, IL-13, SCF (c-kit ligand), Flt-3 ligand, or a combination thereof), preferably within 4 weeks, more preferably within 20 days, even more preferably within 18 days, and even more preferably within 16 days. Examples of macrophage progenitor cells include, but are not limited to, myelomonocytic-macrophage progenitors; or proliferative, conditional, developmentally-arrested, primary macrophage progenitors obtained by overexpressing Hoxb8 in myeloid progenitor cells in medium supplemented with GM-CSF or Flt3L. Phenotypic surface markers expressed by various subsets of macrophage progenitor cells are known in the art and can be used for identification purposes, for example, by flow cytometry or by using immunohistochemical techniques.

[0263] In a preferred embodiment, the population of macrophage progenitor cells is a population of monocyte-macrophage progenitor cells. As used herein, "monocyte-macrophage progenitor cells" includes monocytes with GM-CSF receptors on their surface and other myeloid progenitor cells that respond to GM-CSF. The cells can be obtained from any tissue in which they reside, particularly lymphoid tissues such as the spleen, bone marrow, lymph nodes, and thymus. Monocyte-macrophage progenitor cells can also be isolated from the circulatory system. Peripheral blood is an easily accessible source of monocyte-macrophage progenitor cells. Umbilical cord blood is another source of monocyte-macrophage progenitor cells.

[0264] Monocyte-macrophage progenitors can be obtained from peripheral blood mononuclear cells (PBMCs), either from whole blood diluted 1:1 with buffered saline, or from leukocyte concentrates ("buffy coat" fraction, MSKCC Blood Bank) by standard centrifugation using Ficoll-Paque PLUS (endotoxin-free, #17-1440-03, Amersham Pharmacia Biotech AB, Uppsala, Sweden). Monocyte-macrophage precursors are PBMCs (#35-3003; Falcon, Becton-Dickinson Labware, Franklin Lakes, NJ) that adhere to tissue culture plastic and can be cultured in complete RPMI 1640 containing 1% normal human serum (NHS) in the presence of GM-CSF (1000 IU / ml) and IL-4 (500 IU / ml), replenished every 2 days, as described (Thurner B et al., 1999, J. Immunol. Meth. 223:1-15 and Ratzinger G. et al., 2004, J. Immunol. 173:2780-2791).

[0265] Generally, monocyte-macrophage precursor cells can be identified by the expression of markers such as CD13 and CD33. Myeloid macrophage precursors can differentiate into macrophage cells via the CD14 or CD16 pathway. Therefore, the macrophage precursor cells of the present invention can be differentiated by the expression of markers such as CD14. + CD16 - Macrophage precursor cells, CD14 + CD16 + Macrophage precursor cells or CD14 dim CD16 + In a specific embodiment of the present invention, the myeloid macrophage progenitor cells may be CD34 + CD33 + CD7 - CD10 - In a preferred embodiment, the myeloid macrophage progenitor cells are characterized by a CD14 phenotype. +CD14 is a monocyte. + Monocytes may also express GM-CSF receptors.

[0266] The macrophage progenitor cells used as the starting material for the method of the present invention can be autologous to the subject being treated. In another embodiment, the macrophage progenitor cells used as the starting material for the method of the present invention are heterologous macrophage cells. For example, when treating graft-versus-host disease, the macrophage cells used as the starting material are macrophage cells obtained from a donor. The subject can be, for example, a mouse, rat, dog, chicken, horse, goat, donkey, or primate. Most preferably, the subject is a human.

[0267] As used herein, the phrase "conditions suitable for the formation of a population of immature macrophages" refers to conditions that result in the differentiation of macrophage precursor cells into immature macrophages. Suitable conditions include, for example, culturing for about 6 days in the presence of GM-CSF and / or M-CSF, optionally containing IL-3. In a preferred embodiment, the cells are cultured in the presence of GM-CSF (50 ng / ml) or M-CSF (50 ng / ml).

[0268] This treatment generates immature macrophages. As used herein, the term "immature macrophages" is interchangeable with the terms "undifferentiated macrophages," "naive macrophages," or "M0." Immature macrophages, or undifferentiated macrophages, or naive macrophages or M0, refer to macrophages that have significantly lower T cell activation capacity compared to their mature counterparts. Specifically, immature macrophages may have less than half, preferably less than one-quarter, of the antigen-presenting capacity of macrophages induced to mature by adding LPS (1 μg / ml) and culturing for 2 days. The antigen-presenting ability can be quantified, for example, using the allogeneic T cell activation ability (mixed lymphocyte test: allogeneic T cells and macrophages are co-cultured at macrophage:T cell ratios of 1:10, 1:40, 1:80, and 1:160, preferably at macrophage:T cell ratios of 1:40, 1:80, and 1:160; 3H-thymidine is added 8 hours before the end of the culture, and the T cell proliferation ability is evaluated based on the amount of 3H-thymidine incorporated into the DNA of the T cells (Gene Therapy 7; 249-254) (2000). Alternatively, peptides can be used to assess their ability to induce specific cytotoxic T cells (CTLs), in which a known class I-restricted peptide of a specific antigen is added to macrophages; the macrophages are co-cultured with T cells obtained from the peripheral blood of the same healthy donor from which the macrophages were collected (with IL-2 at 25 U / ml or preferably 100 U / ml from day 3 onwards). The T cells are preferably The effector cells are stimulated with macrophages three times over 21 days, and more preferably twice over 14 days. The resulting effector cells are co-cultured with 51Cr-labeled target cells (peptide-restricted class I-positive tumor cells) at a ratio of 100:1 to 2.5:1 (100:1, 50:1, 25:1, 20:1, 12.5:1, 10:1, 5:1, or 2.5:1), preferably at a ratio of 10:1, for 4 hours; and the 51Cr released from the target cells is quantified (see Arch Dermatol Res 292:325-332 (2000)). Furthermore, immature macrophages preferably have the ability to phagocytose antigens. More preferably, they exhibit low (for example, significantly lower compared to the mature macrophages induced by LPS described above) or negative expression of receptors that induce costimulation of T cell activation.

[0269] In a preferred embodiment, the first step of the method comprises: (i) a polypeptide of the present invention; (ii) a homo-oligomer of the present invention; (iii) a polynucleotide of the invention, and (iv) Vector of the Present Invention The method is carried out in the presence of a composition of matter selected from the group consisting of:

[0270] The terms "polypeptide of the invention", "homo-oligomer of the invention", "polynucleotide of the invention" and "vector of the invention" have been explained in detail above.

[0271] The step performed in the presence of a polypeptide of the invention, a homo-oligomer of the invention, a polynucleotide encoding such a molecule, or a vector containing such a polynucleotide can be performed in vivo or ex vivo. Generally, in these methods, immature macrophages are exposed to a homo-oligomer of the invention at a lower concentration of 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 50, or 100 μg per ml of medium and an upper concentration of 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 50, 100, or 200 μg per ml of medium. Most preferably, macrophages are matured in the presence of a homo-oligomer of the invention at a concentration of 1-10 μg / ml, most preferably 3, 5, or 10 μg / ml, and most preferably 3 μg / ml.

[0272] In a second step, the immature macrophages isolated in the first step are then incubated under conditions suitable for the immature macrophages to mature into tolerogenic mature macrophages, and a composition highly enriched in tolerogenic macrophages is achieved by this method.

[0273] Mature human macrophages are cells that are positive for the expression of one or more of these markers: CD86, CD80, CD68, MHCII, IL-1R, TLR2, TLR4, iNOS, SOCS3, CD163, SR, MMR / CD206, CD200R, TGM2, DecoyR, IL-1RII, TLR1, TLR8 and / or VEGF, depending on the specific type of macrophage. Immature macrophages have moderate or low expression of these markers, preferably weakly positive for these markers, whereas mature macrophage cells are positive. Upon differentiation into M1 and M2, the expression is reduced to CD64. low In tolerogenic macrophages, expression is CD64 - Therefore, in a preferred embodiment, the marker for immature macrophages is CD64 high In a preferred embodiment, the immature macrophages are CD64 high , CD80 - , CD209 - , CD11b + In a preferred embodiment, M1 is CD64 low , CD80 + In a preferred embodiment, M2 is CD209 + , CD11b + is.

[0274] The term "mature macrophages" includes, but is not limited to, both classically activated macrophages (M1) and alternatively activated macrophages (M2).

[0275] M1 macrophages are referred to herein as "classically activated macrophages," also known as "inflammatory macrophages" or "proinflammatory macrophages." M1 macrophages are activated by toll-like receptors (TLRs) and IFNγ, e.g., LPS and IFNγ, and secrete high levels of IL-12 and low levels of IL-10. M1 macrophages exhibit proinflammatory, antimicrobial (mediating resistance to intracellular pathogens), tissue-destructive, antitumor, and phagocytic functional phenotypes. Upon activation, these M1 macrophages express large amounts of proinflammatory cytokines (TNFα, IL-1β, IL-6, IL-12, IL-18, IL-23), chemokines (CXCL-1, 2, 3, 5, 8, 9, 10, CCL-2, 3, 4, 5, 11, 17, and 22), proteases, and ROS / RNS. Thus, M1 macrophages play a crucial role not only in innate responses but also in adaptive antigen-specific responses. M1 macrophages are cells positive for CD64 and CD80.

[0276] M2 macrophages are referred to herein as "alternatively activated macrophages" and are also known as "anti-inflammatory macrophages" or "wound-healing macrophages." M1 macrophages are activated by IL-4 or IL-13. M2 macrophages exhibit a functionally distinct phenotype from M1 macrophages. Their function is generally described as an anti-inflammatory phenotype through the production of anti-inflammatory cytokines, and they also mediate tissue remodeling and repair, as well as resistance to parasites. M2 macrophages produce high levels of IL-10, TGF-β, and low levels of IL-12. M2 macrophages are positive for the expression of CD209 and CD11b markers. M2 macrophages have limited ability to induce antigen-specific responses due to their low HLA expression.

[0277] Mature macrophages lose the ability to take up antigens, and the cells exhibit upregulated expression of costimulatory cell surface molecules and secrete a variety of cytokines.

[0278] As used herein, the phrase "conditions suitable for the maturation of immature macrophages into tolerogenic mature macrophages" refers to a method that allows immature macrophages to mature into tolerogenic mature macrophages. Mature macrophages can be prepared (i.e., matured) by contacting an effective amount or concentration of immature macrophages with a macrophage maturation agent. The macrophage maturation agent varies depending on the type of mature macrophage (M1 or M2) that is obtained. Examples of macrophage maturation agents for obtaining M1 from M0 include IFNγ in combination with inflammatory stimuli such as LPS and TNFα; or GM-CSF and additional polarizing signals such as anoxic environment, β-chemokine, and phorbol myristate acetate (PMA) (Foey AD Chapter 5: Macrophages: Masters of immune activation, suppression, and deviation. In book: Immune response activation. Publisher: InTech, Editors: Guy Huynh Thien Duc, pp. 121-149); or specific TLR agonists that induce both TNF and IFNβ (Mosser DM and Edwards JP 2008. Nat Rev Immunol, 8(12):958-969). In a preferred embodiment, the macrophage maturation agent for M1 is LPS, preferably at a concentration of 40 ng / ml in tissue culture medium. In a more preferred embodiment, the macrophage maturation agent for M1 is a combination of LPS and IFNγ, preferably a combination of 40 ng / ml LPS and 40 ng / ml IFNγ. Macrophage maturation agents for obtaining M2 from M0 include, but are not limited to, IL-4 / IL-13, or M-CSF and IL-14. In a preferred embodiment, the macrophage maturation agent for M2 is IL-4, preferably 40 ng / ml IL-4.

[0279] Immature macrophages are typically contacted with an effective amount of a macrophage maturation agent for about 1 hour to about 48 hours. The immature macrophages can be cultured and matured under appropriate maturation culture conditions. Suitable tissue culture media include AIM-V, RPMI 1640, DMEM, X-VIVO 15 (trademark), and the like. The tissue culture medium can be supplemented with amino acids, vitamins, cytokines, divalent cations, and the like to promote cell maturation.

[0280] In a preferred embodiment, the second step of the method: (i) a polypeptide of the present invention; (ii) a homo-oligomer of the present invention; (iii) a polynucleotide of the invention, and (iv) Vector of the Present Invention The method is carried out in the presence of a composition of matter selected from the group consisting of:

[0281] The maturation of macrophages can be monitored by methods known in the art for macrophages. Cell surface markers can be detected by assays known in the art, such as flow cytometry, immunohistochemistry, etc. Cells can also be monitored for cytokine production (e.g., by ELISA, other immunoassays, or by using oligonucleotide arrays). Mature macrophages of the present invention also lose the ability to uptake antigens, which can be analyzed by uptake assays known to those skilled in the art.

[0282] The term "mature macrophages" refers to macrophages that have significantly stronger antigen-presenting ability to T cells and the like compared with immature macrophages. Specifically, mature macrophages can have half, or preferably the same, antigen-presenting ability as macrophages whose maturation has been induced by adding LPS (1 μg / ml) and incubating for 2 minutes. Furthermore, mature macrophages preferably have weak or no phagocytic ability against antigens and are more preferably positive for the expression of receptors that induce costimulation of T cell activation. Macrophage activation refers to the transition from immature macrophages to mature macrophages; activated macrophages include mature macrophages and macrophages in the transitional process.

[0283] The tolerogenic macrophage population may comprise approximately 50% or more of the cell composition, preferably approximately 75% or more, and even 90% or more. The cells of interest are identified by their surface phenotype, ability to induce tolerance, etc. The enriched cell population may be used immediately or may be frozen at liquid nitrogen temperature for long-term storage and thawed for reuse. Cells are typically stored in 10% DMSO, 50% FCS, and 40% RPMI 1640 medium. The tolerogenic macrophage-enriched cell population can be used in various screening assays and cultures, as described below.

[0284] Macrophages can optionally be further purified by sorting fluorescently labeled cells using antibodies against macrophage markers. Macrophages may also be isolated using antibodies against macrophages, which are linked to magnetic beads.

[0285] The separated cells can be collected in any suitable medium that maintains cell viability, usually with a cushion of serum at the bottom of the collection tube. Various media are commercially available and can be used depending on the nature of the cells. The medium can be liquid or semi-solid, including, for example, agar, methylcellulose, etc. The cell population can be appropriately suspended in an appropriate nutrient medium, such as Iscove's modified dMEM, HBSS, dPBS, RPMI, Iscove's medium, DMEM, or RPMI-1640, typically supplemented with fetal bovine serum (approximately 5-10%), L-glutamine, thiols (especially 2-mercaptoethanol), and antibiotics, such as penicillin and streptomycin.

[0286] Optionally, the presence of macrophages can be confirmed using standard techniques such as morphological observation and immunochemical staining. For example, macrophage purity can be assessed by flow cytometry using fluorochrome-labeled antibodies against one or more characteristic cell surface markers.

[0287] All embodiments disclosed so far for this aspect of the invention may also be applied to this aspect.

[0288] Tolerogenic dendritic cells of the invention obtained using the polypeptides, homo-oligomers, polynucleotides and vectors of the invention The transcriptome profile of tolerogenic dendritic cells is known to vary significantly depending on the approach used to generate them (Navarro-Barriuso et al. 2018. Frontiers in Immunology, 9: 2062).

[0289] Thus, in another aspect, the present invention relates to a tolerogenic dendritic cell of the present invention, which is obtained by differentiating and / or maturing dendritic cells by the method of the present invention in the presence of a polypeptide of the present invention, a homo-oligomer of the present invention, a polynucleotide encoding either the polypeptide or the homo-oligomer, or a vector comprising the polynucleotide, preferably a tolerogenic dendritic cell of the present invention, which is obtained by the method of the present invention in the presence of a homo-oligomer of the present invention, a polynucleotide encoding the homo-oligomer, or a vector comprising the polynucleotide, preferably in the presence of a homo-oligomer of the present invention.

[0290] The tolerogenic dendritic cells of the present invention are characterized by exhibiting one or more of the following characteristics: - Cells are CD83 - , CD86 - , CD80 - , CD40 - and / or CCR7 - The term "negative," when applied to a given marker, indicates that the expression level of a particular cell surface marker on tolerogenic DCs produced by the methods of the invention is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% reduced or undetectable compared to the expression level of the same cell surface marker on appropriate control cells (e.g., naturally occurring mature immunostimulatory DCs or mature immunostimulatory DCs obtained via in vitro maturation, preferably LPS maturation). - if immature, the cells exhibit increased antigen internalization capacity or endocytic activity compared to immature dendritic cells. As used herein, the terms "increased antigen internalization capacity" or "increased endocytic activity" refer to a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 100%, 200%, 300% or more increase in the level of antigen internalization or endocytic activity of immature tolerogenic DCs produced by the methods of the invention compared to the level of antigen internalization capacity or endocytic activity of suitable control cells (e.g., natural immature DCs or immature DCs obtained via in vitro differentiation). The antigen internalization capacity or endocytic activity of tolerogenic dendritic cells can be determined by measuring fluorescent DQ-OVA internalization and processing during differentiation, for example, as described in Materials and Methods, Example 2 and Figure 5 of the present invention. - the cells secrete no or reduced amounts of proinflammatory cytokines, such as IL-12p70 and / or TNF-α, compared to mature dendritic cells. The term "secretes reduced amounts," when applied to a given cytokine, indicates that the secretion level of a particular cytokine in the tolerogenic DCs produced by the methods of the invention is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% reduced or undetectable compared to the secretion level of the same cytokine in an appropriate control cell (e.g., a naturally occurring mature immunostimulatory DC or a mature immunostimulatory DC obtained via in vitro maturation, preferably LPS maturation). -HLA-DR cells + and / or CD14 + The term "positive" as applied to a given marker indicates that the expression level of the particular cell surface marker in the tolerogenic DCs produced by the methods of the invention is substantially equivalent to that of an appropriate control cell (e.g., a naturally occurring mature immunostimulatory DC or a mature immunostimulatory DC obtained via in vitro maturation, preferably LPS maturation). The cells exhibit reduced chemotactic behavior toward CCL21 compared to mature dendritic cells. As used herein, the term "reduced chemotactic behavior" refers to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% reduction or undetectable level of chemotaxis toward CCL21 in tolerogenic DCs produced by the methods of the present invention compared to the level of chemotaxis toward the same cytokine in appropriate control cells (e.g., natural mature immunostimulatory DCs or mature immunostimulatory DCs obtained via in vitro maturation, preferably LPS maturation). The chemotactic behavior of tolerogenic dendritic cells toward CCL21 can be determined, for example, as described in Materials and Methods of the present invention, Example 7, and Figure 12. When immature, the cells exhibit reduced expression at the transcriptional level of PTGER3 and / or EGLN3 compared to immature DCs. The term "reduced expression," when applied to a given gene, indicates that the expression level of a particular gene in tolerogenic immature DCs produced by the methods of the present invention is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% or is undetectable compared to the expression level of the same gene in appropriate control cells (e.g., natural immature DCs or immature DCs obtained via in vitro differentiation). Gene expression levels can be determined, for example, as described in Materials and Methods, Example 8, and Figure 13 of the present invention. In a preferred embodiment, expression is measured on day 4 or 5 of differentiation. When immature, the cells exhibit increased transcriptional expression of olfactory receptors such as "find me" or FPR2, P2RY2, and / or S1PR1, compared to immature dendritic cells. The term "increased expression," when applied to a given gene, indicates that the expression level of a particular gene in tolerogenic immature DCs produced by the methods of the present invention is increased by 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 100%, 200%, 300%, or more, compared to the expression level of the same gene in appropriate control cells (e.g., natural immature DCs or immature DCs obtained via in vitro differentiation). Gene expression levels can be determined, for example, as described in Materials and Methods, Example 8, and Figure 14 of the present invention. In a preferred embodiment, expression is measured on day 5 of differentiation. and / or - The cells remain viable after the differentiation / maturation process, i.e., do not undergo apoptosis. As used herein, the term "viable" refers to a population in which less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% undergo apoptosis after treatment with a maturation stimulus (e.g., LPS). Apoptosis can be measured by any method known in the art, such as Annexin V / 7-ADD staining, caspase-3 activation assay, TUNEL and DNA fragmentation assay, or measurement of mitochondrial membrane potential. Cell viability can be determined, for example, as described in the Materials and Methods of the present invention and Example 2.

[0291] Thus, in a preferred embodiment, the tolerogenic dendritic cells of the present invention are characterized by exhibiting one or more of the characteristics described above.

[0292] The present inventors found that several "find me" receptors, particularly FPR2, P2RY2, and S1PR1, are upregulated at the transcriptional level in immature tolerogenic dendritic cells obtained by the method of the present invention relative to immature dendritic cells. However, when tolerogenic dendritic cells are obtained by other methods, for example, by incubation with the physiological isoform of C4BP (C4BP(α7β0)), which is formed by seven α chains and lacks a β chain (designated PRP-HO7), the above genes are not overexpressed (see FIG. 14).

[0293] Thus, in a preferred embodiment, the tolerogenic dendritic cells of the present invention are characterized by exhibiting overexpression of one or more of the genes FPR2, P2RY2 and / or S1PR1 when compared to immature dendritic cells.

[0294] Therefore, in a preferred embodiment, the tolerogenic dendritic cells of the present invention are FPR2 high , P2RY2 high and / or S1PR1 high In one embodiment, the tolerogenic dendritic cells of the present invention are FPR2 high In another embodiment, the tolerogenic dendritic cells of the present invention are P2RY2 high In another embodiment, the tolerogenic dendritic cells of the present invention are S1PR1 high In another embodiment, the tolerogenic dendritic cells of the present invention are high and P2RY2 high In another embodiment, the tolerogenic dendritic cells of the present invention are high and S1PR1 high In another embodiment, the tolerogenic dendritic cells of the present invention are high and S1PR1 high In a more preferred embodiment, the tolerogenic dendritic cells of the present invention are FPR2 high , P2RY2 high and S1PR1 high is.

[0295] The term "high" when applied to a given gene indicates that the expression level of a particular gene in tolerogenic DCs produced by the methods of the present invention is increased by 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 100%, 200%, 300% or more compared to the expression level of the same gene in appropriate control cells (i.e., natural immature DCs or immature DCs obtained via in vitro differentiation); i.e., the gene is overexpressed compared to immature DCs.

[0296] In another embodiment, the present invention relates to a cell population comprising tolerogenic dendritic cells of the present invention. In a preferred embodiment, the cell population comprises at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% tolerogenic dendritic cells of the present invention. In a preferred embodiment, the cell population comprises at least 80% tolerogenic dendritic cells of the present invention.

[0297] All embodiments disclosed so far for this aspect of the invention may also be applied to this aspect.

[0298] Tolerogenic macrophages of the invention obtained using the polypeptides, homo-oligomers, polynucleotides and vectors of the invention The transcriptome profile of tolerogenic macrophages is known to vary significantly depending on the approach used to generate them (Navarro-Barriuso et al. 2018. Frontiers in Immunology, 9: 2062).

[0299] Thus, in another aspect, the present invention relates to a tolerogenic macrophage of the present invention, which is obtained by differentiating and / or maturing a macrophage by the method of the present invention in the presence of a polypeptide of the present invention, a homo-oligomer of the present invention, a polynucleotide encoding either the polypeptide or the homo-oligomer, or a vector comprising the polynucleotide, preferably a tolerogenic macrophage of the present invention, which is obtained by the method of the present invention in the presence of a homo-oligomer of the present invention, a polynucleotide encoding the homo-oligomer, or a vector comprising the polynucleotide, preferably in the presence of the homo-oligomer of the present invention.

[0300] Tolerogenic macrophages according to the present invention are characterized in that they exhibit one or more of the following characteristics: - Cells are CD64 - and / or CD80 - The term "negative," as applied to the CD64 and CD80 markers, indicates that the expression levels of these particular cell surface markers in the tolerogenic macrophages produced by the methods of the invention are reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% or are undetectable compared to the expression levels of the same cell surface markers in appropriate control cells (e.g., naturally occurring mature M1 macrophages or mature M1 macrophages obtained via in vitro maturation). -Cells are CD209 - and / or CD11b -The term "negative," as applied to the CD209 and CD11b markers, indicates that the expression levels of these particular cell surface markers in the tolerogenic macrophages produced by the methods of the present invention are reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% or are undetectable compared to the expression levels of the same cell surface markers in appropriate control cells (e.g., naturally occurring mature M2 macrophages or mature M2 macrophages obtained via in vitro maturation). When immature, the cells exhibit reduced expression at the transcriptional level of ALCAM, SLC16A1, and / or LMNB1 compared to immature M0 macrophages. The term "reduced expression," when applied to a given gene, indicates that the expression level of a particular gene in the tolerogenic macrophages produced by the methods of the present invention is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% or is undetectable compared to the expression level of the same gene in appropriate control cells (e.g., natural immature M0 macrophages or immature M0 macrophages obtained via in vitro differentiation). Gene expression levels can be determined, for example, as described in Materials and Methods, Example 10, and Figure 16. In a preferred embodiment, expression is measured on days 2, 4, or 6 of differentiation. When immature, the cells exhibit increased transcriptional expression of olfactory receptors such as "find me" or CCR2, CX3CR1, CXCR1, FPR2, and / or P2RY2, compared to immature M0 macrophages. The term "increased expression," when applied to a given gene, indicates that the expression level of a particular gene in the tolerogenic macrophages produced by the methods of the present invention is increased by 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 100%, 200%, 300% or more compared to the expression level of the same gene in appropriate control cells (e.g., natural immature M0 macrophages or immature M0 macrophages obtained via in vitro differentiation). Gene expression levels can be determined, for example, as described in Materials and Methods, Example 10, and Figure 17. In a preferred embodiment, expression is measured on day 6 of differentiation. - the cells secrete reduced amounts of inflammatory cytokines and chemokines, such as CCL1, CCL2, MIP-1, CCL5, CXCL1, IL-6, and / or TNF-α, compared to mature M1 macrophages. When applied to a given cytokine, chemokine, or acute phase protein, the term "secretes reduced amounts" indicates that the secretion level of a particular cytokine or chemokine in the tolerogenic macrophages produced by the methods of the invention is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% reduced or undetectable compared to the secretion level of the same cytokine or chemokine in an appropriate control cell (e.g., a naturally occurring mature M1 macrophage or a mature M1 macrophage obtained via in vitro maturation).

[0301] Thus, in a preferred embodiment, the tolerogenic macrophages of the present invention are characterized by exhibiting one or more of the characteristics described above.

[0302] In a preferred embodiment, the tolerogenic macrophages of the present invention exhibit reduced expression of ALCAM, SLC16A1, and / or LMNB1 at the transcriptional level compared to immature macrophages M0. In a more preferred embodiment, the tolerogenic macrophages of the present invention exhibit reduced expression of ALCAM at the transcriptional level compared to immature macrophages M0, preferably on day 2, 4, or 6 of differentiation, more preferably on day 2 of differentiation. In another embodiment, the tolerogenic macrophages of the present invention exhibit reduced expression of SLC16A1 at the transcriptional level compared to immature macrophages M0, preferably on day 4 or 6 of differentiation. In another embodiment, the tolerogenic macrophages of the present invention exhibit reduced expression of LMNB1 at the transcriptional level compared to immature macrophages M0, preferably on day 2 or 4 of differentiation.

[0303] In a preferred embodiment, the tolerogenic macrophages of the present invention are CD64 - and / or CD80 - In a preferred embodiment, tolerogenic macrophages obtained by differentiating monocytes into M0 in the presence of the homo-oligomer of the present invention and further maturing the M0 with LPS and IFNγ are CD64 - and / or CD80 - In another preferred embodiment, the tolerogenic macrophages of the present invention secrete reduced amounts of CCL1, CCL2, MIP-1, CCL5, CXCL1, IL-6, and / or TNF-α compared to mature M1 macrophages. In a preferred embodiment, the tolerogenic macrophages of the present invention secrete reduced amounts of CCL1, CCL2, and / or MIP-1. In a more preferred embodiment, the tolerogenic macrophages of the present invention secrete reduced amounts of CCL2 compared to mature M1 macrophages.

[0304] In a preferred embodiment, the tolerogenic macrophages of the present invention are CD209 - and / or CD11b -In a preferred embodiment, tolerogenic macrophages obtained by differentiating monocytes into M0 in the presence of the homo-oligomer of the present invention and then maturing the M0 with IL-4 are CD209 - and / or CD11b - is.

[0305] The CD64 and CD11b cell surface markers are further underexpressed when the tolerogenic macrophages are obtained by the methods of the present invention than when the tolerogenic macrophages are obtained by treatment with the physiological isoform C4BP (α7β0) (see Figure 15). Thus, in one embodiment, the tolerogenic macrophages of the present invention are CD64 - and preferably the expression level of CD64 is reduced to 25% compared to the expression level in M1 macrophages. - and preferably the expression level of CD11b is reduced to 18% compared to the expression level in M2 macrophages.

[0306] The tolerogenic macrophages of the present invention secrete even less CCL1 and MIP-1 than tolerogenic macrophages obtained by treatment with the physiological isoform C4BP(α7β0) (see Figure 18). Thus, in one embodiment, the tolerogenic macrophages of the present invention secrete a reduced amount of CCL1 compared to mature M1 macrophages, preferably the expression level of CCL1 is reduced by 70% compared to the expression level in M1 macrophages. In another embodiment, the tolerogenic macrophages of the present invention secrete a reduced amount of MIP-1 compared to mature M1 macrophages, preferably the expression level of MIP-1 is reduced by 63% compared to the expression level in M1 macrophages.

[0307] The present inventors found that several "find me" receptors, particularly CCR2, CX3CR1, CXCR1, FPR2, and P2RY2, are upregulated at the transcriptional level in tolerogenic macrophages obtained by the method of the present invention relative to immature M0 macrophages. However, when tolerogenic macrophages are obtained by other methods, such as incubation with the physiological isoform of C4BP (C4BP(α7β0)) (designated PRP-HO7), which is composed of seven α chains and lacks β chains (designated PRP-HO7), the above genes are not overexpressed (see FIG. 17).

[0308] Thus, in a preferred embodiment, the tolerogenic macrophages of the present invention are characterized by exhibiting overexpression of one or more of the genes CCR2, CX3CR1, CXCR1, FPR2 and / or P2RY2 when compared to immature macrophages M0.

[0309] Thus, in a preferred embodiment, the tolerogenic macrophages of the present invention are CCR2 high , CX3CR1 high , CXCR1 high , FPR2 high and / or P2RY2 high In one embodiment, the tolerogenic macrophages of the present invention are CCR2 high In another embodiment, the tolerogenic macrophages of the present invention are CX3CR1 high In another embodiment, the tolerogenic macrophages of the present invention are CXCR1 high In another embodiment, the tolerogenic macrophage of the present invention is FPR2 high In another embodiment, the tolerogenic macrophages of the present invention are P2RY2 high In another embodiment, the tolerogenic macrophages of the present invention are CCR2 high and CX3CR1 high In another embodiment, the tolerogenic macrophages of the present invention are CCR2 high and CXCR1 highIn another embodiment, the tolerogenic macrophages of the present invention are CCR2 high and FPR2 high In another embodiment, the tolerogenic macrophages of the present invention are CCR2 high and P2RY2 high In another embodiment, the tolerogenic macrophage of the present invention is CX3CR1 high and CXCR1 high In another embodiment, the tolerogenic macrophage of the present invention is CX3CR1 high and FPR2 high In another embodiment, the tolerogenic macrophage of the present invention is CX3CR1 high and P2RY2 high In another embodiment, the tolerogenic macrophage of the present invention is CXCR1 high and FPR2 high In another embodiment, the tolerogenic macrophage of the present invention is CXCR1 high and P2RY2 high In another embodiment, the tolerogenic macrophage of the present invention is FPR2 high and P2RY2 high In another embodiment, the tolerogenic macrophages of the present invention are CCR2 high , CX3CR1 high , CXCR1 high In another embodiment, the tolerogenic macrophages of the present invention are CCR2 high , CX3CR1 high and FPR2 high In another embodiment, the tolerogenic macrophages of the present invention are CCR2 high , CX3CR1 high and P2RY2 high In another embodiment, the tolerogenic macrophages of the present invention are CCR2 high , CXCR1 high , FPR2 high In another embodiment, the tolerogenic macrophages of the present invention are CCR2 high , CXCR1 high and P2RY2high In another embodiment, the tolerogenic macrophages of the present invention are CCR2 high , FPR2 high and / or P2RY2 high In another embodiment, the tolerogenic macrophage of the present invention is CX3CR1 high , CXCR1 high and FPR2 high In another embodiment, the tolerogenic macrophage of the present invention is CX3CR1 high , CXCR1 high and P2RY2 high In another embodiment, the tolerogenic macrophage of the present invention is CX3CR1 high , FPR2 high and P2RY2 high In another embodiment, the tolerogenic macrophage of the present invention is CXCR1 high , FPR2 high and P2RY2 high In another embodiment, the tolerogenic macrophages of the present invention are CCR2 high , CX3CR1 high , CXCR1 high and FPR2 high In another embodiment, the tolerogenic macrophages of the present invention are CCR2 high , CX3CR1 high , CXCR1 high and P2RY2 high In another embodiment, the tolerogenic macrophage of the present invention is CX3CR1 high , CXCR1 high , FPR2 high and P2RY2 high In another embodiment, the tolerogenic macrophages of the present invention are CCR2 high , CX3CR1 high , FPR2 high and P2RY2 high In a more preferred embodiment, the tolerogenic macrophages of the present invention are CCR2 high , CX3CR1 high , CXCR1 high , FPR2high and P2RY2 high is.

[0310] The term "high" as applied to a given gene indicates that the expression level of a particular gene in tolerogenic macrophages produced by the methods of the invention is increased by 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 100%, 200%, 300% or more compared to the expression level of the same gene in an appropriate control cell (e.g., a naturally occurring immature M0 macrophage or an immature M0 macrophage obtained via in vitro differentiation), i.e., the gene is overexpressed compared to immature M0 macrophages.

[0311] In another embodiment, the present invention relates to a cell population comprising the tolerogenic macrophages of the present invention. In a preferred embodiment, the cell population comprises at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% tolerogenic macrophages of the present invention. In a preferred embodiment, the cell population comprises at least 80% tolerogenic macrophages of the present invention.

[0312] All embodiments disclosed so far for this aspect of the invention may also be applied to this aspect.

[0313] Therapeutic Uses of the Tolerogenic Dendritic Cells and Tolerogenic Macrophages of the Invention Tolerogenic dendritic cells and tolerogenic macrophages are useful in treating patients with immunological diseases, particularly autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, and Crohn's disease, as well as in kidney transplantation (Navarro-Barriuso et al. 2018. Frontiers in Immunology, 9: 2062).

[0314] Therefore, in another aspect, the present invention relates to a pharmaceutical composition comprising a tolerogenic dendritic cell of the invention, or a tolerogenic macrophage of the invention, or a cell population of the invention, and a pharmaceutically acceptable carrier.

[0315] All embodiments disclosed so far for this aspect of the invention, in particular all embodiments and definitions disclosed for the pharmaceutical composition of the invention, may also be applied to this aspect.

[0316] In another aspect, the present invention relates to the tolerogenic dendritic cells of the present invention, the tolerogenic macrophages of the present invention, the cell population of the present invention, or pharmaceutical compositions comprising them for use in medicine.

[0317] In another aspect, the present invention relates to the use of the tolerogenic dendritic cells of the present invention, the tolerogenic macrophages of the present invention, the cell population of the present invention, or a pharmaceutical composition comprising them for the manufacture of a medicament.

[0318] In another aspect, the present invention relates to the tolerogenic dendritic cells of the present invention, the tolerogenic macrophages of the present invention, the cell population of the present invention, or a pharmaceutical composition comprising them for use in the prevention and / or treatment of immunological diseases caused by undesired activation of the immune system.

[0319] In another aspect, the present invention relates to the use of the tolerogenic dendritic cells of the present invention, the tolerogenic macrophages of the present invention, the cell population of the present invention, or a pharmaceutical composition comprising them for the manufacture of a medicament for the prevention or treatment of an immunological disease caused by undesired activation of the immune system.

[0320] In another aspect, the present invention relates to a method for preventing and / or treating an immunological disease caused by unwanted activation of the immune system in a subject in need thereof, the method comprising administering to said subject the tolerogenic dendritic cells of the present invention, the tolerogenic macrophages of the present invention, the cell population of the present invention, or a pharmaceutical composition comprising them.

[0321] In a preferred embodiment, the immunological disease is selected from the group consisting of immunoinflammatory diseases, sepsis, autoimmune diseases, transplant rejection, graft-versus-host disease, and hypersensitivity diseases.

[0322] The dosage of the composition for treating immunological disease or disorder can be determined according to the parameters understood by those skilled in the medical field.Therefore, the appropriate dosage can depend on the condition of the patient (e.g., human), that is, the stage of the disease, general health condition, and age, sex and weight, and other factors known to those skilled in the medical field.

[0323] The terms "treatment," "prevention," "target," "immunological disease caused by unwanted activation of the immune system," "immunoinflammatory disease," "sepsis," "autoimmune disease," "graft rejection," "graft-versus-host disease," and "hypersensitivity disease" have been described in detail above and are used interchangeably with respect to the treatment methods of the present invention.

[0324] All embodiments disclosed so far for this aspect of the invention, in particular all embodiments and definitions disclosed for the treatment methods of the invention, may also be applied to this aspect.

[0325] The present invention also relates to: [1] A recombinant polypeptide comprising the CCP6 domain of the C4BP α chain or a functionally equivalent variant thereof, and an oligomerization domain, wherein the polypeptide does not contain any of the CCP1, CCP2, CCP3, CCP4, CCP5, CCP7 and CCP8 domains of the C4BP α chain. [2] The recombinant polypeptide according to [1], wherein the CCP6 domain is the CCP6 domain of the human C4BP α chain. [3] A recombinant polypeptide according to either [1] or [2], wherein the CCP6 domain of the C4BP α chain is SEQ ID NO: 1. [4] The recombinant polypeptide according to any one of [1] to [3], wherein the oligomerization domain is the oligomerization domain of the C4BP α chain or a functionally equivalent mutant thereof. [5] The recombinant polypeptide according to any one of [1] to [4], wherein the oligomerization domain is the oligomerization domain of the human C4BP α chain or a functionally equivalent mutant thereof. [6] The recombinant polypeptide according to any one of [1] to [5], wherein the oligomerization domain is SEQ ID NO: 2 or a functionally equivalent variant thereof, preferably SEQ ID NO: 2. [7] A recombinant polypeptide according to any one of [1] to [6], comprising, from the amino terminus to the carboxy terminus, (a) a CCP6 domain, and (b) an oligomerization domain. [8] The recombinant polypeptide according to any one of [1] to [7], wherein the polypeptide consists of SEQ ID NO: 3 or a functionally equivalent variant thereof, preferably SEQ ID NO: 3. [9] The recombinant polypeptide according to any one of [1] to [8], wherein the polypeptide further comprises a signal peptide.

[10] The recombinant polypeptide according to [9], wherein the signal peptide is the signal peptide of the C4BP α chain, preferably the peptide of SEQ ID NO: 4 or a functionally equivalent variant thereof, preferably SEQ ID NO: 4.

[11] The recombinant polypeptide according to

[10] , wherein the polypeptide consists of SEQ ID NO: 5 or a functionally equivalent variant thereof, preferably SEQ ID NO: 5.

[12] A recombinant polypeptide according to any one of [1] to

[11] , further comprising a peptide that is not a part of the C4BP α chain, preferably a tag peptide.

[13] The recombinant polypeptide according to

[12] , wherein the tag peptide is linked to the N-terminus of the CCP6 domain.

[14] The recombinant polypeptide according to

[12] or

[13] , wherein the tag peptide is a polyhistidine tag.

[15] The recombinant polypeptide according to

[14] , wherein the polypeptide consists of SEQ ID NO: 6 or a functionally equivalent variant thereof, preferably SEQ ID NO: 6.

[16] The recombinant polypeptide according to

[14] , wherein the polypeptide consists of SEQ ID NO: 7 or a functionally equivalent variant thereof, preferably SEQ ID NO: 7.

[17] A homo-oligomer of at least six recombinant polypeptides according to any one of [1] to

[16] .

[18] The homo-oligomer according to

[17] , wherein the homo-oligomer is formed by seven recombinant polypeptides according to any one of [1] to

[16] .

[19] The homo-oligomer according to

[17] , which consists of seven recombinant polypeptides according to any one of [1] to

[16] .

[20] The homo-oligomer according to any one of

[17] to

[19] , wherein the polypeptide is the polypeptide according to

[15] .

[21] A polynucleotide encoding the recombinant polypeptide according to any one of [1] to

[16] .

[22] A vector comprising the polynucleotide described in

[21] .

[23] A host cell containing the vector described in

[22] .

[24] A pharmaceutical composition comprising a recombinant polypeptide according to any one of [1] to

[16] , a homo-oligomer according to any one of

[17] to

[20] , a polynucleotide according to

[21] , a vector according to

[22] , or a host cell according to

[23] , and a pharmaceutically acceptable carrier.

[25] A recombinant polypeptide according to any one of [1] to

[16] , a homo-oligomer according to any one of

[17] to

[20] , a polynucleotide according to

[21] , a vector according to

[22] , a host cell according to

[23] , or a pharmaceutical composition according to

[24] , for use in medicine.

[26] A recombinant polynucleotide according to any one of [1] to

[16] , a homo-oligomer according to any one of

[17] to

[20] , a polynucleotide according to

[21] , a vector according to

[22] , a host cell according to

[23] , or a pharmaceutical composition according to

[24] , for use in the prevention and / or treatment of an immunological disease caused by undesirable activation of the immune system.

[27] A pharmaceutical composition comprising a recombinant polypeptide according to any one of [1] to

[16] , a homo-oligomer according to any one of

[17] to

[20] , a polynucleotide according to

[21] , a vector according to

[22] , a host cell according to

[23] , or

[24] , for use according to

[26] , wherein the immunological disease is selected from the group consisting of immunoinflammatory diseases, sepsis, autoimmune diseases, graft rejection, graft-versus-host disease, and hypersensitivity diseases.

[28] The recombinant polypeptide according to any one of [1] to

[16] , the homo-oligomer according to any one of

[17] to

[20] , the polynucleotide according to

[21] , the vector according to

[22] , the host cell according to

[23] , or the pharmaceutical composition according to

[24] , for use according to

[27] , wherein the autoimmune disease is selected from the group consisting of lupus nephritis, systemic lupus erythematosus, inflammatory bowel disease, and rheumatoid arthritis.

[29] A recombinant polypeptide according to any one of [1] to

[16] , a homo-oligomer according to any one of

[17] to

[20] , a polynucleotide according to

[21] , a vector according to

[22] , a host cell according to

[23] , or a pharmaceutical composition according to

[24] , for use according to

[28] , wherein the autoimmune disease is inflammatory bowel disease.

[30] A method for generating a population of tolerogenic dendritic cells in vitro, comprising: (i) incubating a population of dendritic precursor cells under conditions suitable for the formation of a population of immature dendritic cells; and (ii) incubating the population of immature dendritic cells obtained in step (i) under conditions suitable for the formation of mature dendritic cells. Including, Steps (i) and / or (ii) (a) the polypeptide according to any one of [1] to

[16] ; (b) the homo-oligomer according to any one of

[17] to

[20] ; (c) the polynucleotide according to

[21] , and (d) The vector described in

[22] A method carried out in the presence of a composition of matter selected from the group consisting of:

[31] The method according to

[30] , wherein the population of dendritic precursor cells is a monocyte population.

[32] A method for generating a population of tolerogenic macrophages in vitro, comprising: (i) incubating a population of macrophage progenitor cells under conditions suitable for the formation of a population of immature macrophages; and (ii) incubating the population of immature macrophages obtained in step (i) under conditions suitable for the formation of mature macrophages. Including, Steps (i) and / or (ii) (a) the polypeptide according to any one of [1] to

[16] ; (b) the homo-oligomer according to any one of

[17] to

[20] ; (c) the polynucleotide according to

[21] , and (d) Vector described in

[22] A method carried out in the presence of a composition of matter selected from the group consisting of:

[33] The method according to

[32] , wherein the population of macrophage precursor cells is a monocyte population. Tolerogenic dendritic cells obtained by the method described in either

[34]

[30] or

[31] , or tolerogenic macrophages obtained by the method described in either

[32] or

[33] .

[35] The tolerogenic dendritic cells according to

[34] , wherein the cells are FPR2 high , P2RY2 high and / or S1PR1 high , tolerogenic dendritic cells.

[36] The tolerogenic macrophage according to

[34] , wherein the macrophage is CCR2 high , CX3CR1 high , CXCR1 high , FPR2 high and / or P2RY2 high , tolerogenic macrophages.

[37] A cell population comprising at least 80% tolerogenic dendritic cells as described in

[34] or

[35] , or at least 80% tolerogenic macrophages as described in

[34] or

[36] .

[38] A pharmaceutical composition comprising the tolerogenic dendritic cells described in

[34] or

[35] , the tolerogenic macrophages described in

[34] or

[36] , or the cell population described in

[37] , and a pharmaceutically acceptable carrier.

[39] A tolerogenic dendritic cell according to

[34] or

[35] , a tolerogenic macrophage according to

[34] or

[36] , a cell population according to

[37] , or a pharmaceutical composition according to

[38] for use in medicine.

[40] A tolerogenic dendritic cell according to

[34] or

[35] , a tolerogenic macrophage according to

[34] or

[36] , a cell population according to

[37] , or a pharmaceutical composition according to

[38] for use in the prevention and / or treatment of an immunological disease caused by undesired activation of the immune system.

[41] The tolerogenic dendritic cells described in

[34] or

[35] , the tolerogenic macrophages described in

[34] or

[36] , the cell population described in

[37] , or the pharmaceutical composition described in

[38] for use according to

[41] , wherein the immunological disease is selected from the group consisting of immunoinflammatory diseases, sepsis, autoimmune diseases, graft rejection, graft-versus-host disease, and hypersensitivity diseases.

[42] A method for increasing a tolerogenic dendritic cell population and / or a tolerogenic macrophage population in a subject in need thereof, comprising administering to the subject a polypeptide according to any one of [1] to

[16] , a homo-oligomer according to any one of

[17] to

[20] , a polynucleotide according to

[21] , a vector according to

[22] , a host cell according to

[23] , or a pharmaceutical composition according to

[24] , or a combination thereof. [Example]

[0326] The present invention will be described in detail by the following examples, which should not be construed as limiting the scope of the invention but should be regarded as merely illustrative.

[0327] Examples 1-13 material and method Obtaining and purifying PRP-HE8, PRP-HO7, PRP6-HO7 and PRP6-NO PRP-HE8 is a physiological isoform (C4BP(β)) composed of seven α chains and one β chain purified from human plasma. + )) and is also called C4BP(α7β1) (Olivar et al. 2013. J Immunol, 190(6): 2857-2872).

[0328] PRP-HO7 is a physiological isoform (C4BP(β)) purified from human plasma, which is formed by seven α chains but lacks the β chain. - )) and is also called C4BP(α7β0) (Olivar et al. 2013. J Immunol, 190(6): 2857-2872).

[0329] PRP6-HO7 is a homo-oligomer of seven recombinant polypeptides, each of which is a C4BP (β - ) CCP6 domain and its C-terminus engineered C4BP (β - The immature form of the recombinant polypeptide that forms PRP6-HO7 was formed by fusing the complete oligomerization domain of C4BP (β4) to the mature form of PRP6-HO7. The sequence of the immature form of the recombinant polypeptide that forms PRP6-HO7 is SEQ ID NO:7 and is shown in FIG. 1A. The immature form of the recombinant polypeptide that forms PRP6-HO7 also contained the signal peptide of C4BP, which is excluded in the mature form. The sequence of the mature form of PRP6-HO7 is SEQ ID NO:6 and has a theoretical pI of 5.81 and a theoretical Mw of 14,287.98 Da. The sequence of SEQ ID NO:6 is shown below, with the first six amino acids underlined and corresponding to the histidine tag, followed by the C4BP (β4) signal peptide. - C4BP (β - ) is followed by the sequence of the CCP6 domain, which is double underlined. HHHHHH LCCPEPKLNNGEITQHRKSRPANHCVYFYGDEISFSCHETSRFSAICQGDGTWSPRTPSCGD ETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKEL (SEQ ID NO: 6)

[0330] PRP6-NO inhibits C4BP(β - ) and a mutant C4BP (β) that cannot oligomerize by substituting two cysteine ​​residues with alanine at its C-terminus and deleting the last 13 C-terminal amino acids (Δ537-549 / C498A / C510A). - ) (Kask et al. 2002. Biochemistry, 41:9349-9357). This recombinant polypeptide was engineered by the present inventors. The sequence of the immature form of PRP6-NO is SEQ ID NO: 10 and is shown in Figure 1B. The immature form of PRP6-NO also contained the signal peptide of C4BP, which is excluded in the mature form. The sequence of the mature form of PRP6-NO is SEQ ID NO: 8, and has a theoretical pI of 5.80 and a theoretical Mw of 12,723.23 Da. The sequence of SEQ ID NO: 8 is shown below, with the first six amino acids underlined and corresponding to the histidine tag, followed by the C4BP (β - ) fused to a truncated mutant of C4BP(β - ) is followed by the sequence of the CCP6 domain, which is double underlined. The two residues in the oligomerization domain that were mutated to alanine are shown in bold.

[0331] MHPPKTPSGALHRKRKMAAWPFSRLWKVSDPILFQMTLIAALLPAVLGHHHHHHLCCPEPKLNNGEITQHRKSRPANHCVYFYGDEISFSCHETSRFSAICQGDGTWSPRTPSCGDETPEGAEQVLTGKRLMQALPNPEDVKMALEVYKLSLEIEQLELQ (SEQ ID NO: 10)

[0332] In a preferred embodiment, the polynucleotide encoding SEQ ID NO:10 is SEQ ID NO:11.

[0333] HHHHHH LCCPEPKLNNGEITQHRKSRPANHCVYFYGDEISFSCHETSRFSAICQGDGTWSPRTPSCGD ETPEGAEQVLTGKRLMQALPNPEDVKMALEVYKLSLEIEQLELQ (SEQ ID NO: 8)

[0334] A six-histidine tag was added to the N-terminus of PRP6-HO7 and PRP6-NO to improve purification and detection, and their production was confirmed by SDS-PAGE and peptide mapping analysis (mass spectrometry).

[0335] PRP-HE8 (C4BP(β)) from pooled human plasma supplied by a local blood bank + )) and PRP-HO7 (C4BP(β - Purification involved BaCl precipitation (Dahlback et al. (1983) Biochem. J. 209: 847-856; Blom et al. (2009) Ann. Rheum. Dis. 68: 136-142), followed by four sequential chromatographic steps: heparin chromatography, hydrophobic interaction (butyl) chromatography, anion exchange (Q Sepharose) chromatography, and finally size exclusion (Superdex) chromatography. Both plasma-purified PRP-HE8 and PRP-HO7 were concentrated, dialyzed, and collected in PBS buffer at pH 7.4. The purity of both glycoproteins was greater than 85%, as assessed by Tris-acetate 3-8% SDS-PAGE (NuPAGE precast protein gels; ThermoFisher, Waltham, MA, USA) with 6 μg protein / lane and further Coomassie blue staining.

[0336] The DNA sequences of PRP6-HO7 and PRP6-NO (SEQ ID NOs: 9 and 11, respectively) were cloned into the pCDNA3.1(+) expression vector. Plasmid DNA was amplified and purified using Qiagen's Qiafilter Plasmid MegaKit. Expi293 cells were cultured at 2.5 x 10 6Suspension culture was grown to the desired volume at a cell density of 1000 cells / ml and 98% viability. Cells were transiently transfected with 1 mg of DNA per liter of culture medium and then incubated at 37°C for 7 days, shaking at 125 rpm and supplemented with 8% CO2. Finally, the medium was collected and centrifuged at 6000 g for 30 minutes at 4°C. The supernatant was subjected to protein purification by nickel affinity chromatography (HisTrap FF) according to standard procedures. Both recombinant polypeptides were concentrated, dialyzed, and recovered in PBS buffer at pH 7.4. Purity was assessed by SDS-PAGE and Coomassie blue staining.

[0337] (SEQ ID NO: 9)

[0338] ATGCACCCCCCAAAAACTCCATCTGGGGCTCTTCATAGAAAAAGGAAAATGGCAGCCTGGCCCTTCTCCAGGCTGTGGAAAGTCTCTGATCCAATTCTCTTCCAAATGACCTTGATCGCTGCTCTGTTGCCTGCTGTTCTTGGCCACCACCACCACCACCACTTATGTTGCCCTGAACCAAAGCTAAATAATGGTGAAATCACTCAACACAGGAAAAGTCGTCCTGCCAATCACTGTGTTTATTT CTATGGAGATGAGATTTCATTTTCATGTCATGAGACCAGTAGGTTTTCAGCTATATGCCAAGGAGATGGCACGTGGAGTCCCCGAACACCATCATGTGGAGACGAGACCCCCGAAGGCGCTGA ACAAGTGCTCACAGGCAAAAGACTCATGCAGGCTCTCCCAAACCCAGAGGATGTGAAAATGGCCCTGGAGGTATATAAGCTGTCTCTGGAAATTGAACAACTGGAACTACAGTAA (SEQ ID NO: 11)

[0339] Cell culture and PRP-based treatments Whole blood from healthy donors was obtained at the Blood and Tissue Bank (Barcelona, ​​Spain), and PBMCs were isolated after Ficoll-Paque density centrifugation (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Patients with lupus nephritis experiencing a flare from the nephrology ward of Bellvitge University Hospital underwent blood sampling upon admission, and PBMCs were obtained as described for healthy donors. This study was approved by the Ethics Committee of IDIBELL in accordance with institutional guidelines and the Declaration of Helsinki, and patients provided written informed consent.

[0340] Monocytes (Mo) were purified using colloidal superparamagnetic microbeads conjugated with a monoclonal mouse anti-human CD14 antibody (MACS, Miltenyi Biotec, Auburn, CA, or EasySep® Human CD14 Positive Selection Kit, StemCell Technologies, Grenoble, France) and counted using Perfect Count microspheres (Cytognos SL, Salamanca, Spain). CD14+ cell purity was assessed by CD14 staining and flow cytometry analysis (FACSCanto II flow cytometer with FACSDiva software (Becton Dickinson, Franklin Lakes, NJ)) to assess the number of PBMCs and total CD14+ cells (>90% CD14+).

[0341] Monocytes were cultured at 1 × 10 in 24-well culture plates (Jet Biofil, Guangzhou, China) in RPMI 1640 (Gibco, ThermoFisher, Waltham, MA, USA) supplemented with 100 mg / ml streptomycin, 100 IU / ml penicillin, 2 mM L-glutamine (all from Invitrogen, Carlsbad, CA), and 10% heat-inactivated FBS (Life Technologies, ThermoFisher, Carlsbad, CA) (complete medium) at 37°C under 5% CO . 6 Cells were seeded at 500 μl / well. Monocyte-derived DCs (MoDCs) were generated by supplementing monocyte cultures with complete RPMI 1640 medium (Gentaur, Kampenhout, Belgium) and GM-CSF (800 IU / ml) and IL-4 (500 IU / ml). Monocyte-derived undifferentiated macrophages (M0) were generated by incubation with GM-CSF (50 ng / ml) (Gentaur) or M-CSF (50 ng / ml) (MACS, Miltenyi Biotec, Auburn, CA) for 6 days.

[0342] PRP-based proteins were added to differentiating monocytes at the indicated concentrations on day 0. For DC maturation, untreated or PRP-based molecule-treated iDCs were stimulated with 5 μg / ml LPS (Escherichia coli 055.B5, Sigma-Aldrich, Merck, Darmstadt, Germany) or 10 μg / ml Gardiquimod (an imidazoquinoline compound; TLR7 ligand) (Invivogen, San Diego, CA) for an additional 48 hours on day 5. For M1 (classically activated) and M2 (alternatively activated) macrophage polarization, day 6 M0 cells were further incubated with LPS (40 ng / ml) (Sigma-Aldrich) and γ-IFN (40 ng / ml) (Invitrogen, Carlsbad, CA) for M1 or with IL-4 (40 ng / ml) (Invitrogen) for M2 for 2 days.

[0343] Furthermore, to evaluate the influence of human serum on PRP-HO7 and PRP6-HO7 immunomodulatory activity, human MoDCs were treated with the indicated concentrations of each PRP-based protein and co-cultured with 50% heat-inactivated human serum throughout their differentiation and maturation process (56°C, 1 h).

[0344] Antibodies and flow cytometry Cell surface phenotypes were analyzed using the following mAbs: FITC-conjugated anti-HLA-DR (Immu-357), FITC-conjugated anti-CD83 (HB15a) (Becton Dickinson, Franklin Lakes, NJ), FITC-conjugated anti-CD14 (TUK4), PE-conjugated anti-CD40 (HB14), PE-conjugated anti-CD80 (2D10), PE-conjugated anti-CD86 (FM95), PE-conjugated anti-CD11b (M1 / 70.15.11.5), APC-conjugated anti-CD64 (10.1.1), and APC-conjugated anti-CD209 (REA617) (Miltenyi Biotec, Bergisch Gladbach, Germany), and Alexa Fluor 488-conjugated anti-CCR7 (G043H7) (Biolegend, San Diego, CA). FITC-conjugated anti-IgG1 (IS5-21F5), FITC-conjugated anti-IgG2b (IS6-11E5.11), FITC-conjugated anti-IgG2a (S43.10), PE-conjugated anti-IgG2b (IS6-11E5.11), and APC-conjugated IgG1 (IS5-21F5) (Miltenyi Biotec), PE-conjugated anti-IgG1 (PPV-06) (EuroBioSciences GmbH, Friesoythe, Germany), and Alexa Fluor 488-conjugated anti-IgG2aκ (MOPC-173) (Biolegend) were used as isotype controls.

[0345] After washing with PBS, cells were incubated with 3 μl of MoAb / 10 in 100 ml of FACS buffer (PBS containing 1% BSA and 0.1% sodium azide). 5Cells were stained with PBS for 15 minutes at room temperature. To exclude debris, cells were gated according to forward scatter (FSC) and side scatter (SSC) parameters. They were also stained with 7-aminoactinomycin D (ThermoFischer, Carlsbad, CA) to assess their viability. Stained cells were analyzed using a FACSCanto II flow cytometer (Becton Dickinson). Subsequent analysis was performed using FlowJo software (Flowjo LLC, Ashland, OR).

[0346] Endocytic activity To measure endocytosis of iDCs, 2 x 10 5 Cells / ml were resuspended in 100 μl of PBS and incubated with 4 μl of BODIPY FL-conjugated DQ-ovalbumin (1 mg / ml, DQ-OVA, Molecular Probes, Leiden, Netherlands) at 37°C or 0°C for 30 min (receptor-mediated endocytosis). The incubation was stopped by adding 1 ml of cold FACS buffer. Cells were washed twice with cold FACS buffer and their fluorescence was analyzed using flow cytometry.

[0347] chemotaxis DCs differentiated and matured (LPS for 48 hours) in the presence of PRP-HE8, PRP-HO7, or PRP6-HO7 were tested for migration toward the CCL21 chemokine using a transwell assay. Briefly, the lower chamber of a transwell plate (8.0 μm pore size polycarbonate filter; Costar, Corning, NY) was filled with 400 μl of complete RPMI 1640 medium with or without CCL21 (200 ng / ml). A total of 1.6 × 10 DCs in 100 μl of complete RPMI 1640 medium were cultured. 4DCs were placed in the upper chamber and the cells were incubated at 37°C for 2 hours. Cells that migrated to the lower chamber were collected and counted using a flow cytometer, acquiring events over a 1.5-minute period. Migration assays for all stimulation conditions were performed in duplicate wells. Values ​​are presented as the total number of migrated cells.

[0348] Differential gene expression analysis Total RNA was extracted from untreated and PRP-HE8-, PRP-HO7-, and PRP6-HO7-treated iDC / M0 macrophages using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was performed using the High Capacity cDNA Archive Kit (Applied BioSystems, Carlsbad, CA). Selected gene transcripts were analyzed in individual samples by RT-qPCR using the corresponding inventoried TaqMan Gene Expression Assays (Applied BioSystems). Quantification was performed by the ΔΔCt method. Relative fold changes in mRNA abundance were calculated using the formula 2-ΔΔCt, using PPIA as the endogenous reference transcript.

[0349] DC cytokine secretion Human IL-12p70 and TNF-α concentrations were determined from DC supernatants treated with C4BP isoforms or PRP-based proteins using the respective DuoSet Elisa kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

[0350] SDS-PAGE and Western blot analysis PRP-based proteins were separated by 12% SDS-PAGE under reducing and non-reducing conditions. The gel was soaked in Blue Safe (NZYTech, Lisbon, Portugal) for 15 minutes and destained with water.

[0351] For Western blot analysis, PRP-based proteins were separated by 4-12% gradient SDS-PAGE (NuPAGE Bis-Tris 4-12% Mini Gels, Invitrogen) under reducing and non-reducing conditions and transferred to a PVDF membrane. After blocking, the membrane was probed with primary antibodies: polyclonal PK9008 rabbit anti-C4BP (gift from Anna Blom) at a 1:2000 dilution and 6xHis monoclonal antibody (Clontech, Mountain View, CA, USA) at a 1:4000 dilution, followed by polyclonal goat anti-rabbit IgG HRP (P0448, Dako, Agilent, Santa Clara, CA, USA) at a 1:2000 dilution and polyclonal goat anti-mouse IgG HRP (P0447, Dako, Agilent, Santa Clara, CA, USA) at a 1:2000 dilution, respectively. Detection was performed by enhanced chemiluminescence (ECL) (Pierce, ThermoFischer, Carlsbad, CA, USA) using a ChemiDoc Imager (Bio-Rad, Hercules, CA, USA).

[0352] C4b cofactor activity assay Complement C4b (8.9 μg / ml) and factor I (4.4 μg / ml) (Merck, Darmstadt, Germany) were mixed with C4BP isoforms (0.6 nM or 6 nM) in a total volume of 60 μl of low-salt buffer (25 mM phosphate buffer, pH 7.4 and 25 mM NaCl) and incubated at 37°C for 30 min. Reducing SDS sample buffer was added, and C4b fragments were separated by 4–12% gradient SDS-PAGE (NuPAGE Bis-Tris 4–12% Mini Gels, Invitrogen, ThermoFischer, Carlsbad, CA, USA). Western blot analysis of C4b fragments was performed using a 1:2000 dilution of anti-human C4d MoAb (Quidel, San Diego, CA, USA) followed by a 1:2000 dilution of polyclonal goat anti-mouse IgG HRP (Dako, Agilent Technologies, Santa Clara, CA, USA) in 0.05% TBS-Tween, 1% BSA, and 0.02% NaN3.

[0353] Cytokine Array Representative samples of untreated, PRP-HO7-treated, and PRP6-HO7-treated M1 macrophages (100 μl of cell supernatant) were analyzed using the Proteome Profiler Array "Human Cytokine Array Panel A" kit (R&D Systems) according to the manufacturer's instructions to determine the relative levels of 36 cytokines, chemokines, and acute-phase proteins. The density of each dot was quantified using Quantity One® software and displayed as the normalized mean pixel density for each cytokine and treatment of interest.

[0354] statistical analysis Statistical analysis and scientific graphing were performed using GraphPad Prism 5 software (GraphPad software, Inc., La Jolla, CA). Repeated-measures one-way ANOVA corrected for multiple comparisons using the Dunnett method was performed to contrast MFI and cell count data under different experimental conditions relative to the reference condition. Paired t-tests were used to compare cytokine levels. Relative gene expression levels between treated and untreated cells were analyzed using a one-sample t-test. Two-way ANOVA corrected for multiple comparisons using the Holm-Sidak method was applied to evaluate time-course experiments. Data are presented as mean + SD. In all cases, a P value of <0.05 was considered significant.

[0355] Example 1: Physicochemical characteristics of PRP6-HO7 PRP6-HO7 is derived from the physiological protein PRP-HO7, a minor isoform of the soluble complement regulator human C4BP. It combines the CCP6 domain of the α chain with the oligomerization domain in a spatial arrangement similar to that of PRP-HO7 (Figures 1 and 2). This allows the novel recombinant protein to have a small size (a compact 100 kDa symmetric unit consisting of seven CCP6 domain chains that bind to the C4BP oligomerization domain at their C-terminus and form a radial spider-like homo-oligomer smaller than antibodies), a stability similar to or even superior to that of antibodies in biological fluids (extended plasma half-life), and a cooperative, high-affinity interaction with potential surface receptors with Kds in the low nanomolar range (data not shown), which is thought to be related to the nanoclustering of specific receptors for immunomodulatory signaling. The C-terminal oligomerization scaffold is stabilized by intermolecular disulfide bonds and a layer of electrostatic interactions that contribute to the complex's exceptional thermodynamic stability (Hofmeyer et al. (2013) J. Mol. Biol. 425: 1302-1317). Furthermore, unlike its precursor, PRP-HO7, PRP6-HO7 is not glycosylated, allowing for production and further purification in a variety of expression systems, ranging from eukaryotic cells (shown below) to yeast or bacterial cells (data not shown).

[0356] Finally, due to its simplified structure containing only the CCP6 domain, administration of PRP6-HO7 does not increase susceptibility to infection compared to PRP-HO7. The PRP-HO7 α chain binds to surface proteins of various bacterial pathogens (Blom et al. (2004) Mol. Immunol. 40: 1333-1346), hijacks host complement regulatory factors, and subsequently downregulates complement activation. Therefore, the fact that PRP-HO7 binds to pathogens (mainly via CCP1–CCP4) as a bacterial virulence factor (Blom et al. (2004) Mol. Immunol. 40: 1333–1346) and to plasminogen (via CCP8) (Agarwal et al. (2015) J. Biol. Chem. 290: 18333–18342) is an essential aspect that must be taken into account while planning to use complement inhibitors as immunomodulators for autoimmune diseases.

[0357] In this regard, and to assess whether the CCP6 domain of C4BP retains its anti-inflammatory and immunomodulatory activity attributed to PRP-HO7 when used alone as a monomer or, conversely, in a heptameric oligomerized form resembling its physiological origin (Olivar et al. (2013) J. Immunol. 190: 2857-2872), PRP6-HO7 and PRP6-NO were produced in HEK293 cells and purified from their supernatants (Figure 3). As expected, as shown by both PAGE and Coomassie blue staining and Western blot analysis, PRP6-HO7 folded into a 100 kDa homo-oligomeric structure consisting of seven 14.3 kDa monomer chains, containing the CCP6 domain and the C-terminal oligomerization domain of C4BP. On the other hand, PRP6-NO was unable to oligomerize under non-reducing conditions and remained as a single 12.7 kDa monomer due to the substitution of two essential folding Cys residues (C498A / C510A) and the truncation of 13 C-terminal amino acids from the C-terminus of the C4BP oligomerization domain (Kask et al. (2002) Biochemistry 41: 9349-9357).

[0358] Example 2: PRP6-HO7 downregulates the activation phenotype of DCs Neither PRP-HE8 nor PRP6-NO affected the expression of the DC activation marker CD83 or the DC costimulatory molecule CD86 in LPS-matured DCs. Conversely, PRP6-HO7, like PRP-HO7, significantly downregulated these markers (Figure 4).

[0359] Meanwhile, the antigen internalization capacity of DCs was assessed by flow cytometry for both self-quenching DQ-OVA (a marker of mannose receptor-mediated endocytosis). Thus, the endocytic activity of iDCs was significantly increased by treatment with either PRP-HO7 or PRP6-HO7 (Figure 5), but not by the monomeric PRP6-NO or the inactive molecule PRP-HE8.

[0360] We then evaluated whether the effects of different PRP proteins on DC phenotype were accompanied by changes in their release of cytokines (TNF-α and IL-12p70). Compared with untreated iDCs, the secretion of each inflammatory cytokine was upregulated when iDCs were matured with LPS. DCs pretreated with both PRP-HE8 and PRP6-NO secreted cytokines at the same levels as untreated DCs upon maturation. In contrast, pretreatment with both PRP-HO7 and PRP6-HO7 inhibited the release of IL-12p70 and significantly reduced the release of TNF-α (Figure 6). Thus, the production of Th1 proinflammatory cytokines by LPS-mediated DC stimulation was significantly reduced by DCs treated with PRP6-HO7.

[0361] DCs treated with different PRP molecules maintained high viability throughout the differentiation / maturation process, as assessed by Annexin V / 7-ADD staining, evidenced by less than 10% apoptotic cells 48 h after LPS-mediated DC maturation (data not shown).

[0362] Taken together, these data provide evidence that oligomeric PRP6-HO7, but not monomeric PRP6-NO, may modify the differentiation / maturation of proinflammatory DCs toward an anti-inflammatory and tolerogenic phenotype.

[0363] Example 3: PRP6-HO7 lacks complement inhibitory activity One of the major regulatory functions of C4BP is its function as a cofactor for the serine protease factor I to inactivate C4b, also known as cofactor activity. To confirm that PRP6-HO7 effectively lacks the complement inhibitory activity assigned to the CCP1-CPP3 domain of the PRP-HO7 α chain, we performed a comparative C4BP cofactor activity assay to evaluate its contribution to factor I-mediated cleavage of C4b in solution. Thus, both PRP-HE8 and PRP-HO7, but not PRP6-HO7, promoted factor I cleavage of the α' chain of C4b at all concentrations tested, generating a 70 kDa partial cleavage fragment, α3-C4d, and a smaller fragment, C4d (45 kDa) (Figure 7).

[0364] Example 4: PRP6-HO7 affects the expression of surface activation markers in DCs We evaluated whether PRP6-HO7 could affect the expression of different monocyte and DC surface markers, including CD14, HLA-DR, CD40, CD80, CD83, and CD86. While PRP-HE8 did not affect the expression of these markers in LPS-matured DCs, both PRP-HO7 and PRP6-HO7 significantly downregulated not only CD83 and CD86, as described above, but also the costimulatory molecules CD80 and CD40. Conversely, the expression of HLA-DR and CD14 on DCs was not significantly altered by treatment with either PRP-HO7 or PRP6-HO7 (Figure 8).

[0365] These data support the idea that PRP6-HO7 may alter the differentiation / maturation of proinflammatory DCs, as judged by the expression patterns of various cell surface markers. In contrast, neither PRP6-HO7 alone nor PRP6-HO7 and LPS incubated until days 5–7 (maturation) affected the expression of DC surface markers, indicating that DCs were differentiated (data not shown).

[0366] Example 5: Human serum does not inhibit the immunomodulatory activity of PRP6-HO7 To predict the behavior of PRP6-HO7 in a more complex environment, we analyzed the expression of CD83 and CD86 surface markers in DCs differentiated and LPS-matured in the presence of 50% human serum. Similar to PRP-HO7, PRP6-HO7 was able to significantly down-regulate the above markers in a dose-dependent manner (Figure 9). Thus, under near-physiological conditions, PRP6-HO7 was at least as active as its physiological counterpart, PRP-HO7, in terms of immunomodulation.

[0367] Example 6: PRP6-HO7 regulates TLR expression in DCs of both normal individuals and patients with lupus nephritis When DCs treated with both PRP-HO7 and PRP6-HO7 were matured by exposure to Gardiquimod (a TLR7 analog), similar cell surface marker behavior (e.g., significant downregulation of CD83 and CD86) was observed compared with DCs incubated with the TLR4 ligand LPS (Figure 10). TLR7 plays a key role in disease acceleration in systemic lupus erythematosus (SLE). Importantly, the same trends were observed using DCs isolated from SLE patients experiencing lupus nephritis flares, although statistical significance could not be achieved for some DC markers due to the limited number of available samples (Figure 11). Collectively, these data suggest that PRP6-HO7 positively regulates TLR4 and TLR7 expression in both normal individuals and SLE patients with active disease.

[0368] Example 7: PRP6-HO7 alters DC chemotaxis Maturation signals determine the expression of distinct DC functions, such as migration toward lymph node-directed chemokines. Treatment with both PRP-HO7 and PRP6-HO7 downregulated the expression of the chemokine receptor CCR7. Subsequently, reduced surface CCR7 expression significantly reduced migration of LPS-matured DC toward the chemokine CCL21 (Figure 12). In contrast, LPS maturation of both untreated and PRP-HE8-treated DCs induced maximal migration in response to CCL21.

[0369] Example 8: DCs exposed to PRP6-HO7 upregulate "find me" receptors and downregulate PTGER3 and EGLN3 at the transcriptional level To further evaluate the effects of PRP6-HO7 on several key transcripts consistent with the molecular signature of inflammatory monocyte-derived iDCs, we analyzed the expression of genes encoding PGE2 receptor (EP3; PTGER3), a key mediator of acute inflammation (Kawahara et al. (2015) Biochim. Biophys. Acta 1851: 414-421), and prolyl hydroxylase 3 (EGLN3; PHD3) by RT-qPCR. Recently, loss of PHD3 in myeloid cells has been shown to attenuate inflammatory responses (Beneke et al. (2017) Cell Death Dis. 8: e2976). Accordingly, both PTGER3 and EGLN3 transcripts were preferentially downregulated by PRP6-HO7 (Figure 13). In contrast, transcriptional profiling of all known "find me" receptors in iDCs revealed significant upregulation of FPR2, P2RY2, and S1PR1 under treatment with PRP6-HO7 but not PRP6-HO7, suggesting an increased efferocytic capacity to mediate the tolerance response from PRP6-HO7-treated DCs (Kolb et al. (2017) Trends Immunol. 38: 705-718) (Figure 14).

[0370] Example 9: PRP6-HO7 downregulates the polarized phenotype of monocyte-derived macrophages Macrophages are widely distributed innate immune cells that play a central role in host defense against invading pathogens, as well as maintaining immune homeostasis, contributing to inflammation, and promoting wound healing and tissue repair. They are dynamic cells that mature under the influence of signals from the local microenvironment.

[0371] We differentiated human peripheral blood monocytes into undifferentiated macrophages (M0), then polarized them into M1 (classically activated macrophages) with LPS and IFN-γ, and into M2 (alternatively activated macrophages) with IL-4. M1 and M2 macrophages were identified by flow cytometry as CD80 macrophages, respectively. + CD64 + and CD11b + CD209 + Preincubation with PRP-HO7, especially PRP6-HO7, significantly downregulated the expression of both M1 (CD80 and CD64) and M2 (CD11b and CD209) polarization markers (Figure 15). Thus, the inhibition of macrophage polarization / activation toward the M1 or M2 phenotype, resulting in the maintenance of the undifferentiated-like phenotype of macrophages, was caused by the immunomodulatory activity of PRP6-HO7, but was hardly induced by the immunomodulatory activity of PRP-HO7, and not by the immunomodulatory activity of PRP-HE8.

[0372] Example 10: PRP6-HO7 upregulates "find me" receptors and downregulates the expression of ALCAM, SLC16A1, and LMNB1 in M0 non-differentiated macrophages To examine the effects of PRP6-HO7 on several key transcripts consistent with the molecular signature of monocyte-derived macrophages differentiating toward M0, as in DCs, we performed a time-course assay in which we sequentially analyzed the expression of genes encoding activated leukocyte cell adhesion molecule (ALCAM; CD166), which is upregulated in activated monocytes (Levesque et al. (1998) Arthritis Rheum. 41: 2221-2229); SLC16A1, a lactate transporter induced by autophagy (Bao et al. (2017) Theriogenology 87: 339-348); and LMNB1, a key component of the nuclear lamina that interacts with the nuclear autophagy protein LC3 (Dou et al. (2015) Nature 527: 105-109). These three transcripts were found to be significantly downregulated throughout the differentiation process of monocytes to macrophages, primarily through the action of PRP6-HO7 (Figure 16). Therefore, downregulation of both SLC16A1 and LMNB1 suggests that PRP6-HO7-mediated autophagy is inhibited. In contrast, transcriptional profiling of all known "find me" or olfactory receptors in M0 macrophages revealed that PRP6-HO7 treatment significantly upregulated CCR2, CX3CR1, CXCR1, FPR2, and P2RY2, but not PRP-HO7 or PRP-HE8 treatment (Figure 17). A recent study revealed another interplay between pathways leading to autophagy and phagocytosis. ATG7-deficient macrophages were found to have elevated levels of class A scavenger receptors due to the accumulation of p62 (Cadwell and Philips (2013) Immunity 39: 425-427). Upregulation of these receptors resulted in increased phagocytic uptake rates and increased bacterial engulfment, revealing that loss of autophagy can increase phagocytosis (Bonilla et al. (2013) Immunity 39: 537-547).

[0373] Example 11: PRP6-HO7 modulates the cytokine signature of M1 inflammatory macrophages Finally, we investigated the effects of PRP-HO7 and PRP6-HO7 treatment on cytokine induction in cell supernatants during macrophage polarization from M0 to M1 and on the profile of inflammatory cytokines released by M1 macrophages. Notably, a cytokine array containing 36 cytokines, chemokines, and acute-phase proteins demonstrated differential downregulation of CCL1, CCL2, MIP-1, CCL5, CXCL1, IL-6, and TNF-α by PRP-HO7 and PRP6-HO7 compared with untreated M1 cells. Furthermore, CCL1 and MIP-1 showed stronger downregulation in the presence of PRP6-HO7 (Figure 18). Thus, the upregulation of these inflammatory chemokines during M1 macrophage polarization (Sokol and Luster (2015) Cold Spring Harb. Perspect. Biol. 7: a016303) was inhibited by PRP6-HO7 treatment, but not significantly by PRP-HO7 treatment.

[0374] Example 12: Yeast purification of PRP6-HO7 The corresponding DNA sequence of PRP6-HO7 was codon-optimized for P. pastoris. The synthetic gene was cloned into the pBGZα vector (Bioingenium, Barcelona Science Park, Baldiri Reixac, 15-21 (Helix building) 08028 Barcelona) using the BsaI cloning site. The expression vector pBGZα contained a GAP promoter and an AOX transcription terminator. Additionally, it encoded a Zeocin resistance cassette for clonal selection and was flanked by loxP sites for future reversion of the resistance phenotype, if necessary. Furthermore, the vector contained an ampicillin resistance gene and a pUC origin of replication for plasmid selection and propagation in E. coli, respectively. Finally, an S. cerevisiae α mating factor secretion signal between the promoter and the gene coding sequence enabled protein expression directed to the secretory pathway.

[0375] To avoid the use of methanol in the final industrial-scale production process, it was decided to control gene expression under the constitutive GAP promoter. Taking advantage of the ability of P. pastoris to secrete heterologous proteins, the gene of interest was cloned in frame with the α-mating factor of S. cerevisiae (SEQ ID NO: 62). MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEA (SEQ ID NO: 62)

[0376] The sequence of the recombinant polypeptide including the signal peptide was SEQ ID NO:63. MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAHHHHHHLCCPEPKLNNGEITQHRKSRPANHCVYFYGDEISFSCHETSRFSAICQGDGTWSPRTPSCGDETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKEL (SEQ ID NO: 63)

[0377] The yeast was then cultured and the protein purified from Pichia pastoris. The culture parameters are shown in Table 4. Figure 19 shows the visualized purified protein.

[0378] [Table 4]

[0379] Example 13: Bacterial production of PRP6-HO7 The nucleotide sequence of PRP6-HO7 was codon-optimized for expression in E. coli, subcloned into pUC57 plasmid, and cloned into the expression vector pET32a. The sequence of SEQ ID NO: 61 was expressed in the BL-21(D3) strain. Figure 20 shows the visualized purified protein.

[0380] Example 14 In a classical, chemically induced DSS colitis mouse model (C57BL / 6J), 2–5% DSS (w / v) polymers with molecular weights of 35–50 kDa were dissolved in the drinking water of mice for 5–8 days to induce colitis. From day 9 onwards, animals were sacrificed and evaluated based on various parameters.

[0381] Assessment was performed using the Disease Activity Index (DAI), a cumulative index of ulcerative colitis that quantifies weight loss, rectal bleeding, and stool consistency.

[0382] All of these observations in DSS mice are also seen in human inflammatory bowel disease.

[0383] material and method animal Twenty-six 8-week-old female C57BL / 6 mice weighing approximately 20 g were supplied by Charles River (Sant Cugat del Valles, Barcelona, ​​Spain).

[0384] During the experimental procedure, animals were identified by ear punching.

[0385] Environment and Management Upon arrival, animals were housed in groups of 3-4 per cage. They were kept in an environmentally controlled room (ventilated, 22 ± 2°C temperature, and 35-65% humidity) with a 12-hour light / dark cycle. They underwent a 5-day acclimation period between the day of arrival and the start of the procedure. During this period, animals were observed to ensure their general health. They were fed a maintenance low-fat rodent chow ad libitum. Tap water was provided ad libitum.

[0386] Animal Welfare Animals were maintained in accordance with Legislative Decree 214 / 1997 of 30 July on the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (86 / 609 / EU).

[0387] All experimental procedures were approved by IDIBELL's Ethical Committee on Human and Animal Experimentation (CEEA) (procedure number: 19003) and the Animal Experimentation Commission of the Generalitat de Catalunya (Catalan Government) DAAM: 10561.

[0388] Test material identification and preparation PRP6-HO7 was purified from the bacterial expression system as described above, concentrated to 1.0 mg / mL in Dulbecco's phosphate-buffered saline (DPBS), and stored at −80°C in 1 mL vials.

[0389] PRP-HO7 was purified from human plasma, concentrated to 6.2 mg / mL in Dulbecco's phosphate-buffered saline (DPBS), and stored at −80°C in 1 mL vials.

[0390] Preparation for injection: PRP6-HO7 and PRP-HO7 solutions were prepared by diluting the respective stock solutions with DPBS to the desired concentration of 0.4 mg / mL (60 μg dose / mouse). The formulations were prepared immediately before administration.

[0391] Identification and preparation of reference materials Minocycline was dissolved in 0.1% Tween-80 plus carboxymethylcellulose (CMC) (0.5% w / v in water) at a concentration of 5 mg / mL.

[0392] The formulation was prepared immediately before administration.

[0393] Experimental procedure Colitis Protocol (Days 0-9) (Figure 21): Colitis was induced by adding 2% DSS (w / v) to normal drinking water, which was given to the mice ad libitum. On day 3, the DSS solution was replaced with a fresh 2% DSS solution.

[0394] On day 5, the DSS solution was replaced with tap water until day 9. -Blank animals (non-colitis group) were given water regularly throughout the study (n=4). - Vehicle treatment (n=4): DPBS was injected subcutaneously in a volume of 0.15 mL into control (DSS-colitis group) animals on days 4, 6 and 8. - PRP6-HO7 and PRP-HO7 treatment: 0.15 mL of PRP6-HO7 and PRP-HO7 (60 μg / each) were subcutaneously injected into the animals with this dosing schedule. Groups PRP6-HO7 and PRP-HO7 (n=6 / each): administered on days 4, 6 and 8. Reference compound treatment (n=6) (Days 0-8): Minocycline (50 mg / kg) was administered daily by oral gavage (PO) to the animals in a volume of 10 mL / kg.

[0395] Animal weight, total blood in the stool, and stool consistency were recorded daily for each mouse. Each of these parameters was assigned a score and used to calculate the average daily disease activity index (DAI) for each animal (Table 5) (Melgar et al. 2005. Am J Physiol Gastrointest Liver Physiol. 288:1328-38). On day 9, animals were anesthetized with 2% isoflurane and euthanized by cervical dislocation.

[0396] [Table 5]

[0397] statistical analysis Statistical analysis and scientific graphing were performed using Graphpad Prism 6 software (Graphpad software, Inc., La Jolla, CA, USA). Body weight and clinical score data (DAI) were analyzed using two-way analysis of variance (2-way ANOVA) followed by Bonferroni's post-hoc test. Data are expressed as mean ± SEM. In all cases, a P value <0.05 was considered significant.

[0398] Example 14: Therapeutic effect of PRP6-HO7 in a mouse model of DSS-induced colitis The purpose of this comparative study was to evaluate whether PRP6-HO7 treatment, compared with the wild-type molecule PRP-HO7, could attenuate intestinal inflammation induced in a DSS-induced colitis model. Therefore, both PRP6-HO7 and PRP-HO7 were administered subcutaneously at 60 μg per mouse. The antibiotic and chemical immunomodulator minocycline (Garrido-Mesa et al. 2011. Pharmacol Res. 63:308-19; Garrido-Mesa et al. 2011. Biochem Pharmacol. 82: 1891-1900) was used as a control treatment.

[0399] During the experiment, clinical parameters such as animal weight, occurrence of diarrhea and total blood present in the stool were assessed and collected for each mouse to calculate a disease progression score or DAI.

[0400] After colitis was fully established, repeated subcutaneous administration of PRP6-HO7 at a dose of 60 μg on days 4, 6, and 8 prevented / alleviated inflammatory lesions in DSS-colitis mice.

[0401] Administration of DSS at a concentration of 2% (w / v) for 5 days induced a colonic inflammatory state similar to that previously described (Melgar et al. 2005. Am J Physiol Gastrointest Liver Physiol. 288:1328-38; Garrido-Mesa et al. 2011. Pharmacol Res. 63:308-19; Randhawa et al. 2014. Korean J Physiol Pharmacol. 18:279-88). The DSS-induced colonic inflammatory process was associated with a decrease in mouse weight, likely due to the occurrence of anorexia and diarrhea in the colitis control group compared with non-colitic mice (blank group), which were clearly observable from day 6 after DSS administration. These differences began between days 5 and 6 of the experiment and gradually increased over the course of the experiment, correlating with the severity of inflammation and the progression of colitis (Figure 22).

[0402] Consistent with the weight loss, the DAI scores also progressively increased in control colitic mice throughout the study period (Figure 23).

[0403] All DSS-treated mice experienced moderate constipation during the first 5 days of the experiment. After this period, stools lost consistency and diarrhea persisted in most DDS-treated animals.

[0404] Results obtained after daily administration of minocycline in this experimental protocol revealed that this compound exhibited an anti-inflammatory effect on the intestine, although the effect was not statistically significant on day 8. In contrast, when intestinal damage was already evident in DSS-treated mice, treatment with three subcutaneous doses of PRP-HO7 or PRP6-HO7, starting on day 4 after colitis induction, significantly inhibited weight loss and maintained stool consistency. Importantly, PRP6-HO7 was even more effective than PRP-HO7, as evidenced by a significant decrease in DAI observed 48 hours after the initial PRP6-HO7 treatment compared to untreated colitic mice, which became highly significant by the end of the study (days 8 and 9) (Figure 23), primarily related to the inhibition of weight loss and the improvement of stool consistency. Indeed, the PRP-HO7 and PRP6-HO7 groups showed significantly reduced inflammatory responses compared to the control group, suppressed weight loss, and visible reductions in DAI values ​​(Figures 22 and 23).

[0405] Statistical differences were also evident in the percentage of weight loss (days 8 and 9 vs. day 0) when comparing both PRP-HO7 and PRP6-HO7 to control colitic animals. Thus, therapeutic administration of PRP6-HO7 demonstrated a significant ability to reduce the severity of colitis.

Claims

1. A recombinant polypeptide comprising the CCP6 domain of the C4BPα chain and an oligomerization domain, wherein the recombinant polypeptide does not comprise any of the CCP1, CCP2, CCP3, CCP4, CCP5, CCP7 and CCP8 domains of the C4BPα chain, and wherein the polypeptide comprises SEQ ID NO:

3.

2. 2. The recombinant polypeptide of claim 1, wherein the polypeptide consists of SEQ ID NO:3 or SEQ ID NO:3 and an additional methionine at the N-terminus of the polypeptide.

3. 2. The recombinant polypeptide of claim 1, wherein the polypeptide consists of a sequence selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:

7.

4. 2. The recombinant polypeptide of claim 1, wherein the polypeptide consists of SEQ ID NO:

6.

5. A homo-oligomer formed by six or seven recombinant polypeptides according to any one of claims 1 to 4.

6. The homo-oligomer according to claim 5, wherein the homo-oligomer is formed by seven recombinant polypeptides according to any one of claims 1 to 4.

7. The homo-oligomer according to claim 5 or 6, wherein the polypeptide is a polypeptide consisting of SEQ ID NO:

6.

8. A pharmaceutical comprising a recombinant polypeptide according to any one of claims 1 to 4, a homo-oligomer according to any one of claims 5 to 7, a polynucleotide encoding the recombinant polypeptide according to any one of claims 1 to 4, a vector comprising said polynucleotide, a host cell comprising said vector, or a pharmaceutical composition comprising said recombinant polypeptide, said homo-oligomer, said polynucleotide, said vector or said host cell and a pharmaceutically acceptable carrier.

9. A pharmaceutical for the prevention and / or treatment of an immunological disease caused by undesired activation of the immune system, comprising a recombinant polypeptide according to any one of claims 1 to 4, a homo-oligomer according to any one of claims 5 to 7, a polynucleotide encoding the recombinant polypeptide according to any one of claims 1 to 4, a vector comprising said polynucleotide, a host cell comprising said vector, or a pharmaceutical composition comprising said recombinant polypeptide, said homo-oligomer, said polynucleotide, said vector or said host cell and a pharmaceutically acceptable carrier.

10. 1. A method for generating a population of tolerogenic dendritic cells in vitro, comprising: (i) incubating a population of dendritic precursor cells under conditions suitable for the formation of a population of immature dendritic cells; and (ii) incubating the population of immature dendritic cells obtained in step (i) under conditions suitable for the formation of mature dendritic cells. Including, The steps (i) and / or (ii) are (a) a polypeptide according to any one of claims 1 to 4; (b) the homo-oligomer according to any one of claims 5 to 7; (c) a polynucleotide encoding the recombinant polypeptide of any one of claims 1 to 4, and (d) a vector containing the polynucleotide of (c) The method is carried out in the presence of a composition comprising a substance selected from the group consisting of:

11. 1. A method for generating a population of tolerogenic macrophages in vitro, comprising: (i) incubating a population of macrophage precursor cells under conditions suitable for the formation of a population of immature macrophages; and (ii) incubating the population of immature macrophages obtained in step (i) under conditions suitable for the formation of mature macrophages. Including, The steps (i) and / or (ii) are (a) a polypeptide according to any one of claims 1 to 4; (b) the homo-oligomer according to any one of claims 5 to 7; (c) a polynucleotide encoding the recombinant polypeptide of any one of claims 1 to 4, and (d) a vector containing the polynucleotide of (c) The method is carried out in the presence of a composition comprising a substance selected from the group consisting of:

12. Tolerogenic dendritic cells obtained by the method of claim 10 or tolerogenic macrophages obtained by the method of claim 11, wherein FPR2 ｈｉｇｈ , P2RY2 ｈｉｇｈ and / or S1PR1 ｈｉｇｈ or CCR2 ｈｉｇｈ , CXCR1 ｈｉｇｈ , FPR2 ｈｉｇｈ and / or P2RY2 ｈｉｇｈ The macrophage.

13. A medicament for the prevention and / or treatment of immunological diseases caused by unwanted activation of the immune system, said medicament comprising tolerogenic dendritic cells or tolerogenic macrophages according to claim 12.

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